Preparation method of side chain Fmoc-His (Mtt)
By optimizing the reaction conditions and steps, the method of preparing Fmoc-His (Mtt) solves the problems of high production cost and low efficiency of amino acid side chain protection methods in the prior art, and achieves high yield and high purity product preparation, which is suitable for industrial production.
Patent Information
- Application Number
- CN202411909325.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-28
AI Technical Summary
The existing amino acid side chain protection methods have problems such as high production cost, low efficiency and easy introduction of impurities. Especially when preparing Fmoc-His (Mtt), the operation is complicated and it is difficult to ensure the purity of the product.
A side chain Fmoc-His (Mtt) was used to prepare the side chain Fmoc-His (Mtt), by adding (CH3)3SiCl and Et3N/DIPEA as acid binding agents to the DCM solvent, adding Mtt-Cl in batches after temperature control and reflux, reacting overnight, and then obtaining high purity Fmoc-His (Mtt) through extraction, concentration, recrystallization and other steps.
This method significantly improves the yield of Fmoc-His (Mtt) to more than 96%. The HPLC detection shows that the product purity is higher than 99%, which reduces product losses, improves product quality, and is simple to operate and facilitates industrial production.
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Figure CN119707960A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of molecular sieve dehydration, and in particular to a method for preparing a side chain Fmoc-His (Mtt). Background Art
[0002] With the rapid development of biotechnology, amino acids and their derivatives are increasingly used in drug synthesis, food additives, nutritional supplements and other fields. Especially in peptide synthesis, the protection strategy of amino acids is particularly important. In order to improve the synthesis efficiency of peptides and the purity of products, researchers have adopted a variety of methods to protect the amino groups of amino acid side chains.
[0003] In the prior art, commonly used amino acid protection methods include carboxyl protection and amino protection. Traditional amino acid protection methods usually require the use of expensive protection reagents, and impurities are easily introduced during the reaction process, resulting in reduced purity of the final product. In addition, some traditional methods will produce harmful substances such as heavy metal ions during the reaction process, which will pollute the environment, be difficult to handle, and increase production costs.
[0004] At present, the large-scale production technology of amino acids with protected side chain amino groups mainly utilizes the reaction of amino acids with metal ion compounds (such as copper sulfate, copper sulfide, basic copper carbonate, etc.). After dissolving it in water and heating it, a metal ion chelate of the amino acid can be formed. Then, the excess metal ion compound is removed by suction filtration, and the metal ion chelate of the amino acid is protected, and finally the metal ion chelate of the protected amino acid is obtained. Then, after rejection filtration and purification, EDTA.2Na is used for decoppering, and the target product can be obtained after multiple decoppering.
[0005] Although traditional production technology appears simple on the surface, the operation process is actually complicated, especially when separating metal ion chelates that protect amino acids. This step is particularly difficult and can easily introduce impurities, resulting in impurities in the final product.
[0006] Therefore, developing a simple, efficient and environmentally friendly method for protecting amino acid side chains, especially for the preparation of Fmoc-His(Mtt), has important research value and application prospects. Summary of the invention
[0007] In response to the above technical problems, the present application solves the technical problems of high production cost and low production efficiency in the prior art for preparing amino acid derivatives.
[0008] In order to achieve the above object, the technical solution adopted in the present application is: a method for preparing a side chain Fmoc-His (Mtt), comprising the following steps:
[0009] Step 1: Place Fmoc-His-OH in a reaction bottle, add an appropriate amount of solvent DCM, and add (CH3)3SiCl, control the temperature and reflux for 2 hours, then add base Et3N / DIPEA as an acid binding agent, and react until the solution is clear.
[0010] Step 2: Control the temperature to 10-20°C, add Mtt-Cl and Et3N / DIPEA in batches, raise the temperature to 23-28°C and react overnight to obtain Fmoc-His (Mtt) containing a side chain amino group.
[0011] Step 3: The final product of Fmoc-His (Mtt) is obtained through extraction, concentration, recrystallization, stirring, washing, filtering and drying.
[0012] In order to better implement the present application, further, in the step 1, the molar ratio of Fmoc-His-OH, (CH3)3SiCl and Et3N / DIPEA is 1:1.5:1.2.
