Preparation method of tizpatide

Abstract

According to the preparation method of the tizepatide, conventional solid-phase synthesis and liquid-phase synthesis are combined, Raman spectrum is introduced for on-line monitoring, environmental parameters can be dynamically adjusted, pre-coupled easily-obtained fragment peptide is adopted as a raw material for amino acid at the position with low coupling efficiency in synthesis, and high-purity tizepatide is obtained. Peptide resin folding in synthesis and liquid-phase coupling integral access of side chain functional groups are avoided, and the coupling yield is effectively increased. Compared with the prior art, the method has the advantages that the synthetic route is simple and convenient, the reaction process is real-time and online controllable, the high proportion cost is realized, the side chain consumption is greatly reduced, the generation of impurities such as racemic tizalopatide, [+ 1Gly]-tizalopatide, [-1Gly]-tizalopatide and deletion peptide is reduced, the synthetic yield and purity of the target peptide are effectively improved, the purity of the target peptide is higher than 99.0%, the variety of impurities is few, and the method is suitable for industrial production. Meanwhile, a side chain is connected by adopting a liquid phase method, so that the cost is obviously reduced, and the method has wide practical value and application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of tazeparatide preparation technology and relates to a method for preparing tazeparatide. Background Art

[0002] Tirzepatide is a novel dual agonist of glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor developed by Eli Lilly. Both GIP and GLP-1 are hormones secreted by the intestine that can promote insulin secretion. Therefore, tirzepatide is used clinically to treat type 2 diabetes and obesity.

[0003] The role of Raman analysis in coupling reactions is mainly reflected in the following aspects: (1) Raman spectroscopy can realize real-time monitoring of coupling reactions under in situ conditions, which is of great significance for studying molecular reactions catalyzed by surface plasmons; (2) Surface enhanced Raman spectroscopy (SERS) can effectively display the structural change information of substances, thereby observing the molecular structure change process that cannot be observed by other means such as ultraviolet and mass spectrometry; (3) Raman spectroscopy can be used to explore the mechanism of coupling reactions; (4) Raman spectroscopy can be used to evaluate the catalytic efficiency of catalysts. In summary, Raman analysis plays a vital role in coupling reactions. It can not only provide real-time reaction information, but also help scientists deeply understand the reaction mechanism and optimize the reaction conditions to improve the catalytic efficiency.

[0004] In the existing preparation process of tazeparatide, several fragments are usually obtained by solid phase synthesis, and then the fragments are subjected to condensation reaction and cleavage to obtain crude tazeparatide. However, there are too many fragments in the existing preparation process. For example, there are fragments [1-13], [14-21], [22-29], [30-39] on the market, and there are also fragments [1-14], [15-21], [22-29], [30-39]. There are many defects such as many synthesis steps, high cost, long production time and low purity. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing tazeparatide, which solves the problems of multiple synthesis steps, long production time and low purity in the existing tazeparatide preparation process.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for preparing tazeparatide combines solid phase synthesis with liquid phase synthesis, introduces Raman spectroscopy to monitor the coupling process online, uses pre-coupled readily available protected amino acid fragments and protected amino acids as raw materials, and comprises the following steps:

[0008] Using Sieber resin as a coupling resin carrier, select the corresponding protected amino acids and protected amino acid fragments and couple them one by one on the solid phase to obtain fragments [21-39];

[0009] Solid phase coupling of Fmoc-Lys(Dde)-OH in the protected amino acid and the fragment [21-39] to obtain fragment [20-39];

[0010] Solid phase coupling of the fragment [20-39] with the corresponding protected amino acid and protected amino acid fragment one by one to obtain fragment [1-39];

[0011] Deprotecting the fragment [1-39] with hydrazine hydrate / DMF solution to obtain a de-Dde fragment [1-39];

[0012] Solid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide peptide resin; or, liquid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide;

[0013] The fully protected tazeparatide resin or the fully protected tazeparatide is cleaved using a cleavage agent to obtain a crude tazeparatide;

[0014] Dissolving, filtering, purifying and freeze-drying the crude tazeparatide product to obtain a refined tazeparatide product;

