CN112062813B

CN112062813B - Synthesis method of desmopressin

Info

Publication number: CN112062813B
Application number: CN201910497054.6A
Authority: CN
Inventors: 陈学明; 袁慧星; 宓鹏程; 陶安进; 袁建成
Original assignee: Gansu Changee Bio Pharmaceutical Ltd; Hybio Pharmaceutical Wuhan Co ltd
Current assignee: Gansu Changee Bio Pharmaceutical Ltd; Hybio Pharmaceutical Wuhan Co ltd
Priority date: 2019-06-10
Filing date: 2019-06-10
Publication date: 2022-05-03
Anticipated expiration: 2039-06-10
Also published as: WO2020248372A1; CN112062813A

Abstract

The invention discloses a synthesis method of desmopressin, which comprises the following steps: 1) selecting solid phase carrier resin; 2) synthesizing a backbone peptide resin; 3) cracking, and removing resin and protecting groups; 4) and (3) liquid-phase electrochemical oxidation coupling of the disulfide bond to obtain the desmopressin. The method can avoid the problem of specific selectivity of the reaction, can improve the utilization rate of the fragment peptide resin, the utilization rate of the main chain fragment and the utilization rate of atoms, thereby reducing the production cost, avoiding the use of chemical oxidants of iodine and hydrogen peroxide, being more environment-friendly and more conforming to the concept of green chemistry.

Description

Synthesis method of desmopressin

Technical Field

The invention belongs to the technical field of polypeptide drug production, and relates to a synthesis method of desmopressin.

Background

Desmopressin is a structural analogue of natural argirelin, and is obtained by modifying the chemical structure of natural hormone at two places, namely 1-cysteine is used for removing amino and 8-D-arginine is used for replacing 8-L-arginine. The peptide sequence of desmopressin is shown as formula I, wherein Mpa and Cys form a ring through a disulfide bond.

Desmopressin is mainly used for treating central diabetes insipidus, nocturnal enuresis, hemophilia and the like, is also used for testing the concentrating function of renal urine, and can also be used for hemostasis after operation.

With the increasing development of Fmoc solid-phase peptide synthesis methods and the gradual maturity of preparation processes, currently, liquid-phase synthesis is gradually replaced by solid-phase synthesis in polypeptide synthesis. For the synthesis of desmopressin, the synthesis method mainly adopted is the Fmoc solid-phase polypeptide synthesis strategy. The key to the coupled synthesis of desmopressin in the conventional process is the construction of disulfide bonds, and methods for constructing disulfide bonds used in conventional peptide synthesis include solid phase oxidation and liquid phase oxidation. Wherein the solid-phase oxidant comprises iodine and hydrogen peroxide; the liquid phase oxidation method comprises an air oxidation method, an iodine/acetic acid system oxidation method and a hydrogen peroxide oxidation method, and the methods cannot ensure the specific selectivity of the reaction, inevitably produce mismatched impurities, have low product purity and increase the difficulty of purification. Patent CN101372505A introduces a pseudo-dilution reaction to avoid the problem of polypeptide inter-chain disulfide mismatch in liquid phase reaction, wherein disulfide bond is synthesized by solid phase oxidation, but the solid phase disulfide bond reaction is heterogeneous reaction, the reaction conversion rate is low, and the yield of desmopressin product is low. Meanwhile, iodine is used during oxidation, so that the environment is polluted, and the method is not favorable for environmental protection and sustainable development of the industry.

The development of atom economy reaction and environmental protection become the core content of green chemical research, and from the atom economy point of view, the conventional sequential coupling method cannot avoid the problem of specific selectivity of the construction of the disulfide bond of the linear peptide, so that the effective utilization rate of a main chain fragment is low, namely the atom utilization rate is low when the disulfide bond of the L-cysteine and the mercaptopropionic acid is constructed. In a general solid-phase synthesis operation process, the dosage of amino acid is generally 3-5 eqv, and when the effective utilization rate of a main chain fragment is low, a large amount of corresponding amino acid is wasted.

In view of the above situation, the invention adopts Fmoc/tBu strategy to connect main chain amino acid residues in a solid phase manner, and crude peptide after cracking is coupled with side chains of cysteine and mercaptopropionic acid in a liquid phase manner by an electrochemical method to form a disulfide bond, and finally purified to obtain desmopressin. Therefore, the problem of specific selectivity of the reaction can be avoided, the utilization rate of the main chain fragments is improved, the utilization rate of atoms is improved, the production cost is reduced, the use of oxidants such as iodine and hydrogen peroxide is avoided, the preparation method is more environment-friendly, and the preparation method accords with the concept of green chemistry.

