Preparation method of Fmoc-His
By using Fmoc-His (Trt) with trifluoroacetic acid and catalyst, combined with pH adjustment, the problem of difficulty in removing histidine protective groups is solved, and efficient and low-cost Fmoc-His preparation is achieved, which improves the peptide Stability and efficiency of synthesis.
Patent Information
- Application Number
- CN202411253188.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2025-01-10
AI Technical Summary
In the synthesis of polypeptides, it is difficult to achieve selectivity in the protection of histidine ɑ-amino group and side chain imidazole rings, resulting in difficult conditions for synthesis of Fmoc-His, incomplete reactions, many impurities for product, difficulty in purification, and Fmoc- on the market His(BOC) raw materials are difficult to prepare and are expensive.
Using Fmoc-His (Trt) as raw material, the reaction is carried out by adding a catalyst to the solution of trifluoroacetic acid and dichloromethane, adjusting the pH value to neutral, combining with liquid alkali treatment to obtain high-purity Fmoc-His, which is suitable for a variety of Removal of protective groups reduces costs and improves efficiency.
It realizes Fmoc-His preparation with simple operation, easy control of conditions, stable quality and high yield, reduces production costs, improves the purity and yield of the product, and has environmentally friendly, safe and effective processes.
Smart Images
Figure CN119285552A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of polypeptide synthesis, and in particular to a method for preparing an amino acid derivative. Background Art
[0002] In the field of peptide synthesis, the artificial synthesis of peptides is controlled by different amino acids in a certain order. Protected amino acids are structural fragments that constitute the target product and are key starting materials. The difficulty lies in the fact that the functional groups of amino acids are easily acted upon by reagents for peptide reactions, which results in an increase in by-products and low synthesis efficiency.
[0003] In order to obtain the ideal target peptide, the active groups of amino acids need to be blocked or protected first. Benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (BOC), and fluorenylmethoxycarbonyl (Fmoc) are the preferred ɑ-NH2 protecting groups in peptide synthesis. Histidine has ɑ-amino groups and imino groups on the side chain imidazole ring, and sometimes it is necessary to selectively protect or block the main chain and side chain amino groups. It is difficult to achieve the ideal quality and yield by directly controlling the reaction conditions to synthesize Fmoc-His. The current effective method is to first perform double amino protection and then remove the side chain protecting group to obtain Fmoc-His. There are many side chain imino protecting groups for histidine, such as Fmoc, Tos, Mmt, Mtt, etc. Through experiments using different methods, a synthesis method with simple operation, stable quality, and stable yield was finally obtained.
[0004] The main synthesis method of Fmoc-His is to first synthesize Fmoc-His (Fmoc), and then control the conditions to remove the Fmoc group on the imidazole ring. This synthesis method has the disadvantages of difficult control of conditions, incomplete reaction, and many product impurities that are difficult to purify.
[0005] There are literature reports that using Fmoc-His (BOC) as a raw material, using tin tetrachloride in ethyl acetate to remove the BOC group on the imidazole ring to obtain the target product, or using phosphoric acid in THF to remove the BOC on the imidazole ring to obtain the product, but Fmoc-His (BOC) itself is not easy to prepare and the purchase price on the market is high.
[0006] There are literature reports on using His to directly add Fmoc to the main chain in a weak base, such as sodium carbonate, under controlled conditions, but this condition is difficult to control. I also used baking soda as a base for the reaction, which also produced more Fmoc-His (Fmoc), and the reaction was very small, and many raw materials could not be completely reacted. Summary of the invention
[0007] In view of the above technical problems, the present application solves the deficiencies of the process technology in the prior art and designs a synthesis method with simple operation, easy control of conditions, stable quality, stable yield, and better overall cost and benefits. The present application provides a preparation method of Fmoc-His.
[0008] In order to achieve the above object, the technical solution adopted in the present application is: a method for preparing Fmoc-His, the preparation raw material is Fmoc-His (Trt), and the preparation method comprises the following steps:
[0009] Step 1: Mix trifluoroacetic acid and dichloromethane to prepare a solution.
[0010] Step 2: Dissolve the raw materials in the above solution.
[0011] Step 3: Add the catalyst in batches to react at a certain temperature.
[0012] Step 4: Use liquid alkali to adjust the pH of the solution to neutral and maintain it to obtain a crude product, which is then treated to obtain a qualified product.
[0013] In order to better realize the present invention, further, the preparation method is also applicable to Fmoc-His(TOS), Fmoc-His(BOC), Fmoc-His(Mtt), Fmoc-His(Mmt), Fmoc-His(ONp) and Fmoc-His(Fmoc).
