CN114981285A

CN114981285A - Synthesis of C-terminal protected fragment of peptide

Info

Publication number: CN114981285A
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: 2022-08-30
Anticipated expiration: 2040-12-26
Also published as: WO2022134124A1; CN114981285B

Abstract

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

Description

Synthesis of C-terminal protected fragment of peptide

Technical Field

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

Background

The solid-phase polypeptide synthesis method (SPPS) is firstly proposed by R.BruceMerrifield in 1963, the synthesis is convenient and rapid, the reaction condition is mild, the SPPS can be rapidly and widely used, and more than 85 percent of polypeptide medicines 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 C to N, and finally cutting off the resin and the amino acid protecting group to obtain the target peptide product. The solid phase stepwise coupling method has a good effect on conventional peptides, however, when the peptide chain is long or contains more hydrophobic amino acids, fragment coupling methods including solid phase fragment coupling method and liquid phase fragment coupling method are often used.

CN108059666 reports a solid phase fragment coupling method for synthesizing the somaglutide, but all the peptide fragments used in the method need to be fed in 2-5 times of the substitution degree of the resin, which causes serious waste and thus results in high final synthesis cost. Due to such drawbacks, the conventional fragment condensation method is mostly performed in a liquid phase manner. In contrast, the solution phase fragment coupling method solves the problem of excess dosage rate well, but since peptide fragments are mostly prepared by the solid phase method, the terminal carboxyl group is usually in a naked state when the C-terminal peptide is cleaved off, and an additional protection step is required for the terminal carboxyl group in order to reduce side reactions of coupling between fragments. CN109627317 reports a strategy for synthesizing somaglutide by liquid phase fragment coupling, which finally obtains fully protected somaglutide by liquid phase combination using three fully protected peptide fragments. Cleavage of the protecting group is then performed using a lysis solution to yield the final product. When the C-terminal segment 3 is cut off from the resin, the peptide is protected at the N terminal, the carboxyl at the C terminal is in a naked state, in order to avoid the carboxyl participating in the reaction, the peptide is additionally protected at one step, and then the protecting group at the N terminal is removed, so that the component for segment condensation can be obtained. This strategy adds reaction steps and reduces synthesis efficiency.

Patent EP3205660A1 proposes a one-step preparation method of C-terminal protected peptide fragments, and the method is used for preparing liraglutide. The method utilizes a two-stage connecting arm strategy and a gradient cracking mode to prepare the C peptide fragment with the protected tail end in one step, uses the Wang series connecting arm as a secondary connecting arm, fixes the C peptide fragment on resin containing the acid-hypersensitive primary 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 super-sensitive resin is cracked, the full-protection peptide segment and the Wang secondary connecting arm are simultaneously released, and the target peptide segment with the protection at the tail end is directly obtained. However, the method has the main problems that as the secondary connecting arm adopts the wang connecting arm analogue which is of a benzyl structure, the fragment cannot be treated by catalytic hydrogenation, otherwise, the secondary connecting arm can be removed; at the same time, 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 to be tolerant to nucleophiles by adding an alkyl substituent at the benzyl position of the Wang linker arm to make the secondary linker arm a tertiary alcohol, the secondary linker arm is greatly increased in acid sensitivity and even cannot be distinguished from the primary linker arm. For example, 1-dimethylphenylethanol, which has a similar structure, requires only 2% concentration of TFA to effect cleavage rapidly, which is indistinguishable from first-order cleavage. Thus, the secondary linker arms referred to in this patent cannot achieve all of nucleophile stability, catalytic hydrogenation stability, and acid stability. When site-directed modification of a polypeptide is involved, protecting groups such as Dde, CBZ, Alloc and the like are usually used for forming special protection at a modification site, and nucleophilic reagents or catalytic hydrogenation are usually used for completing site-directed removal of the protecting groups, so that other protecting groups can be kept in a stable state, and the purpose of site-directed modification is achieved. Therefore, when the secondary linker arm is not tolerant to these reagents, the removal of the site-specific protecting group may also result in the removal of the terminal carboxyl protecting group, which affects the selectivity of the modification.

Disclosure of Invention

In order to solve the problems that long peptide synthesis is difficult, solid phase synthesis is easy to cause waste due to excessive feeding, solid-liquid combination synthesis steps are complex, synthesis efficiency is low, and the conventional synthesis method of the peptide fragment with the protection at the C terminal cannot obtain the stability of a nucleophilic reagent, the stability of catalytic hydrogenation, the stability of acid and the like at the same time, the invention provides a synthesis method of the C terminal protection fragment of the peptide, 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, T is selected from compounds with the following structure:

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 composed of 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, R3 are such that T has an aliphatic tertiary alcohol structure, the ester of which with a carboxylic acid is resistant to treatment with low-strength acids and nucleophilic reagents and is capable of breaking upon treatment with medium-high strength acids.

In some embodiments, T is preferably:

preferably, Resin is selected from: 2-chlorotrityl chloride resin, trityl chloride resin, 2-methoxytrityl chloride resin, Sieber resin, Ramage resin, Sasrin resin, dihydropyrane resin and HMBA resin.

Preferably, the cracking reagent in 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 halogenated hydrocarbons, tetrahydrofuran, cyclopentyl methyl ether, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, alcohols, silanes, water, thiols, and phenols. Wherein B1 is used as main component of cracking reagent and is selected from low-strength acid or nucleophilic reagent.

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

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

In some embodiments, the step (1) is specifically performed by: the tertiary alcohol of T reacts with C-terminal carboxyl of the peptide fragment P to obtain T-A, then reacts with Resin to obtain Resin-T-A, and is continuously coupled with protected amino acid or peptide fragment to obtain Resin-T-P. Wherein A is the C-terminal carboxyl component of P.

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

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

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

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

More particularly, the polypeptide is selected from teriparatide, bivalirudin, somaglutide, sinaputide, teduglutide.

