阅读说明：本技术 一种兰瑞肽的制备方法 (Preparation method of lanreotide ) 是由 宓鹏程 王亮 潘俊锋 刘建 于 2021-08-09 设计创作，主要内容包括：本发明涉及多肽合成技术领域,尤其涉及一种兰瑞肽的制备方法。本发明首先采用适宜的保护氨基酸固相逐一合成兰瑞肽树脂,然裂解获得线性肽,利用特定的氧化剂对线性肽进行环化。该方法操作简单,不需要制备全保护肽,收率和收率高,杂质少,几乎不含氧化杂质及二聚体杂质,有效地解决了现有工艺氧化杂质风险大的问题,具有广泛的实用价值和应用前景。(The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of lanreotide. The method firstly adopts a proper protected amino acid solid phase to synthesize the lanreotide resin one by one, then the lanreotide resin is cracked to obtain linear peptide, and the linear peptide is cyclized by using a specific oxidant. The method is simple to operate, does not need to prepare the full-protection peptide, has high yield and few impurities, hardly contains oxidation impurities and dimer impurities, effectively solves the problem of high risk of oxidation impurities in the prior art, and has wide practical value and application prospect.)

Use of N-chlorosuccinimide in the preparation of lanreotide.

2. A preparation method of lanreotide is characterized by comprising the following steps:

according to the amino acid sequence of the lanreotide, coupling the protected amino acids one by one from the C end to the N end on a solid phase carrier to obtain peptide resin;

taking the peptide resin for cracking to obtain a linear peptide;

cyclizing the linear peptide to obtain lanreotide;

the oxidizing agent adopted for cyclization is N-chlorosuccinimide.

3. The method of claim 1, wherein the Resin is an amino Resin comprising Rink Amide Resin, Rink Amide-AM Resin, or Rink Amide-MBHA Resin.

4. The method according to claim 3, wherein the amino acid substitution degree of the amino resin is 0.3 to 0.6 mmol/g.

5. The method of claim 2, wherein the protected amino acid comprises Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Nal-OH.

6. The method of claim 2, wherein the cleaving agent comprises TFA, EDT, and thioanisole and anisole.

7. The method according to claim 2, wherein the volume ratio of TFA, EDT, thioanisole and anisole in the cracking agent is (80-90): (2-4): (4-6): (1-3).

8. The method according to claim 1, wherein the linear peptide further comprises a step of dissolving the linear peptide in water to obtain a solution of the linear peptide having a concentration of 3 to 5mg/ml before cyclization.

9. The method according to claim 1, wherein the mass ratio of the oxidizing agent to the linear peptide is 3 to 5: 1.

10. The method of claim 1, wherein the purification is HPLC purification; the HPLC purification comprises: using reverse-phase octadecylsilane as stationary phase, using 0.1% TFA aqueous solution and acetonitrile as mobile phase, collecting target peak fraction, concentrating and freeze-drying.

Technical Field

The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of lanreotide.

Background

Lanreotide is a drug which is already on the market, mainly for the treatment of acromegaly and patients who are unresponsive to surgery and/or radiotherapy, or patients who are not suitable for surgery and/or radiotherapy treatment.

The Lanreotide peptide sequence is shown as Nal-CY (d-W) KVCT-NH₂2-7Cys ring formation, molecular weight: 1096.32, the structural formula is as follows:

lanreotide contains a pair of disulfide bonds, and for the synthesis of disulfide bonds, in general, on-chain solid phase synthesis is adopted, namely, iodine serving as an oxidant is adopted to directly form disulfide bonds on resin. Or after the linear peptide is cracked from the resin, iodine or hydrogen peroxide is adopted in a liquid phase to form a disulfide bond. CN110330560A introduces a synthesis method of lanreotide, which comprises the steps of firstly synthesizing lanreotide full-protection peptide by a solid phase method, then dissolving the full-protection peptide in a solution, forming a disulfide bond by an iodine oxidation method, and then removing a protection group to obtain the lanreotide, wherein the product contains more than 0.4% of dimer.

