Process for the preparation of serine derivatives

Abstract

This application discloses a method for preparing a serine derivative, comprising the following steps: mixing and stirring N-tert-butoxycarbonyl-L-serine with tetrahydrofuran, cooling and maintaining the temperature to obtain an initial mixture; adding sodium hydrogen in batches based on the initial mixture, and after the addition of sodium hydrogen is complete, adding iodomethane and heating, and performing a reflux reaction; terminating the reaction after TLC detection shows that the reaction is complete to obtain a second-generation mixture; adding hydrochloric acid to adjust the pH value to neutral based on the second-generation mixture, and reacting to obtain crude Boc-Ser(me); and recrystallizing the crude Boc-Ser(me) to obtain qualified Boc-Ser(me); Boc-Ser(me) is prepared by dissolving N-tert-butoxycarbonyl-L-serine in tetrahydrofuran and reacting it with sodium hydrogen and an equimolar amount of iodomethane, resulting in higher purity. Furthermore, this preparation method has a shorter reaction time, lower racemization, more thorough reaction, and produces isomers at levels below 0.1%, making it suitable for scale-up production.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and more specifically to a method for preparing a serine derivative. Background Technology

[0002] In the field of peptide synthesis, protected amino acids are structural fragments that constitute the target product and are key starting materials. However, most amino acids that constitute peptide drugs contain chiral centers. When enantiomers are introduced into the peptide chain, they form diastereopeptide impurities with structures and properties similar to the target product, making purification difficult. Among them, boc-ser(me)-oh is a starting material for peptide synthesis, and its raw material serine is also an amino acid that is prone to racemization. During the synthesis of boc-ser(me)-oh, due to the acid and base conditions used, boc-d-ser(me)-oh isomers are inevitably generated. The content of boc-d-ser(me)-oh isomers varies depending on the degree of control of various synthesis conditions. In the current technology, the synthesis of boc-ser(me)-oh is usually achieved by dissolving Boc-ser in a 30% sodium hydroxide solution, adding tetrabutylammonium bromide, and then adding dimethyl sulfate dropwise. However, this process route has a long reaction time and incomplete reaction. The isomers are more than 10% and are difficult to remove, resulting in very low purity, which is not conducive to production. Summary of the Invention

[0003] To address the technical problems of incomplete reaction in the preparation of boc-ser(me)-oh by existing processes and the resulting high percentage of isomers leading to low purity, this application provides a method for preparing serine derivatives.

[0004] To achieve the above objectives, the technical solution adopted in this application is: a method for preparing a serine derivative, comprising the following steps:

[0005] N-tert-butoxycarbonyl-L-serine and tetrahydrofuran were obtained. The N-tert-butoxycarbonyl-L-serine and tetrahydrofuran were mixed and stirred, cooled and maintained to obtain an initial mixture. Sodium hydrogen and iodomethane were obtained. Sodium hydrogen was added in batches based on the initial mixture. After the addition of sodium hydrogen was completed, iodomethane was added, the temperature was raised, and the reaction was carried out under reflux. The reaction was terminated after TLC detection to obtain a second-generation mixture. Hydrochloric acid was obtained. Based on the second-generation mixture, hydrochloric acid was added to adjust the pH to neutral. The reaction yielded crude Boc-Ser(me). The crude Boc-Ser(me) was recrystallized to obtain qualified Boc-Ser(me).

[0006] As one of the optimized embodiments of the present invention, the mixing ratio of N-tert-butoxycarbonyl-L-serine and tetrahydrofuran is in the range of 1:5-1:6.

[0007] As one of the optimized embodiments of the present invention, N-tert-butoxycarbonyl-L-serine and tetrahydrofuran are mixed and stirred, cooled and maintained to obtain an initial mixture. Specifically, N-tert-butoxycarbonyl-L-serine and tetrahydrofuran are mixed and stirred according to the mixing ratio, and the temperature is controlled within the range of -9 to -10°C for reaction and maintained to obtain an initial mixture.

[0008] As one of the optimized embodiments of the present invention, the sodium hydrogen content is 60%.

[0009] As one of the optimized embodiments of the present invention, the mixing ratio of sodium hydrogen and iodomethane is in the range of 1:1.5-1:2.

