Synthesis method of imine reductase mutant and chiral phosphorus compound

Abstract

The invention discloses an imine reductase mutant and a synthesis method of a chiral phosphorus compound, and belongs to the field of enzyme engineering and organic synthesis, and the imine reductase mutant is obtained by carrying out single-point mutation or double-point mutation on the 1st to 289th sites of an amino acid sequence as shown in SEQ ID NO.1 by wild type imine reductase from Streptomyces chartreus. The wild-type imine reductase, the imine reductase mutant or the genetically engineered bacterium for expressing the imine reductase mutant from streptomyces teaching wine color serve as a biological enzyme catalyst and can be used for catalyzing diaryl phosphine oxide and alpha-substituted aryl ethylene to be subjected to free radical coupling to obtain a chiral phosphorus compound. The synthesis scheme of the current chiral phosphorus compound is enriched, and the method has a wide industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and organic synthesis, and in particular relates to a synthesis method of an imine reductase mutant and a chiral phosphorus compound. Background Art

[0002] Chiral phosphorus compounds, including phosphates, phosphines, etc., are widely used in many fields such as materials science, agricultural chemistry, drug synthesis, and ligand catalysis. The introduction of phosphorus-containing functional groups into organic molecules can effectively change the medicinal properties and catalytic activity of the molecules. In the past 10 years, a variety of therapeutic drugs and chiral catalysts containing chiral phosphorus groups have been discovered and characterized (such as Chinese patent documents with publication numbers CN109476687A, CN118126082A, CN111393243A, etc.), and the important value of these compounds has stimulated the development of methods for synthesizing chiral phosphorus compounds.

[0003] However, although chiral phosphorus compounds have great application prospects, the expensive and complicated synthetic routes limit their further expansion of production. At present, the schemes for synthesizing phosphorus-containing compounds mainly include traditional nucleophilic addition method, transition metal catalysis method, free radical reaction, etc. (Chemical Reviews, 2011, 111, 7981-8006). However, the traditional CP bond formation strategy relying on nucleophilic substitution reaction means has high requirements on substrate stability, harsh reaction conditions and difficult to control chirality. Although the synthesis of phosphorus-containing compounds under mild conditions can be achieved using chiral ligand catalysts or metal photocatalytic pathways, the reaction chirality is controllable, but the reaction cost is high. In fact, in many transition metal-catalyzed CP coupling reactions, the level of stereocontrol is usually not enough to meet the actual application requirements. Therefore, it is of great significance to develop a method that is efficient, universal, and can realize the enantioselective construction of CP bonds under mild conditions.

[0004] Among many chiral synthesis schemes, photoenzyme-catalyzed synthesis has become a strong competitive scheme for realizing green synthesis of chiral compounds because it can directly utilize light energy, is energy-saving and environmentally friendly, clean and efficient, and does not cause side reactions. At present, there are many cases of using photoenzyme reactions to catalyze asymmetric synthesis. For example, the Chinese patent document with publication number CN117987483A discloses a method for preparing chiral-α-benzylamine and its derivatives by photoenzyme-catalyzed asymmetric reduction, and the Chinese patent document with publication number CN116179623A discloses the application of a photoenzyme TPe in the synthesis of chiral compounds by asymmetric catalytic reaction, which provides a new idea for engineering enzymes and their catalytic synthesis of chiral phosphorus compounds. However, there is currently no scheme for photoenzyme-catalyzed synthesis of chiral CP bonds. Therefore, how to explore the photocatalytic ability of non-natural photoenzymes and realize the photoenzyme-catalyzed synthesis of chiral phosphorus compounds is an important issue to be solved. Summary of the invention

[0005] In order to solve the deficiencies in the above-mentioned prior art, the present invention provides an imine reductase mutant and a method for synthesizing a chiral phosphorus compound. The imine reductase mutant has the activity of photocatalytic synthesis of chiral phosphorus compounds, and the corresponding method for synthesizing chiral phosphorus compounds has mild reaction conditions, is green and environmentally friendly, simple and efficient.

