Process for the preparation of 2-amino-3-benzoylpropionic acid compounds

Abstract

The application discloses an enzyme catalytic synthesis method of 2-amino-3-benzoyl propionic acid compounds. The method comprises the following steps: in the presence of a cofactor, a threonine aldolase is used to catalyze a substitution reaction of a compound shown in the following formula a with glycine, and a compound shown in the following formula b is generated. The method can efficiently and selectively synthesize 2-amino-3-benzoyl propionic acid compounds.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, specifically to a method for preparing 2-amino-3-benzoylpropionic acid compounds, and further to an enzyme-catalyzed method for synthesizing 2-amino-3-benzoylpropionic acid compounds. Background Technology

[0002] 2-Amino-3-benzoylpropionic acid compounds are an important class of non-natural amino acids, with the structural formula shown below.

[0003]

[0004] 2-Amino-3-benzoylpropionic acid compounds are commonly used in pharmaceuticals, light industry, and chemical industries. Due to their high structural similarity to natural kynurenine (KYN), they are promising candidates for selective kynurenine-3-monooxygenase (KMO) inhibitors. KMO is located at a key branch point of the kynurenine pathway (KP), the main pathway of tryptophan metabolism in the body, and is predicted to be an attractive drug target for the treatment of neurological diseases, mental illnesses, and cancers, especially neurodegenerative diseases.

[0005] Furthermore, KMO inhibitors can also influence disease progression by modulating inflammation, metabolism, immune responses, and neurological function. Several KMO inhibitors based on the natural substrate kynurenine (KYN) have been summarized in existing technologies, as shown below.

[0006]

[0007] 2-Amino-3-benzoylpropionic acid compounds, as α-amino acids, are currently mainly produced through chemical synthesis. In 2011, Frank Glorius's group at the University of Münster proposed an intermolecular Stetter reaction catalyzed by N-heterocyclic carbene (NHC) ligands, which can accept various aldehydes, but requires a glove box and cumbersome purification steps. In 2012, Tian Shikai et al.'s research showed a highly diastereoselective Mannich decarboxylation reaction of β-keto acids with optically active N-tert-butylsulfinyl-α-imino esters. In 2020, Adrián Gómez-Suárez introduced a method that does not require chiral catalysts and proceeds under very mild conditions: providing acylation or alkylation to octoxazolidinones through a light-mediated free radical decarboxylation process. These methods generally suffer from complex production processes, high production costs, and unavoidable environmental pollution. Therefore, a clean, green, and efficient biological method is urgently needed, which is not only beneficial for industrial production but also of great significance for environmental protection. Summary of the Invention

[0008] Therefore, it is necessary to provide at least one method for preparing 2-amino-3-benzoylpropionic acid compounds.

[0009] In a first aspect of this application, a method for preparing the compound of formula b or a pharmaceutically acceptable salt thereof is provided, the method comprising the steps of: in a reaction system in the presence of a cofactor, using a biocatalyst to catalyze a substitution reaction between the compound of formula a and glycine to generate the compound of formula b;

[0010]

[0011] In formulas b and a: A is selected from C3-C8 saturated or partially unsaturated straight-chain or cyclic alkyl groups, 3-12 saturated or partially unsaturated heterocyclic groups, C6-C 10 The group consisting of aryl and 5-12 heteroaryl groups; each Ra is independently selected from halogen, nitro, carboxyl, hydroxyl, hydroxymethyl, mercapto, thiomethyl, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, -NH-C1-C6 alkyl, mercapto-substituted C1-C6 alkyl, C2-C6 acyl, C3-C8 alicyclic hydrocarbon, 3-12 heterocyclic hydrocarbon, C6-C 10 The group consisting of aryl and 5-12 heteroaryl groups; each of the heterocyclic group and heteroaryl group independently comprises 1 to 4 ring atoms, the ring atoms being selected from the group consisting of N, O and S; n is an integer from 0 to 5; in formula a: X is bromine or iodine; the biocatalyst contains threonine aldolase (LTA).

[0012] In some embodiments, the biocatalyst is selected from the group consisting of LTA, bacterial cells expressing LTA, lysates of the bacterial cells, lyophilized powders of the bacterial cells, and LTA in a cured form.

[0013] In some embodiments, the bacterial cells are selected from a group consisting of Escherichia coli and yeast.

