L-2-chlorophenylalanine and preparation method thereof

Abstract

The invention discloses a preparation method of L-2-chlorophenylalanine. The preparation method comprises the following steps: preparing the L-2-chlorophenylalanine by taking o-chlorocinnamic acid as a raw material; the preparation method of the L-2-chlorophenylalanine comprises the following operation steps: S1, pretreating o-chlorocinnamic acid; s2, carrying out enzyme catalytic reaction; s3, separating enzyme from a product; and S4, crystallizing and centrifuging the product. The invention provides L-2-chlorophenylalanine and a preparation method thereof, which are used for realizing double improvement of product purity and conversion rate and ensuring improvement of chiral purity of a product.

Description

Technical Field

[0001] This invention relates to the technical field of organic compound synthesis, specifically to L-2-chlorophenylalanine and its preparation method. Background Technology

[0002] L-2-Chloro-L-phenylalanine is a phenylalanine derivative, and its background technology has wide applications in fields such as biochemistry, drug development, and analytical chemistry.

[0003] L-2-chlorophenylalanine is commonly used as an intermediate in the synthesis of chiral drugs such as antiepileptics and antidepressants, as its chlorine atom substitution properties can enhance the targeting and metabolic stability of drug molecules. Simultaneously, it is used as an HPLC analytical standard for the detection of drug impurities and the development of methods for the quantification of metabolites.

[0004] Currently, the synthesis of L-2-chlorophenylalanine mainly involves two methods. The first is a chemical synthesis method, which primarily consists of two steps: chlorination and alkaline hydrolysis. First, phenylalanine is used as a starting material and reacts with a chlorinating agent such as thionyl chloride (SO₂Cl₂) to generate a chlorinated derivative. Then, the chlorinated derivative is hydrolyzed under alkaline conditions to finally obtain L-2-chlorophenylalanine. However, this method uses phenylalanine as a starting material, requires chlorinating agents such as thionyl chloride (SO₂Cl₂), has harsh reaction conditions, and is prone to generating byproducts, requiring complex post-processing. Furthermore, the chlorination reaction may destroy the original chiral center, requiring alkaline hydrolysis or resolution techniques to restore the L-configuration, resulting in a complex process and limited yield.

[0005] Another method is biocatalysis. Referring to the literature "A Novel Thermostable Phenylalanine Dehydrogenase for the Efficient Synthesis of Large-Volume Aromatic Amino Acids and Their Derivatives," efficient synthesis is achieved through phenylalanine dehydrogenase catalysis. Specifically, 3-(2-chlorophenyl)-2-oxopropionic acid is used as a substrate. In NH4Cl / NH4OH buffer, QtPDH is coupled with glucose dehydrogenase, and then NAD+ is utilized... + As a coenzyme, it undergoes asymmetric reductive amination at pH 8.5 to yield L-2-chlorophenylalanine. Although biocatalysis can achieve efficient asymmetric synthesis, the long-term stability and large-scale production of the enzyme still need optimization.

[0006] In order to achieve a dual improvement in substrate concentration and conversion rate, while reducing energy consumption and waste emissions, this application provides a solution. Summary of the Invention

[0007] This application provides an L-2-chlorophenylalanine and its preparation method, which can achieve a dual improvement in product purity and conversion rate, while ensuring the improvement of the chiral purity of the product.

[0008] This application provides a method for preparing L-2-chlorophenylalanine, which uses o-chlorocinnamic acid as a raw material to prepare L-2-chlorophenylalanine. The method for preparing L-2-chlorophenylalanine includes the following steps: S1, pretreatment of o-chlorocinnamic acid; S2, enzyme-catalyzed reaction; S3, separation of enzyme and product; S4, crystallization and centrifugation of product.

[0009] By adopting the above technical solution, this application uses o-chlorocinnamic acid as a raw material, and obtains L-2-chlorophenylalanine by transferring the amino group from the amino donor to o-chlorocinnamic acid via transaminase. The principle is that transaminase transfers the amino group from the amino donor to the α-keto acid through an amino transfer reaction, generating the corresponding L-amino acid.

[0010] However, transaminases cannot directly act on o-chlorocinnamic acid (o-chlorophenylacrylic acid), but require the substrate to exist in the form of o-chlorophenylpyruvic acid (α-keto acid) in order to catalyze the formation of L-2-chlorophenylalanine. Therefore, this application requires the pretreatment of o-chlorocinnamic acid to achieve the preparation of L-2-chlorophenylalanine.

