CN113880936B - Solid-phase synthesis method of abamectin - Google Patents

Abstract

The invention provides a solid-phase synthesis method of abamectin, belonging to the field of pharmaceutical chemistry, which comprises the steps of synthesizing 1-9 fragments, 10-20 fragments and 21-34 fragments according to the amino acid sequence from the C end to the N end of a peptide chain of the abamectin, and then coupling 3 fragments by using dimethylformamide and ethylene glycol diglycidyl ether as solvents and using HBTU/HOBT/DIPEA three reagents as a coupling system to obtain the abamectin. The solid-phase synthesis method of the abamectin has the advantages of low cost, simplicity in operation, high yield and high purity.

Description

Solid-phase synthesis method of abamectin

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a solid-phase synthesis method of abamectin.

Background

Abapatide (Abaloperatide) is a novel parathyroid hormone-related peptide developed by Radius Health corporation for the treatment of postmenopausal osteoporosis with a high risk of fracture. Marketed in the united states on 28 th 4 th 2017 under the name TYMLOS. Teriparatide and abapa peptide are parathyroid hormone-related peptide (PTHrP), but abapa peptide is better able to reduce the rate of bone fracture and incidence of hypercalcemia.

The Abapatide consists of 34 amino acids and has a molecular formula of C₁₇₄H₃₀₀N₅₆O₄₉And the molecular weight is 3960.59.

The prior art discloses a solid-phase resin structure as shown in formula I as disclosed in a Chinese patent with the publication number of CN 111944040B; rinkamide Linker-AA_n-AM resin I wherein AA is the same or different side chain protected amino acid: lys, Arg; n is an integer of 2 to 6. The solid phase resin can be used for solid phase synthesis of the abamectin. The method has the bright points and the advantages that the Rink Amide Linker-AA with hydrophobicity is used_nThe AM resin replaces the traditional solid phase coupling initial resin, so that a large amount of deletion impurities caused by beta folding of a peptide sequence in the process of synthesizing the abapa peptide sequence are effectively avoided. Meanwhile, Fmoc-Arg (Pbf) -OH is selected as a protective amino acid of continuous Arg-Arg-Arg, thereby effectively avoiding the generation of a large amount of Arg-deficient impurities caused by difficult sequence coupling of continuous arginine. The method has the advantages of simple process operation, high purity of crude product, high yield and good industrial production prospect.

Disclosure of Invention

The invention aims to provide a solid-phase synthesis method of abapa peptide, which can promote the rapid dissolution of a longer peptide chain in the synthesis process, improve the coupling rate of large steric hindrance amino acids Fmoc-Arg (Pbf) -OH and improve the yield and purity of the abapa peptide.

The technical scheme adopted by the invention for realizing the purpose is as follows:

provides a solid-phase synthesis method of abamectin, which comprises the following specific steps:

A. Fmoc-Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-amino resin is sequentially coupled and synthesized according to the Fmoc protection strategy, and after the Fmoc protection group is removed, apeptide resin segment 1 is obtained;

Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -2CTC resin is coupled and synthesized according to Fmoc protection strategy in sequence, and peptide fragment 2 is obtained after adding lysate and removing 2-CTC resin;

sequentially coupling and synthesizing Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -Gly-Lys (Boc) -Ser (tBu) -2CTC resin according to an Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 3;

B. coupling reaction is carried out on the C end of the peptide fragment 2 and the N end of thepeptide resin fragment 1 to obtain a peptide resin I;

C. removing the N-terminal protecting group of the peptide resin I, and performing coupling reaction with the C terminal of the peptide fragment 3 to obtain a peptide resin II;

D. adding a lysis solution to remove the resin and all protecting groups of the peptide resin II to obtain the abapa peptide;

optionally, step E, reverse phase chromatography preparation and purification to obtain an abamectin pure product;

fmoc is an amino acid N-terminal protecting group, and tBu, Trt, Boc, OtBu and Pbf are amino acid side chain protecting groups.

The Fmoc protection strategy in the step A is as follows: coupling Fmoc-protected amino acid on solid-phase synthetic resin or polypeptide-solid-phase synthetic resin; the terminal Fmoc protecting group is removed and the next Fmoc protected amino acid is coupled until completion.

In certain embodiments, the coupling reactions in steps B and C are coupled using a HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate)/HOBT (1-hydroxybenzotriazole)/DIPEA (N, N-diisopropylethylamine) three-reagent coupling system. Further, the molar ratio of the coupling reagents of the coupling system is as follows: HBTU, HOBT, DIPEA =1-2:1-2: 2-4.

In certain embodiments, the coupling reactions of step B and step C above are performed using DMF (dimethylformamide) and ethylene glycol diglycidyl ether as solvents. Further, the volume ratio of DMF to ethylene glycol diglycidyl ether is 2-4: 1-2. The DMF and the ethylene glycol diglycidyl ether with the volume ratio of 2-4:1-2 are taken as solvents, so that the solubility of the peptide chain of the abapatatin can be increased, and when the solvent is taken as a coupling reaction solvent, the solvent is favorable for quickly dissolving a longer peptide chain in a synthesis process, is favorable for carrying out a condensation reaction, can reduce the reaction time, improve the reaction efficiency, and further can improve the product yield and the product purity, reduce the production cost and simplify the operation steps.

