Method for preparing apremilast intermediate through biological catalysis

Abstract

The invention provides a method for preparing an apremilast intermediate through biological catalysis. According to the method, a novel nitroreductase mutant is used for catalytically reducing 3-nitrophthalic acid to prepare the apremilast intermediate 3-aminphthalic acid, and the method is easy and convenient to operate, mild in reaction condition, high in conversion rate, free of participation of an organic solvent, more environmentally friendly, low in cost and extremely high in industrial application value.

Description

Technical Field

[0001] The invention relates to the field of biocatalysis, and in particular to a method for preparing an Apremilast intermediate by biocatalysis. Background Art

[0002] Biosynthesis refers to a synthesis method that uses biological catalysts (such as yeast, bacteria, fungi, etc.) or biotransformation reactions to convert substrates into desired compounds. It uses the results of natural metabolic activities of organisms, does not require high temperature, high pressure and other conditions, and does not produce toxic and harmful waste and pollution. It is a green and environmentally friendly chemical synthesis method. Nitroreductase is a class of cytoplasmic enzymes that depend on FMN or FAD, and can use NAD(P)H to achieve the reduction metabolism of nitroaromatic compounds and nitroheterocyclic derivatives. Nitroreductase has a wide range of applications, including drug activation, biochemical synthesis, and pollutant biometabolism. However, the current catalytic efficiency of nitroreductase is low, which seriously limits its application. In addition, the modification of nitroreductase faces the challenge of unclear substrates and catalytic mechanisms, which limits the progress of rational modification of the enzyme and further limits its wide application in various fields.

[0003] Apremilast is an oral drug for the treatment of active psoriatic arthritis. It is an oral, selective phosphodiesterase 4 (PDE4) inhibitor for the treatment of plaque psoriasis. 3-Aminophthalic acid is a key intermediate of Apremilast. The synthesis of 3-aminophthalic acid is mainly through catalyzing the nitro group in 3-nitrophthalic acid to amino group.

[0004] In traditional chemical synthesis processes, catalytic reduction of nitro functional groups to amino groups is a common process step, which can often be achieved through a variety of methods such as metal reduction, selective catalytic hydrogenation, catalytic hydrogenation and electrochemical reduction. However, in the industrial production process, there are often problems such as expensive catalysts, flammability and explosion, high pollution, many by-products and demanding equipment requirements.

[0005] Nitroreductase can catalyze the conversion of nitro groups to corresponding hydroxylamine or amino groups under mild reaction conditions using the coenzyme NAD(P)H as an electron donor. The biosynthesis of 3-aminophthalic acid uses the coenzyme cycle NADPH-GDH as a hydrogen donor, and the nitro group of the substrate 3-nitrophthalic acid is reduced to an amino group by catalysis of nitroreductase. However, the existing nitroreductase has low catalytic activity, which limits the biosynthesis of 3-aminophthalic acid. Therefore, it is urgent to develop a method for preparing 3-aminophthalic acid with high yield and high conversion rate. Summary of the invention

[0006] The present invention is based on the inventor's discovery of the following facts and problems:

[0007] Most reactions for reducing nitro compounds to amino compounds involve the use of metal catalysts, hydrogenation reduction and other processes, which have strict equipment requirements, high process costs (metal catalysts are expensive), and require the participation of organic solvents. As an excellent catalytic reaction alternative, nitroreductase has the advantages of mild reaction conditions (catalytic reaction at room temperature and pressure), simpler process, low cost and environmental friendliness (nitroreductase-catalyzed reaction does not require the participation of organic solvents, reducing the use of organic solvents and thus reducing the impact on the environment). However, the relatively low catalytic efficiency of nitroreductase is the main factor limiting its widespread application.

[0008] For example, patent application CN115074263 A discloses nitroreductase BtNRT and recombinant genetic engineering bacteria based on the enzyme, which can be used to reduce 3-nitrophthalic acid, but the enzyme has the disadvantages of low activity under high substrate concentration conditions and is not suitable for industrial production. Further research found that under the condition of 100 g / L of substrate 3-nitrophthalic acid, the substrate conversion rate in the reaction system mediated by BtNRT was only 21.7%, which is not suitable for industrial scale-up production.

[0009] The present invention aims to solve one of the technical problems in the related art to a certain extent.

[0010] The present invention provides a method for preparing a nitro-reduction product. According to an embodiment of the present invention, the method comprises: subjecting a nitro-reduction enzyme substrate to a catalytic treatment in the presence of the nitro-reduction enzyme mutant or the recombinant cell or the recombinant strain of the present invention to obtain a nitro-reduction product. The method has a better application prospect in the preparation of nitro-reduction products, and can significantly reduce reaction costs and reduce environmental pollution.

