A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
By employing a liquid-phase synthesis method and an optimized condensation-deprotection reaction, the problems of low yield and purity in the synthesis of Fmoc-Pro-Pro-Pro-OH were solved, achieving efficient tripeptide synthesis suitable for the industrial production of telpolide.
Patent Information
- Application Number
- CN202511476166.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-16
- Publication Date
- 2026-01-30
- Estimated Expiration
- 2045-10-16
AI Technical Summary
There are few existing methods for synthesizing Fmoc-Pro-Pro-Pro-OH, and the yield and purity are not high, which makes it difficult to meet the industrial production needs of telpolide.
A liquid-phase synthesis method was adopted, which involves the condensation and deprotection reaction of proline reagent and proline derivative. Alkylamine compounds and activating reagent HOBt were used to avoid the use of resin and optimize reaction conditions to improve yield and purity.
The synthesis of Fmoc-Pro-Pro-Pro-OH with high yield and high purity has been achieved, providing a new direction for the optimized production of telpoeptide, which is suitable for industrial applications.
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Figure CN120943886B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide synthesis technology, specifically relating to a method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH. Background Technology
[0002] Tirzepatide (CAS No.: 2023788-19-2) is a dual-target peptide drug for the treatment of diabetes and weight loss. It is suitable for adult patients with type 2 diabetes whose blood sugar is not well controlled despite diet control, exercise, or treatment with metformin or sulfonylureas. Tirzepatide can be administered subcutaneously once a week to achieve the goal of controlling blood sugar.
[0003] The sequence of telpoeptide is: H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-lle-Ala-Gin.{diacid-gamma-Glu-(AEEA)2-Lys}-Ala-Phe-VaGin-Trp-Leu-Ile-Ala-Gly-Gly-Pro-SerSer-Gly-Ala-Pro-Pro-Pro-Ser-NH2. Fmoc-Pro-Pro-Pro-OH can be used as an intermediate in the synthesis of telpoeptide. Currently, there are few publicly available studies on the synthesis of Fmoc-Pro-Pro-Pro-OH. Summary of the Invention
[0004] The purpose of this invention is to provide a method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH with high yield and high purity.
[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:
[0006] A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH includes: preparing Fmoc-Pro-Pro-Pro-OH by a condensation deprotection reaction of a proline reagent and a proline derivative, wherein the proline reagent includes Fmoc-Pro-OH or Fmoc-Pro-Pro-OH, and the proline derivative is H-Pro-OtBu; an activating reagent is used in the condensation deprotection reaction, and the mass ratio of Fmoc-Pro-OH to the activating reagent is 1:0.1-0.9. This invention prepares Fmoc-Pro-Pro-Pro-OH via a liquid-phase synthesis method using a proline reagent and a proline derivative, avoiding the use of resin, reducing raw material consumption, and offering high efficiency and speed, which is beneficial for industrial production. The mass production of Fmoc-Pro-Pro-Pro-OH will provide a new direction for the optimized production of telpolide.
[0007] Preferably, a base is used in the condensation deprotection reaction, including N,N-diisopropylethylamine, N-methylimidazolium, or pyridine.
[0008] Preferably, a base is used in the condensation deprotection reaction. The base includes an alcoholic compound, which is prepared by reacting ethanolamine with ethyl 3-bromopyruvate. In this invention, an alcoholic compound is prepared by reacting an alcoholic amine with ethyl 3-bromopyruvate. This alcoholic compound can be used as a base in conjunction with HOBt in the synthesis of Fmoc-Pro-Pro-Pro-OH or Fmoc-Pro-Pro-OH, resulting in higher yield and purity of Fmoc-Pro-Pro-Pro-OH.
[0009] More preferably, the mass ratio of proline reagent to base used is 1:0.1-0.6.
[0010] More preferably, the mass ratio of the proline reagent to the amino group compound is 1:0.1-0.6.