[0014] In some implementations, the cofactor satisfies one or more of the conditions shown in A1)-A2) below:

[0015] The cofactors mentioned in A1) include pyridoxal phosphate (PLP); and,

[0016] A2) The initial concentration of the cofactor in the reaction system is 0.001 mM-10 mM, based on the amount of feed.

[0017] In some embodiments, the cofactor is initially concentrated in the reaction system at a concentration of 0.02 mM to 0.5 mM.

[0018] In some embodiments, the cofactor is initially concentrated in the reaction system at a concentration of 0.05 mM to 0.2 mM.

[0019] In some embodiments, the reaction system also contains chemical additives.

[0020] In some embodiments, the chemical additive satisfies one or more of the conditions shown in B1)-B2):

[0021] The chemical additives mentioned in B1) include I - Ionic salts; and,

[0022] The initial concentration of the chemical additive described in B2) in the reaction system is ≤40mM.

[0023] In some implementations, the I - Ionic salts include one or more of LiI, NaI, KI, CsI, CaI2, AgI, CuI, CuI2, MgI2, and AlI3.

[0024] In some embodiments, the initial concentration of the chemical additive in the reaction system is 0.01 mM to 40 mM.

[0025] In some embodiments, the initial concentration of the chemical additive in the reaction system is 1 mM-10 mM.

[0026] In some embodiments, the initial concentration of the LTA in the reaction system is 0.05 mg / mL to 60 mg / mL.

[0027] In some embodiments, the initial concentration of the LTA in the reaction system is 0.1 mg / mL to 40 mg / mL.

[0028] In some embodiments, the initial concentration of the LTA in the reaction system is 0.2 mg / mL to 20 mg / mL.

[0029] In some implementations, the LTA satisfies any one of the following conditions C1)-C3):

[0030] C1) is derived from a group composed of Pseudomonas, Escherichia coli, Candida, Nematodes and Leishmania.

[0031] C2) The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; and,

[0032] C3) An amino acid sequence obtained by replacing, deleting and / or inserting one or more amino acids based on the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, while maintaining enzyme activity.

[0033] In some embodiments, the nucleic acid encoding the LTA satisfies any one of the following conditions (D1)-D3):

[0034] D1) The nucleotide sequence is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6;

[0035] A nucleotide sequence complementary to the nucleotide sequence defined by D2) and D1);

[0036] Any polynucleotide sequence or complementary nucleotide sequence that has at least 70% sequence identity with the nucleotide sequence defined by D3) and D1).

[0037] In some embodiments, the reaction system satisfies one or more of the conditions shown in E1)-E2) below:

[0038] E1) Based on the amount of feed, the molar ratio of the compound shown in formula a to the glycine is 1:(1-10000);

[0039] The initial concentration of the compound shown in formula a in E2) is 0.01 mM-1000 mM in the reaction system.

[0040] In some embodiments, the substitution reaction satisfies one or more of the conditions shown in F1)-F2) below:

[0041] F1) is carried out at 10℃-50℃;

[0042] The reaction time for F2 is 1h-48h, preferably 2h-24h for LTA.

[0043] In some embodiments, the substitution reaction is carried out at 15°C-40°C.

[0044] In some embodiments, the substitution reaction is carried out at 20°C-30°C.

[0045] In some embodiments, the reaction system satisfies one or more of the following G1)-G3):

[0046] G1) also includes a buffer solution, wherein the buffer solution is selected from the group consisting of phosphate buffer, Tris-HCl buffer, ammonium acetate buffer and HEPES-NaOH buffer;

[0047] G2) also includes a cosolvent selected from the group consisting of dimethyl sulfoxide, methanol, ethanol, and isopropanol; and,

[0048] G3) pH value is 5-10.

[0049] In some embodiments, the initial amount of the co-solvent is 5%-25% of the volume of the reaction system.

[0050] This application provides an enzymatic method for the production of 2-amino-3-benzoylpropionic acid compounds. Catalyzed by threonine aldolase, the method involves an α-substitution reaction of readily available and inexpensive haloacetophenone compounds with glycine, resulting in a highly efficient one-step synthesis of 2-amino-3-benzoylpropionic acid compounds. This method is a one-step synthesis, simple in process, low in production cost, with mild reaction conditions, easy operation, and high stereoselectivity. In some embodiments, whole-cell catalysis is used, resulting in high catalytic efficiency, high yield, and high selectivity, yielding high-purity single-configuration products with a yield reaching 8.9 g / L. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0052] Figure 1 This is a pET-28a vector structure containing an LTA sequence in one embodiment of this application.