[0011] According to an embodiment of the present invention, the method for preparing a nitro-reduction product further comprises at least one of the following technical features:

[0012] According to an embodiment of the present invention, the nitroreductase substrate includes 3-nitrophthalic acid or its stereoisomers, tautomers or salts.

[0013] According to an embodiment of the present invention, the nitro reduction product is 3-aminophthalic acid.

[0014] According to an embodiment of the present invention, the present invention proposes a method for biocatalytically preparing 3-aminophthalic acid, an intermediate of Apremilast, using a nitroreductase mutant or a recombinant cell carrying the mutant or a recombinant strain expressing the mutant. The method is simple to operate, has mild reaction conditions, a high conversion rate, and does not require the participation of an organic solvent, is more environmentally friendly, has low cost, and has extremely high industrial application value.

[0015] Specifically, the present invention proposes a method for preparing an Apremilast intermediate, comprising reducing 3-nitrophthalic acid using a nitroreductase mutant or a recombinant cell carrying the mutant or a recombinant strain expressing the mutant to prepare 3-aminophthalic acid. According to an embodiment of the present invention, the mutant comprises: compared with the wild-type nitroreductase, at least one amino acid mutation in the following seven sites or functionally equivalent sites: the 40th, 103rd, 201st, 231st, 256th, 258th, 267th. Optionally, the wild-type nitroreductase of the present invention is a wild-type nitroreductase AcNTR.

[0016] According to the embodiments of the present invention, the nitroreductase AcNTR mutant has higher catalytic activity and stability. In industrial applications, the catalytic efficiency can be significantly improved, production costs can be saved, and the preparation method has the characteristics of high conversion rate, high yield, simple operation, low cost and environmental friendliness.

[0017] It should be noted that the equivalent functional sites refer to the amino acid sites corresponding to the same functional domain in a nitroreductase. For example, a certain nitroreductase has two functional domains A and B with the same functions, and the sites in the B domain and the corresponding sites in the A domain are functionally equivalent sites to each other.

[0018] It should be noted that the amino acid positions in the amino acid sequence of the nitroreductase mutant of the present invention are located with reference to the amino acid positions in the amino acid sequence of the wild-type nitroreductase AcNTR.

[0019] According to an embodiment of the present invention, the above-mentioned nitroreductase mutant further comprises at least one of the following technical features:

[0020] According to an embodiment of the present invention, the wild-type nitroreductase has an amino acid sequence as shown in SEQ ID NO: 1;

[0021] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIQSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAG FDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALKGRHLLKQALEKLGFALK(SEQ ID NO:1).

[0022] Wherein, the nucleic acid encoding the wild-type nitroreductase has a nucleotide sequence as shown in SEQ ID NO: 2;

[0023] ATGATGAGCGATACTCTGATTGCCGAAAAGCCCGTCGTGAGCGCGGCTGAAGCCGCGCTGGAGCGGCGTTACCGCGAGCCGCTGGCCGGCGCCGACCACCCCTGGAACGCTGTCATCCAGAGCCTCGTGGACCACCGCACCGTGCGGGCCTTCCTGCCGAAGGCGGTGAGCGAGGAGACGCTGCAGGTGCTGGTGGCCGCCGCCCAATCGGCGCCCACCTCGTCCAATGTGCAGGCCTGGAGCGTGGTGGCGGTGCGCGATCCTGCCCGCAAGGCGCGCCTCGCCCACCTCGCCGGCGACCAGAAGCACATTCTGGAGGCGCCGCTGCTGCTCGTGTGGATCGCCGATCTCTCCCGCGCCGCCGGCATCGGGGAACGCGCGGGCCTGACGCTGGAAGGGCTCGATTTCACGGAGAGCTTCCTCATCGCCGGCTTCGATGCGGCCCTCGCCGCCCAGAATGCGCTGGTGGCGGCCGAATCCCTGGGGCTCGGTACCTGCTATATCGGCGCCTTGCGCAACCACCCCATCGAGGTCGCGGAAACGCTGGGCCTGCCGCGGAAGACCTTCGCCGTATTCGGCCTCGTGGTGGGCCATCCCGATCCCGCGCGCCCGGCGGACGTGAAACCGCGCCTGCCGCAGGCGGCGGTGCTGCATCACGAGACCTACGATCTTGCCGGGCAGGCGGAGGCGGTGGCGCGCCATGATGCCCATAGCCTGGCCTTCCGCGCCGAGCAGAAGCTCGACGCCACCCCCTGGAGCCAGCAACTGATCGGCCGCCTGCATACGGTGGCAGCCCTCAAGGGGCGTCATCTCCTCAAGCAGGCGTTGGAGAAACTCGGCTTTGCGCTGAAATGA(SEQ ID NO:2)。

[0024] According to an embodiment of the present invention, the nitroreductase mutant has at least 80% identity with the wild-type nitroreductase.