[0013] In order to better implement the present application, further, in the step 2, the added Mtt-Cl and Et3N / DIPEA are equimolar amounts.
[0014] In order to better realize the present application, further, in the step three, the organic layer is extracted and washed with alkaline water until it is neutral, and then concentrated at low temperature.
[0015] In order to better realize the present application, further, in the step three, an appropriate amount of MeOH is added during recrystallization.
[0016] In order to better realize the present application, further, in the step three, a mixed solvent with a volume ratio of MeOH:DCM=7:3 and DCM:Pet=3:7 is used during stirring and washing.
[0017] In order to better realize the present application, further, the initial mass of the Fmoc-His-OH is 1.0 mol.
[0018] Compared with the prior art, the technical solution provided by this application has the following beneficial effects:
[0019] 1. By optimizing the reaction conditions, the method provided by the present invention can effectively improve the yield of Fmoc-His (Mtt) to more than 96%, and HPLC detection shows that the product purity is higher than 99%. Compared with the traditional method, the reaction conditions of the present invention significantly reduce the loss of the product and improve the quality of the product.
[0020] 2. By adjusting the ratio of reactants, especially controlling the amount of Mtt-Cl, the present invention can avoid unnecessary side reactions caused by excessive Mtt-Cl and ensure complete attachment of the protecting group to the side chain amino group. In particular, when DIPEA is used as an acid-binding agent, the acidic byproducts can be completely neutralized to prevent the acidic substances from damaging the product, thereby ensuring efficient reaction and high yield.
[0021] 3. The synthesis method of the present application uses common solvents and acid-binding agents, and the operation steps are relatively simple, which is convenient for industrial production. By using conventional operation steps such as rotary evaporation concentration, extraction, and recrystallization, impurities can be effectively removed to obtain Fmoc-His (Mtt) with high purity, reducing the dependence on special equipment and improving the feasibility and economy of production.
[0022] 4. The method of the present application has high reproducibility and is suitable for large-scale production. Experimental data show that the method has high yield, good purity, and is easy to operate, which can meet the strict requirements of raw materials, time and cost in industrial production.
[0023] 5. By comparing the effects of different acid-binding agents (such as DIPEA and Et3N), this application found that the use of DIPEA can more effectively control the reaction process, avoid the acidic effects of the intermediates, and thus improve the final quality of the product. Under this condition, the product of Fmoc-His (Mtt) has higher purity and lower impurities. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Flow chart for the preparation of Fmoc-His(Mtt) for this application;
[0025] Figure 2 This is a schematic diagram of the HPLC test results of Example 2 of the present application;
[0026] Figure 3 This is a schematic diagram of the HPLC test results of Example 3 of the present application;
[0027] Figure 4 This is a schematic diagram of the HPLC test results of Example 4 of the present application;
[0028] Figure 5 This is a schematic diagram of the HPLC test results of Example 5 of the present application. DETAILED DESCRIPTION
[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.
[0030] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0031] Example 1: A method for preparing a side chain Fmoc-His (Mtt), comprising the following steps:
[0032] Step 1: Place Fmoc-His-OH in a reaction bottle, add an appropriate amount of solvent DCM, and add (CH3)3SiCl, control the temperature and reflux for 2 hours, then add base Et3N / DIPEA as an acid binding agent, and react until the solution is clear.
[0033] Step 2: Control the temperature to 10-20°C, add Mtt-Cl and Et3N / DIPEA in batches, raise the temperature to 23-28°C and react overnight to obtain Fmoc-His (Mtt) containing a side chain amino group.
[0034] Step 3: The final product of Fmoc-His (Mtt) is obtained through extraction, concentration, recrystallization, stirring, washing, filtering and drying.
[0035] In the step 1, the molar ratio of Fmoc-His-OH, (CH3)3SiCl and Et3N / DIPEA is 1:1.5:1.2.
[0036] In step 2, the added Mtt-Cl and Et3N / DIPEA are equimolar amounts.
[0037] In step 3, the organic layer is extracted and washed with alkaline water until it is neutral, and then concentrated at low temperature.
