Synthetic method of polypeptide containing flexible joint

Abstract

The invention relates to the field of polypeptide synthesis, in particular to a synthesis method of polypeptide containing a flexible joint. According to the polypeptide synthesis method, resin solid-phase synthesis is adopted, and a mode of condensing amino acids one by one is not adopted in condensation of a flexible joint. The flexible joint is a GS joint or a (G) n joint, and n is equal to 2-10; the flexible joint is preferably a GGS, a GGGS, a GGGGS or a (G) n joint, and n is equal to 6-8. The polypeptide synthesized by the method is a peptide chain-flexible joint or a peptide chain-flexible joint-X, X is an amino acid containing an amino group on a side chain, and the amino group is connected with an active functional group; the peptide chain is preferably GLP1 and a mutant derivative thereof. According to the polypeptide synthesis method disclosed by the invention, the loss of amino acid in the synthesis process is reduced, the resin aggregation phenomenon is avoided, the synthesis yield is improved, the purification difficulty is reduced, and the purification yield and the product purity are improved; and the method is high in repetition rate and stable in multi-batch production yield.

Description

[0001] Cross-reference information

[0002] This application claims priority to a patent application with application number "202311845982.X" filed on December 28, 2023 and invention name "A method for synthesizing polypeptides containing flexible linkers", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to the field of polypeptide synthesis, and in particular, to a method for synthesizing a polypeptide containing a flexible linker. Background Art

[0004] A polypeptide refers to a compound in which multiple amino acids are linked together by peptide bonds. One of the meanings of fusion protein is the expression product of two genes recombined through DNA recombination technology. The principle of constructing fusion protein is to delete the stop codon of the first protein gene and then connect the second protein gene with a stop codon to achieve the fusion expression of the two genes. As an indispensable component of fusion protein recombination, Linker plays an important role in constructing stable and biologically active fusion proteins. Linker is an amino acid chain that connects two fusion proteins, which are Flexible Linker, Rigid Linker and Cleavable Linker.

[0005] Flexible linkers have a certain degree of flexibility to allow the proteins on both sides to perform their respective independent functions. Flexible linkers commonly used in polypeptides have sequences mainly composed of glycine and serine residue fragments ("GS" linkers) or sequences composed of only glycine residue fragments ((G)n linkers). The most widely used flexible linker is the (Gly-Gly-Gly-Gly-Ser)n sequence. By changing the size of n, the distance between the domains can be enlarged or reduced to achieve domain separation (n≥2) or connect the domains to maintain the necessary inter-domain interactions. Another common type of flexible linker is the (Gly)n sequence, where n is greater than or equal to 5. The advantage is that during the yeast expression and purification process, it can resist protease hydrolysis while protecting the functional activity of the protein domains at both ends.

[0006] In solid phase synthesis of peptides, a method of gradual condensation of amino acids is usually used. The applicant of the present application has found that in the synthesis of peptides containing flexible linkers GS or (G)n, if a method of gradual condensation of amino acids is used, resin aggregation and condensation difficulties occur, resulting in the loss of amino acids, poor purity of crude products, and extremely low yields after purification.

[0007] To solve the above problems, the present application provides a method for synthesizing a polypeptide containing a flexible linker, which can reduce the loss of amino acids in the synthesis, improve the synthesis yield, and at the same time reduce the difficulty of purification and improve the purification yield. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present application provides a method for synthesizing a polypeptide containing a flexible linker, which can reduce the loss of amino acids in the synthesis, improve the synthesis yield, and reduce the difficulty of purification, thereby improving the purification yield and product purity. The method can be used for the synthesis of polypeptides containing a "GS" linker or a (G)n linker.

[0009] The present application discloses a method for synthesizing a polypeptide containing a flexible linker, which uses resin solid phase synthesis, and in the condensation of the flexible linker, the method of condensing amino acids one by one is not used. The flexible linker is a "GS" linker or a (G)n linker, where n=2-10, preferably 5-9, more preferably 6-8; specifically, GGS, GGGS, GGGGS, or a (G)n linker.

[0010] When the joint is GGGGS, condensation is performed in one of the following ways:

[0011] (a1) using GGGGS pentapeptide condensation;

[0012] (a2) using S and then GGGG tetrapeptide condensation;

[0013] (a3) condensation using S, GG dipeptide, and GG dipeptide in sequence;

[0014] (a4) using S, G, and GGG tripeptides for condensation in sequence;

[0015] (a5) Condensation is carried out using S, GGG tripeptide and G in sequence.

[0016] When the joint is GGS, condensation is performed in one of the following ways:

[0017] (b1) condensation using GGS tripeptide;

[0018] (b2) Condensation using S and GG dipeptides in sequence.

[0019] When the joint is GGGS, condensation is performed in one of the following ways:

[0020] (c1) using GGGS tetrapeptide condensation;

[0021] (c2) condensation using S and GGG tripeptides in sequence;

[0022] (c3) condensation using S, G, and GG dipeptides in sequence;

[0023] (c4) Condensation is carried out using S, GG dipeptide and G in sequence.

[0024] In the polypeptide synthesis method described in the present application, in the condensation of the flexible joint, the molar amount of the amino acid (such as Gly, Ser) or polypeptide (such as GG dipeptide, GGG tripeptide, GGGG tetrapeptide, GGS tripeptide, GGGS tetrapeptide, GGGGS pentapeptide) is 2-5 times the active group on the resin. In the condensation of the flexible joint, the condensation reagents are OXYMA and DIC; the molar amount of the condensation reagent is preferably the same as the amount of the amino acid or polypeptide used for the condensation. In the condensation of the flexible joint, the amino acid or polypeptide with protected N-terminal amino group and OXYMA are dissolved in an organic solvent, preferably at a concentration of 0.2-0.5 mol / L, and then DIC is added. After ice bath activation, it is added to the reactor for condensation reaction. The ice bath activation time can be 5-20 min, preferably 10-15 min; the reaction time can be 1-4 h, preferably 2-3 h.