[0025] According to an embodiment of the present invention, the nitroreductase mutant has at least 90% or more identity with the wild-type nitroreductase.

[0026] According to an embodiment of the present invention, the nitroreductase mutant has at least 95% or more identity with the wild-type nitroreductase.

[0027] According to an embodiment of the present invention, the nitroreductase mutant has at least 98% or more identity with the wild-type nitroreductase. Generally speaking, the higher the identity of the amino acid sequence, the more similar its structure and function are.

[0028] According to an embodiment of the present invention, the mutant has at least one of the following mutations:

[0029] (1) The amino acid Q at position 40 mutated to R;

[0030] (2) amino acid H at position 103 mutated to Y;

[0031] (3) amino acid P at position 201 mutated to S;

[0032] (4) amino acid V at position 231 mutated to A;

[0033] (5) amino acid L at position 256 mutated to V;

[0034] (6) amino acid G at position 258 mutated to D;

[0035] (7) The amino acid K at position 267 mutated to R.

[0036] According to some specific embodiments of the present invention, different mutation sites produce different catalytic activities of nitroreductases, which can provide more options for actual production needs.

[0037] According to an embodiment of the present invention, the mutant has any one (or combination) of the following mutations (1)-(8):

[0038] (1) The amino acid H at position 103 is mutated to Y; or

[0039] (2) The amino acid P at position 201 is mutated to S; or

[0040] (3) the amino acid V at position 231 is mutated to A; or

[0041] (4) the amino acid Q at position 40 is mutated to R, and the amino acid K at position 267 is mutated to R; or

[0042] (5) the amino acid at position 256 is mutated from L to V, and the amino acid at position 258 is mutated from G to D; or

[0043] (6) the amino acid Q at position 40 is mutated to R, the amino acid K at position 267 is mutated to R, and the amino acid H at position 103 is mutated to Y; or

[0044] (7) the amino acid Q at position 40 mutates to R, the amino acid K at position 267 mutates to R, and the amino acid P at position 201 mutates to S; or

[0045] (8) The amino acid at position 40, Q, mutated to R, the amino acid at position 267, K, mutated to R, and the amino acid at position 231, V, mutated to A.

[0046] According to some specific embodiments of the present invention, when the amino acid sequence of the nitroreductase mutant has the above mutation, the catalytic activity is stronger, the effect of the corresponding preparation method is better, and its value and prospects in industrial applications are significantly improved. In addition, the above mutation sites can also be selectively combined to obtain a nitroreductase mutant suitable for the required catalytic activity, thereby obtaining a preparation method that meets the requirements.

[0047] It should be noted that the above mutant amino acid sequences are all obtained by mutation based on the sequence of SEQ ID NO:1.

[0048] According to an embodiment of the present invention, the mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10;

[0049] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIQSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKYILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAG FDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALKGRHLLKQALEKLGFALK(SEQ ID NO:3);

[0050] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIQSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDSARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALKGRHLLKQALEKLGFALK(SEQ ID NO:4);

[0051] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIQSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAAARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALKGRHLLKQALEKLGFALK(SEQ ID NO:5);

[0052] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIRSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALRGRHLLKQALEKLGFALK(SEQ ID NO:6);

[0053] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIQSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQVIDRLHTVAALKGRHLLKQALEKLGFALK(SEQ ID NO:7);

[0054] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIRSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKYILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALRGRHLLKQALEKLGFALK(SEQ ID NO:8);

[0055] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIRSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAGFDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDSARPADVKPRLPQAAVLHHETYDLAGQAEAVARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALRGRHLLKQALEKLGFALK(SEQ ID NO:9);

[0056] MMSDTLIAEKPVVSAAEAALERRYREPLAGADHPWNAVIRSLVDHRTVRAFLPKAVSEETLQVLVAAAQSAPTSSNVQAWSVVAVRDPARKARLAHLAGDQKHILEAPLLLVWIADLSRAAGIGERAGLTLEGLDFTESFLIAG FDAALAAQNALVAAESLGLGTCYIGALRNHPIEVAETLGLPRKTFAVFGLVVGHPDPARPADVKPRLPQAAVLHHETYDLAGQAEAAARHDAHSLAFRAEQKLDATPWSQQLIGRLHTVAALRGRHLLKQALEKLGFALK(SEQ ID NO:10).

[0057] According to an embodiment of the present invention, the recombinant cell carries a nucleic acid molecule or an expression vector, and is used to express or secrete the nitroreductase mutant of the present invention.

[0058] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following technical features:

[0059] According to an embodiment of the present invention, the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

[0060] According to an embodiment of the present invention, the nucleic acid molecule encodes the nitroreductase mutant of the present invention. The nitroreductase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.