The invention adopts a medium acid-sensitive connecting arm as a secondary connecting arm, and the medium acid-sensitive connecting arm reacts with acid-sensitive resin or nucleophilic-sensitive resin and Fmoc-AA-OH together 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 a strong acid or a nucleophilic reagent, and the protected peptide segment and the secondary connecting arm are released simultaneously as an integral molecule. By using the method, the C peptide protection fragment with the protecting group at the last generation can be directly released from the solid phase, and can be directly used for preparing longer polypeptide or peptide fragment by using a fragment condensation method. The method reduces the preparation steps, improves the synthesis yield and greatly reduces the racemization risk. Meanwhile, the protection fragment end obtained by the method can effectively resist the DKP side reaction and the treatment of a nucleophilic reagent, is stable in catalytic hydrogenation, is low in cost, is easy to control in reaction, and can be used for large-scale production.

Detailed Description

The technical solutions of the present invention will be described below with reference to specific embodiments, and the described embodiments are only a part of embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of teriparatide 1

The synthetic route for fragment I (T-P) is as follows:

remarking: PG represents a protecting group in all examples of the present invention.

Preparation of Resin-T:

10.00g of dihydropyrane resin (SD ═ 1.06mmol/g) was weighed into a reaction flask, 100ml of dichloromethane was added to soak and swell for 1h, the solvent was removed by suction filtration, and the mixture was resuspended in 100ml of dry DCM. 5.51g of 3-methyl-1, 3-butanediol (5eq) was weighed in and stirred at room temperature to dissolve it sufficiently. 0.1g of p-toluenesulfonic acid monohydrate was weighed and added, a drying tube was added to the mixture to react at room temperature for 1 hour, the reaction solution was removed by filtration, the mixture was washed three times with 5% DIEA-DCM solution, three times with DMF, and three times with DCM, and then the mixture was drained, and the mixture was dried in vacuum and weighed as 10.92g, the loading rate was 84.6%, and SD ═ 0.81 mmol/g.

Preparation of Resin-T-A1:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 26.5mmol of Fmoc-Phe-OH and 2.65mmol of DMAP were weighed out and dissolved thoroughly with stirring. Cooled to 0 ℃ in ice bath, and then 29.1mmol DIC was added dropwise. Stirring for 30min at low temperature, moving to room temperature and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM × 3, DMF × 3, and then subjected to end capping treatment.

And (3) measuring 20ml of acetic anhydride and 20ml of pyridine, adding 60ml of DMF, uniformly mixing, adding into the Resin in the step (2), reacting at room temperature for 1h, filtering to remove a reaction liquid after the reaction is finished, washing the Resin with DCM (multiplied by 3), DMF (multiplied by 3) and MeOH (multiplied by 3), and drying in vacuum to obtain Resin-T-A113.75g, wherein the SD (measured substitution degree) is 0.56mmol/g, and the total loading is 7.7 mmol.

Preparation of Resin-T-A2:

Resin-TResin-T was transferred to a reaction flask, suspended in 100mL DCM, and soaked for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 26.5mmol of Fmoc-Asn (Trt) -Phe-OH and 2.65mmol of DMAP were weighed out and dissolved well with stirring. Cooled to 0 ℃ in ice bath, and then 29.1mmol DIC was added dropwise. Stirring for 30min at low temperature, moving to room temperature and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 and then subjected to end capping treatment.

And (2) measuring 20ml of acetic anhydride and 20ml of pyridine, adding 60ml of DMF, uniformly mixing, adding the mixture into the Resin in the step (2), reacting at room temperature for 1h, filtering to remove a reaction liquid after the reaction is finished, washing the Resin with DCM (multiplied by 3), DMF (multiplied by 3) and MeOH (multiplied by 3), and drying in vacuum to obtain Resin-T-A215.78g, wherein the SD (substituted degree test) is 0.42mmol/g, and the total loading is 6.7 mmol.

Preparation of Resin-T-P:

weighing 23.1mmol Fmoc-amino acid and 3.74g HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 3.50g DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying;

wherein the amino acids used for coupling with Resin-T-A1 are sequentially: 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-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-Trt-His (Trt) -OH, Fmoc-Lys (Boc) -OH, and removing the terminal Fmoc protection after the reaction is finished.

The amino acids used for coupling with Resin-T-A2 are in the order: Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-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 (Fmoc) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Trt) -OH, and after the reaction is finished, removing the Fmoc protection at the tail end.

And (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete.

Weighing 150ml of 20% piperidine-DMF solution, adding to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Bubble reaction 10And min, filtering to remove reaction liquid, washing the Resin with DMF for six times, draining, and repeating the coupling step to obtain Resin-T-P after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 400mg of p-toluenesulfonic acid monohydrate is weighed, 40ml of methanol and 160ml of dichloromethane are added, after dissolution, the lysate is added to the resin in one step and cleaved for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect 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 ═ 1.06mmol) was weighed out and suspended in 150mL of DCM for swelling for 1 h. The solvent was filtered off and 150mL of DCM were added again. 31.8mmol of Fmoc-Gly-OH was weighed and added thereto, and stirred to dissolve it sufficiently. 160mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading of the substitution test was 12.3mmol, and the loading yield was 77.4%.

Weighing 36.9mmol Fmoc-amino acid and 11.8g TBTU, dissolving in 150ml DMF, cooling in ice bath, slowly dropping 9.5g DIEA, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: 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 was complete, terminal Fmoc protection was removed.

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

Weighing 150ml of 20% piperidine-DMF solution, adding to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are weighed and uniformly mixed to obtain lysate, and the lysate is added into peptide resin to react for 2 hours at room temperature. Filtering after the reaction is finished, collecting filtrate, washing the resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen ether to precipitate a large amount of solid, centrifuging, collecting the solid, and collecting N ₂ Blow drying gave 28.6g of solid as fragment II (Mr. 2323).

Preparation of teriparatide:

weighing 5.2g of the fragment I and 2.3g of the fragment II, dissolving in 100mL of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4h at room temperature, adding the reaction solution into water after the HPLC monitoring reaction is finished, separating out a large amount of solid, carrying out suction filtration, collecting the solid, repeatedly washing with water, and removing DMF in the solid. And drying the sample and directly using the dried sample for the next reaction.