In certain embodiments, the lysis solution in step D above comprises TFA (trifluoroacetic acid), thioanisole, TIS (triisopropylsilane), and water, corresponding to a volume ratio of 80-90:3-8:3-8: 3-8.

In certain embodiments, the method for synthesizingpeptide resin fragment 1 in step a above specifically comprises:

a1, carrying out coupling reaction on Fmoc-Ala-OH and amino resin under the action of a coupling system to obtain Fmoc-Ala-amino resin;

a2, deprotecting Fmoc protecting group of Fmoc-Ala-amino resin, and then coupling with Fmoc-Thr (tBu) -OH under the action of a coupling system to obtain Fmoc-Thr (tBu) -Ala-amino resin;

3, sequentially and sequentially carrying out amino acid extension coupling on Fmoc-His (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH according to the coupling mode of the step a2, and removing the N-terminal Fmoc protecting group to obtain H-Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-amino resin, namely thepeptide resin segment 1.

Preferably, the amino resin includes, but is not limited to, Rink Amide AM resin or Rink Amide MBHA resin. Further, the degree of substitution of the above resin is 0.5 to 1.0 mmol/g.

Preferably, the coupling system in the synthesis of the abovepeptide resin fragment 1 is HOBT/DIC (N, N-diisopropylcarbodiimide). Further, the molar ratio of HOBT to DIC in the above coupling system is 1:1 to 1.5.

Preferably, DMF is used as a solvent in the coupling reaction in the synthesis of thepeptide resin fragment 1.

In certain embodiments, the method of synthesizing the peptide fragment 2 in the above step a specifically comprises:

b1, carrying out coupling reaction on Fmoc-Glu (OtBu) -OH and 2-CTC resin under the action of a coupling system to obtain Fmoc-Glu (OtBu) -2CTC resin;

b2, carrying out deprotection of Fmoc protecting group on Fmoc-Glu (OtBu) -2CTC resin, and then carrying out coupling reaction with Fmoc-Leu-OH under the action of a coupling system to obtain Fmoc-Leu-Glu (OtBu) -2CTC resin;

3, sequentially coupling Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Ile-OH by amino acid extension according to the coupling method of step b2 to obtain Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -2-CTC-resin, adding lysis solution to remove the Fmoc 2-CTC-CTB-Glu-to obtain Fmoc-Ile-Gln- (Trt) -Asp (Asp-Leu) -Arg (Pbf) -Arg-Glu- (OtBu) -2-Glu-2-CTB-Glu- (Pbf) -2-Leu- (OtBu) -2-Glu- (Pbf) -2-Glu- (Pbf) and (Pbf) -Glu- (Pbf) -2-Glu- (Pbf) are added to obtain Fmoc-Glu-Leu-Leu-Leu-Glu- (Pbf-Glu-E- (Pbf) lysis solution Glu (OtBu) -OH, peptide fragment 2.

Preferably, the coupling system in the synthesis of the above peptide fragment 2 is HOBT/DIC. Further, the molar ratio of HOBT to DIC in the above coupling system is 1:1 to 1.5.

Preferably, the coupling reaction in the synthesis of the peptide fragment 2 is carried out using a mixed solvent of DCM and DMF in a volume ratio of 1:1-3 as a solvent.

More preferably, the coupling system for extension coupling Fmoc-Arg (Pbf) -OH in the synthesis of the peptide fragment 2 further comprises 2, 5-dichlorobenzoxazole and tetraethylthiuram disulfide. Further, the molar ratio of 2, 5-dichlorobenzoxazole to tetraethylthiuram disulfide is 1: 0.08-0.35. Further, the molar ratio of 2, 5-dichlorobenzoxazole to HOBT in the above reaction of extension coupling of the solid phase synthesized peptide fragment 2 to Fmoc-Arg (Pbf) -OH was 1: 4-5. In the process of synthesizing the abapa peptide, when the coupling large-steric-hindrance amino acid Fmoc-Arg (Pbf) -OH is subjected to extension coupling, 2, 5-dichlorobenzoxazole and tetraethylthiuram disulfide are added into a coupling system, so that the coupling rate of Fmoc-Arg (Pbf) -OH can be improved, and the yield and the purity of the abapa peptide are further improved.

Preferably, the lysis solution in the synthesis of the above peptide fragment 2 comprises TFE (trifluoroethanol) and DCM (dichloromethane) in a corresponding volume ratio of 1-2: 7-8.

In certain embodiments, the method of synthesizing the peptide fragment 3 in the above step a specifically comprises:

c1, carrying out coupling reaction on Fmoc-Ser (tBu) -OH and 2-CTC resin under the action of a coupling system to obtain Fmoc-Ser (tBu) -2CTC resin;

c2, deprotecting Fmoc protecting group of Fmoc-Ser (tBu) -2CTC resin, and then carrying out coupling reaction with Fmoc-Lys (Boc) -OH under the action of a coupling system to obtain Fmoc-Lys-Ser (tBu) -2CTC resin;

3, sequentially and sequentially carrying out amino acid extension coupling on Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH and Fmoc-Ala-OH according to the coupling mode of the step c2 to obtain Fmoc-Ala-Val (Ser tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Lys (OtBu) -Boc-Gly-Lys (Boc) (Ser tBu) -2CTC resin, adding lysate to remove 2-CTC resin to obtain Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -Gly-Lys (Boc) -Ser (tBu) -OH, i.e., peptide fragment 3.