[0010] As one of the optimized embodiments of the present invention, sodium hydrogen is added in batches based on the initial mixture. After the sodium hydrogen is added, iodomethane is added, the temperature is raised, and a reflux reaction is carried out. The reaction is terminated after TLC detection to obtain the second-generation mixture. Specifically, 60% sodium hydrogen is added in batches based on the initial mixture, and iodomethane is added according to the mixing ratio range. The temperature range is raised to 15-55°C, and a reflux reaction is carried out. The reaction is detected by TLC to determine whether the reaction is complete. If it is complete, the reaction is terminated to obtain the second-generation mixture.

[0011] As one of the optimized embodiments of the present invention, if the reaction is determined to be incomplete based on TLC detection, the reflux reaction is continued until the reaction is complete as determined by TLC detection.

[0012] As one of the optimized embodiments of the present invention, the concentration of the hydrochloric acid is 3 mol / L.

[0013] Compared with existing technologies, the advantages of this invention are as follows: Boc-Ser(me) is prepared by dissolving N-tert-butoxycarbonyl-L-serine in tetrahydrofuran and reacting it with sodium hydrogen and an equimolar amount of iodomethane. This method yields higher purity, shorter reaction time, lower racemization, and a more complete reaction, producing isomers at levels below 0.1%, making it suitable for large-scale production. First, N-tert-butoxycarbonyl-L-serine is dissolved in tetrahydrofuran, and the temperature is controlled after stirring to provide stable reaction conditions for subsequent steps. Then, sodium hydrogen is added in batches. After the addition is complete, iodomethane is added, and the mixture is heated to reflux. The completeness of the reaction is determined by TLC monitoring. Once the reaction is detected as complete, the reaction is immediately terminated to obtain a second-generation mixture. Then, the pH is adjusted with hydrochloric acid, and the reaction proceeds to obtain crude Boc-Ser(me). Finally, the product is purified and refined by recrystallization to obtain high-quality Boc-Ser(me). Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a flowchart of the preparation method of serine derivatives provided in the first embodiment of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0017] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0018] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0019] In the description of this application, it should be noted that the use of terms such as "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer" to indicate orientation or positional relationships is based on the orientation or positional relationships shown in the accompanying drawings, or the orientation or positional relationships commonly used when the product is in use. These terms are used solely for the convenience of describing this application and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. Furthermore, the use of terms such as "first" and "second" in the description of this application is only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0020] Furthermore, the use of terms such as "horizontal" and "vertical" in the description of this application does not imply that the component is required to be absolutely horizontal or suspended, but rather that it may be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal relative to "vertical," and does not mean that the structure must be completely horizontal, but rather that it may be slightly tilted.

[0021] In the description of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "set up," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0022] Please see Figure 1 The first embodiment of the present invention provides a method for preparing a serine derivative, comprising the following steps:

[0023] Step S1: Obtain N-tert-butoxycarbonyl-L-serine and tetrahydrofuran. Mix N-tert-butoxycarbonyl-L-serine with tetrahydrofuran, stir, cool and maintain to obtain the initial mixture.

[0024] Step S2: Obtain sodium hydrogen and iodomethane. Add sodium hydrogen in batches based on the initial mixture. After the sodium hydrogen is added, add iodomethane, heat and reflux the reaction. Terminate the reaction after TLC detection to obtain the second-generation mixture.

[0025] Step S3: Obtain hydrochloric acid, adjust the pH value to neutral by adding hydrochloric acid based on the second-generation mixture, and react to obtain crude Boc-Ser(me). The crude Boc-Ser(me) is then recrystallized to obtain qualified Boc-Ser(me).

[0026] It is explained that serine derivatives are transformed from serine through a series of chemical reactions. They have hydrophilicity and a variety of biological activities. Serine derivatives have a wide range of functions in organisms, and they usually participate in metabolic processes, regulate cell signaling, and affect gene expression.

[0027] Furthermore, the mixing ratio of N-tert-butoxycarbonyl-L-serine and tetrahydrofuran ranges from 1:5 to 1:6.