1ml of ethanedithiol, 1ml of triisopropylsilane, 1ml of m-cresol and 1ml of water are sucked, and finally TFA is added to 50ml, and the mixture is uniformly mixed to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, pouring the reaction liquid into prefreezing 500ml of diethyl ether, separating out a large amount of solid, centrifugally collecting the solid, drying N2 by blowing to obtain crude product 4.13g, and purifying by RP-HPLC to obtain pure product 2.89g with the yield of 70.0%.

Example 2 preparation of teriparatide 2

The synthetic route for fragment I (T-P) is as follows:

preparation of T-A:

weighing 4.72g (40mmol) of beta-hydroxyisovaleric acid, dissolving in 50ml of dichloromethane, adding 7.74g of DIEA, cooling in an ice bath, slowly dropwise adding 5.20g of trimethylchlorosilane, continuing to react for 30min after the dropwise adding is finished, and keeping the reaction liquid for later use. Separately, 7.75g of Fmoc-Phe-OH was weighed, 50ml of DCM was added, a small amount of DMF was added to dissolve the mixture sufficiently, 4.53g of DCC was added under cooling in ice bath, and the mixture was reacted for 15min in ice bath. Adding the temporarily protected beta-hydroxy isoamyl acid solution, adding 24mg DMAP for catalysis, and reacting for 4 hours at room temperature. 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, evaporated to remove the solvent and purified by column chromatography to give the desired product 6.11g, 62.7% yield.

Preparation of Resin-T-P:

5.00g of Sieber resin (SD ═ 0.69mmol/g) is weighed, soaked in 50ml of DMF for swelling for 30min, and after the reaction is finished, the solution is removed by suction filtration, and deprotection treatment is carried out (step 6) for standby. Weighing 10.35mmol Fmoc-amino acid and 1.68g HOBt, dissolving in 50ml DMF, cooling in ice bath, slowly dropping 1.56g DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin for six times by using DMF (dimethyl formamide), and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: 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-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 removing the terminal Fmoc protection after the reaction is finished.

100ml of 20% piperidine-DMF solution are metered in and added to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying; obtaining Resin-T-P after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Measuring 2ml of TFA, adding dichloromethane to dilute to 200ml to obtain lysate, adding the lysate into resin, cleaving for 1h at room temperature, filtering after the reaction is finished, collecting filtrate, repeating cleavage once, and combining the filtrates. Adding 500ml of frozen ether into the mixture to separate out a large amount of solid, centrifuging and collecting the solid, and then carrying out N ₂ Blow drying gave fragment 17.8g of solid as fragment i (Mr. 5135).

Preparation of teriparatide:

5.4g of fragment I and 2.3g of fragment II from 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 4 hours, the reaction mixture was added to water after monitoring the completion of the reaction by HPLC, and a large amount of solid was precipitated, collected by suction filtration and washed repeatedly with water to remove DMF. And drying the sample and directly using the dried sample for 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 40ml, and a lysate was obtained after uniform mixing. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by decompression and concentration, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, N2 is blown to dry after the solid is collected by centrifugation, 4.13g of crude product is obtained, 2.67g of pure product is obtained after RP-HPLC purification, and the yield is 64.6%.

Example 3 preparation of bivalirudin 1

The synthetic route for fragment I (T-P) is as follows:

preparation of Resin-T:

10.00g of 2-CTC resin (SD ═ 1.17mmol/g) was weighed into a reaction flask, soaked in 100ml of dichloromethane for swelling for 1h, filtered off with suction, and resuspended in 100ml of dry DCM. 8.42g of the compound are weighed

(5eq) was added thereto, and the mixture was stirred at room temperature to be sufficiently dissolved. 30.2g DIEA (20eq) is weighed into the reaction kettle, a drying tube is added to the reaction kettle to react for 48 hours at room temperature, the reaction solution is removed by filtration, the reaction solution is washed three times by DCM solution, washed three times by DMF, washed three times by DCM and then drained, and the reaction solution is weighed as 10.94g after vacuum drying, the theoretical weight is 11.26g, and the load factor is 74.6%. SD ═ 0.80mmol/g, total loading 8.7 mmol. Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 10ml of trichloroacetonitrile was added thereto, and 1ml of DBU was further added thereto for activation. The mixed solution was reacted at room temperature for 4h, filtered to remove the solvent, washed three times with DCM and resuspended in 100ml of dry DCM. 26.1mmol of Fmoc-Leu-OH was weighed and added thereto, and 100. mu.L of BF was dropped ₃ ·Et ₂ And catalyzing by O, reacting for 6 hours at room temperature, filtering to remove reaction liquid after the addition reaction is finished, washing the resin by DCM x 3 and DMF x 3, and carrying out end capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed and added into 60ml of DMF, the mixture is uniformly mixed and added into the resin in the step 2, 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 x 3, DMF x 3 and MeOH x 3, and the resin is dried in vacuum to obtain 13.20g, the substitution degree test SD is 0.51mmol/g, and the total loading is 6.7 mmol.

Preparation of Resin-T-P:

weighing 20.0mmol Fmoc-amino acid and 2.97g HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 2.77g DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin for six times by using DMF (dimethyl formamide), and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Tyr (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, and removing terminal Fmoc protection after the reaction is finished.

Weighing 150ml of 20% piperidine-DMF solution, adding to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the Resin with DMF for six times, pumping to dry, and repeating the coupling step to obtain Resin-T-P after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Measuring 3ml of TFA, adding 297ml of dichloromethane, uniformly mixing to obtain a lysate, adding the lysate to the resin at one time, and performing lysis for 1 h. After the reaction is finished, filtering and collecting filtrate, wherein the weight of the resin is heavyAnd (4) cracking once again, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain segment I (Mr 1732) as segment 11.4g solid.

The synthetic route for fragment ii is as follows:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM and soaked for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Gly-OH was weighed and added thereto, and stirred to dissolve it sufficiently. 88mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading of the substitution test was 7.7mmol, and the loading yield was 65.8%.

Weighing 23.1mmol Fmoc-amino acid and 7.40g TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6.00g DIEA, shaking for dissolving completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Gly-OH, Fmoc-Asn (Trt) -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 are metered in and added to the resin, N ₂ Bubbling deviceReacting for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

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

Preparing bivalirudin:

weighing 1.7g of the fragment I and 1.5g of the fragment II, dissolving in 40mL of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4h at room temperature, monitoring the reaction by HPLC, adding the reaction solution into water after the reaction is completed, precipitating a large amount of solid, filtering by suction, collecting the solid, repeatedly washing by water, and removing DMF. And drying the sample and directly using the sample for the next reaction.