[0011] Preferably, a tetrahydrofuran / hydrochloric acid mixed system is used to remove OtBu in the condensation deprotection reaction.
[0012] Preferably, the mass ratio of tetrahydrofuran to hydrochloric acid in the tetrahydrofuran / hydrochloric acid mixed system is 2-10:1-4.
[0013] Preferably, the reaction solvent in the condensation deprotection reaction includes at least one of N,N-dimethylformamide, dichloromethane, and a tetrahydrofuran / hydrochloric acid mixture.
[0014] Preferably, in the preparation of Fmoc-Pro-Pro-Pro-OH, Fmoc-Pro-Pro-OtBu is first prepared by reacting Fmoc-Pro-OH with H-Pro-OtBu, and then Fmoc-Pro-Pro-OtBu is de-OtBu to obtain Fmoc-Pro-Pro-OH; then Fmoc-Pro-Pro-Pro-OtBu is prepared by reacting Fmoc-Pro-Pro-OH with H-Pro-OtBu, and then Fmoc-Pro-Pro-Pro-OtBu is de-OtBu to obtain Fmoc-Pro-Pro-Pro-OH.
[0015] Preferably, the preparation of Fmoc-Pro-Pro-OtBu uses a base and an activating agent. The base includes at least one of N,N-diisopropylethylamine, N-methylimidazole and pyridine, and the activating agent includes HOBt.
[0016] Preferably, ethyl acetate is also used in the preparation of Fmoc-Pro-Pro-OH.
[0017] Preferably, in the preparation of Fmoc-Pro-Pro-OtBu, Fmoc-Pro-OH and a reaction solvent are mixed, and an alkali, an activating reagent and a proline derivative are added at a temperature of 0-5°C. The mixture is then reacted at 20-30°C for 2-12 hours. After the reaction is completed, Fmoc-Pro-Pro-OtBu is obtained through post-reaction treatment.
[0018] More preferably, in the preparation of Fmoc-Pro-Pro-OtBu, the reaction solvent is at least one of N,N-dimethylformamide and dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent is 1:5-20.
[0019] More preferably, in the preparation of Fmoc-Pro-Pro-OtBu, the base is at least one of N,N-diisopropylethylamine, N-methylimidazolium, and pyridine, and the mass ratio of Fmoc-Pro-OH to the base is 1:0.1-0.6.
[0020] More preferably, in the preparation of Fmoc-Pro-Pro-OtBu, the activating agent is at least one of HATU, HBTU, and HOBt, with HOBt being the preferred activating agent, and the mass ratio of Fmoc-Pro-OH to the activating agent is 1:0.1-0.9.
[0021] More preferably, in the preparation of Fmoc-Pro-Pro-OtBu, the proline derivative is H-Pro-OtBu, and the mass ratio of the amount of Fmoc-Pro-OH to the amount of proline derivative is 1:0.2-1.
[0022] More preferably, in the preparation of Fmoc-Pro-Pro-OtBu, during the post-reaction treatment, water is added to the reaction solution, and after separation, the organic phase is washed sequentially with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer is then dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0023] Preferably, in the preparation of Fmoc-Pro-Pro-OH, Fmoc-Pro-Pro-OtBu and a reaction solvent are mixed, then an acidic reagent is added, and the mixture is reacted at 20-30°C for 0.5-6 hours. After the reaction is completed, Fmoc-Pro-Pro-OH is obtained through post-reaction treatment.
[0024] More preferably, in the preparation of Fmoc-Pro-Pro-OH, the reaction solvent is tetrahydrofuran, the acidic reagent is hydrochloric acid, and the mass ratio of Fmoc-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid is 1:2-10:1-4.
[0025] More preferably, in the preparation of Fmoc-Pro-Pro-OH, during the post-reaction treatment, water and ethyl acetate are added to the reaction solution, the organic layer is washed successively with water and saturated brine, the organic layer is dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-OH.