[0028] Furthermore, step S1 specifically includes:

[0029] N-tert-butoxycarbonyl-L-serine and tetrahydrofuran were mixed and stirred according to a certain mixing ratio, and the reaction was carried out at a controlled temperature range of -9 to -10°C to obtain an initial mixture.

[0030] To clarify, N-tert-butoxycarbonyl-L-serine is an amino acid derivative composed of serine and a tert-butoxycarbonyl (t-Boc) protecting group. The tert-butoxycarbonyl is an amino protecting group that can effectively protect the amino group from modification in chemical reactions, thereby ensuring the stability and reactivity of the serine molecule.

[0031] Tetrahydrofuran, also known as tetrahydrooxocyclopentadiene, is a colorless and transparent liquid with good solubility, capable of dissolving organic compounds, and exhibiting high chemical stability. Dissolving N-tert-butoxycarbonyl-L-serine in tetrahydrofuran, cooling it, and maintaining the initial mixture avoids vigorous molecular motion between the compound and the solvent, thus reducing the instability of the dissolution process.

[0032] Furthermore, the sodium-hydrogen content is 60%, which is the mass ratio of sodium to hydrogen. When the sodium-hydrogen content is moderate, the compound can maintain stable chemical properties and is not prone to decomposition or explosion. The higher the sodium-hydrogen content, the greater the energy density of the compound, which can store more energy and provide a longer service life for various applications.

[0033] Furthermore, the mixing ratio of sodium hydrogen to iodomethane ranges from 1:1.5 to 1:2.

[0034] Furthermore, step S2 specifically includes:

[0035] 60% sodium hydrogen was added in batches to the initial mixture, and iodomethane was added according to the mixing ratio range. The temperature range was raised to 15-55℃ and refluxed. The reaction was detected by TLC to determine whether the reaction was complete. If it was complete, the reaction was terminated to obtain the second-generation mixture.

[0036] Furthermore, if the reaction is determined to be incomplete based on TLC detection, the reflux reaction is continued until the reaction is complete as determined by TLC.

[0037] Sodium hydrogen hydride is a highly reactive hydride. Adding sodium hydrogen hydride in batches can effectively control the reaction rate, avoiding impurities in the product or runaway reaction due to excessively vigorous reaction. This ensures that sodium hydrogen hydride is evenly distributed and fully contacts other components in the initial mixture. Iodomethane is a methylating agent, mainly used in substitution and addition reactions. After adding iodomethane, the temperature is increased to accelerate the reaction rate, and reflux is performed simultaneously. By reintroducing the generated gas into the reaction system, the utilization rate of reactants and the purity of the product can be improved. The reaction progress is monitored in real time by TLC, which allows for timely termination of the reaction when it is complete.

[0038] Furthermore, the concentration of hydrochloric acid is 3 mol / L.

[0039] It should be noted that the concentration of hydrochloric acid solution determines its acidity; the higher the concentration, the higher the degree of ionization of HCl molecules, and the more H+ is produced in the solution. + The higher the ion concentration, the stronger the acidity of the solution.

[0040] The pH of the second-generation mixture was adjusted to neutral by adding hydrochloric acid to achieve optimal acid-base balance. The reaction yielded crude Boc-Ser(me), containing a small amount of Boc-N-Me-Ser. Boc-Ser(me), representing N-tert-butoxycarbonyl-O-methyl-L-serine, has the Boc group as an amino protecting group, which protects the amino group from interference during the chemical reaction. Ser represents serine, providing the amino acid residue. (me) refers to the O-methyl group, indicating that the hydroxyl group in N-tert-butoxycarbonyl-L-serine has been converted to a methyl ether. This signifies the conversion of the hydroxyl group in N-tert-butoxycarbonyl-L-serine into a methyl ether. Boc-N-Me-Ser, i.e., N-methyl-N-tert-butoxycarbonyl-L-serine, affects the purity of Boc-Ser(me).

[0041] As an optional implementation method, the crude Boc-Ser(me) product is prepared by adding hydrochloric acid to adjust the pH value to neutral based on the second-generation mixture, concentrating tetrahydrofuran under vacuum, adding ethyl acetate and continuing to add hydrochloric acid to control the pH value of the solution at 2-3. At this time, the target product is in the ethyl acetate layer. The pH value of the target product is washed to 6-7 using semi-saturated brine, and anhydrous sodium sulfate is added for drying for 2-3 hours. The product is then filtered, the filtrate is concentrated to dryness, crystallized, and then petroleum ether is added for pulping and filtration to obtain the crude Boc-Ser(me) product.