[0025] In the polypeptide synthesis method described in the present application, in the condensation of the flexible linker, an amino acid or polypeptide with Fmoc protection at the N-terminus is used for condensation reaction, and the Fmoc protecting group is removed after condensation, and then the subsequent amino acid or polypeptide is condensed. After the condensation reaction, a piperidine solution is used to remove Fmoc, such as a piperidine / DMF solution, a piperidine / DCM solution, a piperidine / DMSO solution, or a piperidine / NMP solution; preferably a piperidine / DMF solution, more preferably a 10-30% piperidine / DMF solution; the amount of piperidine solution used is preferably 10-40mL / g resin. The piperidine solution is added in two portions, first adding a portion of the piperidine solution and stirring the reaction, removing the liquid, and then adding the remaining piperidine solution and stirring the reaction, and then removing the liquid.

[0026] In the condensation of the flexible linker, an amino acid or polypeptide whose N-terminus is protected by Fmoc and whose side chain active groups are protected is usually used for the condensation reaction; preferably, the side chain hydroxyl of Ser is protected by a tert-butyl group. After the polypeptide condensation is completed, the resin is cleaved and the side face protecting groups are removed.

[0027] In the polypeptide synthesis method described in the present application, the resin is an unmodified CTC resin; the degree of substitution is 0.2-1 mmol / g, preferably 0.35-0.5 mmol / g.

[0028] In the polypeptide synthesis method described in the present application, the number of "GS" linkers in the synthesized polypeptide can be 2, 3, 4, 5, or 6; the number of amino acids in the linker is generally 8-16, preferably 10-15. When the linker in the synthesized polypeptide is GGGGS, the number of linkers is preferably 2-3, when the linker is GGGS, the number of linkers is preferably 3-4, and when the linker is GGS, the number of linkers is preferably 4-5.

[0029] In the polypeptide synthesis method described in the present application, the number of amino acids in the synthesized polypeptide excluding the linker is 4 or more, preferably 10-60, and more preferably 20-50.

[0030] The present application provides a method for synthesizing a polypeptide containing a flexible linker, which comprises the following steps:

[0031] (1) Resin swelling;

[0032] (2) Connecting the first amino acid: Add the first amino acid whose N-terminal amino group is protected and whose side chain active groups are protected to a reactor filled with resin, and add a base to react;

[0033] (3) End-capping;

[0034] (4) removing the N-terminal amino protecting group;

[0035] (5) Condensation of flexible linkers: Condensation is not carried out by condensing amino acids one by one:

[0036] (6) When the flexible joint is a "GS" joint, repeat steps (4)-(5) according to the number of joints;

[0037] (7) Condensation of subsequent amino acids: adding subsequent amino acids in sequence by repeating step (4) and amino acid condensation, using an amino acid whose N-terminal amino group is not protected when condensing the most N-terminal amino acid, and using amino acids whose N-terminal amino group is protected for the remaining amino acids, to obtain a fully protected peptide-resin;

[0038] (8) cracking the resin and removing the side chain protecting groups;

[0039] (9) Purify to obtain the polypeptide product.

[0040] The polypeptide synthesis method described in the present application, step (1) resin swelling is specifically: weigh the resin, add it to the reactor, add the organic solvent, turn on the nitrogen to mix the liquid and the resin from bottom to top so that they are evenly mixed, so that the resin is fully swollen, and a small amount of organic solvent is added during the period to prevent the solution from volatilizing to dryness, and finally, the solvent is filtered out through the sand core. The amount of organic solvent used is 5-20mL / g resin, preferably 8-15mL / g resin, and more preferably 10-12mL / g resin. The mixing time is 5-30min, preferably 10-25min, and more preferably 15-20min. The organic solvent used can be DCM, DMF, DMSO, NMP, DMAc.

[0041] In the polypeptide synthesis method described in the present application, in steps (2) and (7), depending on the different amino acids, the protecting groups on the side chain active groups can be selected from Boc, tert-butyl, trityl, Pbf, and ivdde; the molar amount of the amino acid is 2-5 times, preferably 3-4 times, the amount of the active groups on the resin.

[0042] Step (2) is specifically as follows: using an organic solvent to dissolve the first amino acid whose N-terminal amino group is protected and whose side chain active group is protected, adding the solution to a reactor, dropping a base, and reacting; preferably, the base is an organic amine, more preferably DIEA or TEA; the molar amount of the base is preferably 3-6 times, preferably 4-5 times, of the active group on the resin. The reaction time is 2-8 hours, preferably 3-6 hours, more preferably 4-5 hours. The organic solvent used may be DCM, DMF, DMSO, NMP, or DMAc.

[0043] In step (7), the condensation reagents for amino acid condensation are OXYMA and DIC; the molar amount of the condensation reagent is preferably the same as the amount of the amino acid.

[0044] The specific steps of amino acid condensation in step (7) include: dissolving the amino acid with protected side chain active groups and OXYMA in an organic solvent, preferably at a concentration of 0.2-0.5 mol / L, more preferably 0.3 mol / L, adding DIC, activating in an ice bath, and adding to a reactor for reaction; the ice bath activation time is 5-20 min, preferably 10-15 min; the reaction time is 1-4 h, preferably 2-3 h. After the condensation reaction, the solution is removed using a sand core, and then washed with an organic solvent. The organic solvent used can be DCM, DMF, DMSO, NMP, or DMAc.

[0045] The polypeptide synthesis method described in this application can use a detection reagent to detect whether the condensation reaction is completed, and the detection reagent is ninhydrin. The detection is specifically as follows: take a dozen resins, wash them three times with alcohols such as ethanol and methanol, add ninhydrin detection reagent for detection, heat at 105°C to 120°C for 5 minutes, and colorless is a negative reaction, indicating that the condensation reaction is completed.

[0046] In the polypeptide synthesis method described in the present application, step (3) specifically comprises adding alcohol for end-capping reaction, removing the liquid, and washing with an organic solvent. The alcohol is preferably methanol, ethanol, or isopropanol, the amount of alcohol used is 1-3 mL / g resin, and the end-capping reaction time is 30 min-3 h, preferably 1-2 h. The organic solvent is used for washing 2-6 times, preferably 3-5 times. The organic solvent used may be DCM, DMF, DMSO, NMP, or DMAc.

