A method for the synthesis of abaloparatide

Abstract

The application discloses the technical field of polypeptide medicine preparation and relates to a synthesis method of abaloparatide, which comprises the following steps: step 1, according to the amino acid sequence of the main chain of abaloparatide from the C terminal to the N terminal, protected amino acids are sequentially coupled by a solid-phase synthesis method to obtain a peptide resin of C terminal 1#-33# amino acids; step 2, N terminal 1# Ala is coupled by the solid-phase synthesis method to obtain an abaloparatide peptide resin by using coupling conditions for avoiding carbamate acylation side reactions; and step 3, the abaloparatide peptide resin is cleaved to obtain a crude abaloparatide peptide. The coupling conditions for avoiding carbamate acylation side reactions used in step 2 are as follows: (1) using Ala with an alpha-amino group protected by double protection groups as a reaction raw material; and / or (2) using a combination of a phosphoric acid type condensation reagent and an organic base as a condensation system. The application is optimized from the idea of a carbamate acylation side reaction mechanism, effectively reduces the impurity of added Ala, and reduces the content of the impurity from 1.03% in the crude peptide before optimization to 0.05%.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug preparation technology, specifically relating to a method for synthesizing apapain. Background Technology

[0002] Abaloparatide, developed by Radius Health, is a novel parathyroid hormone-related peptide (PTHrP) for the treatment of osteoporosis in postmenopausal women at high risk of fracture. It was launched in the United States on April 28, 2017, under the brand name TYMLOS. Both teriparatide and abaloparatide are PTHrPs, but abaloparatide is more effective at reducing fracture rates and the incidence of hypercalcemia. The peptide sequence of abaloparatide is as follows:

[0003] H-Ala 1 -Val-Ser-Glu-His-Gln 6 -Leu-Leu-His-Asp-Lys 11 -Gly-Lys-Ser-Ile-Gln 16 -Asp-Leu-Arg-Arg-Arg 21 -Glu-Leu-Leu-Glu-Lys 26 -Leu-Leu-Aib-Lys-Leu 31 -His-Thr-Ala-NH2.

[0004] Apapatide synthesis generates an Ala adduct impurity at the N-terminal 1 position, which is difficult to remove during purification due to its proximity to the main component. Currently, three common methods reported in the literature for avoiding Ala adduct impurities in apapatide preparation are: ① The most common method is to wash and capture residual piperidine to prevent the loss of the Fmoc protecting group during coupling, thus avoiding the formation of adduct impurities. ② Using Boc-Ala-OH as the N-terminal amino acid for coupling avoids the influence of residual piperidine and small amounts of dimethylamine impurities in DMF on the Fmoc protecting group, thus avoiding the formation of adduct impurities. Patents WO1998030590 and WO2020202182 both employ the Boc-Ala-OH coupling method. ③ Using an Ala-Val dipeptide as the N-terminal amino acid for coupling reduces the generation of adduct impurities from single amino acids. Patent CN108047329A uses the Fmoc-Ala-Val-OH dipeptide coupling method to reduce the generation of Ala adduct impurities.

[0005] The methods described above can reduce Ala adduct impurities to some extent, but still do not meet the expected requirements. Increasing the number of washes and using Boc-Ala-OH eliminates the concern that residual piperidine in the reaction solution will remove the Fmoc protecting group and cause adduct impurities. This method can avoid some Ala adduct impurities, but it still cannot avoid the influence of impurities generated by side reactions, and the purification effect does not meet the expected target. The synthesis method using Ala-Val dipeptide raw materials can better avoid the occurrence of Ala adduct impurities, but it is prone to racemic side reactions during dipeptide coupling, introducing and increasing new impurities. At the same time, this Ala adduct impurity occurs at the 34th amino acid at the N-terminus of the long peptide coupling, which can easily cause coupling difficulties and incompleteness when using Ala-Val dipeptides.

[0006] Therefore, it is of great significance to develop a new method for synthesizing apapatide that avoids Ala adduct impurities. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing apapain to effectively avoid Ala adduct impurities.