[0026] Preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, Fmoc-Pro-Pro-OH and a reaction solvent are mixed, and an alkali, an activating reagent and a proline derivative are added at a temperature of 0-5°C. The mixture is then reacted at 20-30°C for 2-12 hours. After the reaction is completed, Fmoc-Pro-Pro-OtBu is obtained through post-reaction treatment.
[0027] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, the reaction solvent is at least one of N,N-dimethylformamide and dichloromethane, and the mass ratio of Fmoc-Pro-Pro-OH to the reaction solvent is 1:5-20.
[0028] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, the base is at least one of N,N-diisopropylethylamine, N-methylimidazolium, and pyridine, and the mass ratio of Fmoc-Pro-Pro-OH to the base is 1:0.1-0.6.
[0029] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, the activating agent is at least one of HATU, HBTU, and HOBt, with HOBt being the preferred activating agent, and the mass ratio of Fmoc-Pro-Pro-OH to the activating agent is 1:0.1-0.9.
[0030] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, the proline derivative is H-Pro-OtBu, and the mass ratio of the amount of Fmoc-Pro-Pro-OH to the amount of proline derivative is 1:0.2-1.
[0031] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OtBu, during the post-reaction treatment, water is added to the reaction solution, and after separation, the organic phase is washed sequentially with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer is then dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-Pro-OtBu.
[0032] Preferably, in the preparation of Fmoc-Pro-Pro-Pro-OH, Fmoc-Pro-Pro-Pro-OtBu and the reaction solvent are mixed, and then an acidic reagent is added. The mixture is reacted at 20-30°C for 0.5-6 hours. After the reaction is completed, water and ethyl acetate are added and mixed. The organic phase is separated and then washed with water and saturated brine in sequence. The organic layer is dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OH.
[0033] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OH, the reaction solvent is tetrahydrofuran, the acidic reagent is hydrochloric acid, and the mass ratio of Fmoc-Pro-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid is 1:2-10:1-4.
[0034] More preferably, in the preparation of Fmoc-Pro-Pro-Pro-OH, in the post-reaction treatment, water and ethyl acetate are added to the reaction solution, the organic layer is washed with water and saturated brine in sequence, the organic layer is dried, filtered and concentrated to obtain the intermediate Fmoc-Pro-Pro-Pro-OH.
[0035] Preferably, in the preparation of the alcoholamine compound, ethanolamine and anhydrous potassium carbonate are added to anhydrous ethanol, and a halogenated compound is added at 60-90°C. The reaction is carried out for 3-24 hours. After the reaction is completed, the filtrate is filtered and the solvent is removed by vacuum distillation. Then, an alkaline solution and cyclohexane are added and mixed. The organic phase is separated and washed with deionized water until neutral. The mixture is dried with anhydrous sodium sulfate and then distilled under reduced pressure to obtain the alcoholamine compound.
[0036] More preferably, in the preparation of the alcoholamine compound, the mass ratio of ethanolamine to anhydrous ethanol used is 1:5-20.
[0037] More preferably, in the preparation of the amino group compound, the mass ratio of anhydrous potassium carbonate to anhydrous ethanol is 1:0.5-3.
[0038] More preferably, in the preparation of the alcoholamine compound, the halogenated compound is ethyl 3-bromopyruvate, and the mass ratio of ethyl 3-bromopyruvate to ethanolamine is 1:0.05-0.5.
[0039] More preferably, in the preparation of the alcoholamine compound, the alkaline solution is a sodium hydroxide solution, the sodium hydroxide content in the sodium hydroxide solution is 5-20 wt%, and the mass ratio of the amount of sodium hydroxide solution used to the amount of anhydrous ethanol is 1:0.5-2.
[0040] More preferably, in the preparation of the amino group compound, the mass ratio of cyclohexane to anhydrous ethanol is 1:0.3-3.
