Non-natural amino acid and synthesis method thereof
By inserting oxygen atoms into the CH bonds of α-amino acid side chains under mild conditions via the ANRORC reaction, the problem of difficult oxygen atom insertion in existing technologies has been solved, enabling efficient, safe, and low-cost synthesis of non-natural amino acids.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-04-10
AI Technical Summary
Existing technologies cannot safely insert oxygen atoms into the CH bonds of α-amino acid side chains under mild conditions, and traditional methods are prone to explosion and are difficult to scale up.
The ANRORC reaction is employed, using inexpensive Boc-L-His-OMe as a raw material. It reacts with oxane solvents, bicarbonates, and water to insert oxygen atoms into the CH bonds of α-amino acid side chains through a multi-stage process. The post-processing is simple, and alcohols and ethers are used to improve purity.
This method enables the safe insertion of oxygen atoms into the side chains of α-amino acids under mild conditions. The reaction is simple to operate, easy to scale up, produces high-purity products, requires simple post-processing, and is inexpensive.
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Figure CN121824428A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of synthesis of compounds, in particular, to unnatural amino acids and a synthesis method thereof. BACKGROUND
[0002] In the structure and function of organisms, proteins and the alpha-amino acids that make it up have played a key role in promoting extensive research on amino acids and alpha-amino acids and analogs thereof. In particular, in order to efficiently and sustainably engineer proteins, the possibility of chemically synthesizing unnatural amino acids and polypeptides is increasing. In polypeptide-based therapeutics, in order to overcome the lack of oral absorption and poor stability, several methods have been developed in recent years. These methods are classified according to whether the side chain is changed or the backbone is modified. Backbone modification includes changes to one of the repeating units NH, CO, and α-CH units. Side chain modification is generally obtained by oxidizing fragments with relatively low redox potential. For example, oxidizing cysteine, methionine, tryptophan, histidine, serine, and tyrosine fragments. Although there has been extensive work on the conversion of side chains of aromatic, heterocyclic, and heteroatom moieties in polypeptides, there is no general method for inserting an oxygen atom into the CH bond present in the side chain of an alpha-amino acid. The main reason is that the chemical bond exhibits low activity. Later, 3,3-dimethyl dioxirane (DMD) was used to introduce an oxygen atom into the CH bond, but this oxidizing agent has explosive properties and is not easy to scale up. Therefore, a method is needed that can insert an oxygen atom into the CH bond present in the side chain of an alpha-amino acid using a safe reagent, under mild reaction conditions, and on a large scale.
[0003] In view of this, the present application is proposed. SUMMARY
[0004] The purpose of the present application is to provide unnatural amino acids and a synthesis method thereof. The synthesis method provided by the present application can insert an oxygen atom into the CH bond present in the side chain of an alpha-amino acid. The synthesis method has mild reaction conditions, is easy to scale up, the raw materials are inexpensive and easy to obtain, the reaction operation is simple, and the post-treatment purification is simple.
[0005] The present application is implemented as follows: In a first aspect, the present application provides a synthesis method of unnatural amino acids, which is synthesized by referring to the following synthesis path:
[0006] In the synthesis of intermediate 2, the steps include: mixing compound 1, compound 2a, an oxane-based solvent, and a bicarbonate to perform a one-stage reaction; then adding water to perform a two-stage reaction, followed by pretreating the reaction liquid of the two-stage reaction to form a crude product, and then mixing the crude product, an alcohol-based solvent, and an amine-based substance to perform a three-stage reaction. The solvent in the reaction solution of the three-stage reaction is removed, and then an ether solvent is added for mixing and filtering; The step of synthesizing the intermediate 3 comprises: performing a hydrolysis reaction on the intermediate 2; The step of synthesizing the intermediate 4 comprises: mixing the intermediate 3, TFA and a chlorinated C1-C3 alkane solvent for reaction; The step of synthesizing the unnatural amino acid comprises: mixing the intermediate 4, a bicarbonate, an Fmoc-containing raw material and a nitrile-water solvent for reaction.