[0008] The main side reaction leading to the addition of Ala impurities to apapatide is a carbamate acylation reaction, primarily resulting from the symmetrical anhydride rearrangement during amino acid activation and condensation. In this reaction, the N-terminus of the secondary amine on one side of the symmetrical anhydride attacks the highly reactive CO bond on the other side, leading to rearrangement to form the Fmoc-(Fmoc-Ala)-Ala-OH structure. Furthermore, the side reaction mechanism for Boc-protected amino acids is consistent with that for Fmoc-protected amino acids. The side reaction mechanism is as follows:

[0009]

[0010] This invention optimizes factors related to the synthesis reaction, such as the reaction substrate, reaction temperature, reaction solvent, condensation reagent, and number of washes, to avoid the generation of Ala adduct impurities from the acylation side reaction of carbamates. The specific scheme is as follows:

[0011] This invention provides a method for synthesizing apapain, comprising the following steps:

[0012] Step 1: Following the amino acid sequence from C-terminus to N-terminus of the abapapeptide backbone, the amino acids are sequentially coupled and protected by solid-phase synthesis to obtain peptide resin containing amino acids 1# to 33# at the C-terminus.

[0013] Step 2: Using a solid-phase synthesis method and coupling conditions that avoid acylation side reactions of carbamate, N-terminal 1#Ala is coupled to obtain abapapeptide peptide resin.

[0014] Step 3: Cleavage the abapapeptide resin to obtain crude abapapeptide peptide;

[0015] The coupling conditions used in step 2 to avoid the acylation side reaction of carbamate are: (1) using Ala with α-amino protected by a double protecting group as the reaction substrate; and / or (2) using a combination of a phosphoric acid condensing agent and an organic base as the condensation system.

[0016] Furthermore, one of the dual protecting groups is Fmoc or Boc, and the other is an amino protecting group that can be removed by acid;

[0017] Preferably, the amino protecting group that can be removed by acid is selected from Hmb or Dmb.

[0018] Furthermore, the α-amino group protected by the double protecting group is selected from Fmoc-N(Hmb)-Ala-OH or Fmoc-N(Dmb)-Ala-OH, preferably Fmoc-N(Hmb)-Ala-OH.

[0019] When the coupling condition used to avoid the acylation side reaction of carbamate is "(1) using Ala with α-amino protected by double protecting groups as the reaction substrate", the condensation system for coupling the N-terminal 1#Ala can be one or more of DIPCDI / HOBt, PyBop / HOBt / organic base, HBTU / HOBt / organic base, DIPCDI / HOAt, HATU / HOAt / organic base, DPPA / organic base, FDPP / organic base, and DEPBT / organic base. Preferably, phosphoric acid condensing agents such as DPPA, DEPBT, and FDPP are selected. Preferably, the condensation system is DEPBT / organic base. The organic base used can be DIPEA, triethylamine, or N-methylmorpholine. Preferably, the organic base is DIPEA. Most preferably, the condensation system is DEPBT / DIPEA.

[0020] When the coupling condition used to avoid the acylation side reaction of carbamate is "(2) using a combination of a phosphoric acid condensing agent and an organic base as the condensation system", the reaction substrate can be an Ala with α-amino protected by two protecting groups, wherein one of the two protecting groups is Fmoc or Boc, and the other is an amino protecting group that can be removed by acid. Alternatively, it can be an Ala with α-amino protected by a single Fmoc or Boc group. For example, it can be Boc-Ala-OH, Fmoc-Ala-OH, Fmoc-N(Hmb)-Ala-OH, or Fmoc-N(Dmb)-Ala-OH. More preferably, it includes, but is not limited to, Fmoc-N(Dmb)-Ala-OH, Fmoc-N(Hmb)-Ala-OH, etc., where the N-terminus is a tertiary amine and a special amino acid with a protecting group that can be removed by acid attached to the N, and most preferably Fmoc-N(Hmb)-Ala-OH.

[0021] Furthermore, the phosphoric acid condensing agent is selected from one or more of DPPA, DEPBT and FDPP, preferably DEPBT;

[0022] The organic base is selected from one or more of DIPEA, triethylamine and N-methylmorpholine, preferably DIPEA.

[0023] Preferably, the condensation system is a composition of DEPBT and DIPEA.

