Deaminase mutants and uses thereof

Abstract

The present application relates to a kind of deaminase mutant and its application, the deaminase mutant is obtained by the mutation of the amino acid sequence shown in SEQ ID NO.1, the mutation at least includes one in following mutation site: the 60th by L mutation A or V, the 100th by S mutation A or T, the 170th by G mutation S or C, the 200th by E mutation K or H.The deaminase mutant is applied to nucleoside compound, catalytic activity is high, stability is good, compared with wild type deaminase, there is different degree of improvement.The catalyzed reaction compared with chemical synthesis method is simple and mild, reaction selectivity is high, preparation cost is low, with good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a deaminase mutant and its applications. Background Technology

[0002] Nucleosides are central biomolecules that play crucial roles in various cellular processes by acting as cofactors for enzymes, building blocks of DNA and RNA, and energy transport systems. Modified nucleosides are indispensable drugs for treating various types of cancer and viral infections. For example, chemically modified purine and pyrimidine nucleoside drugs (nucleoside analogs) account for approximately 20% of all chemotherapy drugs currently used for cancer treatment. For instance, gemcitabine (2',2'-difluoro-2'-deoxycytidine) is widely used to treat various cancers, including pancreatic, lung, breast, and bladder cancer. Modification with 2'-O-MOE increases the Tm value of antisense oligonucleotides, thereby enhancing their ability to bind to complementary RNA, and 2'-O-MOE-modified antisense oligonucleotides exhibit higher nuclease resistance. Based on the above advantages, nucleic acid molecules containing 2'-O-MOE nucleosides have been widely used in the treatment research of various diseases. For example, Nusinersen was approved by the FDA in 2016, Inotersen was approved by the FDA in 2018, and Volanesorsen was approved in Europe in 2019.

[0003] Compound II-1 is an intermediate in the synthesis of nusinersen sodium. Despite significant market demand for this 2'-O-MOE nucleoside, its synthesis remains considered challenging and inefficient (its chemical synthesis methods suffer from long steps, poor selectivity, and high cost). While nucleosides containing ribosyl or 2'-deoxyribosyl groups can be obtained from naturally occurring nucleosides or carbohydrates, the preparation of sugar-modified nucleosides typically suffers from long reaction times and low overall yields. Furthermore, reactions heavily reliant on protecting groups have low overall efficiency because some sugar modifications at the 2' or 4' positions are known to limit the diastereoselectivity of glycosylation methods.

[0004]

[0005] The synthesis of 2'-O-MOE nucleosides can also be achieved through enzymatic methods. Enzymatic methods offer advantages such as high regio- and stereoselectivity, mild reaction conditions, and limited use of contaminant reagents and organic solvents. Enzyme catalysis has been considered a feasible technology for the industrial production of valuable drugs and their intermediates. Its advantages include the ability to perform specific and selective catalytic reactions of substrates in benign solvents under mild reaction conditions. However, enzyme catalysis also has drawbacks. For example, the number of applicable substrates for a specific biological enzyme is relatively small. For instance, natural enzymes may exhibit good catalytic activity for their natural substrates, but their catalytic activity for other specific substrates may be particularly low or even nonexistent. Even so, enzyme catalysts can be modified using various techniques in modern molecular biology, such as directed evolution, random mutation, and protein engineering, to overcome these drawbacks, enabling biocatalysis to be used efficiently and environmentally friendly for the synthesis of chemical molecules (e.g., active pharmaceutical ingredients).

[0006] The aforementioned compound II-1 can be obtained by converting the 4-amino group of the inexpensive and readily available compound I-1 (CAS: 256224-13-2) to a 4-carbonyl group. However, for this non-natural substrate, due to the specificity of enzymes, the catalytic effect of existing deaminases on compounds I-1 is greatly reduced, or even completely absent. Therefore, a suitable deaminase is needed that can selectively convert the 4-amino group of the pyrimidine ring of compound I-1 to a 4-carbonyl group in a single step, which has significant industrial production value.

[0007] Summary of the Invention

[0008] One aspect of the present invention is to provide a deaminase mutant that converts the 4-amino group of a ribose-modified adenosine or its analogue to a 4-carbonyl group. The amino acid sequence of the deaminase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutation includes at least one of the following mutation sites: L to A or V at position 60, S to A or T at position 100, G to S or C at position 170, and E to K or H at position 200; or the amino acid sequence of the deaminase mutant has the mutation site in the mutated amino acid sequence and has more than 85% homology with the mutated amino acid sequence.

[0009] In another preferred embodiment, the amino acid sequence of the deaminase mutant is as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8; or the amino acid sequence of the transaminase mutant has more than 85% homology with the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

[0010] In another preferred embodiment, the amino acid sequence of the deaminase mutant is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0011] In another preferred embodiment, the amino acid sequence of the deaminase mutant is shown in SEQ ID NO.5.

[0012] In another aspect, the present invention provides a polynucleotide that encodes the above-mentioned deaminase mutant.

[0013] In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or

[0014] The nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequences shown in SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.

[0015] In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO.10 or SEQ ID NO.11.

[0016] In another aspect, the present invention provides a recombinant expression vector comprising the aforementioned polynucleotides.

