Imine reductase mutants and method for synthesis of chiral phosphorus compounds

Abstract

The application discloses an imine reductase mutant and a synthesis method of a chiral phosphorus compound, belongs to the field of enzyme engineering and organic synthesis, and is obtained by single-point mutation or double-point mutation of a wild-type imine reductase from Streptomyces chartreusis at positions 1 to 289 of the amino acid sequence shown in SEQ ID NO. 1. The wild-type imine reductase from Streptomyces chartreusis, the imine reductase mutant or the genetically engineered bacteria expressing the imine reductase mutant can be used as a biological enzyme catalyst to catalyze free radical coupling of diaryl phosphine oxide and alpha-substituted aryl ethylene to obtain a chiral phosphorus compound, enriches the current synthesis scheme of the chiral phosphorus compound, and has a wide industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and organic synthesis, specifically relating to methods for synthesizing imine reductase mutants and chiral phosphorus compounds. Background Technology

[0002] Chiral phosphorus compounds, including phosphates and phosphine, are widely used in materials science, agricultural chemistry, drug synthesis, and ligand catalysis. Introducing phosphorus-containing functional groups into organic molecules can effectively alter their medicinal properties and catalytic activity. Over the past decade, numerous therapeutic drugs and chiral catalysts containing chiral phosphorus groups have been discovered and characterized (e.g., Chinese patent documents with publication numbers CN109476687A, CN118126082A, and CN111393243A). The significant value of these compounds has stimulated the development of synthetic methods for chiral phosphorus compounds.

[0003] However, despite the enormous application potential of chiral phosphorus compounds, their expensive and complex synthetic routes limit their further large-scale production. Currently, the main methods for synthesizing phosphorus-containing compounds include traditional nucleophilic addition methods, transition metal catalysis, and free radical reactions. Chemical Reviews (2011, 111, 7981-8006). However, traditional CP bond formation strategies relying on nucleophilic substitution reactions require high substrate stability, stringent reaction conditions, and difficulty in controlling chirality. While chiral ligand catalysts or metal photocatalysis can achieve the synthesis of phosphorus-containing compounds under mild conditions with controllable chirality, the reaction costs are high. In fact, in many transition metal-catalyzed CP coupling reactions, the level of stereocontrol is often insufficient to meet practical application requirements. Therefore, developing an efficient, universal method for achieving enantioselective CP bond construction under mild conditions is of great significance.

[0004] Among various chiral synthesis schemes, photoenzyme-catalyzed synthesis has become a strong competitive option for the green synthesis of chiral compounds due to its direct utilization of light energy, energy efficiency, environmental friendliness, cleanliness, high efficiency, and absence of side reactions. Currently, there are numerous cases of using photoenzymes to catalyze asymmetric synthesis. For example, Chinese patent document CN117987483A discloses a method for photoenzyme-catalyzed asymmetric reduction to prepare chiral α-benzylamine and its derivatives, and Chinese patent document CN116179623A discloses the application of photoenzyme TPe in asymmetric catalytic synthesis of chiral compounds, providing new ideas for the engineering modification of enzymes and their catalytic synthesis of chiral phosphorus compounds. However, no scheme for photoenzyme-catalyzed synthesis of chiral CP bonds has been found. Therefore, how to explore the photocatalytic ability of non-natural photoenzymes and realize photoenzyme-catalyzed synthesis of chiral phosphorus compounds is an important issue that urgently needs to be addressed. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides an imine reductase mutant and a method for synthesizing chiral phosphorus compounds. The imine reductase mutant exhibits photocatalytic activity for synthesizing chiral phosphorus compounds, and the corresponding method for synthesizing chiral phosphorus compounds uses mild reaction conditions, is environmentally friendly, simple, and efficient.