[0053] Figure 2 The yield and enantiomer ratio of the compound are given in one embodiment of this application. Detailed Implementation

[0054] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0056] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0057] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0058] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0059] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0060] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0061] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0062] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0063] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0064] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0065] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0066] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0067] As used in this application, the term "halogen" means fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0068] As used in this application, the term "hydrocarbon group" refers to a straight-chain or branched saturated or unsaturated hydrocarbon group, that is, the group remaining after the corresponding hydrocarbon loses a hydrogen atom (H), including alkyl, alkenyl, and alkynyl groups; preferably alkyl.

[0069] As used in this application, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group. For example, C1-C6 alkyl refers to a straight-chain or branched saturated hydrocarbon group containing 1-6 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, n-hexyl, etc., and optionally, methyl.

[0070] As used in this application, the term "C1-C6 haloalkyl" means a C1-C6 alkyl group that is substituted by one or more halogens;

[0071] As used in this application, the term "alkoxy" refers to a group formed by the connection of a straight-chain or branched alkyl group with an oxygen atom, preferably a C1-C6 alkoxy group, more preferably a C1-C4 alkoxy group, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, etc.

[0072] As used in this application, the term "C1-C6 haloalkoxy" refers to a group in which a C1-C6 alkoxy group is substituted by one or more halogens.

[0073] As used in this application, the term "alkylamine" refers to a group formed by the attachment of a straight-chain or branched alkyl group to -NH2, which may be C1-C2. 10 Alkylamine, further optionally C1-C4 alkylamine, examples of which may include, but are not limited to, methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine, isobutylamine, etc.

[0074] The term "-NH-C1-C6 alkyl" as used in this application indicates that the amino group is replaced by a C1-C6 alkyl group.

[0075] As used in this application, the term "alkyl mercapto" refers to a group formed by connecting a straight-chain or branched alkyl group to a sulfur atom, preferably a C1-C6 alkyl mercapto, more preferably a C1-C4 alkyl mercapto, including but not limited to methyl mercapto, ethmercapto, n-propanyl mercapto, isopropanyl mercapto, n-butanyl mercapto, or isobutanyl mercapto.

[0076] As used in this application, the term "acyl" refers to a group formed by connecting a straight-chain or branched saturated or unsaturated hydrocarbon group to a carbonyl group, preferably an alkyl acyl group, including but not limited to formyl, acetyl, or propionyl.

[0077] As used in this application, the term "alicyclic hydrocarbon group" refers to saturated and unsaturated alicyclic hydrocarbons (alicyclic hydrocarbons containing one or more carbon-carbon double bonds in their molecule), including monocyclic and polycyclic alicyclic hydrocarbons. "C3-C8 alicyclic hydrocarbon group" can include C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C3-C8 cycloynyl, preferably C3-C8 cycloalkyl. "C3-C8 cycloalkyl" refers to a saturated cycloalkyl group containing 3-8 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. "C3-C8 cycloalkenyl" refers to a cycloalkyl group containing 3-8 carbon atoms and one or more carbon-carbon double bonds, including but not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, or cyclohexenyl. "C3-C8 cycloynyl" refers to a cycloalkyl group containing 3-8 carbon atoms and one or more carbon-carbon triple bonds.

[0078] The term "3-12 membered alicyclic hydrocarbon group" as used in this application refers to a group of 3-12 membered rings formed by connecting saturated and unsaturated alicyclic hydrocarbons with oxygen, nitrogen, or sulfur atoms.

[0079] The term "C6-C" is used in this application. 12 "Aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group containing 6-12 carbon atoms and no heteroatoms, such as phenyl or naphthyl.

[0080] As used in this application, the term "5-12-membered heteroaryl" refers to a 5-12-membered aromatic cyclic hydrocarbon group containing 1-4 atoms selected from nitrogen, oxygen, or sulfur, with the remaining atoms being carbon atoms, including monocyclic, bicyclic, or fused rings. Examples of heteroaryl groups include, but are not limited to, pyrroleyl, triazolyl, thiadiazolyl, tetrazolyl, imidazolyl, pyrazolyl, isothiazolyl, thiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, furazonyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, and triazinyl.