[0017] In another preferred embodiment, the vector used to construct the recombinant expression is selected from the pET-28a expression vector, the pET-dute1 expression vector, or the pRSF-dute1 expression vector, with the pET-28a expression vector being more preferred.

[0018] In another aspect, the present invention provides a host cell comprising the above-described recombinant expression vector.

[0019] In another preferred embodiment, the host cell is selected from *Escherichia coli* MG1655, *Escherichia coli* BL21(DE3), or *Escherichia coli* BL21(DE3)pLysS. In another more preferred embodiment, the host cell is selected from *Escherichia coli* BL21(DE3).

[0020] In another aspect, this invention provides the application of the above-mentioned deaminase mutant in the preparation of compound II from compound I.

[0021] The structure of the compound of formula I is as follows:

[0022]

[0023] R1 and R2 are each independently selected from hydrogen, amino, hydroxyl, halogen, nitro, cyano, C1-C8 alkoxy, C1-C8 alkyl, C5-C6 10 cycloalkyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 Heteroaryl groups, wherein the amino group, C1-C8 alkoxy group, C1-C8 alkyl group, C5-C6 alkyl group, C64 alkyl group, C65 alkyl group, C6 ... 10 cycloalkyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 The heteroaryl group may be further selected by one or more groups selected from amino, hydroxyl, amino, halogen, nitro, cyano, C1-C8 alkyl, C1-C8 oxoalkyl, C5-C6 alkyl, C6-C7 alkyl, C6-C8 alkyl, C6-C8 alkyl, C6-C6 ... 10 Cycloalkyl, C2-C6 ester, C2-C8 alkenyl, C2-C8 alkynyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 Substituents of heteroaryl groups;

[0024] The structure of the compound of formula II is as follows:

[0025]

[0026] R1 and R2 are defined as above.

[0027] In another preferred embodiment, in compounds of formula I and formula II, R1 is an amino group and R2 is a 2-methoxyethoxy group, and the structures of compounds of formula I and II are shown in formula I-1 and formula II-1, respectively:

[0028]

[0029] In another aspect, the present invention provides a method for preparing a compound of formula II-1, the method comprising the steps of:

[0030] In the presence of a deaminase, the 4-amino group of the compound shown in formula I-1 is converted to a 4-carbonyl group to give the compound of formula II-1, as shown in the following reaction:

[0031]

[0032] The amino acid sequence of the deaminase is shown in SEQ ID NO.5.

[0033] In another preferred embodiment, the reaction time in the preparation method of compound II-1 is 20–45 °C, more preferably 30–40 °C.

[0034] In another preferred embodiment, in the method for preparing compound II-1, the pH of the reaction system is 6.0 to 9.0, more preferably 7.0 to 8.0.

[0035] In another preferred embodiment, the reaction system in the preparation method of compound II-1 is an aqueous system.

[0036] In another preferred embodiment, the reaction system in the preparation method of compound II-1 is PBS buffer.

[0037] In another preferred embodiment, the reaction system in the preparation method of compound II-1 further includes a co-solvent, which may be selected from dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone or a combination thereof.

[0038] In another preferred embodiment, the concentration of the co-solvent is 1-20%.

[0039] In another preferred embodiment, the method for preparing compound II-1 further includes isolating compound II-1 from the reaction vessel after the reaction. In another preferred embodiment, the isolation is performed by filtering the reaction solution to obtain compound II-1.

[0040] In another preferred embodiment, in the method for preparing compound II-1, the deaminase is an enzyme in free form, an immobilized enzyme, or an enzyme in bacterial cell form.

[0041] The deaminase mutant of the present invention is based on the deaminase shown in SEQ ID NO.1, and is mutated by site-directed mutagenesis to change its amino acid sequence, thereby altering the protein structure and function. Then, through targeted screening, a deaminase with the aforementioned mutation site is obtained. For the deamination of compounds of Formula I, the deaminase mutant of the present invention has the advantage of significantly increased enzyme activity, which is much higher than that of the parent deaminase (wild type). When used to produce purine ketone compounds from aminopurine compounds, it significantly reduces the cost in industrial production. Attached Figure Description

[0042] Figure 1 This is a diagram of the deaminase recombinant expression vector from Example 1.

[0043] Figure 2 The results of polyacrylamide gel electrophoresis for different deaminases are shown. From left to right, the lanes are Marker, wild type (amino acid sequence SEQ ID NO.1), BL21(DE3)-i protease (amino acid sequence SEQ ID NO.3), BL21(DE3)-ii protease (amino acid sequence SEQ ID NO.4), and BL21(DE3)-iii protease (amino acid sequence SEQ ID NO.5). Figure 3 The results shown are polyacrylamide gel electrophoresis results for different deaminases. From left to right, the lanes are Marker, wild type, BL21(DE3)-iv protease (the amino acid sequence of which is SEQ ID NO.6), BL21(DE3)-v protease (the amino acid sequence of which is SEQ ID NO.7), and BL21(DE3)-vi protease (the amino acid sequence of which is SEQ ID NO.8).

[0044] Figure 4 This is the liquid chromatogram of the product obtained in Example 9.