[0024] Further, the specific operation of step 2 is as follows: the α-amino group protected by the double protecting group Ala is dissolved in a solvent, and then a phosphoric acid condensation reagent and an organic base are added for activation treatment to obtain an activated amino acid solution; the activated amino acid solution is added to a peptide resin containing N-terminal deprotected C-terminal 1#~33# amino acids for coupling reaction to obtain apapa peptide resin.

[0025] Preferably, the solvent is selected from chloroform, DCM or DMF, with DCM being the most preferred.

[0026] Furthermore, the activation treatment temperature is 0℃~35℃, preferably 0℃~5℃;

[0027] The temperature of the coupling reaction is 0℃~35℃, preferably 10℃~15℃;

[0028] Preferably, the peptide resin containing the deprotected C-terminal amino acids 1# to 33# is washed 9 times with DMF;

[0029] Preferably, after the coupling reaction is complete, the abapapeptide resin is washed with DMF 6 times.

[0030] Furthermore, the pyrolysis in step 3 uses a pyrolysis solution containing a trapping agent; the trapping agent is selected from one or more of PhSMe, PhOH, H2O, TIS, PhOMe and EDT;

[0031] Preferably, the lysis buffer is a composition of TFA / PhSMe / PhOMe / EDT.

[0032] Furthermore, the solid-state synthesis method described in step 1 is the Fmoc solid-state synthesis method;

[0033] The sequentially coupled protected amino acids are: Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-GIn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(Ot Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-GIn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH.

[0034] Furthermore, the above synthesis method also includes purification of the crude abapapeptide peptide.

[0035] When sequentially coupling protected amino acids using the Fmoc solid-phase synthesis method, an amino resin (Rink Amide Resin, Rink Amide AM Resin, or Rink Amide MBHA Resin) is used as the carrier, with a substitution degree of 0.1–1.0 mmol / g, more preferably 0.3–0.5 mmol / g. The coupling agent is one or more of DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA, and PyAop / HOAt / DIPEA, more preferably DIPCDI / HOBt. The coupling material is 2–8 times the molar amount of the resin, more preferably 3–5 times. In the sequential coupling process, the N-terminal protecting group Fmoc needs to be removed before coupling the next protecting amino acid. The reagent for removing the Fmoc protecting group is a 20% piperidine solution. The solvent used to dissolve the amino acid and the 20% piperidine solution is one or more of NMP, THF, DCM and DMF, more preferably DMF.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. This invention optimizes the process based on the mechanism of acetylation side reactions of carbamates. The main idea is to reduce the generation of symmetrical anhydrides and avoid rearrangement reactions, thereby avoiding acetylation side reactions of carbamates. Specifically, by adopting coupling conditions that avoid acetylation side reactions of carbamates, namely "using Ala with α-amino protected by double protecting groups as the reaction substrate" and / or "using a combination of phosphoric acid condensing reagent and organic base as the condensation system", the amount of added Ala impurities is effectively reduced. The relative percentage mass of added #1 Ala impurity to API is optimized from 1.03% in the crude peptide before optimization to 0.05%.

[0038] 2. This invention uses Ala, with its α-amino group protected by two protecting groups, as the reaction substrate. This effectively avoids the rearrangement side reaction of the α-amino group in the symmetrical anhydride intermediate, thereby reducing the generation of Ala impurities. This overcomes the shortcomings of existing techniques that use Ala-Val dipeptide raw materials, which introduce and increase new impurities and face difficulties in long peptide coupling. Furthermore, the use of a phosphoric acid condensation reagent effectively avoids the formation of symmetrical anhydrides, inhibiting the occurrence of carbamate acylation side reactions at the source.