[0006] The specific technical solution adopted is as follows:

[0007] An imine reductase mutant was developed from *Streptomyces cerevisiae* (…). Streptomyces chartreusis The wild-type imine reductase (IR14) is obtained by single-point 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 one of the following:

[0009] A mutation at position 173;

[0010] A mutation at position 176;

[0011] A mutation at position 240;

[0012] A mutation at position 241;

[0013] The specific sites of the double-point mutation are:

[0014] Combination mutations at positions 176 and 240.

[0015] Furthermore, a specific single-point mutation can be any of the following:

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

[0017] Furthermore, the specific two-point mutation can be any of the following:

[0018] M176L with T240F; M176I with T240F.

[0019] Preferably, the double-point mutation is 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 wild-type imine reductase IR14 is shown in SEQ ID NO.2, and, as an example, 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 comprising a gene encoding the imine reductase mutant.

[0022] The present invention also provides a genetically engineered bacterium that expresses the imine reductase mutant and can be obtained by transformation of the recombinant expression vector into a host microorganism.

[0023] This invention also provides the application of a bio-enzyme catalyst in the catalytic synthesis of chiral phosphorus compounds, wherein the bio-enzyme catalyst is *Streptomyces oryzae*. Streptomyces chartreusis The source is wild-type imine reductase, the imine reductase mutant, or the genetically engineered bacteria.

[0024] Specifically, under blue-violet light irradiation, the diarylphosphine oxide of Formula I and the α-substituted arylethylene of Formula II undergo free radical coupling catalyzed by the aforementioned bio-enzyme catalyst to obtain the chiral phosphorus compound of Formula III; the reaction equation is shown below:

[0025]

[0026] Wherein, Ar is an aromatic group, R2 is hydrogen or an alkyl group, and more 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 to C10 alkyl group.

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

[0028] (1) The organic solution of diarylphosphine oxide shown in Formula I and the organic solution of α-substituted aryl ethylene shown in Formula II are mixed, and a solubilizing solvent and a bio-enzyme catalyst are added to form a reaction system; the bio-enzyme catalyst is Streptomyces oryzae. Streptomyces chartreusis Wild-type imine reductase, the imine reductase mutant, or crude enzyme solution obtained after fermentation culture of the genetically engineered bacteria;

[0029] (2) Under an inert gas atmosphere, the reaction system was subjected to a temperature of 15–50 °C, a rotation speed of 100–300 rpm, and irradiation with 365–450 nm and 20 W blue-violet light for 24–48 h. The chiral phosphorus compound shown in Formula III was then separated and purified.

[0030]

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

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] This invention utilizes Streptomyces cerevisiae to treat Streptomyces cerevisiae. Streptomyces chartreusis The wild-type imine reductase from *Streptomyces cerevisiae* was modified to obtain an imine reductase mutant with photocatalytic activity for the synthesis of chiral phosphorus compounds. The method for synthesizing chiral phosphorus compounds using wild-type imine reductase or its mutant from *Streptomyces cerevisiae* as a photocatalytic enzyme catalyst is simple, efficient, environmentally friendly, and operates under mild reaction conditions. The chirality of the obtained reaction products is highly controllable, and the applicable substrate spectrum is broad. This method enriches the current synthetic schemes for chiral phosphorus compounds, fills the gap in the current photocatalytic synthesis of chiral phosphorus compounds, and has broad prospects for industrial application. Attached Figure Description

[0034] Figure 1 In the diagram, A represents the HPLC chromatogram of racemic product 3a; B represents the product generated by the reaction of wild-type imine reductase with substrate 2a. R HPLC chromatogram of )-3a; C represents the reaction of substrate 1a and substrate 2a catalyzed by the imine reductase mutant "M176I and T240F" to produce ( S HPLC spectrum of )-3a. Detailed Implementation

[0035] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0036] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0037] Wherein, as used herein, the term “AxxB” indicates that amino acid A at position xx is changed to B. For example, “M176I” indicates that methionine (M) at position 176 is mutated to leucine (I); “M176I and T240F” indicates a combination mutation at positions 176 and 240, in which methionine (M) at position 176 is mutated to leucine (I) and threonine (T) at position 240 is mutated to phenylalanine (F), and so on.