[0045] Figure 5 This is the 1H NMR spectrum of the product obtained in Example 9. Detailed implementation method:

[0046] Adenosine deaminase derived from *Escherichia coli* (strain K12 / DH10B) can efficiently catalyze the conversion of adenosine to inosine, but it is inactive against compound (I). The inventors of this invention improved the activity of adenosine deaminase derived from *Escherichia coli* (strain K12 / DH10B) through a rationally designed method. A mutation site was introduced into the deaminase derived from *Escherichia coli* (strain K12 / DH10B) using whole-plasmid PCR, and the activity and stability of the mutants were tested to select mutants with improved activity and stability.

[0047] The deaminase mutant gene provided by this invention is derived from the wild-type gene of *Escherichia coli* (strain K12 / DH10B). The amino acid sequence of this wild-type gene is shown in SEQ ID NO.1, and the codon-optimized gene sequence for *E. coli* is shown in SEQ ID NO.2. Here, "wild-type" refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences present in organisms, which can be isolated from natural sources and have not been intentionally modified by human intervention. Enzymes obtained after expression of these genes exhibit low catalytic activity and poor thermal stability for certain substrates.

[0048] The cDNA of the deaminase derived from Escherichia coli (strain K12 / DH10B), with codon optimization for Escherichia coli, is shown in SEQ ID NO.2.

[0049] The three-dimensional structure of adenosine deaminase from Escherichia coli (strain K12 / DH10B) was obtained using an online protein structure prediction tool. The three-dimensional structure (6n9m.1) of adenosine deaminase from Salmonella typhimurium, which has the highest structural similarity to adenosine deaminase, was obtained using PDB. Then, the binding simulation of the three-dimensional structure of the adenosine deaminase protein with the substrate of Formula I was performed using AutoDock. Finally, Pymol analysis was used to select amino acids that may be related to substrate binding as mutant amino acids.

[0050] Based on Pymol analysis results, multiple pairs of site-directed mutagenesis primers were designed (e.g., L to A or V at position 60 (L60A / V), S to A or T at position 100 (S100T / A), G to S or C at position 170 (G170S / C), and E to K or H at position 200 (E200K / H). Using site-directed mutagenesis with pET-28a as the expression vector, mutant plasmids carrying the target gene were obtained. Site-directed mutagenesis refers to the introduction of desired changes (usually changes indicating a favorable direction) into a target DNA fragment (which can be genomic or plasmid) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the traits and characterization of the target protein expressed by DNA, making it a very useful technique in gene research.

[0051] The deaminase mutant provided by the present invention is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO.1. The mutation includes at least one of the following mutation sites: L at position 60 is mutated to A or V, S at position 100 is mutated to A or T, G at position 170 is mutated to S or C, E at position 200 is mutated to K or H, or the amino acid sequence of the deaminase mutant has the mutation site in the mutated amino acid sequence and has more than 85% homology with the mutated amino acid sequence. In some specific embodiments of the present invention, the deaminase mutant provided by the present invention is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO. 1. The mutation includes at least one of the following mutation sites: L to A at position 60 (L60A), S to T at position 100 (S100T), G to S at position 170 (G170S), E to K at position 200 (E200K), L to V at position 60 (L60V), S to A at position 100 (S100A), G to C at position 170 (G170C), and E to H at position 200 (E200H). Alternatively, the amino acid sequence of the deaminase mutant has the mutation site in the mutated amino acid sequence and has more than 85% homology with the mutated amino acid sequence. Here, "homology" refers to the degree of similarity between two amino acid sequences. The sequences defined by varying degrees of homology in this invention must also simultaneously possess improved deaminase activity. Those skilled in the art, guided by the disclosure of this application, can obtain amino acid sequences that contain the mutation sites in the aforementioned mutated amino acid sequences and possess more than 85% homology with the mutated amino acid sequences.

[0052] In one specific embodiment of the present invention, the above-mentioned deaminase mutant includes mutation sites L60A and S100T, and its amino acid sequence is shown in SEQ ID NO. 3. In one specific embodiment of the present invention, the above-mentioned deaminase mutant includes mutation sites L60A, S100T, and G170S, and its amino acid sequence is shown in SEQ ID NO. 4. In one specific embodiment of the present invention, the above-mentioned deaminase mutant includes mutation sites L60A, S100T, G170S, and E200K, and its amino acid sequence is shown in SEQ ID NO. 5. In one specific embodiment of the present invention, the above-mentioned deaminase mutant includes mutation sites L60V and S100A, and its amino acid sequence is shown in SEQ ID NO. 6. In one specific embodiment of the present invention, the above-mentioned deaminase mutant includes mutation sites L60V, S100A, and G170C, and its amino acid sequence is shown in SEQ ID NO. 7. In one specific embodiment of the present invention, the above-mentioned deaminase mutant contains mutation sites L60V, S100A, G170C, and E200H, and its amino acid sequence is shown in SEQ ID NO: 8.

[0053] The above-mentioned deaminase mutant of the present invention is based on the deaminase shown in SEQ ID NO.1. It is mutated by site-directed mutagenesis to change its amino acid sequence, thereby changing the protein structure and function. Then, by targeted screening, deaminase with the above-mentioned mutation site is obtained. The deaminase mutant of the present invention has significantly improved enzyme activity, thereby greatly reducing the cost in the production of compound II.