[0011] Preferably, in the pretreatment process of o-chlorocinnamic acid, o-chlorocinnamic acid is dissolved in phosphate buffer, FeCl3 catalyst is added, and 30% H2O2 is added dropwise. The mixture is stirred at a certain temperature for a certain time to obtain o-chlorophenylpyruvic acid reaction solution.

[0012] By adopting the above technical solution, this application employs double bond oxidation (H2O2 / Fe). 3+ The Fenton-like reaction converts o-chlorocinnamic acid to o-chlorophenylpyruvic acid to ensure a high conversion rate of o-chlorophenylpyruvic acid.

[0013] Preferably, during the enzyme-catalyzed reaction, an amino donor and a coenzyme are added to the o-chlorophenylpyruvic acid reaction solution, and the reaction time and temperature are controlled to obtain a mixture containing L-2-chlorophenylalanine products.

[0014] By adopting the above technical solution, the one-pot method used in this application directly adds an amino donor and a coenzyme to the oxidized reaction solution to catalyze the amino transfer between α-keto acid (o-chlorophenylpyruvic acid) and the amino donor to generate L-2-chlorophenylalanine.

[0015] Preferably, in the enzyme-product separation step, the mixture containing L-2-chlorophenylalanine product is filtered through a ceramic membrane; the filtrate is passed through an ultrafiltration membrane to remove free enzymes and impurities; and the filtrate is further passed through a membrane distillation system to remove residual amino donors by adsorption with an anion exchange resin, thereby obtaining a concentrated solution containing L-2-chlorophenylalanine product.

[0016] Preferably, in the crystallization centrifugation step of the product, concentrated hydrochloric acid is added dropwise to the concentrated solution containing L-2-chlorophenylalanine product to adjust the pH value, and the mixture is stirred to crystallize for a certain period of time; then the crystals are collected by centrifugation, and finally washed with pure water to obtain the L-2-chlorophenylalanine product.

[0017] By adopting the above technical solution, this application uses isoelectric point crystallization. When the pH is close to the isoelectric point of the product, the net charge of the amino acid is zero and the solubility is the lowest, forming crystals and thus obtaining L-2-chlorophenylalanine product with high purity.

[0018] Preferably, in the crystallization centrifugation step of the product, the mother liquor obtained by centrifugation is used to adsorb unreacted o-chlorocinnamic acid through a cation exchange resin, eluted with NaOH, and then reused in the pretreatment step of the o-chlorocinnamic acid.

[0019] By adopting the above technical solution, this application utilizes the carboxylic acid group of o-chlorocinnamic acid to bind with the cation exchange site of the resin to recover unreacted o-chlorocinnamic acid for recycling.

[0020] Preferably, the amino donor is L-glutamic acid, and the coenzyme is α-transaminase.

[0021] By adopting the above technical solution, α-transaminase catalyzes the amino transfer between o-chlorophenylpyruvic acid and L-glutamic acid to generate L-2-chlorophenylalanine, with α-ketoglutarate as a byproduct.

[0022] Preferably, the amino donor is L-alanine, and the coenzyme is PLP (pyridoxal phosphate).

[0023] By adopting the above technical solution, PLP can catalyze the amino transfer between o-chlorophenylpyruvic acid and L-alanine to generate L-2-chlorophenylalanine, with pyruvic acid as a byproduct.

[0024] Preferably, the amino donor is ammonia, the coenzymes are phenylalanine dehydrogenase and formate dehydrogenase, and an NADH regeneration system is constructed.

[0025] By adopting the above technical solution, using phenylalanine dehydrogenase as a coenzyme, ammonia as an amino donor, and supplementing with an NADH regeneration system, with water as a byproduct, the mechanism is as follows: α-Keto acid + NH3 + NADH + H + →L-amino acid + NAD + +H2O.

[0026] On the other hand, this application provides an L-2-chlorophenylalanine, which is obtained by the preparation method of L-2-chlorophenylalanine with a purity > 99.0% and a chiral purity > 99.5%.

[0027] One or more technical solutions provided in this application have at least the following technical effects or advantages: 1. This application uses o-chlorocinnamic acid as a raw material, and L-2-chlorophenylalanine is obtained by transferring the amino group from the amino donor to o-chlorocinnamic acid via transaminase. The principle is that transaminase transfers the amino group from the amino donor to the α-keto acid through an amino transfer reaction, generating the corresponding L-amino acid.

[0028] 2. This application employs double bond oxidation (H2O2 / Fe). 3+ The Fenton-like reaction converts o-chlorocinnamic acid to o-chlorophenylpyruvic acid to ensure a high conversion rate of o-chlorophenylpyruvic acid.

