A kind of synthetic method of tepote

Abstract

The invention provides a method for synthesizing tepote, comprising the steps of reacting Fmoc-Linker-OH, HBTU and swollen AM Resin to obtain Fmoc-Linker-AM Resin; adding Fmoc-Ser(tBu)-OH and HOBT to a reaction system of resin and PIP / DMF and reacting completely; coupling from the C-terminus to the N-terminus to obtain a fully protected peptide resin; adding a cleavage reagent to the fully protected peptide resin, reacting and collecting the filtrate, cooling and precipitating, and drying to obtain a crude peptide. The method solves the problem that the linker on the MBHA Resin in the prior art will break and generate new impurities; avoids the disadvantage of using heavy metal palladium or genotoxic substance hydrazine hydrate; solves the problem that the C-terminal Pro 36 、Pro 37 、Pro 38 The problem of [‑(Pro‑Pro)] missing impurities produced by step-by-step coupling is solved; the finished product is saved and is conducive to industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and specifically provides a method for synthesizing telpotide. Background Art

[0002] Tirzepatide is a dual agonist of GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) receptors developed by Eli Lilly. It produces a synergistic effect on appetite, caloric intake and metabolic function by activating these two receptors that are closely related to blood sugar regulation. The chemical structure of tirzepatide has been carefully designed to act on both GLP-1 and GIP receptors. This dual agonism helps improve blood sugar control in patients with type 2 diabetes and shows significant effects in weight loss. Tirzepatide is highly selective for human GIP and GLP-1 receptors and has high affinity for both receptors. Its activity on GIP receptors is similar to that of natural GIP hormone, while its activity on GLP-1 receptors is relatively low. Tirzepatide has a molecular weight of 4813.53Da, a molecular weight of C225H348N48O68, and an elimination half-life of approximately 5 days, which allows it to be administered once a week. The peptide sequence is: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -Gln 19 -Lys 20 (AEEA-AEEA-γ-Glu-eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2.

[0003] In Chinese patent CN202211588132.1, MBHA Resin is used as a carrier, and Fmoc Pro Pro ProOH, Fmoc Gly Gly OH, Fmoc Lys(AEEA-AEEA γGlu(α OtBu) Eicosanedioic acid(montBu)) OH, Fmoc Ile Aib OH, Fmoc Thr(tBu) Phe OH, and Boc Tyr(tBu) Aib Glu(OtBu) GlyOH fragments are used as starting materials to synthesize telopoietin resin. When MBHA Resin is used as a carrier, the linker breaks and connects to the Trp side chain during TFA cleavage, which will produce an impurity of [+163]. Although the use of Fmoc-Pro-Pro-Pro-OH fragment solves the problem of missing Pro-Pro, it is a non-commercial product with a high finished product yield, which is not conducive to later industrial production. In the synthesis process, Fmoc-Gly-Gly-OH and Fmoc-Gly-Ala-OH fragments are not used, which will produce an increased impurity of [+Gly].

[0004] In Chinese patent CN202311050210.7, AM Resin is used as a carrier, and Fmoc Gly Gly OH, FmocLys(R1) OH(R1=OAll or Dde), Fmoc Ile Aib OH, Fmoc Thr(tBu) Phe OH, Boc Tyr(tBu)Aib OH, and Fmoc Glu(OtBu) Gly OH fragments are used as starting materials to synthesize telpotide resin. When FmocLys(R1) OH(R1=OAll or Dde) is used, heavy metal palladium or genotoxic substance hydrazine hydrate will be used in the synthesis process; the step-by-step coupling of C-terminal Pro36, Pro37, and Pro38 will produce [-(Pro-Pro)] missing impurities; the step-by-step coupling of the side chain will bring new missing and increase impurities, reduce purity and yield, and is not conducive to industrial production.

