Preparation method of amino acid protected Fmoc-Lys (Trt)-OH containing side chain amino group
By protecting the amino acid side chain and α-amino group with Trt-Cl and Fmoc-osu, the problems of poor protection selectivity and harsh removal conditions in the existing technology are solved, and high-purity and high-yield double-protected amino acids are achieved, which are suitable for peptide synthesis and improve the efficiency and quality of peptide synthesis.
Patent Information
- Application Number
- CN202510850902.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-09-26
AI Technical Summary
Existing technologies make it difficult to effectively protect amino acids containing side chain amino groups, resulting in increased side reactions, reduced purity, and decreased yield. In addition, traditional methods find it difficult to balance the step-by-step protection of side chain amino groups and α-amino groups and the harsh conditions for removing the protecting group, affecting the quality and efficiency of polypeptide synthesis.
The side chain amino group was protected by trityl chloride (Trt-Cl) under ZnCl2 catalysis, and the α-amino group was subsequently protected by Fmoc-osu in the presence of Na2CO3. By using DMF and THF solvent systems, optimizing the reaction conditions and purification steps, a stable double-protected amino acid was formed.
High-purity (≥99.4%) and high-yield (48%) double-protected amino acids are achieved, which are suitable for peptide synthesis, reduce production costs, improve synthesis efficiency and flexibility, and are applicable to a variety of amino acids. The protecting groups can be gently removed without affecting the amino acid main chain structure.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of polypeptide synthesis, and in particular to a method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group. Background Art
[0002] In peptide synthesis, the protection of amino acids containing side chain amino groups is crucial. If the side chain amino groups are not effectively protected, they are prone to side reactions with other reagents during the reaction, resulting in reduced purity and yield of the target product, seriously affecting the efficiency and quality of peptide synthesis.
[0003] There are currently many challenges in this field. On the one hand, commonly used protecting reagents have poor selectivity, making it difficult to accurately protect the side chain amino group. They are prone to react with multiple functional groups of amino acids, making the product complex and increasing the difficulty of separation and purification. On the other hand, it is difficult to achieve step-by-step, efficient and stable protection of the side chain amino group and the α-amino group. Traditional methods cannot take into account the protection effects of both and may have adverse effects on the other amino group or the amino acid main chain structure. In addition, the removal conditions of the protecting group are harsh and may destroy the amino acid main chain structure, limiting its application in peptide synthesis.
[0004] Therefore, developing an efficient, precise, and applicable protection method for a variety of amino acids containing side chain amino groups has become an urgent problem. This method needs to improve protection selectivity, achieve effective step-by-step protection, and optimize removal conditions to provide a universal and efficient amino acid protection strategy for peptide synthesis. Summary of the Invention
[0005] In response to the above technical problems, the present application aims to solve a series of problems faced by amino acids containing side chain amino groups during the protection process, specifically including: improving the selectivity of protecting reagents for side chain amino group protection and reducing the occurrence of side reactions; achieving effective step-by-step protection of side chain amino groups and α-amino groups to ensure that the amino acid main chain structure is not destroyed during the protection process; optimizing the removal conditions of protecting groups so that they can be selectively removed under mild conditions without affecting the amino acid main chain structure, thereby providing a general, efficient, and high-quality amino acid protection strategy for polypeptide synthesis, meeting the requirements of polypeptide synthesis for high purity and high yield of raw materials.
[0006] In order to achieve the above-mentioned object, the technical solution adopted in the present application is: a method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group, comprising the following steps:
[0007] Step 1: Using amino acids containing side chain amino groups and trityl chloride (Trt-Cl) as raw materials, in DMF solvent, with ZnCl2 as catalyst, the reaction is carried out at 65°C to perform Trt protection on the side chain amino groups to generate side chain protected amino acids.
[0008] Step 2: The side chain protected amino acid Lys(Trt)-OH is reacted with Fmoc-osu in a mixed solvent of THF and water with Na2CO3 as a base at room temperature to perform α-amino Fmoc protection, and the double protected amino acid is obtained after purification.
