阅读说明：本技术 一种乌拉立肽的制备方法 (Preparation method of ularitide ) 是由 汪伟 陈永汉 尹传龙 陶安进 余品香 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种乌拉立肽的制备方法,根据其肽序中含有两个Cys的特点,将乌拉立肽分成片段A：H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH2、片段B：H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH2和片段C：Cys-Asn-Ser-Phe-Arg-Tyr-OH三部分,片段中的酰肼基团分别与亚硝酸钠、MPAA反应生成硫酯,其能够选择性地与N端为Cys的多肽片段发生缩合反应。不同于常规片段法,本发明中的片段侧链没有保护基,在水中有较好的溶解度,不存在偶联困难的问题,得到的产品纯度高,易于纯化,操作简单,生产效率高,产品质量高、成本低、适合工业化生产等。(The invention discloses a preparation method of ularitide, which divides the ularitide into a segment A according to the characteristic that the peptide sequence contains two Cys: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH2, fragment B: H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH2 and fragment C: Cys-Asn-Ser-Phe-Arg-Tyr-OH, wherein hydrazide groups in the fragments react with sodium nitrite and MPAA respectively to generate thioester, which can selectively perform condensation reaction with polypeptide fragments with Cys at the N end. Different from the conventional fragment method, the fragment side chain in the invention has no protective group, has better solubility in water, does not have the problem of difficult coupling, and the obtained product has high purity, easy purification, simple operation, high production efficiency, high product quality, low cost, suitability for industrial production and the like.)

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

synthesis of fragment A:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH₂；

synthesis of fragment B:

H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH₂；

synthesis of fragment C:

Cys-Asn-Ser-Phe-Arg-Tyr-OH；

synthesis of fragment D:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH₂；

synthesis of the linear peptide:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH；

and (4) synthesizing a crude peptide.

2. The method for preparing ularitide as claimed in claim 1, wherein the synthesis method of the fragment A or B is as follows: taking 2-Cl-CTC Resin as a carrier, firstly coupling hydrazine hydrate according to a polypeptide solid phase synthesis method, then sequentially coupling amino acids from a C end to an N end according to a peptide sequence, and finally cracking to obtain a fragment A or B, wherein preferably, the substitution degree of the 2-Cl-CTC Resin is 0.1-1.6 mmol/g.

3. The method for preparing ularitide according to claim 1, wherein the fragment C is synthesized by using Wang Resin or 2-Cl-CTC Resin as a carrier, sequentially coupling amino acids from C end to N end according to a peptide sequence according to a polypeptide solid phase synthesis method, and then cracking to obtain the fragment C, wherein preferably, the substitution degree of the Wang Resin is 0.1-3.0 mmol/g, and the substitution degree of the 2-Cl-CTC Resin is 0.1-1.6 mmol/g.

4. The method for preparing ularitide as claimed in claim 1, wherein the fragment D is synthesized by reacting fragment A with sodium nitrite under acidic condition to form azide, reacting with MPAA to form thioester, and reacting with fragment B under neutral condition to obtain fragment D.

5. The method of claim 1, wherein the linear peptide is synthesized by reacting fragment D with sodium nitrite under acidic conditions to form azide, reacting with MPAA to form thioester, and reacting with fragment C under neutral conditions to obtain linear peptide.

6. The method for preparing ularitide as claimed in claim 1, wherein the crude peptide is synthesized by dissolving linear peptide in acetic acid solution and oxidizing with iodine to obtain ularitide crude peptide.

7. The method for producing ularitide as claimed in claim 1, wherein the step of synthesizing the crude peptide is followed by a purification step; the purification step is performed by high pressure liquid chromatography.

8. The method for preparing ularitide as claimed in claim 7, wherein the HPLC uses NOVASEP RP-HPLC system with wavelength of 220nm, reversed phase C18 column as stationary phase, 0.1% TFA solution and acetonitrile as mobile phase system, collects the target fraction, rotary evaporation concentrates and lyophilizes.