[0006] The specific technical solutions adopted are as follows:

[0007] An imine reductase mutant is obtained by subjecting a wild-type imine reductase (IR14) from Streptomyces chartreusis to single-point mutation or double-point mutation at positions 1 to 289 of the amino acid sequence shown in SEQ ID NO. 1;

[0008] The specific single point mutation site is any of the following:

[0009] mutation at position 173;

[0010] mutation at position 176;

[0011] mutation at position 240;

[0012] mutation at position 241;

[0013] The specific double point mutation sites are:

[0014] Combined mutations at positions 176 and 240.

[0015] Further, the specific single point mutation is any one of the following:

[0016] L173A; L173F; M176A; M176L; M176F; M176I; M176N; T240F; T240W; T240Y; M241F.

[0017] Further, the specific double point mutation is any one of the following:

[0018] M176L and T240F; M176I and T240F.

[0019] Preferably, the double point mutations are M176I and T240F, and the corresponding amino acid sequence is shown in SEQ ID NO.3.

[0020] The present invention also provides a gene encoding the imine reductase mutant; specifically, the nucleotide sequence encoding the wild-type imine reductase IR14 is shown in SEQ ID NO.2, and illustratively, the nucleotide sequence encoding the imine reductase mutant "M176I and T240F" is shown in SEQ ID NO.4.

[0021] The present invention also provides a recombinant expression vector, which comprises a gene encoding the imine reductase mutant.

[0022] The present invention also provides a genetically engineered bacterium, wherein the genetically engineered bacterium expresses the imine reductase mutant, which can be obtained by transforming the recombinant expression vector into a host microorganism.

[0023] The present invention also provides an application of a bioenzyme catalyst in catalytic synthesis of chiral phosphorus compounds. The bioenzyme catalyst is a wild-type imine reductase derived from Streptomyces chartreusis, the imine reductase mutant, or the genetically engineered bacteria.

[0024] Specifically, under the condition of blue-violet light irradiation, the bioenzyme catalyst is used to catalyze the free radical coupling of the diarylphosphine oxide shown in formula I and the α-substituted arylethylene shown in formula II to obtain the chiral phosphorus compound shown in formula III; the reaction equation is shown below:

[0025]

[0026] Wherein, Ar is an aromatic group, R2 is hydrogen or an alkyl group, and further preferably, the Ar group is independently selected from phenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-methylphenyl, p-methoxyphenyl, p-ethylphenyl or p-tert-butylphenyl, and R2 is hydrogen or a C1-C10 alkyl group.

[0027] The present invention also provides a method for synthesizing a chiral phosphorus compound, comprising the following steps:

[0028] (1) mixing an organic solution of a diarylphosphine oxide represented by formula I and an organic solution of an α-substituted arylethylene represented by formula II, adding a solubilizing solvent and a biological enzyme catalyst to form a reaction system; the biological enzyme catalyst is a wild-type imine reductase from Streptomyces chartreusis, the imine reductase mutant, or a crude enzyme solution obtained by fermentation and culture of the genetically engineered bacteria;

[0029] (2) Under an inert gas atmosphere, the reaction system is irradiated with 365-450 nm, 20 W blue-violet light at a temperature of 15-50° C. and a rotation speed of 100-300 rpm for 24-48 hours, and then the chiral phosphorus compound represented by formula III is separated and purified;

[0030]