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

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

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

[0041] The amino acid sequence of the wild-type imine reductase is shown in SEQ ID NO.1, and its corresponding nucleotide sequence is shown in SEQ ID NO.2. This nucleotide sequence was totalized and cloned into 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 On BL21(DE3), positive clones are selected to obtain... E.coli BL21(DE3) / pET22b(+)-IR14 genetically engineered bacteria.

[0042] 2. Construction of a carbonyl reductase mutant library

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

[0044] The PCR reaction conditions are as follows: In a PCR reaction system with a total volume of 50 μL, add 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 add sterile distilled water to a final volume of 50 μL. PCR reaction program: (1) 94 ℃ pre-denaturation for 4 min, (2) 98 ℃ denaturation for 30 s, (3) 55 ℃ annealing for 30 s, (4) 68 ℃ extension for 4 min. Steps (2)-(4) are performed for a total of 40 cycles. Store the PCR products at 4 ℃.

[0045] After the PCR products were verified by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37 °C for 2 h. The digested products were then transferred to... E. coli BL21 (DE3) competent cells were plated on plates containing kanamycin sulfate and incubated statically at 37 °C for 12 h. Single clones were picked and sequenced; those with correct sequencing results were the corresponding mutant strains.

[0046] Table 1. Mutant primers (SEQ ID NO.5~SEQ ID NO.16)

[0047]

[0048] Table 2. Mutation sites and mutated amino acids

[0049]

[0050] Example 2: Induced expression of imine reductase mutant

[0051] Single colonies from the mutant library were inoculated into 1 mL of LB liquid medium containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured overnight at 37 °C and 200 rpm to activate the seed culture. The overnight seed culture was then transferred at a 1% inoculation rate to 200 mL of TB medium containing ampicillin and cultured at 37 °C and 200 rpm until OD reached [the desired growth rate]. 600The bacterial culture volume was approximately 0.6–0.8. 0.2 mM IPTG (isopropyl-β-D-thiogalactose) was added, and the mixture was incubated at 18 °C and 200 rpm for 18–20 h. The cells were collected by centrifugation at 4 °C and 8000 rpm, then frozen at -78 °C for 1 h. After thawing, 20 mM Tris-HCl pH 9.0 buffer was added to centrifuge tubes at a ratio of 1 g cells to 5 mL, and the mixture was shaken thoroughly. The cells were lysed using an ultrasonic cell disruptor for 25 min under ice bath conditions. The lysed cell solution was collected and centrifuged at 4 °C and 9600 rpm for 20 min. The supernatant, i.e., the crude enzyme solution, was collected for subsequent reaction analysis.

[0052] Example 3 Synthesis of chiral phosphorus compounds

[0053] Wild-type imine reductase IR14 and imine reductase mutants were induced to express proteins according to the culture and expression method in Example 2. The crude enzyme solution was then collected and used as a biocatalyst to catalyze the radical coupling of diarylphosphine oxide (Formula I) and α-substituted arylethylene (Formula II) to obtain the chiral phosphorus compound (Formula III). The reaction equation is shown below:

[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 shown as follows: .

[0056] Add 100 μL of a 3.2 mg / 100 μL dimethyl sulfoxide solution, 100 μL of a 0.4 mg / 100 μL dimethyl sulfoxide solution, 200 μL of glycerol, and 1600 μL of crude enzyme solution of wild-type imine reductase or its mutant to a 10 mL Shrek tube (buffer was 20 mM pH 9.0 Tris-HCl buffer, and the amount of wild-type imine reductase or its mutant relative to the substrate was 0.5–2 mol%). Purge the system with nitrogen three times to replace the gas. Place the tube on a magnetic stirrer and irradiate with 365 nm 20W blue-violet light at 30 °C, 500 rpm, for 24 h. After the reaction, extract three times with ethyl acetate, then evaporate the solvent and separate and identify the product using HPLC. The yields and ee values ​​of different mutant strains are shown in Table 3.