[0007] In an optional embodiment, the step of synthesizing the intermediate 2 comprises: mixing the compound 1, the compound 2a, an oxane solvent and a bicarbonate for a one-stage reaction; and then adding water for a two-stage reaction. Next, the reaction solution of the two-stage reaction is extracted with an ester solvent, and the organic phase is collected, dried, filtered and concentrated to form a crude product; Then, the crude product, an alcohol solvent and an amine substance are mixed for a three-stage reaction; The solvent in the reaction solution of the three-stage reaction is removed, and then an ether solvent is added for mixing, filtering and drying.
[0008] In an optional embodiment, the conditions for synthesizing the intermediate 2 satisfy the following requirements: (1) the molar ratio of the compound 1 to the compound 2a is 1: (2-5); (2) the molar ratio of the compound 1 to the bicarbonate is 1: (2-5); (3) the molar ratio of the compound 1 to the amine substance is 1: (2-5); (4) the volume ratio of the oxane solvent to the water is (1-1.5): 1; (5) the reaction temperature of the one-stage reaction, the two-stage reaction and the three-stage reaction is 20-30°C.
[0009] In an optional embodiment, the conditions for synthesizing the intermediate 2 satisfy the following requirements: (1) the oxane solvent comprises 1, 4-dioxane; (2) the bicarbonate comprises sodium bicarbonate and potassium sodium carbonate; (3) the alcohol solvent comprises a C1-C3 alcohol solvent; (4) the amine substance comprises triethylamine; (5) the ether solvent comprises methyl tert-butyl ether.
[0010] In an alternative embodiment, the step of synthesizing the intermediate 3 comprises: mixing the intermediate 2 and a hydroxide at 0-25℃ to react; after the reaction is completed, adjusting the pH of the reaction system to 3-4, removing the reaction solvent, and then beating with a nitrile solvent.
[0011] In an alternative embodiment, the hydroxide comprises sodium hydroxide, lithium hydroxide and potassium hydroxide. The nitrile solvent comprises acetonitrile.
[0012] In an alternative embodiment, the volume ratio of TFA and chlorinated C1-C3 alkane solvent is 5:1-20:1.
[0013] In an alternative embodiment, the step of synthesizing the unnatural amino acid comprises: mixing the intermediate 4, bicarbonate, Fmoc-OSu and acetonitrile-water solvent at 20-30℃ to react.
[0014] In an alternative embodiment, the molar ratio of the intermediate 4 and the bicarbonate is 1:(2-5). The molar ratio of the intermediate 4 and the Fmoc-OSu is 1:(0.8-1.2).
[0015] In a second aspect, the present application provides an unnatural amino acid prepared by the method for synthesizing the unnatural amino acid according to any one of the preceding embodiments.
[0016] The present application has the following beneficial effects: the embodiment of the present application uses Boc-L-His-OMe as raw material, which is low in price and easy to obtain, inserts an oxygen atom into the CH bond existing in the branched chain of the alpha-amino acid through ANRORC reaction, and then uses simple synthesis to successfully synthesize the target product. The raw material of the whole reaction is low in price and easy to obtain, the reaction condition is mild and easy to scale up, the reaction operation is simple, the post-treatment and purification are simple, and the product synthesized has high purity. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.
[0018] Figure 1 High-resolution mass spectrum of the intermediate 2 provided for the embodiment 1 of the present application; Figure 2 Mass spectrum of the intermediate 2 provided for the embodiment 1 of the present application; Figure 3The high resolution spectrum of the intermediate 3 provided for the embodiment 1 of the present application; Figure 4 The high resolution spectrum of the intermediate 3 provided for the embodiment 1 of the present application; Figure 5 The mass spectrum of the intermediate 3 provided for the embodiment 1 of the present application; Figure 6 The high resolution spectrum of the intermediate 3 provided for the embodiment 1 of the present application; Figure 7 The high resolution spectrum of the non-natural amino acid provided for the embodiment 1 of the present application; Figure 8 The mass spectrum of the non-natural amino acid provided for the embodiment 1 of the present application; Figure 9 The high resolution spectrum of the non-natural amino acid provided for the embodiment 1 of the present application. DETAILED DESCRIPTION
[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments are not specified by the manufacturers, they are all the conventional products which can be purchased in the market.
