CN115873073A - Preparation method of procatide - Google Patents

Abstract

The invention discloses a preparation method of proca peptide in the field of polypeptide drug preparation, which comprises the steps of coupling 4-hydroxybenzyl alcohol and Fmoc-Leu-OH by taking 2-CTC resin as a solid phase carrier to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin; fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is used as a solid phase carrier, fmoc-Cys (Acm) -OH and 8 peptide fragment B are sequentially coupled by an Fmoc solid phase synthesis method, and 10 peptide fragment C is obtained after Fmoc protection is removed; coupling the 6 peptide fragment A with the 10 peptide fragment C to obtain a peptide fragment D; and (3) the peptide fragment D is subjected to cleavage and two-step oxidation to obtain the crude peptide of the procapsipeptide. The invention adopts a solid-liquid combination method, skillfully utilizes the acid sensitivity difference of Wang resin and 2-CTC resin to prepare Fmoc-Leu-Linker-2-CTC resin as a solid phase carrier, thereby easily obtaining the 10 peptide fragment C protected by the C end. Meanwhile, the problem that in the prior art, when fragment C is synthesized in a liquid phase, fmoc protective reagents including piperidine and triethylamine are removed or a byproduct of fluorenes after Fmoc protection is removed is difficult to remove is solved, the subsequent side reaction caused by residue of the Fmoc protective reagents is avoided, and the purity and the yield of the crude peptide are improved.

Description

Preparation method of procatide

Technical Field

The invention belongs to the field of preparation of polypeptide medicines, and particularly relates to a preparation method of procainatide.

Background

Procatide (plectanatide) is an oral cyclic polypeptide consisting of 16 amino acids, which contains two disulfide bonds in the molecule and is an analog of Uroguanylin (Uroguanylin), the difference between the procatide being Glu at position 3 and Asp at position 3. The composition has the effects of promoting the activation of guanylate cyclase receptor for promoting natriuresis, regulating acid and alkali ions in gastrointestinal tract, inducing liquid to transfer into gastrointestinal tract, and increasing gastrointestinal peristalsis. The peptide plecanatide was originally developed by Callisto pharmaceutical, USA, and later developed and marketed by Synergy pharmaceutical, and is suitable for treating adult chronic idiopathic constipation. The peptide sequence is as follows:

the preparation method of original research patent CN103764672A adopts a fragment liquid phase synthesis method, 16 peptide is split into three fragments of 6+8+2, 6 peptide and 8 peptide are sequentially coupled in a solid phase by adopting a dichloro resin, and 2 peptide is coupled by a liquid phase method; then the fragments are connected in sequence; and finally, positioning the unprotected Cys of the obtained linear peptide by using hydrogen peroxide for first cyclization, removing a second pair of Cys protecting groups Acm from the iodine simple substance, and performing second cyclization to obtain the crude peptide. In the preparation method, both the 2 peptide fragment and the 8 peptide fragment need to be subjected to Fmoc removal, a grinding method is adopted in the post-treatment process, the scale amplification is not suitable, the piperidine or triethylamine and fluorene byproducts subjected to Fmoc removal are not easy to remove, and the residues can cause serious side reactions in the following process, so that the purity of the crude peptide is reduced.

In China, most of procatide is synthesized by Fmoc all-solid-phase sequential coupling, CN104211777A, CN103694320A, CN108440652A adopts an all-solid-phase sequential coupling synthesis method, and procatide is prepared by forming two pairs of disulfide bonds by utilizing orthogonal step-by-step oxidation of different cysteine side chain protecting groups. According to the all-solid-phase sequential coupling synthesis method, due to the fact that an obvious peptide chain aggregation phenomenon occurs at the Val position at the 8 th position in the coupling process, the subsequent amino acid is difficult to couple, and the crude peptide obtained after two-step oxidation is low in purity, content and total yield.

CN108003222A introduces a solid phase fragment synthesis method, solid phase is sequentially coupled with 10 amino acids, then the solid phase is coupled with N-terminal 6 peptide fragment under the action of papain, and then the peptide is subjected to step-by-step oxidation to obtain procaine. According to the synthetic method, the 6 peptide fragment and the 10 peptide fragment are difficult to couple, so that the 10 peptide fragment and the 6 peptide fragment are coupled by adopting papain, but the defects of the enzyme are that the inactivation is easy, and the solid-phase coupling of the 6 peptide fragment is not suitable for realizing scale-up.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a preparation method of procainatide. The specific technical scheme is as follows:

the invention provides a preparation method of procainatide, which comprises the following steps:

step 1: coupling 4-hydroxybenzyl alcohol and Fmoc-Leu-OH by using 2-CTC resin as a solid phase carrier to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin;

step 2: the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is used as a solid phase carrier, fmoc-Cys (Acm) -OH and 8 peptide fragment B are coupled in sequence by an Fmoc solid phase synthesis method, and after Fmoc protection is removed, cleavage is carried out to obtain 10 peptide fragment C with protected side chain;

the 8 peptide fragment B is Fmoc-Cys (Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -OH；

The 10 peptide fragment C is H-Cys (Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -4-OPhCH ₂ OH；

And step 3: under the action of a coupling reagent, the 6 peptide fragment A is coupled with the 10 peptide fragment C to obtain a side chain protected peptide fragment D;

the 6 peptide fragment A is Boc-Asn (Trt) ¹ -Asp(OtBu) ² -Glu(OtBu) ³ -Cys(Trt) ⁴ -Glu(OtBu) ⁵ -Leu ⁶ -OH；

Said peptide fragment D is Boc-Asn (Trt) ¹ -Asp(OtBu) ² -Glu(OtBu) ³ -Cys(Trt) ⁴ -Glu(OtBu) ⁵ -Leu ⁶ -Cys(Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -4-OPhCH ₂ OH；

And 4, step 4: cleaving the peptide fragment D, and deprotecting a portion of the reactive side chain groups to obtain a linear crude peptide;

the linear crude peptide is H-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys(Acm) ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -OH；

And 5: the obtained linear crude peptide is oxidized in the first step and oxidized in the second step to form two pairs of disulfide bonds, and the crude peptide of the procainatide is obtained.