Disclosure of Invention

In order to solve the problems in the background art, the invention aims to provide a synthesis method of desmopressin, which can avoid the problem of specific selectivity of reaction, improve the utilization rate of backbone fragments, improve the utilization rate of atoms, reduce the production cost and protect the environment.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a synthesis method of desmopressin comprises the following steps:

1) selecting solid phase carrier resin;

2) synthesizing a backbone peptide resin;

3) cracking, and removing resin and protecting groups;

4) and (3) liquid-phase electrochemical oxidation coupling of the disulfide bond to obtain the desmopressin.

Further, the solid phase carrier Resin in 1) is selected from Rink Amide AM Resin, Rink Amide MBHA Resin or Rink Amide Resin, and the substitution degree of the solid phase carrier Resin is 0.1-1.0mmol/g, preferably 0.2-0.8mmol/g, more preferably 0.3-0.5 mmol/g.

Further, Fmoc-AA-OH is coupled in the order of C-terminal to N-terminal in step 2) as Fmoc-Gly-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Cys (P) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Phe-OH, Fmoc-Tyr (tBu) -OH and Mpa (Trt) -OH. Wherein P in Fmoc-Cys (P) -OH is Trt, Mmt or Dpm. The solid phase polypeptide synthesis method comprises the following steps: 1) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method; 2) dissolving and activating appropriate amount of amino acid to be coupled and coupling agent in solvent, and adding into solid phase reaction column together until the reaction termination is detected by detection method; 3) repeat 1) and 2). Wherein the Fmoc removal reagent is 20% piperidine/DMF solution (DBLK), i.e. piperidine: DMF (volume ratio) is 1: 4. The detection method used in the reaction is any method known in the art for this purpose, such as chromatography or chemical calibration, preferably using a reagent that can determine the end of the reaction, preferably ninhydrin, when ninhydrin is used, indicating a free amine in the polypeptide if the resin develops color, i.e., no protecting group on the amine.

Further, the reaction of step 2) is carried out in a solid phase reaction column. The solid-phase reaction column used in the present invention is not particularly limited, and any solid-phase reaction column can be used for this purpose. Further, the time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours; the pressure is preferably normal pressure, and may be suitably increased or decreased; the temperature is preferably room temperature (i.e., 20. + -. 5 ℃ C.), and may be suitably elevated or reduced.

Further, the synthesis of the backbone peptide resin in 2) specifically comprises the following steps:

a. adding the solid phase carrier resin into a solid phase reaction column, removing the Fmoc protecting group by using 20% piperidine/N, N-dimethylformamide solution, and then washing;

b. adding Fmoc-Gly-OH and a coupling agent into N, N-dimethylformamide, activating at 0-5 ℃, adding into a solid phase reaction column, reacting at normal pressure and room temperature for 1.5-4h, and washing; then removing Fmoc protecting groups by using 20% piperidine/N, N-dimethylformamide solution and washing; the above process is repeated to couple Fmoc-D-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Cys (P) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Phe-OH, Fmoc-Tyr (tBu) -OH and Mpa (Trt) -OH in sequence, wherein P in Fmoc-Cys (P) -OH is Trt, Mmt or Dpm.

Further, the coupling agent is a combination of DIPCDI and a compound A or a combination of DIPEA and a compound A and a compound B, wherein the compound A is selected from HOBt or HOAt, the compound B is selected from PyBOP, PyAOP, HATU, HBTU or TBTU, and the molar ratio of the components in the coupling agent is DIPCDI: compound a ═ 1.2: 1.1, DIPEA: a compound A: compound B ═ 2.0: 1.1: 1.0. preferably a combination of DIPCDI and Compound A.

Further, the reaction of step 2) is preferably carried out by swelling the resin prior to coupling, washing and swelling steps can be carried out in the art using any reagent that achieves this purpose, including DMF, NMP, dichloromethane, and the like, preferably DMF.

Further, the lysis solution for cleavage is TFA or H₂O, PhOMe, thioanisole, the preferred proportion is TFA: h₂O: PhOMe: benzylsulfide ═ 90: 5: 4: 1(V: V).