The lanreotide sequence has a Tyr residue which is easily iodinated, so that oxidation on resin with iodine and liquid phase oxidation have the risk of Tyr oxidation impurities, and the use of a strong oxidant iodine greatly increases the possibility of dimers. When hydrogen peroxide is used for oxidation, the pH value of an oxidation liquid system needs to be adjusted to be alkaline, but the peptide has poor solubility under alkaline conditions, and when the pH value is more than 7.2, the peptide is precipitated in the solution and cannot be oxidized. The conventional oxidation method is not suitable for the formation of the disulfide bond of lanreotide, and has more dimer impurities and lower purity.

Disclosure of Invention

In view of this, the present invention aims to provide a preparation method of lanreotide, such that the lanreotide obtained by the method has high yield and purity and less impurities.

The invention provides application of N-chlorosuccinimide in preparation of lanreotide.

Research shows that the method adopts N-chlorosuccinimide as an oxidant to cyclize the linear lanreotide, can obviously improve the purity and yield of the lanreotide, has few impurities, and hardly contains oxidation impurities and dimer impurities.

The invention provides a preparation method of lanreotide. The method comprises the following steps:

according to the amino acid sequence of the lanreotide, coupling the protected amino acids one by one from the C end to the N end on a solid phase carrier to obtain peptide resin;

taking the peptide resin for cracking to obtain a linear peptide;

cyclizing the linear peptide to obtain lanreotide;

the oxidizing agent adopted for cyclization is N-chlorosuccinimide.

In the present invention, the resin is an amino resin. In some embodiments, amino resins include Rink Amide Resin, Rink Amide-AM Resin, or Rink Amide-MBHA Resin, including but not limited to.

In the present invention, the amino acid substitution degree of the amino resin is 0.3 to 0.6mmol/g, and specifically may be 0.3mmol/g, 0.5mmol/g or 0.6 mmol/g.

In the present invention, the protected amino acids coupled one by one include Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, and Fmoc-Nal-OH. The coupling process was checked by ninhydrin test to determine if the coupling was complete.

In the present invention, the cleavage agents for cleavage include TFA, EDT, and thioanisole and anisole. In some embodiments, the TFA, EDT, and the volume ratio of thioanisole to anisole is (80-90): (2-4): (4-6): (1-3), in some embodiments 90:3:5: 2.

In the present invention, the linear peptide further comprises a step of dissolving the linear peptide in water to obtain a solution of the linear peptide having a concentration of 3 to 5mg/ml before cyclization. In some embodiments, the concentration of the linear peptide solution may specifically be 3mg/ml, 4mg/ml or 5 mg/ml.

In the present invention, the concentration of the oxidizing agent

The mass ratio of the oxidant to the linear peptide is 3-5: 1. In some embodiments, the mass ratio may specifically be 3:1, 4:1, or 5: 1.

In the present invention, the purification is HPLC purification.

Wherein the HPLC purification comprises: using reverse-phase octadecylsilane as stationary phase, using 0.1% TFA aqueous solution and acetonitrile as mobile phase, collecting target peak fraction, concentrating and freeze-drying.

The method firstly adopts a proper protected amino acid solid phase to synthesize the lanreotide resin one by one, then cracking is carried out to obtain the linear peptide, and the specific oxidant is utilized to carry out cyclization on the linear peptide, so that the obtained lanreotide has higher purity and yield. The preparation method provided by the invention is simple to operate, does not need to prepare the full-protection peptide, is high in yield and yield, contains few impurities, hardly contains oxidation impurities and dimer impurities, effectively solves the problem of high risk of oxidation impurities in the existing process, and has wide practical value and application prospect.

Drawings

FIG. 1 shows a chromatogram of the lanreotide protide of example 9;

fig. 2 shows a chromatogram of the lanreotide protide of comparative example 1.