[0015] Among them, in each coupling reaction process, two methods of central control are used: online Raman analyzer real-time central control and ninhydrin colorimetric reaction assisted central control, and environmental parameters are adjusted in real time according to the dynamic changes of the reaction;

[0016] Wherein, the fragment [21-39] is:

[0017] Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0018] The fragment [20-39] is:

[0019] Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0020] The fragment [1-39] is:

[0021] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(t rt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0022] The de-Dde fragment [1-39] is:

[0023] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0024] The de-Dde fragment [1-39] is obtained after cutting the resin:

[0025] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2;

[0026] The fully protected tazeparatide peptide resin is:

[0027] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0028] The fully protected tazeparatide is:

[0029] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0030] Furthermore, the amount of the protected amino acid and the protected amino acid fragment is 1.5 to 5 of the total mole of the resin fed.

[0031] Furthermore, the environmental parameters include reaction time and environmental temperature.

[0032] Furthermore, in the coupling reaction, the deprotection reagent for removing the Fmoc protection is a PIP / DMF mixed solution, and the mixed solution contains 20% to 25% (V / V) piperidine.

[0033] Furthermore, the degree of substitution of the Sieber resin is in the range of 0.2 to 0.5 mmol / g.

[0034] Further, the reagents for the coupling reaction include one of DIC / HOBT, Pybop / HOBT / DIEA, HATU / HOBT / DIEA or OP / DIC;

[0035] Further, the solvent of the coupling reaction includes one or more of DMF, DMSO or DCM;

[0036] Further, the de-Dde fragment [1-39] resin cutting reagent is 5% TFA / DCM;

[0037] Furthermore, the cracking agent includes water, 90% to 95% of trifluoroacetic acid, 1% to 4% of 1,2-ethanedithiol and 1% to 4% of triisopropylsilane.

[0038] Furthermore, the crude tazeparatide is purified by high performance liquid chromatography, the purification chromatographic column is a reverse octadecyl bonded silica gel; the mobile phases are ammonium acetate solution and acetonitrile solution, respectively; the salt conversion step is carried out by high performance liquid chromatography, and the mobile phase system is ammonium acetate solution-acetonitrile solution.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] In the preparation method of tazeparatide of the present invention, conventional solid phase synthesis is combined with liquid phase, and Raman spectroscopy is introduced for online monitoring, and environmental parameters can be dynamically adjusted, so that online dynamic optimization can be achieved, and pre-coupled easily available fragment peptides are used as raw materials for amino acids with low coupling efficiency in the synthesis, thereby avoiding the folding of peptide resins in the synthesis, and the liquid phase coupling of side chain functional groups is integrated, the amount of side chains used is reduced by more than 50%, and the total cost is reduced by more than 15% year-on-year. Compared with the prior art, the method has the advantages of simple synthesis route, real-time online controllable reaction process, greatly reduced consumption of high-cost side chains, reduced generation of impurities such as racemic tazeparatide, [+1Gly]-tazeparatide, [-1Gly]-tazeparatide and missing peptides, effectively improving the synthesis yield and purity of the target peptide, the purity of the target peptide is higher than 99.0%, and the types of impurities are few. At the same time, the side chains are connected by liquid phase method, which significantly reduces the cost and has broad practical value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative work, among which:

[0042] Figure 1 A process route diagram illustrating a method for preparing tazeparatide according to the present invention;

[0043] Figure 2 An online Raman monitoring spectrum illustrating a method for preparing tazeparatide according to the present invention;

[0044] Figure 3 and Figure 4 The HPLC spectrum of crude tazeparatide is an example of a preparation method of tazeparatide according to the present invention;

[0045] Figure 5 The HPLC spectrum of tazeparatide is shown as an example of a preparation method of tazeparatide according to the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0047] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0048] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0049] As described in the background art, in the existing preparation process of tazeparatide, several fragments are usually first obtained by solid phase synthesis, and then the fragments are subjected to condensation reaction and cleavage to obtain crude tazeparatide. However, there are too many fragments in the existing preparation process. For example, there are fragments [1-13], [14-21], [22-29], [30-39] on the market, and there are also fragments [1-14], [15-21], [22-29], [30-39], which have the defects of many synthesis steps, long production time and low purity.

[0050] Based on this, the inventors have created a method for preparing tazeparatide of the present application to solve the above technical problems.