[0009] As one of the optimized embodiments of the present invention, the amino acids containing side chain amino groups include histidine, lysine, ornithine, 2.3-diaminopropionic acid, 2.4-diaminobutyric acid, tryptophan and other amino acids containing side chain amino group configurations.
[0010] As one of the optimized embodiments of the present invention, in the first step reaction, the molar ratio of amino acid to Trt-Cl is 1:2-1:3, the amount of ZnCl2 is 1-1.5 mol per mole of amino acid, and the amount of DMF is 8-12 mL per gram of amino acid.
[0011] As one of the optimized embodiments of the present invention, the reaction time of the first step is 20-28 h, and the reaction is complete as monitored by TLC (developing solvent C:M=5:1, product Rf=0.2).
[0012] As one of the optimized embodiments of the present invention, the treatment steps after the first step reaction include: adding tap water and EA after cooling, adjusting the pH to 7-8 with 10mol / LNaOH, dissolving the filter cake after filtration and filtering out the Zn(OH)2 precipitate.
[0013] As one of the optimized embodiments of the present invention, in the second step reaction, the molar ratio of the side chain protected amino acid to Fmoc-osu is 1:1-1:1.2, the amount of Na2CO3 used is 1-1.1 mol per mole of side chain protected amino acid, and the volume ratio of THF to water is 1:2-1:3.
[0014] As one of the optimized embodiments of the present invention, the second purification step includes: extracting impurities with PET, stripping the aqueous phase with EA, acidifying to pH = 3-4 and then separating the liquids, washing the organic phase with water and saturated NaCl solution, and concentrating and crystallizing after drying.
[0015] Compared with the prior art, the technical solution provided by this application has the following beneficial effects:
[0016] 1. The double-protected amino acid, Fmoc-Lys(Trt)-OH, prepared by this method in this application has a final product purity of ≥99.4%, a maximum single impurity ≤0.24%, and a considerable yield (32% yield on a small scale and 48% yield on a large scale), which meets the high-purity requirements of peptide synthesis and provides high-quality raw materials for the high-quality synthesis of peptide drugs.
[0017] 2. In the scale-up experiment of this application, the reaction parameters did not need to be significantly adjusted, and the yield and purity data were stable, proving that the process has strong stability and can maintain good results in production of different scales. It has high industrial adaptability and is suitable for large-scale production, reducing production costs and production risks.
[0018] 3. The present method is applicable to a variety of amino acids containing side chain amino groups, such as lysine, histidine, ornithine, and the protecting reagent can be replaced by Cbz-Cl, Fmoc-Cl, etc., providing a universal protection strategy for peptide drug synthesis, reducing the cost and time of developing specific processes for different amino acids and protecting reagents, and improving the flexibility and versatility of the peptide synthesis process.
[0019] 4. This application uses Trt to protect the side chain amino group and Fmoc to protect the α-amino group, achieving precise protection of amino acids containing side chain amino groups, forming a structurally stable "double-protected amino acid." Furthermore, the Fmoc and Trt groups can be selectively removed under mild conditions (such as alkaline hydrolysis and acid hydrolysis) without affecting the amino acid backbone structure, facilitating the smooth progress of subsequent peptide synthesis reactions and improving the efficiency and success rate of peptide synthesis. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Reaction scheme for the preparation of Fmoc-Lys(Trt)-OH for this application;
[0021] Figure 2 This is a schematic diagram of the HPLC test results of Example 2 of the present application;
[0022] Figure 3 This is a schematic diagram of the HPLC test results of Example 3 of the present application; DETAILED DESCRIPTION
[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Generally, the components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.
[0024] 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 protection, but merely represents selected embodiments of the present application. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments in the present application without making any creative efforts shall fall within the scope of protection of the present application.
