CN114981285B

CN114981285B - Synthesis method of C-terminal protection fragment of peptide

Info

Publication number: CN114981285B
Application number: CN202080006758.XA
Authority: CN
Inventors: 王刚; 舒遂智; 张利香; 李新宇; 岳泽乐
Original assignee: SHENZHEN JYMED TECHNOLOGY CO LTD
Current assignee: SHENZHEN JYMED TECHNOLOGY CO LTD
Priority date: 2020-12-26
Filing date: 2020-12-26
Publication date: 2023-08-29
Anticipated expiration: 2040-12-26
Also published as: CN114981285A; WO2022134124A1

Abstract

Relates to the field of polypeptide synthesis, and provides a synthesis method of a C-terminal protection fragment of peptide. The method comprises the following steps: (1) preparing Rcsin-T-P; (2) removing Resin by using a cracking reagent to obtain T-P. The medium acid sensitive connecting arm is used as a secondary connecting arm, and the medium acid sensitive connecting arm and acid sensitive resin or nucleophilic sensitive resin and Fmoc-AA-OH jointly react to realize the loading of amino acid. Peptide chain assembly is then completed. After the peptide chain is assembled, the resin is cracked by using an intensity acid or a nucleophilic reagent, and the protecting peptide segment and the secondary connecting arm are simultaneously released as a whole molecule. By using the method, the C peptide protecting fragment with the protecting group at the end can be directly released from the solid phase, and the protecting fragment can be directly used for synthesizing the polypeptide or the polypeptide fragment with the carboxyl at the C terminal. The method reduces preparation steps, improves synthesis yield, greatly reduces racemization risk, has low cost and easy control of reaction, and can be used for large-scale production.

Description

Synthesis method of C-terminal protection fragment of peptide

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a synthesis method of a C-terminal protection fragment of peptide.

Background

The solid-phase polypeptide synthesis method (SPPS) was proposed by R.BruceMerrifield for the first time in 1963, and has the advantages of convenient and rapid synthesis, mild reaction conditions, rapid and wide use, and more than 85% of polypeptide drugs are obtained by solid-phase synthesis at present. The synthesis method comprises the steps of fixing the C-terminal amino acid of the polypeptide on resin, gradually coupling the amino acid from the C-terminal to the N-terminal, and finally cutting off the resin and the amino acid protecting group to obtain the target peptide product. The solid phase stepwise coupling method works well for conventional peptides, however when the peptide chain is longer or contains more hydrophobic amino acids, it is often necessary to use fragment coupling methods, including solid phase fragment coupling methods, liquid phase fragment coupling methods.

CN108059666 reports a solid phase fragment coupling method for the synthesis of somalundin, but the peptide fragments used in this method all need to be fed with 2-5 times the resin substitution degree, resulting in serious waste and thus higher final synthesis cost. Due to such drawbacks, the current fragment condensation method is mostly performed in a liquid phase manner. In contrast, the liquid phase fragment coupling method well solves the problem of excessive feeding ratio, but because peptide fragments are prepared by a solid phase method, terminal carboxyl groups are usually exposed when C-terminal peptides are cleaved, and in order to reduce side reactions of coupling between fragments, an additional protection step is needed for the terminal carboxyl groups. CN109627317 reports a strategy for synthesizing somalundum by liquid phase fragment coupling method, which finally obtains the full-protection somalundum by liquid phase combination using three full-protection peptide fragments. Cleavage of the protecting group is then performed using a cleavage solution to yield the final product. When the C-terminal fragment 3 is cut off from the resin, the peptide is in N-terminal protection, and the C-terminal carboxyl is in an exposed state, so that the carboxyl is prevented from participating in the reaction, the peptide is additionally protected by one step, and then the N-terminal protecting group is removed, so that the component for fragment condensation can be obtained. This strategy increases the reaction steps and reduces the efficiency of the synthesis.

Patent EP3205660A1 proposes a method for preparing a fragment of a C-terminal protected peptide in one step and using this method for preparing liraglutide. The method utilizes a two-stage connecting arm strategy and a gradient cleavage mode to prepare the C peptide fragment with the end protected in one step, uses Wang series connecting arms as a second-stage connecting arm, fixes the C peptide fragment on resin containing the acid-hypersensitive first-stage connecting arm, and then carries out sequential peptide synthesis on the Wang connecting arm. After the synthesis is finished, the resin is treated by using low-concentration TFA, the hypersensitive resin is cracked, and the full-protection peptide segment and the Wang secondary connecting arm are released simultaneously, so that the target peptide segment with the end protected is directly obtained. However, the main problem caused by the method is that the second connecting arm adopts wang connecting arm analogue which is benzyl structure, so that the fragment cannot be subjected to catalytic hydrogenation treatment, otherwise, the removal of the second connecting arm is caused; meanwhile, wang linker arm analogs are prone to DKP formation and are not tolerant to stronger nucleophiles such as hydrazine. Although the secondary linker arm can be made resistant to nucleophiles by adding an alkyl substituent to the benzyl position of the Wang linker arm to make it tertiary, this in turn results in a greatly increased degree of acid sensitivity of the secondary linker arm and even no differentiation from the primary linker arm. For example, 1-dimethylbenzene ethanol, which is structurally similar thereto, requires only 2% concentration of TFA to allow rapid cleavage, indistinguishable from first order cleavage. Therefore, the secondary linker arm disclosed in this patent cannot achieve both nucleophile stability, catalytic hydrogenation stability and acid stability. When polypeptide site-directed modification is involved, the modification site usually uses protecting groups such as Dde, CBZ, alloc and the like to form special protection, and the protecting groups usually use nucleophilic reagents or catalytic hydrogenation to complete site-directed removal, so that other protecting groups can keep a stable state, thereby achieving the purpose of site-directed modification. Therefore, when the secondary connecting arm cannot tolerate the reagents, the terminal carboxyl protecting group can be removed during the removal of the fixed-point protecting group, and the modification selectivity is affected.

Disclosure of Invention

In order to solve the problems that in the prior art, long peptide synthesis is difficult, excessive solid phase synthesis is easy to cause waste, solid-liquid combination synthesis steps are complex, synthesis efficiency is low, and the existing C-terminal protected peptide fragment synthesis method cannot achieve the stability of nucleophile, catalytic hydrogenation stability, acid stability and the like, the invention provides a peptide C-terminal protected fragment synthesis method, which comprises the following steps:

(1) Preparing Resin-T-P;

(2) Removing Resin by adopting a cracking reagent to obtain T-P;

wherein: resin is acid sensitive Resin, P is peptide fragment, and T is selected from compounds with the following structures:

wherein X is selected from CH ₂ OH,CH ₂ Cl,CH ₂ Br,CH ₂ I,CH ₂ OTs,CH ₂ OMs,CH ₂ OTf, COOH; r1, R2 and R3 are one or more of aliphatic hydrocarbon groups, ether bonds, amide bonds and ester bonds. The X structure in the T structure is used as a handle to be coupled with acid sensitive resin or nucleophilic sensitive resin, and is broken under the treatment of low-strength acid or nucleophilic reagent; r1, R2 and R3 are such that T has an aliphatic tertiary alcohol structure, and the ester of the tertiary alcohol and carboxylic acid is resistant to treatment with low strength acids and nucleophiles and breaks down with medium and high strength acid treatments.