9. The method for preparing ularitide as claimed in claim 1, wherein the method comprises the following steps:

synthesis of fragment A:

adding 2-Cl-CTC Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, and then extracting the solvent; dissolving hydrazine hydrate and triethylamine in a solvent, adding the solution into a solid-phase reaction column under ice bath, reacting for 2 hours at room temperature, extracting the solvent, and washing the solvent; dissolving Fmoc-Ser (tBu) -OH and a coupling agent in a solvent, activating, adding the mixture into a solid-phase reaction column until the reaction is complete, and washing the solvent; removing Fmoc protecting groups, and washing with a solvent; repeating the steps of coupling the amino acids and removing the Fmoc protecting group according to the peptide sequence of fragment A, and sequentially coupling Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH and Fmoc-Thr (tBu) -OH by using a coupling agent; cracking the obtained peptide resin by using a cracking solution to obtain a fragment A;

preferably, the reagent for removing the Fmoc protecting group is a 20% piperidine solution, and the solvent used in the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysate is a TFA solution containing a capture agent, the capture agent is one or more of PhSMe, PhOH, EDT, H2O, TIS and PhOMe, and more preferably, the lysate is TFA/PhSMe/PhOH/EDT/H₂O, wherein TFA, PhSMe, PhOH, EDT, H₂The volume ratio of O is 85:5:5:3: 2;

synthesis of fragment B:

the operation method is similar to the synthesis of the fragment A, hydrazine hydrate is coupled firstly, then Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Met-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH and Boc-Cys (Trt) -OH are coupled in sequence, and the obtained peptide resin is cracked by using a cracking solution to obtain a fragment B;

preferably, the reagent for removing the Fmoc protecting group is a 20% piperidine solution, and the solvent used in the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysate is a TFA solution containing a capture agent, the capture agent is one or more of PhSMe, PhOH, EDT, H2O, TIS and PhOMe, and more preferably, the lysate is TFA/PhSMe/PhOH/EDT/H₂O, wherein TFA, PhSMe, PhOH, EDT, H₂The volume ratio of O is 85:5:5:3: 2;

synthesis of fragment C:

adding Wang Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, then removing the solvent, dissolving and activating Fmoc-Tyr (tBu) -OH and a coupling agent in the solvent, then adding the mixture into the solid phase reaction column until the reaction is complete, and washing the solvent; adding mixed solution of acetic anhydride and pyridine in the volume ratio of 7:6 to seal the Resin for 6 hours, washing the Resin by using a solvent, and contracting and drying the Resin by using methanol to obtain Fmoc-Phe-Wang Resin; referring to the method for synthesizing fragment A, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was cleaved with a cleavage solution to obtain fragment C;

or adding the 2-Cl-CTC Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, and then extracting the solvent; dissolving Fmoc-Tyr (tBu) -OH in a solvent, adding DIPEA under ice bath, adding the solution into a solid phase reaction column, reacting for 2 hours at room temperature, and washing the solvent; adding methanol to seal the Resin for 1 hour, washing by a solvent, and shrinking and drying by methanol to obtain Fmoc-Tyr (tBu) -CTC Resin; referring to the method for synthesizing fragment A, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was cleaved with a cleavage solution to obtain fragment C;

preferably, the reagent for removing the Fmoc protecting group is a 20% piperidine solution, and the solvent used in the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysis solution is a TFA solution containing a capture agent, and the capture agent is PhSMe, PhOH, EDT, H₂One or more of O, TIS and PhOMe, and more preferably, the lysis solution is TFA and H₂O, wherein TFA and H₂The volume ratio of O is 95: 5;

synthesis of fragment D:

dissolving the fragment A in 0.3M disodium hydrogen phosphate solution, adjusting the pH value to 3 by using dilute sodium hydroxide solution, cooling to-10 ℃, slowly dropwise adding sodium nitrite solution, stirring for 30 minutes after dropwise adding, then adding MPAA, adjusting the pH value to 7 by using dilute sodium hydroxide solution, finally adding the fragment B, heating to room temperature for reaction, monitoring the reaction by HPLC (high performance liquid chromatography), adding TCEP (trichloroacetic acid) to terminate the reaction, and removing excessive MPAA and TCEP;

synthesis of the linear peptide:

dissolving the fragment D in 0.3M disodium hydrogen phosphate solution, adjusting the pH value to 3 by using dilute sodium hydroxide solution, cooling to-10 ℃, slowly dropwise adding sodium nitrite solution, stirring for 30 minutes after dropwise adding, then adding MPAA, adjusting the pH value to 7 by using dilute sodium hydroxide solution, finally adding the fragment C, heating to room temperature for reaction, monitoring the reaction by HPLC (high performance liquid chromatography), adding TCEP (trichloropropylacetic acid) to terminate the reaction, and removing excessive MPAA and TCEP;

synthesis of crude peptide:

weighing linear peptide, dissolving the linear peptide in 10% acetic acid solution with the concentration of 5mg/ml, slowly dropwise adding 1% iodine acetic acid solution until the solution turns yellow, stopping dropwise adding, monitoring by HPLC for complete reaction, and adding vitamin C to terminate the reaction to obtain the crude peptide of the ularitide.

10. The method for preparing ularitide as claimed in claim 9, wherein the solvent used in the washing and swelling steps is one or more of NMP, THF, DCM, DMF and DMSO;

the solvent used in the step of dissolving the substance is one or more of NMP, THF, DCM, DMF and DMSO.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a preparation method of ularitide.

Background

Urapide (Ularitide) is a diuretic developed by Cardiomentis AG of Switzerland, has the effects of natriuretic action and blood vessel expansion, can be used for decompensated heart failure, acute renal failure and bronchial asthma, and has a wide market prospect. The peptide sequence is as follows: H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH (positions 11 and 27 form a disulfide bond).

Patent US5449751 discloses two methods for the preparation of ularitide: extracted from urine and synthesized using Boc/Bzl strategy.

CN201610947595 uses Wang resin to couple amino acids from C-terminal to N-terminal one by one, and cleaves TFA to obtain linear peptide, and oxidizes to obtain ularitide.

CN201511029989 adopts pseudo proline dipeptide Fmoc-Asn (Trt) -Ser (psi)^Me,MePro)-OH、Fmoc-Gln(Trt)-Ser(ψ^Me,MePro)-OH、Fmoc-Ser(tBu)-Ser(ψ^Me,MePro)-OH、Fmoc-Arg(pbf)-Ser(ψ^Me,MePro) -OH replaces single amino acid to be coupled one by one, and the urapidine is obtained after cracking and oxidation.

The extraction method introduced in the patent US5449751 has low yield and high cost; the synthesis method has more side reactions, needs a hazardous reagent of hydrofluoric acid, is difficult to purify and has low product purity.

In the method described in CN201610947595 patent, in the coupling process, due to the peptide resin shrinkage phenomenon existing in the interval from Ile at position 19 to Ser at position 10, the reaction of amino acids at multiple sites is incomplete, the purity of the obtained crude peptide is low, and the purification is difficult.

The method described in patent CN201511029989 can inhibit the shrinkage of peptide resin, but has limited effect, cannot avoid a plurality of deletion peptides caused by overlong peptide chain, and the pseudoproline dipeptide is expensive and not easily available.

Aiming at the problems, the invention provides a method for preparing the ularitide by fragment condensation, which divides the ularitide into a fragment A (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Ser-NHNH) according to the characteristic that the peptide sequence of the ularitide contains two Cys₂) Fragment B (H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH)₂) And a fragment C (Cys-Asn-Ser-Phe-Arg-Tyr-OH), wherein a hydrazide group in the fragment reacts with sodium nitrite and MPAA respectively to generate thioester, and the thioester can be selectively subjected to condensation reaction with a polypeptide fragment with Cys at the N end. Different from the conventional fragment method, the fragment side chain in the invention has no protective group, has better solubility in water, does not have the problem of difficult coupling, and the obtained product has high purity, easy purification, simple operation and high production efficiency.