[0025] like Figure 1As shown, a method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group comprises the following steps:
[0026] Step 1: Using amino acids containing side chain amino groups and trityl chloride (Trt-Cl) as raw materials, in DMF solvent, with ZnCl2 as catalyst, the reaction is carried out at 65°C to perform Trt protection on the side chain amino groups to generate side chain protected amino acids.
[0027] Step 2: The side chain protected amino acid Lys(Trt)-OH is reacted with Fmoc-osu in a mixed solvent of THF and water with Na2CO3 as a base at room temperature to perform α-amino Fmoc protection, and the double protected amino acid is obtained after purification.
[0028] By protecting the side chain amino group with Trt and the α-amino group with Fmoc, a "double-protected amino acid" is formed. Its structure is stable and suitable for solid-phase or liquid-phase condensation reactions in subsequent peptide synthesis. The Fmoc and Trt groups can be selectively removed under mild conditions (such as alkaline hydrolysis and acid hydrolysis) without affecting the amino acid main chain structure.
[0029] The amino acids containing side chain amino groups include histidine, lysine, ornithine, 2.3-diaminopropionic acid, 2.4-diaminobutyric acid, tryptophan and other amino acids containing side chain amino group configurations.
[0030] In the first step reaction, the molar ratio of amino acid to Trt-Cl is 1:2-1:3, the amount of ZnCl2 used is 1-1.5 mol per mole of amino acid, and the amount of DMF used is 8-12 mL per gram of amino acid.
[0031] The molar ratio of amino acid to Trt-Cl is 1:2-1:3 to ensure that the side chain amino group is fully reacted, avoid excessive Trt-Cl resulting in increased costs or residual impurities, and prevent incomplete protection when Trt-Cl is insufficient (for example, in Example 2, when Trt-Cl is sufficient, the yield is increased to 48%).
[0032] The ZnCl2 dosage is 1-1.5 mol / mole of amino acid, which acts as a Lewis acid catalyst to activate the chlorine atom of Trt-Cl, accelerate the nucleophilic substitution reaction, shorten the reaction time (such as the reaction is completed within 24 hours in the embodiment), and avoid excessive subsequent Zn(OH)2 precipitation, which affects the filtration efficiency.
[0033] The amount of DMF used is 8-12 mL / g amino acid to ensure the solubility of the amino acid and Trt-Cl, form a homogeneous reaction system, and promote mass transfer and reaction uniformity (for example, the system dissolves from turbid to clear in the embodiment to ensure sufficient reaction).
[0034] The reaction time of the first step was 20-28 h, and the reaction was complete as monitored by TLC (developing solvent C:M=5:1, product Rf=0.2).
[0035] 65°C is the optimal reaction temperature, which not only accelerates the nucleophilic substitution reaction between Trt-Cl and amino groups, but also avoids boiling of DMF solvent (boiling point 153°C) or thermal decomposition of amino acids. For example, after reacting at 65°C for 24 hours, TLC monitoring showed complete conversion (Rf=0.2).
[0036] The reaction time is 20-28 hours, taking into account both efficiency and conversion rate, to avoid incomplete protection due to a too short reaction time, or the induction of byproducts (such as excessive substitution of Trt groups or amino acid degradation) due to a too long reaction time.
[0037] The developing solvent C:M=5:1 was used, and the product was accurately located by the Rf value (0.2), ensuring the reliability of the reaction endpoint judgment and avoiding blind termination of the reaction resulting in yield loss.
[0038] The treatment steps after the first step reaction include: adding tap water and EA after cooling, adjusting the pH to 7-8 with 10 mol / L NaOH, dissolving the filter cake after filtration and filtering out the Zn(OH)2 precipitate.
[0039] After the reaction, adjust the pH to 7-8 to make Zn 2+ It is converted into Zn(OH)2 precipitation, while avoiding the hydrolysis of the Trt group under acidic conditions (Trt is easily deprotected under strong acidic conditions) and ensuring product stability under alkaline conditions.