[0057] Table 3 Results of the synthesis of chiral phosphorus compounds catalyzed by wild-type imine reductase WT and its mutants

[0058]

[0059] Optimally, in a 10 mL Shrek tube, add 100 μL of a dimethyl sulfoxide solution containing 3.2 mg / 100 μL of substrate 1a, 100 μL of a dimethyl sulfoxide solution containing 0.4 mg / 100 μL of substrate 2a, 200 μL of glycerol, and 1600 μL of wild-type imine reductase. Purge the system with nitrogen three times to replace the gas, then place it on a magnetic stirrer and irradiate it with 365 nm 20 W blue-violet light at 30 °C, 500 rpm, for 24 h. After the reaction, extract three times with ethyl acetate, then evaporate the solvent to dryness. Figure 1 Using A as a reference, the product was separated and identified by HPLC. The results showed that using wild-type imine reductase could yield >99% ee in 80% yield. R )-Diphenyl(2-phenylpropyl)phosphine oxide, such as Figure 1 As shown in B in the diagram.

[0060] Optimally, in a 10 mL Shrek tube, add 100 μL of a dimethyl sulfoxide solution containing 3.2 mg / 100 μL of substrate 1a, 100 μL of a dimethyl sulfoxide solution containing 0.4 mg / 100 μL of substrate 2a, 200 μL of glycerol, and 1600 μL of the imine reductase mutant strain “M176I and T240F”. Purge the system with nitrogen three times to replace the gas, place it on a magnetic stirrer, and irradiate with 365 nm 20 W blue-violet light at 30 °C, 500 rpm, for 24 h. After the reaction, extract three times with ethyl acetate, then evaporate the solvent and separate and identify the product using HPLC. >99% ee can be obtained in 75% yield. S )-Diphenyl(2-phenylpropyl)phosphine oxide, such as Figure 1 As shown in C.

[0061] The above embodiments provide a detailed description of the technical solution 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, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

3.

2. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria express the imine reductase mutant of claim 1.

3. The application of a bio-enzyme catalyst in the catalytic synthesis of chiral phosphorus compounds, characterized in that, The bio-enzyme catalyst is the imine reductase mutant of claim 1 or the genetically engineered bacterium of claim 2, and the structural formula of the chiral phosphorus compound is: Where Ar is phenyl and R2 is methyl; When the bio-enzyme catalyst is an imine reductase mutant or a genetically engineered bacterium, the chiral phosphorus compound is in the S configuration.

4. The application according to claim 3, characterized in that, Under blue-violet light irradiation, the diarylphosphine oxide of Formula I and the α-substituted arylethylene of Formula II undergo free radical coupling catalyzed by the aforementioned bio-enzyme catalyst to obtain the chiral phosphorus compound of Formula III; the reaction equation is shown below: ； Where Ar is phenyl and R2 is methyl.

5. A method for synthesizing a chiral phosphorus compound, characterized in that, Includes the following steps: (1) Mix the organic solution of diarylphosphine oxide shown in Formula I and the organic solution of α-substituted aryl ethylene shown in Formula II, add a solubilizing solvent and a bio-enzyme catalyst to form a reaction system; the bio-enzyme catalyst is the imine reductase mutant of claim 1 or the crude enzyme solution obtained after fermentation culture of the genetically engineered bacteria of claim 2. (2) Under an inert gas atmosphere, the reaction system was subjected to a temperature of 15–50 °C and irradiation with 365–450 nm blue-violet light for 24–48 h. The chiral phosphorus compound shown in Formula III was then separated and purified, where Ar is phenyl and R2 is methyl. ； When the bio-enzyme catalyst is an imine reductase mutant or a genetically engineered bacterium, the chiral phosphorus compound is in the S configuration.

6. The method according to claim 5, characterized in that, The cosolvent is glycerol, and the inert gas atmosphere is nitrogen.

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