Disclosure of Invention

In order to solve the problems of the background art, the present invention provides a method for preparing ularitide.

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

synthesis of fragment A:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH₂；

synthesis of fragment B:

H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH₂；

synthesis of fragment C:

Cys-Asn-Ser-Phe-Arg-Tyr-OH；

synthesis of fragment D:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH₂；

synthesis of the linear peptide:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH；

and (4) synthesizing a crude peptide.

Further, the synthesis method of the fragment A or the fragment B comprises the following steps: 2-Cl-CTC Resin is taken as a carrier, hydrazine hydrate is coupled firstly according to a polypeptide solid phase synthesis method, amino acids are coupled in sequence from the C end to the N end according to a peptide sequence, and finally, a fragment A or B can be obtained through cracking.

Furthermore, the substitution degree of the 2-Cl-CTC Resin is 0.1-1.6 mmol/g.

Furthermore, the fragment C is synthesized by using Wang Resin or 2-Cl-CTC Resin as a carrier, sequentially coupling amino acids from the C end to the N end according to a peptide sequence according to a polypeptide solid phase synthesis method, and then cracking to obtain the fragment C.

Furthermore, the substitution degree of the Wang Resin is 0.1-3.0 mmol/g, and the substitution degree of the 2-Cl-CTC Resin is 0.1-1.6 mmol/g.

Further, the synthesis method of the fragment D is that the fragment A firstly reacts with sodium nitrite under acidic condition to generate azide, then reacts with MPAA to generate thioester, and finally reacts with the fragment B under neutral condition to obtain the fragment D.

Further, the linear peptide is synthesized by reacting the fragment D with sodium nitrite under acidic condition to generate azide, reacting with MPAA to generate thioester, and finally reacting with the fragment C under neutral condition to obtain the linear peptide.

Further, the synthesis method of the crude peptide comprises the steps of dissolving the linear peptide in an acetic acid solution, and oxidizing the linear peptide with iodine to obtain the crude peptide of the ularitide.

Further, the step of synthesizing the crude peptide further comprises a purification step; the purification step is performed by high pressure liquid chromatography.

Further, the high pressure liquid chromatography adopts a NOVASEP RP-HPLC system, a wavelength of 220nm, a reversed phase C18 column as a stationary phase, a 0.1% TFA solution and acetonitrile as a mobile phase system, and the target fraction is collected, concentrated by rotary evaporation and freeze-dried.

The preparation method of the ularitide specifically comprises the following steps:

synthesis of fragment A:

adding 2-Cl-CTC Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, and then extracting the solvent; dissolving hydrazine hydrate and triethylamine in a solvent, adding the solution into a solid-phase reaction column under ice bath, reacting for 2 hours at room temperature, extracting the solvent, and washing the solvent; dissolving Fmoc-Ser (tBu) -OH and a coupling agent in a solvent, activating, adding the mixture into a solid-phase reaction column until the reaction is complete, and washing the solvent; removing Fmoc protecting groups, and washing with a solvent; repeating the steps of coupling the amino acids and removing the Fmoc protecting group according to the peptide sequence of fragment A, and sequentially coupling Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH and Fmoc-Thr (tBu) -OH by using a coupling agent; cracking the obtained peptide resin by using a cracking solution to obtain a fragment A;

preferably, the reagent for removing Fmoc protecting groups is 20% piperidine solution, and the solvent used by the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysis solution is TFA solution containing a capture agent, the capture agent is one or more of PhSMe, PhOH, EDT, H2O, TIS and PhOMe, and more preferably, the lysis solution is TFA/PhSMe/PhOH/EDT/H₂O, wherein TFA, PhSMe, PhOH, EDT, H₂The volume ratio of O is 85:5:5:3: 2;