[0031] Furthermore, the solubilizing solvent is glycerol, the inert gas atmosphere is nitrogen atmosphere, and the amount of the imine reductase mutant (the crude enzyme solution is calculated based on the effective amount of the imine reductase mutant) relative to the substrate is 0.5-2 mol%.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The invention transforms the wild-type imine reductase from Streptomyces chartreusis, and the obtained imine reductase mutant has the activity of photocatalytic synthesis of chiral phosphorus compounds; the method of synthesizing chiral phosphorus compounds using the wild-type imine reductase or its mutant from Streptomyces chartreusis as a photocatalytic bioenzyme catalyst is simple, efficient, green and environmentally friendly, and has mild reaction conditions. The chirality of the obtained reaction product is highly controllable and the applicable substrate spectrum range is wide. The corresponding method enriches the current synthesis scheme of chiral phosphorus compounds, fills the gap in the current photoenzyme catalytic synthesis of chiral phosphorus compounds, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A in the figure is the HPLC spectrum of the racemic product 3a; B is the HPLC spectrum of the reaction of substrate 1a and substrate 2a catalyzed by the wild-type imine reductase to generate (R)-3a; C is the HPLC spectrum of the reaction of substrate 1a and substrate 2a catalyzed by the imine reductase mutant "M176I and T240F" to generate (S)-3a. DETAILED DESCRIPTION

[0035] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0036] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0037] Wherein, as used herein, the term "AxxB" means that the amino acid A at position xx is changed to B, for example, "M176I" means that the methionine (M) at position 205 is mutated to leucine (I); "M176I and T240F" means a combined mutation at positions 176 and 240, wherein the methionine (M) at position 176 is mutated to leucine (I), the threonine (T) at position 240 is mutated to phenylalanine (F), and so on.

[0038] In the present invention, the imine reductase mutants constructed in the present invention are all mutants of the wild-type imine reductase (IR14) SEQ ID NO.1 from Streptomyces vinifera.

[0039] Example 1 Construction of the imine reductase IR14 mutant library and screening of mutants

[0040] 1. Construction of pET22b(+)-IR14 plasmid and genetically engineered bacteria E. coli BL21(DE3) / pET22b(+)-IR14

[0041] The amino acid sequence of the wild-type imine reductase is shown in SEQ ID NO. 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 2. The nucleotide sequence was fully synthesized and cloned between the restriction endonuclease sites NdtI and XhoI of the pET-22b(+) vector to obtain the recombinant plasmid pET22b(+)-IR14, which was further transformed into the expression host E. coli BL21(DE3), and the positive clones were picked to obtain the E. coli BL21(DE3) / pET22b(+)-IR14 genetic engineering bacteria.

[0042] 2. Construction of carbonyl reductase mutant library

[0043] Using pET22b(+)-IR14 as a template, the amino acids at positions 173, 176, 240, and 241 in the catalytic active pocket were selected for site-directed mutagenesis. Primers were designed (primer sequences are shown in Table 1), and mutants were constructed using the plasmid rolling circle amplification method. High-fidelity polymerase KOD-plus was used for PCR. The mutation sites and the amino acids after mutation are shown in Table 2.

[0044] The PCR reaction conditions were as follows: 5 μL 10×KOD buffer, 5 μL dNTP (2 mM), 2 μL MgSO4 (25 mM), 20-100 ng template, 2 μL forward primer (10 mM), 2 μL reverse universal primer (10 mM), 1 μL KOD polymerase, and sterile distilled water to 50 μL were added to a PCR reaction system with a total volume of 50 μL. PCR reaction procedure: (1) 94°C pre-denaturation for 4 min, (2) 98°C denaturation for 30 s, (3) 55°C annealing for 30 s, (4) 68°C extension for 4 min, steps (2)-(4) were repeated for a total of 40 cycles. The PCR product was stored at 4°C.

[0045] After the PCR product was verified by agarose gel electrophoresis analysis, restriction endonuclease DpnⅠ was added and digested at 37℃ for 2h. The digestion product was transferred into E.coli BL21(DE3) competent cells and spread on a plate containing kanamycin sulfate, and placed in a 37℃ incubator for 12h. Single clones were selected for sequencing, and the corresponding mutant strains were obtained if the sequencing was correct.