[0054] This invention provides a gene encoding the aforementioned deaminase mutant. This invention obtains a polynucleotide encoding the aforementioned deaminase mutant by mutating the wild-type deaminase gene using rational design (site-directed mutagenesis or other methods to alter individual amino acids in the protein molecule) and methods such as overlap extension PCR and seamless cloning. In one specific embodiment of this invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 9 (encoding the amino acid sequence shown in SEQ ID NO. 3), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 9. In one specific embodiment of this invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 10 (encoding the amino acid sequence shown in SEQ ID NO. 4), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 10. In one specific embodiment of this invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 11 (encoding the amino acid sequence shown in SEQ ID NO. 5), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 11. In one specific embodiment of the present invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 12 (encoding the amino acid sequence shown in SEQ ID NO. 6), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 10. In one specific embodiment of the present invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 13 (encoding the amino acid sequence shown in SEQ ID NO. 7), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 10. In one specific embodiment of the present invention, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 14 (encoding the amino acid sequence shown in SEQ ID NO. 8), or the nucleotide sequence of the polynucleotide has more than 85% homology with the nucleotide sequence shown in SEQ ID NO. 10. Herein, "homology" refers to the degree of similarity between two nucleotide sequences.

[0055] The term "above 85%" as used in this article refers to 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0056] The amino acid sequences of the above deaminase mutants and the nucleotide sequences of the polynucleotides encoding them are shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] The deaminase encoded by the polynucleotides of this invention improves enzyme activity and stability, resulting in higher efficiency and lower cost in the industrial production of nucleoside analogs.

[0066] To meet the requirements of recombination operations, suitable restriction endonuclease cleavage sites can be added to both ends of the above polynucleotides, or additional start codons, stop codons, etc. can be added.

[0067] The vector provided by this invention contains a polynucleotide encoding the deaminase mutant of this invention. This polynucleotide is located at an appropriate position in the recombinant expression vector, enabling the polynucleotide to be correctly and smoothly replicated, transcribed, or expressed. The vector used to construct the recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, including but not limited to the pET-28a expression vector, the pET-dute1 expression vector, or the pRSF-dute1 expression vector.

[0068] The host cell provided by this invention is used to produce the above-mentioned deaminase mutant, which contains the above-mentioned recombinant expression vector. In this invention, the host cell includes, but is not limited to, *Escherichia coli* MG1655, *Escherichia coli* BL21(DE3), or *Escherichia coli* BL21(DE3)pLysS.

[0069] The aforementioned deaminase mutant can be prepared by fermenting the host cells. For example, the deaminase mutant can be prepared industrially under specific fermentation conditions in a production tank. The preferred fermentation conditions in the production tank are: DO ≥ 20% and temperature 20°C.

[0070] In a preferred embodiment of the present invention, a seamless cloning method is used when mutating the deaminase group. The primers on the pET28a plasmid are located upstream and downstream of the deaminase polynucleotide, respectively. Primers with 15 bp homologous arms at both ends are set at the mutation site. The PCR reaction conditions are: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 53-60℃ annealing for 15 s and 72℃ extension for 50 s for 30 cycles; further extension at 72℃ for 10 min, and cooling to 4℃. After PCR amplification according to the above method, the fragment is ligated to pET28a using a seamless cloning kit. The ligated vector is then transformed into E. coli BL21(DE3) to establish a deaminase polynucleotide mutant. Using E. coli BL21(DE3) as the host and the pET28a plasmid as the vector, the host cell expressing the extended mutant deaminase is established.

[0071] The deaminase mutant provided by this invention can catalyze the production of compound II from compound I, wherein the structure of compound I is as follows:

[0072]

[0073] R1 and R2 are each independently selected from hydrogen, amino, hydroxyl, halogen, nitro, cyano, C1-C8 alkoxy, C1-C8 alkyl, C5-C6 10 cycloalkyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 Heteroaryl groups, wherein the amino group, C1-C8 alkoxy group, C1-C8 alkyl group, C5-C6 alkyl group, C64 alkyl group, C65 alkyl group, C6 ... 10 cycloalkyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 The heteroaryl group may be further selected by one or more groups selected from amino, hydroxyl, amino, halogen, nitro, cyano, C1-C8 alkyl, C1-C8 oxoalkyl, C5-C6 alkyl, C6-C7 alkyl, C6-C8 alkyl, C6-C8 alkyl, C6-C6 ... 10 Cycloalkyl, C2-C6 ester, C2-C8 alkenyl, C2-C8 alkynyl, C4-C 11 Heterocyclic group, C6-C 10 Aryl or C5~C 10 Substituents of heteroaryl groups;

[0074] The structure of the compound of formula II is as follows:

[0075]

[0076] R1 and R2 are defined as above.

[0077] The catalytic process is carried out at room temperature. The amount of enzyme used is, for example, 0.5 to 4 wt% of the substrate weight using wet enzyme cells. After 24 hours of reaction, the substrate conversion rate can reach 99.6%. In the description of this invention, the terms "room temperature" or "normal temperature" refer to a temperature of 4-40°C, preferably 35±5°C.