[0014] In order to better realize the present invention, further, in the step 1, dichloromethane can be replaced by tetrahydrofuran or DMF or acetonitrile.
[0015] In order to better realize the present invention, further, the catalyst is specifically trimethylchlorosilane.
[0016] In order to better realize the present invention, further, in the step three, the mixing ratio of trifluoroacetic acid and the catalyst is 1:1.
[0017] In order to better realize the present invention, further, in the step 4, the pH value is adjusted in the range of 6.5-7.0, the pH value is adjusted with liquid alkali, and the temperature is controlled in the range of 20-25°C, and the liquid alkali includes inorganic alkali and organic alkali.
[0018] In order to better realize the present invention, further, the solvent used for treating Fmoc-His is specifically ethyl acetate.
[0019] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:
[0020] 1. The present invention adopts a cheap starting material and prepares Fmoc-His with a content of more than 99% through simple operation, with a yield of more than 85% and low production cost.
[0021] 2. The present invention uses Fmoc-His (Trt) to efficiently remove the Trt protecting group of the imidazole group under acidic conditions, and then alkalizes and purifies to obtain the final product, thereby improving the purity and yield of the product.
[0022] 3. The process of the present invention does not involve high temperature, high pressure, and high pollution, is environmentally friendly, does not involve cumbersome handling, is simple to operate, and is safer and more effective. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is a preparation flow chart of the present invention;
[0024] Figure 2 The HNMR spectrum of Fmoc-His prepared in the present invention;
[0025] Figure 3 This is a schematic diagram of the fluorescent spot detection results of TLC in Example 1 of the present invention;
[0026] Figure 4 This is a schematic diagram of the fluorescent spot detection results of TLC in Example 2 of the present invention;
[0027] Figure 5 Schematic diagram of the detection results of TLC in Example 3 of the present invention; DETAILED DESCRIPTION
[0028] 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.
[0029] 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.
[0030] like Figure 1 As shown, a method for preparing Fmoc-His, the raw material for preparation is Fmoc-His (Trt), and the preparation method comprises the following steps:
[0031] Step 1: Mix trifluoroacetic acid and dichloromethane to form a solution;
[0032] Step 2: dissolving the raw materials in the above solution;
[0033] Step 3: Add the catalyst in batches to react at a certain temperature.
[0034] Step 4: Use liquid alkali to adjust the pH of the solution to neutral and maintain it to obtain a crude product, which is then treated to obtain a qualified product.
[0035] like Figure 2 As shown, specifically, Fmoc-His (Trt) is used as a raw material, and the Trt group is removed under acidic conditions to obtain an Fmoc-His precursor. The target product Fmoc-His-OH is obtained by adjusting the Ph value to be neutral, and then a series of refining treatments are performed to obtain a pure product with an HPLC content of more than 99%, and a residual solvent and water content of less than 1%.
[0036] The preparation method is also applicable to Fmoc-His(TOS), Fmoc-His(BOC), Fmoc-His(Mtt), Fmoc-His(Mmt), Fmoc-His(ONp) and Fmoc-His(Fmoc). Preferably, Fmoc-His(Trt) is used as the raw material for the reaction.
[0037] In the step 1, dichloromethane can be replaced by any one of tetrahydrofuran, DMF and acetonitrile.
[0038] The catalyst is specifically any one of hydrochloric acid, trimethylchlorosilane, and water, preferably trimethylchlorosilane.
[0039] In the step 3, the mixing ratio of trifluoroacetic acid and the catalyst is 1:1.
[0040] In the step 4, the pH value is adjusted to 6.5-7.0, and the pH value is adjusted with solid alkali or liquid alkali, and the temperature is controlled to be 20-25°C, and the liquid alkali includes inorganic alkali and organic alkali. Use a wide range of test paper to measure, and stir evenly and repeatedly confirm the pH value.
[0041] The solvent used for treating Fmoc-His is specifically any one of ethyl acetate, methanol, ethanol, tetrahydrofuran, dichloromethane and water.
[0042] The molar ratio of Fmoc-His (Trt) to trifluoroacetic acid is 1:1-6; the weight ratio of Fmoc-His (Trt) to dichloromethane is 1:1-3; the reaction temperature is 15°C-45°C, preferably 30-35°C; the reaction time is 6 hours-40 hours, preferably 16 hours-20 hours; after the reaction, part of the solid is precipitated from the system, petroleum ether is added, the temperature is lowered to 10-15°C to precipitate the solid, and a crude product is obtained by filtering; the pH value of the crude product is adjusted to desalt, the crude product can be dried before desalting, or the wet product can be desalted directly.