[0005] In Chinese patent CN202011642239.0, MBHA Resin is used as a carrier, BocTyr(tBu) Aib OH, Fmoc Lys(AEEA-AEEA γGlu (α OtBu) Eicosanedioic acid(mon tBu)) OH, Fmoc TyrSer(pro me) OH, and Fmoc (Dmb)Gly OH fragments are used as starting materials to synthesize telpotide resin. When MBHA Resin is used as a carrier, the linker breaks and connects to the Trp side chain during TFA cleavage, which will produce the impurity [+163]. The use of Fmoc Tyr Ser (pro me) OH fragment is a non-commercial product, and the finished product is high, which is not conducive to later industrial production. In the synthesis process, Fmoc Gly Gly OH, Fmoc Ile Aib OH, and Fmoc Thr (tBu) Phe OH fragments are not used, which will produce increased impurities [+Gly] and missing impurities [-Ile] and [-Thr]. The stepwise coupling of C-terminal Pro36, Pro37, and Pro38 will produce missing impurities [-(Pro-Pro)].

[0006] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the invention

[0007] Technical issues

[0008] In order to solve the above problems, the present invention aims to provide a method for synthesizing telopoietin using Fmoc-Pro 36 -Pro 37-OH solves the problem of missing impurities of [-(Pro-Pro)] due to deprotection; using AM Resin as a carrier avoids impurities generated by the Linker during the cleavage process; using the Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH fragment simplifies the coupling of tepote and improves the purity and yield of the crude peptide; using Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Ile-Aib-OH, and Fmoc-Gly-Gly-OH fragments as starting materials greatly reduces the missing impurities of [-Tyr], [-Thr], and [-Ile] and the added impurities of [+Gly], significantly improves the purity of the crude product, is conducive to purification, improves the yield of the product, reduces the production cost, and has good application prospects.

[0009] Technical Solution

[0010] In order to achieve the above-mentioned purpose, the inventors of the present application have conducted in-depth research and fundamentally solved the problem that the linker on the MBHA Resin in the prior art will break and produce new impurities; it also avoids the disadvantages of using heavy metal palladium or genotoxic substance hydrazine hydrate in the prior art; and solves the problem that the C-terminal Pro 36 、Pro 37 、Pro 38 The problem of missing impurities of [-(Pro-Pro)] produced by step-by-step coupling is solved; the finished product is saved, which is conducive to industrial production.

[0011] The present invention includes the following aspects.

[0012] Scheme 1, a method for synthesizing a fully protected peptide resin of telportopeptide, comprising:

[0013] Step S1, Fmoc-Linker-OH and HBTU react with swollen AM Resin in the presence of an activator to prepare Fmoc-Linker-AM Resin;

[0014] Step S2, Fmoc-Ser(tBu)-OH and HOBT are added to the reaction system of Fmoc-Linker-AM Resin and PIP / DMF solution, and reacted under DIC activation conditions until the ninhydrin test is negative;

[0015] Step S3, repeat the above steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment, and complete the peptide resin coupling steps from C-terminus to N-terminus according to the telpotide sequence, wash the resin at least 3 times with an equal volume of methanol to the resin, and vacuum dry to obtain the telpotide fully protected peptide resin.

[0016] In step S1 of some preferred embodiments, the degree of substitution of the AM Resin is between 0.1-1 mmol / L.

[0017] In step S1 of some preferred embodiments, the swelling of AM Resin is specifically performed by swelling the resin with DMF of 2-5 times the volume of AM Resin for 1-2 hours, and then removing the DMF.

[0018] In step S1 of some preferred embodiments, the swollen AM Resin needs to be washed with NMM / DMF solution and DMF in sequence before participating in the reaction to remove the detergent.

[0019] In step S1 of some preferred embodiments, the resin is washed with NMM / DMF solution by washing the resin at least twice with 1-6% NMM / DMF solution of 2-5 times the volume of the swollen AM Resin, and then removing the NMM / DMF solution.

[0020] In step S1 of some preferred embodiments, the resin is washed with DMF by washing the resin at least 5 times with DMF having a volume of 2-5 times that of the swollen AMResin, and then removing the DMF.

[0021] In step S1 of some preferred embodiments, the activating agent is NMM with 2-2.5 equivalents of Fmoc-Linker-OH.

[0022] In some preferred embodiments, step S1 specifically includes: adding Fmoc-Linker-OH and HBTU in a ratio of 3:2.85 into an activation bottle, adding DMF to dissolve, and then adding NMM, and slowly adding to the reactor containing swollen AM Resin after activation, and continuously reacting at 25±15°C. After the reaction is completed, the reaction liquid is extracted and washed with DMF at least 3 times to obtain Fmoc-Linker-AMResin.