[0041] More preferably, the base also includes dimethylaminoethyl ether, and the mass ratio of proline reagent to dimethylaminoethyl ether is 1:0.1-0.6. In this invention, when using an amino group compound, dimethylaminoethyl ether can also be used in conjunction with the amino group compound. Even with a lower amount of amino group compound used, Fmoc-Pro-Pro-Pro-OH can still be prepared, and the yield and purity of Fmoc-Pro-Pro-Pro-OH can be improved.
[0042] This invention prepares Fmoc-Pro-Pro-Pro-OH from a proline reagent and a proline derivative via a condensation-deprotection reaction. The proline reagent includes Fmoc-Pro-OH or Fmoc-Pro-Pro-OH, and the proline derivative is H-Pro-OtBu. An activating reagent and a base are used in the condensation-deprotection reaction, the base including an alcoholic compound, which is prepared by reacting ethanolamine with ethyl 3-bromopyruvate. Therefore, this invention has the following advantages: it can prepare the tripeptide Fmoc-Pro-Pro-Pro-OH with high yield and purity. Thus, this invention provides a high-yield, high-purity synthetic method for the tripeptide Fmoc-Pro-Pro-Pro-OH. Attached Figure Description
[0043] Figure 1 This is the infrared spectrum of an alcoholamine compound.
[0044] Figure 2 The yield graph for the tripeptide Fmoc-Pro-Pro-Pro-OH is shown.
[0045] Figure 3 The purity diagram of the tripeptide Fmoc-Pro-Pro-Pro-OH is shown. Detailed Implementation
[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0047] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0048] Example 1: A method for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH
[0049] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was N-methylimidazole, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-treatment, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0050] Preparation of Fmoc-Pro-Pro-OH: Fmoc-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:6:2. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-OH.
[0051] Preparation of Fmoc-Pro-Pro-Pro-OtBu: Fmoc-Pro-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-Pro-OH to the reaction solvent was 1:12. The base was N-methylimidazole, and the mass ratio of Fmoc-Pro-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-Pro-OH to the proline derivative was 1:0.6. In the post-reaction processing, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-Pro-OtBu.
[0052] Preparation of Fmoc-Pro-Pro-Pro-OH: Fmoc-Pro-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:6:2. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-Pro-OH.
[0053] Example 2: A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
[0054] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was N,N-diisopropylethylamine, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-reaction processing, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0055] Preparation of Fmoc-Pro-Pro-OH: Fmoc-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:6:2. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-OH.
[0056] Preparation of Fmoc-Pro-Pro-Pro-OtBu: Fmoc-Pro-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-Pro-OH to the reaction solvent was 1:12. The base was N,N-diisopropylethylamine, and the mass ratio of Fmoc-Pro-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-Pro-OH to the proline derivative was 1:0.6. In the post-reaction processing, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-Pro-OtBu.
[0057] Preparation of Fmoc-Pro-Pro-Pro-OH: Fmoc-Pro-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:6:2. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-Pro-OH.
[0058] Example 3: A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
[0059] The difference between this embodiment and Embodiment 2 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0060] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was N,N-diisopropylethylamine, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.58. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-reaction processing, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0061] Example 4: A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
[0062] The difference between this embodiment and Example 1 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0063] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was pyridine, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-treatment, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0064] Example 5: A method for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH
[0065] The difference between this embodiment and Example 1 lies in the preparation of Fmoc-Pro-Pro-OH.
[0066] Preparation of Fmoc-Pro-Pro-OH: Fmoc-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:5:1. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-OH.
[0067] Example 6: A method for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH
[0068] The difference between this embodiment and Example 1 lies in the preparation of Fmoc-Pro-Pro-OH.