[0081] As used in this application, the term "pharmaceutically acceptable" means that these compounds, materials, compositions, and / or dosage forms, when used in contact with human and animal tissues with reliable medical judgment, do not cause excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0082] As used in this application, the term "pharmaceutically acceptable salt" refers to the salt form corresponding to the compounds of this application, prepared from compounds with specific substituents available in this application by contacting them with a relatively non-toxic acid or base. When the compounds of this application contain relatively acidic groups, a base addition salt can be obtained by adding a sufficient amount of base to a solution to contact the neutral form of the compound. Pharmaceutically acceptable base addition salts include salts such as sodium, potassium, calcium, ammonium, organic amines, or magnesium salts. When the compounds of this application contain relatively basic groups, an acid addition salt can be obtained by adding a sufficient amount of acid to a solution to contact the neutral form of the compound. Pharmaceutically acceptable acid addition salts include inorganic acid salts and organic acid salts. The inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, and hydrogen sulfate. The organic acids include acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, lactic acid, mandelic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. The compounds of this application contain both basic and acidic functional groups, and can therefore be converted into any acid or base addition salt.

[0083] The term enantiomeric ratio (er) used in this application refers to the ratio of the contents of a pair of enantiomers, and can also be expressed as er = R / S or S / R.

[0084] Through extensive and in-depth research, the inventors unexpectedly discovered that enzymes with specific structures can catalyze the preparation of 2-amino-3-benzoylpropionic acid compounds with high catalytic efficiency. This application was completed based on this discovery.

[0085] This application provides a method for preparing the compound of formula b or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0086] In a reaction system containing a cofactor, a biocatalyst is used to catalyze the substitution reaction between the compound shown in formula a and glycine to generate the compound shown in formula b.

[0087]

[0088] In formulas b and a: A is selected from C3-C8 saturated or partially unsaturated straight-chain or cyclic alkyl groups, 3-12 saturated or partially unsaturated heterocyclic groups, C6-C 10The group consisting of aryl and 5-12 heteroaryl groups; each Ra is independently selected from halogen, nitro, carboxyl, hydroxyl, hydroxymethyl, mercapto, thiomethyl, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, -NH-C1-C6 alkyl, mercapto-substituted C1-C6 alkyl, C2-C6 acyl, C3-C8 alicyclic hydrocarbon, 3-12 heterocyclic hydrocarbon, C6-C 10 The group consisting of aryl and 5-12 heteroaryl groups; each of the heterocyclic group and heteroaryl group independently comprises 1 to 4 ring atoms, wherein the ring atoms are selected from the group consisting of N, O and S; n is an integer from 0 to 5;

[0089] In formula a: X is bromine or iodine;

[0090] The biocatalyst contains threonine aldolase (LTA).

[0091] In this application, the substitution reaction is, for example, a bimolecular nucleophilic substitution reaction (SN2 substitution reaction).

[0092] In some implementations, n is 0, 1, 2, 3, 4, or 5. For example, in some implementations, n is 2.

[0093] In some embodiments, the compound of formula b may be any one or more of b1-b29 listed in Table 2.

[0094] The pharmaceutically acceptable salt can be obtained using conventional salt-forming methods in the art. For example, obtaining a pharmaceutically acceptable salt of the compound shown in formula b involves adding a suitable base, acid, or salt solution to b, followed by vacuum concentration to obtain the corresponding salt solution. Acid solutions include, but are not limited to, formic acid, acetic acid, hydrochloric acid, and nitric acid. Base solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonia. Salt solutions include, but are not limited to, hydrochloride, sulfate, nitrate, or phosphate.

[0095] The salt-forming sites in the compound shown in formula b include, but are not limited to, amino (-NH2) and carboxyl (-COOH) groups. Understandably, when the compound shown in formula b is mixed with an acid solution, the amino group in the compound reacts with the acid in the acid solution to form a salt; when the compound shown in formula b is mixed with a salt solution (e.g., a sodium salt solution), the carboxyl group in the compound shows a reaction with the salt ion to form a salt.