synthesis of fragment B:

the operation method is similar to the synthesis of the fragment A, hydrazine hydrate is coupled firstly, then Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Met-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH and Boc-Cys (Trt) -OH are coupled in sequence, and the obtained peptide resin is cracked by using a cracking solution to obtain a fragment B;

preferably, the reagent for removing Fmoc protecting groups is 20% piperidine solution, and the solvent used by the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysis solution is TFA solution containing a capture agent, the capture agent is one or more of PhSMe, PhOH, EDT, H2O, TIS and PhOMe, and more preferably, the lysis solution is TFA/PhSMe/PhOH/EDT/H₂O, wherein TFA, PhSMe, PhOH, EDT, H₂The volume ratio of O is 85:5:5:3: 2;

synthesis of fragment C:

adding Wang Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, then removing the solvent, dissolving and activating Fmoc-Tyr (tBu) -OH and a coupling agent in the solvent, then adding the mixture into the solid phase reaction column until the reaction is complete, and washing the solvent; adding mixed solution of acetic anhydride and pyridine in the volume ratio of 7:6 to seal the Resin for 6 hours, washing the Resin by using a solvent, and contracting and drying the Resin by using methanol to obtain Fmoc-Phe-Wang Resin; referring to the method for synthesizing fragment A, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was cleaved with a cleavage solution to obtain fragment C;

or adding the 2-Cl-CTC Resin into a solid phase reaction column, washing and swelling the Resin by using a solvent, and then extracting the solvent; dissolving Fmoc-Tyr (tBu) -OH in a solvent, adding DIPEA under ice bath, adding the solution into a solid phase reaction column, reacting for 2 hours at room temperature, and washing the solvent; adding methanol to seal the resin for 1 hour, washing by a solvent, and shrinking and drying by methanol to obtain Fmoc-Tyr (tBu) -CTCResin; referring to the method for synthesizing fragment A, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was cleaved with a cleavage solution to obtain fragment C;

preferably, the reagent for removing Fmoc protecting groups is 20% piperidine solution, and the solvent used by the 20% piperidine solution is one or more of NMP, THF, DCM, DMF and DMSO;

preferably, the coupling agent is one or more of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA;

preferably, the lysis solution is a TFA solution containing a capture reagent, which is PhSMe, PhOH, EDT, H₂One or more of O, TIS and PhOMe, and preferably, the lysis solution is TFA and H₂O, wherein TFA and H₂The volume ratio of O is 95: 5;

synthesis of fragment D:

dissolving the fragment A in 0.3M disodium hydrogen phosphate solution, adjusting the pH value to 3 by using dilute sodium hydroxide solution, cooling to-10 ℃, slowly dropwise adding sodium nitrite solution, stirring for 30 minutes after dropwise adding, then adding MPAA, adjusting the pH value to 7 by using dilute sodium hydroxide solution, finally adding the fragment B, heating to room temperature for reaction, monitoring the reaction by HPLC (high performance liquid chromatography), adding TCEP (trichloroacetic acid) to terminate the reaction, and removing excessive MPAA and TCEP;

synthesis of the linear peptide:

dissolving the fragment D in 0.3M disodium hydrogen phosphate solution, adjusting the pH value to 3 by using dilute sodium hydroxide solution, cooling to-10 ℃, slowly dropwise adding sodium nitrite solution, stirring for 30 minutes after dropwise adding, then adding MPAA, adjusting the pH value to 7 by using dilute sodium hydroxide solution, finally adding the fragment C, heating to room temperature for reaction, monitoring the reaction by HPLC (high performance liquid chromatography), adding TCEP (trichloropropylacetic acid) to terminate the reaction, and removing excessive MPAA and TCEP;

synthesis of crude peptide:

weighing linear peptide, dissolving the linear peptide in 10% acetic acid solution with the concentration of 5mg/ml, slowly dropwise adding 1% iodine acetic acid solution until the solution turns yellow, stopping dropwise adding, monitoring by HPLC for complete reaction, and adding vitamin C to terminate the reaction to obtain the crude peptide of the ularitide.