[0020] The embodiments of the present application provide a synthetic method of a non-natural amino acid (denoted as oxo-Fmoc-L-His-OH), which is synthesized according to the following synthetic path:
[0021] The specific preparation steps are as follows: S1, synthesis of intermediate 2; The compound 1, the compound 2a, the oxane solvent and the bicarbonate are mixed for one-stage reaction; specifically, the compound 1, the compound 2a, the oxane solvent and the bicarbonate are mixed at 0-5℃, and after the mixing is completed, the temperature is raised to 20-30℃ for reaction for 8-15h.
[0022] The molar ratio of the compound 1 to the compound 2a is 1: (2-5); the molar ratio of the compound 1 to the bicarbonate is 1: (2-5); the oxane solvent includes but is not limited to 1, 4-dioxane; and the bicarbonate includes but is not limited to sodium bicarbonate and sodium potassium carbonate.
[0023] The above conditions are beneficial to the reaction, and if the reaction conditions are changed, for example, the oxane solvent is changed to dichloromethane or tetrahydrofuran, etc., the reaction efficiency is slow, and the yield and purity of the obtained product are low.
[0024] Then, water is added to perform a second stage reaction. Here, water is used as a raw material, the reaction temperature is the same as that of the first stage reaction, and the reaction time is 8-15 hours. At this time, the volume ratio of the oxane solvent to the water is (1-1.5):1.
[0025] Next, the reaction solution of the second stage reaction is pre-processed to form a crude product. Specifically, the reaction solution of the second stage reaction is extracted with an ester solvent (e.g., ethyl acetate), the aqueous phase is discarded, and the organic phase is collected. The organic phase is washed with saturated brine, then dried with a drying agent (e.g., anhydrous sodium sulfate), and then filtered and concentrated to dryness to form a crude product.
[0026] The crude product, an alcohol solvent, and an amine substance are mixed to perform a third stage reaction. Specifically, the crude product is dissolved with an alcohol solvent, and then the amine substance is added to react at 20-30°C for 8-15 hours. After the reaction is completed, the reaction solution is concentrated to dryness, an ether solvent is added to precipitate a large amount of solid, the solid is filtered, and then the solid is dried.
[0027] Here, the alcohol solvent includes C1-C3 alcohol solvents, such as, but not limited to, methanol, ethanol, and the like. The amine substance includes, but is not limited to, triethylamine, and the ether solvent includes, but is not limited to, methyl tert-butyl ether.
[0028] The use of the above-mentioned substances, particularly the ether solvent, to treat the reaction solution of the third stage reaction can improve the purity of the product and reduce the residual amine substance and phenol in the product.
[0029] It should be noted that the phenol is a decomposition product of the phenyl chloroformate.
[0030] S2, synthesis of intermediate 3; The intermediate 2 is subjected to a hydrolysis reaction. Specifically, the intermediate 2 and a hydroxide are mixed to react at 0-25°C. After the reaction is completed, the pH of the reaction system is adjusted to 3-4, and then the reaction solvent is removed, and then the intermediate 3 is slurried with a nitrile solvent. Here, the hydroxide includes sodium hydroxide, lithium hydroxide, and potassium hydroxide, and the nitrile solvent includes acetonitrile.
[0031] The use of the above-mentioned conditions can ensure the purity and yield of the intermediate 3. If the slurry solvent is changed, the purity or yield will be reduced.
[0032] S3, synthesis of intermediate 4; The intermediate 3 is mixed with TFA and a chlorinated C1-C3 alkane solvent to react at 20-30°C. Here, the volume ratio of TFA to the chlorinated C1-C3 alkane solvent is 5:1-20:1. The chlorinated C1-C3 alkane solvent includes, but is not limited to, dichloromethane.