500. 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 added to 20ml, and the mixture was uniformly mixed to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, 2.26g of crude product is obtained, and the pure product is obtained after purification by RP-HPLC, wherein the yield is 63.7%.

Example 4 preparation of bivalirudin 2

The synthetic route for fragment I (T-P) is as follows:

preparation of Resin-T:

weighing 10.00g of Sasrin resin (SD is 0.80mmol/g), placing the Sasrin resin in a reaction bottle, adding 100ml of THF, soaking for 1h, filtering to remove the solvent by suction, resuspending in 100ml of dried THF, weighing 2.7g of potassium tert-butoxide, dissolving in 100ml of dried THF, adding the resin, reacting for 1h at room temperature, filtering to remove the reaction liquid by suction, and washing the resin with dried THF for one time for standby. 2.60g of 1-chloro-2-methyl-2-propanol (3eq) was dissolved in 20ml of dry THF and slowly dropped thereto with stirring at room temperature. After the dropwise addition, the reaction mixture was reacted at room temperature over water for 24 hours, filtered to remove the reaction solution, washed three times with THF, washed three times with 5% HOAc-DMF solution, washed three times with DCM, drained, and dried under vacuum to weigh 10.52g, theoretically 10.58g, and the loading rate was 89.7%. SD ═ 0.68mmol/g, total loading 7.2 mmol.

Preparation of Resin-T-P:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 21.6mmol of Fmoc-Leu-OH and 2.16mmol of DMAP were weighed and dissolved thoroughly with stirring. Cooled to 0 ℃ in ice bath, and 23.7mmol DIC was added dropwise slowly. Stirring for 30min at low temperature, moving to room temperature and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 and then subjected to end capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed and added into 60ml of DMF, the mixture is uniformly mixed and added into the resin in the step 2, 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 x 3, DMF x 3 and MeOH x 3, and the resin is dried in vacuum to obtain 12.50g, the substitution degree test SD is 0.47mmol/g, and the total loading is 5.9 mmol.

Weighing 17.7mmol Fmoc-amino acid and 2.63g HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 2.45g DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Tyr (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OHAnd OH, removing terminal Fmoc protection after the reaction is finished.

150ml of 20% piperidine-DMF solution was measured and added to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

preparation of fragment I:

the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Measuring 3ml of TFA, adding 297ml of dichloromethane, uniformly mixing to obtain a lysate, adding the lysate to the resin at one time, and performing lysis for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain segment I (Mr 1732) as segment 11.4g solid. Liquid-phase synthesis of bivalirudin:

1.7g of fragment I and 1.5g of fragment II from 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 4 hours, the reaction mixture was added to water after monitoring the completion of the reaction by HPLC, and a large amount of solid was precipitated, collected by suction filtration and washed repeatedly with water to remove DMF. And drying the sample and directly using the dried sample for the next reaction.

Example 5 preparation of Somarunotide 1

The synthetic route for fragment I (T-P) is as follows:

preparation of T:

weighing 11.8g of beta-hydroxyisovaleric acid, adding 12.7g of HOSu, mixing, dissolving in 100ml of DCM, adding 2.26g of DCC under ice-bath cooling, reacting for 30min, moving to room temperature, adding 10.5g of diglycolamine thereto at one time, and reacting for 12h at room temperature. After the reaction is finished, the filtrate is collected by suction filtration, the filter cake is washed by DCM and then is combined with the filtrate, the organic phase is washed by citric acid solution, NaHCO3 solution and saturated sodium chloride solution for three times respectively, and after being dried by anhydrous MgSO4, the solvent is evaporated to obtain 20.2g of light yellow oily substance, the yield is almost quantitative, and the light yellow oily substance can be directly used for the next reaction without purification.

Preparation of Resin-T-A:

weighing 10.25g (50mmol)

Dissolving in 150ml dichloromethane, adding 9.67g DIEA, cooling in ice bath, slowly dropping 6.50g trimethylchlorosilane, continuing to react for 30min after dropping is finished, and obtaining the reaction solution for later use. Separately, 7.43g Fmoc-Gly-OH (25mmol) was weighed, 150ml DCM was added, a small amount of DMF was added to dissolve it sufficiently, 6.18g DCC was added under cooling in ice bath, and the reaction was carried out in ice bath for 15 min. Will temporarilyThe protected T solution was added, and 30mg DMAP was added to react 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, evaporated to remove the solvent, and purified by column chromatography to give the desired product 9.71g, 75.8% yield.

Synthesis of Resin-T-A:

5.00g of dihydropyrane resin (SD ═ 1.06mmol/g) is weighed into a reaction flask, 100ml of 1, 2-dichloroethane is added and soaked for 1 hour, the solvent is removed by suction filtration, and the mixture is resuspended in 100ml of 1, 2-dichloroethane. 8.15g T-A (3eq) was weighed in and stirred at room temperature to dissolve it sufficiently. Weighing 0.15g of pyridinium p-toluenesulfonate, adding a drying tube for reflux reaction for 6 hours, filtering to remove reaction liquid, washing with DCM solution for three times, washing with DMF for three times, washing with DCM for three times, draining, weighing 6.77g after vacuum drying, and having a load rate of 65.1%. SD was 0.51mmol/g, and the total loading was 3.4 mmol.

Preparation of Resin-T-P:

the Resin-T-A obtained in the above step was suspended in 100ml of DMF and soaked for swelling, followed by deprotection treatment and direct use in the peptide coupling step. Weighing 10.0mmol Fmoc-amino acid and 3.21g TBTU, dissolving in 70ml DMF, slowly dropping 2.60g DIEA at room temperature, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: 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, and the terminal Fmoc group is retained after the coupling is completed.

And (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is completed.

