CN113845587B

CN113845587B - Synthetic method of bivalirudin

Info

Publication number: CN113845587B
Application number: CN202111449570.5A
Authority: CN
Inventors: 刘志国; 魏祝宇; 潘海良; 叶有志; 汪岳斌
Original assignee: Zhejiang Pai Peptide Biology Co ltd Nanjing Branch
Current assignee: Zhejiang Paitide Biological Co ltd Nanjing Branch
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-02-18
Anticipated expiration: 2041-12-01
Also published as: CN113845587A

Abstract

The invention discloses a synthetic method of bivalirudin, belonging to the technical field of preparation of polypeptide medicaments. The synthetic method of bivalirudin comprises the following steps: activating resin, namely activating the resin by adopting activating liquid to obtain the activated resin, wherein the activating liquid comprises dichloromethane, B-butyrolactone and nadic anhydride; synthesizing Leu-resin, namely synthesizing the swelled activated resin and Fmoc-Leu into Leu-resin by adopting a solid-phase synthesis method; step (3), synthesizing bivalirudin-resin, and synthesizing the bivalirudin-resin by adopting a solid-phase synthesis method; and (4) deprotection, namely, deprotection of bivalirudin-resin to obtain bivalirudin. The bivalirudin synthesized by the synthesis method has higher yield.

Description

Synthetic method of bivalirudin

Technical Field

The invention relates to the technical field of preparation of polypeptide medicaments, in particular to a synthetic method of bivalirudin.

Background

Bioactive polypeptides have their unique advantages as pharmaceuticals: firstly, the molecular weight is small, the immunogenicity is avoided, and the preparation method is safe; secondly, the structure is relatively simple, the function is clear, and the effects of specificity and side effect are small; thirdly, the molecular is small, the synthesis is easy, the structure is easy to reform, and the production cost is low; fourth, polypeptide drugs are easily absorbed from multiple routes, so the administration route can be diversified (e.g., oral administration, spray, transdermal absorption, etc.); fifthly, the synthesized polypeptide has high purity and does not have the problem of heat source. For this reason, the research on polypeptide drugs at home and abroad is particularly important.

Bivalirudin is an artificial synthetic anticoagulant based on hirudin, and is a polypeptide chain consisting of 20 amino acids, and the weight of the molecule is 2180 daltons. A pioneering publication of the pharmacological properties of bivalirudin was published in 1989. The drug can reversibly bind fibrin thrombin. Unlike common heparin, bivalirudin does not require a cofactor to assist it in exerting anticoagulant properties, and bivalirudin targets to inhibit thrombin (platelet agonist), thereby effectively reducing platelet reactivity, which is a biological property very beneficial to patients receiving percutaneous coronary intervention.

Currently there are 3 main approaches to peptide synthesis: chemical method, recombinant DNA method, enzyme method. In general, recombinant DNA technology requires a long, expensive research and development stage, and has problems of low expression efficiency and difficult product extraction and recovery in the fermentation stage. Its application is very limited. Enzymatic initiatives are late, and chemical methods are still used most. The chemical synthesis of polypeptides is well established by both liquid phase and solid phase methods. In recent decades, the solid phase method for synthesizing the polypeptide is more time-saving, labor-saving, material-saving, convenient for computer control and convenient for popularization and promotion.

Disclosure of Invention

The invention aims to provide a synthetic method of bivalirudin with high purity; another object of the present invention is to provide a method for activating a resin having a high degree of substitution.

In order to achieve the purpose of the invention, the following technical scheme is adopted.