Further, the liquid-phase electrochemical oxidative coupling disulfide bond is oxidative coupling cysteine and mercaptopropionic acid, the used positive and negative electrodes are platinum electrodes, the used current is 5mA-20mA, preferably 10-15mA, the reaction temperature is 10 ℃ to 100 ℃, preferably 35 ℃, and the electrolyte is tetrabutylammonium perchlorate, tetrabutylammonium tetrafluoroborate or tetrabutylammonium hexafluorophosphate, preferably tetrabutylammonium tetrafluoroborate.

Further, 4) is followed by a purification step, wherein a reversed-phase high-pressure liquid chromatography is adopted for purification to prepare the refined peptide, and the reversed-phase high-pressure liquid chromatography comprises the steps of taking reversed-phase octadecylsilane as a stationary phase and taking 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, collecting target peak fractions, concentrating and freeze-drying.

The invention has the beneficial effects that the invention provides a method for synthesizing desmopressin, which comprises the steps of connecting main chain amino acid residues in a solid phase manner through an Fmoc/tBu strategy, cracking and precipitating to obtain crude peptide, then carrying out liquid-phase oxidative coupling on cysteine through an electrochemical method, and purifying to obtain the desmopressin. The method can avoid the problem of specific selectivity of the reaction, can improve the utilization rate of the fragment peptide resin, the utilization rate of the main chain fragment and the utilization rate of atoms, thereby reducing the production cost, avoiding the use of chemical oxidants of iodine and hydrogen peroxide, being more environment-friendly and more conforming to the concept of green chemistry.

Drawings

FIG. 1 is a reaction scheme of the synthesis of desmopressin according to the present invention.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

The meanings of the abbreviations used in the specification and claims are listed in the following table:

example 1: peptide resin synthesis

Rink Amide resin (52g, substitution 0.48mmol/g) was weighed into a solid phase reaction column, washed with DMF 2 times, the resin was then swelled with DMF for 30 min, DBLK was added for deprotection (5min +7min), and the resin was washed with DMF 6 times.

Fmoc-Gly-OH (22.3g, 75mmol) and HOBT (12.159g, 90mmol) are weighed and added into DMF (250mL) for dissolution, the mixture is cooled to 0-5 ℃ in ice water bath, DIPCDI (12.75mL, 75mmol) is added for activation for 5min, the activated solution is added into a reaction column, nitrogen is bubbled for 2 h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1 h if the resin is colored). After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin.

Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin.

Fmoc-Pro-OH (25.3g, 75mmol) and HOBT (12.159g, 90mmol) are weighed and added into DMF (250mL) for dissolution, cooled to 0-5 ℃ in ice water bath, DIPCDI (12.75mL, 75mmol) is added for activation for 5min, the activated solution is added into a reaction column, nitrogen is bubbled for 2 h at room temperature, and the reaction end point is detected by ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin with ninhydrin.

Fmoc-Cys (Trt) -OH (43.9g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then activated for 5min by adding DIPCDI (12.75mL, 75mmol), and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 h to detect the end point of the reaction with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin with ninhydrin.

Fmoc-Asn (Trt) -OH (44.8g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 h to detect the end point of the reaction with ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color.

Fmoc-Gln (Trt) -OH (45.8g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 h, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects the color of the resin.

Fmoc-Phe-OH (29.1g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, DIPCDI (12.75mL, 75mmol) was added for activation for 5min, the activated solution was added to the reaction column, and the reaction end point was detected by ninhydrin after bubbling nitrogen at room temperature for 2 h. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects the color of the resin.

Fmoc-Tyr (tBu) -OH (34.5g, 75mmol) and HOBT (12.159g, 90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours to detect the end of the reaction with ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color.

Mpa (Trt) -OH (26.1g, 75mmol) and HOBT (12.159g, 90mmol) are weighed and added into DMF (250mL) for dissolution, the mixture is cooled to 0-5 ℃ in ice water bath, DIPCDI (12.75mL, 75mmol) is added for activation for 5min, the activated solution is added into a reaction column, nitrogen is bubbled for 2 h at room temperature, and the reaction end point is detected by ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. After washing with DCM for 3 times, methanol shrinkage and vacuum drying, 57.65g of peptide resin was obtained, the resin weight gain is 85.0%.

Example 2: cleavage peptide resin

57.65g of the peptide resin obtained in example 1 was put into a 2L single-necked flask, and a lysis solution (TFA: H) prefreezed to-15 ℃ was added₂O: PhOMe: benzylsulfide ═ 90: 5: 4: 1(V: V), 600ml), stirred at room temperature for 4 hours, the resin was filtered and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 6L of glacial ethyl ether for precipitation. Centrifugation, washing with glacial ethyl ether 5 times, and nitrogen blow-drying gave 27.80 g of crude peptide with a purity of 88.9% and a yield of 99.9%.