[0046] Table 1 Mutation primers (SEQ ID NO.5 to SEQ ID NO.16)

[0047]

[0048] Table 2 Mutation sites and amino acids after mutation

[0049]

[0050] Example 2 Inducible expression of imine reductase mutants

[0051] The single colonies in the mutant library were inoculated into 1 mL of LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) containing ampicillin antibiotics, and cultured overnight in a shaker at 37°C and 200 rpm for activation to obtain seed liquid. The overnight cultured seed liquid was transferred to 200 mL of TB medium containing ampicillin antibiotics at a 1% inoculation amount and cultured at 37°C and 200 rpm until OD 600The pH value is about 0.6 to 0.8, 0.2mM IPTG (isopropyl-β-D-thiogalactose) is added, and it is induced at 18℃, 200rpm for 18 to 20h. The bacteria are collected by centrifugation at 4℃, 8000rpm, and frozen in a -78℃ refrigerator for 1h, then taken out and thawed. According to the mass of the cells, 20mM Tris-HCl pH 9.0 buffer is added to the centrifuge tube at a ratio of 1g cells to 5mL and shaken evenly. The cells are lysed for 25min using an ultrasonic cell crusher under ice bath conditions. Take out the lysed cell solution, centrifuge it at 4℃, 9600rpm for 20min, and retain the supernatant, i.e., the crude enzyme solution, for subsequent reaction detection.

[0052] Example 3 Synthesis of Chiral Phosphorus Compounds

[0053] The wild-type imine reductase IR14 and the imine reductase mutant were cultured and expressed according to the induction expression method in Example 2, and then the crude enzyme solution was collected. The crude enzyme solution was used as a biological enzyme catalyst to catalyze the free radical coupling of the diarylphosphine oxide shown in formula I and the α-substituted arylethylene shown in formula II to obtain the chiral phosphorus compound shown in formula III; the reaction equation is shown in the following formula:

[0054]

[0055] Substrate 1a is diphenylphosphine oxide (Ar group is phenyl), substrate 2a is methylstyrene (Ar group is phenyl, R2 is methyl), and the structural formula of chiral phosphorus compound 3a is as follows:

[0056]

[0057] In a 10mL Shrek tube, add 100uL of dimethyl sulfoxide solution containing 3.2mg / 100uL substrate 1a, 100uL of dimethyl sulfoxide solution containing 0.4mg / 100uL substrate 2a, 200uL glycerol and 1600uL of crude enzyme solution of wild-type imine reductase or its mutant (the buffer is 20mM pH9.0 Tris-HCl buffer, and the amount of wild-type imine reductase or its mutant relative to the substrate is 0.5-2mol%), and replace the system gas by nitrogen freezing and pumping for 3 rounds. Place it on a magnetic stirrer oscillator, 30°C, 500rpm, 365nm 20W blue-violet light irradiation, and react for 24h. After the reaction, extract it with ethyl acetate 3 times, then spin dry the solvent, and use HPLC to separate and identify the product. The yield and ee value of different mutants are shown in Table 3.

[0058] Table 3 Results of synthesis of chiral phosphorus compounds catalyzed by wild-type imine reductase WT and its mutants

[0059]

[0060] The best solution is to add 100uL of dimethyl sulfoxide solution containing 3.2mg / 100uL substrate 1a, 100uL of dimethyl sulfoxide solution containing 0.4mg / 100uL substrate 2a, 200uL glycerol and 1600uL wild-type imine reductase to a 10mL Shrek tube, replace the system gas by freezing and pumping with nitrogen for 3 rounds, place on a magnetic stirrer, irradiate with 365nm 20W blue-violet light at 30℃, 500rpm, and react for 24h. After the reaction, extract with ethyl acetate 3 times, then spin dry the solvent, and use Figure 1 A in the figure was used as a reference, and the product was separated and identified by HPLC. The results showed that the wild-type imine reductase could obtain (R)-diphenyl (2-phenylpropyl) phosphine oxide with a yield of 80% and >99% ee, such as Figure 1 As shown in B.