[0047] In the polypeptide synthesis method described in the present application, the N-terminal protecting group is removed under the corresponding removal conditions according to the different N-terminal protecting groups, and the liquid is removed and then washed with an organic solvent. When the N-terminal protecting group is an Fmoc protecting group, a piperidine solution can be used for removal, preferably a piperidine / DMF solution, more preferably a 10-30% piperidine / DMF solution, and most preferably a 20% piperidine / DMF solution. The amount of piperidine solution used is 10-40 mL / g resin, preferably 20-30 mL / g resin. The piperidine solution can be added in two times, first adding a portion (such as 1 / 2) of the piperidine solution and stirring the reaction (5-15 min), removing the liquid, and then adding the remaining piperidine solution and stirring the reaction (5-15 min) and then removing the liquid; preferably stirring the reaction under nitrogen protection. After removal, wash with an organic solvent 3-8 times, preferably 4-6 times. The organic solvent used can be DCM, DMF, DMSO, NMP, or DMAc.

[0048] The polypeptide synthesis method described in the present application can use a detection reagent to detect whether the N-terminal amino protecting group removal reaction is completed, and the detection reagent is ninhydrin. The detection is specifically as follows: take a dozen resins, wash them three times with alcohols such as ethanol and methanol, add ninhydrin detection reagent for detection, heat at 105°C to 120°C for 5 minutes, and turn dark blue for a positive reaction, indicating that the removal reaction is complete.

[0049] In the polypeptide synthesis method described in the present application, step (8) cleaves the resin and removes the side chain protecting groups; a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water can be used as a protecting group cleavage solution for removal, and after removal, the resin is filtered out, added to ether and stirred thoroughly, and then precipitated, centrifuged or filtered to obtain a crude polypeptide. Preferably, a mixed solution of TFA:TIS:water=95:2.5:2.5 is used, and after ice bathing, it is added to the fully protected peptide-resin for cleavage. Preferably, glacial ether is used for ether, and the amount used is 5-15 times the volume of the cleavage solution, preferably 10-12 times.

[0050] In the polypeptide synthesis method described in the present application, the purification in step (9) is preferably performed using preparative HPLC, using a mixed solution of water:acetonitrile = 8:2 to dissolve the crude cyclic peptide, and filtering through a microporous filter membrane, such as 0.45 μm, for purification, and the mobile phase is 0.1% TFA and acetonitrile.

[0051] In the polypeptide synthesis method described in the present application, the synthesized polypeptide can be a peptide chain-flexible linker, preferably a peptide chain-flexible linker-X, and X is preferably an amino acid containing an amino group in the side chain, such as Lys, DAB (2,4-diaminobutyric acid), DAP (2,6-diaminopimelic acid), and ORN (ornithine). The side chain amino group of this C-terminal amino acid can be connected to an active functional group, such as a thiol, a halogen, or an alkynyl group, and this active group in the synthesized polypeptide can be combined with a protein, which is beneficial for downstream use. In the polypeptide synthesis method described in the present application, the synthesized polypeptide can be a peptide chain-flexible linker-Lys, preferably a peptide chain-flexible linker-Lys (bromoacetic acid), a peptide chain-flexible linker-Lys (bromopropionic acid), and a peptide chain-flexible linker-Lys (thioglycolic acid). In the polypeptide synthesis method described in the present application, the N-terminal amino acid of the peptide chain in the synthesized polypeptide is preferably histidine, such as the synthesized polypeptide can be H-peptide chain-flexible linker-Lys (bromoacetic acid), more preferably H-peptide chain-(GGGGS)m-Lys (bromoacetic acid). The peptide chain part of the polypeptide synthesized in the present application is preferably GLP1 and its mutant derivatives. In addition to the C-terminal Lys, the protecting groups on the amino acid side chains used in the condensation process can be Boc, tert-butyl, Pbf, Trt.

[0052] The polypeptide synthesis method described in the present application, the synthesized polypeptide is specifically shown in SEQ ID NO:1:

[0053] SEQ ID NO:1

[0054] HXEXFTSDYSSYLEEQAAKEFIAWLXGGGGGGGSGGGGSGGGGSK; 2-X is Aib, i.e. 2-aminoisobutyric acid; 4-X and 26-X represent no amino acid, or any number of amino acids, such as 1-2 random amino acids; 45-K is Lys (Ac-Br).

[0055] The synthetic polypeptide is more specifically shown in SEQ ID NO:2:

[0056] SEQ ID NO:2

[0057] HXEGTFTSDYSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSK; 2-X is Aib, i.e. 2-aminoisobutyric acid, and 47-K is Lys (Ac-Br).