[0023] In some more preferred embodiments, the activation is performed at 0-10°C for 5-10 min.

[0024] In some more preferred embodiments, the end of the reaction means that the reaction is terminated when the ninhydrin test is negative.

[0025] In step S2 of some preferred embodiments, the reaction system of the resin and the PIP / DMF solution comprises: adding an equal volume of the PIP / DMF solution to the resin obtained in step S1 and reacting for 5 minutes, removing the reaction solution and then adding an equal volume of the PIP / DMF solution to the resin and reacting for 15 minutes, and after removing the reaction solution, adding DMF to wash the resin at least 8 times until the ninhydrin test is positive.

[0026] In some more preferred embodiments, the PIP / DMF solution is a 10-40% PIP / DMF solution.

[0027] In some preferred embodiments, step S2 specifically includes: adding Fmoc-Ser(tBu)-OH and HOBT to DMF solution in a ratio of 3:3, adding DIC equivalent to Fmoc-Ser(tBu)-OH for activation under ice-water bath conditions, adding the reaction solution to a reactor, and continuing the reaction at 25±15°C until the ninhydrin test is negative, removing the reaction solution, and washing at least 3 times with an equal volume of DMF to the resin.

[0028] In some more preferred embodiments, the activation time is 5-10 min.

[0029] In step S3 of some preferred embodiments, the step of completing peptide resin coupling from the C-terminus to the N-terminus specifically comprises: coupling from the C-terminus to the N-terminus in sequence, using the following fragments: Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH, Fmoc-Ile-Aib-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH to complete peptide resin coupling.

[0030] In step S3 of some preferred embodiments, the step of completing the peptide resin coupling from the C-terminus to the N-terminus specifically comprises: using Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib- OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH complete the peptide resin coupling.

[0031] In step S3 of some preferred embodiments, the Fmoc-Ile-Aib-OH uses HATU and NMM as coupling reagents, and the feed ratio is Fmoc-Ile-Aib-OH:HATU:NMM=3:2.85:6.

[0032] In step S3 of some preferred implementation schemes, the feed ratio of Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH to HOBT and DIC is 1.5:1.5:1.5.

[0033] According to the present invention, AM Resin resin is used as a carrier. Compared with MBHA Resin, impurities generated due to linker breakage during the cleavage process are avoided; Fmoc-Pro 36 -Pro 37-OH solves the problem of [-(Pro-Pro)] missing impurities caused by deprotection, thereby reducing the difficulty of purification and improving the product yield; compared with the use of Fmoc-Pro-Pro-Pro-OH, the production cost is reduced, which is conducive to industrial production; the use of Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH fragment simplifies the coupling of telpotide and improves the purity and yield of the crude peptide; the use of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Ile-Aib-OH, and Fmoc-Gly-Gly-OH fragments as starting materials greatly reduces [-Tyr], [-Thr], and [-Ile] missing impurities and [+Gly] increased impurities. In addition, a coupling strategy of Fmoc-Pro-Pro-OH and Fmoc-Pro-OH was adopted when coupling Fmoc-Pro-Pro-OH; Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Thr5(tBu)-Phe6-OH, Fmoc-Ile-Aib-OH, Fmoc Lys20[Octadecanedioic(tBu) γ Glu(OtBu)PEG2 PEG2]-OH, and Fmoc-Gly-Gly-OH fragments were also used; impurities, racemic impurities, and missing impurities were reduced, and the purity, net peptide content, and yield of the crude product were significantly improved. The yield of the crude product was not less than 94.5%, and the HPLC purity was not less than 77%, which was beneficial to the subsequent purification, improved the yield of the product, reduced the production cost, was beneficial to industrial production, and had good application prospects.

[0034] Scheme 2. The fully protected peptide resin of telportipeptide prepared by the method described in Scheme 1 is specifically: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu-Eicosanedioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin.