The 6 peptide fragment a and the 8 peptide fragment B were synthesized by Fmoc solid phase synthesis method, which was: connecting Fmoc protected amino acid to a solid phase carrier by using 2-CTC resin as the solid phase carrier, then removing Fmoc protection, sequentially coupling protected amino acid, shrinking the peptide resin by using methanol after the peptide resin is coupled, drying in vacuum, cracking the peptide resin by using a cracking solution, concentrating at 30 ℃ to 20-30% of the volume of the original cracking solution, adding absolute ethyl alcohol to 50% of the volume of the original cracking solution, continuously concentrating to 20-30% of the volume of the original cracking solution, and then adding purified water with the same volume to separate the fully protected peptide to obtain a peptide fragment A or a peptide fragment B. Further, the 2-CTC resin is swollen with a solvent selected from DMF, NMP, dichloromethane, etc., preferably DMF; the substitution degree of the 2-CTC resin is 0.8-1.7 mmol/g, preferably 1.0-1.4 mmol/g; removing the Fmoc protecting group by using 20% piperidine/DMF solution; preferably, the coupling agent is a combination of DIPCDI and compound a or a combination of DIPEA, compound a and compound B, wherein compound a is HOAt or HOBt and compound B is PyAOP, pyBOP, HATU, HBTU or TBTU, preferably DIPCDI/HOBt; the ratio of each component in the protected amino acid and the coupling agent is, in terms of molar ratio, DIPCDI to compound a = 1.2; the amount of each amino acid used is 2 to 5 equivalents, preferably 3 equivalents, and the coupling reaction is usually carried out for 1.5 to 4 hours, preferably 2 to 3 hours; the reaction temperature is 25 +/-10 ℃, and preferably 25 +/-5 ℃; the lysis solution is a 1-5% TFA solution in methylene chloride, preferably 1% lysis solution; or 5% to 99% of a solution of TFE or HFIP in methylene chloride, preferably 20% of a solution of TFE in methylene chloride.

In the technical scheme of the invention, the synthesis method of Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin in the step 1 comprises the following steps: coupling 4-hydroxybenzyl alcohol of Wang resin under the action of organic base by using 2-CTC resin as a solid phase carrier, and then coupling Fmoc-Leu-OH under the action of a coupling agent to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin;

preferably, the substitution degree of the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is 0.21 to 0.81mmol/g, and preferably 0.31 to 0.60mmol/g.

Further, the substitution degree of the 2-CTC resin is 0.30-1.70 mmol/g, preferably 0.70-1.39 mmol/g;

the organic base is selected from DIPEA, pyridine or TMP, preferably DIPEA;

the coupling agent is a combination of DIPCDI and a compound X selected from HOAt or HOBt; preferably the coupling agent is a combination of DIPCDI and HOBt;

preferably, the molar ratio of the 4-hydroxybenzyl alcohol to the organic base is 1:1-5, preferably 1.5; preferably, the 4-hydroxybenzyl alcohol is used in an amount of 2 to 5 equivalents, preferably 3 equivalents;

preferably, the reaction time for the coupling is 1.5 to 4 hours, preferably 2 to 3 hours; the temperature is room temperature (i.e. 25 + -5 deg.C), and can be increased or decreased as appropriate.

Further, when Fmoc-Leu-OH is coupled, a catalyst is also added; preferably, the catalyst is DMAP;

preferably, the molar ratio of Fmoc-Leu-OH, DIPCDI, compound X and DMAP is 1.2; preferably, the Fmoc-Leu-OH is used in an amount of 2 to 5 equivalents, preferably 3 equivalents;

further, the reaction is sealed by acetic anhydride and pyridine, the molar ratio of the acetic anhydride to the pyridine is 1:1, and the dosage is 10 equivalents.

In the above technical scheme of the present invention, the coupling in step 2 is performed under the action of a coupling agent, wherein the coupling agent is a composition of DIPCDI and a compound X or a composition of DIPEA, a compound X and a compound Y; the compound X is selected from HOAt or HOBt, and the compound Y is selected from PyAOP, pyBOP, HATU, HBTU or TBTU;

preferably, the molar ratio of the Fmoc-Cys (Acm) -OH or 8 peptide fragment B, DIPCDI to compound X is 1;

preferably, the molar ratio of Fmoc-Cys (Acm) -OH or 8 peptide fragment B, DIPEA, compound X and compound Y is 1;

preferably, fmoc-Cys (Acm) -OH or 8 peptide fragment B is used in an amount of 2 to 5 equivalents, preferably 3 equivalents;

preferably, when Fmoc-Cys (Acm) -OH is coupled, the coupling agent is a composition of DIPCDI and HOBt;

preferably, when the 8 peptide fragment B is coupled, the coupling agent is a combination of DIPEA, HOBt and HBTU;

preferably, the reaction time for the coupling is 1.5 to 4 hours, preferably 2 to 3 hours; the temperature is room temperature (namely 25 +/-5 ℃), and the operation can also be carried out at a proper increased or reduced temperature;

preferably, the lysis employs as lysis solution 1% of a dichloromethane solution of TFA, 20% of a dichloromethane solution of TFE or 20% of a dichloromethane solution of HFIP, preferably 20% of a dichloromethane solution of TFE;

further, after coupling Fmoc-Cys (Acm) -OH and the fragment B, shrinking the peptide resin by using methanol, drying in vacuum, cracking the peptide resin by using a cracking solution, concentrating at 30 ℃ to 20-30% of the volume of the original cracking solution, adding absolute ethyl alcohol to 50% of the volume of the original cracking solution, continuously concentrating to 20-30% of the volume of the original cracking solution, and then adding the same volume of purified water to separate out the full-protection peptide.

In the above technical solution of the present invention, the step 3 is specifically performed by dissolving the 6 peptide fragment a, the 10 peptide fragment C and the coupling reagent in a reaction solvent, cooling to-15 to 5 ℃, adding DIPEA, and reacting at room temperature (i.e., 25 ± 5 ℃) for 1.5 to 4 hours, preferably 2 to 3 hours, at a suitably increased or decreased temperature;

preferably, the reaction solvent is selected from DMF or NMP, preferably DMF;

preferably, the coupling reagent is a combination of compound a selected from HOAt, HOBt or Cl-HOBt and compound B selected from PyAOP, pyBOP, HATU, HBTU or TBTU; preferably, the coupling agent is a combination of Cl-HOBt and HBTU;

preferably, the molar ratio of the 10 peptide fragment C, the 6 peptide fragment a, the coupling reagent and DIPEA is 1;

preferably, the molar ratio of the 10 peptide fragment C, the 6 peptide fragment A, cl-HOBt, HBTU and DIPEA is 1.

In the technical scheme of the invention, TFA and H are adopted for cleavage in the step 4 ₂ A mixture formed by more than one of O, TIS, EDT, anisole, thioanisole and phenol is used as a cracking solution; preferred are TFA, EDT, H ₂ Mixture of O, phenol and TIS, preferably TFA, EDT, H ₂ The volume ratio of O, phenol and TIS is 85;

preferably, the volume of the lysis solution is 4 to 8 times the weight of the peptide fragment D;

preferably, the cleavage is carried out at room temperature (i.e.25. + -. 5 ℃ C.), also at a suitably elevated or reduced temperature, for a period of 2 to 4 hours, preferably 3 hours.