[0078] In the description of this invention, "C1-C8 alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group comprising 1 to 8 carbon atoms. Preferably, it is a C1–C6 alkyl group, more preferably a C1–C4 alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, etc. The alkyl group may be substituted or unsubstituted.

[0079] In the description of this invention, "C1-C8 alkoxy" refers to a (C1-C8 alkyl-O-) group. The alkyl group is as defined above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, etc.

[0080] In the description of this invention, "C5~C 10 "Cycloalkyl" refers to a saturated or partially saturated monocyclic, fused, bridged, or spirocyclic carbon ring. More preferably, it is a C5-C8 cycloalkyl, and most preferably a C5-C6 cycloalkyl. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl, with cyclopentyl and cyclohexyl being preferred. The cycloalkyl group may be optionally substituted or unsubstituted.

[0081] In the description of this invention, "C4~C 11 "Heterocyclic group" refers to a non-aromatic heterocyclic group in which one or more cyclic atoms are heteroatoms, such as oxygen, nitrogen, sulfur atoms, etc., including monocyclic, polycyclic, fused ring, bridged ring and spirocyclic, and may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and / or sulfur. Examples of "heterocyclic group" include, but are not limited to, morpholino, oxobutyl, thiomorpholino, tetrahydropyrano, 1,1-dioxo-thiomorpholino, piperidinyl, 2-oxo-piperidinyl, pyrrolyl, 2-oxo-pyrrolyl, piperazine-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl, piperazine.

[0082] In the description of this invention, "C6~C 10"Aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be fused together. The term "aryl" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. Aryl groups may be substituted or unsubstituted.

[0083] In the description of this invention, "C5~C 10 "Heteroaryl" refers to an aromatic 5- to 6-membered monocyclic ring or 8- to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen, and / or sulfur. Examples include furanyl, pyridinyl, pyridinyl, pyrimidinyl, pyrazinyl, thiopheneyl, isoxazolyl, oxazolyl, imidazoleyl, pyrroleyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, benzothiopheneyl, benzimidazolyl, indoleyl, isoyindoleyl, quinolinyl, indazoleyl, etc. Heteroaryl groups may be substituted or unsubstituted.

[0084] In the description of this invention, "C2-C6 ester group" refers to an ester group containing 2 to 6 carbons.

[0085] In the description of this invention, "C2-C8 alkenyl" refers to a group containing 2 to 6 carbons and at least one alkene bond.

[0086] In the description of this invention, "C2-C8 alkynyl" refers to a group containing 2 to 6 carbons and at least one alkynyl bond.

[0087] The method for producing compound II from compound I using deaminase catalysis of the present invention has the following advantages and effects:

[0088] 1. The deaminase mutant provided by this invention, especially the deaminase mutant with the amino acid sequence shown in SEQ ID NO.5, exhibits a 24-hour conversion rate greater than 99% for the 4-aminopyrimidine compound shown in Formula I-1; while the wild-type enzyme has essentially zero activity for this type of substrate. The deaminase mutant provided by this invention shows a significant improvement in catalytic activity for the 4-aminopyrimidine compound shown in Formula I-1 compared to the wild type, enabling the synthesis of product II-1 using deaminase catalysis, thus achieving green chemical synthesis.

[0089] 2. For the synthesis of compound II from compound I, the deaminase mutant provided by this invention (especially the deaminase mutant with the amino acid sequence shown in SEQ ID NO.5) has high catalytic activity and good stability; compared with chemical synthesis methods, the reduction reaction it catalyzes is simple and mild, with low preparation cost, and has good application prospects.

[0090] 3. In the reaction system for synthesizing compound II from compound I using the deaminase mutant of the present invention (especially the deaminase mutant having the amino acid sequence shown in SEQ ID NO.5), the substrate feed rate is relatively high, reaching 400-500 g / L, and the specific activity of the enzyme is also relatively high.

[0091] The present invention will be further described in detail below with reference to the embodiments. These embodiments are illustrative of the invention, but the invention is not limited to them. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0092] The LB liquid used in the following examples consisted of: 10 g ± 0.1 g peptone; 5 g ± 0.1 g yeast extract; and 10 g ± 0.1 g NaCl.

[0093] Example 1: Establishment of wild-type deaminase host cells

[0094] The complete gene fragment (nucleotide sequence shown in SEQ ID NO. 2) was artificially synthesized after sequence optimization based on the wild-type amino acid sequence (B1XF88) of Escherichia coli (strain K12 / DH10B) deaminase recorded in UniProt. The gene was then inserted into the pET-28a plasmid by a gene synthesis company using NdeI and XhoI restriction enzymes. Figure 1 The ligated vector was transferred into Escherichia coli BL21(DE3) to establish wild-type deaminase host cells, and the cells were screened for kanamycin resistance and then sequenced for verification.

