CN117305258A - Synthesis method of chiral lactone compound, carbonyl reductase ChKRED20 mutant and application - Google Patents

Synthesis method of chiral lactone compound, carbonyl reductase ChKRED20 mutant and application Download PDF

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CN117305258A
CN117305258A CN202311261575.4A CN202311261575A CN117305258A CN 117305258 A CN117305258 A CN 117305258A CN 202311261575 A CN202311261575 A CN 202311261575A CN 117305258 A CN117305258 A CN 117305258A
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carbonyl reductase
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chkred20
chiral lactone
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CN117305258B (en
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王娜
李静
施明亮
刘艳红
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Sichuan University
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Abstract

The invention discloses a synthesis method of chiral lactone compounds, and a carbonyl reductase ChKRED20 mutant and application thereof, wherein aldehyde compounds and compounds with a structure shown as a formula II are firstly used as initial substrates, and a photocatalyst (TBADT) is used for synthesizing a substrate compound a, so that the substrate spectrum of the carbonyl reductase ChKRED20 mutant is enlarged, and the limitation that one enzyme can only act on one substance or one class of substances with similar molecular structures is broken through.

Description

Synthesis method of chiral lactone compound, carbonyl reductase ChKRED20 mutant and application
Technical Field
The invention relates to the technical field of chemical biology, in particular to a synthesis method of chiral lactone compounds, carbonyl reductase ChKRED20 mutants and application thereof.
Background
Under the guidance of the principle of green chemistry and sustainable development, a strategy for developing novel chiral lactone compounds with high efficiency is needed to be solved. The lactone structure is an important skeleton which forms a plurality of natural compounds and high-value medical intermediates, so that how to efficiently synthesize chiral lactone compounds from simple substrates has practical application significance. Today, the usual chemical synthesis in the preparation of chiral lactone compounds has a number of disadvantages:
1. the chiral catalyst has complex synthesis, harsh catalytic reaction conditions and large pollution;
2. the side reaction is more, the separation from the system is difficult, and the recovery effect is poor;
3. high energy consumption and the need to use a heating reactor or a low-temperature reactor.
Therefore, the synthesis of chiral lactone compounds based on a method combining biocatalysis and photocatalysis becomes a research hot spot, and the biocatalysis method can promote the reaction and precisely control the stereochemical result, so that the method is rapidly popularized in the aspect of synthesizing enantiomer lactones, and the photocatalytic reaction is widely researched due to low energy consumption and small pollution. However, considering that enzymes have instability in industrial production and limited application range, there are still problems to be solved:
1. the coenzyme or cofactor used in the enzyme catalysis process is expensive, and the economic benefit is not high;
2. enzyme catalysis has specificity, and one enzyme can only act on one substance, or a substance with similar molecular structure;
3. many photocatalysts are complex to synthesize and have large pollution in the synthesis process.
Disclosure of Invention
In order to solve the problems, the invention combines a photocatalyst tetrabutylammonium decatungstate (TBADT) with carbonyl reductase ChKRED20, and can generate chiral lactone compounds from simple and easily available aldehyde compounds and compounds with structural formula shown as II as primary substrates by a two-step method.
Based on this, it was an object of the present invention to provide a carbonyl reductase ChKRED20 mutant for the synthesis of chiral lactone compounds.
The technical scheme for solving the technical problems is as follows:
a carbonyl reductase ChKRED20 mutant for synthesizing chiral lactone compounds takes the amino acid sequence of wild carbonyl reductase ChKRED20 as a starting sequence, and mutates amino acids at least two of 97 th, 153 th and 188 th positions;