Example 3: liquid oxidative coupling of disulfide bonds

27.80 g of the crude peptide obtained in example 2 was put into a 1000ml three-necked flask, and 500ml of acetonitrile and 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate were sequentially added, followed by insertion of a platinum electrode (anode: 15 mm. times.15 mm. times.0.3 mm, cathode: 15 mm. times.15 mm. times.0.3 mm)), and the reaction was stirred at 35 ℃ for 48 hours with the current controlled at 12 mA. After the reaction is completed, the energization is stopped.

Example 4: preparation of refined peptide by reversed phase chromatography

The oxidized liquid obtained in example 3 was purified by high performance liquid chromatography, and the desired peak fraction was collected, concentrated and lyophilized using reverse-phase octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase. 20.8g of refined peptide is obtained, the purity is 99.98 percent, and the yield is 74.8 percent.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

Claims

1. A synthesis method of desmopressin is characterized by comprising the following steps:

1) selecting solid phase carrier resin;

2) synthesizing a backbone peptide resin;

3) cracking, and removing resin and protecting groups;

4) performing liquid-phase electrochemical oxidation coupling on the disulfide bond to obtain desmopressin;

2) the synthesis of the backbone peptide resin in (1) specifically comprises the following steps:

b. adding Fmoc-Gly-OH and a coupling agent into N, N-dimethylformamide, activating at 0-5 ℃, adding into a solid phase reaction column, reacting at normal pressure and room temperature for 1.5-4h, and washing; then removing Fmoc protecting groups by using 20% piperidine/N, N-dimethylformamide solution and washing; repeating the above process, sequentially coupling Fmoc-D-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Cys (P) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Phe-OH, Fmoc-Tyr (tBu) -OH and Mpa (Trt) -OH, wherein P in Fmoc-Cys (P) -OH is Trt, Mmt or Dpm;

The liquid-phase electrochemical oxidation coupling disulfide bond is oxidation coupling cysteine and mercaptopropionic acid, the used positive and negative electrodes are platinum electrodes, the used current is 5mA-20mA, the reaction temperature is 10 ℃ to 100 ℃, and the electrolyte is tetrabutylammonium perchlorate, tetrabutylammonium tetrafluoroborate or tetrabutylammonium hexafluorophosphate.

2. The method of claim 1, wherein the solid phase carrier Resin in 1) is selected from Rink Amide AM Resin, Rink Amide MBHA Resin and Rink Amide Resin, and the substitution degree of the solid phase carrier Resin is 0.1-1.0 mmol/g.

3. The method of claim 2, wherein the degree of substitution of the solid support resin is 0.3 to 0.5 mmol/g.

4. The process of claim 1, wherein the coupling agent is a combination of DIPCDI and Compound A or a combination of DIPEA and Compound A and Compound B, wherein Compound A is selected from HOBt or HOAt, Compound B is selected from PyBOP, PyAOP, HATU, HBTU or TBTU, and the ratio of the components in the coupling agent is, in terms of mole ratios, DIPCDI: compound a = 1.2: 1.1, DIPEA: a compound A: compound B = 2.0: 1.1: 1.0.

5. The method of claim 1, wherein step a is carried out by adding the solid phase carrier resin into a solid phase reaction column, swelling with a solvent, and washing after removing Fmoc protecting group with 20% piperidine/N, N-dimethylformamide solution.

6. The method of claim 1, wherein the cleavage lysate is a combination of TFA, H2O, PhOMe, and thioanisole, and the TFA, H2O, PhOMe, and the combination of TFA and H₂O, PhOMe, the volume ratio of the thiobenzol sulfide is 90: 5: 4: 1.

7. the method for synthesizing desmopressin according to claim 1, wherein the current for liquid-phase electrochemical oxidative coupling of disulfide bonds is 10-15mA, the reaction temperature is 35 ℃, and the electrolyte used is tetrabutylammonium tetrafluoroborate.

8. The method for synthesizing desmopressin according to claim 1, wherein the step 4) is followed by a purification step of purifying by reverse-phase high-pressure liquid chromatography, wherein the reverse-phase high-pressure liquid chromatography comprises collecting a target peak fraction by using reverse-phase octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, concentrating and freeze-drying.