The invention discloses a synthetic method of bivalirudin, which comprises the following steps:

activating resin, namely activating the resin by adopting activating liquid to obtain the activated resin, wherein the activating liquid comprises dichloromethane, B-butyrolactone and nadic anhydride;

synthesizing Leu-resin, namely synthesizing the swelled activated resin and Fmoc-Leu into Leu-resin by adopting a solid-phase synthesis method;

step (3), synthesizing bivalirudin-resin, and synthesizing the bivalirudin-resin by adopting a solid-phase synthesis method;

deprotection, namely, deprotection is carried out on bivalirudin-resin to obtain a bivalirudin crude product;

the resin is activated by the activating solution containing B-butyrolactone and nadic anhydride, so that the swelling property of the resin can be enhanced, the sites on the resin for binding amino acid are more fully exposed, the amino acid is more easily connected with the resin, the linking efficiency of the amino acid is increased, and the yield of bivalirudin is increased.

Preferably, in step (1), the resin is Wang resin.

Preferably, in the step (1), the weight ratio of the dichloromethane, the B-butyrolactone and the nadic anhydride in the activating solution is 540-780:30-90: 3-45.

Preferably, the specific operation of step (1) comprises:

adding 30-60g of Wang resin into 1000g of 500-one activating solution, and carrying out oscillation reaction for 1-4h at 120r/min of 100-one; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the weight ratio of the dichloromethane, the B-butyrolactone and the nadic anhydride in the activating solution is 540-780:30-90: 3-45.

Along with the progress of the synthesis reaction, when the substitution degree on the Wang resin is gradually increased, the internal space of the resin is reduced, a certain steric effect is generated to reduce the reaction efficiency, the swelling property of the resin is increased, the internal space of the resin can be effectively increased, the steric effect is further reduced, and the reaction efficiency is prevented from being greatly reduced. The activating solution is adopted to activate the resin, so that the swelling property of the resin is improved, the reaction efficiency of the solid phase reaction is improved, and the yield and the purity are finally improved.

Preferably, in the step (2), the amount ratio of the swollen activated resin to Fmoc-Leu is 30-60g:0.05-0.15 mol.

Preferably, in step (2), the molar ratio of Fmoc-Leu, DCC, HOBt and DMAP is 1:1:1: 0.01-0.1.

Preferably, the specific operation of step (2) comprises:

adding Fmoc-Leu and DMF into the swollen activated resin, placing the swollen activated resin in an ice bath, continuously adding DCC, HOBt and DMAP, and stirring the mixture overnight after the ice bath; after stirring, filtering, washing and drying to obtain Fmoc-Leu-resin; and adding a pyridine/dichloromethane/DMF mixed solution into the Fmoc-Leu-resin, performing suction filtration after reaction, washing and drying to obtain the Leu-resin.

Preferably, the specific operation of step (2) comprises:

adding 0.05-0.15mol of Fmoc-Leu and 0.05-0.15mol of DMF into 30-60g of the swelled activated resin, placing the mixture in an ice bath, and continuously adding 0.05-0.15mol of DCC, 0.05-0.15mol of HOBt and 0.001-0.005mol of DMAP, wherein the molar ratio of Fmoc-Leu, DCC, HOBt and DMAP is 1:1: 0.01-0.1; stirring overnight at 100-120r/min at 2-4 ℃ after ice bath for 1-5 h; after stirring, filtering, washing and drying to obtain Fmoc-Leu-resin; adding 90-120g of pyridine/dichloromethane/DMF mixed solution (V/V/V =0.25-1:1: 1-3) into Fmoc-Leu-resin, reacting for 20-50min, filtering, washing and drying to obtain Leu-resin.

More preferably, the determination of the degree of substitution of the resin is performed after step (2).

More preferably, the specific operation of determining the degree of substitution of the resin comprises:

adding Fmoc-Leu-resin into a piperidine/DMF mixed solution, oscillating at room temperature and filtering; adding methanol into the filtrate, uniformly mixing, diluting and measuring the absorbance at 295 nm; preparing blank control and standard substance according to the above treatment method, and measuring absorbance; the degree of substitution is calculated after the absorbance is measured, and the calculation formula is as follows:

degree of substitution (mmol/g) = OD_{Resin composition}×W_{Standard article}×1000/（OD_{Standard article}×W_{Standard article}×297.3）

The substitution degree of the resin is an important reason for limiting the yield of the solid-phase synthesized polypeptide, the degree of substitution also influences the amount of impurities, and whether the reaction is sufficient or not can be judged in time by measuring the degree of substitution, so that the high efficiency of polypeptide synthesis is ensured.