[0035] Scheme 3, a method for synthesizing telpotide, comprising adding a cleavage reagent to the telpotide fully protected peptide resin described in Scheme 2 above, reacting and collecting the filtrate, and then adding it to cold methyl tert-butyl ether, centrifuging, collecting the precipitate, and drying to obtain a crude telpotide peptide.

[0036] In some preferred embodiments, the cleavage reagent is a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 88-95:6-3:3-1:3-1.

[0037] In some preferred embodiments, the added amount of the lysate is 10-15 mL / g in volume mass ratio to the telpotide fully protected peptide resin.

[0038] In some preferred embodiments, the reaction time is at least 2 hours.

[0039] In some preferred embodiments, the reaction temperature is 25±5°C.

[0040] In some preferred embodiments, the filtrate includes the filtrate collected after the reaction and the filtrate obtained by washing the resin with TFA at least three times.

[0041] In some preferred embodiments, after collecting the precipitate by centrifugation, the precipitate is washed at least three times with cold methyl tert-butyl ether.

[0042] In some preferred embodiments, the crude yield of the fully protected telpoxetine peptide resin is not less than 94.5%, and the HPLC purity is not less than 71%.

[0043] Furthermore, the synthesis method of telpotide also includes: dissolving the crude telpotide peptide in a phosphate buffer of pH 6.5 containing 0.08-0.1 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain the pure telpotide peptide. The isoelectric point of tepote is about 4.6±0.1. The use of a phosphate buffer solution with a pH of 6.8 to dissolve the crude tepote peptide helps to improve the solubility of tepote peptide, so that it is fully dissolved, and the crude concentration is not less than 55 g / L. The crude peptide solution after dissolution is more stable and is not prone to precipitation, thereby avoiding peptide aggregation, adsorption, and separation. The inventors further found that adding a trace amount of sulfobutyl-β-cyclodextrin to the pH of 6.5 phosphate buffer solution for dissolving the crude tepote peptide can significantly improve the separation effect, so that after only conventional chromatographic purification, a pure tepote peptide with a purity of not less than 99.4% and a maximum single impurity of not more than 0.2% can be obtained, which greatly simplifies the purification steps and is greatly improved compared to the complicated preparation and purification process of the prior art.

[0044] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0045] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0046] Beneficial Effects

[0047] According to the synthesis method of tepote peptide of the present invention, by using Fmoc-Pro 36 -Pro 37 -OH solves the problem of missing impurities of [-(Pro-Pro)] due to deprotection; using AM Resin as a carrier avoids impurities generated by the Linker during the cleavage process; using the Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH fragment simplifies the coupling of tepote and improves the purity and yield of the crude peptide; using Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Ile-Aib-OH, and Fmoc-Gly-Gly-OH fragments as starting materials greatly reduces the missing impurities of [-Tyr], [-Thr], and [-Ile] and the added impurities of [+Gly], significantly improves the purity of the crude product, is conducive to purification, improves the yield of the product, reduces the production cost, and has good application prospects.

[0048] Adding a trace amount of sulfobutyl-β-cyclodextrin to the pH 6.5 phosphate buffer used to dissolve the crude telpotide can significantly improve the separation effect, so that a pure telpotide peptide with a purity of not less than 99.4% and a maximum single impurity of not more than 0.2% can be obtained after only conventional chromatographic purification, which greatly simplifies the purification steps and is a great improvement over the complicated preparation and purification process of the prior art.

[0049] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0051] Figure 1 It represents the technical roadmap of the synthesis method of telpotide;

[0052] Figure 2 A schematic diagram showing the yield of crude tepoxetine peptide;

[0053] Figure 3 Schematic diagram showing the HPLC purity of crude tepoxetine peptide;

[0054] Figure 4 A schematic diagram showing the purity of pure tepoxetine peptide;

[0055] Figure 5 Schematic diagram showing the maximum single impurity content of pure tepoxetine peptide. DETAILED DESCRIPTION

[0056] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0057] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0058] Unless otherwise specified, all percentages, parts, ratios, etc. are by weight; otherwise specified, including but not limited to, "%", "wt%", "mass%" means weight percentage, "mol%" means molar percentage, and "vol%" means volume percentage.