[0042] Understandably, Boc-Ser(me) is prepared by dissolving N-tert-butoxycarbonyl-L-serine in tetrahydrofuran and reacting it with sodium hydrogen and an equimolar amount of iodomethane. This method yields higher purity, shorter reaction time, lower racemization, and a more complete reaction, producing isomers at levels below 0.1%, making it suitable for scale-up production. First, N-tert-butoxycarbonyl-L-serine is dissolved in tetrahydrofuran, and the temperature is controlled with stirring to provide stable reaction conditions for subsequent steps. Then, sodium hydrogen is added in batches. After the addition is complete, iodomethane is added, and the mixture is heated to reflux. The completeness of the reaction is determined by TLC monitoring. Once the reaction is detected as complete, the reaction is immediately terminated to obtain a second-generation mixture. The pH is then adjusted with hydrochloric acid, and the reaction proceeds to obtain crude Boc-Ser(me). Finally, the product is purified and refined by recrystallization to obtain high-quality Boc-Ser(me).

[0043] Specifically, in this embodiment, 205 g of N-tert-butoxycarbonyl-L-serine was added to a clean, dry 5 L three-necked flask, followed by 2 L of tetrahydrofuran. Stirring was initiated, and the internal temperature was lowered to -10°C and maintained. 80 g of 60% sodium hydroxide was added in batches. After the sodium hydroxide addition was complete, 142 g of methyl iodide was added. The temperature was then slowly raised to 45°C, and the reaction was weakly refluxed for 1 hour. The reaction was terminated immediately after TLC detection confirmed completeness. The pH was adjusted to neutral by adding 3N hydrochloric acid, and the tetrahydrofuran was concentrated under vacuum. 1 L of ethyl acetate was added, and the pH was adjusted to 2-3 by adding 3N hydrochloric acid. The product was in the ethyl acetate layer. The product solution was washed with 200 ml of semi-saturated saline solution each time until the pH reached 6-7. The product solution was dried with anhydrous sodium sulfate for 2-3 hours, filtered, and the filtrate was concentrated to dryness. After induced crystallization, petroleum ether was added and the mixture was stirred and filtered to obtain crude Boc-Ser(me), containing a small amount of Boc-N-Me-Ser. After recrystallization with ethyl acetate and petroleum ether, qualified Boc-Ser(me) was obtained. 180 grams of Boc-Ser(me) were recovered, with a purity of 98.2%, 0.09% isomers, and a yield of 82.19%.

[0044] It can be noted that the chemical equation for preparing Boc-Ser(me) based on sodium hydrogen and iodomethane is as follows:

[0045]

[0046] It is explained that, in order to obtain the best experimental data, the corresponding experimental data were obtained based on the method of controlling variables.

[0047] Firstly, 60g, 80g, and 100g of 60% sodium hydride were added in batches for the reaction. The final experimental data were as follows: 60g of sodium hydride yielded 95.5g of Boc-Ser(me) with a purity of 96.5%, 0.12% isomers, and a yield of 43.56%; 80g of sodium hydride yielded 180g of Boc-Ser(me) with a purity of 98.2%, 0.09% isomers, and a yield of 82.19%; 100g of sodium hydride yielded... 83 g of Boc-Ser(me) was obtained with a purity of 94.5%, 0.25% isomers, and a yield of 38.2%. It can be seen that the amount of sodium hydrogen added has a significant impact on the final yield and purity of Boc-Ser(me). Less than twice the molar amount of sodium hydrogen results in incomplete reaction and reduced product yield; more than twice the molar amount of sodium hydrogen leads to more side reactions and the formation of more isomers, which is not conducive to purification. Therefore, twice the molar amount of sodium hydrogen is preferred.