[0029] 3. The one-pot method used in this application involves directly adding an amino donor and a coenzyme to the oxidized reaction solution to catalyze the amino transfer between α-keto acid (o-chlorophenylpyruvic acid) and the amino donor, generating L-2-chlorophenylalanine.

[0030] 4. This application uses isoelectric point crystallization. When the pH is close to the isoelectric point of the product, the net charge of the amino acid is zero and the solubility is the lowest, forming crystals and thus obtaining L-2-chlorophenylalanine product with high purity. Detailed Implementation

[0031] This application provides an L-2-chlorophenylalanine and its preparation method, which can achieve a dual improvement in product purity and conversion rate, while ensuring the improvement of the chiral purity of the product.

[0032] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0033] It should be noted that the terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or modules not explicitly listed or inherent to such processes, methods, products, or devices.

[0034] raw material o-chlorocinnamic acid CAS No.: 3752-25-8 Molecular weight: 182.60 Purity: 99.0%; Ferric chloride CAS No.: 10225-77-1 Molecular weight: 476.475 Purity: 90.0% Hydrogen peroxide CAS No.: 7722-84-1 Molecular weight: 34.015 Concentration: 30.0%; L-Glutamic Acid CAS No.: 56-86-0 Molecular Weight: 147.129 Purity: 98.0% L-Alanine CAS No.: 56-41-7 Molecular Weight: 89.093 Purity: 98.0% Ammonia solution CAS number: 1336-21-6 Molecular weight: 35.046 Concentration: 28%. Example Example 1

[0035] S1. Pretreatment of o-chlorocinnamic acid: o-chlorocinnamic acid (250 g / L) was dissolved in pH 7.5 phosphate buffer, 0.5% FeCl3 catalyst was added, and 30% H2O2 (molar ratio 1:1.2) was added dropwise. The mixture was stirred at 50 °C for 3 h to obtain o-chlorophenylpyruvic acid reaction solution.

[0036] S2. Enzyme-catalyzed reaction: L-glutamic acid (amino donor, molar ratio 1:1.2) and coenzyme α-transaminase were added to the o-chlorophenylpyruvic acid reaction solution. In this application, aspartate transaminase was used. The pH was controlled at 8.5, the reaction temperature was 35℃, and the reaction time was 8 hours to obtain a mixture containing L-2-chlorophenylalanine products.

[0037] S3. Separation of enzyme and product: The mixture containing L-2-chlorophenylalanine product was filtered through a 0.1 μm ceramic membrane; the filtrate was passed through a 10 kDa ultrafiltration membrane to remove free enzyme and impurities; the filtrate was further concentrated to a solid content of 30% by a membrane distillation system (PTFE hydrophobic membrane, 40 °C), and residual amino donors were removed by adsorption through an anion exchange resin (D201) to obtain a concentrated solution containing L-2-chlorophenylalanine product.

[0038] S4. Crystallization and centrifugation of the product: Concentrated hydrochloric acid was added dropwise to the concentrated solution containing L-2-chlorophenylalanine to adjust the pH value to a certain level. The pH value was dynamically controlled to 2.8-3.0 using an online pH sensor, and the mixture was stirred for 2 hours to induce crystallization. Then, the product was centrifuged (3000 rpm, 10 min) to collect the crystals. Finally, the product was washed with pure water to obtain L-2-chlorophenylalanine. The purity of L-2-chlorophenylalanine was measured to be >99.0%, and the chirality was >99.5%.

[0039] In the post-treatment cycle, the mother liquor obtained by centrifugation is passed through a D001 cation exchange resin to adsorb unreacted o-chlorocinnamic acid, eluted with 0.5M NaOH, and then reused in the pretreatment step of o-chlorocinnamic acid. It should be noted that the α-transaminase in Example 1 of this application can be either aspartate transaminase or alanine transaminase; this application uses aspartate transaminase. Example 2

[0040] The difference between Example 2 and Example 1 is that the amino donor and the coenzyme used are different. The amino donor is L-alanine and the coenzyme is PLP (pyridoxal phosphate). Example 3

[0041] The difference between Example 3 and Example 1 is that the amino donor and coenzyme used are different. The amino donor is ammonia water, and the coenzymes are phenylalanine dehydrogenase and formate dehydrogenase. In addition, an NADH regeneration system is constructed during the reaction.

[0042] Comparative Example Comparative Example 1 L-2-chlorophenylalanine was synthesized using existing chemical synthesis methods. The specific steps are as follows: phenylalanine was used as the starting material, and thionyl chloride (SO2Cl2) was selected as the chlorinating agent.