[0059] When a quantity, concentration or other numerical value or parameter is given as a range, a preferred range or a series of upper preferred values ​​and lower preferred values, it should be understood that it specifically discloses all ranges formed by any pair of numerical values ​​of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether the range is disclosed separately. For example, when describing the range of "1 to 5 (1-5)", the described range should be understood to include the range of "1 to 4 (1-4)", "1 to 3 (1-3)", "1 to 2 (1-2)", "1 to 2 (1-2) and 4 to 5 (4-5)", "1 to 3 (1-3) and 5" etc. Unless otherwise stated, where a numerical range is described herein, the range includes the range end values ​​and all integers and fractions within the range.

[0060] Unless specifically stated, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are still described herein.

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0062] In order to facilitate the understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention are first explained or defined. The relevant explanations or definitions do not absolutely limit the meanings of the abbreviations and key terms. For undefined abbreviations or key terms, they are commonly understood by those skilled in the art.

[0063] GLP-1: glucagon-like peptide-1;

[0064] GIP: glucose-dependent insulinotropic polypeptide;

[0065] Fmoc: 9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, is an amino protecting group commonly used in organic synthesis;

[0066] HBTU: full name O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, is a condensation agent commonly used in peptide synthesis;

[0067] AM Resin: amino resin;

[0068] HOBT: 1-Hydroxybenzotriazole, an amide condensation agent commonly used in peptide synthesis;

[0069] PIP: piperidine;

[0070] DMF: N,N-dimethylformamide;

[0071] DIC: 1,3-diisopropylcarbodiimide, N,N'-Diisopropylcarbodiimide;

[0072] NMM: N-Methylmorpholine.

[0073] The present invention is described in detail below

[0074] Embodiment 1:

[0075] Provided is a method for synthesizing a telpotide fully protected peptide resin, comprising:

[0076] (1) Weigh AM Resin with a degree of substitution of about 0.5 mmol / g, add DMF twice the volume of the resin to swell the resin for 1-2 hours, remove the DMF, add 5% NMM / DMF solution to wash the resin twice, remove the 5% NMM / DMF solution, then wash the resin five times with DMF, and remove the DMF solution.

[0077] (2) Weigh Fmoc-Linker-OH and HBTU in a ratio of Fmoc-Linker-OH:HBTU = 3:2.85 and add them to an activation bottle, then add DMF to dissolve, then add NMM twice the equivalent of Fmoc-Linker-OH, and activate at 0-10℃ for 5-10min. Finally, slowly add it to AM Resin, and continue to react at 25±15℃ until the ninhydrin test is negative, remove the reaction solution, and wash with DMF 3 times to obtain Fmoc-Linker-AM Resin. (3) Add 20% PIP / DMF solution to the reactor, react for 5min, remove the reaction solution, add 20% PIP / DMF solution again, react for 15min, remove the reaction solution, and then wash the resin with DMF 8 times. The ninhydrin test is positive. Weigh Fmoc-Ser(tBu)-OH and HOBT in a ratio of Fmoc-Ser(tBu)-OH:HOBT=3:3, then add DMF solution, add DIC equivalent to Fmoc-Ser(tBu)-OH in an ice-water bath for activation for 5-10 minutes, add the activated reaction solution to the reactor, and continue the reaction at 25±15℃ until the ninhydrin test is negative. After the reaction is completed, remove the reaction solution and wash the resin with DMF 3 times.

[0078] (4) Repeat the above steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment, and according to the sequence of telportin, use Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu )-Phe-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH to complete the peptide resin coupling, and the resin was washed with methanol for 3 times. The peptide resin was vacuum dried to obtain the fully protected peptide resin of telport peptide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu-Eicosanedioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin, in which Fmoc-Ile-Aib-OH uses HATU and NMM as coupling reagents, and the feed ratio is Fmoc-Ile-Aib-OH:HATU:NMM=3:2.85:6, Fmoc-L-Lys[19-(t-butoxycarbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH feeding ratio Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH:HOBT:DIC=1.5:1.5:1.5. .

