A method of synthesis of daptomycin

Abstract

The application discloses a synthesis method of daptomycin, and the daptomycin is prepared through a full liquid-phase fragment method; two full-protection fragments are prepared through the liquid-phase method by using two glycine sites contained in the daptomycin; the two full-protection peptide segments are condensed to form full-protection daptomycin; and after the protection groups are removed through acid cutting, the daptomycin is formed through an STL ring closing technology. The application solves the problems of low ring closing efficiency, low product purity after ring closing and many polymeric impurities of other ring closing technologies, so that the efficient preparation of the daptomycin is realized, and the application has the characteristics of less reagent and amino acid usage, less by-product and impurity production in the preparation process, high product purity and high yield compared with other preparation methods.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, and specifically to a method for synthesizing daptomycin. Background Technology

[0002] Since the discovery of penicillin in 1928, new antibiotics have been continuously developed, making previously serious illnesses seem much simpler. This has led to the misconception that antibiotics are omnipotent, resulting in the overuse of antibiotics. Data shows that antibiotic overuse accounts for as much as 50% worldwide. This overuse has led to drug resistance, resulting in the emergence of superbugs. Consequently, previously effective antibiotics become ineffective when encountering pathogens, requiring stronger antibiotics. This creates a vicious cycle that could ultimately lead to a shortage of effective antibiotics.

[0003] Antimicrobial peptides are considered ideal alternatives to antibiotics. They are small cationic polypeptides produced by organisms with strong antibacterial activity and are important components of the body's innate immune system. Antimicrobial peptides have a relatively small molecular weight, generally composed of 20-60 amino acid residues, approximately 2k-7kDa; they exhibit good thermal stability and maintain strong activity even under high ionic strength and low or high pH conditions. Antimicrobial peptides not only have strong inhibitory effects on Gram-positive and Gram-negative bacteria but also show significant antibacterial activity against certain drug-resistant strains. They possess broad-spectrum antibacterial activity as well as highly effective antifungal, viral, and protozoal activity. Furthermore, the unique antibacterial mechanism of antimicrobial peptides makes them less prone to inducing drug resistance.

[0004] Daptomycin is an antibacterial natural product, chemically named N-decanoyl-tryptophan. 1 -D-Asparagine 2 -Aspartic acid 3 -Threonine 4 -glycine 5 -ornithine 6 -Aspartic acid 7 -D-alanine 8 -Aspartic acid 9 -glycine 10 -D-serine 11 3-Methyl-L-glutamic acid 12 -L-kynurenine 13ε1-lactone is the first clinically approved calcium-dependent cyclic lipopeptide antibiotic. Daptomycin was approved in the United States in 2003 for the treatment of skin and skin structure infections caused by Gram-positive bacteria. In 2006, daptomycin was approved for the treatment of bacteremia and right endocarditis caused by methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, daptomycin shows strong in vivo activity against clinically important Gram-positive cocci, such as vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant Enterococcus faecalis (VREF). In 2018, the World Health Organization recognized daptomycin as an extremely important antibiotic. Daptomycin has 13 amino acid residues, 10 of which comprise a macrocycle surrounded by an ester bond between the Thr4 side chain and the α-COOH of kynurenine (Kyn)13. The remaining amino acids form an exocyclic tripeptide, acylated with decanoic acid at its N-terminus. Daptomycin contains three unusual amino acids: Orn6, (2S,3R)-3-methylglutamic acid (3MeGlu)12, and Kyn13, as well as three D-amino acids: D-Asn2, D-Ala8, and D-Ser11. The presence of these unusual amino acids, lipid tails, and ester bonds makes the chemical synthesis of daptomycin a challenge. The chemical structure of daptomycin is as follows:

[0005] Daptomycin was isolated from the fermentation products of *Streptomyces roseum* in the 1980s. However, methods for obtaining the product from *Streptomyces roseum* fermentation have been hampered by patent protection and the difficulty in obtaining *Streptomyces roseum*, hindering the development of preparation processes. Dr. Xu Hongyan and colleagues at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, designed a solid-liquid phase synthesis strategy for daptomycin (CN101235080B). This method has been proven to rapidly synthesize the target molecule. However, the liquid-phase cyclization followed by cleavage method used in the solid-liquid phase synthesis results in low product yield and purity. Furthermore, the monomers kynurenine and 3-methylglutamic acid are scarce in natural sources, and their chemical synthesis is extremely difficult. The excess ratio of more than 3 times used in this patent significantly increases the preparation cost. Northwestern Polytechnical University has designed a method for preparing daptomycin and its analogues using all-solid-phase synthesis (CN101696235A). This method involves total synthesis and cyclization on a solid-phase support to form the target molecule. However, the high reagent consumption during resin washing and the high amino acid excess ratios of 4 to 8 times during solid-phase synthesis greatly increase the cost of preparing the target molecule. Furthermore, the cyclization efficiency on the resin is only 60-80%, and the incomplete cyclization will put great pressure on the subsequent purification of the target product. Summary of the Invention

[0006] In view of this, the present invention provides a method for synthesizing daptomycin. The protected amino acid used in the all-liquid phase synthesis method provided by the present invention is reduced from an excess of 3-5 times or even 8 times in solid phase synthesis to 1-1.5 times. Moreover, the washing reagents used in all-liquid phase synthesis are greatly reduced compared to solid phase synthesis, making it more environmentally friendly. Furthermore, the cyclization reaction of the deprotected daptomycin precursor has less steric hindrance, resulting in a more complete reaction when using STL technology for cyclization, thereby reducing subsequent purification costs.

[0007] The objective of this invention is achieved through the following technical solution: Step 1: Synthesize the protected fragment A' of fragment A and the protected fragment B' of fragment B; Fragment A is: H-ornithine 6 -Aspartic acid 7 -D-alanine 8 -Aspartic acid 9 -glycine 10 -OH; Fragment B is: N-decanoyl-tryptophan 1 -D-Asparagine 2 -Aspartic acid 3 -Threonine 4 (HD-serine) 11 3-Methyl-L-glutamic acid 12 -L-kynurenine 13 )-glycine 5 -OH; Step 2: Fragment A' and fragment B' condense to form a fully protected daptomycin peptide; Step 3: Remove the side chain protecting groups of the fully protected daptomycin peptide to obtain the cyclized precursor; Step 4: The cyclization precursor is subjected to a cyclization reaction to form an N,O-benzyl acetal-linked cyclized daptomycin intermediate product. Step 5: By treating with an organic acid solution, the O-to-N acyl transfer is triggered to convert the imine of the daptomycin intermediate product into an amide bond, forming crude daptomycin, which is then purified to obtain the daptomycin.

[0008] Fragment A' is H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethylacetal.

[0009] Fragment B' is Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu-Kyn(Boc)]-Gly-OH.

[0010] Step 2 specifically involves: Fragments A' and B' condense via DIC / HOAt to form the fully protected daptomycin peptide Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal.

[0011] Step 3 specifically involves cleaving the fully protected daptomycin peptide fragment Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal using TFA / Phenol / H2O to remove the side-chain protecting groups, yielding Dec-Trp-D-Asn-Asp-Thr[HD-Ser-3-Me-Glu-Kyn]-Gly-Orn-Asp-D-Ala-Asp-Gly-salicylaldehyde (SAL).

[0012] In step 5, the organic acid solution is TFA / TIS / H2O.