[0033] The above conditions are conducive to the formation of intermediate 4, and changing the volume ratio of TFA and chlorinated C1-C3 alkane solvent can lead to incomplete reaction with a large amount of raw material remaining.
[0034] S4, synthesis of unnatural amino acids; The intermediate 4, bicarbonate, Fmoc-containing raw material and nitrile-water solvent are mixed for reaction. Specifically, the intermediate 4, bicarbonate, Fmoc-OSu and acetonitrile-water solvent are mixed for reaction at 20-30°C. The molar ratio of intermediate 4 to bicarbonate is 1:(2-5); the molar ratio of intermediate 4 to Fmoc-OSu is 1:(0.8-1.2). The volume ratio of acetonitrile to water in the acetonitrile-water solvent is 1:(2-3). The above conditions are conducive to the synthesis of unnatural amino acids, improving their purity and yield.
[0035] The features and performance of the application are further described in detail below in conjunction with the examples.
[0036] Example 1 The examples of the present application provide a method for synthesizing unnatural amino acid (denoted as oxo-Fmoc-L-His-OH), which is synthesized by referring to the following synthesis path:
[0037] The specific preparation steps are as follows: S1, synthesis of intermediate 2; In a 3000 mL three-necked flask, compound 1 Boc-His-OMe (95g, 352.77 mmol), 1,4-dioxane (1425 mL) were added, mechanical stirring was started and the resulting solution was cooled to 0-5°C. Sodium bicarbonate (237.09 g, 2822.16 mmol), phenyl chloroformate (276.17 g, 1763.85 mmol) were added slowly. After addition, the ice water bath was immediately removed. The temperature was raised to 25°C and stirred for 12 hours.
[0038] Then, water (1000 mL) was added to the above reaction solution, and stirring was continued at 25°C for 12 hours.
[0039] Next, 1000 mL of EA was added for extraction, and the aqueous phase was discarded. The organic phase was washed once with saturated brine (300 mL), dried over anhydrous sodium sulfate (500 g), filtered, and concentrated to dryness under reduced pressure to form a crude product. The crude product was dissolved in MeOH (2090 mL), and TEA (110 g, 1087.06 mmol) was added at 25°C. After addition, stirring was carried out at room temperature. Stirring was carried out for 12 hours.
[0040] TLC spot check (eluent: DCM: MeOH = 10:1, Rf = 0.70) showed the reaction was complete.
[0041] After the reaction was completed, the reaction solution was concentrated to dryness at 45 °C, 500 mL MTBE was added, and a large amount of solid was precipitated, and stirring was continued for 3 h. Filtration, and the solid was collected. Drying at 45 °C under reduced pressure for 2 h gave 96 g of off-white solid; purity: 99%, yield: 95% (based on the mass of compound 1).
[0042] The characterization spectra of intermediate 2 are shown in Figures 1 to 3 .
[0043] S2, synthesis of intermediate 3; In a 1000 mL three-necked flask, intermediate 2 (40 g, 140.20 mmol) was dissolved in MeOH (400 mL), and stirring was started, and an aqueous solution of LiOH H2O (11.77 g, 280.4 mmol) (160 mL) was added at 0-5 °C. After the addition was completed, stirring was continued at 0-5 °C. The reaction was carried out at 20 °C for 4 h.
[0044] TLC spot check (the reaction solution was directly spotted, eluent: DCM: MeOH = 10:1, the Rf of the raw material was 0.55, the reaction was complete, and the Rf of the product was 0.02) showed that the reaction was complete.
[0045] The pH was adjusted to 3-4 with 70 mL of 2 N HCl, and then MeOH was removed by concentration under reduced pressure at 45 °C. The solid was slurried with 150 mL of MeCN and stirred for 12 h. Filtration, and the solid was collected. Then, drying was carried out under reduced pressure at 50 °C. 20 g of white solid was obtained, with a purity of 99.2% and a yield of 52% (based on the mass of intermediate 2).
[0046] The characterization spectra of intermediate 3 are shown in Figures 4 to 6 .