[0061] According to an embodiment of the present invention, the expression vector comprises the nucleic acid molecule of the present invention. According to an embodiment of the present invention, the expression vector can express a nitroreductase mutant in vitro.

[0062] According to an embodiment of the present invention, the above expression vector may further include at least one of the following technical features:

[0063] According to an embodiment of the present invention, the expression vector is selected from at least one of pET series vectors, shuttle vectors, phage and virus vectors.

[0064] According to an embodiment of the present invention, the pET series vectors include at least one of pET-3a, pET-11a, pET-15a, pET-28a, pET-30a, pET-32a and pET-22b.

[0065] According to an embodiment of the present invention, the pET series vector is pET-28a. According to a specific embodiment of the present invention, pET-28a is used as an expression vector, and the relative activity and volume enzyme activity of the nitroreductase mutant are high.

[0066] According to an embodiment of the present invention, the recombinant strain expresses the nitroreductase mutant of the present invention. The nitroreductase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.

[0067] According to an embodiment of the present invention, the preparation method further comprises an oxidoreductase system.

[0068] According to an embodiment of the present invention, the oxidoreductase system includes glucose dehydrogenase, glucose and coenzyme NADP, or lactate dehydrogenase, lactate and coenzyme NADP.

[0069] According to an embodiment of the present invention, the reaction of reducing 3-nitrophthalic acid is carried out in a phosphate buffer.

[0070] According to an embodiment of the present invention, the pH of the phosphate buffer is 6.5-10.

[0071] According to an embodiment of the present invention, the pH of the phosphate buffer is 8. Experimental verification has found that when the pH of the phosphate buffer is 8, the nitroreductase mutant has a high catalytic efficiency.

[0072] According to an embodiment of the present invention, the reduction reaction of 3-nitrophthalic acid is carried out at 15-50°C.

[0073] According to an embodiment of the present invention, the preparation method includes that the reduction of 3-nitrophthalic acid is carried out in the presence of: phosphate buffer, a glucose dehydrogenase coenzyme circulation system (including glucose dehydrogenase, glucose and coenzyme) or a lactate dehydrogenase coenzyme circulation system (including lactate dehydrogenase, lactic acid and coenzyme) in which NAD(P) is a coenzyme.

[0074] According to an embodiment of the present invention, the preparation method includes: a glucose dehydrogenase coenzyme circulation system (including glucose dehydrogenase, glucose and coenzyme) or a lactate dehydrogenase coenzyme circulation system (including lactate dehydrogenase, lactic acid and coenzyme) with NAD (P) as a coenzyme, 3-nitrophthalic acid as a substrate, a buffer solution with a pH of 6.5-10 as a reaction medium, reacting at 15-50°C, and after the reaction is completed, separating and purifying the reaction solution to obtain 3-aminophthalic acid.

[0075] According to an embodiment of the present invention, the preparation method also includes a method for preparing a nitroreductase mutant, comprising: culturing the recombinant cell or recombinant strain of the present invention under conditions suitable for protein expression to obtain the nitroreductase mutant. According to a specific embodiment of the present invention, a large number of nitroreductase mutants with high catalytic activity can be cultured and obtained by this method.

[0076] According to an embodiment of the present invention, the preparation method also includes a method for preparing a nitroreductase mutant, which comprises: performing mutation treatment on a wild-type nitroreductase to obtain a nitroreductase mutant library; performing a first catalytic treatment on a nitroreductase substrate in the presence of the nitroreductase mutant library; and performing a screening treatment on the first catalytic treatment product to obtain the nitroreductase mutant of the present invention. According to a specific embodiment of the present invention, a nitroreductase mutant with high catalytic activity can be obtained by this method.

[0077] According to an embodiment of the present invention, the method for preparing a nitroreductase mutant may further include at least one of the following technical features:

[0078] According to an embodiment of the present invention, the mutation treatment is selected from at least one of random mutation and site-directed mutation. According to a specific embodiment of the present invention, the mutation treatment includes but is not limited to methods such as using chemical mutagens or PCR error introduction.

[0079] According to an embodiment of the present invention, the nitroreductase substrate includes 3-nitrophthalic acid or its stereoisomers, tautomers or salts.

[0080] According to an embodiment of the present invention, the nitroreductase substrate is 3-nitrophthalic acid.

[0081] According to an embodiment of the present invention, the first catalytic treatment is carried out in the presence of NADPH.

[0082] According to an embodiment of the present invention, the screening process includes obtaining a nitroreductase mutant having an absorbance value higher than that of a control group (eg, 2 times or more).

[0083] According to an embodiment of the present invention, the control group is wild-type nitroreductase.