[0096] Exemplarily, a method for synthesizing 2-amino-3-benzoylpropionic acid compounds catalyzed by an enzyme is provided, which is achieved by the above method.

[0097] In some embodiments, the biocatalyst is selected from the group consisting of LTA, bacterial cells expressing LTA, lysates of the bacterial cells, lyophilized powders of the bacterial cells, and LTA immobilized in a solid form (such as embedding, loading, etc.) to a solid support.

[0098] In this application, the initial concentration of LTA in the reaction system can be between 0.05 mg / mL and 60 mg / mL. Exemplarily, it can be 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, or any range or value between two such values. In some embodiments, the initial concentration of LTA in the reaction system is between 0.1 mg / mL and 40 mg / mL. In some embodiments, the initial concentration of LTA in the reaction system is between 0.2 mg / mL and 20 mg / mL.

[0099] The sources of LTA in this application include, but are not limited to, natural organisms such as Pseudomonas, Escherichia coli, Candida, nematodes, and protozoa. In some embodiments, the LTA is a coding sequence derived from Pseudomonas, Leishmania, or other similar organisms.

[0100] The amino acid sequence of the LTA may include the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 or a variant thereof.

[0101] The variant, for example, is an amino acid sequence obtained by replacing, deleting, and / or inserting one or more amino acids based on the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, while maintaining enzyme activity.

[0102] It will be apparent to those skilled in the art that such substitution can occur in regions other than the aforementioned sites while retaining the corresponding activity. In some embodiments, the mutant following conserved substitution has at least one conserved amino acid substitution. Examples of conserved substitution are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine).

[0103] In some embodiments, the conservative substitutions are selected from the group consisting of substitutions G to A, A to G or S, V to I, L, A, T or S, I to V, L or M, L to I, M or V, M to L, I or V, P to A, S or N, F to Y, W or H, Y to F, W or H, W to Y, F or H, R to K, E or D, K to R, E or D, H to Q, N or S, D to N, E, K, R or Q, E to Q, D, K, R or N, S to T or A, T to S, V or A, C to S, T or A, N to D, Q, H or S, Q to E, N, H, K or R, and their opposite interchanges.

[0104] Proteins that have a certain degree of amino acid homology with the LTA as described above, such as homology between 70% and 99%, further homology between 80% and 99%, further homology between 90% and 99%, and homology of 99%, should also fall within the scope of protection of this application.

[0105] The "homology" (sequence identity percentage) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools can be used, such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., *Bioinformatics* (Cambridge, UK), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software can be used to perform alignments to determine the percentage of identity between amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters as needed for the alignment, for example by selecting a suitable algorithm.

[0106] As used in this application, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized below.

[0107] Amino acid name Three-letter code Single-letter code alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F proline Pro P Serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0108] The nucleotide sequence of the nucleic acid encoding the LTA may be:

[0109] 1) The nucleotide sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;

[0110] 2) A nucleotide sequence complementary to the nucleotide sequence defined in 1); or,

[0111] 3) Any polynucleotide sequence or complementary nucleotide sequence having at least 70% (optionally at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity with the nucleotide sequence defined in 1).

[0112] This application also provides a recombinant expression vector carrying the above-mentioned genes, into which the encoding gene of the LTA enzyme is introduced using molecular cloning technology. Heterologous protein expression can be performed using *E. coli* or yeast as the host, and the expression vectors include, but are not limited to, *E. coli* expression vectors such as pETDuet, pACYCDuet, pRSFDuet, pET28a, or pET22b.

[0113] In this application, a plasmid containing the threonine aldolase gene sequence is transformed into competent E. coli cells, and protein expression is induced by induction at 18°C ​​for 20 hours using 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). The purified LTA enzyme and bacterial cells exhibit catalytic activity at temperatures ranging from 20°C to 50°C, for example, from 25°C to 30°C.

[0114] The substitution reaction is catalytically active at pH 5.0–pH 10.0. In some embodiments, the pH is, for example, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or any range or value between two values.

[0115] The reaction time can be from 1 hour to 48 hours. For example, it can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or 48 hours, or any range or value between two such values. In some implementations, the reaction time is 2 hours to 24 hours.

[0116] In this application, the cofactor may include or be pyridoxal phosphate (PLP).