[0047] S3, synthesis of intermediate 4; In a 250 mL round-bottom flask, intermediate 3 (13 g, 47.92 mmol) was added to DCM (50 mL), and TFA (5 mL) was added, and the reaction was carried out at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure at 45 °C to obtain a solid. Drying was carried out under reduced pressure at 50 °C. 15 g of gray solid was obtained. The crude product was directly used in the next step without further purification.
[0048] S4, synthesis of unnatural amino acids; In a 100 mL round bottom flask, add intermediate 4 (15 g, 52.60 mmol), MeCN (70 mL), water (70 mL), NaHC03(13.26 g, 157.8 mmol), Fmoc-OSu (17.74 g, 52.6 mmol) and react at 25 °C for 12 hours. Cool the reaction system to 0 °C, filter, collect the solid. Slowly add the solid to 2 M HC1 600 mL. Note that the gas production is very serious, continue to stir for 1 hour after adding. Filter, collect the solid. The solid is slurried with MeCN 300 mL for 12 hours. Filter, collect the solid. Dry at 50 °C under reduced pressure to obtain 10 g of white solid; purity 98%, yield: 48% (based on the mass of intermediate 4).
[0049] The characterization spectrum of the unnatural amino acid is shown in Figures 7 to 9 .
[0050] Process optimization example 1 Referring to the operation of synthesizing intermediate 2 in S1 provided in Example 1, Boc-His-OMe and phenyl chloroformate are used as reagents, TEA and NaHC03 are used as bases in different reaction stages, 1, 4-dioxane and anhydrous methanol are used as solvents in different reaction stages, and the purification of the crude product is studied, mainly to remove TEA or phenol in the final reaction system. The results are shown below:
[0051] It should be noted that the volume ratio of good solvent and poor solvent is 1:1, and the amount of good solvent is 500 mL. Specifically, 500 mL of good solvent is mixed with the crude product, and then the poor solvent is added to precipitate the solid, which is filtered and dried. The dried material is detected to determine whether TEA and phenol are present, and the yield and purity of intermediate 2.
[0052] According to the above table, if the solvent for post-treatment is changed to other good solvent-poor solvent combinations instead of methyl tert-butyl ether, it will result in the presence of TEA or phenol in intermediate 2, which will also significantly reduce the yield and purity of intermediate 2. Using a single solvent, methyl tert-butyl ether, not only has good impurity removal effect, but also is the preferred choice for yield and product purity, and the operation is more convenient.
[0053] Process optimization example 2 Referring to the operation of synthesizing intermediate 3 in S2 provided in Example 1, the selection of the slurry solvent for the crude product after removing MeOH is optimized. For example, 200 mg of crude product is used, 5 times the volume of each solvent is used for testing, and the slurry time is 12 hours as the standard. The results are as follows:
[0054] According to the result, it can be known that changing the beating solvent can cause the beating purity and yield to decrease obviously, and the beating purity is high when acetonitrile is used.
[0055] Process optimization example 3 According to the operation of synthesizing intermediate 4 in S3 provided in Example 1, the influence of the amount of TFA on the reaction was studied, and the amount of TFA was calculated according to the volume ratio of TFA and DCM, and the specific results are as follows:
[0056] According to the above results, it can be known that the reaction speed increases with the increase of the content of TFA, and the reaction has a tendency to deteriorate when the ratio is 1:1, and the raw materials are not completely reacted when pure TFA or DCM is used.
[0057] Process optimization example 4 According to the operation of synthesizing unnatural amino acids in S4 provided in Example 1, the influence of the reaction solvent on the reaction product was studied, and the results are as follows:
[0058] According to the above results, it can be known that using acetonitrile and water to form a reaction solvent can improve the yield of the product, so that the raw materials can be completely reacted. If the reaction solvent is changed, the raw materials cannot be completely reacted, and the product content is also significantly reduced, which further shows that only the solvent provided in the examples of the present application can obtain a product with high yield and purity.