[0043] Reaction conditions: Phenylalanine is dissolved in anhydrous dichloromethane or tetrahydrofuran, and thionyl chloride (molar ratio 1:1.2) is slowly added dropwise under ice bath conditions. Then the temperature is raised to 30-40℃ and the reaction is stirred for 6-8 hours.

[0044] The chlorinated derivative was then dissolved in an aqueous sodium hydroxide solution (2-4 M concentration), and hydrolyzed by stirring at 50-60 °C for 4-6 hours. The pH was adjusted to neutral (pH 7.0), and the D-type byproduct was removed by chiral resolution (using enzymatic resolution) to finally obtain the L-configuration product.

[0045] Finally, the hydrolysate was cooled to 0-5°C, and concentrated hydrochloric acid was added to adjust the pH to the isoelectric point (pH 5.6). White crystals precipitated, which were filtered and washed with ice water. The solution was then recrystallized using an ethanol / water mixed solvent to obtain the L-2-chlorophenylalanine product.

[0046] Comparative Example 2 L-2-chlorophenylalanine was synthesized using a biocatalytic method based on existing technology. The specific steps are as follows, including enzyme selection: thermostable phenylalanine dehydrogenase (QtPDH) was used and expressed in Escherichia coli through genetic engineering. The coenzyme system was coupled with glucose dehydrogenase (BmGDH) to achieve NAD⁺ recycling and regeneration, reducing the amount of coenzyme required.

[0047] Substrate preparation: 3-(2-chlorophenyl)-2-oxopropionic acid (CPOA) was used as the substrate and dissolved in NH4Cl / NH4OH buffer (pH 8.5).

[0048] Reaction conditions: temperature 30℃, stirring speed 200rpm, reaction time 3 hours (substrate conversion rate >99%); enzyme loading was QtPDH and BmGDH added in a 1:2 ratio (total enzyme activity 1kU and 2kU / L, respectively).

[0049] The pH was maintained at 8.5 by real-time monitoring with online sensors and the addition of ammonia. After the reaction, enzymes and bacterial cells were removed by filtration using a ceramic membrane (0.1 μm pore size). The filtrate was then concentrated by nanofiltration to remove the amino donor (NH4). + The solution was then adjusted to its isoelectric point (pH 5.6) with concentrated hydrochloric acid, and the crystals were collected by centrifugation to obtain the L-2-chlorophenylalanine product.

[0050] Data Analysis This application analyzes the L-2-chlorophenylalanine products prepared in Examples 1-3 and Comparative Examples 1-2, specifically focusing on the purity of the products, the conversion rate of L-2-chlorophenylalanine, the chiral purity of L-2-chlorophenylalanine, and the analysis of the process costs in each scheme.

[0051] Table 1. Purity of products from Examples 1-3 and Comparative Examples 1-3

[0052] Conclusion Analysis An analysis was conducted between Example 1 and Comparative Example 1. Comparative Example 1 synthesized L-2-chlorophenylalanine using a conventional chemical synthesis method, with the following specific steps: phenylalanine was used as the starting material, and thionyl chloride (SO2Cl2) was selected as the chlorinating agent. The purity, conversion rate, and chiral purity of its product were all lower than those of Example 1. Furthermore, in industrial production, due to the cumbersome procedures and the price of raw materials, the estimated production cost is higher than that of Example 1.

[0053] Next, Comparative Example 2 and Example 1 will be compared and analyzed. In Comparative Example 2, L-2-chlorophenylalanine was synthesized using a conventional biocatalytic method with 3-(2-chlorophenyl)-2-oxopropionic acid (CPOA) as the substrate and thermostable phenylalanine dehydrogenase (QtPDH) as the enzyme. The resulting product, L-2-chlorophenylalanine, showed improved purity compared to the traditional chemical synthesis method in Comparative Example 1, but slightly lower. Furthermore, the production cost was comparable to that of Example 1.

[0054] A comparative analysis was conducted between Examples 1-3. When ammonia was used as the amino donor, and phenylalanine dehydrogenase and formate dehydrogenase were used as coenzymes, and an NADH regeneration system was constructed during the reaction, the production cost was lower than that of Examples 1 and 2. Regarding chiral purity, Example 3 was superior to Examples 1 and 2, but its purity and conversion rate were slightly lower. Analysis showed that Example 3 is suitable for large-scale production. Specifically, Example 3 used phenylalanine dehydrogenase as the coenzyme, ammonia as the amino donor, and an NADH regeneration system, with water as the byproduct. The mechanism was analyzed. α-Keto acid + NH3 + NADH + H + →L-amino acid + NAD + +H2O.