In the above technical scheme of the present invention, the first oxidation in step 5 is to use an oxidant to make Cys in the linear crude peptide ⁴ And Cys ¹² A first pair of disulfide bonds;

preferably, in the first step of oxidation, the linear crude peptide is dissolved in water, the pH value is adjusted to 8-10, preferably 8.5-9.0 by using concentrated ammonia water, and the concentration of the linear crude peptide is 1-10 mg/mL, preferably 1mg/mL;

preferably, the oxidant for the first step oxidation is selected from DMSO, oxygen or hydrogen peroxide oxidation, and is preferably hydrogen peroxide; preferably, the dosage of the hydrogen peroxide is 0.5 to 1 equivalent;

preferably, the reaction time of the first oxidation step is 0.5 to 2 hours, preferably 1 hour;

the second oxidation in step 5 is to use iodine to make Cys in the linear crude peptide ⁷ And Cys ¹⁵ Form a second pair of disulfide bonds therebetween;

preferably, in the second oxidation step, glacial acetic acid is used for adjusting the pH value to 2-5, preferably 3.50-4.00;

preferably, the iodine is used in an amount of 2 to 4 equivalents, preferably 2 equivalents;

preferably, the reaction time of the second oxidation step is 0.5 to 2 hours, preferably 1 hour.

The preparation method provided by the invention also comprises the steps of purifying, concentrating and freeze-drying the obtained procainatide crude peptide;

preferably, the purification adopts reversed-phase high-pressure liquid chromatography, and further adopts reversed-phase octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as a mobile phase.

The invention has the beneficial effects that:

1. the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is used as a solid phase carrier, fmoc-Cys-OH and 8 peptide fragment B are sequentially coupled and then subjected to Fmoc protection removal, fmoc removal is performed after solid phase synthesis, the problem that in the prior art, piperidine and triethylamine which are used for Fmoc protection removal and fluorenes which are a byproduct of Fmoc protection removal after liquid phase synthesis of a peptide fragment C are difficult to remove is solved, side reaction caused by residue on subsequent steps is avoided, and the purity of crude peptide is improved.

2. The invention adopts Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin as a solid phase carrier, obtains 10 peptide fragment C protected at the C end by utilizing the acid sensitivity difference of Wang resin and 2-CTC resin, can avoid side reaction caused by the C end of the 10 peptide fragment in a limited way when reacting with 6 peptide fragment A under the condition of liquid phase, provides favorable conditions for coupling among fragments, overcomes the problems of scale limitation and difficult coupling among large fragments in solid phase synthesis in the prior art, improves the purity and yield of crude peptide, and finally improves the yield of products.

Drawings

FIG. 1 is a scheme for the synthesis of procainatide;

FIG. 2 is a mass spectrum of procainatide;

FIG. 3 is an HPLC chromatogram of procatide.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Abbreviations used in the present invention and their meanings are as follows:

the invention provides a preparation method of procainatide, the synthetic route is shown as figure 1, and the steps are as follows:

(1) Coupling 4-hydroxybenzyl alcohol and Fmoc-Leu-OH by using 2-CTC resin as a solid phase carrier to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin;

(2) The Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is used as a solid phase carrier, fmoc-Cys (Acm) -OH and 8 peptide fragment B are coupled in sequence by an Fmoc solid phase synthesis method, and after Fmoc protection is removed, cleavage is carried out to obtain 10 peptide fragment C with protected side chain;

(3) Under the action of a coupling reagent, the 6 peptide fragment A is coupled with the 10 peptide fragment C to obtain a side chain protected peptide fragment D;

(4) Cleaving the peptide fragment D, and deprotecting a portion of the reactive side chain groups to obtain a linear crude peptide;

(5) The obtained linear crude peptide is oxidized in the first step and oxidized in the second step to form two pairs of disulfide bonds, and the crude peptide of the procapsipeptide is obtained.

Example 1:6 Synthesis of peptide fragment A

143.8g of dichloro resin with a substitution of 200mmol of 1.39mmol/g was weighed, swollen with dichloromethane for half an hour, then pumped out, fmoc-Leu-OH (212.0g, 600mmol) was dissolved in 1L of DMF, and added to a solid phase reaction column, followed by addition of DIPEA (154.8g, 1200mmol) for reaction for 2 hours, addition of 100mL of methanol for further reaction for 2 hours, and washing with DMF three times in an amount of 1L each. Washing with methanol for three times, wherein the dosage is 1L each time and 10 minutes each time, vacuumizing and drying until the resin is in a quicksand shape to obtain 197.8g of Fmoc-Leu-2-CTC resin, and measuring the substitution degree of the Fmoc-Leu-2-CTC resin to be 0.92mmol/g.

195.6g of Fmoc-Leu-2-CTC resin is taken, the synthesis scale is 180mmol, the Fmoc protecting group is removed by 20% piperidine DMF solution, and the deprotection is carried out twice, each time for 5 minutes, and the dosage is 1L. DMF was washed 6 times in 1L each time. The protected amino acid (540 mmol) and HOBt (80.2g, 594mmol) are dissolved in 1LDMF, added into a solid phase reaction column, nitrogen is bubbled uniformly, DIC (81.6g, 648mmol) and 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is transparent to carry out the next amino acid coupling.

After the peptide resin was coupled, the peptide resin was washed with methanol three times, 1L each time, for 10 minutes, vacuum dried until the resin became a quicksand to give 360.6g, the peptide resin was cleaved with 20% TFE dichloromethane solution (3L), filtered, and the residue was washed with dichloromethane (1L) to collect the filtrate. Concentrating at 30 deg.C to 600mL, adding anhydrous ethanol to 1.5L, concentrating to 600mL, adding 600mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 600mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to give 242.4g of the fully protected peptide fragment A in 91.2% yield and 92.0% purity.

Example 2:8 Synthesis of peptide fragment B

287.6g of a dichloro resin having a substitution of 1.39mmol/g was weighed out, swollen with dichloromethane for half an hour, and then drained, fmoc-Gly-OH (356.4g, 1200 mmol) was dissolved in 2L of DMF, and the solution was charged into a solid-phase reaction column, followed by addition of DIPEA (309.6 g,2400 mmol) and reaction for 2 hours. Then, 200mL of methanol was added, the reaction was continued for 2 hours, and the reaction solution was washed with DMF three times in an amount of 2L each time. Washing with methanol for three times, wherein the dosage is 2L each time and 10 minutes each time, vacuumizing and drying until the resin is in a quicksand shape to obtain 398.6g of Fmoc-Gly-2-CTC resin, and measuring the substitution degree of the Fmoc-Gly-2-CTC resin to be 1.00mmol/g.

And taking 360g of Fmoc-Gly-2-CTC resin, wherein the synthesis scale is 360mmol, removing the Fmoc protecting group by using 20% piperidine DMF solution, and deprotecting twice for 5 minutes each time, wherein the dosage is 2L each time. DMF was washed 6 times in 2L portions. Protected amino acid (1080 mmol) and HOBt (160.4g, 1188mmol) are dissolved in 2LDMF, added into a solid phase reaction column, nitrogen is bubbled uniformly, DIC (163.2g, 1296 mmol) and 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is transparent to carry out the next amino acid coupling.