[0084] The nitroreductase mutant used in the preparation method of the present invention has at least the characteristics of high catalytic activity, high conversion rate, high catalytic rate and high stability; further, the nitroreductase mutant still has a high conversion rate under high substrate concentration conditions. In other words, the preparation method of the present invention has the characteristics of high conversion rate, high yield, simple operation and low cost, and is particularly suitable for preparing 3-aminophthalic acid under high concentration conditions. For example, compared with the method for preparing 3-aminophthalic acid using wild-type nitroreductase AcNTR and known nitroreductase BtNRT, the preparation method of the present invention has higher catalytic activity and higher catalytic rate, especially under high substrate concentration reaction conditions, faster reaction and higher conversion rate.

[0085] The preparation method of the present invention has mild reaction conditions (such as room temperature), can directly catalyze the conversion of the substrate 3-nitrophthalic acid into 3-aminophthalic acid, does not require the participation of an organic solvent, is more environmentally friendly, is easier to operate, has a high conversion rate / yield, is low in cost, and has extremely high industrial application value.

[0086] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0088] Figure 1 is the electrophoresis result of the AcNTR error-prone PCR mutation product according to Example 1 of the present invention; wherein M is a DNA molecular weight marker, DL2000;

[0089] Figure 2 It is the electrophoresis result of the pet28a plasmid fragment after double restriction digestion and the AcNTR error-prone PCR mutant product after double restriction digestion according to Example 1 of the present invention; wherein M is a DNA molecular weight marker, DL5000. DETAILED DESCRIPTION

[0090] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0091] definition

[0092] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0093] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0094] In the present invention, unless otherwise specified, the term "amino acid" is represented by a single-letter or three-letter code and has the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0095] In the present invention, unless otherwise specified, the term "nitroreductase" refers to an enzyme that can catalyze the reduction reaction of nitroaromatic compounds to aromatic amines.

[0096] In the present invention, "homology" has the conventional meaning in the art, and refers to the "identity" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared after the best alignment, and the differences between the two sequences are randomly distributed over their entire length. In the present invention, the mutants are described according to their mutations at specific residues, and their positions are determined by comparison with the wild-type enzyme sequence SEQ ID NO:1 or the reference enzyme sequence SEQ ID NO:1.

[0097] In the present invention, unless otherwise indicated, the terms "mutant" and "variant" can be used interchangeably, "substitution" or "mutation" can be used interchangeably, and these expressions refer to amino acids relative to wild-type proteins, such as the wild-type sequence SEQ ID NO: 1 nitroreductase AcNTR, or derived from nitroreductase AcNTR, including changes at one or more positions, i.e., substitutions, insertions and / or deletions, and still retaining their activity. Mutants can be obtained by various techniques known in the art, such as products of chemical synthesis, or produced from prokaryotic or eukaryotic hosts using recombinant technology. In particular, exemplary techniques for modifying DNA sequences encoding wild-type proteins for recombinant technology include, but are not limited to, directed mutagenesis, random mutagenesis, and the construction of synthetic oligonucleotides.

[0098] In the present invention, unless otherwise indicated, the term "substitution" with respect to an amino acid position or residue means that the amino acid at a particular position has been replaced by another amino acid. The substitution may be conservative or non-conservative.

[0099] In the present invention, unless otherwise specified, the form of "XaY" is used to represent the mutation or substitution of an amino acid, wherein a represents the position of the amino acid in SEQ ID NO: 1, X represents the wild-type amino acid species at position a in SEQ ID NO: 1, and Y represents the mutated amino acid species at position a in SEQ ID NO: 1. For example, "T15K" means that, compared with SEQ ID NO: 1, the threonine T at position 15 of SEQ ID NO: 1 is replaced by lysine K.

[0100] In the present invention, unless otherwise specified, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of foreign DNA into cells.

[0101] In the present invention, unless otherwise specified, the term "relative activity" refers to the enzyme activity of each nitroreductase relative to the pET28a-AcNTR enzyme calculated when the enzyme activity of pET28a-AcNTR is 100%.

[0102] In the present invention, unless otherwise specified, the term "volumetric enzyme activity" refers to the rate of enzyme-catalyzed substrate conversion per unit time under certain conditions.

[0103] In the present invention, unless otherwise specified, the term "stereoisomer" refers to compounds with the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0104] In the present invention, unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (protontautomers) (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0105] In the present invention, unless otherwise specified, the term "salt" refers to organic salts and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.