Detailed Description

The invention provides a preparation method of lanreotide. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the present invention, the abbreviations and meanings of English are as follows:

TABLE 1

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 preparation of Fmoc-Thr (tBu) -Rink Amide Resin with a Segregation of 0.60mmol

Weighing 100g of Rink Amide Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out nitrogen bubbling for swelling for 60 minutes; Fmoc-Thr (tBu) -OH 39.8 g (100mmol) and HOBt16.2 g (120mmol) were weighed, dissolved in DMF, 20.3mL (120mmol) of DIC was added at 0 ℃ and activated for 5 minutes, and then the mixture was loaded onto a reaction column. After reacting for two hours, adding 70mL of acetic anhydride and 60mL of pyridine, mixing and sealing for 24 hours, washing with DCM for three times, shrinking with methanol, and then pumping out the Resin to obtain Fmoc-Thr (tBu) -Rink Amide Resin, wherein the detection substitution degree is 0.62 mmol/g.

Example 2 preparation of Fmoc-Thr (tBu) -Rink Amide-MBHA Resin with a degree of substitution of 0.50mmol

Weighing 100g of Rink Amide-MBHA Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; Fmoc-Thr (tBu) -OH 31.7 g (70mmol) and HOBt 11.3 g (84mmol) were weighed, dissolved in DMF, and 14.2mL (84mmol) of DIC was added at 0 ℃ to activate for 5 minutes and then the mixture was loaded onto a reaction column. After reacting for two hours, adding 70mL of acetic anhydride and 60mL of pyridine, mixing and sealing for 24 hours, washing with DCM for three times, shrinking with methanol, and then pumping out the Resin to obtain Fmoc-Thr (tBu) -Rink Amide-MBHA Resin, wherein the detection substitution degree is 0.50 mmol/g.

Example 3 preparation of Fmoc-Thr (tBu) -Rink Amide-AM Resin with a degree of substitution of 0.30mmol

Weighing 100g of Rink Amide-AM Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; Fmoc-Thr (tBu) -OH 20.4 g (45mmol) and HOBt 7.3 g (54mmol) were weighed, dissolved in DMF, and 9.1mL (54mmol) of DIC was added at 0 ℃ to activate for 5 minutes and then the mixture was loaded onto a reaction column. After reacting for two hours, adding 70mL of acetic anhydride and 60mL of pyridine, mixing and sealing for 24 hours, washing with DCM for three times, shrinking with methanol, and then pumping out the Resin to obtain the Fmoc-Thr (tBu) -Rink Amide-AM Resin, wherein the detection substitution degree is 0.31 mmol/g.

Example 4 preparation of Nal-Cys (Trt) -Tyr (tBu) -D-Trp (Boc) -Lys (Boc) -Val-Cys (Trt) -Thr (tBu) -Rink Amide-MBHA Resin

Weighing 100g of Fmoc-Thr (tBu) -Rink Amide-MBHA Resin with the substitution degree of 0.50mmol/g prepared in example 2 into a solid phase reaction column, adding DMF, and carrying out bubbling and swelling for 60 minutes by nitrogen; then deprotected with DBLK for 6min +8min, washed 6 times with DMF. 87.8g (150mmol) of Fmoc-Cys (Trt) -OH and 24.3g (180mmol) of HOBT are weighed and dissolved in DMF, 28.1mL (180mmol) of DIPCDI is added under ice-water bath for activation for 3min, the mixture is added into a reaction column and reacted for 2h 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 for 3 times, adding DBLK to perform deprotection for 6min +8min, washing the resin with DMF for 6 times, and detecting the color of the resin by ninhydrin. The above coupling operation was repeated to sequentially couple Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH and Fmoc-Nal-OH in the order of peptide sequence to obtain 178.9g of peptide resin

Example 5 Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂Preparation of crude Linear peptides

178.9g of the peptide resin from example 4 was added to a 3L three-necked flask, and pre-cooled to below 0 ℃ TFA EDT-thioanisole: anisole 90: 5: 3: 2(V: V)1.79L, reacting at room temperature for 2 hours, filtering the resin, and collecting the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 17.9L of ethyl acetate, precipitated, centrifuged, washed 5 times with ethyl acetate, and dried under reduced pressure to give 55.5g of crude peptide, 101.6% weight yield.