[0058] When synthesizing H-peptide chain-flexible linker-Lys (bromoacetic acid), H(Trt)-peptide chain-flexible linker-Lys(ivdde)-resin can be obtained according to steps (1)-(7) in the above method; (8-1) using Boc anhydride to protect the N-terminal amino group, preferably using an organic solvent such as DCM, DMF, DMSO, NMP, DMAc for dissolution, preferably with a concentration of 0.2-0.5 mol / L, preferably reacting in the presence of an organic amine such as DIEA, TEA, more preferably using 3-6 eq of an organic amine. (8-2) adding a hydrazine hydrate solution to remove the ivdde side chain protecting group on Lys, preferably a hydrazine hydrate / DMF solution, more preferably a 5-10% hydrazine hydrate / DMF solution. The amount of hydrazine hydrate solution used is 10-40 mL / g resin, preferably 20-30 mL / g resin. The hydrazine hydrate solution can be added in two times. First, a portion (such as 1 / 2) of the piperidine solution is added and stirred for reaction (10-40 minutes, preferably 20-30 minutes). After removing the liquid, the remaining piperidine solution is added and stirred for reaction (10-40 minutes, preferably 20-30 minutes) and then the liquid is removed; preferably, the reaction is stirred under nitrogen protection. After removal, it is washed with an organic solvent for 3-8 times, preferably 4-6 times. The organic solvent can be DCM, DMF, DMSO, NMP, DMAc. (8-3) Lys side chain amino bromoacetylation, using bromoacetic acid as a raw material, the molar amount is preferably 2-5 times that of the product of the previous step, more preferably 3-4 times; the condensation reagent is OXYMA and DIC; the molar amount of the condensation reagent is preferably the same as the amount of bromoacetic acid; specifically, bromoacetic acid and OXYMA are dissolved in an organic solvent, the concentration is preferably 0.2-0.5 mol / L, more preferably 0.3 mol / L, and then DIC is added, activated in an ice bath, and then added to a reactor for reaction; the ice bath activation time is 5-20 min, preferably 10-15 min; the reaction time is 1-4 h, preferably 2-3 h. After the condensation reaction, the solution is removed using a sand core, and then washed with an organic solvent. The organic solvent used can be DCM, DMF, DMSO, NMP, or DMAc. (8-4) Cleavage of the resin and removal of other side chain protecting groups; a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water can be used as a protecting group cleavage solution for removal. After removal, the resin is filtered out, added to ether and stirred thoroughly, and then precipitated. Centrifugation or filtration is performed to obtain a crude polypeptide. Preferably, a mixed solution of TFA:TIS:water = 95:2.5:2.5 is used, and after an ice bath, it is added to the fully protected peptide-resin for cleavage. Preferably, glacial ether is used as ether, and the amount used is 5-15 times the volume of the cleavage solution, preferably 10-12 times. (9) Purification is performed to obtain a polypeptide product.

[0059] After research, it is found that compared with the prior art, the present invention has at least the following beneficial effects:

[0060] 1. In the condensation of the flexible linker during polypeptide synthesis, the present application does not adopt the method of condensing amino acids one by one, which reduces the loss of amino acids in the synthesis process, improves the synthesis yield, and reduces the difficulty of purification, thereby improving the purification yield and product purity.

[0061] 2. Especially in the condensation of GGGGS linkers, the synthesis methods of GG dipeptide, GGG tripeptide, GGGG tetrapeptide and GGGGS pentapeptide are mainly used to avoid resin aggregation and improve the synthesis yield and purity of the polypeptide product.

[0062] 3. In the condensation of flexible joints, the reaction concentration and equivalent of amino acids or polypeptides in the synthesis process are controlled to ensure the yield and purity of the product. In addition, the method has a high reproducibility and stable yield in multiple batches of production. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 HPLC and MS charts of the pure polypeptide obtained in Example 1.

[0064] Figure 2 The MS and HPLC images of the crude polypeptide obtained in Example 2 are shown.

[0065] Figure 3 The MS and HPLC images of the crude polypeptide obtained in Comparative Example 1 are shown. DETAILED DESCRIPTION

[0066] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps used herein are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0067] “Polypeptide” or “peptide” has its ordinary meaning in the art and refers to a compound covalently linked together by condensation dehydration of the carboxyl group of one amino acid molecule with the amino group of another amino acid molecule to form an amide bond (peptide bond).

[0068] The nomenclature used to define peptides is that commonly used in the art, with the amino group at the N-terminus appearing on the left and the carboxyl group at the C-terminus appearing on the right.

[0069] "Amino acid residue" also has its ordinary meaning in the art and refers to the composition of an amino acid (as a single amino acid or as part of a peptide) after it is combined with a peptide, another amino acid, or an amino acid residue. Typically, when an amino acid is combined with another amino acid or amino acid residue, water is removed, and the amino acid that remains is referred to as an amino acid residue.

[0070] "Amino acid" has its ordinary meaning in the art and may include natural amino acids and unnatural amino acids. The abbreviations for amino acid residues in the present invention are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common amino acids.

[0071] Amino acid residues or amino acids have isomeric forms including L-form and D-form.

[0072] The suffixes "-OH" and "-NH2" after a hyphen or bracket refer to the free acid and amide forms of a peptide or amino acid, respectively. For example, Fmoc-Thr-OH refers to the free acid form of a Thr amino acid whose N-terminal amino group is protected by Fmoc. Peptide-NH2 refers to the amino acid at the C-terminal end of the peptide chain in the amide form.

[0073] "Linker", also known as conjugate, is an amino acid chain that connects two fusion proteins. It has a certain flexibility to achieve proper separation of functional domains, allowing the proteins on both sides to perform their independent functions or maintain necessary inter-domain interactions.

[0074] "Protecting group" or "protecting group" has its ordinary meaning in the art. Protecting groups include chemical moieties that are attached to or configured to be attached to a reactive group (i.e., a protected group) within a molecule (e.g., a peptide) so that the protecting group prevents or otherwise inhibits the protected group from participating in a reaction. Protection can be performed by attaching the protecting group to the molecule. Deprotection can occur when the protecting group is removed from the molecule, for example, by a chemical transformation that removes the protecting group.

[0075] When a protective group is indicated in brackets after an amino acid, it means that the reactive side chain group of the amino acid is protected by the protective group in the brackets. For example, Thr(tBu) means that the side chain active group OH is protected by a tert-butyl group as a tert-butyl ether, and Glu(tBu) means that the side chain active group carboxyl is protected by a tert-butyl group as a tert-butyl ester.

[0076] In the synthesis of peptides, suitable protecting groups are often used to protect the reactive side chain groups of various amino acid moieties, which will prevent chemical reactions from occurring at that position until the protecting group is finally removed. "Fully protected peptides" and "fully protected polypeptides" refer to peptides (polypeptides) in which all side chain active groups are protected by protecting groups. In addition, when the whole reacts at the carboxyl group, it is also common to protect the α-amino group of the amino acid or fragment, and then selectively remove the α-amino protecting group to allow subsequent reactions to occur at that position. Although specific protecting groups have been disclosed for solid phase synthesis methods, it should be noted that each amino acid can be protected by various amino acid protecting groups commonly used in liquid phase synthesis.