[0117] In some embodiments, the initial concentration of the cofactor in the reaction system is 0.001 mM to 10 mM. In some embodiments, the initial concentration is 0.02 mM to 0.5 mM. In some embodiments, the initial concentration is 0.02 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, or a range or value between any two values. For example, in some embodiments, the initial concentration is 0.05 mM to 0.2 mM. In other embodiments, the initial concentration is 0.1 mM to 1 mM.

[0118] The reaction system described in this application may also contain chemical additives.

[0119] For example, the chemical additive may include I - Ionic salt. In some embodiments, the I... - Ionic salts include one or more of LiI, NaI, KI, CsI, CaI2, AgI, CuI, CuI2, MgI2, and AlI3.

[0120] The amount of chemical additive used, such as its initial concentration in the reaction system, can be below 40 mM, for example, 0.01 mM to 40 mM, such as 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or any range or value between two values. In some embodiments, the initial concentration of the chemical additive (e.g., KI) is 1 mM to 10 mM. In other embodiments, the initial concentration of the chemical additive (e.g., KI) is 0.2 mM to 5 mM.

[0121] In the reaction system, the molar ratio of the compound represented by formula a (a 2-bromoacetophenone compound) to glycine, based on the amount of feed, can be 1:(1-10000), for example 1:1, 1:10, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or any range or value between two ratios. In some embodiments, the initial concentration of the compound represented by formula a in the reaction system is 0.01 mM-1000 mM, for example 20 mM.

[0122] The reaction system can use buffers including, but not limited to, the following: phosphate buffer, Tris-HCl buffer, ammonium acetate buffer, and HEPES-NaOH buffer.

[0123] In some embodiments, the buffer solution used in the reaction system contains 50 mM Na2HPO4 and 50 mM NaCl.

[0124] The reaction system may contain a co-solvent to enhance substrate solubility. The co-solvent may be an organic solvent such as dimethyl sulfoxide, methanol, ethanol, or isopropanol. In some embodiments, the co-solvent is dimethyl sulfoxide.

[0125] The initial amount of the co-solvent can be 5%-25% of the volume of the reaction system. Exemplarily, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or any range or value between two values.

[0126] In some embodiments, the method further includes isolating a class B compound or a pharmaceutically acceptable salt thereof from the reaction system.

[0127] In some embodiments, the reaction system comprises 20 mM 2-bromoacetophenone compounds, 0.2 M glycine (Gly), 0.1 mM PLP, 4 mg / mL LmLTA from Leishmania major, and 5 mM KI.

[0128] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0129] The following are some examples.

[0130] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0131] Main reagents and consumables

[0132] The gene sequences of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 were synthesized by GENEBIZ and cloned into the commonly used E. coli plasmid pET28a. The competent cells of the expression strain E. coli BL21(DE3) were purchased from E. coli Sangon Biotech.

[0133] The antibiotics (kanamycin), culture medium components Tryptone, Yeast extract, and NaCl, agar, and isopropyl β-D-thiogalactoside (IPTG), the protein expression inducer used in this application, were purchased from Sangon Biotech. Glycine was purchased from Bio-Pharmaceuticals, and the cofactor pyridoxal phosphate (PLP) was purchased from J&K Scientific Ltd.; the deuteration reagent was purchased from J&K Scientific Ltd. NiNTA and Unionrose 6FF used for protein purification were purchased from Union Biotech.

[0134] Example 1: Gene Acquisition, Expression, and Purification

[0135] The amino acid sequences of LmLTA from Leishmania major, PpLTA-2 from Pseudomonas putida NBRC 14164, and PhLTA from Porcisia hertigi were obtained from the PDB database (as shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5). After codon optimization and gene sequence synthesis (nucleic acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively), the gene was cloned into the vector pET28a to obtain the recombinant plasmid. See the diagram below. Figure 1The recombinant plasmid was then transformed into competent E. coli BL21(DE3) cells and cultured at 37°C for 12 h. A single colony was picked using a sterile inoculation loop in a laminar flow hood and inoculated into 100 ml of liquid LB medium containing the corresponding antibiotic (50 μg / ml kanamycin or 100 μg / ml ampicillin). After overnight culture at 37°C and 220 rpm, the colony was transferred at a 1% (v / v) inoculation rate to 1 L of liquid LB medium containing the corresponding antibiotic and cultured at 37°C and 220 rpm until OD200. 600 When the concentration was 0.8–1.0, the temperature was lowered to 18°C ​​and 180 rpm, and IPTG was added to a final concentration of 0.1 mM to induce expression for 20 h. The cells were then collected by centrifugation at 9000 rpm for 15 minutes.