[0043] Example 1: Add 30 ml of dichloromethane to a 250 ml three-necked flask, then add 6 g of trifluoroacetic acid, then add 10 g of Fmoc-His (Trt), and stir to dissolve. Add 3 g of trimethylsilyl chloride at room temperature. After adding the raw materials, react at room temperature for 26 hours. TLC (DCM: MeOH = 5: 1) shows that there is no raw material, and post-treatment is performed.
[0044] like Figure 3 As shown, Fmoc-His (Trt) is in the middle and the reaction solutions are on both sides.
[0045] The reaction solution was concentrated to dryness, and then dried with 10 ml DCM to precipitate solids. The solids were stirred and dissolved with 15 ml THF, and THF was concentrated. 20 ml petroleum ether was added to the residue, and then 7 ml EA was added and stirred to precipitate solid powder. Filter to obtain a wet product, and dry the wet product to obtain 5.8 g of dry product. 5.8 g of solid was finely ground and added to a 250 ml three-necked flask, 30 ml of water was added to the flask, and then the pH was adjusted to 7 with an equal amount of caustic soda to precipitate solids. Filter, wash the filter cake with 25 ml of water once, and filter to dry. The product was dried to obtain 4.7 g of dry product, HPLC96.5%, yield 78%.
[0046] Example 2: Add 66 ml of DCM to a 250 ml three-necked flask, then add 41 g of Fmoc-His (Trt) and stir to dissolve. Add 15 g of trifluoroacetic acid to the reaction flask and keep the temperature at 30 ° C to react overnight. The next morning, there was a lot of raw material remaining on the plate (DCM: MeOH = 5: 1), and 7.5 g of trifluoroacetic acid was added to continue the reaction at 30 ° C overnight. On the third morning, the reaction was still not complete, and 15 g of trifluoroacetic acid was added, and the temperature was raised to 40 ° C to react until the reaction was basically complete on the fifth morning, and post-treatment was performed.
[0047] like Figure 4 As shown, DCM:MeOH=5:1, UV color development, the left side is the reaction solution, and the right side is Fmoc-His (Trt).
[0048] Concentrate DCM, add 100ml petroleum ether to the residue, slurry, filter, wash the filter cake with petroleum ether once, and filter dry. Dry the filter cake, grind it into fine powder, and then add it to a 250ml three-necked flask, add 120ml water, add an equal amount of solid caustic soda to adjust the pH value to neutral, and precipitate solid. Filter, wash the filter cake with 120ml water twice, filter dry, wash the solid with EA twice, and filter dry. Dry the wet product to obtain 19g of dry product, HPLC97%, yield 76%.
[0049] Example 3: Add 300 ml of dichloromethane and 60 g of trifluoroacetic acid to a 1000 ml three-necked flask, then add 100 g of Fmoc-His (Trt), stir to dissolve, add 20 ml of concentrated hydrochloric acid at room temperature to react, and react at room temperature overnight.
[0050] The next morning, there was still a lot of raw materials on the plate. 10 ml of concentrated hydrochloric acid was added to continue the reaction for 4 hours until the reaction was complete. At this time, a small amount of solid precipitated out of the system and separated into two layers. Filter the solid, separate the filtrate into layers, and then Figure 5 As shown, the upper layer is basically impurities and is discarded. The lower layer is concentrated and dried at 45°C, and 30 ml of DCM is added twice to dry. No solid is precipitated. EA is added to the concentrated residual liquid, and solid is precipitated. It is slurried with EA and filtered. The above solids are combined and dried to obtain 60 g of dry product, HPLC 97%, yield 76%. Nuclear magnetic HNMR detection of the product shows that the structure is correct.
[0051] Example 4: Add 2.5L of dichloromethane and 696g of trifluoroacetic acid to a 5L three-necked flask, then add 1240g of Fmoc-His (Trt) in batches, and stir to dissolve. Cool with tap water, add 458g of trimethylsilyl chloride in batches, and maintain the reaction at room temperature overnight. The next morning, some raw materials were not reacted completely when the plate was turned on, and 196g of trimethylsilyl chloride was added to continue the reaction at room temperature for one day until overnight.
[0052] On the third day, the reaction was complete. A large amount of solids were precipitated from the system. Filter, wash the solids with petroleum ether twice, filter and dry, and dry the solids to obtain 900g of crude product. Add 900g of crude powder to a 10L three-necked flask, add 9L of water, and then adjust the Ph value to neutral with an equal amount of solid caustic soda. Filter, it is not easy to filter. Wash the solids with 7L of water twice, filter and dry, then wash with 7LEA twice, filter and dry, and dry the solids to obtain 643g of dry product. HPLC 99.1%, yield 85%.