[0013] The method for synthesizing fragment A' includes the following steps: S1, Fmoc-D-Ala-OH and HOSu form Fmoc-D-Ala-OSu; or, Fmoc-D-Ala-OH and HPcp form Fmoc-D-Ala-OPcp; or, Fmoc-D-Ala-OH and HPfp form Fmoc-D-Ala-OPfp; or, Fmoc-D-Ala-OH and HONp form Fmoc-D-Ala-ONp; Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH was prepared by coupling one of S2, Fmoc-D-Ala-OSu, Fmoc-D-Ala-OPcp, Fmoc-D-Ala-OPfp, and Fmoc-D-Ala-ONp with H-Asp(OtBu)-(Dmb)Gly-OH. S3. Remove Fmoc from Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH to obtain HD-Ala-Asp(OtBu)-(Dmb)Gly-OH; S4, HD-Ala-Asp(OtBu)-(Dmb)Gly-OH is coupled with one of Fmoc-Asp(OtBu)-OPfp, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-ONp, and Fmoc is removed to obtain H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH; Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH and Troc-Orn(Boc)-OPcp, Troc-Orn(Boc)-OPfp, Troc-Orn(Boc)-ONp, Troc-Orn(Boc)-OSu are coupled together to obtain Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH; S6, Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH reacts with 2-(dimethoxymethyl)phenol to form Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal; S7. Removing the N-terminal Troc group from Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal yields fragment A', namely H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal.

[0014] The method for synthesizing fragment B' includes the following steps: 1) One of Fmoc-Thr(TBS)-ONp, Fmoc-Thr(TBS)-OPfp, Fmoc-Thr(TBS)-OSu, and Fmoc-Thr(TBS)-OPcp is coupled with H-Gly-OH and Fmoc is removed to prepare H-Thr(TBS)-Gly-OH; 2) One of Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OPfp, and Fmoc-Asp(OtBu)-ONp is coupled with H-Thr(TBS)-Gly-OH to prepare Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH; 3) Protect Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH with an allyl All group and remove Fmoc to prepare H-Asp(OtBu)-Thr(TBS)-Gly-OAll; 4) H-Asp(OtBu)-Thr(TBS)-Gly-OAll is coupled with Fmoc-D-Asn(Trt)-OH and Fmoc is removed to obtain HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 5) HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll is coupled with Fmoc-Trp(Boc)-OH and Fmoc is removed to obtain H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 6) H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll was prepared by coupling with Dec-Cl to obtain Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 7) The TBS protecting group of Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll is removed and then coupled with Fmoc-Kyn(Boc)-F to obtain Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll.

[0015] 8) Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll is prepared by removing Fmoc and then coupling it with Fmoc-3-Me-Glu(OtBu)-OH. 9) Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll was prepared by removing Fmoc. 10), Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll and Boc-D-Ser(tBu) -OH coupling forms Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll 11) The fragment B' is obtained by removing All from Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll, i.e., Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OH.

[0016] The cyclization process employs STL cyclization technology.

[0017] The daptomycin prepared by the aforementioned synthesis method also falls within the scope of protection of this invention.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) In its research, this invention discovered that traditional all-liquid-phase synthesis suffers from excessively long peptide chains and low condensation efficiency. In particular, the conventional activated condensation method for condensing the C-terminus and N-terminus of daptomycin often results in the formation of numerous dimer and polymeric impurities. Even when using STL condensation to form an ester with 2-(dimethoxymethyl)phenol after synthesizing the fully protected sequence, this process is often incomplete, leading to incomplete cyclization and excessive impurities in the product. Therefore, this invention forms an ester with 2-(dimethoxymethyl)phenol during the synthesis of the short-chain fragment A'. This reaction is very rapid and complete due to the shorter peptide chain and higher C-terminal carboxylic acid reactivity, thus avoiding the shortcomings of traditional all-liquid-phase synthesis and efficiently preparing daptomycin.

[0019] 2) Compared with solid-phase or solid-liquid combined synthesis methods for daptomycin, the all-liquid phase synthesis method provided by this invention reduces the amount of protected amino acids used from 3-5 times or even 8 times the excess in solid-phase synthesis to 1-1.5 times. In particular, daptomycin contains several special amino acids, such as fully protected kynurenine, 3-methylglutamic acid, and D-type amino acids, which have very high preparation and purchase costs. Applying the low amino acid excess multiple of all-liquid phase synthesis can save a lot of raw material costs. This is because it is very difficult for the protected kynurenine to form an ester bond with the threonine side chain hydroxyl group after activation in solid-phase synthesis, resulting in an 8-10 times excess of protected kynurenine for coupling. However, in this invention, the fluorinated product of protected kynurenine is used to couple the hydroxyl group under DMAP catalysis. From the effect, the condensation excess only needs to be 2 times, reducing the amount of protected kynurenine used and saving preparation costs.

[0020] 3) Compared with solid-phase or solid-liquid combination synthesis methods for daptomycin, which use a large amount of organic solvents to wash the resin carrier during the preparation process, the all-liquid phase synthesis method provided by this invention can save a lot of organic washing solvent costs, and the preparation process only generates a small amount of organic waste liquid and wastewater, which is more environmentally friendly.

[0021] 4) This invention avoids the risk of aspartimide formation side reactions caused by frequent alkali treatment (piperidine) during daptomycin synthesis, and avoids impurities generated at the Asp-Gly positions during solid-phase or liquid-phase synthesis. Simultaneously, this invention avoids the diketopiperazine (DKP) side reaction formed during alkali treatment of dipeptide lipids, improving product purity and reducing purification difficulty.

[0022] 5) This invention cleverly utilizes the two non-racemic glycines (Gly) in daptomycin to decompose the daptomycin sequence into two fragments, which are tandemly connected to form the product in solid-phase synthesis, reducing the risk of misconnection during the synthesis process. This provides a feasible industrial preparation scheme for the synthesis and preparation of daptomycin.

[0023] 6) In the final cyclization of daptomycin, the present invention employs N-terminal serine / threonine chemical linking (STL) cyclization technology. This cyclization strategy is more complete than cyclization on a solid support or cyclization of fully protected daptomycin, with a cyclization degree greater than 99%. After cyclization, the natural peptide bond of the coupling site is restored by acid removal of the acetal group. The presence of unprotected side chain functional groups does not adversely affect the cyclization process, so no dimer, oligomer, or high-polymer impurities or other byproducts are generated during the cyclization process.

[0024] 7) The total yield of daptomycin prepared from the protected amino acid activated ester to the final cyclization product using the all-liquid phase synthesis method was 36.2%, which is a significant improvement over the less than 20% total yield of the solid phase method or the solid-liquid combination method. Therefore, the all-liquid phase method is a process route with low raw material and solvent costs and high yield. This process route can be applied to the large-scale production of daptomycin. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is the HPLC chromatogram of daptomycin prepared in Example 3 of the present invention; Figure 2 This is the HPLC chromatogram of daptomycin prepared in Example 3 of the present invention. Detailed Implementation

[0026] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0027] This invention provides a method for synthesizing daptomycin, firstly by preparing two fragments, wherein: Fragment A: H-ornithine 6 -Aspartic acid 7 -D-alanine 8 -Aspartic acid 9 -glycine 10 -OH; Fragment B: N-decanoyl-tryptophan 1 -D-Asparagine 2 -Aspartic acid 3 -Threonine 4 (HD-serine) 11 3-Methyl-L-glutamic acid 12-L-kynurenine 13 )-glycine 5 -OH.