[0077] "Solid phase synthesis" refers to a synthesis method in which the reactants are attached to an insoluble solid support.

[0078] Solid phase synthesis of peptides refers to first connecting an amino acid to the insoluble solid phase carrier, and then connecting other amino acids to the amino acids connected to the solid phase carrier in sequence. In the solid phase synthesis of peptides, resins are usually used as solid phase carriers. The process of solid phase synthesis of peptides is usually to protect the amino group of the first amino acid at the C-terminus of the target peptide, covalently connect it to the solid phase carrier through the C-terminal carboxyl group, and then use the N-terminus of the amino acid as the starting point of synthesis. After removing the amino protecting group, it undergoes a condensation reaction with the second amino acid (or polypeptide fragment) protected by the amino group, and the peptide chain is extended. The operation is repeated to achieve the ideal length of the synthetic peptide chain. Finally, the peptide chain is cleaved from the resin, separated and purified to obtain the target polypeptide.

[0079] As is known in the art, resins used for solid phase synthesis of polypeptides include, but are not limited to, polystyrene-phenylene crosslinked resins, polyacrylamide resins, polyethylene-glycol resins and derivatives, which can be directly connected to the (first) amino acid after the reactive groups are introduced into these resins. Depending on the reactive groups introduced, these resins and resin derivatives can be divided into chloromethyl resins, carboxyl resins, amino resins or hydrazide resins. Currently, commonly used resins in the art include, but are not limited to, CTC resins, PAM resins, MBHA resins, Wang resins, Rink resins, TRT resins, etc.

[0080] In the process of peptide synthesis, the connection reaction between the first amino acid and the resin is different from the access of subsequent amino acids, especially the degree of substitution of the access is directly related to the synthesis of subsequent peptide chains. In some embodiments of the present invention, the resin used is an unmodified CTC resin. In peptide synthesis, the first amino acid is first connected to the CTC resin, and then a capping reaction is performed to convert the active end groups of the unconnected amino acids on the resin into stable end groups.

[0081] "Condensation" refers to the process of adding new amino acids to bound amino acids or peptides. In the embodiment of the present invention, the preparation of the polypeptide adopts a solid phase method, and the condensation is one by one except for the flexible linker.

[0082] "Condensation reagent", "coupling reagent" or "condensing agent" refers to a reagent used for amino acid condensation in polypeptide solid phase synthesis. In polypeptide solid phase synthesis, different condensation methods and / or different condensation reagents can be selected according to different resins, different amino acid sequences and / or different protecting groups. Condensation reagents include but are not limited to carbodiimide type and onium salt type. Carbodiimide type mainly includes EDCI, DCC, DIC, EDC, etc. Carbodiimide type condensation reagents can be used alone or in combination with other condensation reagents. Carbodiimide type condensation reagents can be used in combination with OXYMA. Onium salt type condensation reagents mainly include HBTU, HATU, PyBOP, etc. During use, onium salt type condensation reagents can be added with organic bases for activating amino acids, such as DIPEA, etc. When multiple condensation reagents are used, these condensation reagents can be used in combination. It should be understood by those skilled in the art that the combined use of two or more condensation reagents means adding the two or more condensation reagents in one condensation reaction. How to use two or more condensation reagents in combination is within the knowledge of those skilled in the art. Those skilled in the art know how to add the two or more condensation reagents in a coupling reaction, such as their amounts, timing and order of addition, etc.

[0083] The present invention is further described below with reference to the examples; however, these examples do not limit the scope of the present invention. Unless otherwise stated, all reactants used in the examples are obtained from commercial sources; the instruments and equipment used in the synthesis experiments and product analysis and detection are conventional instruments and equipment commonly used in organic synthesis.

[0084] Table 1 Commonly used chemical reagents

[0085] Fmoc: fluorenylmethoxycarbonyl

[0086] TFA: trifluoroacetic acid

[0087] TIS: Triisopropylsilane

[0088] MeOH: Methanol

[0089] DCM: dichloromethane

[0090] DMF: N,N-dimethylformamide

[0091] DMSO: dimethyl sulfoxide

[0092] NMP: N-methylpyrrolidone

[0093] DMAc: N,N-dimethylacetamide

[0094] PIP: Piperidine

[0095] DIEA: N,N-diisopropylethylamine

[0096] TEA: triethylamine

[0097] ACN: Acetonitrile

[0098] DIC: N,N'-diisopropylcarbodiimide

[0099] OXYMA: Ethyl 2-oximecyanoacetate

[0100] Boc anhydride: di-tert-butyl dicarbonate

[0101] DAB: 2,4-diaminobutyric acid

[0102] DAP: 2,6-diaminopimelic acid

[0103] ORN: Ornithine

[0104] Trt: trityl

[0105] ivdde: (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylene)-3-methylbutyl

[0106] Pbf: 2,2,4,6,7-pentamethylbenzodihydrofuran-5-sulfonyl

[0107] Aib: 2-aminoisobutyric acid

[0108] Fmoc-Lys(ivdde)-OH: N-fluorenylmethoxycarbonyl-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl]-L-lysine

[0109] Fmoc-Gln(Trt)-OH:N-Fluorenylmethoxycarbonyl-N-trityl-L-glutamine

[0110] Fmoc-Glu(tBu)-OH:N-Fluorenylmethoxycarbonyl-L-glutamic acid-5-tert-butyl ester

[0111] Fmoc-Gly-OH:N-Fluorenylmethoxycarbonyl-glycine

[0112] Fmoc-His(Trt)-OH:N-Fmoc-N'-trityl-L-histidine

[0113] Fmoc-Ile-OH:N-Fluorenylmethoxycarbonyl-L-isoleucine

[0114] Fmoc-Leu-OH:N-Fluorenylmethoxycarbonyl-L-leucine

[0115] Fmoc-Lys(Boc)-OH:N-Fluorenylmethoxycarbonyl-N-tert-butyloxycarbonyl-L-lysine

[0116] Fmoc-Phe-OH:N-Fluorenylmethoxycarbonyl-L-phenylalanine

[0117] Fmoc-Ser(tBu)-OH:N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-serine