[0053] Example 5: Add 2.8L of dichloromethane and 783g of trifluoroacetic acid to a 5L three-necked flask, then add 1395g of Fmoc-His (Trt), and stir to dissolve. Cool down with tap water, add 736g of trimethylchlorosilane in batches, and keep warm at 30-35°C to react overnight. TLC (DCM: MeOH = 5: 1) at noon the next day showed no raw materials, and a large amount of solids precipitated from the system. The reaction was completed after 16 hours of reaction. After filtering, it was found that there was a layer of oil at the bottom of the filtrate. No solids were precipitated when petroleum ether was added to the oil. The filtered solid was stirred and washed twice with petroleum ether in a beaker, and filtered to obtain a crude product.
[0054] Add the above solid to a 10L three-necked flask, add 5L water, add 2L THF, stir, adjust pH to 7 with liquid alkali. Stir for one hour and filter, it is difficult to filter. The filter cake is stirred and washed once with a mixture of 2L THF and 6L water, filtered, and the solid is stirred and washed twice with EA, filtered and dried. The dry product is 740g, HPLC99.5%, and the yield is 87%.
[0055] The above 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 5 experiments.
[0056] Comparative Example for Data Analysis:
[0057] Comparing Example 1 and Example 2, during the experiment, by adding a catalyst (trimethylsilyl chloride) to Example 1, the time required for the reaction to be complete was recorded. From the experimental results, Example 1 required 26 hours; Example 2 required 5 days. It is concluded that adding trimethylsilyl chloride can significantly reduce the time required for the reaction to be complete.
[0058] Comparing Example 3 and Example 4, Example 3 was catalyzed by concentrated hydrochloric acid, and Example 4 was catalyzed by trimethylchlorosilane. From the experimental results, Example 3 obtained 60g of dry product, HPLC 97%, and a yield of 76%; Example 4 obtained 643g of dry product, HPLC 99.1%, and a yield of 85%. It is concluded that the product obtained by adding trimethylchlorosilane to the catalytic reaction is more conducive to purification, and the product quality and yield are higher.
[0059] Compare Example 4 and Example 5, control the ratio of trimethylsilyl chloride to trifluoroacetic acid, and judge whether the ratio affects the reaction time. From the experimental results, no matter at what temperature, in order to complete the reaction, the ratio of trimethylsilyl chloride to trifluoroacetic acid needs to be maintained at about 1:1; under the condition that the ratio of trimethylsilyl chloride to trifluoroacetic acid is maintained at about 1:1, the temperature is raised to 30-35°C during the reaction, which can increase the reaction speed and save time.
[0060] Through the analysis of the above comparative experiments, it is better to use trimethylchlorosilane to catalyze the reaction than to add concentrated hydrochloric acid; heating to 30-35°C can effectively increase the reaction rate; the ratio of trimethylchlorosilane to trifluoroacetic acid is preferably 1:1.
[0061] 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 Fmoc-His, characterized in that: The preparation raw material is Fmoc-His (Trt), and the preparation method comprises the following steps: Step 1: Mix trifluoroacetic acid and dichloromethane to prepare a solution; Step 2: dissolving the raw materials in the above solution; Step 3: Add the catalyst in batches to react at a certain temperature. Step 4: Use liquid alkali to adjust the pH of the solution to neutral and maintain it to obtain a crude product, which is then treated to obtain a qualified product.
2. The method for preparing Fmoc-His according to claim 1, characterized in that: The preparation method is also applicable to Fmoc-His(TOS), Fmoc-His(BOC), Fmoc-His(Mtt), Fmoc-His(Mmt), Fmoc-His(ONp) and Fmoc-His(Fmoc).
3. The method for preparing Fmoc-His according to claim 1, characterized in that: In the step 1, dichloromethane can be replaced by tetrahydrofuran, DMF or acetonitrile.
4. The method for preparing Fmoc-His according to claim 2, characterized in that: The catalyst is specifically trimethylchlorosilane.
5. The method for preparing Fmoc-His according to claim 2, characterized in that: In the step 3, the mixing ratio of trifluoroacetic acid and the catalyst is 1:
1.
6. The method for preparing Fmoc-His according to claim 1, characterized in that: In the step 4, the pH value is adjusted in the range of 6.5-7.0, and the pH value is adjusted with liquid alkali, and the temperature is controlled in the range of 20-25° C. The liquid alkali includes inorganic alkali and organic alkali.
7. The method for preparing Fmoc-His according to claim 1, characterized in that: The solvent used for treating Fmoc-His is specifically ethyl acetate.