Preferably, the coupling system in the synthesis of the above peptide fragment 3 is HOBT/DIC. Further, the molar ratio of HOBT to DIC in the above coupling system is 1:1 to 1.5.

Preferably, the coupling reaction in the synthesis of the peptide fragment 3 is performed using DMF as a solvent.

Preferably, the cleavage solution in the synthesis of the above peptide fragment 3 comprises TFE and DCM in a corresponding volume ratio of 1-2: 7-8.

In certain embodiments, the degree of substitution of the above 2-CTC resin is from 0.3 to 1.2 mmol/g.

In certain embodiments, the molar ratio of the single protected amino acid of the extensional coupling to HOBT in the coupling system is from 1:1 to 2 in the synthesis ofpeptide resin fragment 1, peptide fragment 2, and peptide fragment 3 described above.

In certain embodiments, the molar ratio ofpeptide resin fragment 1, peptide fragment 2, and peptide fragment 3 in step B above is from 1:1 to 3.

In certain embodiments, the deprotecting of the Fmoc protecting group described above is performed using a 20v/v% piperidine in dimethylformamide.

The invention also provides an application of the abamectin solid-phase synthesis method in preparing a medicament for treating osteoporosis.

The invention takes DMF and ethylene glycol diglycidyl ether with the volume ratio of 2-4:1-2 as solvent when synthesizing the peptide resin I and the peptide resin II, thereby having the following beneficial effects: the solubility of the peptide chain of the abapatatin can be increased, when the abapatatin is used as a coupling reaction solvent, the rapid dissolution of a longer peptide chain in a synthetic process is facilitated, the condensation reaction is facilitated, the reaction time can be shortened, the reaction efficiency is improved, the product yield and the product purity can be improved, the production cost is reduced, and the operation steps are simplified.

According to the invention, when the large steric hindrance amino acid Fmoc-Arg (Pbf) -OH is coupled, 2, 5-dichlorobenzoxazole and tetraethylthiuram disulfide are added into the coupling system, so that the coupling system has the following beneficial effects: the coupling rate of Fmoc-Arg (Pbf) -OH can be improved, and the yield and the purity of the abapa peptide are further improved.

Therefore, the invention is the solid-phase synthesis method of the abamectin with lower cost, simple operation, high yield and high purity.

Drawings

FIG. 1 is a mass spectrum of an abapa peptide pure product in example 1 of the present invention;

FIG. 2 shows the results of the solubility test in test example 1 of the present invention;

FIG. 3 shows the coupling ratio R in test example 1 of the present invention₁The measurement result of (1);

FIG. 4 shows the coupling ratio R in test example 1 of the present invention₂The measurement result of (1);

FIG. 5 shows the coupling ratio R in test example 1 of the present invention₃The measurement result of (1);

FIG. 6 shows the coupling ratio S in test example 1 of the present invention₁The measurement result of (1);

FIG. 7 shows the coupling ratio S in test example 1 of the present invention₂The measurement result of (1);

FIG. 8 shows the results of measurement of the purity of abamectin in test example 1 of the present invention;

FIG. 9 shows the results of measurement of the yield of abapa peptide in test example 1 of the present invention.

Detailed Description

The present invention is further described in detail with reference to the following examples:

example 1:

1. a solid phase synthesis method of abamectin specifically comprises the following steps:

1.1 Synthesis of peptide resin fragment 1:

1.1.1 g Rink Amide AM resin (crosslinked 1m/m% divinylbenzene, degree of substitution 0.8mmol/g, 200 mesh, available from Shanghai Aladdin Biotechnology Ltd.) was added to a solid phase reaction column, swollen with 300mL DMF for 30min and then dried to obtain an activated resin.

1.1.2 adding 350mL of 20v/v% piperidine DMF solution into the activated resin, oscillating and reacting at 25 ℃ for 12min, filtering, and washing with DMF for 4 times to obtain the resin with the Fmoc protecting group removed; dissolving 50mmol Fmoc-Ala-OH and 75mmol HOBT in 300mL DMF, adding 90mmol DIC under the condition of ice-water bath to activate for 5min, adding into a solid phase reaction column, reacting at 25 ℃ for 2h, and detecting the reaction end point by ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, which indicates that the reaction is incomplete, and the coupling reaction is required for 1 h). After the reaction, the resin was washed 4 times with DMF to obtain Fmoc-Ala-Rink Amide AM resin.

1.1.3 adding 350mL of 20v/v% piperidine DMF solution into Fmoc-Ala-Rink Amide AM resin, oscillating and reacting for 12min at 25 ℃, filtering, and washing with DMF for 4 times to obtain H-Ala-Rink Amide AM resin; adding 50mmol Fmoc-Thr (tBu) -OH and 75mmol HOBT into 300mL DMF for dissolving, adding 90mmol DIC for activation for 5min under the condition of ice-water bath, adding into a solid phase reaction column, reacting for 2h at 25 ℃, and detecting the reaction end point by ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, the reaction is incomplete, and the coupling reaction is required for 1 h). After the reaction is finished, washing the mixture for 4 times by using DMF to obtain Fmoc-Thr (tBu) -Ala-Rink Amide AM resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, sequentially and respectively carrying out amino acid extension coupling on Fmoc-His (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, finally carrying out oscillation reaction for 12min at 25 ℃ by using 350mL of 20v/v% piperidine solution, and carrying out suction filtration to obtain H-Lys (Boc) -Leu-Aib-Lys (Boc) -Leu-His Trt) -Thr (tBu) -Ala-Rink Amide AM resin, namely thepeptide resin fragment 1.