In some embodiments, T is preferably:

preferably, the Resin is selected from: 2-chlorotrityl chloride resin, trityl chloride resin, 2-methoxytrityl chloride resin, sieber resin, ramage resin, sasrin resin, dihydropyran resin, HMBA resin.

Preferably, the cleavage reagent of step (2) consists of B1 and B2, wherein B1 is selected from one or more compounds of TFA, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, RNH2, and B2 is selected from one or more compounds of the group consisting of halogenated hydrocarbons, tetrahydrofuran, cyclopentyl methyl ether, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, alcohols, silanes, water, thiols, phenols. Wherein B1 is selected from low-strength acids or nucleophiles as the main component of the lysis reagent.

In some embodiments, the cleavage reagent B1 of step (2) is selected from TFA, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, and has a concentration of 1-5% of the cleavage reagent. When low-strength acid is selected as the cleavage reagent, the Resin can be broken from Resin-T-P in the concentration range of 1-5%, and the peptide chain can not be affected.

In some embodiments, step (1) is specifically performed as follows: the X group of T reacts with Resin to synthesize Resin-T, the Resin-T reacts with C-terminal carboxyl of peptide fragment P to obtain Resin-T-A, and the Resin-T-P is obtained by coupling protective amino acid or peptide fragment. Wherein A is the C-terminal carboxy component of P.

In some embodiments, step (1) is specifically performed as follows: and (3) reacting tertiary alcohol of T with C terminal carboxyl of the peptide fragment P to obtain T-A, reacting with Resin to obtain Resin-T-A, and continuing coupling to protect amino acid or peptide fragment to obtain Resin-T-P. Wherein A is the C-terminal carboxy component of P.

The C-terminal of the peptide fragment P may be reacted with Resin-T to obtain Resin-T-A, wherein A is the C-terminal carboxyl component of P (e.g., a peptide fragment consisting of a single amino acid or a plurality of amino acids)

T has a bifunctional structure, one end is used for connecting resin, and the other end is used for connecting amino acid, so that the synthesis sequence can be adjusted by means of some chemical synthesis means, and the final result is not affected.

Use of a C-terminal protecting fragment as described above in the synthesis of a polypeptide or polypeptide fragment.

In particular, the polypeptide or polypeptide fragment is terminated with a carboxyl group.

More particularly, the polypeptide is selected from teriparatide, bivalirudin, somalundin, cinacacet, tedirudin.

The invention adopts a medium acid sensitive connecting arm as a secondary connecting arm, and jointly reacts with acid sensitive resin or nucleophilic sensitive resin and Fmoc-AA-OH to realize the loading of amino acid. Peptide chain assembly is then completed. After the peptide chain is assembled, the resin is cracked by using an intensity acid or a nucleophilic reagent, and the protecting peptide segment and the secondary connecting arm are simultaneously released as a whole molecule. By utilizing the method, the C peptide protection fragment with the end group with the protection group can be directly released from the solid phase, and can be directly used for preparing longer polypeptide or peptide fragment by a fragment condensation method. The method reduces preparation steps, improves synthesis yield, and greatly reduces racemization risk. Meanwhile, the end protection of the protection fragment obtained by the method can effectively resist DKP side reaction and nucleophilic reagent treatment, is relatively stable to catalytic hydrogenation, has low cost and easy control of reaction, and can be used for large-scale production.

Detailed Description

The technical solution of the present invention will be described below with reference to specific embodiments, and the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 preparation of teriparatide 1

The synthesis route of fragment I (T-P) is as follows:

remarks: PG represents a protecting group in all embodiments of the invention.

Preparation of Resin-T:

10.00g of dihydropyran resin (SD=1.06 mmol/g) was weighed into a reaction flask, soaked in 100ml of dichloromethane and swelled for 1h, the solvent was removed by suction filtration, and resuspended in 100ml of dry DCM. 5.51g of 3-methyl-1, 3-butanediol (5 eq) was weighed into it and stirred at room temperature to dissolve it thoroughly. 0.1g of p-toluenesulfonic acid monohydrate was weighed and added, a drying tube was added thereto for reaction at room temperature for 1 hour, the reaction solution was filtered off, washed three times with 5% diea-DCM solution, three times with DMF, three times with DCM, and then dried by suction, and after vacuum drying, the weighing was 10.92g, the loading rate was 84.6%, sd=0.81 mmol/g.

Preparation of Resin-T-A1:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 26.5mmol Fmoc-Phe-OH,2.65mmol DMAP was weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, and 29.1mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the Resin in the step 2 after being uniformly mixed, the reaction is carried out for 1 hour at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the Resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, and Resin-T-A113.75g is obtained after vacuum drying, and the substitution degree test SD=0.56 mmol/g and the total load is 7.7mmol.

Preparation of Resin-T-A2:

Resin-TResin-T was transferred to the reaction flask, suspended in 100mL DCM, and soaked and swelled for 1h. After filtering off the solvent, 100mL of DCM was added again. 26.5mmol Fmoc-Asn (Trt) -Phe-OH and 2.65mmol DMAP were weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, and 29.1mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the Resin in the step 2 after being uniformly mixed, the reaction is carried out for 1 hour at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the Resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 215.78 g of Resin-T-A is obtained after vacuum drying, and the substitution degree test SD=0.42 mmol/g and the total load is 6.7mmol.

Preparation of Resin-T-P:

weighing 23.1mmol Fmoc-amino acid, dissolving 3.74g HOBt in 100ml DMF, cooling in ice bath, slowly dripping 3.50g DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying;

wherein the amino acids used for coupling with Resin-T-A1 are in sequence: fmoc-Asn (Trt) -OH, fmoc-His (Trt) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Glu (OtBu) -OH, fmoc-Val-OH, fmoc-Arg (Pbf) -OH, fmoc-Glu (OtBu) -OH, fmoc-Met-OH, fmoc-Ser (OtBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Leu-OH, fmoc-His (Boc) -OH, and removing the end of the reaction.

The amino acids used for coupling with Resin-T-A2 were in the following order: fmoc-His (Trt) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Glu (OtBu) -OH, fmoc-Val-OH, fmoc-Arg (Pbf) -OH, fmoc-Glu (OtBu) -OH, fmoc-Met-OH, fmoc-Ser (OtBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Leu-OH, fmoc-His (Trt) -OH, fmoc-Lys (Boc) -OH, and Fmoc-Lys (Boc) -OH).

