阅读说明：本技术 一种肽的固相合成方法 (Solid-phase synthesis method of peptide ) 是由 王刚 舒遂智 李新宇 邱心敏 付玉清 林艳霞 于 2020-05-31 设计创作，主要内容包括：本发明涉及多肽合成领域,特别涉及一种多肽的固相合成方法。该方法包括以下步骤：1)羟基树脂经活化剂活化成X-CO-O-Resin,其中X为活化基团；2)X-CO-O-Resin与NH-(2)NH-(2)反应合成NH-(2)NH-CO-O-Resin,或与Fmoc-NHNH-(2)反应合成Fmoc-NHNH-CO-O-Resin再脱除Fmoc得到NH-(2)NH-CO-O-Resin；3)NH-(2)NH-CO-O-Resin与氨基酸或肽片段按肽序偶联成肽酰肼树脂；4)肽酰肼树脂经裂解试剂裂解得到肽酰肼；5)肽肼在水中能转化为可用于偶联的酰基叠氮或肼末端结构,从而制备一些碳末端具有特殊修饰的多肽。该发特别适用碳末端为乙胺基或氨基脲的瑞林类多肽。利用肽肼方法制备瑞林类多肽,能够极大程度的降低成本,减少操作步骤,减少三废的排放。且合成过程安全稳定,无不良副反应发生,适合工业放大生产。(The invention relates to the field of polypeptide synthesis, in particular to a solid-phase synthesis method of polypeptide. The method comprises the following steps: 1) activating the hydroxyl Resin into X-CO-O-Resin by an activating agent, wherein X is an activating group; 2) X-CO-O-Resin and NH 2 NH 2 Reaction for synthesis of NH 2 NH-CO-O-Resin, or with Fmoc-NHNH 2 Fmoc-NHNH-CO-O-Resin is synthesized by reaction and then Fmoc is removed to obtain NH 2 NH‑CO‑O‑Resin；3)NH 2 Coupling NH-CO-O-Resin with amino acids or peptide fragments into peptide hydrazide Resin in peptide order; 4) cracking the peptide hydrazide resin by a cracking reagent to obtain peptide hydrazide; 5) peptide hydrazines can be converted in water to acyl azide or hydrazine terminal structures useful for conjugation, thereby producing some polypeptides with specific modifications at the carbon terminus. The method is particularly suitable for the relin polypeptide of which the carbon terminal is an ethylamino group or semicarbazide.The method for preparing the relin polypeptide by using the peptide hydrazine can greatly reduce the cost, reduce the operation steps and reduce the discharge of three wastes. And the synthesis process is safe and stable, no adverse side reaction occurs, and the method is suitable for industrial large-scale production.)

1. A method for solid phase synthesis of a peptide, comprising the steps of:

step 1: activating the hydroxyl Resin by an activating agent to form active carbonyl Resin X-CO-O-Resin, wherein X is an activating group;

step 2: active carbonyl Resin X-CO-O-Resin and NH₂NH₂Reaction for synthesis of NH₂NH-CO-O-Resin, or with Fmoc-NHNH₂Fmoc-NHNH-CO-O-Resin is synthesized by reaction and then Fmoc is removed to obtain NH₂NH-CO-O-Resin；

And step 3: NH (NH)₂Coupling NH-CO-O-Resin with amino acid or peptide fragment in peptide order to form peptide hydrazide Resin Fmoc- (AA) n-CONH-NH-CO-O-Resin;

and 4, step 4: fmoc- (AA) n-CONH-NH-CO-O-Resin is cracked by a cracking reagent to obtain peptide hydrazide (AA) n-CONH-NH₂；

And 5: peptide hydrazides are treated to provide peptides.

2. The method of claim 1, wherein the hydroxyl resin of step 1 is selected from the group consisting of: wang Resin, HMBA Resin, HMPA Resin.

3. The method of claim 1, wherein the activator of step 1 is selected from the group consisting of: phosgene, diphosgene, triphosgene, N '-carbonyl diimidazole, 4-nitrophenyl chloroformate and N, N' -disuccinimidyl carbonate.

4. The method of claim 3, wherein when the activator is phosgene, diphosgene, or triphosgene, X is Cl.

5. The process of claim 3 wherein when the activator is N, N' -carbonyldiimidazole, X is imidazolyl.

6. The process of claim 3 wherein X is 4-nitrophenyl when the activator is 4-nitrophenylchloroformate.

7. The method of claim 3, wherein when the activator is N, N' -disuccinimidyl carbonate, X is a succinimide group.

8. A method according to any one of claims 1 to 7, wherein the peptide has the structure: H-Pyr-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y, wherein X is selected from: D-Leu, D-Ser (tBu), D-Ala, D-Trp, Y is selected from: NHC₂H₅、NHNHCONH₂。

9. The method of claim 8, wherein when X is D-Ser (tBu), the cleavage reagent of step 4 is a mixed solution of TFA, TIS and DCM.

10. The method of claim 9, wherein the volume ratio of TFA, TIS and DCM is 5-20: 5-10: 70-90.

11. The method of claim 8, wherein Y is NHNHCONH₂Then, the step 5 is: the peptide hydrazide is treated with cyanate to obtain the peptide.