[0038] In step 3, an appropriate amount of MeOH is added during recrystallization.
[0039] In step 3, a mixed solvent with a volume ratio of MeOH:DCM=7:3 and DCM:Pet=3:7 was used for stirring and washing.
[0040] The initial mass of the Fmoc-His-OH was 1.0 mol.
[0041] Embodiment 2:
[0042] Weigh 1.0 mol of Fmoc-His-OH 377.4g and dissolve it in 2L of DCM, add (CH3)3SiCl130.4g, heat to 40℃ and reflux for 2h, add DIPEA 155.1g, and react to make it clear. Control the temperature to about 15℃ and add 307.4g Mtt-Cl and 135.7g DIPEA in batches, heat to about 25℃ and react. TLC monitors the progress of the reaction, showing that the reaction is complete, and the target product Fmoc-His (Mtt) is obtained; sodium bicarbonate aqueous solution is added to the product solution for extraction until the water layer is neutral. Concentrate by rotary evaporation at 40℃, and add 1LMeOH to precipitate solid. The mixture was stirred and washed with 1400 mL of MeOH and 600 mL of DCM for 20 min, filtered to remove large polar impurities, and stirred and washed with 600 mL of DCM and 1400 mL of Pet for 20 min, filtered to remove small polar impurities. The pure Fmoc-His (Mtt) was obtained, and the sample was sent for testing after drying. 583.7 g of Fmoc-His (Mtt) was obtained, with a yield of 92.1%. Figure 2 As shown, the content was 99.04% by HPLC.
[0043] Embodiment 3:
[0044] Weigh 1.0 mol of Fmoc-His-OH 377.4g and dissolve it in 2L of DCM, add (CH3)3SiCl162.9g, heat to 40℃ and reflux for 2h, add DIPEA 155.1g, and react to make it clear. Control the temperature to about 15℃ and add 307.4g Mtt-Cl and 135.7g DIPEA in batches, heat to about 25℃ and react. TLC monitors the progress of the reaction, showing that the reaction is complete, and the target product Fmoc-His (Mtt) is obtained; sodium bicarbonate aqueous solution is added to the product solution for extraction until the water layer is neutral. Concentrate by rotary evaporation at 40℃, and add 1LMeOH to precipitate solid. The mixture was stirred and washed with 1400 mL of MeOH and 600 mL of DCM for 20 min, filtered to remove large polar impurities, and stirred and washed with 600 mL of DCM and 1400 mL of Pet for 20 min, filtered to remove small polar impurities. The pure Fmoc-His (Mtt) was obtained, and the sample was sent for testing after drying. 597.6 g of Fmoc-His (Mtt) was obtained, with a yield of 94.3%. Figure 3 As shown, the content was 99.11% by HPLC.
[0045] Embodiment 4:
[0046] Weigh 1.0 mol of Fmoc-His-OH 377.4g and dissolve it in 2L of DCM, add (CH3)3SiCl162.9g, heat to 40℃ and reflux for 2h, add DIPEA 155.1g, and react to make it clear. Control the temperature to about 15℃ and add 336.7g Mtt-Cl and 148.6g DIPEA in batches, heat to about 25℃ and react. TLC monitors the progress of the reaction, showing that the reaction is complete, and the target product Fmoc-His (Mtt) is obtained; sodium bicarbonate aqueous solution is added to the product solution for extraction until the water layer is neutral. Concentrate by rotary evaporation at 40℃, and add 1LMeOH to precipitate solid. The mixture was stirred and washed with 1400 mL of MeOH and 600 mL of DCM for 20 min, filtered to remove large polar impurities, and stirred and washed with 600 mL of DCM and 1400 mL of Pet for 20 min, filtered to remove small polar impurities. The pure Fmoc-His (Mtt) was obtained, and the sample was sent for testing after drying. 612.8 g of Fmoc-His (Mtt) was obtained, with a yield of 96.7%. Figure 4 As shown, the content was 99.32% by HPLC.