After the peptide resin was coupled, the peptide resin was washed with methanol three times in an amount of 2L each for 10 minutes, vacuum dried until the resin became a quicksand to give 758.4g, the peptide resin was cleaved with 20% TFE dichloromethane solution (6L), filtered, and the residue was washed with dichloromethane (2L) to collect the filtrate. Concentrating at 30 deg.C to 1200mL, adding anhydrous ethanol to 3000mL, concentrating to 1200mL, adding 1200mL of purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 1200mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to give 468.8g of the fully protected peptide fragment in 81.5% yield and 92.9% purity.

Example 3: synthesis of Fmoc-Leu-Linker-2-CTC resin

Weighing 29.4g of 50mmol2-CTC resin with substitution degree of 1.7mmol/g, swelling with dichloromethane for half an hour, draining, adding 4-hydroxybenzyl alcohol (12.4g, 100mmol) into DMF, stirring to dissolve, adding into a solid phase reaction column, adding DIPEA (29.0g, 225mmol), and reacting for 2 hours. Additional 25mL of methanol was added and the reaction was continued for 2 hours. DMF was washed three times in 150mL portions, and the solvent was drained. Fmoc-Leu-OH (35.3g, 100mmol), HOBT (14.9g, 110mmol) and DMAP (3.7g, 30mmol) were dissolved in 200mL of DMF, and the solution was introduced into a solid phase reaction column, nitrogen was bubbled uniformly, and DIC (15.1g, 120mmol) was added thereto and the reaction was carried out for 2 hours. The solution was washed three times with 250mL of DMF. Acetic anhydride (50.1g, 0.5 mol) and pyridine (39.5 g,0.5 mol) are added into 200mLDMF to be uniformly mixed, and the mixed solution is added into a reaction column to carry out closed reaction for 2 hours. Washed three times with 150mL each time of DMF. Washing with methanol for three times, wherein the dosage is 150mL each time and 10 minutes each time, vacuumizing and drying until the resin is in a flowing sand shape to obtain 42.8g of Fmoc-Leu-Linker-2-CTC resin, and measuring the substitution degree of the resin to be 0.81mmol/g.

Example 4: synthesis of Fmoc-Leu-Linker-2-CTC resin

Weighing 36.0g of 50mmol2-CTC resin with the substitution degree of 1.39mmol/g, swelling with dichloromethane for half an hour, pumping off, adding 4-hydroxybenzyl alcohol (12.4g, 100mmol) into DMF, stirring and dissolving, adding into a solid phase reaction column, and then adding DIPEA (29.0g, 225mmol) for reacting for 2 hours. Additional 25mL of methanol was added and the reaction was continued for 2 hours. DMF was washed three times in 250mL portions and the solvent was drained. Fmoc-Leu-OH (35.3g, 100mmol), HOBT (14.9g, 110mmol) and DMAP (3.7g, 30mmol) were dissolved in 250mL of DMF, and the solution was introduced into a solid phase reaction column, nitrogen was bubbled uniformly, and DIC (15.1g, 120mmol) was added and the reaction was carried out for 2 hours. Washed three times with 250mL of DMF. Acetic anhydride (50.1g, 0.5 mol) and pyridine (39.5g, 0.5 mol) were added to 200mL of DMF and mixed uniformly, and the mixture was added to a reaction column and reacted for 2 hours in a closed state. The solution was washed three times with 250mL of DMF. Washing with methanol for three times, wherein the dosage is 250mL each time and 10 minutes each time, vacuumizing and drying until the resin is in a flowing sand shape to obtain 50.1g of Fmoc-Leu-Linker-2-CTC resin, and measuring the substitution degree of the resin to be 0.60mmol/g.

Example 5: synthesis of Fmoc-Leu-Linker-2-CTC resin

45.5g of 50mmol2-CTC resin with substitution degree of 1.10mmol/g was weighed, swollen with dichloromethane for half an hour and then drained, 4-hydroxybenzyl alcohol (18.6 g, 150mmol) was added to DMF and dissolved with stirring, and added to a solid phase reaction column, followed by the addition of DIPEA (29.0 g, 225mmol) and reacted for 2 hours. Additional 25mL of methanol was added and the reaction was continued for 2 hours. DMF was washed three times in 250mL portions and the solvent was drained. Fmoc-Leu-OH (53.0g, 150mmol), HOBT (22.3g, 165mmol), DMAP (5.5g, 45mmol) were dissolved in 250ml of DMF, and the solution was introduced into a solid-phase reaction column, nitrogen gas was bubbled uniformly, and DIC (27.7g, 180mmol) was added thereto and the reaction was carried out for 2 hours. Washed three times with 250mL of DMF. Acetic anhydride (50.1g, 0.5 mol) and pyridine (39.5g, 0.5 mol) were added to 200mL of DMF and mixed uniformly, and the mixture was added to a reaction column and reacted for 2 hours in a closed state. Washed three times with 250mL of DMF. Washing with methanol for three times, wherein the dosage is 250mL each time and 10 minutes each time, vacuumizing and drying until the resin is in a flowing sand shape to obtain 55.4g of Fmoc-Leu-Linker-2-CTC resin, and measuring the substitution degree of the resin to be 0.46mmol/g.

Example 6: synthesis of Fmoc-Leu-Linker-2-CTC resin

71.4g of 50mmol2-CTC resin with a substitution degree of 0.7mmol/g was weighed, and was swelled with dichloromethane for half an hour and then taken out, 4-hydroxybenzyl alcohol (31.0g, 250mmol) was added to DMF and stirred to dissolve, and the solution was added to a solid phase reaction column, followed by addition of DIPEA (161.25g, 1250mmol) and reaction for 2 hours. Additional 25mL of methanol was added and the reaction was continued for 2 hours. DMF was washed three times in 250mL portions and the solvent was drained. Fmoc-Leu-OH (88.5g, 250mmol), HOBT (37.2g, 275mmol), DMAP (9.2g, 75mmol) were dissolved in 250mL DMF, and the mixture was introduced into a solid-phase reaction column, nitrogen gas was bubbled uniformly, and DIC (37.8g, 300mmol) was added thereto and reacted for 2 hours. The solution was washed three times with 250mL of DMF. Acetic anhydride (50.1g, 0.5 mol) and pyridine (39.5 g,0.5 mol) are added into 200mLDMF to be uniformly mixed, and the mixed solution is added into a reaction column to carry out closed reaction for 2 hours. Washed three times with 250mL of DMF. Washing with methanol for three times, wherein the dosage is 250mL each time and 10 minutes each time, vacuumizing and drying until the resin is in a flowing sand shape to obtain 85.6g of Fmoc-Leu-Linker-2-CTC resin, and measuring the substitution degree of the resin to be 0.31mmol/g.