12. The method of claim 8, wherein Y is NHC₂H₅Then, the step 5 is: the peptide hydrazide is prepared into acyl azide by a nitroso compound, and then the acyl azide reacts with an ethylamine aqueous solution to obtain the peptide.

13. A process according to claim 11, wherein the cyanate is selected from: potassium cyanate, sodium cyanate and isocyanate.

14. The method according to claim 9, characterized in that the arginine used for the synthesis of said peptide is selected from the group consisting of: Fmoc-Arg-OH HF, Fmoc-Arg-OH HCl, Fmoc-Arg-OH HBr, Fmoc-Arg-OH CF₃COOH、Fmoc-Arg-OH·TosOH、Fmoc-Arg-OH·H₂SO₄。

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a solid-phase synthesis method of polypeptide.

Background

The polypeptide synthesis method mainly comprises two modes of solid-phase synthesis and liquid-phase synthesis. The liquid phase synthesis method is mainly used for short peptide synthesis and has the defects of complex intermediate purification operation, complex post-treatment operation, long synthesis period and the like. BruceMerrifield first proposed a solid-phase polypeptide synthesis method (SPPS) in 1963, which is convenient and rapid to synthesize, mild in reaction conditions, and widely used rapidly, and more than 85% of polypeptide drugs are obtained by solid-phase synthesis at present. Peptide hydrazide polypeptide coupling in solid phase polypeptide synthesis is an emerging chemoselective polypeptide coupling method reported by professor Liuliu in 2011, and the method uses a C-terminal hydrazide group as a precursor of thioester. It is worth emphasizing that the peptide hydrazide precursor can be very stably present in the SPPS process and the system of coupling of various polypeptides. After the corresponding peptide hydrazide is prepared by SPPS, the peptide hydrazide can be oxidized by sodium nitrite under an acidic condition to generate acyl azide and then reacts with aryl mercaptan, so that the corresponding high-activity aromatic polypeptide thioester can be prepared quickly and equivalently. The polypeptide thioester can selectively react with cysteine at the N-terminal of another polypeptide to generate a natural peptide bond. Currently, the synthesis of polypeptide C-terminal hydrazide mainly comprises CTC resin and hydrazine hydrate/Fmoc-NHNH₂And (4) reaction. However, CTC resins are very unstable, both in storage and in reaction. The stored CTC resin absorbs moisture and releases HCl as the storage time increases, so that the degree of substitution decreases. Meanwhile, the reaction is unstable, and the yield fluctuation of the product is large. Therefore, the preparation of hydrazide from CTC resin is only suitable for laboratory research and is not suitable for large-scale industrial production.

Most of carbon terminals of the relin polypeptide are ethylamino and semicarbazide, the existing synthesis mode is slightly different from that of the common peptide, US2010311946A1 utilizes Merrifield resin for coupling, corresponding peptide hydrazine is obtained in a hydrazinolysis mode after the reaction is finished, a dilute TFA solution is used for cutting off related acid-sensitive protecting groups, and finally, a semicarbazide structure is constructed in an aqueous solution through cyanate treatment to obtain the relin polypeptideTo goserelin crude product. However, in this patent Fmoc-OSu and hydrazine hydrate were synthesized as activated Fmoc-NHNH₂The experiment shows that the activated Fmoc-NHNH₂In DMF, it is difficult to dissolve, which causes great difficulty in solid phase synthesis. And the hydrazinolysis step has long reaction time and low yield, and is easy to cause hydrazine residue. CN102653555 uses Sieber Resin as solid phase carrier, uses N, N-disuccinimidyl carbonate to couple with it, then adds hydrazine hydrate to obtain Azagly-Resin, then sequentially makes peptide chain assembly to obtain Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-Arg (NO)₂) -Pro-Azagly-Resin, which is cleaved off after the synthesis using 10% TFA-DCM solution, part of the side chain protecting groups are removed, and Bzl and NO are finally removed by catalytic hydrogenation₂And obtaining the goserelin finished product by the protective group. This patent fails to avoid the hydrogenation step due to the Arg side chain NO₂The hydrogenation process is stable, and can be completely deprotected after being continued for dozens of hours, which greatly affects the productivity. And the process needs a high-pressure environment and uses palladium carbon as a catalyst, so that huge potential safety hazards and heavy metal residue risks exist. CN 106146622 takes resin as a solid phase carrier, adopts a one-by-one coupling mode to connect amino acid with side chain protecting groups, and synthesizes to obtain side chain full-protection leuprorelin precursor peptide resin; aminolysis cutting the side chain full-protection peptide resin by adopting an ethylamine-THF solution, and removing a side chain protecting group by using a deprotection reagent to obtain leuprorelin; or removing side chain protecting groups by using a deprotection reagent, and then carrying out aminolysis and cutting by adopting an ethylamine-THF solution to obtain the leuprorelin. The method disclosed by the patent needs to prepare a free ethylamine-THF solution in advance, and on the other hand, the aminolysis time is long and generally needs 24 hours, and the method for constructing the carbon-terminal ethylamine group by aminolysis by using the ethylamine-THF solution is quite common in the synthesis of the relin polypeptide, but the method is easy to generate obvious racemization reaction.