[0047] Embodiment 5:
[0048] Weigh 1.0 mol of Fmoc-His-OH 377.4g and dissolve it in 2L of DCM, add (CH3)3SiCl162.9g, heat to 40℃ and reflux for 2h, add Et3N 121.2g, and react to make it clear. Control the temperature to about 15℃ and add 336.7g Mtt-Cl and 116.2g Et3N in batches, heat to about 25℃ and react. TLC monitors the progress of the reaction, showing that the reaction is complete, and the target product Fmoc-His (Mtt) is obtained; sodium bicarbonate aqueous solution is added to the product solution for extraction until the water layer is neutral. Concentrate by rotary evaporation at 40℃, and add 1LMeOH to precipitate solid. The mixture was stirred and washed with 1400 mL of MeOH and 600 mL of DCM for 20 min, filtered to remove large polar impurities, and stirred and washed with 600 mL of DCM and 1400 mL of Pet for 20 min, filtered to remove small polar impurities. The pure Fmoc-His (Mtt) was obtained, and the sample was sent for testing after drying. 586.2 g of Fmoc-His (Mtt) was obtained, with a yield of 92.5%. Figure 5 As shown, the content was 99.02% detected by HPLC.
[0049] The above description is only a preferred embodiment of the present application, and is only selected as a control condition. The impact on product yield and quality does not mean that this patent technology has only been verified by four experiments.
[0050] Comparing Example 2 with Example 3, during the experiment, when the amount of (CH3)3SiCl was 1.5eq, the effect was the best, and the carboxyl group could be completely protected, thus avoiding the reaction with Mtt-Cl and causing losses.
[0051] Comparing Example 3 with Example 4, the excess Mtt-Cl promotes the reaction to proceed in the forward direction, reduces the loss of the product during the post-treatment process, saves the cost of raw materials, and makes the product yield higher and the test data in various aspects better.
[0052] Comparing Example 4 and Example 5, when different bases are used as acid-binding agents, it is found that the same equivalent of DIPEA is better than Et3N, and the raw material reaction is more thorough by TCL monitoring. Not only does it save the cost of raw materials, but it also reduces the loss of products in the post-treatment process, making the product yield higher and the test data in various aspects better.
[0053] Through the analysis of the above comparative experiments, it is concluded that the best effect is achieved when DIPEA is selected as the acid-binding agent and the molar ratio of the main raw materials Fmoc-His-OH: (CH3)3SiCl: DIPEA: Mtt-Cl is 1:1.5:2.35:1.15. The final yield of Fmoc-His (Mtt) can reach more than 96%, and the purity is higher than 99%, which meets the needs of high-standard applications. The optimal reactant ratio is determined to ensure the efficiency and stability of the reaction and avoid waste of resources.
[0054] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing a side chain Fmoc-His (Mtt), characterized in that: The following steps are involved: Step 1: Place Fmoc-His-OH in a reaction bottle, add an appropriate amount of solvent DCM, and add (CH3)3SiCl, reflux at controlled temperature for 2 hours, then add base Et3N / DIPEA as an acid-binding agent, and react until the solution is clear; Step 2: Control the temperature to 10-20°C, add Mtt-Cl and Et3N / DIPEA in batches, raise the temperature to 23-28°C and react overnight to obtain Fmoc-His (Mtt) containing a side chain amino group; Step 3: The final product of Fmoc-His (Mtt) is obtained through extraction, concentration, recrystallization, stirring, washing, filtering and drying.
2. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: In the step 1, the molar ratio of Fmoc-His-OH, (CH3)3SiCl and Et3N / DIPEA is 1:1.5:1.
2.
3. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: In the step 2, the added Mtt-Cl and Et3N / DIPEA are in equimolar amounts.
4. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: In the step 3, the organic layer is extracted and washed with alkaline water until it is neutral, and then concentrated at low temperature.
5. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: In the step 3, an appropriate amount of MeOH is added during recrystallization.
6. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: In the step 3, a mixed solvent with a volume ratio of MeOH:DCM=7:3 and DCM:Pet=3:7 is used during stirring and washing.
7. The method for preparing a side chain Fmoc-His (Mtt) according to claim 1, characterized in that: The initial mass of the Fmoc-His-OH was 1.0 mol.