[0051] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0052] Raman spectroscopy was introduced to monitor the coupling process online, using pre-coupled readily available protected amino acid fragments and protected amino acids as raw materials, including the following steps:

[0053] Using Sieber resin as a coupling resin carrier, the corresponding protected amino acids and protected amino acid fragments are selected and coupled one by one on a solid phase to obtain fragments [21-39]. For example, the protected amino acids and protected amino acid fragments may include Fmoc-Lys(Dde)-OH, and also include Fmoc-Ser(tbu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tbu)-Gly-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, Boc-Eicosanedioic acid-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(tb u)-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Asp(otbu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(otbu)-Gly-OH and Boc-Tyr(tbu)-Aib-OH. The amino acid fragment is a peptide fragment containing Tyr-Aib, -Glu-Gly-, -Ile-Aib-, -Gly-Gly-, -Ser-Gly-, -Pro-Pro-Pro-, -X- groups; wherein X is the side chain functional group of tezetapeptide: Eicosanedioic acid-γ-Glu-AEEA-AEEA. For example, the protected amino acids and protected amino acid fragments are purchased from the market or prepared by independent synthesis. The fragment [21-39] is: Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0054] The Fmoc-Lys(Dde)-OH in the protected amino acid is solid-phase coupled with the fragment [21-39] to obtain fragment [20-39].

[0055] Solid phase coupling of the fragment [20-39] with the corresponding protected amino acid and protected amino acid fragment one by one to obtain fragment [1-39];

[0056] Deprotecting the fragment [1-39] with hydrazine hydrate / DMF solution to obtain a de-Dde fragment [1-39];

[0057] Solid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide peptide resin; or, liquid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide;

[0058] The fully protected tazeparatide resin or the fully protected tazeparatide is cleaved using a cleavage agent to obtain a crude tazeparatide;

[0059] Dissolving, filtering, purifying and freeze-drying the crude tazeparatide product to obtain a refined tazeparatide product;

[0060] Among them, in each coupling reaction process, two methods of central control are used: online Raman analyzer real-time central control and ninhydrin colorimetric reaction assisted central control, and environmental parameters are adjusted in real time according to the dynamic changes of the reaction;

[0061] Wherein, the fragment [21-39] is:

[0062] Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0063] The fragment [20-39] is:

[0064] Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0065] The fragment [1-39] is:

[0066] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(t rt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0067] The de-Dde fragment [1-39] is:

[0068] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin;

[0069] The de-Dde fragment [1-39] is obtained after cutting the resin:

[0070] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2;

[0071] The fully protected tazeparatide peptide resin is:

[0072] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0073] The fully protected tazeparatide is:

[0074] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0075] In the preparation method of tazeparatide of the present invention, conventional solid phase synthesis is combined with liquid phase, and Raman spectroscopy is introduced for online monitoring, and environmental parameters can be dynamically adjusted. The pre-coupled easily available fragment peptide is used as a raw material for the amino acid at the position with low coupling efficiency in the synthesis, thereby avoiding the folding of the peptide resin in the synthesis, and the liquid phase coupling of the side chain functional group is integrated, effectively improving the coupling yield. Compared with the prior art, the method has the advantages of simple synthesis route, real-time online controllable reaction process, greatly reduced consumption of high-cost side chains, reduced generation of impurities such as racemic tazeparatide, [+1Gly]-tazeparatide, [-1Gly]-tazeparatide and missing peptides, effectively improving the synthesis yield and purity of the target peptide, the purity of the target peptide is higher than 99.0%, and the types of impurities are few. At the same time, the side chain is connected by liquid phase method, which significantly reduces the cost, and has a wide range of practical value and application prospects.

[0076] In another embodiment, the amount of the protected amino acid and the protected amino acid fragment is 1.5 to 5 times, preferably 3 to 4 times, the total molar number of the resin fed.

[0077] In another embodiment, the environmental parameters include reaction time and ambient temperature. As shown in Table 1 below, such settings can effectively reduce the generation of impurities during the synthesis process.