Disclosure of Invention

Aiming at the defects of the prior art, the invention establishes a novel polypeptide hydrazide method for synthesizing the polypeptide, in particular to a ruin polypeptide with an ethylamino group or semicarbazide at the carbon terminal. The method has simple route and low reaction difficulty, can effectively reduce the production cost, and is beneficial to the mass production of the polypeptide.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for solid phase synthesis of a peptide comprising the steps of:

step 1: activating the hydroxyl Resin by an activating agent to form active carbonyl Resin X-CO-O-Resin, wherein X is an activating group;

step 2: active carbonyl Resin X-CO-O-Resin and NH₂NH₂Reaction for synthesis of NH₂NH-CO-O-Resin, or with Fmoc-NHNH₂Fmoc-NHNH-CO-O-Resin is synthesized by reaction and then Fmoc is removed to obtain NH₂NH-CO-O-Resin；

And step 3: NH (NH)₂NH-CO-O-Resin is coupled with amino acid or peptide fragment according to peptide sequence to form Fmoc- (AA) n-CONH-NH-CO-O-Resin;

and 4, step 4: fmoc- (AA) n-CONH-NH-CO-O-Resin is cracked by a cracking reagent to obtain peptide hydrazide (AA) n-CONH-NH₂；

And 5: peptide hydrazides are treated to provide peptides.

Preferably, the hydroxyl resin is selected from: wang Resin, HMBA Resin, HMPA Resin. Compared with hydroxyl resin, the activated active carbonyl resin has better acid sensitivity, can be broken in a TFA-DCM system with the concentration of more than 5 percent, and the selected hydroxyl resin has low price, high storage stability, simple step operation of activating the resin, only reaction before use and contribution to industrial large-scale production.

Preferably, the activator of step 1 may be selected from: phosgene, diphosgene, triphosgene, N '-carbonyl diimidazole, 4-nitrophenyl chloroformate and N, N' -disuccinimidyl carbonate.

In some embodiments, the activating agent is phosgene, diphosgene or triphosgene, X is Cl, that is, the active carbonyl Resin is Cl-CO-O-Resin, and the specific structure is as follows:in some embodiments, the activator is N, N' -carbonyldiimidazole, and X is imidazolyl, i.e., the active carbonyl Resin is imidazole-CO-O-Resin, having the specific structure:in some embodiments, the activator is 4-nitrophenylchloroformate, X is 4-nitrophenyl, i.e., the activated carbonyl Resin is 4-nitrophenyl-CO-O-Resin, having the following specific structure:

in some embodiments, the activating agent is N, N' -disuccinimidyl carbonate, X is a succinimide group, i.e., the active carbonyl Resin is a succinimide group-CO-O-Resin, and the specific structure is as follows:

in some embodiments, the method can be used with a polypeptide having the structure H-Pyr-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y, wherein X is selected from the group consisting of: D-Leu, D-Ser (tBu), D-Ala, D-Trp, Y is selected from: NHC₂H₅、NHNHCONH₂. X is D-Leu, Y is NHC₂H₅When the polypeptide is leuprorelin; x is D-Ser (tBu), Y is NHC₂H₅When the polypeptide is buserelin; x is D-Ala and Y is NHC₂H₅When the polypeptide is alarelin; x is D-Trp and Y is NHC₂H₅When the polypeptide is the cererelin; x is D-Ser (tBu), Y is NHCONH₂When the polypeptide is goserelin, the polypeptide is goserelin.

Preferably, in the embodiment of preparing goserelin or buserelin, the volume ratio of TFA, TIS and DCM in the step 4 is 5-20: 5-10: 70-90. By controlling the concentration of the acid, the resin and part of the protecting groups can be guaranteed to be accurately cracked; meanwhile, as the cracking process adopts a solvent which is not soluble with water, and compared with the cracking system, the peptide hydrazide product has better solubility in water, and can be directly extracted by water after the cracking is finished, the whole synthesis process does not need to use settling agents such as diethyl ether and isopropyl ether to remove capture reagents, trifluoroacetic acid and other reagents in the cracking solution, the operation steps are saved, and the production efficiency is improved; and because the use of ether reagents is avoided, the cost is reduced, and the production safety is improved.

Preferably, in an embodiment of the preparation of goserelin, said step 5 is: treating peptide hydrazide with cyanate to obtain goserelin, wherein the cyanate is selected from: potassium cyanate, sodium cyanate and isocyanate. The applicant has surprisingly found that in the example of preparation of goserelin, the use of hydrazine is at the beginning of the process, and that, being a solid phase synthesis, the excess hydrazine hydrate can be removed by filtration and repeated washing, greatly reducing the possibility of residues. In the method for constructing the hydrazide by using the activated resin instead of using expensive Sieber resin in the selection of the resin, the activated resin is used for constructing the hydrazide, and the final cracking step can use TFA solution with relatively low concentration for cracking, so that the release of the target peptide hydrazine is ensured while the side chain of D-Ser (tBu) is not removed. In addition, because the final construction step is carried out in water, a settling agent such as diethyl ether is not needed, and the water phase is directly reserved after water extraction for the next step. And (4) directly purifying after the reaction is finished to obtain the target peptide.