Example 7: synthesis of Fmoc-Leu-Linker-2-CTC resin

166.7g of 50mmol2-CTC resin (substitution: 0.30 mmol/g) was weighed, swelled with dichloromethane for half an hour and then drained, 4-hydroxybenzyl alcohol (12.4g, 100mmol) was added to DMF and dissolved by stirring, and the mixture was added to a solid phase reaction column, followed by the addition of DIPEA (29.0 g, 225mmol) and reacted for 2 hours. Additional 25mL of methanol was added and the reaction was continued for 2 hours. DMF was washed three times in 450mL portions, and the solvent was drained. Fmoc-Leu-OH (35.3g, 100mmol), HOBT (14.9g, 110mmol) and DMAP (3.7g, 30mmol) were dissolved in 450ml of DMF, and the solution was introduced into a solid phase reaction column, nitrogen was bubbled uniformly, and DIC (15.1g, 120mmol) was added and the reaction was carried out for 2 hours. Washed three times with 250mL of DMF. Adding acetic anhydride (50.1g, 0.5 mol) and pyridine (39.5 g,0.5 mol) into 450mLDMF, mixing uniformly, adding the mixed solution into a reaction column, and carrying out a closed reaction for 2 hours. Washed three times with 450mL of DMF. Washing with methanol for three times, wherein the dosage is 450mL each time and 10 minutes each time, vacuumizing and drying until the resin is in a flowing sand shape to obtain 180.5g of Fmoc-Leu-Linker-2-CTC resin, and measuring the substitution degree of the resin to be 0.21mmol/g.

Example 8: synthesis of 10 peptide fragment C

Fmoc-Leu-Linker-2-CTC (12.2g, 15mmol, alternative 0.81 mmol/g) resin obtained in example 3 was weighed as a solid phase carrier, fmoc protecting group was removed with 20% piperidine/DMF solution, deprotected twice, 5 minutes each time, 75mL each time, and washed 6 times with DMF, 75mL each time. Fmoc-Cys (Acm) -OH (12.5g, 30mmol) and HOBt (4.5g, 33.0mmol) are weighed and dissolved in 75mLDMF, the solution is added into a solid phase reaction column, nitrogen is bubbled uniformly, DIC (4.6 g, 36mmol) and a 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is required to be transparent. DMF was washed 3 times in 100mL portions.

The Fmoc protecting group was removed twice with 5 min each time using 75mL of 20% piperidine/DMF. DMF was washed 6 times in 75mL portions. Fragment B (48.0g, 30mmol), HOBt (4.9g, 36mmol) and HBTU (11.4g, 30mmol) are weighed and dissolved in 150mLDMF, and the solution is added into a solid phase reaction column, nitrogen is bubbled uniformly, DIPEA (7.8g, 60mmol) and 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is required. DMF was washed 3 times in 150mL portions. The Fmoc protecting group was removed twice with 5 min each time using 150mL of 20% piperidine/DMF. DMF was washed 6 times in 150mL portions.

Washing the peptide resin with methanol three times at a rate of 150mL each time for 10 minutes, vacuum drying until the resin is quicksand to give 40.4g of peptide resin, cleaving the peptide resin with 20% (w/w) TFE methylene chloride solution (320 mL), filtering, washing the residue with methylene chloride (100 mL), and collecting the filtrate. Concentrating at 30 deg.C to 64mL, adding anhydrous ethanol to 160mL, concentrating to 64mL, adding 64mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 100mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to obtain 21.1g of the fully protected peptide fragment C in 81.8% yield and 85.5% purity.

Example 9: synthesis of 10 peptide fragment C

Fmoc-Leu-Linker-2-CTC (25.0g, 15mmol, alternative 0.60 mmol/g) resin obtained in example 4 was weighed as a solid phase carrier, fmoc protecting group was removed with 20% piperidine/DMF solution, deprotection was performed twice, 5 minutes each time, 150mL each time, and DMF was washed 6 times, 150mL each time. Weighing Fmoc-Cys (Acm) -OH (12.5g, 30mmol) and HOBt (4.5g, 33.0mmol) to dissolve in 100mLDMF, adding into a solid phase reaction column, bubbling nitrogen gas uniformly, then adding DIC (4.6 g, 36mmol) and 5% ninhydrin ethanol solution to monitor the reaction, and requiring the resin to be transparent. DMF was washed 3 times in 150mL portions.

The Fmoc protecting group was removed twice with 5 min each time using 150mL of 20% piperidine/DMF. DMF was washed 6 times in 150mL portions. Fragment B (72.0g, 45mmol), HOBt (7.3g, 54mmol) and HBTU (17.1g, 45mmol) were weighed and dissolved in 250mL DMF, and the solution was added into a solid phase reaction column, nitrogen was bubbled uniformly, DIPEA (11.6g, 90mmol) and 5% ninhydrin ethanol solution were added to monitor the reaction, and the resin was required. DMF was washed 3 times in 150mL portions. The Fmoc protecting group was removed twice with 5 min each time using 150mL of 20% piperidine/DMF. DMF was washed 6 times in 150mL portions.

The peptide resin was washed with methanol three times at 150mL each for 10 minutes, vacuum dried until the resin became a quicksand to give 53.6g of peptide resin, which was cleaved with 20% TFE in dichloromethane (400 mL), filtered, and the residue was washed with dichloromethane (150 mL) to collect the filtrate. Concentrating at 30 deg.C to 100mL, adding anhydrous ethanol to 200mL, concentrating to 100mL, adding 100mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 100mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to give 24.6g of the fully protected peptide fragment C in 95.0% yield and 91.2% purity.

Example 10: synthesis of 10 peptide fragment C

Fmoc-Leu-Linker-2-CTC (32.6 g,15mmol, alternative 0.46 mmol/g) resin obtained in example 5 was weighed as a solid support, the Fmoc protecting group was removed with 20% piperidine/DMF solution, deprotected twice for 5 minutes each time in 150mL each time, and washed 6 times with DMF in 150mL each time. Fmoc-Cys (Acm) -OH (18.6g, 45mmol) and HOBt (6.9g, 49.5mmol) are weighed and dissolved in 150mLDMF, the solution is added into a solid phase reaction column, nitrogen is bubbled uniformly, and DIC (6.8g, 54mmol) and 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is required. DMF was washed 3 times in 150mL portions.

The Fmoc protecting group was removed twice with 5 min twice using 150mL of 20% piperidine/DMF. DMF was washed 6 times in 150mL portions. Fragment B (72.0g, 45mmol), HOBt (7.3g, 54mmol) and HBTU (17.1g, 45mmol) are weighed and dissolved in 250ml of DMF, the solution is added into a solid phase reaction column, nitrogen is bubbled uniformly, DIPEA (11.6g, 90mmol) and 5% ninhydrin ethanol solution are added to monitor the reaction, and the resin is required. DMF was washed 3 times in 150mL portions. The Fmoc protecting group was removed twice with 5 min each time using 150mL of 20% piperidine/DMF. DMF was washed 6 times in 150mL portions.