[0039] 3. In the coupling of the N-terminal 1#Ala, the activation temperature of the reaction solution is further specified as 0℃~35℃, preferably 0℃~5℃, and the coupling reaction temperature is specified as 0℃~35℃, preferably 10℃~15℃. At these temperatures, complete coupling of the Ala amino acid can be achieved, and the occurrence of carbamate acylation side reactions is suppressed. The reaction solvent is preferably the aprotic solvent DCM, which can suppress electron migration and thus inhibit the rearrangement phenomenon of carbamate acylation side reactions. Attached Figure Description

[0040] Figure 1 The detection spectrum of apapain synthesized in Example 2;

[0041] Figure 2 The detection spectrum of apapain synthesized in Example 3;

[0042] Figure 3 The detection spectrum of apapain synthesized in Example 4;

[0043] Figure 4 The detection spectrum of apapain synthesized in Example 5;

[0044] Figure 5 The detection spectrum of apapain synthesized in Comparative Example 1;

[0045] Figure 6 This is the mass spectrum of abapapeptide synthesized in Example 5. Detailed Implementation

[0046] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer. The meanings of the abbreviations used in this invention are as follows:

[0047]

[0048] Example 1: C-terminus 1 of apapatinide # ~33 # Preparation of amino acid peptide resin

[0049] Weigh 33.3 g of Rink Amide resin (substitution degree 0.30 mmol / g, 10 mmol), swell the resin with DMF under nitrogen purge for 30 minutes, dry the resin, and wash twice with DMF. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir at room temperature for 10 minutes, dry the resin, repeat the operation once, and then wash with DMF 6 times. Separately weigh 12.45 g (40 mmol) of Fmoc-Ala-OH, 6.49 g (48 mmol) of HOBT, and 60 ml of DMF, stir to dissolve, add 8.13 ml (52 mmol) of DIPCDI under ice bath for activation, and then add to the resin and react at room temperature for 2 hours. Use the Kaiser method to determine whether the reaction is complete. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and the above operation needs to be repeated. This criterion applies to the following content, and the reaction endpoint will continue to be determined by the Kaiser method in subsequent reactions. Repeat the steps above to remove the Fmoc protecting group and couple the corresponding amino acid, sequentially coupling Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, and Fmoc-As. p(OtBu)-OH, Fmoc-GIn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-GIn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH. After removing the Fmoc protecting group, the mixture was washed six times with DMF to obtain the C-terminal 1 of abapapeptide. # ~33 # Amino acid peptide resin.

[0050] Example 2: Synthesis of abapapeptide under the reaction conditions of Fmoc-N(Hmb)-Ala-OH / DEPBT / DIPEA / DCM

[0051] To 10 mmol of apapatin C-terminus 1 # ~33 # The amino acid peptide resin was added to the Ala amino acid reaction solution. 17.88 g (40 mmol) of Fmoc-N(Hmb)-Ala-OH, 11.9 g (40 mmol) of DEPBT, and 13.3 mL (80 mmol) of DIPEA were dissolved in 60 mL of DCM and added to the resin to initiate the reaction. The reaction was carried out at 10-15 °C for 2 h. After the reaction was complete, the Fmoc protecting group was removed with DBLK reagent, and the mixture was washed 6 times with DMF. The resin was then washed with methanol, shrunk 3 times, and dried under vacuum overnight to obtain the apapatitin peptide resin.

[0052] The above-mentioned abapapeptide resin was added to lysis buffer (TFA:PhOMe:PhSMe:EDT=90:2:5:3) at a ratio of 10 ml / g. The mixture was stirred at room temperature for 3 hours, filtered, and the filtrate was poured into frozen methyl ether to precipitate. After centrifugation, the precipitate was washed three times with methyl ether. The precipitate was then dried under vacuum to obtain 61.9 g of crude abapapeptide peptide with a purity of 80.05%.

[0053] After purification and quality testing, the crude abapapeptide produced using the Fmoc-N(Hmb)-Ala-OH / DEPBT / DIPEA / DCM reaction starter at 10-15℃ contained 0.05% Ala impurities. The detection chromatogram is shown below. Figure 1 .

[0054] Example 3: Synthesis of abapapeptide under Boc-Ala-OH / DEPBT / DIPEA / DMF reaction conditions

[0055] Take the C-terminus 1 of the abapapeptide obtained in Example 1 # ~33 # 10 mmol of amino acid peptide resin was added to Ala amino acid reaction solution. 7.56 g (40 mmol) of Boc-Ala-OH, 11.9 g (40 mmol) of DEPBT, and 13.3 mL (80 mmol) of DIPEA were dissolved in 60 mL of DMF and added to the resin to initiate the reaction. The reaction was carried out at 25 °C for 2 h. After the reaction was complete, the Fmoc protecting group was removed with DBLK reagent, and the mixture was washed 6 times with DMF. The resin was then washed with methanol, shrunk 3 times, and dried under vacuum overnight to obtain apapatitin peptide resin.