Preferably, the specific operation of step (3) comprises:

and (3) sequentially coupling Try, Glu, Pro, Ile, Glu, Phe, Asp, Gly, Asn, Gly, Pro, Arg, Pro and Phe according to the operation method in the step (2) until obtaining the bivalirudin-resin, namely Phe-Pro-Arg-Pro-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-resin. The solid-state polypeptide synthesis method is used for connecting amino acids from a carboxyl terminal to an amino terminal in sequence, which is beneficial to improving the synthesis efficiency.

Preferably, the specific operation of step (4) comprises:

adding the TFA solution into the bivalirudin-resin prepared in the step (3), stirring in the dark and filtering; adding anhydrous ether into the filtrate, standing after white precipitate is separated out, taking the precipitate and freeze-drying after the solution is layered and the upper layer is clear to obtain bivalirudin.

More preferably, the specific operation of step (4) comprises:

adding 100-150 parts by weight of 90-98% TFA solution into 10-20 parts by weight of bivalirudin-resin prepared in the step (3), stirring for 10-60min in the dark, and filtering; and adding 100-200 parts of anhydrous ether into the filtrate, standing at the temperature of minus 20-10 ℃ after white precipitate is separated out, taking the precipitate after the solution is layered and the upper layer is clarified, and freeze-drying to obtain the bivalirudin.

Still further preferably, the specific operation of step (4) includes:

taking 100-150 parts by weight of 90-98% TFA solution, 10-30 parts by weight of (S) -glycidol and 5-15 parts by weight of 10-50% 4-bromobutyric acid solution, uniformly mixing, adding 10-20 parts by weight of bivalirudin-resin prepared in the step (3), stirring for 10-60min in dark and filtering; and adding 100-200 parts of anhydrous ether into the filtrate, standing at-20 to-10 ℃ after white precipitate is separated out, taking the precipitate after the solution is layered and the upper layer is clarified, and freeze-drying to obtain bivalirudin.

During deprotection, unnecessary impurities may be generated due to the residue of a protective group or the breakage of a peptide bond, and by adding (S) -glycidol and a 4-bromobutyric acid solution during deprotection, the residue of the protective group can be reduced, the purity of bivalirudin is improved, impurities are further reduced, and the subsequent purification step is facilitated. In addition, the resin used in the invention has better swelling property, although the utilization rate of the resin is improved, impurities are likely to be generated more easily, which causes difficulty in purification, and the addition of the (S) -glycidol and 4-bromobutyric acid solution in the deprotection step can avoid the generation of a large amount of impurities, thereby reducing the difficulty in the purification step.

The invention also discloses an activating solution which comprises dichloromethane, B-butyrolactone and nadic anhydride in a weight ratio of 540-.

The invention also discloses the application of the activating solution in improving the substitution degree of the resin.

Compared with the prior art, the invention has the beneficial effects that:

the method for synthesizing bivalirudin uses the pretreated resin, and the addition of B-butyrolactone and nadic anhydride during pretreatment can increase the swelling degree of the resin and make the swelling ratio reach more than 1.6, thereby better exposing the site for combining amino acid, making the amino acid more easily combined with the resin, improving the utilization rate of the resin and increasing the yield of the bivalirudin. In addition, the (S) -glycidol and 4-bromobutyric acid solution are used in the deprotection step, so that the deprotection is more thorough, and the generation of impurities is reduced, thereby increasing the yield of bivalirudin, and the yield can reach more than 70%. In addition, the B-butyrolactone, nadic anhydride, (S) -glycidol and 4-bromobutyric acid solution added in the synthesis process do not influence the activity of bivalirudin.