Further, the solvent adopted in the steps of washing and swelling is one or more of NMP, THF, DCM, DMF and DMSO;

the solvent used in the step of dissolving the substance is one or more of NMP, THF, DCM, DMF and DMSO.

The invention has the beneficial effects that: the invention provides a method for preparing ularitide by fragment condensation, which divides the ularitide into three parts of a fragment A (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Ser-Ser-NHNH 2), a fragment B (H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH2) and a fragment C (Cys-Asn-Ser-Phe-Arg-Tyr-OH) according to the characteristic that the peptide sequence of the ularitide contains two Cys, wherein hydrazide groups in the fragments respectively react with sodium nitrite and MPAA to generate thioester, and the thioester can selectively react with a polypeptide fragment with Cys at the N terminal. Different from the conventional fragment method, the fragment side chain in the invention has no protective group, has better solubility in water, does not have the problem of difficult coupling, and the obtained product has high purity, easy purification, simple operation and high production efficiency. The purity of the crude peptide of the ularitide obtained by the invention can be 74.64%, and the total yield can reach 50.2%. The purity of the refined peptide purified by HPLC can reach 99.87%. Compared with the prior art, the invention has the characteristics of high product quality, low cost, suitability for industrial production and the like.

Drawings

FIG. 1 is a scheme of synthesis of ularitide;

FIG. 2 is an HPLC chromatogram of a crude peptide of ularitide;

FIG. 3 is an HPLC chromatogram of urotropin protamine.

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

abbreviations and English	Means of
		HOAt	1-hydroxy-7-azobenzotriazol
DIPCDI	Diisopropylcarbodiimide
		HOBt	1-hydroxybenzotriazoles
HATU	2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
		HBTU	benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
DIPEA	N, N-diisopropylethylamine
		PyBOP	Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates
PyAOP	(3H-1,2, 3-triazolo [4,5-b ]]Pyridin-3-yloxy) tris-1-pyrrolidinophosphonium hexafluorophosphate
		EDT	Ethanedithiol
DMF	N, N-dimethylformamide
		NMP	N-methyl pyrrolidone
THF	Tetrahydrofuran (THF)
		DCM	Methylene dichloride
DMSO	Dimethyl sulfoxide
		PG	Protecting group
AA	Amino acids
		TFA	Trifluoroacetic acid
Fmoc	Fmoc group
		PhSMe	Thioanisole
PhOH	Phenol and its preparation
		TIS	Tri-isopropyl silane
PhOMe	Phenylmethyl ether
		MPAA	4-mercaptophenylacetic acid
TCEP	Tris (2-carboxyethyl) phosphine

A method of preparing ularitide, comprising the steps of:

1. fragment A (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH)₂) And fragment B (H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH)₂) Condensing to obtain fragment D (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH)₂): namely, thioester is formed at the C end of the segment A, and then the thioester is condensed with the segment B to obtain a segment D.

2. Condensing the fragment C (Cys-Asn-Ser-Phe-Arg-Tyr-OH) and the fragment D to obtain a linear peptide (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH): namely, thioester is formed at the C end of the segment D, and then the thioester is condensed with the segment C to obtain the ularitide linear peptide.

Example 1:

fragment A (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-NHNH)₂) Synthesis of (2)