[0056] The above-mentioned abapapeptide resin was added to lysis buffer (TFA:PhOMe:PhSMe:EDT=90:2:5:3) at a ratio of 10 ml / g. The mixture was stirred at room temperature for 3 hours, filtered, and the filtrate was poured into frozen methyl ether to precipitate. After centrifugation, the precipitate was washed three times with methyl ether. The precipitate was then dried under vacuum to obtain 60.5 g of crude abapapeptide with a purity of 79.57%.

[0057] After purification and quality testing, the crude abapapeptide produced using Boc-Ala-OH / DEPBT / DIPEA / DMF reaction raw materials at 25°C contained 0.11% Ala impurities. The detection chromatogram is shown below. Figure 2 .

[0058] Example 4: Synthesis of abapapeptide under the reaction conditions of Fmoc-Ala-OH / DEPBT / DIPEA / DMF

[0059] Take the C-terminus 1 of the abapapeptide obtained in Example 1 # ~33 # 10 mmol of amino acid peptide resin was added to Ala amino acid reaction solution. 12.45 g (40 mmol) of Fmoc-Ala-OH, 11.9 g (40 mmol) of DEPBT, and 13.3 mL (80 mmol) of DIPEA were dissolved in 60 mL of DMF and added to the resin to initiate the reaction. The reaction was carried out at 25 °C for 2 h. After the reaction was complete, the Fmoc protecting group was removed with DBLK reagent, and the mixture was washed 6 times with DMF. The resin was then washed with methanol, shrunk 3 times, and dried under vacuum overnight to obtain apapatitin peptide resin.

[0060] The above-mentioned abapapeptide resin was added to lysis buffer (TFA:PhOMe:PhSMe:EDT=90:2:5:3) at a ratio of 10 ml / g. The mixture was stirred at room temperature for 3 hours, filtered, and the filtrate was poured into frozen methyl ether to precipitate. After centrifugation, the precipitate was washed three times with methyl ether. The precipitate was then dried under vacuum to obtain 60.5 g of crude abapapeptide with a purity of 79.57%.

[0061] After purification and quality testing, the crude abapapeptide produced using Fmoc-Ala-OH / DEPBT / DIPEA / DMF reaction raw materials at 25°C contained 0.23% Ala impurities. The detection chromatogram is shown below. Figure 3 .

[0062] Example 5: Synthesis of apapatide under the reaction conditions of Fmoc-N(Hmb)-Ala-OH / HOBt / DMF

[0063] Take the C-terminus 1 of the abapapeptide obtained in Example 1 # ~33 #10 mmol of amino acid peptide resin was added to Ala amino acid reaction solution. 17.88 g (40 mmol) of Fmoc-N(Hmb)-Ala-OH, 6.49 g (48 mmol) of HOBT, and 60 ml of DMF were added to the resin to initiate the reaction, which was carried out at 25 °C for 2 h. After the reaction was complete, the Fmoc protecting group was removed with DBLK reagent, and the mixture was washed 6 times with DMF. The resin was then washed with methanol, shrunk 3 times, and dried under vacuum overnight to obtain apapatitin peptide resin.

[0064] The above-mentioned abapapeptide resin was added to lysis buffer (TFA:PhOMe:PhSMe:EDT=90:2:5:3) at a ratio of 10 ml / g. The mixture was stirred at room temperature for 3 hours, filtered, and the filtrate was poured into frozen methyl ether to precipitate. After centrifugation, the precipitate was washed three times with methyl ether. The precipitate was then dried under vacuum to obtain 60.5 g of crude abapapeptide with a purity of 79.57%.

[0065] After purification and quality testing, the crude abapapeptide produced using Fmoc-Ala-OH / HOBt / DMF reaction raw materials at 25°C contained 0.24% Ala impurities. The detection chromatogram is shown below. Figure 4 .