Drawings

Fig. 1 is a mass spectrum result of bivalirudin prepared in example 1.

Detailed Description

The exemplary embodiments will be described herein in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Synthesis of bivalirudin

1. Activation of the resin

Adding 45g of Wang resin into 690g of activating solution, and carrying out oscillation reaction at 100r/min for 2 h; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the activating solution comprises 600g of dichloromethane, 60g of B-butyrolactone and 30g of nadic anhydride.

2. Synthesis of Leu-resin

Taking 50g of the swelled activated resin and 0.08mol of Fmoc-Leu, adding 0.08mol of DMF, swelling, then carrying out ice bath, continuously adding 0.08mol of DCC, 0.08mol of HOBt and 0.0025mol of DMAP, carrying out ice bath for 2h, then stirring at 4 ℃ at 100r/min overnight; after stirring, filtering, washing and drying to obtain Fmoc-Leu-resin; and adding 100g of pyridine/dichloromethane/DMF mixed solution (V/V/V =1:1: 3) into the Fmoc-Leu-resin, reacting for 30min, filtering, washing and drying to obtain the Leu-resin.

3. Synthesis of bivalirudin-resin

And (3) sequentially coupling amino acids according to the operation method in the step 2 to prepare bivalirudin-resin. The sequence of amino acid couplings is shown in table 1.

TABLE 1 amino acid coupling sequences

4. Deprotection of the amino acid

Taking 120g of 95% TFA solution, 20g of (S) -glycidol and 10g of 20% 4-bromobutyric acid solution, uniformly mixing, adding 15g of bivalirudin-resin prepared in the step 4, stirring for 30min in the dark, and filtering; and adding 150g of anhydrous ether into the filtrate, standing at the temperature of-20 ℃ after white precipitate is separated out, taking the precipitate and drying after the solution is obviously layered and the upper layer is clear to obtain bivalirudin.

5. Purification of bivalirudin

Purification was performed using a high performance liquid system, reverse phase C18 chromatography column, mobile phase a as phosphate buffer (ph 2.4), mobile phase B as acetonitrile: buffer =1:1, flow rate 1mL/min, purification conditions as shown in table 2, purity was checked while detecting wavelength 210 nm. The preparation method of the phosphate buffer solution comprises the following steps: 20mL of phosphoric acid and 25mL of triethylamine are uniformly mixed and then the volume is adjusted to 2000mL by ultrapure water.

TABLE 2 HPLC purification conditions

Example 2

Synthesis of bivalirudin

1. Activation of the resin

Adding 30g of Wang resin into 573g of activating solution, and carrying out oscillation reaction for 1h at 100 r/min; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the activating solution comprises 540g of dichloromethane, 30g of B-butyrolactone and 3g of nadic anhydride.

2. Synthesis of Leu-resin

Taking 30g of the swelled activated resin and 0.05mol of Fmoc-Leu, adding 0.05mol of DMF, swelling, then carrying out ice bath, continuously adding 0.05mol of DCC, 0.05mol of HOBt and 0.001mol of DMAP, carrying out ice bath for 1h, then stirring at 2 ℃ at 100r/min and standing overnight; after stirring, filtering, washing and drying to obtain Fmoc-Leu-resin; and adding 90g of pyridine/dichloromethane/DMF mixed solution (V/V/V =0.25:1: 1) into the Fmoc-Leu-resin, reacting for 20min, performing suction filtration, washing and drying to obtain the Leu-resin.

3. Synthesis of bivalirudin-resin

The same as in example 1.

4. Deprotection of the amino acid

Uniformly mixing 100g of 90% TFA solution, 10g of (S) -glycidol and 5g of 10% 4-bromobutyric acid solution, adding 10g of bivalirudin-resin prepared in the step 4, stirring for 10min in the dark, and filtering; and adding 100g of anhydrous ether into the filtrate, standing at the temperature of-20 ℃ after white precipitate is separated out, and taking the precipitate and drying after the solution is obviously layered and the upper layer is clear to obtain bivalirudin.