[0079] Embodiment 2:

[0080] Based on Example 1, a method for synthesizing crude telpotide peptide is provided, comprising:

[0081] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent (trifluoroacetic acid / 1,2-ethanedithiol / triisopropylsilane / water = 90.0 / 5.0 / 2.5 / 2.5) was added at a ratio of 15 mL / g resin, and the reaction was continued for 4.0 h at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was washed with a small amount of TFA for 3 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 3 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide, with a crude yield of 94.8% and an HPLC purity of 77.17%.

[0082] Embodiment 3:

[0083] Based on Example 1, a method for synthesizing crude telpotide peptide is provided, comprising:

[0084] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10 mL / g resin, wherein the cleavage reagent was a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 90.5:4.5:2.5:2.5, and the reaction was continued for 2 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was then washed with a small amount of TFA for 5 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 5 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide.

[0085] Embodiment 4:

[0086] Based on Example 1, a method for synthesizing crude telpotide peptide is provided, comprising:

[0087] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10-15 mL / g resin, wherein the cleavage reagent was a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 95:3:1:1, and the reaction was continued for 3 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was then washed with a small amount of TFA for 4 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 3 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide.

[0088] Embodiment 5:

[0089] Based on Example 1, a method for synthesizing crude telpotide peptide is provided, comprising:

[0090] The obtained telpotide fully protected peptide resin was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10-15 mL / g resin, the cleavage reagent being a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 88:6:3:3, and the reaction was continued for 4 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, the filtrate was collected, and the resin was washed with a small amount of TFA 4 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, the precipitate was collected by centrifugation, the precipitate was washed 4 times with cold methyl tert-butyl ether, and the solid was obtained by vacuum drying, i.e., the crude telpotide peptide.

[0091] Embodiment 6:

[0092] Based on Example 2, a method for synthesizing crude telpotide peptide is provided, comprising:

[0093] The obtained telpotide fully protected peptide resin was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 15 mL / g resin, the cleavage reagent being a mixed solution of trifluoroacetic acid, 1,8-octanedithiol, triisopropylsilane and water in a volume ratio of 90.0 / 5.0 / 2.5 / 2.5, and the reaction was continued for 4.0 h at 25±2°C. The reaction mixture was filtered with a sand core funnel, the filtrate was collected, and the resin was washed with a small amount of TFA for 3 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, the precipitate was collected by centrifugation, the precipitate was washed 3 times with cold methyl tert-butyl ether, and the solid was obtained by vacuum drying, i.e., the crude telpotide peptide.

[0094] Embodiment 7:

[0095] Based on Example 3, a method for synthesizing crude telpotide peptide is provided, comprising:

[0096] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10 mL / g resin, wherein the cleavage reagent was a mixed solution of trifluoroacetic acid, 1,8-octanedithiol, triisopropylsilane and water in a volume ratio of 90.5:4.5:2.5:2.5, and the reaction was continued for 2 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was then washed with a small amount of TFA for 5 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 5 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide.

[0097] Embodiment 8:

[0098] Based on Example 4, a method for synthesizing crude telpotide peptide is provided, comprising:

[0099] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10-15 mL / g resin, wherein the cleavage reagent was a mixed solution of trifluoroacetic acid, 1,8-octanedithiol, triisopropylsilane and water in a volume ratio of 95:3:1:1, and the reaction was continued for 3 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was then washed with a small amount of TFA for 4 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 3 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide.

[0100] Embodiment 9:

[0101] Based on Example 5, a method for synthesizing crude telpotide peptide is provided, comprising:

[0102] The obtained fully protected peptide resin of telpotide was placed in a cleavage reactor, and a cleavage reagent was added at a ratio of 10-15 mL / g resin, wherein the cleavage reagent was a mixed solution of trifluoroacetic acid, 1,8-octanedithiol, triisopropylsilane and water in a volume ratio of 88:6:3:3, and the reaction was continued for 4 hours at 25±2°C. The reaction mixture was filtered with a sand core funnel, and the filtrate was collected. The resin was then washed with a small amount of TFA for 4 times, and the combined filtrate was added to pre-cooled methyl tert-butyl ether, and the precipitate was collected by centrifugation. The precipitate was washed 4 times with cold methyl tert-butyl ether, and vacuum dried to obtain a solid, i.e., crude telpotide peptide.