[0040] Extraction with water and EA is performed to achieve preliminary separation of the aqueous phase (containing zinc salt) and the organic phase (containing the product), and the filter cake is subsequently dissolved by THF and Zn(OH)2 is filtered to reduce residual inorganic impurities (for example, after filtering out Zn(OH)2 in the embodiment, the product purity reaches more than 99%).
[0041] The temperature should be controlled below 45°C to avoid a sudden rise in system temperature caused by heat release when adjusting pH value by NaOH, and to prevent boiling and volatilization of EA to ensure operational safety in industrial production.
[0042] In the second step reaction, the molar ratio of the side chain protected amino acid to Fmoc-osu is 1:1-1:1.2, the amount of Na2CO3 used is 1-1.1 mol per mole of side chain protected amino acid, and the volume ratio of THF to water is 1:2-1:3.
[0043] The molar ratio of the side chain protected amino acid to Fmoc-osu is 1:1-1:1.2 to ensure that the α-amino group is fully acylated. An excess of 10%-20% Fmoc-osu can compensate for the hydrolysis loss and improve the reaction conversion rate (for example, the yield is 48% and the purity is 99.8% at a 1:1 molar ratio in Example 2).
[0044] The amount of Na2CO3 used is 1-1.1 mol / mole of substrate, which acts as a base catalyst to neutralize the HCl generated in the reaction, maintain the alkaline environment of the system, promote the nucleophilic substitution reaction between Fmoc-osu and amino groups, and avoid excessive base causing hydrolysis of the Fmoc group (Fmoc is easily deprotected under strong alkaline conditions).
[0045] The volume ratio of THF to water is 1:2-1:3, forming a homogeneous reaction system: THF dissolves the side chain protected amino acids, and water dissolves Na2CO3 and Fmoc-osu. The mixing of the two phases promotes reaction contact and increases the reaction rate (for example, the reaction is completed at room temperature within 4 hours in the embodiment).
[0046] The second purification step comprises: extracting impurities with PET, stripping the aqueous phase with EA, acidifying to pH=3-4 and then separating the liquids, washing the organic phase with water and saturated NaCl solution, drying, and concentrating to crystallize.
[0047] PET is used to extract impurities, and the difference in solubility of impurities in non-polar solvents is used to remove unreacted Trt-Cl and other fat-soluble impurities; when EA is used to strip the aqueous phase, the pH is adjusted to 3-4 (under acidic conditions, the product is salted and dissolved in the aqueous phase, and after acidification, it is freed and precipitated into the EA organic phase) to achieve separation of the product and water-soluble impurities.
[0048] Wash with water and saturated NaCl solution to remove residual alkali, salt and water-soluble impurities, and dry with Na2SO4 to remove trace moisture to avoid moisture causing product hydrolysis or crystallization difficulties.
[0049] During the concentration and crystallization process, EA is added for cooling and stirring to promote the precipitation of the product and form uniform crystals, reduce residual impurities in the mother liquor, and ultimately achieve high purity (99.4%-99.8%) and low single impurity (≤0.24%), meeting the high standard requirements for raw materials for polypeptide synthesis.
[0050] The compounds covered by this patent include amino acids with side chain amino groups, such as histidine, lysine, ornithine, 2.3-diaminopropionic acid, 2.4-diaminobutyric acid, and tryptophan. The protective reagents covered by this patent include Cbz-Cl, Trt-Cl, Fmoc-Cl, and Mtt-Cl. The following uses lysine as an example, and Trt-Cl as an example protective reagent to illustrate this patent.
[0051] Example 1: Figure 2 As shown, the small-scale synthesis of Fmoc-Lys(Trt)-OH
[0052] The first step is to protect the side chain amino group Trt:
[0053] To a 3 L reaction flask, 14.6 g of Lys, 120 mL of DMF, 13.6 g of ZnCl2, and 33.4 g of Trt-Cl3 were added in sequence.
[0054] The temperature was raised to 65°C and stirred for 24 h. The system gradually became clear from turbid and then turbid.