100ml of 20% piperidine-DMF solution was weighed out and added to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

100mL of DCM was added, 10mL of phenylsilane was added, the reaction was carried out for 3min, and 2.2g of Pd (PPh) was added ₃ ) ₄ And reacting for 45min at room temperature, pumping out the reaction liquid, and detecting the ninhydrin to be positive, wherein the result shows that Alloc is removed. Fmoc-AEEA-OH, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester are coupled in sequence according to standard coupling steps, after the reaction is finished, the terminal Fmoc is removed by DBLK, then the step of cracking is carried 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 times with dichloromethane, drained and transferred to a round bottom flask. 400mg of p-toluenesulfonic acid monohydrate is weighed, 40ml of methanol and 160ml of dichloromethane are added, after dissolution, the lysate is added to the resin in one step and cleaved for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, concentrating under reduced pressure to 1/2 volume, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain segment 11.33g of solid, which is segment I (Mr is 3333.18)

The synthetic routes for fragments II and C are as follows:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Gly-OH (for preparation of fragment II) or Fmoc-Ser (OtBu) -OH (for preparation of fragment C) were weighed in and stirred to dissolve it sufficiently. 88mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading was 7.7mmol or 7.1mmol, and the loading yield was 65.8% (Fmoc-Gly-OH) or 60.7% (Fmoc-Ser (OtBu) -OH).

Weighing 3eq Fmoc-amino acid and 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6eq DIEA, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling the fragment II are sequentially as follows: 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 acids used for coupling the fragment C are 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 are metered in and added to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are weighed and uniformly mixed to obtain lysate, and the lysate is added into peptide resin to react for 2 hours at room temperature. After the reaction, the filtrate was collected by suction filtration, and the resin was washed with methylene chlorideMixing with the filtrate after three times, adding vacuum concentrating to remove most solvent, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain 10.62g (or 10.15g) of solid as segment II (or segment C) (Mr: 1371/1430).

Liquid-phase synthesis of somaltulin:

weighing 3.4g of the fragment I and 1.4g of the fragment II, dissolving in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4h at room temperature, adding reaction liquid into water after the HPLC monitoring reaction is finished, separating out a large amount of solid, carrying out suction filtration, collecting the solid, repeatedly washing with water, and removing DMF in the solid. The sample is dried and dissolved in 30ml of 20% DBLK solution, stirred for 30min at room temperature, added with 200ml of ether to precipitate solid, centrifuged to collect the solid, washed twice with ether, and dried for the next coupling.

And (3) dissolving the AB full-protection peptide obtained in the previous step and 1.5g of the fragment C in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4 hours at room temperature, adding reaction liquid into water after HPLC monitoring reaction is finished, precipitating a large amount of solid, carrying out suction filtration to collect the solid, repeatedly washing with water, and removing DMF in the solid. The sample is dried and then directly used 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 added to 20ml, followed by uniform mixing to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, 4.30g of crude product is obtained, and 2.52g of pure product is obtained after RP-HPLC purification, and the yield is 58.6%.

Example 6 preparation of Somarunotide 2

The synthetic route for fragment I (T-P) is as follows:

preparation of Resin-T:

10.00g of Trt-Cl resin (SD ═ 1.06mmol/g) was weighed into a reaction flask, 100ml of dichloromethane was added to the flask to swell for 1 hour, the solvent was removed by suction filtration, and the mixture was resuspended in 100ml of dry DCM. Weighing 12.52g

(5eq) was added thereto, and the mixture was stirred at room temperature to be sufficiently dissolved. 27.3g DIEA (20eq) was weighed into it, a drying tube was added to react for 48h at room temperature, the reaction solution was removed by filtration, washed three times with DCM solution, three times with DMF, three times with DCM and then with suction dried, 11.64g was weighed after vacuum drying, 12.12g theoretically, load factor 77.4%. SD-0.80 mmol/g, total loading 8.2 mmol.

Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 24.6mmol Fmoc-Gly-OH and 2.46mmol DMAP were weighed and stirred to dissolve them sufficiently. The mixture was cooled to 0 ℃ in an ice bath, and 27.1mmol DIC was added dropwise slowly. Stirring at low temperature for 30min, moving to room temperature, and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM × 3, DMF × 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 uniformly mixed and added into the resin in the step 2, the reaction is carried out for 1h at room temperature, after the reaction is finished, the reaction liquid is removed by suction filtration, the resin is washed by DCM x 3, DMF x 3 and MeOH x 3, and after vacuum drying, 13.94g of resin is obtained, the substitution degree test SD is 0.59mmol/g, the total loading is 8.2mmol, and the reaction is considered to be quantitative. Preparation of Resin-T-P:

weighing 10.0mmol Fmoc-amino acid and 3.21g TBTU, dissolving in 70ml DMF, slowly dropping 2.60g DIEA at room temperature, shaking to dissolve completely, adding activated reaction solution into resin, N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: 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 the coupling is complete.

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 400mg of p-toluenesulfonic acid monohydrate is weighed, 40ml of methanol and 160ml of dichloromethane are added, after dissolution, the lysate is added to the resin at a time and is cracked for 1 hour. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, concentrating under reduced pressure to 1/2 volume, adding 500ml of frozen diethyl ether to precipitate a large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying gave fragment 27.3g of solid as fragment i (Mr. 3333.18).

Liquid-phase synthesis of somaltulin:

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

The fully protected 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 room temperature, and stirred at room temperature for 4 hours, after the reaction was monitored by HPLC, the reaction solution was added to water, and a large amount of solid was precipitated, and the solid was collected by suction filtration and washed with water repeatedly to remove DMF therefrom. The sample is dried and then directly used for the next reaction.

500. 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 added to 20ml, and the mixture was uniformly mixed to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by decompression and concentration, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, N2 is blown to dry after the solid is collected by centrifugation, 4.37g of crude product is obtained, 2.74g of pure product is obtained after RP-HPLC purification, and the yield is 62.7%.