[0118] Fmoc-Thr(tBu)-OH:N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine

[0119] Fmoc-Trp(Boc)-OH:N-Fluorenylmethoxycarbonyl-N-in-tert-butyloxycarbonyl-L-tryptophan

[0120] Fmoc-Tyr(tBu)-OH:N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine

[0121] Fmoc-Val-OH:N-Fmoc-L-Valine

[0122] Fmoc-Ala-OH: N-Fmoc-L-alanine

[0123] Fmoc-Aib-OH: N-Fmoc-2-aminoisobutyric acid

[0124] Embodiment 1:

[0125] The polypeptide sequence is the sequence shown in SEQ ID NO: 2

[0126] H{Aib}EGTFTSDYSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGS{Lys(Ac-Br)}

[0127] (1) Resin swelling: Weigh 5 g of unmodified 2-chlorotrityl chloride (CTC) resin with a degree of substitution of 0.35 mmol / g, add it to the reactor, add 50 mL of DCM, turn on the nitrogen gas to mix the liquid and resin from bottom to top so that they are evenly mixed for 15 minutes to allow the resin to fully swell. During this period, pay attention to adding a small amount of DCM to prevent the solution from evaporating to dryness, and filter out the solvent through a sand core.

[0128] (2) Connecting the first amino acid: Weigh 3 times the molar amount (3 times the molar amount of the active groups on the resin) of Fmoc-Lys(ivdde)-OH, add DCM to dissolve, add to the reactor, add 5 eq (5 times the molar amount of the active groups on the resin) of DIEA dropwise, and react for 4 hours.

[0129] (3) End-capping: 5 mL of methanol was then added for end-capping reaction for 1 h, the liquid was removed, and the mixture was washed three times with 50 mL of DMF.

[0130] (4) Removal of the N-terminal Fmoc protecting group: Add 50 mL of 20% piperidine / DMF solution to the reactor, stir and react for 5 min under nitrogen protection, remove the liquid, add 50 mL of 20% piperidine / DMF, stir and react for 5 min under nitrogen protection, remove the liquid, and wash with 50 mL of DMF 6 times. Take a dozen resins, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 105°C to 110°C for 5 min, and turn dark blue for a positive reaction.

[0131] (5) Condensation: Weigh 3 times the moles (3 times the moles of the active groups on the resin) of Fmoc-Gly-Gly-Gly-Gly-Ser(tBu)-OH pentapeptide amino acid and 3 times the moles (3 times the moles of the active groups on the resin) of OXYMA, add DMF to dissolve, control the concentration to 0.3 mol / L, then add 3 times the moles (3 times the moles of the active groups on the resin) of DIC to activate in an ice bath for 10 minutes, put into the reactor, and react for 2 hours. Draw out the solution through the sand core and wash 3 times with 50 mL of DMF. Take a dozen resins, wash three times with ethanol, add ninhydrin detection reagent for detection, heat at 120°C for 2 minutes, and colorless is a negative reaction.

[0132] (6) Repeat steps (4)-(5): add subsequent amino acids in sequence by repeatedly removing the Fmoc protecting group and adding amino acids to react, wherein the GGGGS flexible linker part uses Fmoc-Gly-Gly-Gly-Gly-Ser(tBu)-OH pentapeptide and the N-terminal His uses H(Trt)-OH until H(Trt){Aib}E(tBu)GT(tBu)FT(tBu)S(tBu)D(tBu)Y(tBu)S(tBu)S(tBu)Y(tBu)LE(tBu)E(tBu)Q(Trt)AAK(Boc)E(tBu)FIAW(Boc)LVK(Boc)GGGGGGGS(tBu)GGGGS(tBu)GGGGS(tBu){Lys(ivdde)}-resin is synthesized.

[0133] (7) Protecting the N-terminal amino group: Weigh 3 times the molar amount (3 times the molar amount of the active group on the resin) of Boc anhydride, add DMF to dissolve, control the concentration to 0.3 mol / L, add 6 eq of DIEA, put it into the reactor, and react for 2 h.

[0134] (8) Removal of the ivdde protecting group on Lys: Add 50 mL of 5% hydrazine hydrate / DMF solution, stir and react for 30 min under nitrogen protection, remove the liquid, add 50 mL of 5% hydrazine hydrate / DMF solution again, stir and react for 30 min under nitrogen protection, remove the liquid, repeat 3 times, and wash 6 times with 50 mL DMF. Take a dozen resins, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 120°C for 5 min, and turn dark blue for positive reaction.

[0135] (9) Lys side chain amino bromoacetylation: Weigh 3 times the mole of bromoacetic acid and 3 times the mole of OXYMA, add DMF to dissolve, control the concentration to 0.3 mol / L, then add 3 times the mole of DIC to activate in an ice bath for 10 minutes, put it into the reactor, and react for 2 hours. Draw out the solution through the sand core, wash 3 times with 50 mL of DMF, take a dozen resins, wash three times with ethanol, add the detection reagent for detection, heat at 120 ° C for 2 minutes, and colorless is a negative reaction. Add 50 ml of methanol and wash repeatedly 4 times and then drain. The drained resin weighs 14.75 g.

[0136] (10) Resin cleavage and removal of side chain protecting groups: A cleavage solution of 147 ml was prepared with trifluoroacetic acid: TIS: water in a ratio of 90:8:2. After an ice bath for 1 h, the solution was added to the resin and cleaved for 3 h. After cleavage, the resin was filtered through a sand core and the filtrate was slowly added to 10 times the volume of ice ether. The mixture was stirred thoroughly and then allowed to settle. The mixture was centrifuged at 3000 rpm and washed three times with ether to obtain a solid centrifugal precipitate.

[0137] (11) Purification: Add a mixed solution of pure water and acetonitrile (8:2) to the crude polypeptide, stir with ultrasonic oscillation until completely dissolved, filter the polypeptide solution through a microporous filter membrane with a pore size of 0.45 μm, use 0.1% TFA solution as mobile phase A and acetonitrile as mobile phase B, and purify by preparative HPLC; the prepared solution is freeze-dried to obtain 1.56 mg of white powder with a yield of 19.5%; HPLC-MS detection shows a purity of 98.5%.