[0069] Preparation of Fmoc-Pro-Pro-OH: Fmoc-Pro-Pro-OtBu and a reaction solvent were mixed, and then an acidic reagent was added. The mixture was reacted at 25°C for 3 hours. After the reaction was completed, Fmoc-Pro-Pro-OH was obtained through post-treatment. The reaction solvent was tetrahydrofuran, and the acidic reagent was hydrochloric acid. The mass ratio of Fmoc-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid was 1:5:3. In the post-treatment, water and ethyl acetate were added to the reaction solution. The organic layer was separated and washed successively with water and saturated brine. The organic layer was dried, filtered, and concentrated to obtain the intermediate Fmoc-Pro-Pro-OH.
[0070] Example 7: A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
[0071] The difference between this embodiment and Example 1 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0072] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was an amino acid compound, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.3. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-treatment, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0073] Preparation of the alkanolamine compound: Ethanolamine and anhydrous potassium carbonate were added to anhydrous ethanol, and a halogenated compound was added at 80°C. The reaction was allowed to proceed for 12 hours. After the reaction was complete, the filtrate was filtered, and the solvent was removed by vacuum distillation. Then, an alkaline solution and cyclohexane were added and mixed. The organic phase was separated and washed with deionized water until neutral. The mixture was dried over anhydrous sodium sulfate and then distilled under reduced pressure to obtain the alkanolamine compound. The mass ratio of ethanolamine to anhydrous ethanol was 1:10, the mass ratio of anhydrous potassium carbonate to anhydrous ethanol was 1:1.5, the halogenated compound was ethyl 3-bromopyruvate, and the mass ratio of ethyl 3-bromopyruvate to ethanolamine was 1:0.25. The alkaline solution was sodium hydroxide solution with a sodium hydroxide content of 10 wt%, and the mass ratio of sodium hydroxide solution to anhydrous ethanol was 1:1. The mass ratio of cyclohexane to anhydrous ethanol was 1:1.
[0074] Example 8: A method for synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH
[0075] The difference between this embodiment and Example 7 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0076] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base consisted of an amino group compound and a dimethylaminoethyl ether, with a mass ratio of Fmoc-Pro-OH to the base of 1:0.2 and a mass ratio of amino group compound to dimethylaminoethyl ether of 1:1. The activating reagent was HOBt, with a mass ratio of Fmoc-Pro-OH to the activating reagent of 1:0.1. The proline derivative was H-Pro-OtBu, with a mass ratio of Fmoc-Pro-OH to the proline derivative of 1:0.6. In the post-reaction processing, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0077] Comparative Example 1: A method for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH
[0078] The difference between this comparative example and Example 7 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0079] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was an amino acid compound, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.05. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.5. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-treatment, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0080] Comparative Example 2: A method for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH
[0081] The difference between this comparative example and Example 7 lies in the preparation of Fmoc-Pro-Pro-OtBu.
[0082] Preparation of Fmoc-Pro-Pro-OtBu: Fmoc-Pro-OH and a reaction solvent were mixed, and a base, an activating reagent, and a proline derivative were added at 0°C. The mixture was reacted at 25°C for 6 hours. After the reaction was complete, Fmoc-Pro-Pro-OtBu was obtained through post-treatment. The reaction solvent was dichloromethane, and the mass ratio of Fmoc-Pro-OH to the reaction solvent was 1:12. The base was an amino acid compound, and the mass ratio of Fmoc-Pro-OH to the base was 1:0.1. The activating reagent was HOBt, and the mass ratio of Fmoc-Pro-OH to the activating reagent was 1:0.05. The proline derivative was H-Pro-OtBu, and the mass ratio of Fmoc-Pro-OH to the proline derivative was 1:0.6. In the post-treatment, water was added to the reaction solution, and after separation, the organic phase was washed successively with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried, filtered, and concentrated to obtain Fmoc-Pro-Pro-OtBu.
[0083] Experimental example:
[0084] The amino alcohol compounds prepared in Example 8 were characterized by infrared radiation, as shown below. Figure 1 As shown, at 3328cm -1 The infrared absorption peak of the hydroxyl group is at 2858 cm⁻¹. -1 2963cm -1 The infrared absorption peaks for methylene and methyl groups are located at 1662 cm⁻¹. -1 The infrared absorption peak for the carbonyl group is at 1461 cm⁻¹. -1 The infrared absorption peak at this point indicates the presence of a carbon-nitrogen bond, suggesting the formation of an alcohol-amine compound.