Taking 1-5 mg of resin, placing the resin into a centrifuge tube, washing the resin with ethanol for three times, washing the supernatant, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating the mixture on a metal bath at 100 ℃ for 5min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be performed; if the mixture is blue-black, the mixture indicates that part of the mixture is still unreacted, and the feeding is repeated until the reaction is complete.

150ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into resin, adding N ₂ Bubbling reaction is carried out for 10min, the reaction liquid is filtered and removed, the Resin is washed by DMF for six times, the coupling step is repeated after the Resin is pumped out, and the Resin-T-P is obtained after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane and transferred to a round bottom flask after draining. 400mg of p-toluenesulfonic acid monohydrate was weighed, 40ml of methanol and 160ml of methylene chloride were added, and after dissolution, the lysate was added to the resin at once and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 38.8g of solid as fragment i (mr=5135). The synthetic route for fragment II is as follows:

15g of 2-CTC resin (SD=1.06 mmol) was weighed out and suspended in 150mL DCM for swelling by soaking for 1h. After filtering off the solvent, 150mL of DCM was added again. 31.8mmol Fmoc-Gly-OH was weighed and added thereto and stirred to dissolve thoroughly. 160mmol DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 12.3mmol and the load yield was 77.4%.

36.9mmol Fmoc-amino acid, 11.8g TBTU are weighed, dissolved in 150ml DMF, cooled in ice bath, 9.5g DIEA is slowly dripped into the mixture, the activated reaction solution is added into the resin after shaking to dissolve the whole, and N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Leu-OH, fmoc-Asn (Trt) -OH, fmoc-His (Trt) -OH, fmoc-Met-OH, fmoc-Leu-OH, fmoc-Gln (Trt) -OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Ser (OtBu) -OH, fmoc-Val-OH, fmoc-Ser (OtBu) -OH. After the reaction is completed, the terminal Fmoc protection is removed.

150ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into resin, adding N ₂ Bubbling reaction is carried out for 10min, reaction liquid is filtered and removed, resin is washed for six times by DMF, and coupling steps are repeated after the resin is pumped out;

the peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. Weighing 40ml of trifluoroethanol and 160ml of dichloromethane, uniformly mixing to obtain a lysate, and adding the lysate into the peptide resinThe reaction was carried out at room temperature for 2 hours. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-drying gave 28.6g of solid as fragment ii (mr=2323).

Preparation of teriparatide:

5.2g of fragment I and 2.3g of fragment II were weighed, dissolved in 100mL of DMF, added with 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirred at room temperature for 4h, after the reaction was monitored by HPLC, the reaction solution was added to water to precipitate a large amount of solid, the solid was collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and then used directly in the next reaction.

1ml of ethanedithiol, 1ml of triisopropylsilane, 1ml of m-cresol and 1ml of water were aspirated, and finally TFA was added to 50ml, and the mixture was mixed well to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is poured into pre-frozen 500ml diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation, then N2 is dried, 4.13g of crude product is obtained, 2.89g of pure product is obtained after purification by RP-HPLC, and the yield is 70.0%.

Example 2 preparation of teriparatide 2

The synthesis route of fragment I (T-P) is as follows:

preparation of T-A:

4.72g (40 mmol) of beta-hydroxyisovaleric acid is weighed, dissolved in 50ml of dichloromethane, 7.74g of DIEA is added, 5.20g of trimethylchlorosilane is slowly added dropwise after the ice bath is properly cooled, and the reaction is continued for 30min after the dropwise addition is finished, so that the reaction liquid is ready for use. Another 7.75g Fmoc-Phe-OH was weighed, 50ml DCM was added, a small amount of DMF was added to make it fully dissolved, 4.53g DCC was added under ice-bath cooling, and the reaction was carried out in ice-bath for 15min. The temporarily protected beta-hydroxyisovalerate solution was added thereto, and then 24mg DMAP was added for catalysis, and the reaction was carried out at room temperature for 4 hours. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was collected, and 80ml of methylene chloride was added to dilute the reaction solution. The organic phase was washed three times with 2N HCl, washed with saturated NaCl, dried over anhydrous MgSO4, and the solvent was distilled off, followed by purification by column chromatography to give 6.11g of the desired product in 62.7% yield.

Preparation of Resin-T-P:

5.00g Sieber resin (SD=0.69 mmol/g) is weighed, placed in 50ml DMF for soaking and swelling for 30min, and after the reaction is finished, the solution is removed by suction filtration, and deprotection treatment (step 6) is carried out for standby. Weighing 10.35mmol Fmoc-amino acid, dissolving 1.68g HOBt in 50ml DMF, cooling in ice bath, slowly dripping 1.56g DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: T-A, fmoc-Asn (Trt) -OH, fmoc-His (Trt) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Glu (OtBu) -OH, fmoc-Val-OH, fmoc-Arg (Pbf) -OH, fmoc-Glu (OtBu) -OH, fmoc-Met-OH, fmoc-Ser (OtBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Leu-OH, fmoc-His (Trt) -OH, and removing the end of the reaction.

100ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into resin, adding N ₂ Bubbling reaction is carried out for 10min, reaction liquid is filtered and removed, resin is washed for six times by DMF, and coupling steps are repeated after the resin is pumped out; and (3) after the coupling is finished, obtaining Resin-T-P.

Preparation of fragment I:

Resin-T-P is washed three times with DMF and then three times with dichloromethane, and is drained and transferredMove to a round bottom flask. 2ml of TFA is measured, methylene dichloride is added to dilute to 200ml to obtain a lysate, the lysate is added into resin, the resin is cleaved for 1h at room temperature, after the reaction is finished, the filtrate is collected by suction filtration, the cleavage is repeated once again, and the filtrates are combined. Adding 500ml frozen diethyl ether, precipitating a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 17.8g of solid as fragment i (mr=5135).

Preparation of teriparatide:

5.4g of fragment I and 2.3g of fragment II obtained in example 1 were weighed, dissolved in 100mL of DMF, 0.63g of PyBOP and 0.30g of DIEA were added at room temperature, stirred at room temperature for 4h, after the reaction was monitored by HPLC, the reaction solution was added to water to precipitate a large amount of solid, and the solid was collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and then used directly in the next reaction.

1ml of ethanedithiol, 1ml of triisopropylsilane, 1ml of m-cresol and 1ml of water were aspirated, and TFA was finally added to 40ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 4.13g of crude products are obtained, and 2.67g of pure products are obtained after purification by RP-HPLC, and the yield is 64.6%.