[0138] Specific conditions of HPLC: SHIMADZU high performance liquid chromatography was used to analyze the purity of the pure peptide, the analytical column was Inertsil ODSSP (4.6×250 mm×5 μm), the injection volume was 30 μL, the mobile phase A was 0.1% TFA / water, B was 0.1% TFA / acetonitrile, the flow rate was 1 mL / min, and the detection wavelength was 220 nm.

[0139] Embodiment 2:

[0140] The polypeptide sequence is the sequence shown in SEQ ID NO: 2

[0141] H{Aib}EGTFTSDYSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGS{Lys(Ac-Br)}

[0142] (1) Resin swelling: Weigh 3 g of unmodified 2-chlorotrityl chloride (CTC) resin with a degree of substitution of 0.35 mmol / g, add it to the reactor, add 30 mL of DCM, turn on the nitrogen gas to mix the liquid and resin from bottom to top so that they are evenly mixed for 15 minutes to allow the resin to fully swell. During this period, pay attention to adding a small amount of DCM to prevent the solution from evaporating to dryness, and filter out the solvent through a sand core.

[0143] (2) Connecting the first amino acid: Weigh 3 times the molar amount (3 times the molar amount of the active groups on the resin) of Fmoc-Lys(ivdde)-OH, add DCM to dissolve, add to the reactor, add 5 eq (5 times the molar amount of the active groups on the resin) of DIEA dropwise, and react for 4 hours.

[0144] (3) Capping: 3 mL of methanol was added for capping reaction for 1 h, the liquid was removed, and 30 mL of DMF was used for washing 3 times.

[0145] (4) Removal of the N-terminal Fmoc protecting group: Add 30 mL of 20% piperidine / DMF solution to the reactor, stir and react for 3 min under nitrogen protection, remove the liquid, add 30 mL of 20% piperidine / DMF, stir and react for 5 min under nitrogen protection, remove the liquid, and wash with 30 mL of DMF 6 times. Take a dozen resins, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 105°C to 110°C for 5 min, and turn dark blue for a positive reaction.

[0146] (5) Condensation: Weigh 3 times the moles (3 times the moles of the active groups on the resin) of Fmoc-Ser(tBu)-OH and 3 times the moles (3 times the moles of the active groups on the resin) of OXYMA, add DMF to dissolve, control the concentration to 0.3 mol / L, then add 3 times the moles (3 times the moles of the active groups on the resin) of DIC to activate in an ice bath for 10 minutes, put into the reactor, and react for 2 hours. Draw out the solution through the sand core, wash 3 times with 30 mL of DMF. Take a dozen resins, wash three times with ethanol, add ninhydrin detection reagent for detection, heat at 120°C for 2 minutes, and colorless is a negative reaction.

[0147] (6) Repeat steps (4)-(5): add subsequent amino acids in sequence by repeatedly removing the Fmoc protecting group and adding amino acids to the reaction, wherein the GGGGS flexible linker part uses Fmoc-Ser(tBu)-OH, Fmoc-Gly-Gly-OH, and Fmoc-Gly-Gly-OH, and the N-terminal His uses H(Trt)-OH, until H(Trt){Aib}E(tBu)GT(tBu)FT(tBu)S(tBu)D(tBu)Y(tBu)S(tBu)S(tBu)Y(tBu)LE(tBu)E(tBu)Q(Trt)AAK(Boc)E(tBu)FIAW(Boc)LVK(Boc)GGGGGGS(tBu)GGGGS(tBu)GGGGS(tBu){Lys(ivdde)}-resin is synthesized.

[0148] (7) Protecting the N-terminal amino group: Weigh 3 times the molar amount (3 times the molar amount of the active group on the resin) of Boc anhydride, add DMF to dissolve, control the concentration to 0.3 mol / L, add 6 eq of DIEA, put it into the reactor, and react for 2 h.

[0149] (8) Removal of the ivdde protecting group on Lys: Add 30 mL of 5% hydrazine hydrate / DMF solution, stir and react for 30 min under nitrogen protection, remove the liquid, add 30 mL of 5% hydrazine hydrate / DMF solution again, stir and react for 30 min under nitrogen protection, remove the liquid, repeat 3 times, and wash 6 times with 30 mL of DMF. Take a dozen resins, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 120°C for 5 min, and turn dark blue for positive reaction.

[0150] (9) Lys side chain amino bromoacetylation: Weigh 3 times the mole of bromoacetic acid and 3 times the mole of OXYMA, add DMF to dissolve, control the concentration to 0.3 mol / L, then add 3 times the mole of DIC to activate in an ice bath for 10 minutes, put it into the reactor, and react for 2 hours. Draw out the solution through the sand core, wash 3 times with 30 mL of DMF, take a dozen resins, wash three times with ethanol, add the detection reagent for detection, heat at 120 ° C for 2 minutes, and colorless is a negative reaction. Add 30 ml of methanol and wash repeatedly for 4 times, then drain. The dried resin weighs 10.8 g.

[0151] (10) Resin cleavage and removal of side chain protecting groups: trifluoroacetic acid: TIS: water = 90:8:2 to prepare cleavage solution, a total of 108 ml, after ice bath for 1 hour, add to the resin, cleave for 3 hours, filter the resin through a sand core after cleavage, slowly add the filtrate to 10 times the volume of ice ether, stir well and then settle, centrifuge at 3000 rpm, wash with ether 3 times, and obtain a solid centrifugal precipitate. Its MS and HPLC graphs are shown in the following figure. Figure 2 shown.

[0152] (11) Purification: Add a mixed solution of pure water and acetonitrile (8:2) to the crude polypeptide, stir with ultrasonic oscillation until completely dissolved, filter the polypeptide solution through a microporous filter membrane with a pore size of 0.45 μm, use 0.1% TFA solution as mobile phase A and acetonitrile as mobile phase B, and purify by preparative HPLC; the prepared solution is freeze-dried to obtain 0.73 mg of white powder with a yield of 15.2%; HPLC-MS detection shows that the purity is greater than 98%.