[0106] The following is a detailed description of the embodiments. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0107] Example 1: Construction of a nitroreductase random mutation library

[0108] 1) Using the recombinant plasmid pET28a-AcNTR as a template (nitroreductase AcNTR is derived from Azorhizobiumcaulinodans ORS571, the enzyme gene size is 854bp, encoding 284 amino acids, and the accession number in NCBI is: BAF89174), a mutation library was constructed using error-prone PCR technology; wherein, error-prone PCR mutation primers are as follows:

[0109] F:CATGCCATGGATGATGAGCGATACTCTGATTGCCGAAAA(5'→3');

[0110] R:CCGCTCGAGTCATTTCAGCGCAAAGCCGAGTTTCTCCAA(5'→3');

[0111] 2) Amplifying the mutant gene fragment using the above primers, the amplification system is shown in Table 1;

[0112] Table 1:

[0113]

[0114] The amplification procedure is shown in Table 2 ;

[0115] Table 2:

[0116]

[0117] The amplification results are as follows: Figure 1 As shown, the AcNTR error-prone PCR mutant gene fragment was recovered by gel excision. The recovered fragment and plasmid pet28a were both subjected to double enzyme digestion, and the double enzyme digestion system is shown in Table 3;

[0118] Table 3:

[0119]

[0120]

[0121] 3) After mixing, place at 37°C for 30 minutes, and then cut and recover the gel ( Figure 2 );

[0122] 4) The recovered target band and pet28a vector fragment were connected using T4 DNA ligase, and the connection product was directly mixed with BL21 (DE3) electroporation competent cells for electroporation transformation. Use a gun to gently blow and mix the competent cell-DNA, then quickly transfer it to the electroporation cup to avoid bubbles and cover the cup. Start the Bio-rad electroporator, adjust to fixed parameters, quickly put the electroporation cup into the electroporation tank, and quickly insert it into ice after the electroporation is completed. After 2 minutes, take out the electroporation cup from the ice, put it at room temperature, add 700μL of antibiotic-free LB culture medium (room temperature), blow and suck the bottom of the electroporation cup several times with a gun to mix, and then transfer it to a 1.5mL EP tube. Resuscitate and culture at 37℃, 220rpm for 1h. Centrifuge at 5000rpm for 1min, resuspend and take 200μL and add it to the LB plate containing kanamycin, and gently spread the cells evenly with sterile glass beads. Incubate at room temperature for 10 min. After the liquid is absorbed, invert the plate and incubate overnight at 37°C for 13 to 17 h.

[0123] Example 2: Cultivation and initial screening of positive mutants

[0124] The error-prone mutants of AcNTR grown on the plate were picked up and cultured in a sterile 96-well plate (about 1200 strains were picked up), each well contained 300 μL of LB medium, and cultured overnight at 30°C. The next day, 150 μL of bacterial solution was transferred to the corresponding new 96-well plate (each well contained 850 μL of LB medium, and the LB medium contained kanamycin resistance + IPTG), mixed and placed at 30°C for culture. The remaining 150 μL of bacterial solution in each well was mixed with 100 μL of 50% glycerol and stored in a -20°C refrigerator. After the bacterial cells were cultured for 24 hours, the bacterial cells in the 96-well plate were collected by centrifugation at 3500 rpm for 20 minutes, and the supernatant was removed. Pretreatment of bacterial cell samples: Prepare a sodium phosphate buffer (0.05M) with a pH of 7.5, then add 5U / mL DNase I and 1mg / mL lysozyme to 500mL of the buffer, mix well, dispense 400μL into each well of a 96-well plate, mix well by pipetting, and incubate at 30℃, 800rpm for 1h. Centrifuge at 3500rpm for 15min, collect the supernatant, and store at room temperature.

[0125] The constructed high-throughput screening method was then used to obtain positive mutant strains:

[0126] (a) Substrate treatment: Weigh 0.12 g of 3-nitrophthalic acid sample and dissolve it in 300 mL of PBS buffer (0.05 mM, pH = 7.5) to prepare a sample with a concentration of 0.4 g / L. Ultrasonic treatment for 15 min, followed by filtration using a 0.22 μm membrane, and the sample solution was used for high-throughput screening in a 96-well plate;

[0127] (b) Coenzyme NADPH treatment: Prepared into a 50 mM solution;

[0128] (c) Microplate reader assay process and system: 158 μL of 3-nitrophthalic acid solution + 50 μL of bacterial lysis supernatant + 2.5 μL of NADPH, mix well and place in a microplate reader, read the absorbance values ​​at 340 nm at 0 min, 5 min, 10 min and 15 min. By comparing the changes in absorbance values, the enzyme activity is determined (high activity consumes more NADPH), and then the clones with absorbance changes much higher than the control strain (more than twice that of the wild-type strain) are screened.