Example 3 preparation of bivalirudin 1

The synthesis route of fragment I (T-P) is as follows:

preparation of Resin-T:

10.00g of 2-CTC resin (SD=1.17 mmol/g) was weighed into a reaction flask, soaked in 100ml of dichloromethane and swelled for 1h, the solvent was removed by suction filtration, and resuspended in 100ml of dry DCM. 8.42g of the compound was weighed out(5 eq) was added thereto and stirred at room temperature to be sufficiently dissolved. Weighing 30.2g DIEA (20 eq) was added thereto, the reaction was carried out at room temperature for 48h with the addition of a drying tube, the reaction solution was filtered off, washed three times with DCM solution, three times with DMF, three times with DCM, dried by suction after three times with DCM, weighed 10.94g after vacuum drying, 11.26g theoretical, and the loading ratio was 74.6%. Sd=0.80 mmol/g, total loading 8.7mmol. Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 10ml of trichloroacetonitrile was pipetted into it and 1ml of DBU was added for activation. The mixed solution was reacted at room temperature for 4h, the solvent was removed by filtration, washed three times with DCM and resuspended in 100ml of dry DCM. 26.1mmol Fmoc-Leu-OH was weighed and added thereto, and 100. Mu.L of BF was dropped ₃ ·Et ₂ O is catalyzed, the reaction is carried out for 6 hours at room temperature, the reaction solution is removed by suction filtration after the reaction is completed, and the resin is subjected to end capping treatment after being washed by DCM multiplied by 3 and DMF multiplied by 3.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 13.20g of resin is obtained after vacuum drying, and the substitution degree test SD=0.51 mmol/g and the total load is 6.7mmol.

Preparation of Resin-T-P:

weighing 20.0mmol Fmoc-amino acid, dissolving 2.97g HOBt in 100ml DMF, cooling in ice bath, slowly dripping 2.77g DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Tyr (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Pro-OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, and terminal Fmoc protection is removed after the reaction is completed.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 3ml of TFA was measured, 297ml of methylene chloride was added and mixed uniformly to obtain a lysate, and the lysate was added to the resin at one time and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 11.4g of solid as fragment i (mr=1732).

The synthetic route for fragment II is as follows:

10g of 2-CTC resin (SD=1.17 mmol) was weighed out and suspended in 100mL of DCM for swelling by soaking for 1h. After filtering off the solvent, 100mL of DCM was added again. 17.5mmol Fmoc-Gly-OH was weighed and added thereto and stirred to dissolve thoroughly. 88mmol of DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 7.7mmol and the load yield was 65.8%.

23.1mmol Fmoc-amino acid, 7.40g TBTU are weighed, dissolved in 100ml DMF, cooled in ice bath, 6.00g DIEA is slowly dripped into the mixture, the activated reaction solution is added into the resin after shaking to dissolve the whole, and N is added ₂ Bubbling reaction for 2h, pumpingFiltering to remove reaction liquid, washing resin with DMF for six times, and performing ninhydrin detection after pumping; wherein the amino acids used for coupling are in turn: fmoc-Gly-OH, fmoc-Asn (Trt) -OH, fmoc-Gly-OH, fmoc-Gly-OH, fmoc-Gly-OH, fmoc-Gly-OH, fmoc-Pro-OH, fmoc-Arg (Pbf) -OH, fmoc-Pro-OH, boc-D-Phe-OH.

100ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into resin, adding N ₂ Bubbling reaction is carried out for 10min, reaction liquid is filtered and removed, resin is washed for six times by DMF, and coupling steps are repeated after the resin is pumped out;

The peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are measured and mixed uniformly to obtain a lysate, and the lysate is added into peptide resin and reacted for 2 hours at room temperature. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-drying gave 11.6g of solid as fragment ii (mr=1509).

Preparation of bivalirudin:

1.7g of fragment I and 1.5g of fragment II are weighed, dissolved in 40mL of DMF, added with 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirred at room temperature for 4h, after the reaction is monitored by HPLC, the reaction solution is added into water to precipitate a large amount of solid, the solid is collected by suction filtration and repeatedly washed with water, and DMF in the solid is removed. The sample was dried and then used directly in the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml of diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 2.26g of crude products are obtained, and 1.44g of pure products are obtained after purification by RP-HPLC, and the yield is 63.7%.

Example 4 preparation 2 of bivalirudin

The synthesis route of fragment I (T-P) is as follows:

preparation of Resin-T:

10.00g Sasrin resin (SD=0.80 mmol/g) was weighed into a reaction flask, 100ml THF was added for soaking and swelling for 1h, the solvent was removed by suction filtration, resuspended in 100ml dry THF, 2.7g potassium tert-butoxide was weighed, dissolved in 100ml dry THF, added to the resin, reacted for 1h at room temperature, the reaction solution was removed by suction filtration, and the resin was washed once with dry THF for use. 2.60g of 1-chloro-2-methyl-2-propanol (3 eq) were weighed into 20ml of dry THF and stirred at room temperature and slowly dropped into it. After the dripping is completed, the reaction is carried out at the water-stop room temperature for 24 hours, the reaction solution is filtered and removed, the reaction solution is washed three times by THF, washed three times by 5 percent HOAc-DMF solution, pumped down after DCM washing three times, and the load factor is 89.7 percent after vacuum drying, and the weighing is 10.52g and theoretical 10.58 g. Sd=0.68 mmol/g, total loading 7.2mmol.

Preparation of Resin-T-P:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 21.6mmol Fmoc-Leu-OH,2.16mmol DMAP was weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, and 23.7mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 12.50g of resin is obtained after vacuum drying, and the substitution degree test SD=0.47 mmol/g and the total load is 5.9mmol.

Weighing 17.7mmol Fmoc-amino acid, dissolving 2.63g HOBt in 100ml DMF, cooling in ice bath, slowly dripping 2.45g DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Tyr (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Pro-OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, and terminal Fmoc protection is removed after the reaction is completed.

preparation of fragment I:

the peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 3ml of TFA was measured, 297ml of methylene chloride was added and mixed uniformly to obtain a lysate, and the lysate was added to the resin at one time and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying to obtain 11.4g of solid fragmentsBody, fragment i (mr=1732). Liquid phase synthesis of bivalirudin:

1.7g of fragment I and 1.5g of fragment II obtained in example 3 were weighed, dissolved in 40mL of DMF, 0.63g of PyBOP and 0.30g of DIEA were added at room temperature, stirred at room temperature for 4h, after the reaction was monitored by HPLC, the reaction solution was added to water to precipitate a large amount of solid, and the solid was collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and then used directly in the next reaction.