Weighing 20.0g (10mmol) of 2-Cl-CTC Resin with the substitution degree of 0.5mmol/g, adding the 2g (10mmol) of 2-Cl-CTC Resin into a solid phase reaction column, washing the solid phase reaction column with DMF for 2 times, swelling the Resin with DMF for 30 minutes, then removing the solution, weighing 5.0g (100mmol) of hydrazine hydrate and 5.1g (50mmol) of triethylamine, dissolving the 5.0g (100mmol) of hydrazine hydrate and the 5.1g (50mmol) of triethylamine into DMF, adding the mixture into the solid phase reaction column under ice bath, reacting at room temperature for. 19.17g (50mmol) of Fmoc-Ser (tBu) -OH, 8.1g (60mmol) of HOBt and 18.96g (50mmol) of HBTU were weighed out and dissolved in DMF, and 12.9g (100mmol) of DIPEA was added to the solution in ice bath, and the solution was charged into a solid phase reaction column, reacted at room temperature for 2 hours, and washed 3 times with DMF. The Fmoc protecting group was removed in 20% piperidine (reaction time 5+7 min) and washed 6 times with DMF.

Repeating the steps of amino acid coupling and Fmoc protection group removal according to the peptide sequence of fragment A, and sequentially coupling Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Thr (tBu) -OH, Fmoc-Leu-OH and Fmoc-Thr (tBu) -OH by using coupling agents HOBt/DIPCDI or HOBt/PyBop/DIPEA or HBTU/HOBt/DIPEA or HOAt/PyAop/DIPEA.

The resulting peptide resin was treated with TFA/PhSMe/PhOH/EDT/H₂O (85:5:5:3:2, vol.) was cleaved for 2 hours, and the fragment A12.0g, 85% purity and 105% weight yield were obtained by ether precipitation, centrifugation and vacuum drying.

Example 2:

fragment B (H-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH)₂) Synthesis of (2)

The procedure was carried out in a manner similar to that of example 1, by coupling hydrazine hydrate, followed by coupling Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Met-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH and Boc-Cys (Trt) -OH in this order, and obtaining a peptide resin using TFA/PhSMe/PhOH/EDT/H₂O (85:5:5:3:2, volume ratio) for 2 hours, ether settling, centrifuging, and vacuum drying to obtain fragment B17.0g with purity of 80% and weight yield of 104%.

Example 3:

synthesis of fragment C (Cys-Asn-Ser-Phe-Arg-Tyr-OH)

62.5g (50mmol) of Wang Resin with a substitution degree of 0.8mmol/g was weighed, added to a solid phase reaction column, washed 2 times with DMF, after swelling the Resin with DMF for 30 minutes, 45.96g (100mmol) of Fmoc-Tyr (tBu) -OH, 16.2g (120mmol) of HOBt and 12.2g (10mmol) of DMAP were weighed, dissolved in DMF, added 16.4g (130mmol) of DIPCDI under ice bath, added to the solid phase reaction column, reacted at room temperature for 2 hours, and washed 6 times with DMF. The Resin was blocked for 6 hours by adding a mixture of acetic anhydride and pyridine (7:6 by volume), washed with DMF for 6 times, and then contracted and drained with methanol to give 81.4g of Fmoc-Phe-Wang Resin, which showed a degree of substitution of 0.4 mmol/g.

Referring to the method of example 1, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was treated with TFA/H₂O (95:5, vol.) was cleaved for 2 hours and ether precipitated to give fragment C90% pure in 102% weight yield.

Or taking 45.45g (50mmol) of 2-Cl-CTC Resin with the substitution degree of 1.1mmol/g, adding the 2-Cl-CTC Resin into a solid phase reaction column, washing the solid phase reaction column for 2 times by DMF, swelling the Resin by DMF for 30 minutes, then pumping out the solution, weighing 45.96g (100mmol) of Fmoc-Tyr (tBu) -OH, dissolving the mixture in DMF, adding 51.7g (400mmol) of DIPEA under ice bath, adding the mixture into the solid phase reaction column, reacting for 2 hours at room temperature, and washing by DMF for 6 times. Then 15ml of methanol was added to block the Resin for 1 hour, DMF was used for 6 times, and methanol was contracted and drained to obtain 62.2g of Fmoc-Tyr (tBu) -CTC Resin, the detection substitution was 0.8 mmol/g.