Preferably, in the embodiment of preparing a polypeptide with an ethylamino group at the carbon terminal, such as leuprorelin, buserelin, alarelin, dorelin, etc., the step 5 is: the peptide hydrazide is prepared into acyl azide by a nitroso compound, and then the acyl azide reacts with an ethylamine aqueous solution to obtain the polypeptide. The applicant unexpectedly found that when such polypeptides are prepared by peptide hydrazine polypeptide coupling, acidic aqueous solutions of peptide hydrazine can be directly subjected to structural transformation to a certain extent to obtain acyl azide, which is then directly coupled with aqueous ethylamine solution in an aqueous phase. In all coupling methods known to date, the degree of racemization in the azide process is minimal, thus minimizing the racemization effect of the final coupling step. Meanwhile, acyl azide is very mild, and NH in a peptide chain is removed₂Other groups may be unprotected and coupling can be achieved in the aqueous phase. Secondly, compared with the prior art, the method does not need to prepare an ethylamine-THF solution in advance, can finish the condensation of the final stage only by using an aqueous solution, greatly reduces the synthesis cost, and is suitable for industrial mass production.

In some embodiments, the arginine used to synthesize goserelin or buserelin using the present invention may be selected from the group consisting of: Fmoc-Arg-OH HF, Fmoc-Arg-OH HCl, Fmoc-Arg-OH HBr, Fmoc-Arg-OH CF₃COOH、Fmoc-Arg-OH·TosOH、Fmoc-Arg-OH·H₂SO₄. Protonating the guanidine group instead of protecting group Fmoc-Arg (Pbf) -OH or Fmoc-Arg (NO)₂) And the-OH can avoid a high-concentration acid environment or catalytic hydrogenation reaction required in the removal of a protecting group, and reduce the reaction difficulty and potential safety hazard of industrial production.

The invention establishes a peptide hydrazine method process for synthesizing polypeptide, which takes cheap and easily obtained hydroxyl resin as an initial carrier to synthesize the corresponding peptide hydrazine resin through conventional reaction. The resin is sensitive to acid, and can release peptide hydrazine products while retaining partial protecting groups by selecting TFA lysates with different concentrations. Peptide hydrazine can be converted into acyl azide or hydrazine terminal structure for coupling in water, thereby preparing some polypeptides with special modification at the carbon terminal, in particular to the polypeptide of the relin class with ethylamine group or semicarbazide group at the carbon terminal. The compound is prepared by a peptide hydrazine method, so that the cost can be greatly reduced, the operation steps are reduced, and the discharge of three wastes is reduced. And the synthesis process is safe and stable, no adverse side reaction occurs, and the method is suitable for industrial large-scale production.

Detailed Description

The present invention will be described in further detail with reference to specific examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Example 1 activation of Wang Resin 1

5.00g of Wang resin (degree of substitution: 0.84mmol/g) was weighed out, and was soaked in 50ml of dichloromethane for 15min to sufficiently swell, and then the solvent was removed by filtration, and 50ml of dichloromethane was added again, and then the mixture was cooled in an ice bath. 1.27g triethylamine (3eq) was added, 1.25g triphosgene was added in portions, held at low temperature for 30min, and moved to room temperature for further reaction for 2 h. After the reaction was completed, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with methylene chloride and then dried under vacuum to give 5.27g of resin.

Example 2 activation of Wang Resin 2

5.00g of Wang resin (degree of substitution: 0.84mmol/g) was weighed out, and soaked in 50ml of THF for 15min to swell sufficiently, the solvent was removed by filtration, 50ml of THF was added again, 2.05g of carbonyldiimidazole was added at room temperature, and the reaction was stirred for 2 hours. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 5.40g of resin.

Example 3 activation of Wang Resin 3

5.00g of Wang resin (degree of substitution: 0.84mmol/g) was weighed out, and was soaked in 50ml of dichloromethane for 15min to sufficiently swell, and then the solvent was removed by filtration, and 50ml of dichloromethane was added again, and then the mixture was cooled in an ice bath. 1.27g triethylamine (3eq) was added, then 2.54g p-nitrophenyl chloroformate was added in portions, kept at low temperature for 30min, and then moved to room temperature for further reaction for 2 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 5.69g of resin.

Example 4 activation of Wang Resin 4

5.00g of Wang resin (degree of substitution 0.84mmol/g) was weighed, 50ml of DMF was added and soaked for 15min to fully swell, the solvent was removed by filtration, 50ml of DMF was added again, 5.34g (5eq) of N, N' -disuccinimidyl carbonate was added at room temperature, 0.26g of DMAP was added for catalysis, and the reaction was stirred at room temperature for 6 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 5.60g of resin.