[0078] Experimental comparison:

[0079] Table 1

[0080] Central control method Crude product purity% Ninhydrin color development 71.30 Online Raman Analyzer Real-time Central Control Solid Phase Synthesis + Ninhydrin Color Development 83.25

[0081] In another embodiment, in the coupling reaction, the deprotection reagent for removing the Fmoc protection is a PIP / DMF mixed solution, and the mixed solution contains 20% to 25% (V / V) piperidine. For example, the amount of the deprotection reagent is 1 to 1.2 times the volume of the peptide resin. The deprotection times are two, the first deprotection takes 5 to 10 minutes, and the second deprotection takes 20 to 30 minutes, which can ensure that the Fmoc is removed with higher efficiency.

[0082] In another embodiment, the degree of substitution of the Sieber resin is in the range of 0.2 to 0.5 mmol / g. For example, in practice, a Sieber resin with a degree of substitution of 0.2 to 0.5 mmol / g is selected as a carrier, and finally 85.87 g of a peptide resin is obtained, while a Sieber resin with a degree of substitution of 0.6-0.8 mmol / g is selected as a carrier, and finally 61.11 g of a peptide resin is obtained, so it can be concluded that the synthesis yield of the Sieber resin with a degree of substitution of 0.2-0.5 mmol / g is significantly higher than that of the Sieber resin with a degree of substitution of 0.6-0.8 mmol / g, that is, the synthesis yield of the degree of substitution in this range is higher.

[0083] In another embodiment, the reagent for the coupling reaction includes one of DIC / HOBT, Pybop / HOBT / DIEA, HATU / HOBT / DIEA or OP / DIC. The preferred coupling reagent for coupling a single protected amino acid is DIC / HOBT, and the preferred coupling reagent for coupling a protected amino acid fragment is Pybop / HOBT / DIEA, as shown in Table 2 below, and the crude product obtained by the latter has significantly higher purity. The amount of the coupling reagent used is 3 to 8 times the molar number of the carrier resin; preferably 4 to 6 times.

[0084] Experimental comparison:

[0085] Table 2

[0086]

[0087]

[0088] In other embodiments, the solvent for the coupling reaction includes one or more of DMF, DMSO or DCM.

[0089] In another embodiment, the side chain coupling method includes overall protection side chain fragment coupling and side chain one by one coupling. As shown in Table 3 below, the purity of the crude product obtained by the three coupling methods is the highest with overall access, and the cost of the solid phase side chain overall coupling raw material is 100%. The cost of the three coupling methods is the lowest with the side chain liquid phase coupling cost, and the reagents used for subsequent cleavage are less, and the total batch production cost is reduced by about 15%.

[0090] Table 3

[0091]

[0092] In another embodiment, the cleavage agent includes water, 90% to 95% trifluoroacetic acid, 1% to 4% 1,2-ethanedithiol and 1% to 4% triisopropylsilane. The amount of cleavage agent consumed per gram of tazeparatide resin is 7 to 15 ml; preferably, 10 ml of cleavage agent is required per gram of tazeparatide resin, and the cleavage time is 2 to 4 hours at room temperature, preferably 3 hours. The experiment is shown in Table 4 below. The crude product obtained with 10 ml of cleavage agent and 3 hours of cleavage time is large in quantity and high in purity.

[0093] Experimental comparison (with 1g peptide resin as the experimental unit):

[0094] Table 4

[0095]

[0096]

[0097] In another embodiment, the crude tazeparatide is purified by high performance liquid chromatography, and the purification chromatographic column is a reverse octadecyl bonded silica gel; the mobile phases are ammonium acetate solution and acetonitrile solution, respectively; the salt conversion step is carried out by high performance liquid chromatography, and the mobile phase system is 0.1% ammonia solution-acetonitrile solution, as shown in Table 5 below. Such an arrangement can effectively improve the purity of the product.

[0098] Experimental comparison:

[0099] Table 5

[0100]

[0101] The following is a specific example to illustrate this application. Figure 1 .

[0102] Step 1:

[0103] Preparation of Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0104] Sieber resin (0.3mmol / g, 20mmol) was added to the reactor and washed with DMF three times, 10min / time. Fmoc protection was removed twice with 20% PIP / DMF solution. After detection, the product was washed with DMF 6 times. Fmoc-Ser(tbu)-OH (60mmol), HOBt (120mmol), and DIC (120mmol) were dissolved in DMF and added to the solid phase reactor. The online Raman analyzer controlled the reaction time in real time. The ninhydrin color reaction assisted in the reaction endpoint. After the reaction was completed, the product was washed with DMF 6 times, 2-3min / time. Fmoc-Ser(tbu)-NH-resin was prepared.