[0095] Example 2: Design of deaminase mutants

[0096] According to the Pymol analysis results, at least one site in the amino acid sequence of the wild-type deaminase shown in SEQ ID NO.1 was mutated: position 60, position 100, position 170, and position 200, with position 60 being mutated from L to A or V, position 100 from S to A or T, position 170 from G to S or C, and position 200 from E to K or H. The amino acid sequences with the following combined mutation sites were finally obtained: (i) L60A, S100T (corresponding to the amino acid sequence shown in SEQ ID NO.3); (ii) L60A, S100T, G170S (corresponding to the amino acid sequence shown in SEQ ID NO.4); (iii) L60A, S100T, G170S, E200K (corresponding to the amino acid sequence shown in SEQ ID NO.5); (iv) L60V, S100A (corresponding to the amino acid sequence shown in SEQ ID NO.6); (v) L60V, S100A, G170C (corresponding to the amino acid sequence shown in SEQ ID NO.7); (vi) L60V, S100A, G170C, E200H (corresponding to the amino acid sequence shown in SEQ ID NO.8).

[0097] Example 3: Construction of recombinant expression vector:

[0098] Table 2. Forward and reverse primers for mutation points.

[0099]

[0100]

[0101] Expression vector pET28a(+) (see) Figure 1 The sample was double-digested with restriction endonucleases NdeI and XhoI, respectively. The digestion system consisted of 84 μL (approximately 30 μg) of pET28a(+), 3 μL of NdeI, and 3 μL of XhoI. After digestion at 37°C for 1.5 h, agarose gel electrophoresis was performed. The target band was located at 1 kb, and the digested fragments were recovered using a DNA recovery kit.

[0102] The coding sequences for the wild-type deaminases were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Using the synthesized gene in SEQ ID NO.2 as a template, PCR amplification was performed using primers P1 / P2 and P3 / P4 listed in Table 1. A band of approximately 1 kb was separated by agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO.9. The adenosine deaminase containing the combined mutation site (ii) was amplified by PCR using the synthesized polynucleotide shown in SEQ ID NO.9 as a template, and PCR amplification was performed using primers P5 / P6 listed in Table 1. A band of approximately 0.1 kb was separated by agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO.10. The adenosine deaminase containing the combined mutation site (iii) was amplified by PCR using the synthesized polynucleotide sequence shown in SEQ ID NO.10 as a template, and PCR amplification was performed using primers P7 / P8 listed in Table 1. A band of approximately 0.1 kb was separated by agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO.11. The coding sequence of the adenosine deaminase containing the combined mutation site (iv) was obtained using the synthesized polynucleotide sequence shown in SEQ ID NO.2 as a template, and the primers P9 / P listed in Table 1 were used. 10 P 11 / P 12 PCR amplification was performed, and a band of approximately 0.1 kJ was separated after agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO. 12. Adenosine deaminase containing the combined mutation site (v) was amplified by PCR using the synthesized polynucleotide shown in SEQ ID NO. 12 as a template, using primers P listed in Table 1. 13 / P 14 PCR amplification was performed, and a band of approximately 0.1 kJ was separated after agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO. 13. Adenosine deaminase containing the combined mutation site (vi) was synthesized using the polynucleotide sequence shown in SEQ ID NO. 13 as a template, and primer P was used. 15 / P 16 PCR amplification was performed, and a band of approximately 0.1 kJ was separated after agarose gel electrophoresis, which is the nucleotide sequence shown in SEQ ID NO.14. The six PCR amplification products were recovered using a DNA recovery kit.

[0103] Linearized vectors double-digested with NdeI and XhoI were ligated with PCR amplification products using a seamless cloning kit to construct recombinant expression vectors containing the above six mutant combinations of deaminase genes and wild-type genes. Ligation was performed according to the following reaction system: 50 ng of linearized vector pET28a(+), 100 ng of primer P1 / P2 amplification product, and 5 μL of 2* seamless cloning buffer. The mixture was incubated at 50°C for 20 min to obtain the recombinant expression vectors. PCR verification using T7 / T7t universal primers and sequencing analysis yielded the recombinant expression vectors pET28a-i, pET28a-ii, pET28a-iii, pET28a-iv, pET28a-v, pET28a-vi, and pET28a-0.

[0104] Example 4: Obtaining the recombinant expression transformant

[0105] The recombinant expression vectors pET28a-i, pET28a-ii, pET28a-iii, pET28a-iv, pET28a-v, pET28a-vi, and pET28a-0 were transformed into BL21(DE3) to obtain the recombinant expression transformants. The transformation method utilized the heat shock method: competent cells were removed from a -80°C freezer and placed on ice. Approximately 200-500 ng of expression vector was added, followed by an ice bath for 15 min, a heat shock at 42°C for 90 s, an ice bath for 3 min, the addition of 500 μL of LLB culture medium, and incubation at 37°C and 220 rpm for 45 min. 50 μL of the incubator was then evenly spread onto LB solid medium containing 50 μg / ml, inverted and cultured overnight to obtain single clones. After expansion, the clones were stored at -80°C with 20% final concentration glycerol. After sequencing analysis confirmed their correctness, they were identified as recombinant expression transformants, named BL21(DE3)-i, BL21(DE3)-ii, BL21(DE3)-iii, BL21(DE3)-iv, BL21(DE3)-v, BL21(DE3)-vi, and BL21(DE3)-0.