[0055] After the reaction is complete (C:M = 5:1, product Rf = 0.2), cool the reaction system and add 100 mL of tap water and 100 mL of EA. Rapidly add 10 mol / L NaOH (liquid caustic soda) to adjust the pH to 7-8 (not below 7). Maintain the temperature below 45°C. Stir continuously for 10 minutes and filter.
[0056] The filtered solid was dissolved in 1 L of THF and 1 L of tap water, and the Zn(OH)2 solid was removed by suction filtration.
[0057] The THF was removed by rotary evaporation, and a white solid precipitated, which was directly filtered to obtain Lys(Trt)-OH solid. After drying, 29 g of solid was obtained.
[0058] The second step is α-amino Fmoc protection:
[0059] 30 mL of THF and 60 mL of tap water were added, followed by 10.6 g of 0.1 mol of Na2CO3 and 3.7 g of 0.1 mol of Fmoc-osu3. The mixture was stirred at room temperature for 4 h. The reaction was completed after monitoring by TLC (C:M=10:1, product Rf=0.3).
[0060] Add 100 mL of PET to extract impurities from the reaction system. The bottom of the aqueous phase becomes viscous, and 50 mL of THF is added. This extraction process is repeated three times. Discard the organic phase from the first run. A larger amount of product is present in the subsequent two runs. Add 50 mL of 5% sodium carbonate solution to strip the organic phase. Most of the product from the runs enters the aqueous phase, which is then combined. Add 150 mL of EA twice to the aqueous phase to extract the product. Add 100 mL of water to the EA layer, followed by hydrochloric acid (approximately 30 mL of 6 M) to acidify to a pH of 3-4.
[0061] After separation, the organic phase was washed twice with 100 mL of H2O and twice with 100 mL of a saturated NaCl solution.
[0062] The organic phase was washed to neutrality, dried over Na2SO4, and 100 mL of THF was added.
[0063] The mixture was filtered and concentrated to a small volume to separate out a solid which was filtered and 1 L of EA was added and the mixture was stirred under cooling for 2 h.
[0064] The product was filtered to obtain a pale yellow solid, which was then dried to give 20 g of authentic Fmoc-Lys(Trt)-OH with a yield of 32%, a purity of 99.4%, and a maximum single impurity of 0.24%.
[0065] Example 2: Figure 3 As shown, the scale-up synthesis of Fmoc-Lys(Trt)-OH
[0066] The first step is to protect the side chain amino group Trt:
[0067] To a 3 L reaction flask, 146 g of Lys, 1.2 L of DMF, 136 g of ZnCl, and 334 g of Trt-Cl were added in sequence.
[0068] The temperature was raised to 65°C and stirred for 24 h. The system gradually became clear from turbid and then turbid.
[0069] The reaction was complete (C:M = 5:1, product Rf = 0.2) as monitored by TLC. After completion, the temperature was lowered, 1.2 L of tap water and 1.2 L of LEA were added to the reaction system, and 10 mol / L NaOH (liquid caustic soda) was quickly added to adjust the pH to 7-8 (not below 7), maintaining the temperature below 45°C. Stirring was continued for 10 min, and the mixture was filtered.
[0070] The filtered solid was dissolved in 1 L of THF and 1 L of tap water, and the Zn(OH)2 solid was removed by suction filtration.
[0071] The THF was removed by rotary evaporation, and a white solid precipitated, which was directly filtered to obtain Lys(Trt)-OH solid. After drying, 312 g of solid was obtained.
[0072] The second step is α-amino Fmoc protection:
[0073] 300 mL THF and 600 mL tap water were added, followed by 1 mol Na 2 CO 3 106 g and 1 mol Fmoc-osu 337 g. The mixture was stirred at room temperature for 4 h. The reaction was completed under TLC monitoring (C:M=10:1, product Rf=0.3).