[0028] Two protective fragments were synthesized via all-liquid phase synthesis: The protected fragment A' corresponding to fragment A is H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal, and its synthetic route is as follows:

[0029] The protected fragment B' corresponding to fragment B is Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu-Kyn(Boc)]-Gly-OH, and its synthetic route is as follows:

[0030] Fragments A' and B' condense via DIC / HOAt to form the fully protected peptide Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal; The ester Dec-Trp-D-Asn-Asp-Thr[HD-Ser-3-Me-Glu-Kyn]-Gly-Orn-Asp-D-Ala-Asp-Gly-salicylaldehyde (SAL) was obtained by cleavage with trifluoroacetic acid. Then, the sample was treated with pyridine acetate buffer via STL cyclization to form an N,O-benzyl acetal-linked cyclization product. After purification and separation, the O-to-N acyl transfer was triggered by treatment with trifluoroacetic acid / water solution to convert the imine to an amide bond. The final product was obtained by HPLC purification. The synthetic route is as follows:

[0031] Example 1 Preparation of protected fragment A' corresponding to fragment A: H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal 1. Coupling of Fmoc-D-Ala-OSu with H-Asp(OtBu)-(Dmb)Gly-OH 1.1 Preparation of Fmoc-D-Ala-OSu

[0032] Weigh 12.5g of Fmoc-D-Ala-OH (purchased from GL Biochemical) and 5.52g of N-hydroxysuccinimide (HOSu) (purchased from GL Biochemical), add 80ml of tetrahydrofuran, dissolve under stirring at room temperature, and cool in an ice bath to 2-8℃. Add 6.05g of DIC (126.1) dropwise over 2 minutes. Stir in an ice bath for 0.5hr, remove the ice bath, and stir at room temperature. Monitor the reaction until completion by TLC. Concentrate under reduced pressure to a small volume, add 300ml of ethyl acetate, mix well, and place in a separatory funnel. Wash the ester layer with 100ml of 5% citric acid, then wash the ester layer with 3*100ml of water. Dry the ethyl acetate solution with anhydrous sodium sulfate for 1hr. Concentrate the dried ethyl acetate under reduced pressure to a small volume, add n-heptane under stirring to precipitate a white solid. After stirring at room temperature for 1 hour, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 15.8 g, with a yield of 96.7%. The purity was 99.6% as determined by HPLC, thus preparing Fmoc-D-Ala-OSu.

[0033] 1.2. Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH was prepared by coupling Fmoc-D-Ala-OSu with H-Asp(OtBu)-(Dmb)Gly-OH.

[0034] Dissolve 15.8 g of Fmoc-D-Ala-OSu in 100 ml of tetrahydrofuran solution with stirring, then add 50 ml of tetrahydrofuran / water (4:6) solution containing 17.4 g of H-Asp(OtBu)-(Dmb)Gly-OH. Adjust the pH to 8.0 with 1 M sodium carbonate solution and react at room temperature with stirring until the reaction is complete. Concentrate under reduced pressure to a small volume, add 500 ml of ethyl acetate to completely dissolve, and place in a separatory funnel. Wash the ester layer once with 150 ml of 5% potassium bisulfate, and measure the pH to 3.5. Then wash the ester layer with 3 x 150 ml of water. Separate the ethyl acetate solution and dry it with anhydrous sodium sulfate for 1 hour. Concentrate the dried ethyl acetate under reduced pressure to a small volume, and add n-hexane with stirring to precipitate a white solid. After stirring at room temperature for 1-2 hours, the mixture was filtered. The filter cake was washed with n-hexane and dried to obtain 25.5 g, with a yield of 95.6%. The purity was 99.3% as determined by HPLC. This yielded Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH.

[0035] 2. Removal of Fmoc from Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH

[0036] To 25.5 g of Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH, 200 ml of 25% diethylamine / ethyl acetate (v / v) was added. The mixture was kept at 25-28 °C with stirring to remove Fmoc until complete removal. The solution was concentrated under reduced pressure to remove diethylamine and ethyl acetate. Methyl tert-butyl ether was added, and the solid was collected, washed with methyl tert-butyl ether, and dried to obtain 17.0 g of HD-Ala-Asp(OtBu)-(Dmb)Gly-OH, with a purity of 99.7% as determined by HPLC.

[0037] 3. Coupling of Fmoc-Asp(OtBu)-OPfP with HD-Ala-Asp(OtBu)-(Dmb)Gly-OH and removal of Fmoc 3.1 Preparation of Fmoc-Asp(OtBu)-OPfP

[0038] Weigh 16.5g of Fmoc-Asp(OtBu)-OH (purchased from GL Biochemical) and 8.84g of pentafluorophenol (HPfp) (purchased from Sinopharm Chemical Testing), add 100ml of tetrahydrofuran, dissolve under stirring at room temperature, and cool in an ice bath to 2-8℃. Add 20ml of 9.6g of EDC.HCl in THF solution dropwise over 2 minutes. Stir in an ice bath for 0.5hr, remove the ice bath, and stir at room temperature. Monitor the reaction until completion by TLC. Concentrate under reduced pressure to a small volume, add 300ml of ethyl acetate, mix well, and place in a separatory funnel. Wash the ester layer with 100ml of 5% citric acid, then wash the ester layer with 3*100ml of water. Dry the ethyl acetate solution with anhydrous sodium sulfate for 1hr. Concentrate the dried ethyl acetate under reduced pressure to a small volume, add n-heptane under stirring to precipitate a white solid. After stirring at room temperature for 1 hour, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 21.95 g, with a yield of 95.0%. The purity was 99.1% as determined by HPLC, thus preparing Fmoc-Asp(OtBu)-OPfp.

[0039] 3.2 Coupling of Fmoc-Asp(OtBu)-OPfP with HD-Ala-Asp(OtBu)-(Dmb)Gly-OH and removal of Fmoc to prepare H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH.

[0040]

[0041] Weigh 21.0 g of Fmoc-Asp(OtBu)-OPfp and add it to a 150 ml tetrahydrofuran solution. Then add 50 ml of a tetrahydrofuran / water (5:5) solution containing 17.0 g of HD-Ala-Asp(OtBu)-(Dmb)Gly-OH. Stir at room temperature until the reaction is complete. Concentrate under reduced pressure to a small volume, add 500 ml of ethyl acetate to completely dissolve the ester layer, and place it in a separatory funnel. Wash the ester layer once with 150 ml of 5% citric acid. The pH of the aqueous phase is measured to be 3.5. Then wash the ester layer with 3 x 150 ml of water. Dry the separated ethyl acetate layer with anhydrous sodium sulfate for 1 hour. Collect the dried ethyl acetate and concentrate it under reduced pressure to 200-250 ml. The purity is determined to be 99.1% by HPLC. Diethylamine was added with stirring until its concentration reached 25% (v / v). Then, Fmoc was removed by stirring at a temperature of 25-28℃ until complete removal. The mixture was concentrated under reduced pressure to remove diethylamine and ethyl acetate. Heptane was added, the solid was collected, washed with heptane, and dried to obtain 20.6 g, with a yield of 97.6%. The purity was determined to be 99.0% by HPLC, yielding H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH.