[0085] The yields of Fmoc-Pro-Pro-Pro-OH prepared by the methods of the various embodiments and comparative examples of the present invention are as follows: Figure 2As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, and D2 is Comparative Example 2. In this invention, Fmoc-Pro-Pro-OtBu is first prepared by reacting Fmoc-Pro-OH and H-Pro-OtBu under the action of an alkali and an activating reagent. Then, OtBu is removed under acidic conditions to obtain Fmoc-Pro-Pro-OH. The activating reagent is then used to further prepare Fmoc-Pro-Pro-Pro-OtBu. For HOBt, the base includes at least one of N,N-diisopropylethylamine, N-methylimidazolium, and pyridine. The use of N,N-diisopropylethylamine is more effective than that of N-methylimidazolium or pyridine, resulting in a higher yield of Fmoc-Pro-Pro-Pro-OH. In the preparation of Fmoc-Pro-Pro-OH from Fmoc-Pro-Pro-OtBu by removing OtBu, the yield can also be improved by adjusting the mixing amounts of Fmoc-Pro-Pro-OtBu with tetrahydrofuran and hydrochloric acid. The mass ratio of c-Pro-Pro-OtBu to tetrahydrofuran and hydrochloric acid of 1:6:2 is more effective than that of 1:5:3 and 1:5:1. This invention also prepares an alcoholic compound from ethanolamine and ethyl 3-bromopyruvate. The alcoholic compound and the activating reagent HOBt can be used together to prepare Fmoc-Pro-Pro-OtBu or Fmoc-Pro-Pro-Pro-OtBu, and further to obtain Fmoc-Pro-Pro-OH or Fmoc-Pro-Pro-Pro-OH. However, the alcoholic compound needs to meet certain appropriate quantity limits; otherwise, the desired product cannot be prepared. Fmoc-Pro-Pro-OtBu could not be prepared, and Fmoc-Pro-Pro-OH could not be prepared if the amount of activating reagent HOBt was too small. This invention found that when the amount of activating reagent HOBt is too small, if an alcoholic compound and a dimethylaminoethyl ether are used together, Fmoc-Pro-Pro-OtBu or Fmoc-Pro-Pro-Pro-OtBu can be prepared, and then Fmoc-Pro-Pro-OH or Fmoc-Pro-Pro-Pro-OH can be prepared. Comparative Examples 1-2 could not prepare Fmoc-Pro-Pro-Pro-OH, therefore the yield of Comparative Examples 1-2 is recorded as 0.
[0086] The purity of Fmoc-Pro-Pro-Pro-OH prepared by the methods of the various embodiments and comparative examples of the present invention is as follows: Figure 3 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, and D2 is Comparative Example 2. In this invention, Fmoc-Pro-Pro-OtBu is first prepared by reacting Fmoc-Pro-OH and H-Pro-OtBu under the action of an alkali and an activating reagent. Then, OtBu is removed under acidic conditions to obtain Fmoc-Pro-Pro-OH. The activating reagent is HOBt, and the alkali includes at least one of N,N-diisopropylethylamine, N-methylimidazole, and pyridine. An alcoholic compound was also prepared from ethanolamine and ethyl 3-bromopyruvate. The alcoholic compound, used in conjunction with the activating reagent HOBt, can be used to prepare Fmoc-Pro-Pro-OtBu or Fmoc-Pro-Pro-Pro-OtBu, and further to obtain Fmoc-Pro-Pro-OH or Fmoc-Pro-Pro-Pro-OH. In this invention, the alcoholic compound and dimethylaminoethyl ether can also be used as a base, together with HOBt, to prepare Fmoc-Pro-Pro-OtBu or Fmoc-Pro-Pro-Pro-OtBu, and further to obtain Fmoc-Pro-Pro-OH or Fmoc-Pro-Pro-Pro-OH. The Fmoc-Pro-Pro-Pro-OH prepared by the method of this invention has a high purity, all above 95%, while Comparative Examples 1-2 could not prepare Fmoc-Pro-Pro-Pro-OH; therefore, the purity of Comparative Examples 1-2 is recorded as 0.