[0105] Fmoc-Pro-Pro-Pro-OH (60 mmol), HOBt (60 mmol), Pybop (120 mmol), and DIEA (160 mmol) were dissolved in DMF and added to a solid phase reactor. The online Raman analyzer controlled the reaction time in real time. The ninhydrin color development reaction assisted in proving the reaction endpoint. After the reaction was completed, the mixture was washed with DMF for 6 times, 2-3 min / time, to obtain Fmoc-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0106] The same coupling reaction method was used to couple single protected amino acids and protected amino acid fragments: Fmoc-Ala-OH, Fmoc-Ser(tbu)-Gly-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, and Fmoc-Ala-OH to prepare Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0107] Step 2:

[0108] Preparation of Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0109] The Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin prepared in step 1 was deprotected twice with 20% PIP / DMF solution. After detection, the resin was washed with DMF 6 times. Fmoc-Lys(Dde)-OH (60 mmol), HOBt (120 mmol), and DIC (120 mmol) were dissolved in DMF and added to a solid phase reactor. The online Raman analyzer controlled the reaction time in real time. The ninhydrin color reaction assisted in confirming the reaction endpoint. After the reaction was completed, the resin was washed with DMF 6 times, 2-3 min / time. Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin was prepared.

[0110] Step 3:

[0111] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(t Preparation of rt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0112] In the Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin of step 2, Fmoc protection was removed twice with 20% PIP / DMF solution. After the protection was completely removed, DMF was washed 6 times. Fmoc-Gln(trt)-OH (60 mmol), HOBt (120 mmol), and DIC (120 mmol) were dissolved in DMF and added to a solid phase reactor. The reaction time was controlled in real time by an online Raman analyzer. The reaction endpoint was confirmed by ninhydrin color development (see Figure 2 ), the reaction was completed, and the mixture was washed with DMF for 6 times to prepare Fmoc-Gln(trt)-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0113] The same coupling reaction method as step 1 was used to couple single protected amino acids and protected amino acid fragments one by one on the solid phase: 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-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(otbu)- Gly-OH, Boc-Tyr(tbu)-Aib-OH, thereby preparing Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Fmoc-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(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0114] Step 4:

[0115] Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)- Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0116] 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)- in step 3 Lys(boc)-Ile-Ala-Gln(trt)-Lys(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-G The resin was deprotected with 5% hydrazine hydrate / DMF solution. After detection, the resin was washed with DMF 8 times and DCM 4 times to obtain 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-Glu(otbu)-Al a-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin.

[0117] Step 5:

[0118] Take 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-Glu(otbu)-Al Half of the a-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin (theoretical 10 mmol) was added to a reactor, and the Dde protection was removed with a hydrazine hydrate / DMF solution to obtain a de-Dde fragment [1-39]; the de-Dde fragment [1-39] was washed with DMF for 3 times, Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH (30 mmol), HOBt (30 mmol), Pybop (60 mmol), and DIEA (80 mmol) were dissolved in DMF and added to a solid phase reactor, and the reaction time was controlled in real time by an online Raman analyzer, and the reaction end point was assisted by a ninhydrin color development reaction. After the reaction was completed, the fragment was washed with DMF for 8 times and with DCM for 4 times. 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(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin was prepared. The resin was placed in a vacuum desiccator and dried overnight. On the next day, 85.87 g of fully protected tazeparatide peptide resin was weighed.

[0119] Step 6:

[0120] Take 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-Glu(otbu)-Al The other half of the a-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin (theoretical 10 mmol) was added to the reactor, and the resin cutting reagent 5% TFA / DCM was added. After the reaction was completed, the reaction was filtered, the filtrate was washed to remove TFA, and the organic phase was concentrated, precipitated, and dried to obtain Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-P he-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otb u)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH (11 mmol), HOBt (11 mmol), Pybop (20 mmol), and DIEA (80 mmol) were activated in the solution, and after activation, 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-Glu(otbu)-Ala-Phe-Val-G 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(Boc-Eicosane dioicacid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2. The amount of side chain material in the liquid phase side chain coupling method is about 1 / 3 of the amount of side chain material in the solid phase coupling method, which has a greater cost advantage.