1.2 Synthesis of peptide fragment 2:

1.2.1 50g of 2-CTC resin (crosslinked 1m/m% divinylbenzene, degree of substitution 0.6mmol/g, 200 mesh, available from Shanghai Aladdin Biotechnology Ltd.) was charged into a solid phase reaction column, swollen with 500mL of DMF for 30min and then dried by suction to obtain an activated resin.

1.2.2 adding 600mL of 20v/v% piperidine DMF solution into the activated resin, oscillating and reacting at 25 ℃ for 12min, filtering, and washing with DMF for 4 times to obtain the resin with the Fmoc protecting group removed; dissolving 110mmol Fmoc-Glu (OtBu) -OH and 150mmol HOBT in a mixed solvent of 150mL DCM and 350mL DMF, adding 170mmol DIC under ice-water bath condition for activation for 5min, adding into a solid phase reaction column, and performing N-phase reaction at 25 deg.C₂Stirring for 2h, and detecting the end point of the reaction with ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, indicating that the reaction is incomplete, and the coupling reaction is required for 1 h). After the reaction was completed, the resin was washed 4 times with DMF to obtain Fmoc-Glu (OtBu) -2CTC resin.

1.2.3 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, and washing with DMF for 4 times to obtain H-Ala-amino resin; 110mmol Fmoc-Glu (OtBu) -OH and 150mmol HOBT are added into a mixed solvent of 150mL DCM and 350mL DMF for dissolution, 170mmol DIC is added under the condition of ice-water bath for activation for 5min, then the mixture is added into a solid phase reaction column for reaction for 2h at 25 ℃, and the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, the reaction is incomplete, and the coupling reaction is needed for 1 h). After the reaction is finished, washing the mixture for 4 times by using DMF to obtain Fmoc-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially carrying out amino acid extension coupling on Fmoc-Leu-OH and Fmoc-Glu (OtBu) -OH one by one to obtain Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin.

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol of Fmoc-Arg (Pbf) -OH, 150mmol of HOBT, 30mmol of 2, 5-dichlorobenzoxazole and 6.6mmol of tetraethylthiuram disulfide were dissolved in a mixed solvent of 150mL of DCM and 350mL of DMF, and 170mmol of DIC was added under the condition of ice-water bath to activate for 5min, and then the mixture was put into a solid-phase reaction column and reacted at 25 ℃ for 2 h. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin.

1.2.5 repeating 1.2.3 steps of removing Fmoc protection and adding corresponding amino acid for coupling, sequentially and sequentially carrying out amino acid extension coupling on Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Ile-OH to obtain Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -2CTC resin, adding 800mL of lysate (TFE and dichloromethane in a volume ratio of 1: 4), reacting for 3h at 20 ℃, finishing the reaction, filtering the resin, collecting filtrate, spirally steaming the filtrate to about 400mL, dropwise adding the filtrate into 4500mL of diethyl ether, centrifuging, washing the diethyl ether, and drying in vacuum to obtain the 2 peptide fragment.

1.3 Synthesis of peptide fragment 3:

1.3.1 50g of 2-CTC resin (crosslinked 1m/m% divinylbenzene, degree of substitution 0.6mmol/g, 200 mesh, available from Shanghai Aladdin Biotechnology Ltd.) was charged into a solid phase reaction column, swollen with 500mL of DMF for 30min and then dried by suction to obtain an activated resin.

1.3.2 adding 600mL of 20v/v% piperidine DMF solution into the activated resin, oscillating and reacting at 25 ℃ for 12min, filtering, and washing with DMF for 4 times to obtain the resin with the Fmoc protecting group removed; 110mmol Fmoc-Ser (tBu) -OH and 150mmol HOBT are added into a mixed solvent of 150mL DCM and 350mL DMF for dissolving, 170mmol DIC is added under the condition of ice-water bath for activation for 5min, then the mixture is added into a solid phase reaction column for reaction for 2h at 25 ℃, and the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, the reaction is incomplete, and the coupling reaction needs to be carried out for 1 h). After the reaction is finished, washing the reaction product for 4 times by using DMF to obtain Fmoc-Ser (tBu) -2CTC resin.

1.3.3 to Fmoc-Ser (tBu) -2CTC resin, adding 600mL of 20v/v% piperidine DMF solution, oscillating and reacting at 25 ℃ for 12min, filtering, and washing with DMF for 4 times to obtain H-Ala-amino resin; 110mmol Fmoc-Lys (Boc) -OH and 150mmol HOBT are added into a mixed solvent of 150mL DCM and 350mL DMF for dissolution, 170mmol DIC is added under the condition of ice-water bath for activation for 5min, then the mixture is added into a solid phase reaction column for reaction for 2h at 25 ℃, and the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is complete, the resin is colored, the reaction is incomplete, and the coupling reaction is required for 1 h). After the reaction is finished, washing the reaction product for 4 times by using DMF to obtain Fmoc-Lys-Ser (tBu) -2CTC resin; repeating the above steps of removing Fmoc protection and adding corresponding amino acid for coupling, sequentially and sequentially subjecting Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH and Fmoc-Ala-OH to amino acid extension coupling to obtain Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His-Trt) -Asp (OtBu) -Boc-Gly-Lys (Boc) -2CTC resin, adding 800mL of lysate (TFE and dichloromethane in a volume ratio of 1: 4), reacting at 20 ℃ for 3h, filtering the resin after the reaction is finished, collecting the filtrate, evaporating the volume of the filtrate to about 400mL, dropwise adding the filtrate into 4500mL of diethyl ether, centrifuging, washing with anhydrous diethyl ether, and drying in vacuum to obtain peptide fragment 3.