[0042] 4. Coupling of Troc-Orn(Boc)-OPcp with H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH 4.1 Preparation of Troc-Orn(Boc)-OPcp

[0043] Weigh 13.5g of Troc-Orn(Boc)-OH (purchased from GL Biochemical) and 9.59g of pentachlorophenol (HPcp) (purchased from Sinopharm Chemical Testing), add 120ml of tetrahydrofuran, dissolve under stirring at room temperature, and cool in an ice bath to 2-8℃. Add 5.05g of DIC dropwise over 2 minutes, stir in an ice bath for 0.5hr, remove the ice bath and stir at room temperature. Monitor the reaction by TLC until completion, concentrate under reduced pressure to a small volume, add 300ml of ethyl acetate, mix well, and place in a separatory funnel. Wash the ester layer with 100ml of 5% citric acid, then wash the ester layer with 3*100ml of water. Dry the ethyl acetate solution with anhydrous sodium sulfate for 1hr. Concentrate the dried ethyl acetate under reduced pressure to a small volume, add n-heptane under stirring to precipitate a white solid. After stirring at room temperature for 1 hour, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 20.5 g, with a yield of 94.4%. The purity was 99.0% as determined by HPLC, thus preparing Troc-Orn(Boc)-OPcp.

[0044] 4.2 Preparation of Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH by coupling Troc-Orn(Boc)-OPcp with H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH.

[0045]

[0046] Weigh 20.3 g of Troc-Orn(Boc)-OPcp and dilute it with 150 ml of tetrahydrofuran. Then add 150 ml of a tetrahydrofuran / water (5:5) solution containing 20.6 g of H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH. React under stirring at room temperature, and detect the end of the reaction by HPLC. Concentrate under reduced pressure to a small volume, add 1 L of ethyl acetate to completely dissolve it, and place it in a separatory funnel. Wash the ester layer once with 300 ml of 5% potassium bisulfate. The pH of the aqueous phase is measured to be 3.5. Then wash the ester layer with 3 x 300 ml of water. The separated ethyl acetate layer is dried with anhydrous sodium sulfate for 1 hour. Collect the dried ethyl acetate and concentrate it under reduced pressure to a small volume. Add petroleum ether under stirring at low temperature (2-8℃), and a white solid precipitates. After stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with petroleum ether and dried to obtain 30.2 g, with a yield of 93.5%. The purity was 98.8% as determined by HPLC. The Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH was thus prepared.

[0047] 5. Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH forms an ester with 2-(dimethoxymethyl)phenol.

[0048] Dissolve 30.2 g of Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH and 5.9 g of 2-(dimethoxymethyl)phenol in 220 ml of THF / NMP (4:1) with stirring until completely dissolved. Then, add dropwise 100 ml of a solution containing 18.2 g of PyBOP (Mw: 520.4, 35 mmol) in THF / NMP (4:1). After the addition is complete, stir the reaction at room temperature until the reaction is finished. Concentrate under reduced pressure to a small volume, add 1 L of ethyl acetate, and place in a separatory funnel. Wash the ester layer with 3 x 300 ml of 25% sodium chloride. Dry the separated ethyl acetate layer with anhydrous sodium sulfate for 1 hour. Collect the dried ethyl acetate and concentrate under reduced pressure to a small volume. Add n-heptane with stirring at low temperature (2-8℃), and a white solid precipitates. After stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 33.1 g, with a yield of 96.0%. The purity was 97.1% as determined by HPLC. Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal was thus prepared.

[0049] 6. Removal of the N-terminal Troc group from the peptide to form H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal (fragment A')

[0050] Dissolve 33.1 g of Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal (27.73 mmol) in 500 ml of THF. After complete dissolution, add 18.0 g of freshly activated zinc powder, followed by 50 ml of 1 M potassium dihydrogen phosphate. Stir at room temperature until the 2,2,2-trichloroethoxycarbonyl protecting group (Troc) is completely removed. Filter to remove the solid. Wash the solid with 2 x 50 ml of THF. Combine the filtrates and washings and concentrate under reduced pressure to a small volume. Add 800 ml of dichloromethane and wash with 3 x 200 ml of 15% sodium chloride aqueous solution. Separate the dichloromethane layer and dry it with anhydrous calcium chloride for 2-3 hours. The solid was concentrated under reduced pressure to a small volume, and n-heptane was added to precipitate it. The solid was filtered, and the filter cake was dried to obtain 24.3 g, with a yield of 87.3%. H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal (fragment A') was prepared.

[0051] Example 2 The preparation of protected fragment B' corresponding to fragment B is as follows: Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Kyn(Boc)-3-Me-Glu(OtBu-D-Ser(tBu-Boc)]-Gly-OH. 1. Coupling of Fmoc-Thr(TBS)-ONp with H-Gly-OH and de-Fmoc 1.1 Preparation of Fmoc-Thr(TBS)-ONp

[0052] Weigh 23.7g of Fmoc-Thr(TBS)-OH (purchased from GL Biochemical) and 7.65g of p-nitrophenol (HONp) (purchased from Sinopharm Chemical Reagent). Add 100ml of tetrahydrofuran, dissolve under stirring at room temperature, and cool in an ice bath to 2-8℃. Add 25ml of 11.5g of EDC.HCl THF solution dropwise over 2 minutes. Stir in an ice bath for 0.5hr, then remove the ice bath and stir at room temperature. Monitor the reaction by TLC until completion. Concentrate under reduced pressure to a small volume and add 300ml of... After mixing 1 part of ethyl acetate, the mixture was placed in a separatory funnel. The ester layer was washed with 100 ml of 5% citric acid, followed by 3 x 100 ml of water. The ethyl acetate solution was dried over anhydrous sodium sulfate for 1 hour. The dried ethyl acetate was concentrated under reduced pressure to a small volume, and n-heptane was added with stirring to precipitate a white solid. After stirring at room temperature for 1 hour, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 28.84 g, with a yield of 96.7%. The purity was determined to be 98.5% by HPLC, thus preparing Fmoc-Thr(TBS)-ONp.

[0053] 1.2 Coupling of Fmoc-Thr(TBS)-ONp with H-Gly-OH and removal of Fmoc to prepare H-Thr(TBS)-Gly-OH

[0054] Weigh 28.8 g of Fmoc-Thr(TBS)-ONp and dissolve it in 300 ml of tetrahydrofuran solution. Then add 30 ml of a sodium carbonate aqueous solution (pH 8.0) containing 4.32 g of H-Gly-OH. React at room temperature with stirring until the reaction is complete. Concentrate under reduced pressure to a small volume, add 500 ml of ethyl acetate to completely dissolve the ester layer, and place it in a separatory funnel. Wash the ester layer once with 150 ml of 5% citric acid, and determine the pH to be 4.0. Then wash the ester layer with 3 x 150 ml of water. Separate the ethyl acetate solution and dry it with anhydrous sodium sulfate for 1 hour. Concentrate the dried ethyl acetate under reduced pressure to 200 ml, and determine the purity to be 99.7% by HPLC. Add diethylamine under stirring in an ice bath (2-8℃) until the concentration reaches 25% (v / v). Then remove Fmoc under stirring at 25-28℃ until complete. Concentrate under reduced pressure to remove diethylamine and ethyl acetate. Methyl tert-butyl ether was added, the solid was collected, washed with methyl tert-butyl ether, and dried to obtain 14.1 g, with a yield of 96.8%. The purity was determined to be 99.4% by HPLC, and H-Thr(TBS)-Gly-OH was prepared.