[0121] Step 7: Preparation of crude tezetribotepeptide.

[0122] The fully protected tazeparatide resin (theoretical 10 mmol) was added to a 2000 ml round-bottom flask, and 1000 ml of cleavage reagent (940 ml of TFA, 20 ml of EDT, 20 ml of TIS, and 20 ml of H2O) was prepared. The cleavage reagent was poured into the resin and reacted at room temperature for 2 hours. After the reaction was completed, the resin was filtered and the filtrate was collected. The resin was washed 3 times with a small amount of TFA, the filtrates were combined, the filtrates were concentrated under reduced pressure, and precipitated with frozen ether, and then washed 3 times with frozen ether, filtered, and vacuum dried to obtain tazeparatide crude peptide ①37.08 g (the crude product HPLC purity was 84.57%, please refer to Figure 3 ).

[0123] Fully protected tazeparatide (theoretical 10 mmol) was added to a 1000 ml round-bottom flask, and 300 ml of cleavage reagent (282 ml of TFA, 6 ml of EDT, 6 ml of TIS, and 6 ml of H2O) was prepared. The cleavage reagent was poured into the resin and reacted at room temperature for 2 hours. After the reaction was completed, the cleavage solution was concentrated and precipitated with ice-cold ether, then washed with ice-cold ether for 3 times, filtered, and vacuum dried to obtain tazeparatide crude peptide ②38.12 g (the crude product HPLC purity was 83.93%, please refer to Figure 4 ).

[0124] Step 8: Purification, salt conversion, concentration and lyophilization of tazeparatide.

[0125] (1) Dissolve two portions of crude tazeparatide in ammonium acetate solution, filter the solution with a 0.45 μm mixed microporous filter membrane, and purify for later use. (2) Use high performance liquid chromatography for purification, wherein the purification column is an octadecyl bonded silica gel column with a diameter of 5 cm and a filler with a particle size of 10 μm, the mobile phases are ammonium acetate aqueous solution and acetonitrile, respectively, the flow rate is 60 ml / min, the sample load is 5 to 10 g, and the detection wavelength of the chromatograph is 220 nm; take the tazeparatide purified intermediate concentrated solution, filter it with a 0.45 μm filter membrane and set aside. (3) The salt was exchanged by high performance liquid chromatography, the mobile phase system was 0.1% ammonia / water solution-acetonitrile solution, and the flow rate was 60 mL / min; gradient elution and cyclic loading method were used, the sample was loaded on the chromatographic column, the mobile phase elution was started, the spectrum was collected, the change of absorbance was observed, the main peak of the salt exchange was collected and the purity was detected by analytical liquid phase, the main peak solution of the salt exchange was combined, and the solution was concentrated under reduced pressure to obtain a tazeparatide solution, which was freeze-dried to obtain tazeparatide fine product ① 18.12 g, with a total yield of 37.64%; tazeparatide fine product ② 18.27 g, with a total yield of 37.96%; the purity of the tazeparatide finished product was 99.44%, please refer to Figure 5 .