1.4 Synthesis of peptide resin I:

1.4.1 dissolving the peptide fragment 2, 40mmol HBTU and 40mmol HOBT prepared in 1.2 in a mixed solvent of 450mL DMMF and 150mL ethylene glycol diglycidyl ether, activating with 90mL DIPEA under ice bath conditions for 8min, adding into the solid phase reaction column of thepeptide resin fragment 1 prepared in 1.1, reacting at 25 ℃ for 3h, blocking with 400mL blocking solution (DIPEA, methanol and DCM at a volume ratio of 1:2: 17) three times for 3min each, washing with DMF 4 times to obtain Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Boc-Lys (Leu-His (Trt) -Thr-Ala-Ambi-AM resin, namely peptide resin I.

1.5 Synthesis of peptide resin II:

1.5.1 adding 700mL of 20v/v% piperidine solution in DMF to peptide resin I, reacting at 25 ℃ for 12min with shaking, filtering, and washing 4 times with DMF to obtain H-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr tBu (Ala-Rink Amide AM resin;

1.5.2 dissolving the peptide fragment 3, 40mmol HBTU and 40mmol HOBT obtained from 1.3 in a mixed solvent of 450mL DMMF and 150mL ethylene glycol diglycidyl ether, activating with 90mL DIPEA under ice bath conditions for 8min, adding into a solid phase reaction column of peptide resin I obtained from 1.4, reacting at 25 deg.C for 3h, blocking with 400mL blocking solution (DIPEA, methanol and DCM at a volume ratio of 1:2: 17) three times for 3min each, and washing with DMF 4 times to obtain Fmoc-Ala-Val-Ser tBu (Glu OtBu) -His (trt) -Gln Tr (t) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Gly-Lys) (Boc) -Ser (tBu) -Ile-Gln Glu (Tru) -Asp (Leu-Arg (Pbf) -Boc-Leu-Leu-Leu (Trt) (Pbf) (Boc-Arg-Pbf) -Lys- (Boc-Leu) (Met-Leu- (OtBu-Leu-Glu-Leu) (Trt) (Pbf) (s-Arg (Pbf) and Pbf) Lys (Boc-Leu-Leu-OtBu-Leu-Leu-Leu-4) obtained by (4-4) and (4-Glu-4-Glu-Arg-4-Arg-His (Trt-Arg-4 (Trt-Glu-4-Arg-4 (Trb-Arg-4-Glu-4-Arg-Glu-Arg-Glu-4 (Trt-Arg-Glu-Arg-4 (Trt) (Trt-Arg-4 (Trp-Arg-4 (Trp-Arg-4 (Trb-Arg-4 (Trp-Arg-Glu-4 (Trp-4 (Trb-4 (Trp-Arg-Glu-Arg-4 (Trp-Arg-4 (Tab-Arg-4 (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-Rink Amide AM resin, peptide resin II.

1.6 adding 800mL of lysate (TFA, thioanisole, TIS and water in a volume ratio of 85:5:7: 3) into the peptide resin II prepared in the step 1.5, reacting at 25 ℃ for 3 hours, filtering the resin after the reaction is finished, collecting the filtrate, evaporating the filtrate to about 450mL in volume, dropwise adding the filtrate into 5000mL of diethyl ether, centrifuging, washing with anhydrous diethyl ether, and drying in vacuum to obtain the crude product of abamectin. The crude abapatatin solution was filtered through a 0.45um microfiltration membrane. The detection proves that the purity of the abapatatin crude product is 81.2%. Purifying the crude abapatatin by using a reversed-phase high performance liquid chromatography (RP-HPLC) method: a chromatographic column: 50 x 250mm reversed phase C18 column, using mobile phase A as 0.1v/v% trifluoroacetic acid water solution, mobile phase B as 0.1v/v% trifluoroacetic acid acetonitrile solution, eluting, purifying, collecting and concentrating the needed components, obtaining abapatatin; then, eluting and transferring salt by adopting a mobile phase A as an acetic acid aqueous solution with the volume fraction of 0.1% and a mobile phase B as an acetic acid-acetonitrile solution with the volume fraction of 0.1%; both steps were gradient elution: the flow rate is 80mL/min, the elution time is 60min, the volume percentage of phase B of the elution gradient mobile phase is 10% -60%, the wavelength of ultraviolet detection is 220nm, the required components are collected, concentrated and freeze-dried to obtain the pure abamectin product with the purity of 99.1%, the mass spectrogram of the pure abamectin product is shown in figure 1, wherein h is 832.12, i is 1069.25, j is 1425.69, k is 1788.68, l is 1979.89, m is 3959.64, n is 4149.64, and o is 4273.54. As can be seen from FIG. 1, the molecular weight of the pure Abapatide is 3959.64, which is basically consistent with the theoretical molecular weight of Abapatide, confirming that Abapatide is Abapatide.