[0055] 2. Coupling of Fmoc-Asp(OtBu)-OSu with H-Thr(TBS)-Gly-OH 2.1 Preparation of Fmoc-Asp(OtBu)-OSu

[0056] Weigh 19.8 g of Fmoc-Asp(OtBu)-OH (purchased from GL Biochemical) and 5.76 g of N-hydroxysuccinimide (HOSu) (purchased from GL Biochemical), add 100 ml of tetrahydrofuran, dissolve under stirring at room temperature, and cool in an ice bath to 2-8 °C. Add 6.94 g of DIC dropwise over 2 minutes. Stir in an ice bath for 0.5 hours, remove the ice bath, and stir at room temperature. Monitor the reaction by TLC until completion. Concentrate under reduced pressure to a small volume, add 300 ml of ethyl acetate, mix well, and transfer to a separatory funnel. Wash the ester layer with 100 ml of 5% citric acid, then wash with 3 x 100 ml of water. Dry the ethyl acetate solution with anhydrous sodium sulfate for 1 hour. Concentrate the dried ethyl acetate under reduced pressure to a small volume, add n-heptane while stirring, and a white solid precipitates. After stirring at room temperature for 1 hour, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 22.8 g, with a yield of 93.2%. The purity was 99.0% as determined by HPLC, thus preparing Fmoc-Asp(OtBu)-OSu.

[0057] 2.2 Preparation of Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH by coupling Fmoc-Asp(OtBu)-OSu with H-Thr(TBS)-Gly-OH.

[0058]

[0059] Dissolve 22.8 g of Fmoc-Asp(OtBu)-OSu in 220 ml of tetrahydrofuran, then add 50 ml of a tetrahydrofuran / water (1:1) solution containing 14.1 g of H-Thr(TBS)-Gly-OH. Stir at room temperature until the reaction is complete. Concentrate under reduced pressure to a small volume, add 500 ml of ethyl acetate to completely dissolve, and place in a separatory funnel. Wash the ester layer once with 150 ml of 5% potassium bisulfate, and determine the pH to be 3.0. Then wash the ester layer with 3 x 150 ml of water. Separate the ethyl acetate solution and dry it with anhydrous sodium sulfate for 1 hour. Concentrate the dried ethyl acetate under reduced pressure to a small volume, and add petroleum ether under low temperature (2-8℃) with stirring. A white solid precipitates out. After stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with petroleum ether and dried to obtain 30.1 g, with a yield of 98.2%. The purity was 99.3% as determined by HPLC. Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH was thus prepared.

[0060] 3. Protection of allyl (All) groups and removal of Fmoc

[0061] Weigh 30.1 g of Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH and dissolve it in 200 ml of a THF:DMF (3:1) mixture. Add 50 ml of a THF solution containing 14.7 g of diallyl pyrocarbonate. While stirring in an ice bath (2-8℃), slowly add 10 ml of a THF solution containing 1 g of 4-dimethylaminopyridine (DMAP) over 10 minutes. Transfer to room temperature (25-30℃) and stir to react. Detect the reaction as complete by HPLC. Terminate the diallyl pyrocarbonate reaction by adding acetic acid. After THF removal by vacuum concentration, the remaining solvent was added to 500 ml of ethyl acetate and mixed well. The mixture was then placed in a separatory funnel and washed once with 150 ml of 5% potassium bisulfate, followed by 3 x 150 ml of water, then 150 ml of 5% sodium bicarbonate, and finally 3 x 150 ml of water until neutral. The ethyl acetate solution was separated and dried over anhydrous sodium sulfate for 1 hour. The purity was determined to be 99.5% by HPLC. Diethylamine was added under stirring in an ice bath (2-8℃) until the concentration reached 25% (v / v). Then, Fmoc was removed by stirring at 25-28℃ until complete. The mixture was concentrated under vacuum to remove diethylamine and ethyl acetate. Methyl tert-butyl ether was added, and the solid was collected, washed with methyl tert-butyl ether, and dried to obtain 21.8 g, with a yield of 99.1%. The purity was determined to be 99.5% by HPLC, yielding H-Asp(OtBu)-Thr(TBS)-Gly-OAll.

[0062] 4. Coupling of H-Asp(OtBu)-Thr(TBS)-Gly-OAll with Fmoc-D-Asn(Trt)-OH and removal of Fmoc

[0063] Add 23.9g of Fmoc-D-Asn(Trt)-OH and 25.0g of PyBOP to 200ml of THF / DMF (4:1) and dissolve with stirring. After complete dissolution, place the solution in an ice bath (2-8℃) and dropwise add 25ml of THF solution containing 6.2g of DIEA. After the addition is complete, stir in the ice bath for 30min. Remove the ice bath and mix with 150ml of THF solution containing 21.8g of H-Asp(OtBu)-Thr(TBS)-Gly-OAll. React at room temperature with stirring until the reaction is complete. Concentrate the solution under reduced pressure to a small volume, add 500 ml of ethyl acetate to completely dissolve it, and place it in a separatory funnel. Wash the ester layer once with 150 ml of 5% potassium bisulfate solution, and determine the pH to be 3.0. Then wash the ester layer with 3 x 150 ml of water, then wash it once with 150 ml of 5% sodium bicarbonate solution, and wash the ester layer with 3 x 150 ml of water until neutral. Separate the ethyl acetate solution and dry it with anhydrous sodium sulfate for 1 hour. Remove the sodium sulfate and determine the purity of the ester layer by HPLC. The purity is 99.2%. Add diethylamine to the ester layer with stirring in an ice bath (2-8℃) until the concentration reaches 25% (v / v). Then remove Fmoc with stirring while maintaining the temperature (25-28℃) until complete. Concentrate under reduced pressure to remove diethylamine and ethyl acetate. Methyl tert-butyl ether was added, the solid was collected, washed with methyl tert-butyl ether, and dried to obtain 33.9 g, with a yield of 98.8%. The purity was determined to be 99.3% by HPLC, thus preparing HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll.

[0064] 5. Coupling of HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll with Fmoc-Trp(Boc)-OH and removal of Fmoc

[0065] Weigh 20.0g of Fmoc-Trp(Boc)-OH and 17.1g of HBTU and dissolve them in 180ml of THF / NMP (4:1) mixture. After complete dissolution, place the mixture in an ice bath (2-8℃) and dropwise add 20ml of THF solution containing 6.8g of DIEA. After the addition is complete, stir in the ice bath for 30min. Remove the ice bath and mix with 200ml of THF / NMP (9:1) solution containing 33.9g of HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll. Stir at room temperature until the reaction is complete. THF was removed by concentration under reduced pressure. Then, 600 ml of DCM was added to completely dissolve the DCM layer, which was then placed in a separatory funnel. The DCM layer was washed once with 150 ml of 10% citric acid, and the pH was measured to be 3.0. The DCM layer was then washed with 3 x 150 ml of saturated sodium chloride, followed by 150 ml of 10% sodium bicarbonate. The DCM layer was then washed with 3 x 150 ml of saturated sodium chloride until neutral. The DCM solution was separated and dried over anhydrous sodium sulfate for 1 hour. After removing the sodium sulfate, the purity of the DCM layer was determined by HPLC to be 99.0%. Diethylamine was added to the DCM layer under stirring in an ice bath (2-8℃) until its concentration reached 25% (v / v). Then, Fmoc was removed completely under stirring at 25-28℃. The solution was concentrated under reduced pressure to remove diethylamine and DCM. Methyl tert-butyl ether was added, the solid was collected, washed with methyl tert-butyl ether, and dried to obtain 42.6 g, with a yield of 97.9%. The purity was determined to be 99.5% by HPLC, thus preparing H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll.