Example 6 Nal-Cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂Preparation of

Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH in example 5 was weighed₂55.5g (50mmol) of the linear crude peptide was dissolved in 18.5L of purified water to prepare a 3mg/ml aqueous solution. Measuring 12.1ml (150mmol) NCS, adding into the reaction solution, stirring for 60min, and detecting the presence of wireless peptide raw material by HPLC to obtain Nal-cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂And (3) solution.

Example 7 Nal-Cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂Preparation of

Weighing 50mmol Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂The linear crude peptide was dissolved in purified water to prepare an aqueous solution of 5 mg/ml. 20.2ml (250mmol) of NCS was measured and added to the reaction mixture,stirring for reaction for 60min, and detecting the existence of wireless peptide raw material by HPLC to obtain Nal-cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 solution.

Example 8 Nal-Cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂Preparation of

Weighing 50mmol Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂The linear crude peptide was dissolved in purified water to prepare a 4mg/ml aqueous solution. Measuring 16.2ml (200mmol) NCS, adding into the reaction solution, stirring for 60min, and detecting the presence of wireless peptide raw material by HPLC to obtain Nal-cyclo [ Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂And (3) solution.

Example 9 preparation of lanreotide fine peptide

The reaction solution obtained in example 6 was directly purified by high performance liquid chromatography. And (3) purifying by using reverse-phase octadecylsilane as a stationary phase (a purification system is that phase A is 0.1% TFA, phase B is acetonitrile, and gradient is that phase B is 21% -41% (60min)), collecting target peak fractions, concentrating and freeze-drying to obtain a pure product 33.9g, wherein the purity is higher than 99.5%, the total yield is 61.9%, no dimer is detected, and a chromatogram is shown in figure 1.

Example 10 preparation of lanreotide fine peptide

The reaction solution obtained in example 7 was directly purified by high performance liquid chromatography. And (3) purifying by using reverse-phase octadecylsilane as a stationary phase (a purification system is that phase A is 0.1% TFA, phase B is acetonitrile, and gradient is that phase B is 21% -41% (60min)), collecting target peak fractions, concentrating and freeze-drying to obtain a pure product 33.9g, wherein the purity is 99.6%, the total yield is 59.7%, and dimers are not detected.

Example 11 preparation of lanreotide fine peptide

The reaction solution obtained in example 8 was directly purified by high performance liquid chromatography. And (3) purifying by taking reverse-phase octadecylsilane as a stationary phase (the purification system is that the A phase is 0.1% TFA, the B phase is acetonitrile, the gradient is that the B% is 21% -41% (60min)), collecting target peak fractions, concentrating and freeze-drying to obtain a pure product 33.9g, wherein the purity is 99.8%, the total yield is 58.9%, and dimers are not detected.

Comparative example 1

A linear crude Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 peptide (50mmol) was prepared according to the procedure of example 1 to 5, and the crude peptide was dissolved in water to prepare an aqueous solution of 0.1 g/ml. Weighing (50mmol) iodine, dissolving in methanol to prepare an oxidation solution, then dropwise adding the oxidation solution into the crude peptide aqueous solution, carrying out oxidation reaction for 2h, and monitoring the reaction by HPLC until the reaction is finished. After the reaction was completed, ascorbic acid was added to quench the reaction. The reaction solution was purified by the method of example 9 to obtain lanreotide refined peptide, and the chromatogram thereof is shown in fig. 2, with a purity of 98.98%, and a dimer of 0.45% (peak with a retention time of 17.333) was detected.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.