[0129] Example 3: Shake flask culture of positive mutants and enzyme activity determination

[0130] The positive mutant strains obtained in the initial screening were removed from the 96-well plate and transferred to 10 mL of LB medium, and cultured overnight at 30°C and 220 rpm. The next morning, the inoculation volume was transferred to 60 mL of fermentation medium containing kanamycin resistance (10 g / L yeast powder, 20 g / L peptone, 15 g / L glycerol, 10 g / L NaCl, 3 g / L KNO3, 1.74 g / L K2HPO4, 1.36 g / L KH2PO4, and 0.5 g / L MgCl2), and cultured at 37°C and 220 rpm for 2 h;

[0131] IPTG (final concentration of 0.2 mM) was then added and cultured at 22°C, 220 rpm for 22 h to induce protein expression. After fermentation, the bacterial cells were collected by centrifugation (8000 rpm, 5 min). The bacterial cells were resuspended in PB buffer (pH = 7.5) and the bacterial concentration was adjusted to 5 g / L. Ultrasonic disruption was then performed, working for 3 s, stopping for 3 s, and the working time was 3 min;

[0132] After the crushing, centrifuge (10000rpm, 5min), take the supernatant, and use a microplate reader to measure the enzyme activity (the measurement system is: 188μL substrate solution (0.4g / L 3-nitrophthalic acid) + 10μL bacterial lysis supernatant + 2.5μL NADPH, mix well and place in a microplate reader, read the absorbance values ​​of 0min and 3min at 340nm), and calculate the relative activity according to the absorbance value (the measurement results are shown in Table 4). Plasmids were extracted from the 5 mutants with positive mutations in the rescreening results, and the introduced mutation sites were determined by DNA sequencing to be H103Y, P201S, V231A, Q40R / K267R, and L256V / G258D, respectively.

[0133] Table 4: Activity comparison of nitroreductase mutants

[0134] Enzyme name Amino acid sequence number Relative activity (%) AcNTR SEQ ID NO.1 100 H103Y SEQ ID NO.3 121 P201S SEQ ID NO.4 124 V231A SEQ ID NO.5 172 Q40R / K267R SEQ ID NO.6 312 L256V / G258D SEQ ID NO.7 126

[0135] Note: At 30°C, the amount of enzyme that reduces 1 μmol of NADPH per minute is defined as one unit of enzyme activity.

[0136] Example 4: Superposition and combination mutation of mutant strain Q40R / K267R

[0137] In this example, the mutant strain Q40R / K267R was used as the parent strain, and mutations at different sites were superimposed to construct the combined mutant strains Q40R / K267R / H103Y, Q40R / K267R / P201S and Q40R / K267R / V231A. The primers used are shown in Table 5:

[0138] Table 5: Primer design table for combined mutagenesis

[0139]

[0140]

[0141] Site-directed mutagenesis was performed using the primers in Table 5. The mutation system is shown in Table 6.

[0142] Table 6:

[0143] Components volume 2×Premix (Primestar max) 20μL stencil 0.2μL Forward / reverse primers 0.2μL each DMSO 2μL water 17.4μL (i.e. make up to 40μL)

[0144] The amplification procedure is shown in Table 7;

[0145] Table 7:

[0146]

[0147] After agarose gel electrophoresis was performed to confirm that the amplification results were correct, 10 μL of the PCR product was mixed with 0.5 μL of Dpn I and placed at 37°C for 30 min. The treated sample was then heat-shock transformed (known and used) into E. coli BL21 (DE3) competent cells, ice-bathed, revived, and plated. The transformants were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0148] The mutant strains with correct sequencing were cultured in shake flasks and the enzyme activity was determined (see Example 3 for the method). The results showed that the activities of the three mutants Q40R / K267R / H103Y, Q40R / K267R / P201S and Q40R / K267R / V231A obtained in this round of mutation were 1.58 times, 2.99 times and 1.25 times that of the wild-type AcNTR control, respectively.

[0149] Example 5: Preparation of nitroreductase bacterial powder

[0150] All nitroreductase mutants Q40R / K267R were inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C, 200 rpm for 8 h. Subsequently, the seed solution was transferred to fresh fermentation medium containing kanamycin (10 g / L yeast powder, 20 g / L peptone, 15 g / L glycerol, 10 g / L NaCl, 3 g / L KNO3, 1.74 g / L K2HPO4, 1.36 g / L K2HPO4, and 0.5 g / L MgCl2) at a 2% inoculum and cultured at 37°C, 200 rpm until the bacterial concentration reached OD 600The expression of nitroreductase protein was induced by adding IPTG at a final concentration of 0.2mM, and the culture conditions were 25°C, 200rpm, and 15h. The bacterial cells were collected by centrifugation at 4°C and 12000rpm for 10min. Subsequently, the collected bacterial cells were washed with 0.85% saline, centrifuged again, and the bacterial cells were collected and placed at -20°C for precooling. Subsequently, the precooled bacterial cells were vacuum freeze-dried to finally obtain the bacterial powder of nitroreductase for subsequent research.