[0066] 6. H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll coupled with Dec-Cl

[0067] Dissolve 42.6 g of H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll in 300 ml of THF / NMP (9:1) solution, then mix with 50 ml of THF solution containing 10.6 g of Dec-Cl, and then add dropwise 20 ml of THF solution containing 7.2 g of DIEA. Stir the reaction at room temperature until the reaction is complete, concentrate under reduced pressure to remove THF, then add 600 ml of DCM to completely dissolve the solution and place it in a separatory funnel. Wash the DCM layer once with 150 ml of 10% citric acid, and measure the pH to 3.0. Then wash the DCM layer with 3 x 150 ml of water, and then with 150 ml of... The DCM layer was washed once with 10% sodium bicarbonate, and then washed with 3 x 150 ml of water until neutral. The separated DCM solution was dried with anhydrous sodium sulfate for 1 hour. The dried DCM was concentrated under reduced pressure to a small volume, and n-heptane was added under low temperature (2-8℃) with stirring, resulting in the precipitation of a white solid. After stirring at low temperature for another 2-3 hours, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 48.0 g, with a yield of 99.4%. The purity was determined to be 99.7% by HPLC, yielding Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll.

[0068] 7. Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll removes the TBS protecting group and forms an ester bond with Fmoc-Kyn(Boc)-F.

[0069] 48 g of Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll was weighed and dissolved in 240 ml of tetrabutylammonium fluoride (TBAF) in DMF solution. The reaction was stirred at room temperature until tert-butyldimethylsilyl ether (TBS) was completely removed. The reaction solution was cooled in an ice bath and 1 L of ethyl acetate was added. A large amount of white solid precipitated out. After stirring in an ice bath for 1 hr, the mixture was filtered. The filter cake was washed three times with ethyl acetate and dried to obtain 42.6 g of white solid, with a yield of 97.3%. The purity was 99.0% as determined by HPLC.

[0070]

[0071] The above 42.6g of Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr-Gly-OAll was dissolved in 250ml of THF / DMF (9:1) solution and mixed with 150ml of THF solution containing 38.3g of Fmoc-Kyn(Boc)-F. 40ml of THF solution containing 0.98g of 4-dimethylaminopyridine (DMAP) and 9.3g of DIEA was added dropwise while stirring at room temperature. After the addition was complete, the reaction was stirred at room temperature until the reaction was complete. After adding H-Lys(NH2)-OMe to consume unreacted Fmoc-Kyn(Boc)-F, the solution was concentrated under reduced pressure to remove THF. Then, 1 liter of chloroform was added to completely dissolve the solution, and the solution was placed in a separatory funnel. The chloroform layer was washed once with 300 ml of 10% citric acid and the pH was measured to be 3.0. The chloroform layer was then washed with 3 x 300 ml of water, and then once with 300 ml of 10% sodium bicarbonate. The chloroform layer was washed with 3 x 300 ml of water until neutral. The separated chloroform solution was dried with anhydrous sodium sulfate for 1 hour. The dried chloroform was concentrated under reduced pressure to a small volume, and petroleum ether was added under stirring at low temperature (2-8℃), resulting in the precipitation of a white solid. After stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with petroleum ether and dried to obtain 60.2 g, with a yield of 98.2%. The purity was 98.0% as determined by HPLC. The product was Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll.

[0072] 8. Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll removes Fmoc and its coupling with Fmoc-3-Me-Glu(OtBu)-OH.

[0073] Weigh 60.2g of Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll and dissolve it in 400ml of THF. Add diethylamine to the solution in an ice bath (2-8℃) with stirring until the concentration reaches 35% (v / v). Then, remove Fmoc by stirring at 25-28℃ until complete. Concentrate under reduced pressure to remove diethylamine and THF. Disperse the resulting oil in 150ml of THF for later use.

[0074] Dissolve 15.2g of Fmoc-3-Me-Glu(OtBu)-OH and 15.2g of HATU in 150ml of THF / DMF (8:1). After complete dissolution, place the solution in an ice bath (2-8℃) and dropwise add 20ml of THF solution containing 5.8g of DIEA. After the addition is complete, stir in the ice bath for 30min. Remove the ice bath and combine the solution with 150ml of THF dispersion containing Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H2N-Kyn(Boc)]-Gly-OAll. Stir the solution at room temperature until the reaction is complete. THF was removed by concentration under reduced pressure. Then, 1 liter of chloroform was added to dissolve the chloroform completely. The solution was placed in a separatory funnel and washed once with 300 ml of 10% citric acid. The pH was measured to be 3.0. The chloroform layer was then washed with 3 x 300 ml of water, followed by 300 ml of 10% sodium bicarbonate solution. The chloroform layer was washed with 3 x 300 ml of water until neutral. The separated chloroform solution was dried with anhydrous sodium sulfate for 1 hour. The dried chloroform was concentrated under reduced pressure to a small volume. Heptane was added under low temperature (2-8℃) with stirring, and a white solid precipitated out. After stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with n-heptane and dried to obtain 64.4 g, with a yield of 98.4%. The purity was 97.8% as determined by HPLC. The product was Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll.

[0075] 9. Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll removes Fmoc

[0076] Weigh 64.4 g of Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll and dissolve it in 400 ml of THF / DMF (4:1). Add diethylamine to the solution in an ice bath (2-8℃) with stirring until the concentration reaches 50% (v / v). Then, remove Fmoc by stirring at 25-28℃ until complete. Concentrate under reduced pressure to remove diethylamine and THF. Disperse the resulting oil in 150 ml of THF / DMF (4:1) and set aside for later use to prepare Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll.

[0077] 10. Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll and Boc-D-Ser(tBu)- OH coupling forms Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll

[0078] 15.5 g of Boc-D-Ser(tBu)-OH.DCHA was suspended in 150 ml of ethyl acetate. Then, 75 ml of 5% potassium hydrogen sulfate solution was added to remove the DCHA salt. The mixture was stirred at room temperature for 30 min, and then the aqueous layer was separated in a separatory funnel. The ester layer was washed with 3 x 75 ml of 5% sodium chloride solution. The separated ester layer was dried with anhydrous sodium sulfate for 1 hour. After drying, the ester layer was concentrated under reduced pressure to a small volume. 100 ml of THF was added and mixed well, followed by 12.8 g of solid TBTU. The mixture was dissolved with stirring at room temperature. After complete dissolution, the mixture was placed in an ice bath (2-8℃), and 20 ml of solution containing 5.7 g of TBTU was added dropwise. After adding the THF solution of DIEA dropwise, stir in an ice bath for 30 minutes. Remove the ice bath and mix with 150 ml of a THF / DMF (4:1) solution containing Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll. Stir the reaction at room temperature until the reaction is complete. Concentrate under reduced pressure to remove THF, then add 1 liter of chloroform to completely dissolve the solution. Place the solution in a separatory funnel and wash the chloroform layer once with 300 ml of 10% citric acid. The pH is measured to be 3.0. Then wash the chloroform layer with 3 x 150 ml of water, and then with 300 ml of... The chloroform layer was washed once with 10% sodium bicarbonate, and then washed with 3 x 300 ml of water until neutral. The separated chloroform solution was dried with anhydrous sodium sulfate for 1 hour. The dried chloroform was concentrated under reduced pressure to a small volume, and hexane was added under low temperature (2-8℃) with stirring, resulting in the precipitation of a white solid. After stirring at low temperature for another 2-3 hours, the mixture was filtered. The filter cake was washed with hexane and dried to obtain 64.1 g, with a yield of 98.5%. The purity was determined to be 97.6% by HPLC. The prepared product was Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll.