Example 5 activation of HMBA Resin 1

5.00g of HMBA resin (degree of substitution 0.69mmol/g) was weighed out, and was soaked in 50ml of dichloromethane for 15min to sufficiently swell, and then the solvent was removed by filtration, and 50ml of dichloromethane was added again, and the mixture was cooled in an ice bath. 1.05g of triethylamine (3eq) was added, 1.02g of triphosgene was added in portions, the mixture was kept at low temperature for 30min, and the mixture was allowed to cool to room temperature for further reaction for 2 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 5.22g of resin.

EXAMPLE 6 activation of HMBA Resin 2

10.00g of HMBA resin (degree of substitution 0.69mmol/g) was weighed out, 100ml of THF was added and soaked for 15min to fully swell, the solvent was removed by filtration, 100ml of THF was added again, 3.36g (3eq) of N, N' -carbonyldiimidazole was added at room temperature, and the reaction was stirred for 2 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 10.66g of resin.

Example 7 activation of HMPA Resin 1

3.00g of HMPA resin (degree of substitution 0.54mmol/g) was weighed out, soaked in 30ml of dichloromethane for 15min to fully swell, filtered to remove the solvent, added with 30ml of dichloromethane again, and cooled in an ice bath. 0.49g N-methylmorpholine (3eq) was added, followed by 0.98g of 4-nitrophenyl chloroformate in portions, which was kept at low temperature for 30min, and then allowed to cool to room temperature for further reaction for 2 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 3.27g of resin.

Example 8 activation of HMPA Resin 2

5.00g of HMPA resin (degree of substitution 0.54mmol/g) was weighed, 50ml of DMF was added and soaked for 15min to fully swell, the solvent was removed by filtration, 50ml of DMF was added again, 3.43g (5eq) of N, N' -disuccinimidyl carbonate was added at room temperature, 0.16g of DMAP was added for catalysis, and the reaction was stirred at room temperature for 6 h. After the reaction, the solution was removed by suction filtration, and the resin was washed three times with DMF and three times with dichloromethane and then dried under vacuum to give 5.38g of resin.

Example 9 NH₂Synthesis of NH-CO-O-Resin 1

2.5ml of hydrazine hydrate was measured and DMF was added to 50ml to give a 5% hydrazine-DMF solution. Suspending the resin in example 1 in 50ml DMF, soaking and swelling, removing the solvent by suction filtration, adding the 5% hydrazine-DMF prepared in advance, reacting for 1.5h at room temperature, collecting the resin by suction filtration after the reaction is finished, washing the resin three times by DMF, and directly using the resin for subsequent reaction.

Example 10 NH₂Synthesis of NH-CO-O-Resin 2

The resin from example 6 was suspended in 100ml DMF and soaked for swelling, after the solvent was removed by suction filtration, 100ml DMF was added again and 17.55g Fmoc-NHNH was weighed₂(10eq) and 8.90g (10eq) DIPEA were added and reacted at room temperature for 2 h. After the reaction, the resin was collected by suction filtration, washed three times with DMF, and then subjected to deprotection treatment. 100ml of 20% piperidine-DMF solution are metered in and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N₂Bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and directly using the resin for subsequent reaction after being dried by suction.

Example 11 NH₂Synthesis of NH-CO-O-Resin 3

1.5ml of hydrazine hydrate was measured and DMF was added to 30ml to give a 5% hydrazine-DMF solution. Suspending the resin in example 7 in 30ml DMF, soaking and swelling, removing the solvent by suction filtration, adding the 5% hydrazine-DMF prepared in advance, reacting for 1.5h at room temperature, collecting the resin by suction filtration after the reaction is finished, washing the resin three times by DMF, and directly using the resin for subsequent reaction.

EXAMPLE 12 Synthesis of Fmoc-NHNH-CO-O-Resin 4

The resin of example 4 was suspended in 50ml DMF and soaked for swelling, after the solvent was removed by suction filtration, 50ml DMF was added again, and 10.68g Fmoc-NHNH was weighed₂(10eq) and 5.40g (10eq) of DIPEA were added and reacted at room temperature for 2 hours. After the reaction, the resin was collected by suction filtration, washed three times with DMF, and then subjected to deprotection treatment. 50ml of 20% piperidine-DMF solution are weighed out and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 50ml 20% piperidine-DMF solution again, adding into the resin, N₂Bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and directly using the resin for subsequent reaction after being dried by suction.

Example 13 NH₂Synthesis of NH-CO-O-Resin 5

The resin of example 3 was suspended in 50ml DMF and soaked for swelling, after the solvent was removed by suction filtration, 50ml DMF was added again, and 10.68g Fmoc-NHNH was weighed₂(10eq) and 5.40g (10eq) of DIPEA were added and reacted at room temperature for 2 hours. After the reaction, the resin was collected by suction filtration, washed three times with DMF, and then subjected to deprotection treatment. 50ml of 20% piperidine-DMF solution are weighed out and added to the resin, N₂Bubbling deviceReacting for 5min, filtering to remove reaction solution, adding 50ml 20% piperidine-DMF solution, adding into resin, and adding N₂Bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and directly using the resin for subsequent reaction after being dried by suction.