[0087] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0088] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method of synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH, comprising: Fmoc-Pro-Pro-Pro-OH is prepared by condensation and deprotection reaction of proline reagent and proline derivative, the proline reagent is Fmoc-Pro-OH or Fmoc-Pro-Pro-OH, and the proline derivative is H-Pro-OtBu; an activating reagent is used in the condensation and deprotection reaction, and the mass ratio of the use amount of Fmoc-Pro-OH to the use amount of the activating reagent is 1:0.1-0.9; An alkali is used in the condensation and deprotection reaction, and the alkali is an alcohol amine-based compound, which is prepared by reaction of ethanolamine and ethyl 3-bromopyruvate; In the preparation of the alcohol amine-based compound, ethanolamine and anhydrous potassium carbonate are added to anhydrous ethanol, a halogenated compound is added at 60-90℃, and the reaction is carried out for 3-24h; after the reaction is completed, the filtrate is obtained by filtration, the solvent is removed by distillation under reduced pressure, then a basic solution and cyclohexane are added for mixing, the organic phase is obtained by liquid separation, and then deionized water is used for washing until neutral, anhydrous sodium sulfate is used for drying, and the alcohol amine-based compound is obtained by distillation under reduced pressure; the mass ratio of the use amount of ethanolamine to the use amount of anhydrous ethanol is 1:5-20; the halogenated compound is ethyl 3-bromopyruvate, and the mass ratio of the use amount of ethyl 3-bromopyruvate to the use amount of ethanolamine is 1:0.05-0.
5.
2. The method of claim 1, wherein the method of synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH is characterized by: The mass ratio of the use amount of the proline reagent to the use amount of the alkali is 1:0.1-0.
6.
3. The method of claim 1, wherein the method of synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH is characterized by: In the condensation and deprotection reaction, tetrahydrofuran / hydrochloric acid mixed system is used for removing OtBu.
4. The method of claim 3, wherein the method of synthesizing the tripeptide Fmoc-Pro-Pro-Pro-OH is characterized by: In the tetrahydrofuran / hydrochloric acid mixed system, the mass ratio of tetrahydrofuran to hydrochloric acid is 2-10:1-4.
5. The method of claim 1, wherein the method of synthesis of tripeptide Fmoc-Pro-Pro-Pro-OH is characterized by: At least one of N,N-dimethylformamide, dichloromethane and tetrahydrofuran / hydrochloric acid mixed system is used as the reaction solvent in the condensation and deprotection reaction.
6. The method of claim 1, wherein the method of synthesis of tripeptide Fmoc-Pro-Pro-Pro-OH is characterized by: In the preparation of Fmoc-Pro-Pro-Pro-OH, Fmoc-Pro-Pro-OtBu is first prepared by reaction of Fmoc-Pro-OH and H-Pro-OtBu, and then Fmoc-Pro-Pro-OtBu is deprotonated to obtain Fmoc-Pro-Pro-OH; then Fmoc-Pro-Pro-Pro-OtBu is prepared by reaction of Fmoc-Pro-Pro-OH and H-Pro-OtBu, and then Fmoc-Pro-Pro-Pro-OtBu is deprotonated to obtain Fmoc-Pro-Pro-Pro-OH.
7. The method of claim 6, wherein the method is for synthesizing a tripeptide Fmoc-Pro-Pro-Pro-OH. In the preparation of Fmoc-Pro-Pro-OH, ethyl acetate is also used.