[0103] Experimental Example 1:

[0104] The yield and HPLC purity of crude telportipeptide peptide obtained in Examples 2 to 9 were respectively calculated, and the results were as follows: Figure 2 and Figure 3 As shown, Figure 2 The yield of crude telpotide peptide is shown. Figure 3 The HPLC purity of the crude tepote peptide is shown. From the yield and HPLC purity of the tepote peptide obtained in Examples 2-5, it can be seen that the tepote peptide fully protected peptide resin is subjected to a cleavage reaction using the cleavage reagents of the present invention including trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water, and a simple purification operation can be performed to obtain a crude tepote peptide with a yield of not less than 94.5% and an HPLC purity of not less than 77%. The cleavage operation is simple, and the yield and purity can meet the requirements of general processes. On this basis, the inventors of the present application can further improve the cleavage efficiency on the basis of the original cleavage reagent by replacing 1,2-ethanedithiol in the above-mentioned cleavage reagent with 1,8-octanedithiol, and more efficiently break the peptide bonds on the resin, and more thoroughly release the polypeptide. The yield of the crude peptide is not less than 95.5%, and the HPLC purity is not less than 78%, thereby further improving the yield and purity to meet the needs of higher-demand application scenarios.

[0105] Embodiment 10:

[0106] On the basis of the above-mentioned embodiment, this embodiment provides a method for preparing pure telpotide peptide, which is further purified on the basis of embodiment 2, comprising: dissolving the crude telpotide peptide in a phosphate buffer solution of pH 6.5 containing 0.08 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide.

[0107] The phosphate buffer solution of pH 6.5 is as follows: take 0.68 g of potassium dihydrogen phosphate (KH2PO4), add 15.2 mL of 0.1 mol / L sodium hydroxide solution, and dilute with water to 100 mL to obtain a phosphate buffer solution of pH 6.5.

[0108] Chromatographic purification: stationary phase: octadecylsilane bonded silica gel filler, column diameter and length: 150mm*250mm; phase A: 2% acetic acid aqueous solution; phase B: 100% acetonitrile; flow rate: 500mL / min; detection wavelength: 210nm and 254nm; linear gradient phase A 50-70%, elution 80min. Purification process: the sample load is 20g, the target peak is collected, and the components with a purity greater than 93% and a single impurity less than 1% are collected.

[0109] Embodiment 11:

[0110] Based on the above examples, this example provides a method for preparing pure telpotide peptide, which is further purified on the basis of Example 2, comprising: dissolving the crude telpotide peptide in a phosphate buffer of pH 6.5 containing 0.1 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide. The phosphate buffer of pH 6.5 and the chromatographic purification are the same as those in Example 10.

[0111] Embodiment 12:

[0112] Based on the above examples, this example provides a method for preparing pure telpotide peptide, which is further purified on the basis of Example 2, comprising: dissolving the crude telpotide peptide in a phosphate buffer of pH 6.5 containing 0.085 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide. The phosphate buffer of pH 6.5 and the chromatographic purification are the same as those in Example 10.

[0113] Embodiment 13:

[0114] Based on the above examples, this example provides a method for preparing pure telpotide peptide, which is further purified on the basis of Example 2, comprising: dissolving the crude telpotide peptide in a phosphate buffer of pH 6.5 containing 0.06 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide. The phosphate buffer of pH 6.5 and the chromatographic purification are the same as those in Example 10.

[0115] Embodiment 14:

[0116] Based on the above examples, this example provides a method for preparing pure telpotide peptide, which is further purified on the basis of Example 2, comprising: dissolving the crude telpotide peptide in a phosphate buffer of pH 6.5 containing 0.12 mmol / L sulfobutyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide. The phosphate buffer of pH 6.5 and the chromatographic purification are the same as those in Example 10.

[0117] Embodiment 15:

[0118] Based on the above embodiments, this embodiment provides a method for preparing pure telpotide peptide, which is further purified on the basis of embodiment 2, including: dissolving crude telpotide peptide in a phosphate buffer at pH 6.5, passing through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain pure telpotide peptide. The phosphate buffer at pH 6.5 and the chromatographic purification are the same as those in embodiment 10.