Example 5 preparation of Somalin peptide 1

The synthesis route of fragment I (T-P) is as follows:

preparation of T:

11.8g of beta-hydroxyisovaleric acid is weighed, 12.7g of HOSu is added, the mixture is dissolved in 100ml of DCM, 2.26g of DCC is added under ice bath cooling, the reaction is carried out for 30min and then the reaction is carried out to room temperature, 10.5g of diglycolamine is added into the mixture at one time, and the reaction is carried out for 12h at room temperature. After the reaction was completed, the filtrate was collected by suction filtration, the filter cake was washed with DCM and combined with the filtrate, the organic phase was washed three times with citric acid solution, naHCO3 solution, saturated sodium chloride solution, dried over anhydrous MgSO4 and the solvent was distilled off to give 20.2g of a pale yellow oil, which was almost quantitative in yield and used directly in the next reaction without purification.

Preparation of Resin-T-A:

weigh 10.25g (50 mmol)Dissolving in 150ml of dichloromethane, adding 9.67g of DIEA, cooling in an ice bath, slowly dripping 6.50g of trimethylchlorosilane, and continuing to react for 30min after the dripping is finished, wherein the reaction solution is ready for use. Another 7.43g Fmoc-Gly-OH (25 mmol) was weighed, 150ml DCM was added, a small amount of DMF was added to make it fully dissolved, 6.18g DCC was added under ice-bath cooling, and the reaction was carried out in ice-bath for 15min. The temporarily protected T solution was added thereto, and 30mg of DMAP was added thereto for catalysis, and reacted at room temperature for 4 hours. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was collected, and 80ml of DCMA was added to dilute the reaction solution. The organic phase was washed three times with 2N HCl, washed with saturated NaCl, dried over anhydrous MgSO4, and the solvent was distilled off, followed by purification by column chromatography to give 9.71g of the desired product in 75.8% yield.

Synthesis of Resin-T-A:

5.00g of dihydropyran resin (SD=1.06 mmol/g) are weighed into a reaction flask, 100ml of 1, 2-dichloroethane are added for swelling for 1h, the solvent is removed by suction filtration, and the mixture is resuspended in 100ml of 1, 2-dichloroethane. 8.15. 8.15g T-A (3 eq) was weighed into it and stirred at room temperature to dissolve thoroughly. 0.15g of pyridine p-toluenesulfonate is weighed, a drying pipe is added for reflux reaction for 6 hours, the reaction liquid is filtered and removed, the reaction liquid is washed three times by DCM solution and three times by DMF, the reaction liquid is pumped out after the DCM is washed three times, the weighing is 6.77g after vacuum drying, and the loading rate is 65.1%. Sd=0.51 mmol/g, total loading of 3.4mmol.

Preparation of Resin-T-P:

the Resin-T-A prepared in the previous step is suspended in 100ml of DMF for soaking and swelling, and then deprotection treatment is carried out, and the Resin-T-A is directly used for the peptide coupling step. Weighing 10.0mmol Fmoc-amino acid, dissolving 3.21g TBTU in 70ml DMF, slowly dripping 2.60g DIEA at room temperature, shaking for dissolving completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Alloc) -OH, fmoc-Ala-OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, the terminal Fmoc group is retained after coupling.

100mL of DCM was added, followed by 10mL of phenylsilane, and the reaction was carried out for 3min, followed by 2.2g of Pd (PPh) ₃ ) ₄ The reaction was carried out at room temperature for 45min, the reaction solution was withdrawn, and ninhydrin detection was positive, indicating that Alloc had been removed. Fmoc-AEEA-OH, fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester are sequentially coupled according to standard coupling steps, after the reaction is finished, the tail end Fmoc is removed by DBLK, then the reaction is transferred to a cleavage step, and the coupling is finished to obtain Resin-T-P.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 400mg of p-toluenesulfonic acid monohydrate was weighed, 40ml of methanol and 160ml of methylene chloride were added, and after dissolution, the lysate was added to the resin at once and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to 1/2 volume, adding 500ml frozen diethyl ether, precipitating solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 11.33g of solid as fraction I (Mr= 3333.18)

The synthetic route for fragments II and C is as follows:

10g of 2-CTC resin (SD=1.17 mmol) was weighed out and suspended in 100mL of DCM for swelling by soaking for 1h. After filtering off the solvent, 100mL of DCM was added again. 17.5mmol Fmoc-Gly-OH (for preparing fragment II) or Fmoc-Ser (OtBu) -OH (for preparing fragment C) was weighed and added thereto and stirred to dissolve it sufficiently. 88mmol of DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 7.7mmol or 7.1mmol, and the load yield was 65.8% (Fmoc-Gly-OH) or 60.7% (Fmoc-Ser (OtBu) -OH).

Weighing 3eq Fmoc-amino acid, 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 6eq DIEA, shaking to dissolve completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for fragment II coupling are in sequence: DVSSYLEG, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (OtBu) -OH, fmoc-Ser (OtBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH; the amino acid used for coupling the fragment C is His-Aib-Gly-Glu-Thr-Phe-Thr-Ser in sequence; fmoc-Ser (OtBu) -OH, fmoc-Thr (OtBu) -OH, fmoc-Phe-OH, fmoc-Thr (OtBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Gly-OH, boc-His (Trt) -Aib-OH.

100ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into resin, adding N ₂ Bubbling reaction for 10min, filtering to remove reaction liquid, and collecting treeWashing the grease with DMF for six times, and repeating the coupling step after pumping;

the peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are measured and mixed uniformly to obtain a lysate, and the lysate is added into peptide resin and reacted for 2 hours at room temperature. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-dried to give 10.62g (or 10.15 g) of solid as fragment ii (or fragment C) (mr=1371/1430).

Liquid phase synthesis of somalupeptide:

3.4g of fragment I and 1.4g of fragment II were weighed, dissolved in 40ml of DMF, added with 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirred at room temperature for 4h, after the reaction was monitored by HPLC, the reaction solution was added to water to precipitate a large amount of solid, the solid was collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and dissolved in 30ml of 20% DBLK solution, stirred at room temperature for 30min, 200ml of diethyl ether was added to precipitate a solid, which was collected by centrifugation and washed twice with diethyl ether, dried and used for the next coupling.

The AB full-protection peptide obtained in the previous step and 1.5g of fragment C are dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA are added at a temperature, stirring is carried out for 4 hours at room temperature, after the reaction is monitored by HPLC, the reaction solution is added into water, a large amount of solids are separated out, the solids are collected by suction filtration and repeatedly washed by water, and DMF in the solids is removed. The sample was dried and used directly for the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 4.30g of crude products are obtained, and 2.52g of pure products are obtained after purification by RP-HPLC, and the yield is 58.6%.