Example 14 NH₂Synthesis of NH-CO-O-Resin 5

The resin from example 5 was suspended in 50ml DMF and swollen, after removing the solvent by suction filtration, 50ml DMF was added again and 8.78g Fmoc-NHNH was weighed₂(10eq) and 4.40g (10eq) of DIPEA were added and reacted at room temperature for 2 hours. After the reaction, the resin was collected by suction filtration, washed three times with DMF, and then subjected to deprotection treatment. 50ml of 20% piperidine-DMF solution are weighed out and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 50ml 20% piperidine-DMF solution again, adding into the resin, N₂Bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and directly using the resin for subsequent reaction after being dried by suction.

Example 15

2.5ml of hydrazine hydrate was measured and DMF was added to 50ml to give a 5% hydrazine-DMF solution. Suspending the resin in example 2 in 50ml DMF, soaking and swelling, filtering to remove the solvent, adding the prepared 5% hydrazine-DMF, reacting for 1.5h at room temperature, filtering to collect the resin after the reaction is finished, washing with DMF for three times, and directly using the resin for the subsequent reaction.

Example 16

2.5ml of hydrazine hydrate was measured and DMF was added to 50ml to give a 5% hydrazine-DMF solution. Suspending the resin in example 8 in 50ml DMF, soaking and swelling, removing the solvent by suction filtration, adding the 5% hydrazine-DMF prepared in advance, reacting for 1.5h at room temperature, collecting the resin by suction filtration after the reaction is finished, washing the resin three times by DMF, and directly using the resin for subsequent reaction.

Example 17 Synthesis of leuprorelin peptide hydrazide

Transferring the resin obtained in example 9 to a solid phase polypeptide synthesis tube, adding 50ml of DMF to soak and swell, washing off the solvent, and sequentially performing coupling operation;

a coupling step: weighing 12.6mmol Fmoc-amino acid, 2.04g HOBt, dissolving in 50ml DMF, and placingCooling in ice bath, slowly dropping 1.58g DIC, activating for 5min, adding activated reaction solution into resin, and adding N₂Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Tyr (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp-OH, Fmoc-His (Trt) -OH, H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete. Deprotection step: 50ml of 20% piperidine-DMF solution are weighed out and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 50ml 20% piperidine-DMF solution again, adding into the resin, N₂Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 90ml of TFA, 5ml of TIS and 5ml of H2O were weighed out, mixed and pre-frozen at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. After the reaction is finished, filtering and collecting filtrate, washing the resin with dichloromethane for three times, mixing the resin with the filtrate, decompressing and concentrating to remove most of the solvent, adding 300ml of frozen ether, washing out a large amount of solid, centrifuging and collecting the solid, and then carrying out N₂Drying by blowing to obtain 5.12g of crude peptide hydrazine.

Example 18 Synthesis of goserelin/buserelin peptide hydrazide 1

Transferring the resin obtained in the example 10 into a solid phase polypeptide synthesis tube, adding 100ml of DMF to soak and swell, washing away the solvent, and sequentially performing coupling operation;

a coupling step: weighing 20.7mmol Fmoc-amino acid and 3.35g HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 2.87g DIC, activatingAdding the activated reaction solution into resin for 5min, N₂Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Pro-OH, Fmoc-Arg-OH HCl, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr-OH, Fmoc-Ser-OH, Fmoc-Trp-OH, Fmoc-His (Trt) -OH, H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete. Deprotection step: 100ml of 20% piperidine-DMF solution are metered in and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 100ml 20% piperidine-DMF solution again, adding into the resin, N₂Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 30ml of TFA, 15ml of TIS and 105ml of methylene chloride were weighed out, mixed and pre-frozen at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. After the reaction is finished, filtering and collecting filtrate, washing the resin with dichloromethane for three times, mixing the resin with the filtrate, decompressing and concentrating to remove most of the solvent, adding 600ml of frozen ether to wash out a large amount of solid, centrifuging and collecting the solid, and then carrying out N₂Drying by blowing to obtain 8.31g of crude peptide hydrazine.

Example 19 Synthesis of goserelin/buserelin peptide hydrazide 2

Transferring the resin obtained in example 11 to a solid phase polypeptide synthesis tube, adding 30ml of DMF to soak and swell, washing off the solvent, and sequentially performing coupling operation;

a coupling step: weighing 4.9 mmole Fmoc-amino acid and 0.79g HOBt, dissolving in 30ml DMF, cooling in ice bath, slowly dropping 0.67g DIC, activating for 5min, adding activated reaction solution into resin,N₂carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Pro-OH, Fmoc-Arg-OH & HBr, Fmoc-Leu-OH, Fmoc-D-Ser (tBu) -OH, Fmoc-Tyr-OH, Fmoc-Ser-OH, Fmoc-Trp-OH, Fmoc-His (Trt) -OH, H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete. Deprotection step: 30ml of 20% piperidine-DMF solution are metered in and added to the resin, N₂Bubbling for 5min, filtering to remove the reaction solution, adding 30ml of 20% piperidine-DMF solution again, adding into the resin, and adding N₂Carrying out bubbling reaction for 10min, filtering to remove reaction liquid, washing the resin with DMF for six times, and repeatedly carrying out the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 5ml of TFA, 2.5ml of TIS and 42.5ml of methylene chloride were weighed out, mixed and pre-frozen at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. And after the reaction is finished, adding 30ml of pure water, stirring at room temperature for 10min, separating liquid to remove an organic phase, adding 30min of pure water into the organic phase, stirring for 10min, separating liquid to remove the organic phase, combining water phases, adding sodium acetate solid, and adjusting the pH to 3-4 for later use. To obtain the goserelin/buserelin peptide hydrazide aqueous solution.