The peptide resin was washed with methanol three times at 150mL each for 10 minutes, vacuum dried until the resin became a quicksand to give 63.6g of peptide resin, which was cleaved with 20% TFE in dichloromethane (500 mL), filtered, and the residue was washed with dichloromethane (150 mL) to collect the filtrate. Concentrating at 30 deg.C to 100mL, adding anhydrous ethanol to 200mL, concentrating to 100mL, adding 100mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 100mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to obtain 25.6g of the fully protected peptide fragment C in 99.2% yield and 93.5% purity.

Example 11: synthesis of 10 peptide fragment C

Fmoc-Leu-Linker-2-CTC (48.4 g,15mmol, alternative 0.31 mmol/g) resin obtained in example 6 was weighed as a solid phase carrier, fmoc protecting group was removed with 20% piperidine/DMF solution, deprotected twice for 5 minutes each time at 250mL each time, and washed 6 times with DMF at 250mL each time. Fmoc-Cys (Acm) -OH (31.1g, 75mmol) and HOBt (11.1g, 82.5mmol) were weighed, dissolved in 250mLDMF, added to a solid phase reaction column, nitrogen bubbled uniformly, and DIC (11.4g, 90mmol) and 5% ninhydrin ethanol solution were added to monitor the reaction, requiring the resin. DMF was washed 3 times in 250mL portions.

The Fmoc protecting group was removed twice with 5 min each time using 250mL of 20% piperidine/DMF. DMF was washed 6 times in 250mL portions. Fragment B (95.9g, 60mmol), HOBt (9.7g, 72mmol) and HBTU (22.8g, 60mmol) were weighed and dissolved in 300mL DMF, and the solution was added to a solid phase reaction column, nitrogen was bubbled uniformly, DIPEA (15.5g, 120mmol) and 5% ninhydrin ethanol solution were added to monitor the reaction, and the resin was called. DMF was washed 3 times in 250mL portions. The Fmoc protecting group was removed twice with 5 min each time using 250mL of 20% piperidine/DMF. DMF was washed 6 times in 250mL portions.

The peptide resin was washed with methanol three times at 150mL each for 10 minutes, vacuum dried until the resin became a quicksand to give 77.7g of peptide resin, which was cleaved with 20% TFE in dichloromethane (600 mL), filtered, and the residue was washed with dichloromethane (150 mL) to collect the filtrate. Concentrating at 30 deg.C to 120mL, adding anhydrous ethanol to 300mL, concentrating to 120mL, adding 120mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with purified water twice with 100mL each time. The filter cake was air-dried at 40 ℃ for 30 hours to obtain 24.8g of the fully protected peptide fragment C in a yield of 96.2% and a purity of 92.8%.

Example 12: synthesis of 10 peptide fragment C

Fmoc-Leu-Linker-2-CTC (71.4g, 15mmol, alternative 0.21 mmol/g) resin obtained in example 7 was weighed as a solid phase carrier, fmoc protecting group was removed with 20% piperidine/DMF solution, deprotected twice, 5 minutes each time, 250mL each time, and washed 6 times with DMF, 250mL each time. Fmoc-Cys (Acm) -OH (31.1g, 75mmol) and HOBt (11.1g, 82.5mmol) were weighed, dissolved in 250mLDMF, added to a solid phase reaction column, nitrogen bubbled uniformly, and DIC (11.4g, 90mmol) and 5% ninhydrin ethanol solution were added to monitor the reaction, requiring the resin. DMF was washed 3 times in 250mL portions.

The Fmoc protecting group was removed twice with 5 min each time using 250mL of 20% piperidine/DMF. DMF was washed 6 times in 250mL portions. Fragment B (95.9g, 60mmol), HOBt (9.7g, 72mmol) and HBTU (22.8g, 60mmol) were weighed and dissolved in 300mL DMF, and the solution was added to a solid phase reaction column, nitrogen was bubbled uniformly, DIPEA (15.5g, 120mmol) and 5% ninhydrin ethanol solution were added to monitor the reaction, and the resin was called. DMF was washed 3 times in 250mL portions. The Fmoc protecting group was removed twice with 5 min each time using 250mL of 20% piperidine/DMF. DMF was washed 6 times in 250mL portions.

The peptide resin was washed with methanol three times at 150mL each for 10 minutes, vacuum dried until the resin became a quicksand to give 103.2g of peptide resin, which was cleaved with 20% TFE in dichloromethane (800 mL), filtered, and the residue was washed with dichloromethane (250 mL) to collect the filtrate. Concentrating at 30 deg.C to 160mL, adding anhydrous ethanol to 400mL, concentrating to 160mL, adding 160mL purified water to precipitate total protective peptide, filtering, and washing the filter cake with 120mL purified water twice. The filter cake was air-dried at 40 ℃ for 30 hours to obtain 24.5g of the fully protected peptide fragment C in 95.0% yield and 93.2% purity.

Example 13: synthesis of linear bold

Fragment A (10.3g, 7.0mmol) obtained in example 1, fragment C (12.3g, 7.0mmol) obtained in example 8, cl-HOBt (1.19g, 7 mmol) and HBTU (2.66g, 7 mmol) were weighed out and dissolved in 75mL of DMF, the temperature was reduced to-15 to 5 ℃ or lower, DIPEA (1.8g, 14mmol) was slowly added thereto with stirring, and after completion of dropwise addition, the reaction mixture was slowly warmed to room temperature and the reaction was further stirred for 1.5 hours. HPLC monitoring reactionShould be used. The reaction mixture was poured into 375mL of purified water with stirring, filtered, and the filter cake was washed with 0.1N HCl, saturated NaHCO ₃ Washing with 100mL of aqueous solution (2 times), purified water (3 times), diethyl ether (2 times), and drying under vacuum, yielded fragment D20.5g, yield 92.0% (the fully protected peptide was not detected by an appropriate analytical method due to poor solubility and low polarity).

Fragment D was treated with 100mL of lysate TFA EDT H ₂ Phenol: TIS = 85. The cleavage was carried out for 4 hours at room temperature, precipitated with 1L of precooled methyl tert-butyl ether, centrifuged and washed twice with methyl tert-butyl ether. After drying with nitrogen, 10.6g of linear crude peptide with a purity of 74.2% was obtained.