Example 7 preparation of Sinapsin 1

The synthetic route for fragment I (T-P) is as follows:

synthesis of Resin-T:

weighing 10.00g of Sasrin resin (SD ═ 0.80mmol/g) and placing in a reaction bottle, adding 100ml of THF, soaking for 1h, filtering to remove the solvent by suction, resuspending in 100ml of dried THF, weighing 2.7g of potassium tert-butoxide, dissolving in 100ml of dried THF, adding into the resin, reacting for 1h at room temperature, filtering to remove the reaction solution by suction, and washing the resin once with dried THF for standby. 4.71g of Compound T (3eq) was dissolved in 20ml of dry THF and added dropwise slowly with stirring at room temperature. After the dropwise addition, the reaction mixture was reacted at room temperature over water for 24 hours, filtered to remove the reaction solution, washed with THF three times, washed with 5% HOAc-DMF three times, washed with DCM three times, drained, dried under vacuum, weighed to 10.80g, and the load ratio was quantitative. SD ═ 0.74mmol/g, total loading 8.0 mmol. Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 24.0mmol of Fmoc-Lys (Boc) -OH, 2.4mmol of DMAP were weighed and dissolved well with stirring. The mixture was cooled to 0 ℃ in an ice bath, and 26.4mmol DIC was added dropwise slowly. Stirring at low temperature for 30min, moving to room temperature, and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 and then subjected to end capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed and added into 60ml of DMF, the mixture is uniformly mixed and added into the resin in the step 2, 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 x 3, DMF x 3 and MeOH x 3, and the resin is dried in vacuum to obtain 13.80g, the substitution degree test SD is 0.48mmol/g, and the total loading is 6.7 mmol.

Preparation of Resin-T-P:

weighing 20.0mmol Fmoc-amino acid and 22.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 22.0mmol DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, and removing the terminal Fmoc protection after the reaction is finished.

Measuring 150ml of 20% piperazinepyridine-DMF solution, added to the resin, N ₂ Bubbling for 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into the resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the Resin with DMF for six times, pumping to dry, and repeating the coupling step to obtain Resin-T-P after the coupling is finished.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Measuring 3ml of TFA, adding 297ml of dichloromethane, uniformly mixing to obtain a lysate, adding the lysate to the resin at one time, and performing lysis for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying gave fragment 13.7g of solid as fragment i (Mr. 1708.33).

Preparation of fragment II:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM and soaked for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Lys (Boc) -OH was weighed and added thereto, and stirred to dissolve it sufficiently. 58.5mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading of the substitution test was 7.4mmol, and the loading yield was 63.2%.

Weighing 3eq Fmoc-amino acid and 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6eq DIEA, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Bubbling for 2h, removing reaction liquid by suction filtration, washing the resin with DMF for six times, and carrying out ninhydrin after suction dryingDetecting; wherein the amino acids used for coupling the fragment II are sequentially as follows: 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 coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are weighed and uniformly mixed to obtain a lysate, and the lysate is added into the peptide resin and reacts for 2 hours at room temperature. Filtering after the reaction is finished, collecting filtrate, washing the resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen ether to precipitate a large amount of solid, centrifuging, collecting the solid, and collecting N ₂ Blow drying to obtain 10.95g of solid as fragment II (Mr ═ 1480).

Liquid phase synthesis of sinapultide:

dissolving 1.7g of the fragment I and 1.5g of the fragment II in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4h at room temperature, adding the reaction solution into water after the reaction is monitored by HPLC, precipitating a large amount of solid, collecting the solid by suction filtration, repeatedly washing with water, and removing DMF in the solid. The sample is dried and then directly used for the next reaction.

500. 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 added to 20ml, and the mixture was uniformly mixed to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, 2.55g of crude product is obtained, and the pure product is obtained after purification by RP-HPLC, wherein the yield is 67.5%.

Example 8 preparation of Sinapsin 2

The synthetic route for fragment I (T-P) is as follows:

synthesis of Resin-T:

weighing 10.00g of Sasrin resin (SD ═ 0.80mmol/g) and placing in a reaction bottle, adding 100ml of THF, soaking for 1h, filtering to remove the solvent by suction, resuspending in 100ml of dried THF, weighing 2.7g of potassium tert-butoxide, dissolving in 100ml of dried THF, adding into the resin, reacting for 1h at room temperature, filtering to remove the reaction solution by suction, and washing the resin once with dried THF for standby. 6.54g of Compound T are weighed out

(3eq) was dissolved in 20ml of dry THF and added dropwise slowly with stirring at room temperature. After the dropwise addition, the reaction mixture was reacted at room temperature over water for 24 hours, filtered to remove the reaction solution, washed with THF three times, washed with 5% HOAc-DMF three times, washed with DCM three times, drained, dried under vacuum, weighed to 10.76g, and the load ratio was quantitative. SD ═ 0.71mmol/g, total loading 7.6 mmol.

Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 22.8mmol of Fmoc-Lys (Boc) -OH, 2.3mmol of DMAP were weighed out and dissolved well with stirring. Cooled to 0 ℃ in ice bath, and then gradually added dropwise with 25.1mmol DIC. Stirring for 30min at low temperature, moving to room temperature and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM × 3, DMF × 3, and then subjected to end capping treatment.

20ml of acetic anhydride and 20ml of pyridine are weighed and added into 60ml of DMF, the mixture is uniformly mixed and added into the resin in the step 2, 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 x 3, DMF x 3 and MeOH x 3, and the resin is dried in vacuum to obtain 13.78g, the substitution degree test SD is 0.48mmol/g, and the total loading is 6.7 mmol.

Preparation of Resin-T-P:

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Measuring 3ml of TFA, adding 297ml of dichloromethane, uniformly mixing to obtain a lysate, adding the lysate to the resin at one time, and performing lysis for 1 h. After the reaction is finished, filtering and collecting filtrate, wherein the weight of the resin is heavyAnd (4) cracking once again, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml of frozen diethyl ether, precipitating large amount of solid, centrifuging, collecting solid, and collecting N ₂ Blow drying gave fragment 11.4g of solid as fragment i (Mr. 1708.33).

Preparation of fragment II:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM and soaked for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Lys (Boc) -OH was weighed and added thereto, and stirred to dissolve it sufficiently. 58.5mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total amount of the substitution test load was 7.4mmol, and the yield was 63.2%.

Weighing 3eq Fmoc-amino acid and 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6eq DIEA, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin for six times by using DMF (dimethyl formamide), and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling the fragment II are sequentially as follows: 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.