EXAMPLE 6 preparation of Somali Lupeptide 2

The synthesis route of fragment I (T-P) is as follows:

preparation of Resin-T:

10.00g of Trt-Cl resin (SD=1.06 mmol/g) was weighed into a reaction flask, soaked in 100ml of dichloromethane and swelled for 1h, the solvent was removed by suction filtration, and resuspended in 100ml of dry DCM. Weigh 12.52g(5 eq) was added thereto and stirred at room temperature to be sufficiently dissolved. 27.3g DIEA (20 eq) was weighed and added to it, and the reaction was carried out for 48h at room temperature with a drying tube, the reaction solution was filtered off, washed three times with DCM solution, three times with DMF, three times with DCM, dried by suction after three times with DCM, and weighed 11.64g, 12.12g theoretical, and 77.4% loading. Sd=0.80 mmol/g, total loading 8.2mmol.

Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 24.6mmol Fmoc-Gly-OH,2.46mmol DMAP was weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, 27.1mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride, 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added to the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 13.94g of resin is obtained after vacuum drying, the substitution degree test SD=0.59 mmol/g, the total load amount is 8.2mmol, and the reaction is considered to be quantitative. Preparation of Resin-T-P:

Weighing 10.0mmol Fmoc-amino acid, dissolving 3.21g TBTU in 70ml DMF, slowly dripping 2.60g DIEA at room temperature, shaking for dissolving completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Alloc) -OH, fmoc-Ala-OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, the terminal Fmoc group remaining after coupling.

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 400mg of p-toluenesulfonic acid monohydrate was weighed, 40ml of methanol and 160ml of methylene chloride were added, and after dissolution, the lysate was added to the resin at once and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to 1/2 volume, adding 500ml frozen diethyl ether, precipitating solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave fragment 27.3g of solid as fragment i (mr= 3333.18).

Liquid phase synthesis of somalupeptide:

3.4g of fragment I and 1.4g of fragment II obtained in example 5 were weighed, dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA were added at room temperature, stirred at room temperature for 4h, after the reaction was monitored by HPLC, the reaction solution was added to water, a large amount of solids was precipitated, the solids were collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and dissolved in 30ml of 20% DBLK solution, stirred at room temperature for 30min, 200ml of diethyl ether was added to precipitate a solid, which was collected by centrifugation and washed twice with diethyl ether, dried and used for the next coupling.

The full protection peptide obtained in the above step and 1.5g of fragment C obtained in example 5 were dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA were added at a temperature, stirred for 4 hours at room temperature, after the completion of the HPLC monitoring reaction, the reaction solution was added to water to precipitate a large amount of solid, and the solid was collected by suction filtration and repeatedly washed with water to remove DMF. The sample was dried and used directly for the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml of diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 4.37g of crude products are obtained, 2.74g of pure products are obtained after purification by RP-HPLC, and the yield is 62.7%.

EXAMPLE 7 preparation of cinacacetin 1

The synthesis route of fragment I (T-P) is as follows:

synthesis of Resin-T:

10.00g Sasrin resin (SD=0.80 mmol/g) was weighed into a reaction flask, 100ml THF was added for soaking and swelling for 1h, the solvent was removed by suction filtration, resuspended in 100ml dry THF, 2.7g potassium tert-butoxide was weighed, dissolved in 100ml dry THF, added to the resin, reacted for 1h at room temperature, the reaction solution was removed by suction filtration, and the resin was washed once with dry THF for use. 4.71g of Compound T (3 eq) are weighed out in 20ml of dry THF, stirred at room temperature and slowly added thereto. After the completion of the dropwise addition, the reaction was carried out at a water-stop temperature for 24 hours, the reaction solution was removed by filtration, washed three times with THF, three times with 5% HOAc-DMF solution, dried by suction after three times with DCM, and then weighed to 10.80g after vacuum drying, and the load factor was quantified. Sd=0.74 mmol/g, total loading 8.0mmol. Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 24.0mmol Fmoc-Lys (Boc) -OH,2.4mmol DMAP was weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, and 26.4mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 13.80g of resin is obtained after vacuum drying, and the substitution degree test SD=0.48 mmol/g and the total load is 6.7mmol.

Preparation of Resin-T-P:

weighing 20.0mmol Fmoc-amino acid, 22.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 22.0 mmole DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, and terminal Fmoc protection is removed after the reaction.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 3ml of TFA was measured, 297ml of methylene chloride was added and mixed uniformly to obtain a lysate, and the lysate was added to the resin at one time and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 13.7g of solid as fragment i (mr= 1708.33).

Preparation of fragment II:

10g of 2-CTC resin (SD=1.17 mmol) was weighed out and suspended in 100mL of DCM for swelling by soaking for 1h. After filtering off the solvent, 100mL of DCM was added again. 17.5mmol Fmoc-Lys (Boc) -OH was weighed into it and stirred to dissolve it thoroughly. 58.5mmol of DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 7.4mmol and the load yield was 63.2%.

Weighing 3eq Fmoc-amino acid, 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 6eq DIEA, shaking to dissolve completely, and activatingAdding the reaction solution into the resin, N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for fragment II coupling are in sequence: fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH.

The peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are measured and mixed uniformly to obtain a lysate, and the lysate is added into peptide resin and reacted for 2 hours at room temperature. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-drying gave 10.95g of solid as fragment ii (mr=1480).

Liquid phase synthesis of cinacaprin:

1.7g of fragment I and 1.5g of fragment II are dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA are added at a temperature, stirring is carried out at room temperature for 4h, after the reaction is monitored by HPLC, the reaction solution is added into water, a large amount of solid is separated out, the solid is collected by suction filtration and repeatedly washed by water, and DMF in the solid is removed. The sample was dried and used directly for the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 2.55g of crude product is obtained, and 1.72g of pure product is obtained after purification by RP-HPLC, and the yield is 67.5%.

EXAMPLE 8 preparation of cinacacetin 2

The synthesis route of fragment I (T-P) is as follows:

synthesis of Resin-T:

10.00g Sasrin resin (SD=0.80 mmol/g) was weighed into a reaction flask, 100ml THF was added for soaking and swelling for 1h, the solvent was removed by suction filtration, resuspended in 100ml dry THF, 2.7g potassium tert-butoxide was weighed, dissolved in 100ml dry THF, added to the resin, reacted for 1h at room temperature, the reaction solution was removed by suction filtration, and the resin was washed once with dry THF for use. 6.54g of Compound T are weighed out(3 eq) was dissolved in 20ml of dry THF and stirred at room temperature and slowly added thereto. After the completion of the dropwise addition, the reaction was carried out at a water-stop temperature for 24 hours, the reaction solution was removed by filtration, washed three times with THF, three times with 5% HOAc-DMF solution, dried by suction after three times with DCM, and then weighed to 10.76g after vacuum drying, and the load factor was quantified. Sd=0.71 mmol/g, total loading 7.6mmol.

Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked and swollen for 1h. After filtering off the solvent, 100mL of DCM was added again. 22.8mmol Fmoc-Lys (Boc) -OH,2.3mmol DMAP was weighed out and stirred to dissolve thoroughly. Cooled to 0 ℃ in ice bath, and 25.1mmol DIC was slowly dropped. Stirring for 30min at low temperature, and after the reaction time is shifted to room temperature, continuing to react for 8h. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM. Times.3 and DMF. Times.3 and then subjected to end-capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 13.78g of resin is obtained after vacuum drying, and the substitution degree test SD=0.48 mmol/g and the total load is 6.7mmol.

Preparation of Resin-T-P:

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 3ml of TFA was measured, 297ml of methylene chloride was added and mixed uniformly to obtain a lysate, and the lysate was added to the resin at one time and cleaved for 1 hour. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking resin onceThe two lysates were combined. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave 11.4g of solid as fragment i (mr= 1708.33).

Preparation of fragment II:

10g of 2-CTC resin (SD=1.17 mmol) was weighed out and suspended in 100mL of DCM for swelling by soaking for 1h. After filtering off the solvent, 100mL of DCM was added again. 17.5mmol Fmoc-Lys (Boc) -OH was weighed into it and stirred to dissolve it thoroughly. 58.5mmol of DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 7.4mmol load yield was 63.2%.

Weighing 3eq Fmoc-amino acid, 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 6eq DIEA, shaking to dissolve completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for fragment II coupling are in sequence: fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH.

150ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to remove reaction solution, adding 150ml 20% piperidine-DMF solution again, addingInto the resin, N ₂ Bubbling reaction is carried out for 10min, reaction liquid is filtered and removed, resin is washed for six times by DMF, and coupling steps are repeated after the resin is pumped out;

Liquid phase synthesis of cinacaprin:

EXAMPLE 9 preparation of Tidollutide 1

The synthesis route of fragment I (T-P) is as follows:

synthesis of Resin-T:

10.00g of Sasrin resin (SD=0.80 mmol/g) was weighed into a reaction flask,adding 100ml of THF, soaking and swelling for 1h, removing the solvent by suction filtration, re-suspending in 100ml of dry THF, weighing 2.70g of potassium tert-butoxide, dissolving in 100ml of dry THF, adding into resin, reacting for 1h at room temperature, removing the reaction liquid by suction filtration, and washing the resin once with dry THF for later use. Weigh 6.54g(3 eq) was dissolved in 20ml of dry THF and stirred at room temperature and slowly added thereto. After the completion of the dropwise addition, the reaction was carried out at a water-stop temperature for 24 hours, the reaction solution was removed by filtration, washed three times with THF, three times with 5% HOAc-DMF solution, dried by suction after three times with DCM, and weighed 11.04g after vacuum drying, and the load factor was quantified. Sd=0.72 mmol/g, total loading 8.0mmol.

Preparation of Resin-T-A:

20ml of acetic anhydride and 20ml of pyridine are weighed, 60ml of DMF is added, the mixture is added into the resin in the step 2 after being uniformly mixed, the reaction is carried out for 1h at room temperature, the reaction liquid is removed by suction filtration after the reaction is finished, the resin is washed by DCM multiplied by 3, DMF multiplied by 3 and MeOH multiplied by 3, 13.83g of resin is obtained after vacuum drying, and the substitution degree test SD=0.51 mmol/g and the total load is 7.1mmol.

Preparation of Resin-T-P:

weighing 21.0mmol Fmoc-amino acid, 23.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 23.0 mmole DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Thr (OtBu) -OH, fmoc-Ile-OH, fmoc-Lys (Boc) -OH, fmoc-Thr (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Asn (Trt)) -OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH; and removing the end Fmoc protection after the reaction is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 3ml of TFA was measured, 297ml of methylene chloride was added and mixed uniformly to obtain a lysate, and the lysate was added to the resin at one time and cleaved for 1 hour. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave fragment 20.56g of solid as fragment i (mr= 2895.63).

Preparation of fragment II:

10g of 2-CTC resin (SD=1.17 mmol) was weighed out and suspended in 100mL of DCM for swelling by soaking for 1h. After filtering off the solvent, 100mL of DCM was added again. 17.5mmol Fmoc-Ala-OH was weighed and added thereto and stirred to dissolve it sufficiently. 58.5mmol of DIEA was added and the reaction was carried out at room temperature for 2h. After the reaction was completed, 20ml of methanol was added for capping, and the reaction was performed for 1 hour. After the reaction was completed, the reaction mixture was removed by suction filtration, and the resin was washed with DCM×3 and DMF×3 and then coupled. The total amount of substitution test load was 8.7mmol and the load yield was 74.4%.

Weighing 3eq Fmoc-amino acid, 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 6eq DIEA, shaking to dissolve completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for fragment II coupling are in sequence: fmoc-Ala-OH, fmoc-Leu-OH, fmoc-Asn (Trt) -OH, fmoc-Asp (OtBu) -OH, fmoc-Leu-OH, fmoc-Ile-OH, fmoc-Thr (OtBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Met-OH, fmoc-Glu (OtBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (OtBu) -OH, fmoc-Phe-OH, fmoc-Ser (OtBu) -OH, fmoc-Gly-OH, fmoc-Asp (OtBu) -OH, fmoc-Gly-OH, boc-His (Trt) -OH.

the peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are measured and mixed uniformly to obtain a lysate, and the lysate is added into peptide resin and reacted for 2 hours at room temperature. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-drying gave 27.45g of solid as fragment ii (mr=3155.47)。

Liquid phase synthesis of tidolutetin:

2.9g of fragment I and 3.2g of fragment II were dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA were added at room temperature and stirred for 4h at room temperature, after the completion of the HPLC monitoring reaction, the reaction solution was added to water, a large amount of solids were precipitated, the solids were collected by suction filtration and repeatedly washed with water to remove DMF therefrom. The sample was dried and used directly for the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml of diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 3.83g of crude product is obtained, and 2.61g of pure product is obtained after purification by RP-HPLC, and the yield is 69.6%.

EXAMPLE 10 preparation of Tidollutide 2

The synthesis route of fragment I (T-P) is as follows:

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weigh 10.25g (50 mmol)Dissolving in 150ml of dichloromethane, adding 9.67g of DIEA, cooling in an ice bath, slowly dripping 6.50g of trimethylchlorosilane, and continuing to react for 30min after the dripping is finished, wherein the reaction solution is ready for use. Another 25mmol Fmoc-Asp (OtBu) -OH was weighed, 150ml DCM was added, a small amount of DMF was added to make it fully dissolved, 6.18g DCC was added under ice-bath cooling and the ice-bath reaction was carried out for 15min. The temporarily protected T solution was added thereto, and 30mg of DMAP was added thereto for catalysis, and reacted at room temperature for 4 hours. Filtering to remove precipitate after the reaction is completed, collecting filtrate, and addingThe reaction was diluted with 80ml of DCMA. The organic phase was washed three times with 2N HCl, washed with saturated NaCl, dried over anhydrous MgSO4, and the solvent was distilled off, followed by purification by column chromatography to give 9.71g of the desired product in 75.8% yield.