Referring to the method of example 1, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH and Boc-Cys (Trt) -OH were coupled in this order according to the peptide sequence of fragment C, and the resulting peptide resin was treated with TFA/H₂O (volume ratio, 95:5) was cleaved for 2 hours and ether precipitated to give fragment C92% pure and 92% weight yield.

Example 4:

fragment D (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-NHNH)₂) Synthesis of (2)

Weighing 12.0g (10mmol) of fragment A12 in 0.3M disodium hydrogen phosphate solution, adjusting pH to 3 with dilute sodium hydroxide solution, cooling to-10 ℃, slowly adding dropwise sodium nitrite solution (34.5g, 500mmol), stirring for 30 minutes after dropwise addition, adding 100.8g (600mmol) of MPAA, adjusting pH to 7 with dilute sodium hydroxide solution, finally adding 17.0g (10mmol) of fragment B17, heating to room temperature for reaction, monitoring by HPLC to complete the reaction, and adding TCEP to terminate the reaction.

The solution is subjected to HPLC simple purification to remove excessive MPAA and TCEP, namely a NOVASEP RP-HPLC system with the wavelength of 220nm and the stationary phase of a reversed phase C18 column and the mobile phase system of 0.1% TFA solution and acetonitrile are adopted, a target fraction is collected, and the fragment D28.7 g with the purity of 75.3% is obtained by rotary evaporation and concentration and freeze-drying.

Example 5:

synthesis of a linear peptide (H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH)

Weighing 28.7g (10mmol) of segment D, dissolving in 0.3M disodium hydrogen phosphate solution in a three-necked flask, adjusting pH to 3 with dilute sodium hydroxide solution, cooling to-10 ℃, slowly adding dropwise sodium nitrite solution (20.3g, 300mmol), stirring for 30 minutes after dropping, adding 67.2g (400mmol) of MPAA, adjusting pH to 7 with dilute sodium hydroxide solution, adding 8.0g (10mmol) of segment C, heating to room temperature for reaction, monitoring by HPLC for reaction completion, and adding TCEP to terminate the reaction.

And (3) carrying out simple HPLC purification on the solution to remove excessive MPAA and TCEP, namely adopting a NOVASEP RP-HPLC system, wherein the wavelength is 220nm, the stationary phase is a reversed phase C18 column, the 0.1% TFA solution and acetonitrile are mobile phase systems, collecting target fractions, carrying out rotary evaporation and concentration, and freeze-drying to obtain 36.4g of linear peptide with the purity of 72.4%.

Example 6:

synthesis of crude peptide

Weighing linear peptide, dissolving the linear peptide in 10% acetic acid solution with the concentration of 5mg/ml, slowly dropwise adding 1% iodine acetic acid solution, stopping dropwise adding when the solution turns yellow, monitoring the reaction by HPLC (high performance liquid chromatography), and adding vitamin C to terminate the reaction. The crude peptide of the ularitide is obtained, and the purity is 74.64%.

And (3) purifying the solution by HPLC, namely adopting a NOVASEP RP-HPLC system, wherein the wavelength is 220nm, the stationary phase is a reversed phase C18 column, the 0.1% TFA solution and acetonitrile are used as a mobile phase system, collecting target fractions, performing rotary evaporation and concentration, and performing freeze-drying to obtain 20.0g of the ularitide refined peptide, wherein the purity is 99.87%, and the total yield is 50.2%. The table below shows the comparison between the purity of the crude peptide, the purity of the refined peptide and the total yield in the preparation method of the urotropine protected by CN201610947595 and CN 201511029989.

Patent name	Purity of crude peptide	Purity of refined peptide	Overall yield of
				CN201610947595	～19％	98％	15％
CN201511029989	～50％	99.2％	40％
				The invention	74.64％	99.87％	50.2％

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.

SEQUENCE LISTING

<110> Shenzhen Hanyu pharmaceutical stockings Limited

<120> preparation method of ularitide

<130> CP11901943C

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 32

<212> PRT

<213> Artificial sequence

<400> 1

Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met

1 5 10 15

Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr

20 25 30