[0079] 11. Removing All to form B' Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OH

[0080] 64.1 g of fragment B was weighed and dissolved in 500 ml of DCM / NMP (4:1). Then, 50 ml of phenylsilane was added and stirred until homogeneous. Next, 300 ml of DCM solution containing 80 g of Pd(PPh3)4 was added, and the mixture was stirred at room temperature until all components were completely removed. The solution was transferred to a separatory funnel, and the DCM layer was washed once with 250 ml of 5% citric acid and shaken. The DCM layer was then washed with 3 x 250 ml of 10% sodium chloride until neutral. The separated DCM solution was dried over anhydrous sodium sulfate for 1 hour. The dried DCM was concentrated under reduced pressure to a small volume, and petroleum ether was added under stirring at low temperature (2-8°C), resulting in the precipitation of a white solid. After continuing stirring at low temperature for 2-3 hours, the mixture was filtered. The filter cake was washed with petroleum ether and dried to obtain 54.6 g (29.1 mmol) of a pale yellow solid, with a yield of 87.1%. HPLC analysis showed a purity of 98.1%, thus preparing B'. Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OH.

[0081] Example 3 1. Condensation coupling of fragments A' and B' Weigh 54.6g of fragment B' and 5.9g of HOAt and add them to 500ml of THF / NMP (5:1) to dissolve. Stir until completely dissolved. Place the solution in an ice bath (2-8℃) and add 20ml of THF solution containing 5.6g of DIC dropwise over 10min. Then stir in the ice bath for 0.5hr. Remove the ice bath and add 300ml of THF / NMP (5:1) solution containing 24.3g of fragment A'. Stir the reaction at room temperature until the reaction is complete. The solution was concentrated under reduced pressure to a small volume and placed in an ice bath (2-8℃). 1.5 L of ethyl acetate was added and mixed well. The ester layer was washed once with 500 ml of 10% citric acid and the pH was measured to be 3.0. The ester layer was then washed with 3 x 500 ml of water, followed by 500 ml of 10% sodium bicarbonate. The ester layer was washed with 3 x 500 ml of water until neutral. The separated ester solution was dried with anhydrous sodium sulfate for 1 hour. The dried ester layer was concentrated under reduced pressure to a small volume and petroleum ether was added under stirring at low temperature (2-8℃). A slightly yellow solid precipitated out. After stirring at low temperature for another 2-3 hours, the mixture was filtered. The filter cake was washed with petroleum ether and dried to obtain 64.9 g, with a yield of 93.7%. The purity was 95.6% as determined by HPLC. This yielded Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal (fully protected daptomycin). The reaction formula is as follows:

[0082] 2. Preparation of daptomycin by cyclizing amide bonds 2.1 Removal of protecting groups and formation of daptomycin C-terminal salicylaldehyde ester Prepare 500 ml of TFA / Phenol / H2O (95:4:1) and cool it in an ice bath for 1 hour. Then add it to a mixture containing 64.9 g of fully protected daptomycin solid. Stir at room temperature for 2-3 hours to remove the protecting group. Under the same conditions, the C-terminal 2-(dimethoxymethyl)phenyl ester is converted to C-terminal salicylaldehyde ester. Then transfer the reaction solution to an ice bath and add 1.5 L of methyl tert-butyl ether to precipitate the solid. Stir in an ice bath for 1-2 hours and filter. Wash the filter cake with methyl tert-butyl ether and dry to obtain 36.4 g of daptomycin C-terminal salicylaldehyde ester Dec-Trp-D-Asn-Asp-Thr[HD-Ser-3-Me-Glu-Kyn]-Gly-Orn-Asp-D-Ala-Asp-Gly-salicylaldehyde (SAL) with a yield of 92.1%.

[0083]

[0084] 2.2. The C-terminal salicylaldehyde ester and the N-terminal D-serine ester cyclize to form an N,O-benzyl acetal-linked cyclodaptomycin intermediate product. 36.4 g of daptomycin C-terminal salicylaldehyde ester was dissolved in 100 ml of acetic acid / pyridine (1:1 mol:mol) solution and stirred at room temperature. The reaction was then processed by STL cyclization technology to form an N,O-benzyl acetal-linked cyclized daptomycin intermediate. The reaction was monitored by HPLC until the C-terminal salicylaldehyde ester and the N-terminus of serine were completely cyclized to form an imine cyclic peptide intermediate.

[0085] The STL (Ser / Thr ligation) refers to the process of linking the C-terminal salicylaldehyde ester of a polypeptide with the N-terminal serine / threonine residues to form a cyclization product under certain conditions.

[0086]

[0087] 2.3 Synthesis of daptomycin After freeze-drying to remove the solvent and obtaining a solid, 100 ml of TFA / TIS / H2O (95:4:1) was added to treat the resulting N,O-benzyl acetal imine cyclic peptide to form daptomycin.

[0088]

[0089] Then, 500 ml of methyl tert-butyl ether was added to precipitate the solid. The precipitated solid was washed with methyl tert-butyl ether and dried. Further purification by HPLC yielded 23.5 g of a white solid, with a yield of 69.4%. HPLC analysis conditions: Waters Arc TM HPLC; Column: XBridge Peptide BEH C18 Column, 300 Å, 5 µm, 4.6 × 250 mm, Detection wavelength: 226 nm, Column temperature: 60 ℃, Flow rate: 1.0 mg / ml, Mobile phase A: containing 0.05% TFA / H2O, Mobile phase B: containing 0.05% TFA / ACN, Elution gradient: 0-15 min, 35-95% B, Peak time for daptomycin detection was 6.764 min, Purity was 98.65% ( Figure 1 Its molecular weight, analyzed by mass spectrometry (Thermo Scientific™ Q Exactive™ Combined Quadrupole Orbitrap™ Mass Spectrometer), was 1619.715. Figure 2 It was confirmed to be the final product of daptomycin.

[0090] This invention discloses a method for synthesizing daptomycin. Daptomycin is prepared via a total liquid-phase fragmentation method. Two fully protected fragments are prepared using two glycine sites in daptomycin via liquid-phase processing. These two fully protected peptides are then condensed to form fully protected daptomycin. After acid cleavage to remove the protecting group, daptomycin is cyclized using STL cyclization technology to form daptomycin. This invention solves the problems of low cyclization efficiency, low purity of the cyclized product, and high levels of polymeric impurities associated with other cyclization techniques, thus achieving highly efficient preparation of daptomycin. Compared with other preparation methods, this method has the advantages of using less reagent and amino acid, generating fewer byproducts and impurities during the preparation process, and achieving higher product purity and yield.