Example 14: synthesis of linear bold

Fragment A (10.3g, 7.0mmol) obtained in example 1, fragment C (12.3g, 7.0mmol) obtained in example 9, cl-HOBt (1.19g, 7 mmol) and HBTU (2.66g, 7 mmol) were weighed out and dissolved in 75mL of DMF, the temperature was reduced to-15 to 5 ℃ or lower, DIPEA (1.8g, 14mmol) was slowly added thereto with stirring, and after completion of dropwise addition, the reaction mixture was slowly warmed to room temperature and the reaction was further stirred for 1.5 hours. The reaction was monitored by HPLC. The reaction mixture was poured into 375mL of purified water with stirring, filtered, and the filter cake was washed with 0.1N HCl, saturated NaHCO ₃ Washing with aqueous solution (2 times), pure water (3 times), diethyl ether (2 times) in 100mL each time, and drying under vacuum gave fragment d20.5g in 92.0% yield (fully protected peptide due to poor solubility and low polarity, no suitable analytical method detected).

Fragment D was treated with 100mL of lysate TFA EDT H ₂ Phenol: TIS (volume ratio) = 85. The cleavage was carried out at room temperature for 4 hours, precipitated with 1L of precooled methyl tert-butyl ether, centrifuged and washed twice with methyl tert-butyl ether. After drying with nitrogen, 11.3g of linear crude peptide with purity of 78.8% was obtained.

Example 15: synthesis of crude Linear peptide

Fragment A (10.3g, 7.0mmol) obtained in example 1, fragment C (12.3g, 7.0mmol) obtained in example 10, cl-HOBt (1.19g, 7 mmol) and HBTU (2.66g, 7 mmol) were weighed out and dissolved in 75mL of DMF, the temperature was reduced to-15 to 5 ℃ or lower, DIPEA (1.8g, 14mmol) was slowly added thereto with stirring, and after completion of dropwise addition, the reaction mixture was slowly warmed to room temperature and further stirredThe reaction was carried out for 2 hours. The reaction was monitored by HPLC. The reaction mixture was poured into 375mL of purified water with stirring, filtered, and the filter cake was washed with 0.1N HCl, saturated NaHCO ₃ The fragment was obtained in a yield of 96.0% by washing with 100mL of aqueous solution (2 times), pure water (3 times), diethyl ether (2 times) and drying under vacuum, yielding D21.4g (fully protected peptide was not detected by suitable analytical methods due to poor solubility and low polarity).

Fragment D was treated with 150mL of lysate TFA EDT H ₂ Phenol: TIS (volume ratio) = 85. The cleavage was carried out for 3 hours at room temperature, precipitated with 1.5L of precooled methyl tert-butyl ether, centrifuged and washed twice with methyl tert-butyl ether. After drying with nitrogen, 11.1g of crude linear peptide with a purity of 81.8% was obtained.

Example 16: synthesis of crude Linear peptide

Fragment A (10.3g, 7.0mmol) obtained in example 1, fragment C (12.3g, 7.0mmol) obtained in example 11, cl-HOBt (1.19g, 7 mmol) and HBTU (2.66g, 7 mmol) were weighed and dissolved in 75mL DMF, the temperature was reduced to-15 to 5 ℃ or below, DIPEA (1.8g, 14mmol) was slowly added with stirring, and after completion of dropwise addition, the reaction solution was slowly warmed to room temperature and stirred for further reaction for 4 hours. The reaction was monitored by HPLC. The reaction mixture was poured into 375mL of purified water with stirring, filtered, and the filter cake was washed with 0.1N HCl, saturated NaHCO ₃ Washing with aqueous solution (2 times), pure water (3 times), diethyl ether (2 times) in 100mL portions, and drying under vacuum gave fragment D21.2 g, 95.2% yield (full protection peptide due to poor solubility and low polarity, no suitable analytical method to detect).

Fragment D was treated with 170mL of lysate TFA EDT H ₂ Phenol: TIS (volume ratio) = 85. The cleavage was carried out at room temperature for 2 hours, precipitated with 1.7L of precooled methyl tert-butyl ether, centrifuged and washed twice with methyl tert-butyl ether. After drying with nitrogen, 11.6g of linear crude peptide with a purity of 81.5% was obtained.

Example 17: synthesis of crude Linear peptide

Fragment A (10.3g, 7.0mmol) obtained in example 1, fragment C (12.3g, 7.0mmol) obtained in example 12, cl-HOBt (1.19g, 7 mmol) and HBTU (2.66g, 7 mmol) were weighed out and dissolved in 75mL of DMF, the temperature was reduced to-15 to 5 ℃ or lower, DIPEA (1.8g, 14mmol) was slowly added thereto with stirring, and after dropwise addition, the mixture was addedThe reaction was allowed to slowly warm to room temperature and the reaction was stirred for an additional 4 hours. The reaction was monitored by HPLC. The reaction mixture was poured into 375mL of purified water with stirring, filtered, and the filter cake was washed with 0.1N HCl, saturated NaHCO ₃ Washing with aqueous solution (2 times), pure water (3 times), diethyl ether (2 times) in 100mL each time, and drying under vacuum gave fragment D21.2 g, yield 95.2% (fully protected peptide was not detected by appropriate analytical methods due to poor solubility and low polarity).

Fragment D was treated with 90mL of lysate TFA EDT H ₂ Phenol: TIS (volume ratio) = 85. The cleavage was carried out for 2 hours at room temperature, precipitated with 900mL of precooled methyl tert-butyl ether, centrifuged and washed twice with methyl tert-butyl ether. After drying with nitrogen, 10.9g of linear crude peptide with purity of 82.3% was obtained.

Example 18: synthesis of procapsipeptide

Any of the linear crude peptides obtained in examples 13 to 17 was dissolved in 10L of purified water; adjusting pH to 8.5 with concentrated ammonia water, first pair of disulfide bonds (Cys) ⁴ And Cys ¹² ) During oxidation, 1.25mL of 30% hydrogen peroxide is added, the oxidation time is 1 hour, and the reaction end point is judged by HPLC. Second pair of disulfide bonds (Cys) ⁷ -Cys ¹⁵ ) During oxidation, the pH value is adjusted to 3.5 by glacial acetic acid, 3.5g of iodine is added, oxidation is carried out for 1 hour, the end point of the reaction is judged by HPLC, and vitamin C is added to quench excessive iodine after the reaction is finished. Using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing. Adopting NOVASEPRP-HPLC system, wavelength is 220nm, chromatographic column is reversed phase C18 column, TFA water solution with volume fraction of 0.1% is mobile phase A, and acetonitrile is mobile phase B. Purifying, desalting, collecting the desired peak fraction, concentrating by rotary evaporation, and lyophilizing to obtain purified procainatide (refined procainatide). The mass spectrum of the procatide is 1682.0 as shown in figure 1, the molecular weight of the procatide is consistent with that of a monoisotope, the molecular weight of the procatide is +1, and the purity of the refined peptide can reach 99.78 percent as shown in figure 2.