[0126] After analysis of experimental data, the peptide resin synthesis process of the preparation method of tazeparatide of the present invention adopts an online Raman analyzer to control the coupling process in real time, and the solid phase synthesis time is strictly controlled by the auxiliary evidence of the ninhydrin color development reaction, thereby avoiding the false positive and false negative results existing in the single ninhydrin color development reaction control, reducing the generation of impurities such as racemic impurities and missing peptides, and adopting partial small fragment coupling, with fewer synthesis steps one by one, which can reduce the production time and improve the product yield, and solve the problems of many synthesis steps, long production time and low purity of crude products in the existing tazeparatide preparation process. The target peptide amounts obtained by the two coupling side chain fragments in the implementation example are not much different, and both can be scaled up for production. Considering the material production cost factor, the liquid phase fragment side chain synthesis method has lower cost and higher economic benefits.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made by any technician familiar with the field within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing tazeparatide, characterized in that: The method uses pre-coupled readily available protected amino acid fragments and protected amino acids as raw materials to synthesize the target product by combining solid phase synthesis main chain and liquid phase coupling side chain under online monitoring of Raman spectroscopy, including the following steps: Using Sieber resin as a coupling resin carrier, select the corresponding protected amino acids and protected amino acid fragments and couple them one by one on the solid phase to obtain fragments [21-39]; Solid phase coupling of Fmoc-Lys(Dde)-OH in the protected amino acid and the fragment [21-39] to obtain fragment [20-39]; Solid phase coupling of the fragment [20-39] with the corresponding protected amino acid and protected amino acid fragment one by one to obtain fragment [1-39]; Deprotecting the fragment [1-39] with hydrazine hydrate / DMF solution to obtain a de-Dde fragment [1-39]; Solid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide peptide resin; or, liquid phase coupling of the de-Dde fragment [1-39] with Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-OH in the protected amino acid fragment to obtain fully protected tazeparatide; The fully protected tazeparatide resin or the fully protected tazeparatide is cleaved using a cleavage agent to obtain a crude tazeparatide; Dissolving, filtering, purifying and freeze-drying the crude tazeparatide product to obtain a refined tazeparatide product; Among them, in each coupling reaction process, two methods of central control are used: online Raman analyzer real-time central control and ninhydrin colorimetric reaction assisted central control, and environmental parameters are adjusted in real time according to the dynamic changes of the reaction; Wherein, the fragment [21-39] is: Fmoc-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin; The fragment [20-39] is: Fmoc-Lys(Dde)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin; The fragment [1-39] is: 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(Dde)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin; The de-Dde fragment [1-39] is: Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin; The de-Dde fragment [1-39] is obtained after cutting the resin: Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Tr p(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2; The fully protected tazeparatide peptide resin is: Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH-resin. The fully protected tizepatide is as follows: Boc-Tyr(tbu)-Aib-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tb u)-Ile-Aib-Leu-Asp(otbu)-Lys(boc)-Ile-Ala-Gln(trt)-Lys(Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-AEEA-)-Glu(otbu)-Ala-Phe-Val-Gln(trt)-Trp(boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

2. The method for preparing tazeparatide according to claim 1, characterized in that: The protected amino acids and protected amino acid fragments include Fmoc-Lys(Dde)-OH, and also include Fmoc-Ser(tbu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tbu)-Gly-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, Boc-Eicosanedioic acid-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(tb u)-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Asp(otbu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(otbu)-Gly-OH and Boc-Tyr(tbu)-Aib-OH.

3. The method for preparing tazeparatide according to claim 1, characterized in that: The dosage of the protected amino acid and the protected amino acid fragment is 1.5 to 5 times the total molar number of the resin fed.

4. The method for preparing tazeparatide according to claim 1, characterized in that: The environmental parameters include reaction time and environmental temperature.

5. The method for preparing tazeparatide according to claim 1, characterized in that: In the coupling reaction, the deprotection reagent for removing Fmoc protection is a PIP / DMF mixed solution, and the mixed solution contains 20% to 25% (V / V) of piperidine.

6. The method for preparing tazeparatide according to claim 1, characterized in that: The degree of substitution of the Sieber resin is in the range of 0.2 to 0.5 mmol / g.

7. The method for preparing tazeparatide according to claim 1, characterized in that: The reagents for the coupling reaction include one of DIC / HOBT, Pybop / HOBT / DIEA, HATU / HOBT / DIEA or OP / DIC.

8. The method for preparing tazeparatide according to claim 1, characterized in that: The solvent for the coupling reaction includes one or more of DMF, DMSO or DCM.

9. The method for preparing tazeparatide according to claim 1, characterized in that: The cracking agent comprises water, 90% to 95% of trifluoroacetic acid, 1% to 4% of 1,2-ethanedithiol and 1% to 4% of triisopropylsilane.

10. The method for preparing tazeparatide according to claim 1, characterized in that: The crude product of tazeparatide is purified by high performance liquid chromatography, the purification chromatographic column is a reverse octadecyl bonded silica gel; the mobile phases are ammonium acetate solution and acetonitrile solution respectively, the salt conversion step is carried out by high performance liquid chromatography, and the mobile phase system is ammonia solution-acetonitrile solution.

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