[0059] It should be noted that the product here refers to the percentage of the reaction solution after the reaction without purification.
[0060] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A method for synthesizing non-natural amino acids, characterized in that, Perform the synthesis according to the following synthesis path: The steps for synthesizing intermediate 2 include: mixing compound 1, compound 2a, an oxane solvent and bicarbonate for a first-stage reaction; then adding water for a second-stage reaction; then pretreating the reaction solution of the second-stage reaction to form a crude product; and then mixing the crude product, an alcohol solvent and an amine substance for a third-stage reaction. The solvent in the reaction solution of the three-stage reaction is then removed, and then an ether solvent is added, mixed, and filtered. The steps for synthesizing intermediate 3 include: hydrolyzing intermediate 2; The steps for synthesizing intermediate 4 include: reacting intermediate 3 with TFA and a chlorinated C1-C3 alkane solvent; The steps for synthesizing non-natural amino acids include: mixing the intermediate 4, bicarbonate, Fmoc-containing raw material, and nitrile-water solvent and reacting them.
2. The method for synthesizing non-natural amino acids according to claim 1, characterized in that, The steps for synthesizing intermediate 2 include: mixing compound 1, compound 2a, an oxane solvent, and bicarbonate for a first-stage reaction; and then adding water for a second-stage reaction. Next, the reaction solution of the second-stage reaction was extracted with ester solvents and the organic phase was collected, then dried, filtered and concentrated to form the crude product; Then, the crude product, alcohol solvent, and amine are mixed and subjected to a three-stage reaction. The solvent in the reaction solution of the three-stage reaction is then removed, and then an ether solvent is added, mixed, filtered, and dried.
3. The method for synthesizing non-natural amino acids according to claim 1 or 2, characterized in that, The conditions for synthesizing intermediate 2 shall meet the following requirements: (1) The molar ratio of compound 1 to compound 2a is 1:(2-5); (2) The molar ratio of compound 1 to the bicarbonate is 1:(2-5); (3) The molar ratio of compound 1 to the amine is 1:(2-5); (4) The volume ratio of the oxane solvent to the water is (1-1.5):1; (5) The reaction temperatures for the first-stage reaction, the second-stage reaction, and the third-stage reaction are all 20-30℃.
4. The method for synthesizing non-natural amino acids according to claim 1 or 2, characterized in that, The raw materials required for synthesizing intermediate 2 are as follows: (1) The oxane solvents include 1,4-dioxane; (2) The bicarbonate includes sodium bicarbonate and potassium sodium carbonate; (3) The alcohol solvents include C1-C3 alcohol solvents; (4) The amines include triethylamine; (5) The ether solvents include methyl tert-butyl ether.
5. The method for synthesizing non-natural amino acids according to claim 1, characterized in that, The steps for synthesizing intermediate 3 include: mixing intermediate 2 and hydroxide and reacting at 0-25°C; after the reaction is completed, adjusting the pH of the reaction system to 3-4, removing the reaction solvent, and then slurrying with a nitrile solvent.
6. The method for synthesizing non-natural amino acids according to claim 5, characterized in that, The hydroxides include sodium hydroxide, lithium hydroxide, and potassium hydroxide; The nitrile solvents include acetonitrile.
7. The method for synthesizing non-natural amino acids according to claim 1, characterized in that, The volume ratio of TFA to chlorinated C1-C3 alkane solvent is 5:1 to 20:
1.
8. The method for synthesizing non-natural amino acids according to claim 1, characterized in that, The steps for synthesizing non-natural amino acids include: mixing the intermediate 4, bicarbonate, Fmoc-OSu, and acetonitrile-water solvent and reacting them at 20-30°C.
9. The method for synthesizing non-natural amino acids according to claim 8, characterized in that, The molar ratio of intermediate 4 to the bicarbonate is 1:(2-5). The molar ratio of intermediate 4 to Fmoc-OSu is 1:(0.8-1.2).
10. A non-natural amino acid, characterized in that, It is prepared by the method for synthesizing non-natural amino acids as described in any one of claims 1-9.