[0048] Secondly, the reaction was carried out with iodomethane additions of 120g, 142g, and 160g, respectively. The experimental data obtained were as follows: 120g of iodomethane yielded 120g of Boc-Ser(me) with a purity of 97.1%, 0.12% isomers, and a yield of 55.3%; 142g of iodomethane yielded 180g of Boc-Ser(me) with a purity of 98.2%, 0.09% isomers, and a yield of 82.19%; 160g of iodomethane yielded 141g of Boc-Ser(me) with a purity of 97.4%, 0.09% isomers, and a yield of 64.9%. It can be seen that adding less or more than one molar amount of iodomethane will affect the yield, generate more impurities, and result in greater product loss during recrystallization. Therefore, adding one molar amount of iodomethane is the optimal amount.

[0049] Thirdly, the temperature was increased after the addition of iodomethane to 25℃, 45℃, and 55℃, and the corresponding reactions were observed. The experimental data obtained were as follows: at 25℃, 100 g of Boc-Ser(me) was obtained with a purity of 96.3%, an isomer of 0.09%, and a yield of 46%; at 45℃, 180 g of Boc-Ser(me) was obtained with a purity of 98.2%, an isomer of 0.09%, and a yield of 82.19%; at 55℃, 110 g of Boc-Ser(me) was obtained with a purity of 96.3%, an isomer of 0.21%, and a yield of 50.7%. It can be seen that the yield and purity are lower at lower or higher temperatures, so 45℃ is the optimal temperature.

[0050] Fourth, after adding 142g of iodomethane and raising the temperature to 45℃, the reflux reaction time was controlled. The reaction times were 0.5h, 1h, and 2h, respectively. The experimental data were as follows: after reflux for 0.5h, 52g of Boc-Ser(me) was obtained with a purity of 96.3%, isomers of 0.08%, and a yield of 23.9%; after reflux for 1h, 180g of Boc-Ser(me) was obtained with a purity of 98.2%, isomers of 0.09%, and a yield of 82.19%; after reflux for 2h, 182g of Boc-Ser(me) was obtained with a purity of 98.2%, isomers of 0.32%, and a yield of 83.9%. It can be seen that a shorter reflux reaction time results in a lower yield and lower purity. After 1h, the formation of the target product no longer significantly increases, but the isomer index increases, which is not conducive to purification. Therefore, controlling the reflux reaction time to 1h is the preferred method.

[0051] Based on the above experimental data, it can be seen that the optimal reaction conditions for preparing products with higher purity and lower isomers are: two molar amounts of sodium hydrogen, one molar amount of iodomethane, a reaction temperature of 45°C, and a reflux reaction time of 1 hour. These conditions result in the best quality and yield of the target product.

[0052] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a serine derivative, characterized in that, The serine derivative is N-tert-butoxycarbonyl-O-methyl-L-serine (Boc-Ser(Me)), comprising the following steps: Step 1: Add 205 g of N-tert-butoxycarbonyl-L-serine to a clean, dry 5 L three-necked flask, add 2 L of tetrahydrofuran, start stirring, cool the internal temperature to -10 °C and maintain it, add 80 g of 60% sodium hydrogen in batches, after the sodium hydrogen is added, add 142 g of iodomethane, after the addition is complete, slowly raise the temperature to 45 °C, and react under weak reflux for 1 h. Stop the reaction immediately after TLC detection shows that the reaction is complete. Step 2: Add 3N hydrochloric acid to adjust the pH to neutral, concentrate the tetrahydrofuran under vacuum, add 1L of ethyl acetate, and add 3N hydrochloric acid to adjust the pH to 2-3. Step 3: The product is in the ethyl acetate layer. The product solution is washed with 200 ml of semi-saturated saline solution each time until the pH reaches 6-7. The product solution is dried with anhydrous sodium sulfate for 2-3 hours, filtered, and the filtrate is concentrated to dryness. After inducing crystallization, petroleum ether is added and the mixture is stirred. The mixture is then filtered to obtain crude Boc-Ser(me) containing a small amount of Boc-N-Me-Ser. Step 4: After recrystallization with ethyl acetate and petroleum ether, qualified Boc-Ser(me) can be obtained, and 180 grams of Boc-Ser(me) are collected.

2. The method for preparing a serine derivative according to claim 1, characterized in that, The N-tert-butoxycarbonyl-O-methyl-L-serine was prepared with a purity of 98.2%, an isomer of 0.09%, and a yield of 82.19%.

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