wherein, taking the amino acid sequence of wild carbonyl reductase ChKRED20 as a starting sequence, mutating the 97 th glutamine into leucine;
taking the amino acid sequence of wild carbonyl reductase ChKRED20 as a starting sequence, and mutating 153 th serine into leucine;
the amino acid sequence of wild carbonyl reductase ChKRED20 is taken as a starting sequence, and the 188 th tyrosine is mutated into serine.
Another object of the present invention is the use of carbonyl reductase ChKRED20 mutants for the synthesis of chiral lactone compounds.
The invention also provides a synthesis method of the chiral lactone compound, which comprises the following steps:
step 1, under the atmosphere of protective gas, dissolving an aldehyde compound and a compound with a structural formula shown as II in a solvent, adding a photocatalyst tetrabutylammonium decatungstate, and then reacting for 10-14 hours in a photoreactor to obtain a substrate compound a;
step 2, dispersing a substrate compound a and a carbonyl reductase ChKRED20 mutant in a solvent for enzyme catalytic reaction to prepare a chiral lactone compound;
wherein, formula II's structural formula is:wherein R is 1 Is->And->Any one of them; which is a kind ofThe synthetic process route of the chiral lactone compound is shown in figure 2. Preferably, the compound shown as II in the present invention may be methyl acrylate, methyl methacrylate, methyl crotonate, etc.
The beneficial effects of the invention are as follows: in the invention, an aldehyde compound and a compound with a structural formula shown as II are firstly used as initial substrates, and a photocatalyst (tetrabutylammonium decatungstate, TBADT) is used for synthesizing a substrate compound a, so that the substrate spectrum of a carbonyl reductase ChKRED20 mutant is enlarged, and the limitation that one enzyme can only act on one substance or one kind of substances with similar molecular structures is broken through.
Further, the structural formula of the aldehyde compound is shown as a formula I, and the structural formula of the formula I is as follows:wherein R is Any one of them;
R 2 h, F, NC, cl, br, me, meO, CF of a shape of H, F, NC, cl, br, me, meO, CF 3 、Et、tBu、CF 3 O、CF 3 Any one of S and EtO;
R 3 f, me, CN, br and Cl;
R 4 is Me or CF 3 。
Further, in the step 2, the molar ratio of the aldehyde compound to the compound with the structural formula shown as II is 1:1-5:4; the protective gas is nitrogen; the mass ratio of the photocatalyst to the compound with the structural formula shown as II is 3:2-2:1; preferably, the molar ratio of the aldehyde compound to the compound shown in the structural formula II is 2:1.
Further, the conditions for the enzyme-catalyzed reaction in step 3 are: reacting for 42-54 h at 35-38 ℃. Preferably, the conditions of the enzyme-catalyzed reaction are: the reaction was carried out at 37℃for 48h.
Further, the preparation method of the photocatalyst tetrabutylammonium decatungstate is as follows:
the tungstate and tetrabutylammonium bromide are dissolved in a solvent and heated, and the pH value is regulated to be 2-3, and then the reaction is continued for 20-40 min to prepare the photocatalyst. Preferably, the pH is 2 and the reaction time is 30min.
Further, the molar ratio of tungstate to tetrabutylammonium bromide is 25-32:10-17; the tungstate is sodium tungstate; the solvent is deionized water; the temperature after heating is 85-95 ℃.
The invention has the following beneficial effects:
according to the invention, a photocatalyst butylammonium decatungstate (TBADT) and a carbonyl reductase ChKRED20 mutant are combined with each other, and a chiral lactone compound is synthesized by using a simple and easily available aldehyde compound and a compound with a structural formula shown as II;
in addition, the invention designs the wild ChKRED20 (namely, takes the amino acid sequence of the wild carbonyl reductase ChKRED20 as a starting sequence and mutates the amino acids at least two of the 97 th, 153 th and 188 th amino acids) to resist a large volume of gamma-keto ester substrate, and uses the substrate compound a obtained after photocatalysis to successfully and efficiently synthesize a series of chiral lactone compounds with high enantioselectivity;