[0074] 1L of PET was added to extract impurities from the reaction system. The bottom of the aqueous phase became viscous, and 500mL of THF was added. This extraction process was repeated three times. The organic phase was discarded from the first run. A large amount of product was present in the two subsequent runs. 500mL of 5% sodium carbonate solution was added to strip the organic phase. Most of the product from the runs entered the aqueous phase, which was then combined. 1.5L of EA was added to the aqueous phase twice to extract the product. After adding 1L of water to the EA layer, hydrochloric acid (6M, approximately 300mL) was added to acidify to a pH of 3-4.
[0075] After separation, the organic phase was washed twice with 1 L of H2O and twice with 1 L of saturated NaCl solution.
[0076] The organic phase was washed to neutrality, dried over Na2SO4, and 1 L THF was added.
[0077] The mixture was filtered and concentrated to a small volume to separate out solids, which were filtered and 1 L of EA was added, followed by cooling and stirring for 2 h.
[0078] Filtration yielded a white to off-white solid, which was then dried to give 300 g of authentic Fmoc-Lys(Trt)-OH with a yield of 48%, a purity of 99.8%, and a maximum single impurity of 0.08%.
[0079] The above description is only a preferred embodiment of the present application, which is only selected as a control condition. The impact on product yield and quality does not mean that this patented technology has been verified by only two experiments.
[0080] Comparing Examples 1 and 2, the final product had a purity of ≥99.4% and a maximum single impurity of ≤0.24%, meeting the high-purity requirements of peptide synthesis. Reaction parameters did not require significant adjustment, and yield and purity data remained more stable, demonstrating strong process stability, high industrial adaptability, and suitability for large-scale production. This method is applicable to a variety of amino acids containing side chain amino groups (such as lysine and histidine), and the protective reagent can be replaced with Cbz-Cl, Fmoc-Cl, etc., providing a universal protection strategy for peptide drug synthesis and reducing process development costs.
[0081] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.
Claims
1. A method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group, characterized in that: The following steps are involved: Step 1: Using amino acids containing side chain amino groups and trityl chloride (Trt-Cl) as raw materials, in DMF solvent, with ZnCl2 as catalyst, the reaction is carried out at 65°C to perform Trt protection on the side chain amino groups to generate side chain protected amino acids; Step 2: The side chain protected amino acid Lys(Trt)-OH is reacted with Fmoc-osu in a mixed solvent of THF and water with Na2CO3 as a base at room temperature to perform α-amino Fmoc protection, and the double protected amino acid is obtained after purification.
2. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: The amino acids containing side chain amino groups include histidine, lysine, ornithine, 2.3-diaminopropionic acid, 2.4-diaminobutyric acid, tryptophan and other amino acids containing side chain amino group configurations.
3. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: In the first step reaction, the molar ratio of amino acid to Trt-Cl is 1:2-1:3, the amount of ZnCl2 used is 1-1.5 mol per mole of amino acid, and the amount of DMF used is 8-12 mL per gram of amino acid.
4. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: The reaction time of the first step was 20-28 h, and the reaction was complete as monitored by TLC (developing solvent C:M=5:1, product Rf=0.2).
5. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: The treatment steps after the first step reaction include: adding tap water and EA after cooling, adjusting the pH to 7-8 with 10 mol / L NaOH, dissolving the filter cake after filtration and filtering out the Zn(OH)2 precipitate.
6. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: In the second step reaction, the molar ratio of the side chain protected amino acid to Fmoc-osu is 1:1-1:1.2, the amount of Na2CO3 used is 1-1.1 mol per mole of side chain protected amino acid, and the volume ratio of THF to water is 1:2-1:
3.
7. The method for preparing Fmoc-Lys(Trt)-OH protected by an amino acid containing a side chain amino group according to claim 1, wherein: The second purification step comprises: extracting impurities with PET, stripping the aqueous phase with EA, acidifying to pH=3-4 and then separating the liquids, washing the organic phase with water and saturated NaCl solution, drying, and concentrating to crystallize.