5. Purification of bivalirudin

The same as in example 1.

Example 3

Synthesis of bivalirudin

1. Activation of the resin

Adding 60g of Wang resin into 915g of activating solution, and carrying out oscillation reaction at 120r/min for 4 h; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the activating solution comprises 780g of dichloromethane, 90g of B-butyrolactone and 45g of nadic anhydride.

2. Synthesis of Leu-resin

Taking 60g of the swelled activated resin and 0.15mol of Fmoc-Leu, adding 0.15mol of DMF, swelling, then carrying out ice bath, continuously adding 0.15mol of DCC, 0.15mol of HOBt and 0.005mol of DMAP, carrying out ice bath for 5h, stirring at the temperature of 4 ℃ at 120r/min and standing overnight; after stirring, filtering, washing and drying to obtain Fmoc-Leu-resin; and adding 120g of dichloromethane/DMF mixed solution (V/V/V =1:1: 3) into the Fmoc-Leu-resin, reacting for 50min, filtering, washing and drying to obtain the Leu-resin. (modified according to the summary of the invention)

3. Synthesis of bivalirudin-resin

The same as in example 1.

4. Deprotection of the amino acid

Uniformly mixing 150g of 98% TFA solution, 30g of (S) -glycidol and 15g of 50% 4-bromobutyric acid solution, adding 20g of bivalirudin-resin prepared in the step 4, stirring for 60min in the dark, and filtering; and adding 200g of anhydrous ether into the filtrate, standing at-10 ℃ after white precipitate is separated out, taking the precipitate and drying after the solution is obviously layered and the upper layer is clear to obtain bivalirudin.

5. Purification of bivalirudin

The same as in example 1.

Example 4

Synthesis of bivalirudin

This example differs from example 1 in that no (S) -glycidol and 4-bromobutyric acid solution was added in step 4.

Example 5

Synthesis of bivalirudin

This example differs from example 2 in that no (S) -glycidol and 4-bromobutyric acid solution was added in step 4.

Example 6

Synthesis of bivalirudin

This example differs from example 3 in that no (S) -glycidol and 4-bromobutyric acid solution was added in step 4.

Comparative example 1

Synthesis of bivalirudin

1. Activation of the resin

Adding 45g of Wang resin into 660g of activating solution, and carrying out oscillation reaction for 2h at 100 r/min; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the activating solution comprises 600g of dichloromethane and 60g of B-butyrolactone.

2. Synthesis of Leu-resin

The same as in example 1.

3. Synthesis of bivalirudin-resin

The same as in example 1.

4. Deprotection of the amino acid

Adding 120g of 95% TFA solution into 15g of bivalirudin-resin prepared in the step 3, stirring for 30min in the dark, and filtering; and adding 150g of anhydrous ether into the filtrate, standing at the temperature of-20 ℃ after white precipitate is separated out, taking the precipitate and drying after the solution is obviously layered and the upper layer is clear to obtain bivalirudin.

5. Purification of bivalirudin

The same as in example 1.

Comparative example 2

Synthesis of bivalirudin

This comparative example differs from comparative example 1 in that step 1 is as follows:

activation of the resin

Adding 45g of Wang resin into 630g of activating solution, and carrying out oscillation reaction for 2h at 100 r/min; filtering out the resin after reaction, and washing and drying to obtain the swollen activated resin; wherein the activating solution comprises 600g of dichloromethane and 30g of nadic anhydride.

Comparative example 3

Synthesis of bivalirudin

1. Activation of the resin

Adding 45g of Wang resin into 600g of activating solution, and carrying out oscillation reaction for 2h at 100 r/min; filtering out the resin after reaction, and washing and drying to obtain activated resin; wherein the activating solution is 600g of dichloromethane.

2. Synthesis of Leu-resin

The same as in example 1.

3. Synthesis of bivalirudin-resin

The same as in example 1.