Example 2:

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol of Fmoc-Arg (Pbf) -OH, 150mmol of HOBT, 30mmol of 2, 5-dichlorobenzoxazole and 3.5mmol of tetraethylthiuram disulfide were dissolved in a mixed solvent of 150mL of DCM and 350mL of DMF, and 170mmol of DIC was added under the condition of ice-water bath to activate for 5min, and then the mixture was put into a solid-phase reaction column and reacted at 25 ℃ for 2 h. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin. The rest of the process was identical to example 1.

Example 3:

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol of Fmoc-Arg (Pbf) -OH, 150mmol of HOBT, 30mmol of 2, 5-dichlorobenzoxazole and 9.9mmol of tetraethylthiuram disulfide were dissolved in a mixed solvent of 150mL of DCM and 350mL of DMF, and 170mmol of DIC was added under the condition of ice-water bath to activate for 5min, and then the mixture was put into a solid-phase reaction column and reacted at 25 ℃ for 2 h. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin. The rest of the process was identical to example 1.

Example 4:

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol of Fmoc-Arg (Pbf) -OH, 150mmol of HOBT and 6.6mmol of tetraethylthiuram disulfide are added into a mixed solvent of 150mL of DCM and 350mL of DMF for dissolution, 170mmol of DIC is added under the condition of ice-water bath for activation for 5min, and then the mixture is added into a solid phase reaction column for reaction for 2h at 25 ℃. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin. The rest of the process was identical to example 1.

Example 5:

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol of Fmoc-Arg (Pbf) -OH, 150mmol of HOBT and 30mmol of 2, 5-dichlorobenzoxazole are dissolved in a mixed solvent of 150mL of DCM and 350mL of DMF, 170mmol of DIC is added under the condition of ice-water bath to activate for 5min, and then the mixture is added into a solid phase reaction column to react for 2h at 25 ℃. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin. The rest of the process was identical to example 1.

Example 6:

1.2.4 adding 600mL of 20v/v% piperidine DMF solution into Fmoc-Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin, oscillating and reacting at 25 ℃ for 12min, filtering, washing 4 times with DMF to obtain H-Ala-amino resin; 110mmol Fmoc-Arg (Pbf) -OH and 150mmol HOBT were dissolved in a mixed solvent of 150mL DCM and 350mL DMF, and 170mmol DIC was added thereto in an ice-water bath to activate the mixture for 5min, and then the mixture was put on a solid phase reaction column and reacted at 25 ℃ for 2 h. Adding a confining liquid (pyridine (20mL) and acetic anhydride (22mL)), reacting for 2h, draining, and after the reaction is finished, washing with DMF for 4 times to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin; repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially performing amino acid extension coupling on Fmoc-Arg (Pbf) -OH and Fmoc-Arg (Pbf) -OH one by one to obtain Fmoc-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -2CTC resin. The rest of the process was identical to example 1.

Example 7:

the reaction solvents used for the synthesis of the peptide resin I and the peptide resin II are 480mL of DMF and 120mL of ethylene glycol diglycidyl ether. The rest of the process was identical to example 1.

Example 8:

the reaction solvents used for the synthesis of the peptide resin I and the peptide resin II are 400mL of DMF and 200mL of ethylene glycol diglycidyl ether. The rest of the process was identical to example 1.

Example 9:

the reaction solvents used for the synthesis of the peptide resin I and the peptide resin II are 500mL of DMF and 100mL of ethylene glycol diglycidyl ether. The rest of the process was identical to example 1.

Example 10:

the reaction solvents used for the synthesis of the peptide resin I and the peptide resin II are 200mL of DMF and 400mL of ethylene glycol diglycidyl ether. The rest of the process was identical to example 1.

Example 11:

the reaction solvent used for synthesizing the peptide resin I and the peptide resin II is 600mL of ethylene glycol diglycidyl ether. The rest of the process was identical to example 1.

Example 12:

the reaction solvent used for the synthesis of peptide resin I and peptide resin II was 600mL DMF. The rest of the process was identical to example 1.

Test example 1:

1.1 solubility test:

organic solvent 1: DMF and ethylene glycol diglycidyl ether in a volume ratio of 3: 1;

organic solvent 2: DMF and ethylene glycol diglycidyl ether in a volume ratio of 4: 1;

organic solvent 3: DMF and ethylene glycol diglycidyl ether in a volume ratio of 2: 1;

organic solvent 4: DMF and ethylene glycol diglycidyl ether in a volume ratio of 5: 1;

organic solvent 5: DMF and ethylene glycol diglycidyl ether in a volume ratio of 1: 2;

organic solvent 6: ethylene glycol diglycidyl ether;

organic solvent 7: DMF.

To 200mL of the above organic solvent was added 2g of abamectin, respectively, and the mixture was dissolved with stirring at 25 ℃ to determine the total dissolution time of abamectin in the solvent. The results of the solubility test are shown in fig. 2, wherein a is anorganic solvent 1, b is an organic solvent 2, c is an organic solvent 3, d is an organic solvent 4, e is an organic solvent 5, f is an organic solvent 6, and g is an organic solvent 7.