[0136] The collected bacterial cells were resuspended in cell lysis buffer and homogenized using a high-pressure homogenizer to release the target protein. The supernatant was collected by centrifugation at 4°C and 15,000 rpm for 30 min. The protein was purified using NiNTA Unionrose 6FF, and the loading was repeated 4-5 times. Impurities were washed away with lysis buffer containing 20 mM imidazole, and the target protein was eluted with lysis buffer containing 250 mM imidazole. The target protein was obtained after dialysis.

[0137] Cell lysis buffer used for LTA: 50mM Na2HPO4, 300mM NaCl, pH 8.0; Dialysis buffer: 50mM Na2HPO4, 50mM NaCl, pH 7.4.

[0138] Example 2: Synthesis of 2-amino-3-benzoylpropionic acid compounds catalyzed by threonine aldolase

[0139] Based on the amount of the target compound required for subsequent experiments, the reaction volume was determined to be 10 mL. Through a review of relevant literature, the reaction system was determined as follows:

[0140] Table 1. Reaction system of threonine aldolase (LTA)

[0141]

[0142] The reaction buffer contained 50 mM Na2HPO4 and 50 mM NaCl, pH 7.4; the reaction was carried out at room temperature on a magnetic stirrer for 2-6 hours.

[0143] Example 3: Purification, yield calculation, and characterization of the target product

[0144] (1) Centrifuge the reaction solution after the reaction is complete and collect the supernatant. Use a rapid preparative liquid chromatography column. The product was purified by gradient elution using a methanol / water system (both methanol and aqueous phases containing 0.1% formic acid). The fraction containing the product was collected, and the solvent was removed by rotary evaporation. The product was weighed, and the yield was calculated. The yield calculation formula is: Product yield = m / M × 100%, where m is the actual mass of the obtained product, and M is the theoretical mass of the product.

[0145] (2) Take a small amount of the purified product, add about 600 μL of deuterated reagent, centrifuge, collect the supernatant, and collect the compound-like substances. 1 H NMR, 13 C NMR and fluorine-containing compounds 19 F NMR spectra were used, and the structures of similar compounds were identified by combining high-resolution mass spectrometry and other methods. Compound yield and enantiomeric ratio (er) were as follows: Figure 2 As shown.

[0146] The NMR and high-resolution mass spectra of the 29 compounds obtained above are summarized in Table 2:

[0147] Table 2 NMR and HRMS (ESI)

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Example 4: Development of whole-cell catalysts for gram-scale preparation

[0155] According to the method described in Example 1, wet bacterial cells were collected, and water was removed using a freeze dryer to obtain lyophilized powder. A phosphate buffer system was used, with 9.76 g / L of 3'-nitro-2-bromoacetophenone added, and the lyophilized powder added at a rate of 20 mg / mL. The stirring speed was 550–600 rpm, and the reaction was carried out at room temperature for 24 h. The reaction solution was centrifuged, and the supernatant was collected. The product was purified by gradient elution using an ODS column with a methanol / water (both methanol and aqueous phases contain 0.1% formic acid) system. The fraction containing the product was collected, and the solvent was removed by rotary evaporation to obtain the product. The yield was calculated after weighing the product (same as in Example 3), with a yield of 93.5% and a production rate of 8.9 g / L.