Synthesis of Resin-T-A:

5.00g HMBA resin (SD = 1.02 mmol/g) was weighed into a reaction flask, and 100ml DCM was added to soak and swell for 1h, the solvent was removed by suction filtration and resuspended in 100ml DCM. 15.3mmol of T-A (3 eq) was weighed into it and stirred at room temperature to dissolve it sufficiently and then transferred to ice bath for cooling. 16.8mmol DIC was weighed, added thereto in portions, 1.7mmol DMAP was added thereto for catalytic reaction, a drying tube was added thereto for 30min at low temperature, and the mixture was allowed to stand at room temperature and stirred overnight. After the reaction was completed, the reaction mixture was removed, washed three times with DCM solution, washed three times with DMF, dried by suction after three times with DCM, and dried in vacuo to give 5.52g of Resin-T-A, which was used quantitatively. Sd=0.92 mmol/g, total loading of 5.1mmol.

Synthesis of Resin-T-P:

weighing 15.0mmol Fmoc-amino acid, 17.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 17.0 mmole DIC, activating for 15min, adding the activated reaction solution into resin, and adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for coupling are in turn: fmoc-Thr (OtBu) -OH, fmoc-Ile-OH, fmoc-Lys (Boc) -OH, fmoc-Thr (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Trp-OH, fmoc-Asn (Trt) -OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH; and removing the end Fmoc protection after the reaction is finished.

150ml of 20% piperidine-DMF solution was taken and added to the resin, N ₂ Bubbling reaction for 5min, filtering to removeThe reaction mixture was again added to 150ml of 20% piperidine-DMF solution and added to the resin, N ₂ Bubbling reaction is carried out for 10min, the reaction liquid is filtered and removed, the Resin is washed by DMF for six times, the coupling step is repeated after the Resin is pumped out, and the Resin-T-P is obtained after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three more times with THF, drained and transferred to a round bottom flask. 80ml THF was added to the resin and 40ml 4M NH was added ₃ Cleavage with MeOH solution for 1h. And after the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two cracking solutions. Washing resin with dichloromethane three times, mixing with the filtrate, concentrating under reduced pressure to remove most of solvent, adding 500ml frozen diethyl ether to precipitate a large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow-drying gave fragment 15.50g of solid as fragment i (mr= 3039.63).

Preparation of fragment II:

Weighing 3eq Fmoc-amino acid, 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dripping 6eq DIEA, shaking to dissolve completely, adding the activated reaction solution into resin, adding N ₂ Bubbling reaction is carried out for 2 hours, reaction liquid is removed by suction filtration, resin is washed for six times by DMF, and ninhydrin detection is carried out after suction drying; wherein the amino acids used for fragment II coupling are in sequence: fmoc-Ala-OH, fmoc-Leu-OH, fmoc-Asn (Trt) -OH, fmoc-Asp (OtBu) -OH, fmoc-Leu-OH, fmoc-Ile-OH, fmoc-Thr (OtBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Met-OH, fmoc-Glu (OtBu))-OH,Fmoc-Asp(OtBu)-OH,Fmoc-Ser(OtBu)-OH,Fmoc-Phe-OH,Fmoc-Ser(OtBu)-OH,Fmoc-Gly-OH,Fmoc-Asp(OtBu)-OH,Fmoc-Gly-OH,Boc-His(Trt)-OH。

The peptide resin after coupling was washed three times with DMF and three more times with dichloromethane, dried and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are measured and mixed uniformly to obtain a lysate, and the lysate is added into peptide resin and reacted for 2 hours at room temperature. Filtering and collecting filtrate after the reaction is finished, washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging and collecting solid N ₂ Blow-dried to give 27.45g of solid as fragment ii (mr= 3155.47).

Liquid phase synthesis of tidolutetin:

3.0g of fragment I and 3.2g of fragment II are dissolved in 40ml of DMF, 0.63g of PyBOP and 0.30g of DIEA are added at a temperature, stirring is carried out at room temperature for 4h, after the reaction is monitored by HPLC, the reaction solution is added into water, a large amount of solid is separated out, the solid is collected by suction filtration and repeatedly washed by water, and DMF in the solid is removed. The sample was dried and used directly for the next reaction.

mu.L of ethanedithiol, 500. Mu.L of triisopropylsilane, 500. Mu.L of m-cresol and 500. Mu.L of water were aspirated, and TFA was finally added to 20ml, followed by mixing to obtain a lysate. After pre-freezing for 30min in a refrigerator, adding the crude fully protected peptide into the mixture, and stirring the mixture for 1h at room temperature. After the reaction, the reaction solution is concentrated under reduced pressure to remove most of the reaction solution, the residue is poured into pre-frozen 200ml diethyl ether, a large amount of solids are separated out, the solids are collected by centrifugation and then dried by N2, 3.77g of crude product is obtained, 2.52g of pure product is obtained after purification by RP-HPLC, and the yield is 67.2%.

Claims

1. A synthesis method of a C-terminal protected fragment of a peptide, comprising the steps of:

(1) Preparing Resin-T-P;

(2) Removing Resin by adopting a cracking reagent to obtain T-P;

wherein: resin is acid sensitive Resin or nucleophilic sensitive Resin, selected from: 2-CTC resin, trt-Cl resin, sieber resin, sasrin resin, dihydropyran resin, HMBA resin;

p is a peptide fragment;

t is selected from:，/>，/>，/>，/>，，/>，/>，。

2. the synthesis of a C-terminal protected fragment of a peptide according to claim 1, wherein the cleavage reagent of step (2) consists of B1 and B2, wherein B1 is selected from TFA, p-toluene sulfonic acid or THF; b2 is selected from methanol, dichloromethane and NH ₃ -one or more compounds in MeOH.

3. The method for synthesizing a C-terminal protected fragment of a peptide according to claim 2, wherein the cleavage reagent B1 in the step (2) is selected from TFA and p-toluenesulfonic acid, and the concentration thereof is 1 to 5% of the cleavage reagent.

4. Use of the C-terminal protection fragment of claim 1 in the synthesis of a polypeptide or polypeptide fragment.

5. The use of claim 4, wherein the polypeptide or polypeptide fragment is carboxy-terminated.

6. The use according to claim 4 or 5, wherein the polypeptide is selected from the group consisting of teriparatide, bivalirudin, somalundin, cinacacet and tidulcin.