1.2 measurement of condensation ratio:

first, a standard curve of Fmoc concentration was prepared: 59.3mg of Fmoc-Gly-OH is weighed and dissolved in 25v/v% piperidine DMF solution, the mixture is evenly mixed and shaken for 10min, the mixture is filtered, the filtrate is combined and is positioned in a 10mL volumetric flask, and the concentration of the Fmoc protecting group is 20 mmol/L. The solution was diluted to 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L, 0.6mmol/L, 0.7mmol/L, 0.8mmol/L, 0.9mmol/L and standard solutions, absorbance of the standard solutions was measured at 300nm using an ultraviolet spectrophotometer and a standard curve was drawn based on the data. According to numberThe values yield a regression equation. The absorbance is taken as an axis y, the concentration is taken as an axis x, and the established Fmoc standard curve equation is y =5.57x +0.11, R²=0.996。

Accurately weighing 0.005g of resin sample connected with amino acid, placing the resin sample into a centrifuge tube, adding 5ml of 25v/v% piperidine DMF solution, stirring for 5min to ensure that Fmoc falls off, standing, sucking supernatant liquid, adding the supernatant liquid into a cuvette, and measuring the absorbance of the sample and blank at 300 nm. Calculated according to the following formula:

content of amino group (mmol/g) = (sample absorbance-blank absorbance) × preparation volume/weight of resin sample;

theoretical content (mmol/g) = (substitution degree resin weight of resin × resin weight)/{ resin weight + [ (substitution degree of resin × molecular weight of resin weight) × (molecular weight of Fmoc group of Fmoc-AA-molecular weight-H)₂Molecular weight of O)/1000]}；

Condensation rate = (content of amino group/theoretical content) × 100%.

The coupling ratio R of Fmoc-Arg (Pbf) -OH at the first time in the synthesis of the peptide fragment 2 was calculated according to the above formula₁Second coupling ratio R of Fmoc-Arg (Pbf) -OH₂Third coupling ratio R of Fmoc-Arg (Pbf) -OH₃(ii) a And calculating the coupling ratio S of the peptide fragment 2 during the synthesis of the peptide resin I₁Coupling ratio S of peptide fragment 3 during peptide resin II Synthesis₂. Coupling ratio R₁The results of the measurements are shown in FIG. 3, where A is example 1, B is example 2, C is example 3, D is example 4, E is example 5, and F is example 6. Coupling ratio R₂The results of the measurements are shown in FIG. 4, where A is example 1, B is example 2, C is example 3, D is example 4, E is example 5, and F is example 6. Coupling ratio R₃The results of the measurements are shown in FIG. 5, where A is example 1, B is example 2, C is example 3, D is example 4, E is example 5, and F is example 6. Coupling ratio S₁The results are shown in FIG. 6, where A is example 1, G is example 7, H is example 8, I is example 9, J is example 10, K is example 11, and L is example 12. Coupling ratio S₂The results of the measurement are shown in FIG. 7, wherein A is example 1 and G isExample 7, H is example 8, I is example 9, J is example 10, K is example 11, and L is example 12.

1.3 purity and yield determination: and (3) detecting the abapatulin crude product obtained in the above embodiment by using an RP-HPLC method, calculating the purity, purifying the abapatulin crude product, transferring salt, calculating the theoretical yield according to the substitution degree of the resin, and calculating the yield of the abapatulin. The results of the purity determination of abapatatine are shown in fig. 8, where a is example 1, B is example 2, C is example 3, D is example 4, E is example 5, F is example 6, G is example 7, H is example 8, I is example 9, J is example 10, K is example 11, and L is example 12. The results of the measurement of the yield of abapatatine are shown in fig. 9, wherein a is example 1, B is example 2, C is example 3, D is example 4, E is example 5, F is example 6, G is example 7, H is example 8, I is example 9, J is example 10, K is example 11, and L is example 12.

As can be seen from FIG. 2, the dissolution time of the abamectin by the organic solvents a, b and c is obviously shorter than that of the organic solvents d, e, f and g, and as can be seen from FIG. 6, FIG. 7, FIG. 8 and FIG. 9, the coupling rates S of the examples 1, 7 and 8 are shown₁Coupling ratio S₂The purity and the yield of the abapa peptide are obviously higher than those of the examples 9, 10, 11 and 12, which shows that the solubility of the peptide chain of the abapa peptide can be increased by using DMF and ethylene glycol diglycidyl ether with the volume ratio of 2-4:1-2 as solvents, and when the solvents are used as coupling reaction solvents, the solvents are favorable for quickly dissolving longer peptide chains in the synthesis process, are favorable for carrying out condensation reaction, can reduce the reaction time and improve the reaction efficiency, and further can improve the product yield and the product purity, reduce the production cost and simplify the operation steps.