[0061] Optimally, 100uL of a dimethyl sulfoxide solution containing 3.2mg / 100uL substrate 1a, 100uL of a dimethyl sulfoxide solution containing 0.4mg / 100uL substrate 2a, 200uL glycerol, and 1600uL imine reductase mutant strain "M176I and T240F" were added to a 10mL Shrek tube, and nitrogen was passed through the tube for 3 rounds of replacement of the system gas, and the mixture was placed on a magnetic stirrer, 30°C, 500rpm, 365nm 20W blue-violet light irradiation, and reacted for 24h. After the reaction was completed, the mixture was extracted with ethyl acetate 3 times, and then the solvent was dried by spin drying, and the product was separated and identified by HPLC. (S)-diphenyl (2-phenylpropyl) phosphine oxide with an ee of >99% can be obtained with a yield of 75%, such as Figure 1 As shown in C.

[0062] The above-described embodiments provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that: The wild-type imine reductase from Streptomyces chartreusis is obtained by subjecting the wild-type imine reductase from Streptomyces chartreusis to single-point mutation or double-point mutation at positions 1 to 289 of the amino acid sequence shown in SEQ ID NO.1; The specific single point mutation site is any of the following: mutation at position 173; mutation at position 176; mutation at position 240; mutation at position 241; The specific double point mutation sites are: Combined mutations at positions 176 and 240.

2. The imine reductase mutant according to claim 1, characterized in that: The specific single point mutation is any of the following: L173A; L173F; M176A; M176L; M176F; M176I; M176N; T240F; T240W; T240Y; M241F; And / or, the specific double point mutation is any one of the following: M176L and T240F; M176I and T240F.

3. A gene encoding the imine reductase mutant according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene according to claim 3.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express the imine reductase mutant according to claim 1 or 2.

6. Use of a bio-enzyme catalyst in the catalytic synthesis of chiral phosphorus compounds, characterized in that: The bio-enzyme catalyst is a wild-type imine reductase from Streptomyces chartreusis, an imine reductase mutant according to claim 1 or 2, or a genetically engineered bacterium according to claim 5.

7. The use according to claim 6, characterized in that: Under the condition of blue-violet light irradiation, the bioenzyme catalyst is used to catalyze the free radical coupling of the diaryl phosphine oxide shown in formula I and the α-substituted aryl ethylene shown in formula II to obtain the chiral phosphorus compound shown in formula III; the reaction equation is shown in the following formula: Wherein, Ar is an aromatic group, and R2 is hydrogen or an alkyl group.

8. The use according to claim 7, characterized in that: The Ar group is independently selected from phenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-methylphenyl, p-methoxyphenyl, p-ethylphenyl or p-tert-butylphenyl, and R2 is hydrogen or a C1-C10 alkyl group.

9. A method for synthesizing a chiral phosphorus compound, characterized in that: The following steps are involved: (1) An organic solution of a diarylphosphine oxide represented by formula I and an organic solution of an α-substituted arylethylene represented by formula II are mixed, and a solubilizing solvent and a biological enzyme catalyst are added to form a reaction system; the biological enzyme catalyst is a wild-type imine reductase from Streptomyces chartreusis, an imine reductase mutant described in claim 1 or 2, or a crude enzyme solution obtained by fermentation culture of the genetically engineered bacteria described in claim 5; (2) Under an inert gas atmosphere, the reaction system is subjected to a temperature of 15 to 50° C. and irradiated with 365 to 450 nm blue-violet light for 24 to 48 hours, and then the chiral phosphorus compound represented by formula III is separated and purified, wherein Ar and R2 have the same definitions as above; 10. The method according to claim 9, characterized in that: The dissolving solvent is glycerol, and the inert gas atmosphere is nitrogen.

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