[0156] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0157] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing the compound of formula b or a pharmaceutically acceptable salt thereof, characterized in that, The process includes the following steps: in a reaction system containing a cofactor, a biocatalyst is used to catalyze a substitution reaction between a compound of formula a and glycine to generate a compound of formula b. In formulas b and a: A is selected from C3-C8 saturated or partially unsaturated straight-chain or cyclic alkyl groups, 3-12 saturated or partially unsaturated heterocyclic groups, C6-C 10 The group consisting of aryl and 5-12 heteroaryl groups; each Ra is independently selected from halogen, nitro, carboxyl, hydroxyl, hydroxymethyl, mercapto, thiomethyl, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, -NH-C1-C6 alkyl, mercapto-substituted C1-C6 alkyl, C2-C6 acyl, C3-C8 alicyclic hydrocarbon, 3-12 heterocyclic hydrocarbon, C6-C 10 The group consisting of aryl and 5-12 heteroaryl groups; each of the heterocyclic group and heteroaryl group independently comprises 1 to 4 ring atoms, wherein the ring atoms are selected from the group consisting of N, O and S; n is an integer from 0 to 5; In formula a: X is bromine or iodine; The biocatalyst contains threonine aldolase (LTA).

2. The method as described in claim 1, characterized in that, The biocatalyst is selected from the group consisting of LTA, bacterial cells expressing LTA, lysate of the bacterial cells, lyophilized powder of the bacterial cells, and LTA in a solidified form. Optionally, the bacterial cells are selected from a group consisting of Escherichia coli and yeast.

3. The method as described in claim 1 or 2, characterized in that, The auxiliary factor satisfies one or more of the following conditions (A1)-A2): The cofactors mentioned in A1) include pyridoxal phosphate (PLP); and, A2) Based on the amount of feed, the initial concentration of the cofactor in the reaction system is 0.001 mM-10 mM; optionally, the initial concentration is 0.02 mM-0.5 mM; further optionally, the initial concentration is 0.05 mM-0.2 mM.

4. The method according to any one of claims 1-3, characterized in that, The reaction system also contains chemical additives; Optionally, the chemical additive satisfies one or more of the conditions shown in B1)-B2) below: The chemical additives mentioned in B1) include I - Ionic salts, optionally including one or more of LiI, NaI, KI, CsI, CaI2, AgI, CuI, CuI2, MgI2, and AlI3; and, The initial concentration of the chemical additive described in B2) in the reaction system is ≤40mM; optionally 0.01mM-40mM; further optionally 1mM-10mM.

5. The method according to any one of claims 1-4, characterized in that, The initial concentration of LTA in the reaction system is 0.05 mg / mL to 60 mg / mL; Optionally, the concentration can be 0.1 mg / mL to 40 mg / mL; Further, optionally, the concentration is 0.2 mg / mL to 20 mg / mL.

6. The method according to any one of claims 1-5, characterized in that, The LTA satisfies any one of the following conditions C1)-C3): C1) is derived from a group composed of Pseudomonas, Escherichia coli, Candida, Nematodes and Leishmania. C2) The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; and, C3) An amino acid sequence obtained by replacing, deleting and / or inserting one or more amino acids based on the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, while maintaining enzyme activity.

7. The method as described in claim 6, characterized in that, The nucleic acid encoding the LTA satisfies any one of the following conditions (D1)-D3): D1) The nucleotide sequence is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; A nucleotide sequence complementary to the nucleotide sequence defined by D2) and D1); Any polynucleotide sequence or complementary nucleotide sequence that has at least 70% sequence identity with the nucleotide sequence defined by D3) and D1).

8. The method according to any one of claims 1-7, characterized in that, The reaction system described below satisfies one or more of the conditions shown in E1)-E2): E1) Based on the amount of feed, the molar ratio of the compound shown in formula a to the glycine is 1:(1-10000); The initial concentration of the compound shown in formula a in E2) is 0.01 mM-1000 mM in the reaction system.

9. The method according to any one of claims 1-8, characterized in that, The substitution reaction satisfies one or more of the conditions shown in F1)-F2) below: F1) The test is performed at 10℃-50℃; alternatively, at 15℃-40℃; further alternatively, at 20℃-30℃. The reaction time for F2 is 1 h to 48 h; alternatively, the reaction time for LTA is 2 h to 24 h.

10. The method according to any one of claims 1-9, characterized in that, The reaction system described satisfies one or more of the following G1)-G3): G1) also includes a buffer solution, wherein the buffer solution is selected from the group consisting of phosphate buffer, Tris-HCl buffer, ammonium acetate buffer and HEPES-NaOH buffer; G2) also includes cosolvents selected from the group consisting of dimethyl sulfoxide, methanol, ethanol and isopropanol; Optionally, the initial amount of the co-solvent is 5%-25% of the volume of the reaction system; and, G3) pH value is 5-10.

Images