[0066] Comparative Example 1: Synthesis of abapapeptide under the reaction conditions of Fmoc-Ala-OH / HOBt / DIC / DMF

[0067] Take the C-terminus 1 of the abapapeptide obtained in Example 1 # ~33 # 10 mmol of amino acid peptide resin was added to Ala amino acid reaction solution. 12.45 g (40 mmol) of Fmoc-Ala-OH, 6.49 g (48 mmol) of HOBT, and 60 ml of DMF were added to the resin to initiate the reaction, which was carried out at 25 °C for 2 h. After the reaction was complete, the Fmoc protecting group was removed with DBLK reagent, and the mixture was washed 6 times with DMF. The resin was then washed with methanol, shrunk 3 times, and dried under vacuum overnight to obtain apapatitin peptide resin.

[0068] The above-mentioned abapapeptide resin was added to lysis buffer (TFA:PhOMe:PhSMe:EDT=90:2:5:3) at a ratio of 10 ml / g. The mixture was stirred at room temperature for 3 hours, filtered, and the filtrate was poured into frozen methyl ether to precipitate. After centrifugation, the precipitate was washed three times with methyl ether. The precipitate was then dried under vacuum to obtain 60.3 g of crude abapapeptide peptide with a purity of 78.23%.

[0069] After purification and quality testing, the crude abapapeptide produced using Fmoc-Ala-OH / HOBt / DIC / DMF reaction raw materials at 25°C contained 1.03% Ala impurities. The detection chromatogram is shown below. Figure 5 .

[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method of synthesizing abaloparatide, characterized by, Includes the following steps: Step 1: Following the amino acid sequence from C-terminus to N-terminus of the abapapeptide backbone, the amino acids are sequentially coupled and protected by solid-phase synthesis to obtain peptide resin containing amino acids 1# to 33# at the C-terminus. Step 2: Using a solid-phase synthesis method and coupling conditions that avoid acylation side reactions of carbamate, N-terminal 1#Ala is coupled to obtain abapapeptide peptide resin. Step 3: Cleavage the abapapeptide resin to obtain crude abapapeptide peptide; The coupling conditions used in step 2 to avoid the acylation side reaction of carbamate are: (1) using Ala with α-amino protected by a double protecting group as the reaction substrate; and (2) using a combination of a phosphoric acid condensing agent and an organic base as the condensation system; The α-amino group protected by the double protecting group is selected from Fmoc-N(Hmb)-Ala-OH; The condensation system is a composition of DEPBT and DIPEA; The coupling reaction temperature is 10℃~15℃.

2. The method of synthesis of claim 1, wherein, The specific operation of step 2 is as follows: Ala, whose α-amino group is protected by a double protecting group, is dissolved in a solvent, and then a phosphoric acid condensation reagent and an organic base are added for activation treatment to obtain an activated amino acid solution; the activated amino acid solution is added to a peptide resin containing N-terminal deprotected C-terminal 1#~33# amino acids for coupling reaction to obtain apapa peptide resin. The activation treatment temperature is 0℃~5℃.

3. The method of synthesis of claim 2, wherein, The solvent is selected from chloroform, DCM or DMF.

4. The synthesis method according to claim 3, characterized in that, The solvent is DCM.

5. The synthesis method according to claim 4, characterized in that, The peptide resin containing the N-terminal deprotected C-terminal amino acids 1#~33# was washed 9 times with DMF.

6. The synthesis method according to claim 5, characterized in that, After the coupling reaction was completed, the abapapeptide resin was washed 6 times with DMF.

7. The synthesis method according to claim 1, characterized in that, In step 3, the pyrolysis uses a pyrolysis solution containing a trapping agent; the trapping agent is selected from one or more of PhSMe, PhOH, H2O, TIS, PhOMe and EDT.

8. The synthesis method according to claim 7, characterized in that, The lysis solution is a composition of TFA, PhSMe, PhOMe and EDT.

9. The synthesis method according to claim 1, characterized in that, The solid-phase synthesis method described in step 1 is the Fmoc solid-phase synthesis method; The sequentially coupled protected amino acids are: Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-GIn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(Ot Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-GIn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH.

10. The synthesis method according to claim 1, characterized in that, It also includes the purification process of crude abapapeptide.

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