[0091] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for synthesizing daptomycin, characterized in that, The synthesis method includes the following steps: Step 1: Synthesize protected fragment A' of fragment A and protected fragment B' of fragment B using liquid phase method; Fragment A is: H-ornithine 6 -Aspartic acid 7 -D-alanine 8 -Aspartic acid 9 -glycine 10 -OH; Fragment B is: N-decanoyl-tryptophan 1 -D-Asparagine 2 -Aspartic acid 3 -Threonine 4 (HD-serine) 11 3-Methyl-L-glutamic acid 12 -L-kynurenine 13 )-glycine 5 -OH; Fragment A' is H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethylacetal; The fragment A' is synthesized by a method including the following steps: (a1) Fmoc-D-Ala-OH and HOSu form Fmoc-D-Ala-OSu; or, Fmoc-D-Ala-OH and HPcp form Fmoc-D-Ala-OPcp; or, Fmoc-D-Ala-OH and HPfp form Fmoc-D-Ala-OPfp; or, Fmoc-D-Ala-OH and HONp form Fmoc-D-Ala-ONp; (a2) Fmoc-D-Ala-OSu, Fmoc-D-Ala-OPcp, Fmoc-D-Ala-OPfp, and Fmoc-D-Ala-ONp are condensed with H-Asp(OtBu)-(Dmb)Gly-OH to obtain Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH; (a3) Remove Fmoc from Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH to obtain HD-Ala-Asp(OtBu)-(Dmb)Gly-OH; (a4) HD-Ala-Asp(OtBu)-(Dmb)Gly-OH is sequentially condensed with one of Fmoc-Asp(OtBu)-OPfp, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-ONp, and one of Troc-Orn(Boc)-OPcp, Troc-Orn(Boc)-OPfp, Troc-Orn(Boc)-ONp, and Troc-Orn(Boc)-OSu, and 2-(dimethoxymethyl)phenol is attached to the C-terminus. The N-terminal 2,2,2-trichloroethoxycarbonyl protecting group Troc of the peptide is removed to obtain the fragment A'. Fragment B' is Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OH; The protected fragment B' is synthesized by a method including the following steps: (b1) One of Fmoc-Thr(TBS)-ONp, Fmoc-Thr(TBS)-OPfp, Fmoc-Thr(TBS)-OSu, and Fmoc-Thr(TBS)-OPcp is coupled with H-Gly-OH and Fmoc is removed to prepare H-Thr(TBS)-Gly-OH; (b2) Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OPfp, and Fmoc-Asp(OtBu)-ONp are coupled with H-Thr(TBS)-Gly-OH to prepare Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH; The C-terminus of Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH was protected with an allyl All, and after removing Fmoc, H-Asp(OtBu)-Thr(TBS)-Gly-OAll was obtained. (b3) Connect the protected D-asparagine and the protected tryptophan sequentially, and connect the decyl group at the N-terminus to obtain Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; (b4) Remove the TBS protecting group from the product of step (b3) to release the Thr side chain hydroxyl group; (b5) The product obtained in step (b4) is coupled with Fmoc-Kyn(Boc)-F, so that Kyn is linked to the Thr side chain via an ester bond; (b6) Connect the protected 3-methylglutamic acid and the protected D-serine in sequence, and finally remove the C-terminal allyl (All) protecting group to obtain the fragment B'; Step 2: Fragment A' and fragment B' condense to form a fully protected daptomycin peptide; Step 3: Remove the side chain protecting groups of the fully protected daptomycin peptide to obtain the cyclized precursor; Step 4: The cyclization precursor is subjected to a cyclization reaction in an acetic acid / pyridine solution to form an N,O-benzyl acetal-linked cyclized daptomycin intermediate product; Step 5: By treating with an organic acid solution, the O-to-N acyl transfer is triggered to convert the imine of the daptomycin intermediate product into an amide bond, forming crude daptomycin, which is then purified to obtain the daptomycin.

2. The method for synthesizing daptomycin according to claim 1, characterized in that, Step 2 specifically involves: Fragments A' and B' condense via DIC / HOAt to form the fully protected daptomycin peptide Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal.

3. The method for synthesizing daptomycin according to claim 1, characterized in that, Step 3 specifically involves cleaving the fully protected daptomycin peptide fragment Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal using TFA / Phenol / H2O to remove the side-chain protecting groups, yielding Dec-Trp-D-Asn-Asp-Thr[HD-Ser-3-Me-Glu-Kyn]-Gly-Orn-Asp-D-Ala-Asp-Gly-salicylaldehyde (SAL).

4. The method for synthesizing daptomycin according to claim 1, characterized in that, In step 5, the organic acid solution is TFA / TIS / H2O.

5. The method for synthesizing daptomycin according to claim 1, characterized in that, The method for synthesizing fragment A' includes the following steps: S1, Fmoc-D-Ala-OH, and HOSu form Fmoc-D-Ala-OSu; or, Fmoc-D-Ala-OH and HPcp form Fmoc-D-Ala-OPcp; or, Fmoc-D-Ala-OH and HPfp form Fmoc-D-Ala-OPfp; or, Fmoc-D-Ala-OH and HONp form Fmoc-D-Ala-ONp; Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH was prepared by coupling one of S2, Fmoc-D-Ala-OSu, Fmoc-D-Ala-OPcp, Fmoc-D-Ala-OPfp, and Fmoc-D-Ala-ONp with H-Asp(OtBu)-(Dmb)Gly-OH. S3. Remove Fmoc from Fmoc-D-Ala-Asp(OtBu)-(Dmb)Gly-OH to obtain HD-Ala-Asp(OtBu)-(Dmb)Gly-OH; S4, HD-Ala-Asp(OtBu)-(Dmb)Gly-OH is coupled with one of Fmoc-Asp(OtBu)-Opfp, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-ONp, and Fmoc is removed to obtain H-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH; Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH and Troc-Orn(Boc)-OPcp, Troc-Orn(Boc)-OPfp, Troc-Orn(Boc)-ONp, Troc-Orn(Boc)-OSu are coupled together to obtain Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH; S6, Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-OH reacts with 2-(dimethoxymethyl)phenol to form Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal; S7. Removing the N-terminal Troc group from Troc-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehydedimethyl acetal yields fragment A', namely H-Orn(Boc)-Asp(OtBu)-D-Ala-Asp(OtBu)-(Dmb)Gly-salicylaldehyde dimethyl acetal.

6. The method for synthesizing daptomycin according to claim 1, characterized in that, The method for synthesizing fragment B' includes the following steps: 1) One of Fmoc-Thr(TBS)-ONp, Fmoc-Thr(TBS)-OPfp, Fmoc-Thr(TBS)-OSu, and Fmoc-Thr(TBS)-OPcp is coupled with H-Gly-OH and Fmoc is removed to prepare H-Thr(TBS)-Gly-OH; 2) One of Fmoc-Asp(OtBu)-OSu, Fmoc-Asp(OtBu)-OPcp, Fmoc-Asp(OtBu)-OPfp, and Fmoc-Asp(OtBu)-ONp is coupled with H-Thr(TBS)-Gly-OH to prepare Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH; 3) Protect Fmoc-Asp(OtBu)-Thr(TBS)-Gly-OH with an allyl All group and remove Fmoc to prepare H-Asp(OtBu)-Thr(TBS)-Gly-OAll; 4) H-Asp(OtBu)-Thr(TBS)-Gly-OAll is coupled with Fmoc-D-Asn(Trt)-OH and Fmoc is removed to obtain HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 5) HD-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll is coupled with Fmoc-Trp(Boc)-OH and Fmoc is removed to obtain H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 6) H-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll was prepared by coupling with Dec-Cl to obtain Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll; 7) Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr(TBS)-Gly-OAll is prepared by removing the TBS protecting group and then coupling it with Fmoc-Kyn(Boc)-F. 8) Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-Kyn(Boc)]-Gly-OAll is prepared by removing Fmoc and then coupling it with Fmoc-3-Me-Glu(OtBu)-OH. 9) Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Fmoc-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll was prepared by removing Fmoc. 10), Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[H-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll and Boc-D-Ser(tBu) -OH coupling forms Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll 11) The fragment B' is obtained by removing All from Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OAll, i.e., Dec-Trp(Boc)-D-Asn(Trt)-Asp(OtBu)-Thr[Boc-D-Ser(tBu)-3-Me-Glu(OtBu)-Kyn(Boc)]-Gly-OH.

7. The method for synthesizing daptomycin according to claim 1, characterized in that, In step 4, the circumduction is performed using STL circumduction technology.

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