As can be seen from FIGS. 3, 4, 5, 8 and 9, the coupling ratios R in examples 1, 2 and 3 were those in examples₁The coupling ratio R₂The coupling ratio R₃The purity and yield of the abamectin are obviously higher than those of the examples 4, 5 and 6, which shows that 2, 5-di-n is added into a coupling system when the coupling large-steric-hindrance amino acid Fmoc-Arg (Pbf) -OH is extended during the synthesis of the abamectinThe chlorobenzoxazole and the tetraethylthiuram disulfide can improve the coupling rate of Fmoc-Arg (Pbf) -OH and further improve the yield and the purity of the abamectin.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims (8)

1. A solid phase synthesis method of abamectin is characterized by comprising the following steps:

A. Fmoc-Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-amino resin is sequentially coupled and synthesized according to the Fmoc protection strategy, and after the Fmoc protection group is removed, a peptide resin segment 1 is obtained;

Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -2CTC resin is coupled and synthesized according to Fmoc protection strategy in sequence, and peptide fragment 2 is obtained after adding lysate and removing 2-CTC resin;

sequentially coupling and synthesizing Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -Gly-Lys (Boc) -Ser (tBu) -2CTC resin according to an Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 3;

B. coupling reaction is carried out on the C end of the peptide fragment 2 and the N end of the peptide resin fragment 1 to obtain a peptide resin I;

C. removing the N-terminal protecting group of the peptide resin I, and performing coupling reaction with the C terminal of the peptide fragment 3 to obtain a peptide resin II;

D. adding a lysis solution to remove the resin and all protecting groups of the peptide resin II to obtain the abapa peptide;

the Fmoc is an amino acid N-terminal protecting group, and tBu, Trt, Boc, OtBu and Pbf are amino acid side chain protecting groups;

coupling reaction in the step B and the step C by using a HBTU/HOBT/DIPEA three-reagent coupling system, wherein the coupling reaction in the step B and the step C uses dimethylformamide and ethylene glycol diglycidyl ether as solvents;

the volume ratio of the dimethylformamide to the ethylene glycol diglycidyl ether is 2-4: 1-2; the amino resin used in the process of coupling and synthesizing Fmoc-Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-amino resin in the step A is crosslinked 1m/m% divinylbenzene and Rink Amide AM resin with the substitution degree of 0.8mmol/g, the coupling reaction temperature in the step B and the step C is 25 ℃, the reaction time is 3h, the volume-mass ratio of the solvent of the coupling reaction in the step B to the Rink Amide AM resin in the step A is 40mL:1g, and the volume-mass ratio of the solvent of the coupling reaction in the step C to the Rink Amide AM resin in the step A is 40mL:1 g;

the coupling system in the synthesis of the peptide fragment 2 in the step A is HOBT/DIC, and the molar ratio of HOBT to DIC is 1: 1-1.5; the coupling system for extension coupling Fmoc-Arg (Pbf) -OH in the synthesis of the peptide fragment 2 further comprises 2, 5-dichlorobenzoxazole and tetraethylthiuram disulfide, wherein the molar ratio of the 2, 5-dichlorobenzoxazole to the tetraethylthiuram disulfide is 1:0.08-0.35, and the molar ratio of the 2, 5-dichlorobenzoxazole to the HOBT is 1: 4-5.

2. The solid phase synthesis method of claim 1, wherein: the molar ratio of the coupling reagents of the three-reagent coupling system in the step B and the step C is as follows: HBTU, HOBT, DIPEA =1-2:1-2: 2-4.

3. The solid phase synthesis method of claim 1, wherein: the cracking liquid in the step D comprises trifluoroacetic acid, thioanisole, triisopropylsilane and water, and the corresponding volume ratio is 80-90:3-8:3-8: 3-8.

4. The solid phase synthesis method of claim 1, wherein: the method for synthesizing the peptide fragment 2 in the step a specifically comprises:

b1, carrying out coupling reaction on Fmoc-Glu (OtBu) -OH and 2-CTC resin under the action of a coupling system to obtain Fmoc-Glu (OtBu) -2CTC resin;

b2, carrying out deprotection of Fmoc protecting group on Fmoc-Glu (OtBu) -2CTC resin, and then carrying out coupling reaction with Fmoc-Leu-OH under the action of a coupling system to obtain Fmoc-Leu-Glu (OtBu) -2CTC resin;

3, sequentially coupling Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Ile-OH by amino acid extension according to the coupling method of step b2 to obtain Fmoc-Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Glu (OtBu) -2-CTC-resin, adding lysis solution to remove the Fmoc 2-CTC-CTB-Glu-to obtain Fmoc-Ile-Gln- (Trt) -Asp (Asp-Leu) -Arg (Pbf) -Arg-Glu- (OtBu) -2-Glu-2-CTB-Glu- (Pbf) -2-Leu- (OtBu) -2-Glu- (Pbf) -2-Glu- (Pbf) and (Pbf) -Glu- (Pbf) -2-Glu- (Pbf) are added to obtain Fmoc-Glu-Leu-Leu-Leu-Glu- (Pbf-Glu-E- (Pbf) lysis solution Glu (OtBu) -OH, peptide fragment 2.

5. The solid phase synthesis method of claim 4, wherein: the substitution degree of the 2-CTC resin is 0.3-1.2 mmol/g.

6. The solid phase synthesis method of claim 4, wherein: the cleavage solution in the step b3 of the synthesis of the peptide fragment 2 comprises trifluoroethanol and dichloromethane, and the corresponding volume ratio is 1-2: 7-8.

7. The solid phase synthesis method of claim 4, wherein: the coupling reaction in the synthesis of the peptide fragment 2 takes a mixed solvent of DCM and DMF with the volume ratio of 1:1-3 as a solvent.

8. Use of a solid phase synthesis method as claimed in any one of claims 1 to 7 in the manufacture of a medicament for the treatment of osteoporosis.

Images