Weighing 150ml of 20% piperidine-DMF solution, adding to the resin, N ₂ Bubbling reaction 5min, filtering to remove the reaction solution, adding 150ml 20% piperidine-DMF solution again, adding into resin, N ₂ Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

Liquid phase synthesis of sinapultide:

dissolving 1.7g of the fragment I and 1.5g of the fragment II in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4 hours at room temperature, monitoring the reaction by HPLC, adding the reaction solution into water after the reaction is completed, precipitating a large amount of solid, collecting the solid by suction filtration, repeatedly washing with water, and removing the DMF. The sample is dried and then directly used 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 added to 20ml, followed by uniform mixing to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, 2.55g of crude product is obtained, and the pure product is obtained after purification by RP-HPLC, wherein the yield is 67.5%.

Example 9 preparation of teduglutide 1

The synthetic route for fragment I (T-P) is as follows:

synthesis of Resin-T:

weighing 10.00g of Sasrin resin (SD ═ 0.80mmol/g) and placing in a reaction bottle, adding 100ml of THF, soaking for 1h, filtering to remove the solvent by suction, resuspending in 100ml of dried THF, weighing 2.70g of potassium tert-butoxide, dissolving in 100ml of dried THF, adding into the resin, reacting for 1h at room temperature, filtering to remove the reaction solution by suction, and washing the resin once with dried THF for standby. 6.54g of

(3eq) was dissolved in 20ml of dry THF and added dropwise with stirring at room temperature. After the dropwise addition, the reaction mixture was reacted at room temperature over water for 24 hours, filtered to remove the reaction solution, washed with THF three times, washed with 5% HOAc-DMF three times, washed with DCM three times, drained, dried under vacuum, weighed to 11.04g, and the load ratio was quantitative. SD ═ 0.72mmol/g, total loading 8.0 mmol.

Preparation of Resin-T-A:

Resin-T was transferred to a reaction flask, suspended in 100mL DCM and swelled for 1 h. The solvent was filtered off and 100mL of DCM were added again. 24.0mmol of Fmoc-Lys (Boc) -OH and 2.4mmol of DMAP were weighed and stirred to dissolve sufficiently. The mixture was cooled to 0 ℃ in an ice bath, and 26.4mmol DIC was added dropwise slowly. Stirring for 30min at low temperature, moving to room temperature and continuing to react for 8 h. After the reaction, the reaction solution was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 and then subjected to end capping treatment.

Weighing 20ml of acetic anhydride and 20ml of pyridine, adding 60ml of DMF, uniformly mixing, adding into the resin in the step 2, reacting at room temperature for 1h, filtering to remove the reaction liquid by suction after the reaction is finished, washing the resin by DCM × 3, DMF × 3 and MeOH × 3, and drying in vacuum to obtain 13.83g of resin, wherein the substitution degree test SD is 0.51mmol/g, and the total loading is 7.1 mmol.

Preparation of Resin-T-P:

weighing 21.0mmol Fmoc-amino acid and 23.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 23.0mmol DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Bubbling reaction for 2h, suction-filtering to remove reaction liquid, washing the resin with DMF for six times, and suction-dryingNinhydrin detection is carried out; wherein the amino acids used for coupling are in sequence: 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 after the reaction is finished, removing the Fmoc protection at the tail end.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with methylene chloride, drained and transferred to a round bottom flask. Measuring 3ml of TFA, adding 297ml of dichloromethane, uniformly mixing to obtain a lysate, adding the lysate to the resin at one time, and performing lysis for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain 20.56g of solid as fragment I (Mr: 2895.63).

Preparation of fragment II:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM and soaked for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Ala-OH was weighed in and stirred to dissolve it sufficiently. 58.5mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading of the substitution test is 8.7mmol, and the loading yield is 74.4%.

Weighing 3eq Fmoc-amino acid and 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6eq DIEA, shaking to dissolve completely, adding activated reaction solution into resin, N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin for six times by using DMF (dimethyl formamide), and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling the fragment II are sequentially as follows: 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 coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Weighing 40ml of trifluoroethanol and 160ml of dichloromethane, uniformly mixing to obtain lysate, and adding the lysate to the peptideThe reaction was carried out for 2 hours at room temperature in the resin. Filtering after the reaction is finished, collecting filtrate, washing the resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen ether to precipitate a large amount of solid, centrifuging, collecting the solid, and collecting N ₂ Blow drying gave 27.45g of solid as fragment II (Mr. 3155.47).

Liquid phase synthesis of teduglutide:

dissolving 2.9g of the fragment I and 3.2g of the fragment II in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4h at room temperature, adding the reaction solution into water after the reaction is monitored by HPLC, precipitating a large amount of solid, collecting the solid by suction filtration, repeatedly washing with water, and removing DMF in the solid. The sample is dried and then directly used for the next reaction.

500. 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 added to 20ml, and the mixture was uniformly mixed to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, crude product 3.83g is obtained, and pure product 2.61g is obtained after RP-HPLC purification, and the yield is 69.6%.

Example 10 preparation of teduglutide 2

The synthetic route for fragment I (T-P) is as follows:

weighing 10.25g (50mmol)

Dissolved in 150ml of dichloromethaneAdding 9.67g of DIEA, slowly dripping 6.50g of trimethylchlorosilane after the ice bath is properly cooled, continuing to react for 30min after the dripping is finished, and keeping the reaction liquid for later use. In addition, 25mmol of Fmoc-Asp (OtBu) -OH was weighed, 150ml of DCM was added, a small amount of DMF was added to dissolve it sufficiently, 6.18g of DCC was added under cooling in ice bath, and the reaction was carried out in ice bath for 15 min. The temporarily protected T solution was added to the reaction mixture, and 30mg of DMAP was added thereto to perform a reaction 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, evaporated to remove the solvent, and purified by column chromatography to give the desired product 9.71g, 75.8% yield.