[0119] Experimental Example 2:

[0120] The purity and maximum single impurity of the pure telportin peptide obtained in Examples 10-15 were statistically analyzed, and the statistical results were as follows: Figure 4 and Figure 5 As shown, Figure 4 The purity of the pure peptide of tepoxetine is shown. Figure 5 The maximum single hybrid of pure telopoietin peptide is shown. Figure 4 and Figure 5 It can be seen that after the crude telpotide peptide is dissolved in a phosphate buffer solution at pH 6.5, the purity of the pure peptide can be increased to not less than 99% by conventional chromatographic purification, and the maximum single impurity is controlled below 0.3%. Further, after adding a trace amount of sulfobutyl-β-cyclodextrin to the buffer solution, the separation effect can be further improved, so that after only conventional chromatographic purification, a pure telpotide peptide with a purity of not less than 99.4% and a maximum single impurity of not more than 0.2% can be obtained, which greatly simplifies the purification steps and has a great improvement over the complicated preparation and purification process of the prior art. After simple preparation and purification steps, a pure telpotide peptide that is sufficient to meet high purity requirements can be obtained.

[0121] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0122] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0123] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0124] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0125] Matters not covered in the present invention are all known technologies.

Claims

1. A method for synthesizing a fully protected peptide resin of tepote, characterized in that include: Step S1, Fmoc-Linker-OH and HBTU react with swollen AM Resin in the presence of an activator to prepare Fmoc-Linker-AM Resin; Step S2, Fmoc-Ser(tBu)-OH and HOBT are added to the reaction system of Fmoc-Linker-AM Resin and PIP / DMF solution, and reacted under DIC activation conditions until the ninhydrin test is negative; Step S3, repeating the above steps of removing Fmoc protection and coupling with the corresponding amino acid or polypeptide fragment, completing the peptide resin coupling steps from the C-terminus to the N-terminus according to the telpotide sequence, washing the resin at least 3 times with an equal volume of methanol to the resin, and vacuum drying to obtain the telpotide fully protected peptide resin; In step S3, the steps of completing peptide resin coupling from the C-terminus to the N-terminus in sequence specifically include: using Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib- OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH complete the peptide resin coupling.

2. The method according to claim 1, characterized in that: In step S1, the degree of substitution of the AM Resin is between 0.1-1 mmol / L.

3. The method according to claim 1, characterized in that: Step S1 specifically includes: adding Fmoc-Linker-OH and HBTU in a ratio of 3:2.85 into an activation bottle, adding DMF to dissolve, then adding NMM, and slowly adding to the reactor containing swollen AM Resin after activation, and continuously reacting at 25±15° C. After the reaction is completed, the reaction liquid is extracted and washed with DMF at least 3 times to obtain Fmoc-Linker-AM Resin.

4. The method according to claim 1, characterized in that: In step S2, the reaction system of the resin and the PIP / DMF solution comprises: adding an equal volume of the PIP / DMF solution to the resin obtained in step S1 and reacting for 5 minutes, removing the reaction solution and then adding an equal volume of the PIP / DMF solution to the resin and reacting for 15 minutes, and removing the reaction solution and adding DMF to wash the resin at least 8 times until the ninhydrin test is positive.

5. The method according to claim 1, characterized in that: Step S2 specifically includes: adding Fmoc-Ser(tBu)-OH and HOBT to DMF solution in a ratio of 3:3, adding DIC equivalent to Fmoc-Ser(tBu)-OH for activation under ice-water bath conditions, adding the reaction solution to a reactor, continuing the reaction at 25±15° C. until the ninhydrin test is negative, removing the reaction solution, and washing with DMF at least 3 times.

6. The method according to claim 1, characterized in that: In step S3, the Fmoc-Ile-Aib-OH uses HATU and NMM as coupling reagents, and the feed ratio is Fmoc-Ile-Aib-OH:HATU:NMM=3:2.85:6; and / or The feeding ratio of Fmoc-L-Lys[19-(t-butoxy carbonyl)-nonadecanoyl-γ-Glu(OtBu)-AEEA-AEEA]-OH to HOBT and DIC is 1.5:1.5:1.5.

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