[0151] Example 6: Application of recombinant nitroreductase mutant in catalytic synthesis of 3-aminophthalic acid

[0152] The freeze-dried bacterial powders of the recombinant nitroreductase mutant Q40R / K267 and the wild-type strain AcNTR obtained in Example 5 were used as biocatalysts to catalyze the synthesis of 3-aminophthalic acid.

[0153] The reaction system is as follows:

[0154] 1.0 g 3-nitrophthalic acid (100 g / L), 0.5 g bacterial powder (50 g / L), lactate dehydrogenase coenzyme circulation system (2 g lactate (200 g / L), 30 mg lactate dehydrogenase (3 g / L) and 30 mg NADP coenzyme (3 g / L)) and 30 mg vanadium pentoxide (3 g / L) were added to 10 mL phosphate buffer (pH = 8.0). The reaction conditions were: 30°C, 250 rpm.

[0155] The experimental results showed that the space-time yield of the mutant Q40R / K267 in converting the substrate 3-nitrophthalic acid was greatly improved compared with the wild-type strain, and 50 g / L of bacterial powder could completely convert 100 g / L 3-nitrophthalic acid into 3-aminophthalic acid within 4 hours. In contrast, the wild-type strain AcNTR needed 12 hours.

[0156] In addition, the mutant strain can completely convert 160 g / L 3-nitrophthalic acid into 3-aminophthalic acid within 12 h.

[0157] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0158] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an apremilast intermediate, comprising reducing 3-nitrophthalic acid using a nitroreductase mutant or a recombinant cell carrying the mutant or a recombinant strain expressing the mutant to prepare 3-aminophthalic acid; in, The nitroreductase mutant includes: compared with the wild-type nitroreductase, at least one amino acid mutation in the following seven positions or functionally equivalent positions: position 40, position 103, position 201, position 231, position 256, position 258, and position 267; Wherein, the wild-type nitroreductase has an amino acid sequence as shown in SEQ ID NO:

1.

2. The preparation method according to claim 1, wherein The nitroreductase mutant has at least 80% or 90% or 95% or 98% identity to the wild-type nitroreductase.

3. The preparation method according to claim 1 or 2, wherein The mutant has at least one of the following mutations: (1) The amino acid Q at position 40 mutated to R; (2) amino acid H at position 103 mutated to Y; (3) amino acid P at position 201 mutated to S; (4) amino acid V at position 231 mutated to A; (5) amino acid L at position 256 mutated to V; (6) amino acid G at position 258 mutated to D; (7) The amino acid K at position 267 mutated to R.

4. The preparation method according to claim 3, characterized in that: The mutant has any one of the following mutations (1)-(8): (1) The amino acid H at position 103 is mutated to Y; or (2) The amino acid P at position 201 is mutated to S; or (3) the amino acid V at position 231 is mutated to A; or (4) the amino acid Q at position 40 is mutated to R, and the amino acid K at position 267 is mutated to R; or (5) the amino acid at position 256, L, mutates to V, and the amino acid at position 258, G, mutates to D; or (6) the amino acid Q at position 40 is mutated to R, the amino acid K at position 267 is mutated to R, and the amino acid H at position 103 is mutated to Y; or (7) the amino acid Q at position 40 mutates to R, the amino acid K at position 267 mutates to R, and the amino acid P at position 201 mutates to S; or (8) The amino acid Q at position 40 mutated to R, the amino acid K at position 267 mutated to R, and the amino acid V at position 231 mutated to A; Optionally, the mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 3-10.

5. The preparation method according to any one of claims 1 to 4, wherein The recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

6. The preparation method according to any one of claims 1 to 5, comprising an oxidoreductase system; Optionally, the oxidoreductase system comprises glucose dehydrogenase, glucose and the coenzyme NADP or lactate dehydrogenase, lactate and the coenzyme NADP.

7. The preparation method according to any one of claims 1 to 6, wherein The reduction reaction of 3-nitrophthalic acid is carried out in a phosphate buffer; Optionally, the pH of the phosphate buffer is 6.5-10 or 8.

8. The method according to any one of claims 1 to 7, wherein: The reduction reaction of 3-nitrophthalic acid is carried out at 15-50°C.

9. The preparation method according to any one of claims 1 to 8, further comprising a preparation method for a nitroreductase mutant, comprising: The wild-type nitroreductase is subjected to mutation treatment to obtain a nitroreductase mutant library; subjecting 3-nitrophthalic acid to a first catalytic treatment in the presence of the nitroreductase mutant library; The first catalytic treatment product is subjected to screening treatment to obtain the nitroreductase mutant.

10. The preparation method according to claim 9, wherein: The mutation treatment is selected from at least one of random mutation and site-directed mutation; Optionally, the first catalytic treatment is carried out in the presence of NADPH; The screening process includes obtaining a nitroreductase mutant having an absorbance value higher than that of a control group; optionally, the control group is a wild-type nitroreductase.

Images