[0055] Phenylalanine dehydrogenase is a type of oxidoreductase whose core function is to catalyze the oxidative deamination reaction of L-phenylalanine or its reverse reaction. In this application, phenylalanine dehydrogenase is used as a coenzyme catalyst, utilizing its reductive amination ability to react o-chlorophenylpyruvic acid (α-keto acid) with ammonia (NH3 / NH4). + The two molecules combine to form an imine intermediate. NADH acts as a reducing agent to reduce the imine to an L-configuration amino acid. The consumed NADH needs to be recycled through a coenzyme regeneration system (such as formate dehydrogenase + sodium formate).

[0056] It should be noted that the order of the embodiments described above is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. Furthermore, specific embodiments have been described above. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps described in the claims can be performed in a different order than those shown in the embodiments and still achieve the desired results. In some implementations, multitasking and parallel processing are also possible or may be advantageous.

[0057] The above description is only a preferred embodiment of this application and is not intended to limit this application. 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.

[0058] This specification is merely an illustrative description of this application and is intended to cover any and all modifications, variations, combinations, or equivalents within the scope of this application. Clearly, those skilled in the art can make various alterations and modifications to this application without departing from its scope. Therefore, if such modifications and modifications fall within the scope of this application and its equivalents, this application intends to include such modifications and modifications.

Claims

1. A method for preparing L-2-chlorophenylalanine, characterized in that, L-2-chlorophenylalanine was prepared using o-chlorocinnamic acid as a raw material. The preparation method of L-2-chlorophenylalanine includes the following steps: S1, pretreatment of o-chlorocinnamic acid; S2, enzyme-catalyzed reaction; S3, separation of enzyme and product; S4, crystallization and centrifugation of product.

2. The method for preparing L-2-chlorophenylalanine according to claim 1, characterized in that, In the pretreatment process of o-chlorocinnamic acid, o-chlorocinnamic acid is dissolved in phosphate buffer, FeCl3 catalyst is added, and 30% H2O2 is added dropwise. The mixture is stirred at a certain temperature for a certain time to obtain o-chlorophenylpyruvic acid reaction solution.

3. The method for preparing L-2-chlorophenylalanine as described in claim 2, characterized in that, During the enzyme-catalyzed reaction, an amino donor and a coenzyme are added to the o-chlorophenylpyruvic acid reaction solution, and the reaction time and temperature are controlled to obtain a mixture containing L-2-chlorophenylalanine products.

4. The method for preparing L-2-chlorophenylalanine as described in claim 3, characterized in that, In the enzyme-product separation step, the mixture containing L-2-chlorophenylalanine product is filtered through a ceramic membrane; the filtrate is then passed through an ultrafiltration membrane to remove free enzymes and impurities; the filtrate is further passed through a membrane distillation system to remove residual amino donors by adsorption with an anion exchange resin, resulting in a concentrated solution containing L-2-chlorophenylalanine product.

5. The method for preparing L-2-chlorophenylalanine as described in claim 4, characterized in that, In the crystallization centrifugation step of the product, concentrated hydrochloric acid is added dropwise to the concentrated solution containing L-2-chlorophenylalanine product to adjust the pH value, and the mixture is stirred to crystallize for a certain period of time; then the crystals are collected by centrifugation and finally washed with pure water to obtain the L-2-chlorophenylalanine product.

6. The method for preparing L-2-chlorophenylalanine as described in claim 5, characterized in that, In the crystallization centrifugation step of the product, the mother liquor obtained by centrifugation is passed through a cation exchange resin to adsorb unreacted o-chlorocinnamic acid, eluted with NaOH, and then reused in the pretreatment step of the o-chlorocinnamic acid.

7. The method for preparing L-2-chlorophenylalanine as described in claim 3, characterized in that, The amino donor is L-glutamic acid, and the coenzyme is α-transaminase.

8. The method for preparing L-2-chlorophenylalanine as described in claim 3, characterized in that, The amino donor is L-alanine, and the coenzyme is PLP (pyridoxal phosphate).

9. The method for preparing L-2-chlorophenylalanine as described in claim 3, characterized in that, The amino donor is ammonia, and the coenzymes are phenylalanine dehydrogenase and formate dehydrogenase, and an NADH regeneration system is constructed.

10. An L-2-chlorophenylalanine, characterized in that, The L-2-chlorophenylalanine obtained by the method for preparing L-2-chlorophenylalanine according to any one of claims 1-9 has a purity > 99.0% and a chiral purity > 99.5%.

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