4. Deprotection of the amino acid

Adding 120g of 95% TFA solution, 20g of (S) -glycidol and 10g of 20% 4-bromobutyric acid solution into 15g of bivalirudin-resin prepared in the step 3, stirring for 30min in the dark, and filtering; and adding 150g of anhydrous ether into the filtrate, standing at the temperature of-20 ℃ after white precipitate is separated out, taking the precipitate and drying after the solution is obviously layered and the upper layer is clear to obtain bivalirudin.

5. Purification of bivalirudin

The same as in example 1.

Comparative example 4

Synthesis of bivalirudin

This comparative example differs from comparative example 3 in that step 4 does not add 4-bromobutyric acid solution.

Comparative example 5

Synthesis of bivalirudin

This comparative example differs from comparative example 3 in that step 4 does not add (S) -glycidol.

Comparative example 6

This comparative example differs from comparative example 3 in that step 4 does not add (S) -glycidol and 4-bromobutyric acid solution.

Test example 1

Resin Property measurement

Measurement of resin swelling Property

The swelling properties of the Wang resin after activation in examples 1, 2 and 3 and comparative examples 1 and 2 were determined by the following specific methods:

taking 1g of activated Wang resin, completely immersing the Wang resin in DMF (dimethyl formamide) with the volume of 20 times at room temperature, oscillating for 6 hours at 100r/min to achieve a full saturation state, filtering the resin, wiping off residual solution on the surface of the resin, and measuring the mass; after the measurement, the swelling ratio was calculated as follows:

swelling ratio = (mass-dry weight after swelling)/dry weight

The measurement results are shown in Table 3.

TABLE 3 measurement results of resin swelling Properties

As can be seen from Table 3, the swelling ratios of the resins in examples 1-3 are all larger than those of the comparative examples, which shows that the resins can be more fully swelled after being activated by the activating solution containing B-butyrolactone and nadic anhydride, so that the amino acid binding sites can be more fully exposed, the amino acid binding is easier, and the binding efficiency is improved; comparative examples 1 to 3 show that the swelling ratio of the resin in example 3 is the highest, and that the swelling effect of the resin is affected by the ratio of each component in the activating solution, and that the swelling ratio is increased more when the ratio of B-butyrolactone and nadic anhydride in the activating solution is larger.

Secondly, measuring the degree of substitution:

the Wang resin used in the experiment is purchased from Shanghai drum minister biotechnology GmbH, and the substitution degree is 0.4-0.6 mmol/g. 20mg of Fmoc-Leu-resin prepared in step 2 of examples 1-3 and comparative examples 1-3 were added to 5mL of 20% piperidine/DMF mixed solution (V/V =1: 3), shaken at 100r/min at room temperature for 30min and then filtered; adding methanol into the filtrate to 50mL, uniformly mixing, taking 1mL of solution, then using methanol to fix the volume to 25mL, uniformly mixing, and measuring the absorbance at 295 nm; a blank was prepared and absorbance was measured using 5mL of a 20% piperidine/DMF mixed solution (V/V =1: 3) according to the above treatment method; preparing a standard substance by using 20mg of Fmoc-Leu-OH according to the above treatment method and measuring absorbance; the degree of substitution is calculated after the absorbance is measured, and the calculation formula is as follows:

The measurement results are shown in Table 4.

TABLE 4 measurement results of degree of substitution of resin

As is clear from Table 4, the degree of substitution of the resins in examples 1 to 3 and comparative examples 1 to 3 was in the range of 0.4 to 0.6mmol/g, which was in accordance with the degree of substitution set before the experiment, and therefore the synthesis in the present invention was continued with high efficiency. The measured degree of substitution was consistent with the degree of substitution set before the experiment, which shows that the degree of substitution of the resin was not reduced while the swellability of the resin was improved by adding B-butyrolactone and nadic anhydride simultaneously when the resin was activated, and that the degree of substitution was not greatly affected by the amounts of both.