[0106] Example 5: Shake-flask culture and fermentation of recombinant expression transformants

[0107] The recombinant expression transformants (BL21(DE3)-i, BL21(DE3)-ii, BL21(DE3)-iii, BL21(DE3)-iv, BL21(DE3)-v, BL21(DE3)-vi, and BL21(DE3)-0) obtained in Example 4 were inoculated into LB liquid medium (with 100 μg / ml kanamycin added) and cultured overnight at 37°C and 220 rpm. The cultures were then transferred at a 1:100 ratio into 50 mL of fresh LB medium (containing 10 g ± 0.1 peptone, 5 g ± 0.1 yeast extract, and 10 g ± 0.1 NaCl in a 250 mL shake flask) and grown at 37°C. The optical density (OD) at 600 nm was measured. 600 When the concentration reached approximately 0.6, isopropyl galactothioglycoside (IPTG) was added to bring the final concentration to 1 mM. Cells were grown at 25°C for 16 hours. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was discarded. The cell pellet was resuspended in pre-chilled 10 mM PBS buffer (pH 7.5) at 200 g / L, sonicated, and then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C. The crude enzyme solution was subjected to polyacrylamide gel electrophoresis; the results are shown below. Figure 2 and Figure 3 .in Figure 2 The lanes from left to right are: Marker, wild type (amino acid sequence SEQ ID NO.1), BL21(DE3)-i protease (amino acid sequence SEQ ID NO.3), BL21(DE3)-ii protease (amino acid sequence SEQ ID NO.4), and BL21(DE3)-iii protease (amino acid sequence SEQ ID NO.5). Figure 3 The lanes from left to right are wild type, BL21(DE3)-iv protease (the amino acid sequence of which is SEQ ID NO.6), BL21(DE3)-v protease (the amino acid sequence of which is SEQ ID NO.7), and BL21(DE3)-vi protease (the amino acid sequence of which is SEQ ID NO.8).

[0108] Example 6: Transformer for substrate conversion

[0109] The recombinant expression transformants BL21(DE3)-i, BL21(DE3)-ii, BL21(DE3)-iii, BL21(DE3)-iv, BL21(DE3)-v, BL21(DE3)-vi, and BL21(DE3)-0 obtained by fermentation in Example 5 were used to catalyze the conversion of compound I-1 to compound II-1 in the following manner:

[0110] 7 ml of 0.01 M PBS buffer (pH = 7.5), 3 g of substrate (compound shown in Formula I-1), and 3 ml of lysate containing 0.6 g of wet bacterial cells (the cell pellet was obtained by centrifuging the shake-flask fermentation broth at 12000 rpm and 4°C for 10 min, discarding the supernatant, and resuspending each 0.6 g of wet bacterial cells in 3 ml of 0.01 M PBS buffer (pH = 7.5), followed by sonication and centrifugation at 12000 rpm and 4°C for 30 min; the supernatant was collected as the lysate). The reaction was carried out at 35°C / 200 rpm for 24 hours, and samples were taken for analysis (the pH of the system was controlled at 7.5 during the reaction, for example, using sodium hydroxide solution). The reaction solutions of the reduction reactions of the four recombinant expression transformants were analyzed by HPLC, and the results are shown in Table 3 below.

[0111] Table 3.

[0112]

[0113] Note: 1wt means that the weight of the wet cell lysate is 1 times the weight of the substrate.

[0114] As can be seen from Table 3, the transformants (BL21(DE3)-ii and BL21(DE3)-iii) containing mutant combinations (ii) and (iii) exhibit ideal catalytic activity in forming compound II-1 from substrate I-1. Among them, the transformant (BL21(DE3)-iii) containing mutant combination (iii) shows the best catalytic activity, achieving a substrate conversion rate of 99.5% within 24 hours.

[0115] Example 7: High-density fermentation preparation of recombinant expression transformant BL21(DE3)-iii

[0116] The recombinant mutant strain (BL21(DE3)-iii) obtained in Example 5 was inoculated into 3 mL of liquid LB medium and cultured overnight at 37°C with shaking at 220 rpm. Then, it was inoculated into 400 mL of liquid LB medium at a ratio of approximately 1% and cultured until OD500. 600 When the pH reached 4, it was used as seed culture and inoculated into 2L of fermentation medium for high-density fermentation. The initial temperature was 37℃, the stirring speed was 300rpm, the aeration rate was 1.5vvm, and the pH was 6.8. Subsequently, the stirring speed was continuously increased to a maximum of 1000rpm. The fermentation culture was divided into two stages. In the first stage, after inoculation, the culture was incubated for about 4 hours until the carbon source in the medium was completely consumed, and then fed back according to the DO (dissolved oxygen) principle. After feeding back, the temperature was reduced to 25℃, and the dissolved oxygen was maintained above 30%. Eight hours after feeding back, isopropyl thiogalactoside (IPTG) was added for induction. After 12 hours of induction, the culture was transferred to the fermentation tank. The cells were centrifuged at 8000rpm for 10min, and the supernatant was discarded to obtain wet bacterial cells.