[0153] The HPLC conditions were the same as in Example 1.

[0154] Comparative Example 1:

[0155] The other steps remain the same as in Example 1, except that in step (6), the single amino acids are gradually condensed throughout the entire process. The MS and HPLC images of the resulting crude polypeptide are as follows: Figure 3 The test results showed that the resin aggregated severely, and multiple peaks were found during purification, which were corresponding to the peaks of amino acid deletions of 1 G, 2 Gs, and 3 Gs, indicating that the amino acid G was severely deleted and the deletion position was different.

[0156] As can be seen from the examples and comparative examples, in the condensation process of the flexible linker, the present application does not use the method of condensing amino acids one by one, but uses GG dipeptide, GGG tripeptide, GGGG tetrapeptide, or GGGGS pentapeptide for condensation, which reduces the number of amino acids in the synthesis process, improves the synthesis yield, reduces the difficulty of purity, and improves the purification efficiency. Moreover, the applicant has found that the method has a high synthesis repetition rate and a stable yield in multiple batches of production.

[0157] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0158] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0159] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for synthesizing a polypeptide containing a flexible linker, characterized in that: Resin solid phase synthesis is used, and the condensation of the flexible linker is not carried out by condensing amino acids one by one.

2. The method for synthesizing a polypeptide according to claim 1, characterized in that: The flexible linker is a "GS" linker or a (G)n linker, where n=2-10; the flexible linker is preferably GGS, GGGS, GGGGS, or a (G)n linker, where n=6-8.

3. The method for synthesizing a polypeptide according to claim 2, characterized in that: When the joint is GGGGS, condensation is performed in one of the following ways: (a1) using GGGGS pentapeptide condensation; (a2) using S and then GGGG tetrapeptide condensation; (a3) condensation using S, GG dipeptide, and GG dipeptide in sequence; (a4) using S, G, and GGG tripeptides for condensation in sequence; (a5) condensation using S, GGG tripeptide, and G in sequence; When the joint is GGS, condensation is performed in one of the following ways: (b1) condensation using GGS tripeptide; (b2) condensation using S and GG dipeptides in sequence; When the joint is GGGS, condensation is performed in one of the following ways: (c1) using GGGS tetrapeptide condensation; (c2) condensation using S and GGG tripeptides in sequence; (c3) condensation using S, G, and GG dipeptides in sequence; (c4) Condensation is carried out using S, GG dipeptide and G in sequence.

4. The method for synthesizing a polypeptide according to any one of claims 1 to 3, characterized in that: In the condensation of the flexible linker, the molar amount of the amino acid or polypeptide is 2-5 times that of the active groups on the resin.

5. The method for synthesizing a polypeptide according to any one of claims 1 to 3, characterized in that: In the condensation of the flexible linker, the condensation reagents are OXYMA and DIC; the molar amount of the condensation reagents is preferably the same as the amount of the amino acid or polypeptide used in the condensation.

6. The method for synthesizing a polypeptide according to claim 5, characterized in that: In the condensation of the flexible linker, the amino acid or polypeptide with protected N-terminal amino group and OXYMA are dissolved in an organic solvent, preferably at a concentration of 0.2-0.5 mol / L, and then DIC is added. After activation in an ice bath, the mixture is added to a reactor for condensation reaction.

7. The method for synthesizing a polypeptide according to claim 6, characterized in that: The ice bath activation time is 5-20 min, preferably 10-15 min; the reaction time is 1-4 h, preferably 2-3 h.

8. The method for synthesizing a polypeptide according to any one of claims 1 to 3, characterized in that: In the condensation of the flexible linker, an amino acid or polypeptide with Fmoc protection at the N-terminus is used for the condensation reaction, and after the condensation, the Fmoc protecting group is removed, and then the subsequent amino acid or polypeptide is condensed.

9. The method for synthesizing a polypeptide according to claim 8, characterized in that: After the condensation reaction, Fmoc is removed using a piperidine solution; preferably a piperidine / DMF solution, a piperidine / DCM solution, a piperidine / DMSO solution, a piperidine / NMP solution; more preferably a 10-30% piperidine / DMF solution; and the amount of the piperidine solution used is preferably 10-40 mL / g resin.

10. The method for synthesizing a polypeptide according to claim 9, characterized in that: The piperidine solution is added in two portions. First, a portion of the piperidine solution is added, stirred for reaction, and then the liquid is removed. Then, the remaining piperidine solution is added, stirred for reaction, and then the liquid is removed.

11. The method for synthesizing a polypeptide according to any one of claims 1 to 3, characterized in that: The resin is an unmodified CTC resin; the degree of substitution is 0.2-1 mmol / g, preferably 0.35-0.5 mmol / g.

12. The method for synthesizing a polypeptide according to any one of claims 2 to 11, characterized in that: The number of "GS" linkers in the synthesized polypeptide can be 2, 3, 4, 5, or 6; the number of amino acids in the linker is generally 8-16, preferably 10-15.

13. The method for synthesizing a polypeptide according to claim 12, characterized in that: In the synthesized polypeptide, when the linker is GGGGS, the number of linkers is 2-3, when the linker is GGGS, the number of linkers is 3-4, and when the linker is GGS, the number of linkers is 4-5.

14. The method for synthesizing a polypeptide according to any one of claims 1 to 13, characterized in that: The number of amino acids in the synthesized polypeptide excluding the linker is 4 or more, preferably 10-60, more preferably 20-50.

15. The method for synthesizing a polypeptide according to any one of claims 1 to 13, characterized in that: The synthesized polypeptide is a peptide chain-flexible linker, or a peptide chain-flexible linker-X, wherein X is an amino acid containing an amino group on the side chain, and the amino group is connected to an active functional group; preferably, the peptide chain is GLP1 and its mutant derivatives; preferably, the active functional group is a thiol, a halogen, or an alkynyl.

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