Test example 2

Bivalirudin performance assay

Bivalirudin molecular weight determination

Bivalirudin prepared in example 1 was measured using a mass spectrometer, and the measurement result is shown in fig. 1, and the molecular weight is 2180, which is in line with the theoretical value.

Second, bivalirudin yield determination

The weights of bivalirudin and high-purity bivalirudin prepared in step (4) and step (5) of examples 1 to 6 were measured, respectively, and the yields were calculated; the purity determined in step (5) in examples 1-6 was also recorded;

yield (%) = high-purity bivalirudin mass/bivalirudin mass × 100%

The results are shown in Table 5.

TABLE 5 Bivalirudin purity

As can be seen from table 5, the high-purity bivalirudin prepared in examples 1 to 6 all had higher purity, wherein the highest yield was the bivalirudin prepared in example 1, which reached 76.33%; the reason is probably that the resin with good swelling property is used, the generation of impurities is reduced during deprotection, the utilization rate of the resin is increased, and the difficulty of purification is reduced; it is demonstrated that the addition of B-butyrolactone and nadic anhydride during resin activation and the addition of (S) -glycidol and 4-bromobutyric acid solution during deprotection can effectively improve the yield of bivalirudin.

Determination of bivalirudin Activity

The activity of bivalirudin is determined by an anticoagulation test, and the specific operation steps are as follows:

bivalirudin prepared in examples 1 to 6 and comparative examples 1 to 6, respectively, was prepared into a sample solution of 5mg/mL using 50mmol/L Tris-HCl (pH = 7.8); preparing a thrombin reagent chromogenic substrate Chromozym TH into a 1.3mg/mL chromogenic solution by using ultrapure water; preparing calf serum and NaCl into a calf serum solution of 30mg/mL by using 50mmol/L Tris-HCl (pH = 7.8); mixing 1IU of thrombin solution and 50 mul of sample solution respectively, adding into a 96-well plate, and carrying out water bath at 37 ℃ for 40 min; after water bath, 20 mu L of the mixture is taken out and added into a new 96-well plate, and 70 mu L of calf serum solution and 10 mu L of developing solution are added simultaneously, and after uniform mixing, the light absorption value is measured at 402nm by using an enzyme-labeling instrument. The measurement results are shown in Table 6.

TABLE 6 measurement results of bivalirudin activity

As can be seen from Table 6, the activity of bivalirudin obtained in all examples and comparative examples is close to that of the standard, indicating that neither the addition of B-butyrolactone and nadic anhydride when activating the resin nor the addition of (S) -glycidol and 4-bromobutyric acid solution only when deprotecting had any effect on the activity of bivalirudin.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A synthetic method of bivalirudin comprises the following steps:

deprotection, namely performing deprotection on bivalirudin-resin to obtain bivalirudin;

the resin is Wang resin;

the weight ratio of the dichloromethane, the B-butyrolactone and the nadic anhydride in the activating solution is 540-780:30-90: 3-45;

the specific operation of the step (4) comprises the following steps:

2. The method of claim 1, wherein in the step (2), the swollen activated resin and Fmoc-Leu are used in a ratio of 30-60g:0.05-0.15 mol.

3. The synthesis method according to claim 1, wherein the specific operation of the step (3) comprises:

and (3) sequentially coupling Try, Glu, Pro, Ile, Glu, Phe, Asp, Gly, Asn, Gly, Pro, Arg, Pro and Phe according to the operation method in the step (2) until obtaining the bivalirudin-resin.

4. The synthetic method of claim 1, wherein bivalirudin is purified using a high performance liquid system to obtain high purity bivalirudin.

5. Use of the synthesis method according to claim 1 for increasing the purity and/or yield of bivalirudin.

6. The Wang resin activating liquid comprises dichloromethane, B-butyrolactone and nadic anhydride in a weight ratio of 540-.

7. Use of the Wang resin activating solution according to claim 6 for improving the swellability of Wang resins.