[0117] Example 8: Study on the dosage of wet bacterial cells

[0118] The bacterial wet cells obtained in Example 7 were used to deaminate the substrate compound I-1 according to the methods and steps described in Example 6. The conversion results of the compound I-1 were shown in Table 4 below when different amounts of bacterial wet cell lysis buffer were used (each 0.6 g of bacterial wet cells was resuspended in 3 ml of 0.01 M PBS (pH = 7.5) buffer, sonicated, centrifuged at 12000 rpm and 4°C for 30 min, and the supernatant was collected).

[0119] Table 4.

[0120] The amount of breaking fluid used is 0.5 wt. 1wt of breaking fluid 2wt% of breaking fluid 6-hour conversion rate 50.5％ 65.5％ 67.9％ 12h conversion rate 90.2％ 96.8％ 98.5％ 24-hour conversion rate 93.6％ 99.5％ 99.6％

[0121] Note: 0.5wt means that the weight of the wet cell lysis buffer is 0.5 times the weight of the substrate. 1wt means that the weight of the wet cell lysis buffer is 1 times the weight of the substrate; 2wt means that the weight of the wet cell lysis buffer is 2 times the weight of the substrate.

[0122] Table 4 shows that the higher the amount of wet cell disruption solution used, the faster the catalytic efficiency, but 1 wt is sufficient to meet production requirements.

[0123] Example 9 Catalytic reaction system of deaminase

[0124] At room temperature, 30 g of substrate (compound of formula I-1, 88.15 mmol), 70 ml of 0.01 M PBS buffer, and 30 ml of BL21(DE3)-iii adenosine deaminase cell lysis buffer (the preparation process and concentration of the lysis buffer were the same as in Example 8, i.e., 0.6 g of wet cells were resuspended in 3 ml of 0.01 M PBS buffer, sonicated, centrifuged at 12000 rpm and 4°C for 30 min, and the supernatant was collected) were mixed thoroughly and reacted at 35°C / 200 rpm for 24 hours before sampling and analysis. The pH of the system was controlled at 7.5 during the reaction (e.g., using sodium hydroxide solution), and the mixture was sampled for HPLC analysis. After 24 h, the conversion rate reached 99.5%; after 30 h, the conversion rate reached 99.7%.

[0125] After the reaction was complete, the filtrate was cooled to 0-5℃ to allow the product to crystallize. After filtration, the product was rinsed with 100 ml of water and dried to obtain compound II-1. Compound II-1 was verified by liquid chromatography and mass spectrometry. The target compound yielded 25.1 g, with a molar yield of approximately 83.2% and a purity of 99.7%. The HPLC chromatogram and 1H NMR spectrum of the product are shown below. Figure 4 and Figure 5 .

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A deaminase mutant, characterized in that, The amino acid sequence of the deaminase mutant is shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

2. The deaminase mutant according to claim 1, characterized in that, The amino acid sequence of the deaminase mutant is shown in SEQ ID NO.4 or SEQ ID NO.

5.

3. The deaminase mutant according to claim 1, characterized in that, The amino acid sequence of the deaminase mutant is shown in SEQ ID NO.

5.

4. A polynucleotide, characterized in that, The polynucleotide encodes the deaminase mutant of claim 1.

5. The polynucleotide according to claim 4, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO.

11.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 6.

8. The use of the deaminase mutant according to any one of claims 1 to 4 in the preparation of compound II from compound I, The structure of the compound of formula I is as follows: ， R1 and R2 are each independently selected from hydrogen, amino, hydroxyl, halogen, nitro, cyano, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C5-C6. 10 Cycloalkyl, substituted or unsubstituted C4~C 11 Heterocyclic group, substituted or unsubstituted C6~C 10 Aryl, or substituted or unsubstituted C5~C 10 Heteroaryl, wherein the substituted C1-C8 alkoxy, substituted C1-C8 alkyl, substituted C5-C 10 Cycloalkyl, substituted C4~C 11 Heterocyclic groups, substituted C6~C 10 Aryl or substituted C5~C 10 The heteroaryl group is one or more selected from amino, hydroxyl, halogen, nitro, cyano, C1-C8 alkyl, C1-C8 oxoalkyl, C5-C6 alkyl, C6-C7 alkyl, C6-C8 alkyl, C6-C8 alkyl, C6-C6 ... 10 Cycloalkyl, C2-C6 ester, C2-C8 alkenyl, C2-C8 alkynyl, C4-C 11 Heterocyclic group, C6~C 10 Aryl or C5~C 10 The C1-C8 alkoxy, C1-C8 alkyl, and C5-C substituents of the heteroaryl group 10 cycloalkyl, C4~C 11 Heterocyclic group, C6~C 10 Aryl or C5~C 10 Mixed aromatics; The structure of the compound of formula II is as follows: ， R1 and R2 are defined as above.

9. The application according to claim 8, characterized in that, In compounds of formula I and formula II, R1 is an amino group and R2 is a 2-methoxyethoxy group. The structures of compounds of formula I and II are shown in formula I-1 and formula II-1, respectively: 。 10. A method for preparing a compound of formula II-1, characterized in that, The preparation method includes the following steps: In the presence of a deaminase, the 4-amino group of the compound shown in formula I-1 is converted to a 4-carbonyl group to give the compound of formula II-1, as shown in the following reaction: ， The amino acid sequence of the deaminase is shown in SEQ ID NO.5.

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