Examples 13-17 prucalotide obtained after purification of crude linear peptide was designated as products 1-5, respectively, and the results of each set of experiments are as follows:

product(s)	Pracannapeptide protamine	Overall yield of	Purity of
				1	4.2g	33.2％	98.89％
2	4.5g	35.2％	99.78％
				3	5.0g	40.2％	99.77％
4	4.7g	37.3％	99.52％
				5	4.8g	37.8％	99.40％

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. A preparation method of procainatide is characterized by comprising the following steps:

step 1: coupling 4-hydroxybenzyl alcohol and Fmoc-Leu-OH by taking 2-CTC resin as a solid phase carrier to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin;

and 2, step: the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is used as a solid phase carrier, fmoc-Cys (Acm) -OH and 8 peptide fragment B are sequentially coupled by an Fmoc solid phase synthesis method, and after Fmoc protection is removed, cleavage is carried out to obtain 10 peptide fragment C with protected side chain;

the 8 peptide fragment B is Fmoc-Cys (Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -OH；

The 10 peptide fragment C is H-Cys (Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -4-OPhCH ₂ OH；

And 3, step 3: under the action of a coupling reagent, the 6 peptide fragment A is coupled with the 10 peptide fragment C to obtain a side chain protected peptide fragment D;

the 6 peptide fragment A is Boc-Asn (Trt) ¹ -Asp(OtBu) ² -Glu(OtBu) ³ -Cys(Trt) ⁴ -Glu(OtBu) ⁵ -Leu ⁶ -OH；

Said peptide fragment D is Boc-Asn (Trt) ¹ -Asp(OtBu) ² -Glu(OtBu) ³ -Cys(Trt) ⁴ -Glu(OtBu) ⁵ -Leu ⁶ -Cys(Acm) ⁷ -Val ⁸ -Asn(Trt) ⁹ -Val ¹⁰ -Ala ¹¹ -Cys(Trt) ¹² -Thr(tBu) ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -4-OPhCH ₂ OH；

And 4, step 4: cleaving the peptide fragment D, and deprotecting a portion of the reactive side chain groups to obtain a linear crude peptide;

the linear crude peptide is H-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys(Acm) ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys(Acm) ¹⁵ -Leu ¹⁶ -OH；

And 5: the obtained linear crude peptide is oxidized in the first step and oxidized in the second step to form two pairs of disulfide bonds, and the crude peptide of the procapsipeptide is obtained.

2. The method of preparing as claimed in claim 1, wherein the 6 peptide fragment a and the 8 peptide fragment B are synthesized by Fmoc solid phase synthesis method.

3. The method of claim 1, wherein the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin of step 1 is synthesized by: coupling 4-hydroxybenzyl alcohol of Wang resin under the action of organic base by using 2-CTC resin as a solid-phase carrier, and then coupling Fmoc-Leu-OH under the action of a coupling agent to obtain Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin;

preferably, the substitution degree of the Fmoc-Leu-4-hydroxybenzyl alcohol-2-CTC resin is 0.21 to 0.81mmol/g, and preferably 0.31 to 0.60mmol/g.

4. The preparation process according to claim 3, characterized in that the degree of substitution of the 2-CTC resin is between 0.30 and 1.70mmol/g, preferably between 0.70 and 1.39mmol/g;

the organic base is selected from DIPEA, pyridine or TMP, preferably DIPEA;

the coupling agent is a combination of DIPCDI and a compound X selected from HOAt or HOBt; preferably the coupling agent is a combination of DIPCDI and HOBt;

preferably, the molar ratio of the 4-hydroxybenzyl alcohol to the organic base is 1:1-5, preferably 1.5; preferably, the 4-hydroxybenzyl alcohol is used in an amount of 2 to 5 equivalents, preferably 3 equivalents.

5. The method according to claim 4, wherein a catalyst is further added when Fmoc-Leu-OH is coupled; preferably, the catalyst is DMAP;

preferably, the molar ratio of Fmoc-Leu-OH, DIPCDI, compound X and DMAP is 1.2; preferably, fmoc-Leu-OH is used in an amount of 2 to 5 equivalents, preferably 3 equivalents.

6. The method of claim 1, wherein the coupling in step 2 is performed with a coupling agent which is a combination of DIPCDI and Compound X or a combination of DIPEA, compound X and Compound Y; the compound X is selected from HOAt or HOBt, and the compound Y is selected from PyAOP, pyBOP, HATU, HBTU or TBTU;

preferably, the molar ratio of Fmoc-Cys (Acm) -OH or 8 peptide fragment B, DIPCDI and compound X is 1;

preferably, the molar ratio of Fmoc-Cys (Acm) -OH or 8 peptide fragment B, DIPEA, compound X and compound Y is 1;

preferably, fmoc-Cys (Acm) -OH or 8 peptide fragment B is used in an amount of 2 to 5 equivalents, preferably 3 equivalents;

preferably, when Fmoc-Cys (Acm) -OH is coupled, the coupling agent is a composition of DIPCDI and HOBt;

preferably, when the 8 peptide fragment B is coupled, the coupling agent is a combination of DIPEA, HOBt and HBTU;

preferably, the cleavage is carried out using a 1-% TFA in dichloromethane, 20-% TFE in dichloromethane, or 20-% HFIP in dichloromethane as the cleavage solution, preferably 20-% TFE in dichloromethane.

7. The method according to claim 1, wherein the step 3 comprises dissolving the 6-peptide fragment A, the 10-peptide fragment C and the coupling reagent in a reaction solvent, cooling to-15-5 ℃, adding DIPEA, and performing a coupling reaction;

preferably, the reaction solvent is selected from DMF or NMP, preferably DMF;

preferably, the coupling reagent is a combination of compound a selected from HOAt, HOBt or Cl-HOBt and compound B selected from PyAOP, pyBOP, HATU, HBTU or TBTU; preferably, the coupling agent is a combination of Cl-HOBt and HBTU;

preferably, the molar ratio of the 10 peptide fragment C, 6 peptide fragment a, coupling reagent and DIPEA is 1;

preferably, the molar ratio of the 10 peptide fragment C, the 6 peptide fragment A, cl-HOBt, HBTU and DIPEA is 1.

8. The method of claim 1, wherein the cleavage in step 4 is performed with TFA or H ₂ A mixture formed by more than one of O, TIS, EDT, anisole, thioanisole and phenol is used as a lysate; preferred are TFA, EDT, H ₂ Mixture of O, phenol and TIS, preferably TFA, EDT, H ₂ Volume ratio of O, phenol and TIS is 85.

9. The method according to claim 1, wherein the first oxidation in step 5 is to oxidize Cys in the linear crude peptide with an oxidizing agent ⁴ And Cys ¹² A first pair of disulfide bonds; the oxidant for the first step of oxidation is selected from DMSO, oxygen or hydrogen peroxide, and is preferably hydrogen peroxide;

the second oxidation in step 5 is to use iodine to make Cys in the linear crude peptide ⁷ And Cys ¹⁵ Forming a second pair of disulfide bonds therebetween.

10. The method according to claim 1, further comprising purifying and lyophilizing the crude procapsipeptide;

preferably, the purification is performed by reverse phase high pressure liquid chromatography.

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