Example 20 Synthesis of goserelin/buserelin peptide hydrazide 3

Transferring the resin obtained in example 12 to a solid phase polypeptide synthesis tube, adding 50ml of DMF to soak and swell, washing off the solvent, and sequentially performing coupling operation;

a coupling step: weighing 12.6mmol Fmoc-amino acid and 2.04g HOBt, dissolving in 50ml DMF, cooling in ice bath, slowly dropping 1.75g DIC, activating for 5min, adding activated reaction solution into resin, and adding N₂Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Pro-OH, Fmoc-Arg-OH 1/2H₂SO₄,Fmoc-Leu-OH,Fmoc-D-Ser(tBu)-OH,Fmoc-Tyr-OH,Fmoc-Ser-OH,Fmoc-Trp-OH,Fmoc-His(Trt)-OH,H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete. Deprotection step: measuring 50ml of 20% piperidine-DMF solution, adding the solution into resin, carrying out bubbling reaction for 5min by N2, filtering to remove reaction liquid, adding 50ml of 20% piperidine-DMF solution again, adding the solution into the resin, carrying out bubbling reaction for 10min by N2, filtering to remove the reaction liquid, washing the resin with DMF for six times, and repeating the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. Weighing 7.5ml of TFA, 4ml of TIS and 38.5ml of dichloromethane in a ratio of 5-20: 5-10: 70-90, confirming that no problem exists at once), mixing, and pre-freezing at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. And after the reaction is finished, adding 30ml of pure water, stirring at room temperature for 10min, separating liquid to remove an organic phase, adding 30min of pure water into the organic phase, stirring for 10min, separating liquid to remove the organic phase, combining water phases, adding sodium acetate solid, and adjusting the pH to 3-4 for later use. To obtain the goserelin/buserelin peptide hydrazide aqueous solution.

Example 21 Synthesis of Alarelin peptide hydrazide

Transferring the resin obtained in example 13 to a solid phase polypeptide synthesis tube, adding 50ml of DMF to soak and swell, washing off the solvent, and sequentially performing coupling operation;

a coupling step: weighing 15.1mmol Fmoc-amino acid and 2.45g HOBt, dissolving in 60ml DMF, cooling in ice bath, slowly dropping 2.10g DIC, activating for 5min, and mixingAdding the activated reaction solution into resin, N₂Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; wherein the amino acids used for coupling are in sequence: Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-D-Ala-OH, Fmoc-Tyr-OH, Fmoc-Ser-OH, Fmoc-Trp-OH, Fmoc-His (Trt) -OH, H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete.

Deprotection step: measuring 60ml of 20% piperidine-DMF solution, adding the solution into resin, carrying out bubbling reaction for 5min by N2, filtering to remove reaction liquid, adding 60ml of 20% piperidine-DMF solution again, adding the solution into the resin, carrying out bubbling reaction for 10min by N2, filtering to remove the reaction liquid, washing the resin with DMF for six times, and repeating the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 54ml of TFA, 3ml of TIS and 3ml of H were weighed out₂And O, mixing, and pre-freezing at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. After the reaction, the reaction mixture was concentrated under reduced pressure, and 300ml of ethyl ether was added to precipitate a large amount of solid. After drying in vacuo, the crude peptide hydrazine was obtained as a white block solid 5.94g, crude yield 102.06% Synthesis of the peptide hydrazide example 22 Deserelin

Transferring the resin obtained in example 16 to a solid phase polypeptide synthesis tube, adding 30ml of DMF to soak and swell, washing off the solvent, and sequentially performing coupling operation;

a coupling step: weighing 16.2 mmole Fmoc-amino acid and 2.62g HOBt, dissolving in 100ml DMF, cooling in ice bath, slowly dropping 2.24g DIC, activating for 5min, adding activated reaction solution into resin, and adding N₂Carrying out bubbling reaction for 2h, carrying out suction filtration to remove reaction liquid, washing the resin with DMF for six times, and carrying out ninhydrin detection after suction drying; it is composed ofThe amino acids used for the intermediate coupling are sequentially: Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-D-Trp-OH, Fmoc-Tyr-OH, Fmoc-Ser-OH, Fmoc-Trp-OH, Fmoc-His (Trt) -OH, H-Pyr-OH.