Synthesis of Resin-T-A:

5.00g of HMBA resin (SD ═ 1.02mmol/g) was weighed into a reaction flask, 100ml of DCM was added and soaked for swelling for 1h, the solvent was removed by suction filtration and resuspended in 100ml of DCM. 15.3mmol of T-A (3eq) was added thereto, stirred at room temperature to dissolve it sufficiently, and then transferred to an ice bath to cool it sufficiently. Weighing 16.8mmol DIC, adding into the mixture in batches, adding 1.7mmol DMAP for catalytic reaction, adding a drying tube for reacting at low temperature for 30min, moving to room temperature, and stirring overnight. After the reaction is finished, the reaction solution is removed, washed by DCM solution for three times, washed by DMF for three times, washed by DCM for three times, drained, dried in vacuum, weighed to be 5.52g, Resin-T-A is obtained, and the reaction is quantitative. SD was 0.92mmol/g, and the total loading was 5.1 mmol.

Synthesis of Resin-T-P:

weighing 15.0mmol Fmoc-amino acid and 17.0mmol HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 17.0mmol DIC, activating for 15min, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: 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 after the reaction is finished, removing the Fmoc protection at the tail end.

Preparation of fragment I:

Resin-T-P was washed three times with DMF and three times with THF, drained and transferred to a round bottom flask. 80ml of THF were weighed into the resin, and 40ml of 4M NH were added ₃ Cleavage with MeOH for 1 h. After the reaction is finished, filtering and collecting filtrate, repeatedly cracking the resin once, and combining the two lysates. Washing resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentrating to remove most solvent, adding 500ml frozen diethyl ether to precipitate large amount of solid, centrifuging to collect solid, and collecting N ₂ Blow drying to obtain 15.50g of solid as fragment I (Mr: 3039.63).

Preparation of fragment II:

10g of 2-CTC resin (SD ═ 1.17mmol) was weighed out and suspended in 100mL of DCM for swelling for 1 h. The solvent was filtered off and 100mL of DCM were added again. 17.5mmol of Fmoc-Ala-OH was weighed and added thereto, and stirred to dissolve it sufficiently. 58.5mmol DIEA was added and the reaction was carried out at room temperature for 2 h. After the reaction is finished, 20ml of methanol is added for end capping, and the reaction is carried out for 1 h. After the reaction, the reaction mixture was removed by suction filtration, and the resin was washed with DCM X3 and DMF X3 for coupling. The total loading of the substitution test was 8.7mmol, and the loading yield was 74.4%.

Weighing 3eq Fmoc-amino acid and 3eq TBTU, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 6eq DIEA, shaking to dissolve completely, adding activated reaction solution into resin, and adding N ₂ Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling the fragment II are sequentially as follows: 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 coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 40ml of trifluoroethanol and 160ml of dichloromethane are weighed and uniformly mixed to obtain lysate, and the lysate is added into peptide resin to react for 2 hours at room temperature. Filtering after the reaction is finished, collecting filtrate, washing the resin with dichloromethane for three times, mixing with the filtrate, adding vacuum concentration to remove most of solvent, adding 500ml of frozen ether to precipitate a large amount of solid, centrifuging, collecting the solid, and collecting N ₂ Blow drying gave 27.45g of solid as fragment II (Mr. 3155.47).

Liquid phase synthesis of teduglutide:

dissolving 3.0g of the fragment I and 3.2g of the fragment II in 40ml of DMF, adding 0.63g of PyBOP and 0.30g of DIEA at room temperature, stirring for 4 hours at room temperature, monitoring the reaction by HPLC, adding the reaction solution into water after the reaction is completed, precipitating a large amount of solid, collecting the solid by suction filtration, repeatedly washing with water, and removing the DMF. The sample is dried and then directly used 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 added to 20ml, followed by uniform mixing to obtain a lysate. Placing in a refrigerator for pre-freezing for 30min, adding the crude full-protection peptide, and stirring at room temperature for 1 h. After the reaction is finished, most reaction liquid is removed by concentration under reduced pressure, the residue is poured into prefreezed 200ml of diethyl ether, a large amount of solid is separated out, the solid is collected by centrifugation and dried by N2, crude product 3.77g is obtained, and pure product 2.52g is obtained after RP-HPLC purification, and the yield is 67.2%.

Claims

A method for synthesizing 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, P is peptide fragment, 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 respectively composed of one or more of aliphatic hydrocarbon groups, ether bonds, amide bonds and ester bonds.
The method for synthesizing a C-terminal protected fragment of a peptide according to claim 1, wherein T is selected from the group consisting of:
the method for synthesizing a C-terminal protected fragment of a peptide according to claim 1, wherein Resin is selected from the group consisting of: 2-chlorotrityl chloride resin, trityl chloride resin, 2-methoxytrityl chloride resin, Sieber resin, Ramage resin, Sasrin resin, dihydropyrane resin and HMBA resin.
The method for synthesizing C-terminal protected fragment of peptide according to claim 1, wherein said cleavage reagent in step (2) is composed of B1 and B2, wherein B1 is selected from one or more compounds selected from TFA, p-toluenesulfonic acid, pyridinium p-toluenesulfonic acid, RNH2, and B2 is selected from one or more compounds selected from halogenated hydrocarbons, tetrahydrofuran, cyclopentyl methyl ether, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, alcohols, silanes, water, thiols, and phenols.
The method for synthesizing C-terminal protected fragment of peptide according to claim 4, wherein said cleavage reagent B1 in step (2) is selected from TFA, p-toluenesulfonic acid, and pyridinium p-toluenesulfonate, and its concentration is 1-5% of the cleavage reagent.
The method for synthesizing C-terminal protected fragment of peptide according to claim 1, wherein the specific process of step (1) is: reacting the X group of the T with Resin to synthesize Resin-T, reacting the Resin-T with the C-terminal carboxyl of the peptide fragment P to obtain Resin-T-A, and continuously coupling and protecting the amino acid or the peptide fragment to obtain Resin-T-P;

wherein A is the C-terminal carboxyl component of P.
The method for synthesizing C-terminal protected fragment of peptide according to claim 1, wherein the specific process of step (1) is: 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 continuously coupling and protecting amino acid or peptide fragment to obtain Resin-T-P;

wherein A is the C-terminal carboxyl component of P.
Use of the C-terminal protective fragment of claim 1 in the synthesis of a polypeptide or polypeptide fragment.
The use of claim 8, wherein the polypeptide or polypeptide fragment terminates with a carboxyl group.
Use according to claim 8 or 9, characterized in that said polypeptide is selected from teriparatide, bivalirudin, somaglutide, sinapultide, teduglutide.