Ninhydrin detection: and (3) putting 1-5 mg of resin into a centrifugal tube, washing with ethanol for three times, washing off supernate, adding a plurality of drops of 5% ninhydrin-ethanol solution, and heating on a metal bath at 100 ℃ for 5 min. If the resin and the detection liquid are yellow, the reaction is complete, and deprotection operation can be carried out; if the reaction product is blue black, the reaction product still has part unreacted, and the feeding is repeated until the reaction is complete.

Deprotection step: measuring 100ml of 20% piperidine-DMF solution, adding the solution into resin, carrying out bubbling reaction for 5min by N2, filtering to remove reaction liquid, adding 100ml of 20% piperidine-DMF solution again, adding the solution into the resin, carrying out bubbling reaction for 10min by N2, filtering to remove the reaction liquid, washing the resin with DMF for six times, and repeating the coupling step after drying;

a cracking step: the coupled peptide resin was washed three times with DMF and three times with dichloromethane, drained and transferred to a round bottom flask. 90ml TFA, 5ml TIS, 5ml H were measured₂And O, mixing, and pre-freezing at-20 ℃ for 30 min. Adding the lysate into the resin at one time, and performing pyrolysis for 1.5 h. After the reaction, the reaction mixture was concentrated under reduced pressure, and 600ml of ethyl ether was added to precipitate a large amount of solid. After vacuum drying, crude peptide hydrazine was obtained as a white massive solid 6.89g with a crude yield of 98.43%.

EXAMPLE 23 Synthesis of leuprolide

The peptide hydrazide obtained in example 17 was dissolved in 200ml of a 30% methanol-water solution, and the pH was adjusted to about 3 using dilute hydrochloric acid. Cooling at-10 deg.C. Weighing 1.45g NaNO₂Dissolving in 20ml water, slowly dropping into the peptide hydrazine solution, reacting at low temperature for 30 min. 2.7ml of ethylamine water solution (70 percent, 10eq) is sucked and slowly dropped into the reaction system, the reaction is continuously carried out for 6 hours in a low-temperature environment, HPLC (high performance liquid chromatography) monitors that the reaction is finished, and sampling detection shows that the purity of the crude leuprorelin is 80.6 percent and the synthesis yield is 65.2 percent.

EXAMPLE 24 Synthesis of buserelin

The peptide hydrazine solution (60 ml) obtained in example 18 was diluted with methanol (30 ml) and then cooled at-10 ℃, and 0.56g of NaNO2 was dissolved in water (10 ml) and slowly dropped into the reaction system. Keeping the temperature at low temperature for 30min, then adding 1ml of ethylamine aqueous solution (70 percent, 10eq), reacting at low temperature for 6h, monitoring the reaction by HPLC, sampling and detecting, wherein the purity of the buserelin crude product is 55.4 percent, and the synthesis yield is 53.1 percent.

Example 25 Synthesis of goserelin

The peptide hydrazine solution obtained in example 19 was stirred at room temperature, 1.02g of potassium cyanate was weighed and dissolved in 10ml of water, and slowly dropped into the peptide hydrazine solution, stirred at room temperature for 20min, the reaction was monitored by HPLC, and the purity of the goserelin crude product was 60.25% by sampling and detection, and the synthesis yield was 50.8%.

Example 26 Synthesis of goserelin

The peptide hydrazine solution obtained in example 20 was stirred at room temperature, 1.45g of trimethylsilyl isocyanate was absorbed, and slowly dropped into the peptide hydrazine solution, stirred at room temperature for 20min, and the reaction was monitored by HPLC, and the purity of the goserelin crude product was 57.3% and the synthesis yield was 46.2% by sampling detection.

Example 27 Synthesis of alarelin

The peptide hydrazide of example 21 was dissolved in 150ml of 30% methanol-water solution, and the pH was adjusted to about 3 using dilute hydrochloric acid. Cooling at-10 deg.C. Weighing 1.04g NaNO₂Dissolving in 20ml water, slowly dropping into the peptide hydrazine solution, reacting at low temperature for 30 min. 3.2ml of ethylamine water solution (70 percent, 10eq) is sucked and slowly dropped into the reaction system, the reaction is continuously carried out for 6 hours in a low-temperature environment, HPLC (high performance liquid chromatography) monitors that the reaction is finished, and sampling detection shows that the purity of the crude alarelin is 78.7 percent and the synthesis yield is 61.5 percent.

Example 28 Synthesis of Deserelin

The peptide hydrazide of example 22 was dissolved in 150ml of 30% methanol-water solution, and the pH was adjusted to about 3 using dilute hydrochloric acid. Cooling at-10 deg.C. Weighing 1.12g NaNO₂Dissolving in 20ml water, slowly dropping into the peptide hydrazine solution, reacting at low temperature for 30 min. 3.5ml of ethylamine water solution (70 percent, 10eq) is sucked and slowly dropped into the reaction system, the reaction is continuously carried out for 6 hours in a low-temperature environment, HPLC (high performance liquid chromatography) is used for monitoring the completion of the reaction, sampling and detection are carried out, and Deserelin is crudeThe product purity was 72.6% and the synthesis yield was 69.3%.