thus, the combination of the photocatalyst butylammonium decatungstate (TBADT) with the carbonyl reductase ChKRED20 mutant successfully expands the substrate spectrum, while the mutant strain M3C1 (i.e., the amino acid sequence of the carbonyl reductase ChKRED20 is taken as a starting sequence, and the amino acids at the 97 th, 153 th and 188 th positions are mutated) has better substrate universality.
Drawings
FIG. 1 is a schematic diagram of the synthesis route of substrate compound a;
FIG. 2 is a synthetic scheme for chiral lactone compound e;
FIG. 3 is a hydrogen spectrum of substrate compound 2 a;
FIG. 4 is a carbon spectrum of substrate compound 2 a;
FIG. 5 is a liquid phase spectrum of racemate 2e standard;
FIG. 6 is a liquid phase spectrum of chiral lactone compound 2 e;
FIG. 7 is a schematic diagram of the structure of a photo-reactor;
FIG. 8 is a hydrogen spectrum of substrate compound 1 a;
FIG. 9 is a hydrogen spectrum of substrate compound 5 a;
FIG. 10 is a hydrogen spectrum of substrate compound 10 a;
FIG. 11 is a hydrogen spectrum of substrate compound 12 a;
FIG. 12 is a hydrogen spectrum of substrate compound 15 a;
FIG. 13 is a hydrogen spectrum of substrate compound 16 a;
FIG. 14 is a hydrogen spectrum of chiral lactone compound 5 e;
FIG. 15 is a hydrogen spectrum of chiral lactone compound 10 e;
FIG. 16 is a hydrogen spectrum of chiral lactone compound 17 e;
FIG. 17 is a hydrogen spectrum of chiral lactone compound 26e.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Synthesis of tetrabutylammonium decatungstate (TBADT), a photocatalyst synthesized by:
sodium tungstate (30.3 mmol) and tetrabutylammonium bromide (14.9 mmol) are dissolved in deionized water (100 mL) to form a reaction system, then the reaction system is heated to 90 ℃ and the pH value of the reaction system is adjusted to 2 by using a 2M hydrochloric acid solution and is quickly mixed to form a suspension, the reaction is continued at 90 ℃ for 30min, after the reaction is finished, the reaction is cooled to room temperature and filtered to obtain white solid, and the obtained white solid is washed by water and then is dried in vacuum to obtain a crude product.
The crude product was dissolved in hot acetonitrile and then left at-20 ℃ for 12 hours, the solid particles were collected again with a filter and washed with a small amount of cold acetonitrile to obtain pure TBADT. In addition, the filtrate may be concentrated again to prepare TBADT.
Example 2
Constructing a carbonyl reductase ChKRED20 mutant, wherein the amino acid sequence of the wild carbonyl reductase ChKRED20 is taken as a starting sequence, and amino acids at least two of 97 th, 153 th and 188 th are mutated; wherein the wild-type ChKRED20 sequence is as follows: ATG GGA ATT TTA GAC AAC AAA GTA GCA CTT GTT ACA GGA GCAGGA TCC GGA ATC GGA TTA GCT GTT GCT CAT TCG TAT GCA AAAGAA GGC GCC AAA GTT ATT GTA TCC GAT ATT AAT GAA GAT CACGGT AAC AAA GCA GTC GAA GAC ATT AAA GCA CAA GGC GGG GAAGCG TCT TTT GTA AAA GCA GAT ACT TCA AAC CCT GAA GAA GTGGAA GCT TTA GTA AAA AGA ACA GTA GAA ATC TAC GGA AGA CTTGAT ATT GCA TGT AAT AAT GCG GGA ATC GGT GGC GAA CAG GCGCTG GCA GGC GAT TAC GGT CTC GAC AGC TGG CGA AAA GTA TTAAGC ATA AAT CTT GAT GGC GTA TTC TAC GGG TGC AAA TAT GAGTTA GAA CAA ATG GAA AAA AAC GGG GGC GGC GTT ATT GTG AATATG GCC TCT ATT CAT GGT ATT GTT GCT GCA CCG CTT TCC TCAGCC TAC ACT TCT GCA AAG CAC GCA GTG GTA GGG CTT ACT AAAAAT ATA GGA GCA GAA TAC GGA CAG AAA AAT ATC CGT TGC AATGCG GTG GGG CCT GCT TAT ATT GAA ACC CCG CTG TTG GAA AGCCTG ACA AAG GAA ATG AAG GAA GCA CTG ATT TCA AAA CAT CCGATG GGA AGA CTG GGA AAA CCT GAA GAA GTA GCA GAA CTG GTGTTG TTC CTG AGT TCA GAA AAA TCA TCT TTT ATG ACG GGA GGCTAT TAT CTT GTA GAT GGT GGC TAC ACG GCA GTT TAA (SEQ ID NO. 1).
1. The construction of mutant M1B (Q97L) comprises the following steps: :
step 1, taking wild ChKRED20 as a DNA template sequence, and carrying out site-directed mutagenesis on 97 th glutamine by utilizing a PCR technology to obtain leucine. Selecting a forward primer: 5'-ggaatcggtggcgaacttgcgctggcaggcgat-3' (SEQ ID NO. 2), reverse primer: 3'-atcgcctgccagcgcaagttcgccaccgattcc-5' (SEQ ID NO. 3). PCR conditions: pre-denaturation at 95℃for 2min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 6min, 25 cycles total, and extension at 72℃for 10min. After completion of PCR, 1. Mu.L of DpnI was added thereto and digested at 37℃for 1 hour. Then 10. Mu.L of E.coli DH 5. Alpha. Competent cells were transferred into 50. Mu.L by heat shock, and then the monoclonal strain was picked up and sent to the company for sequencing verification.
Step 2, collecting the mutant library clone in the step 1, extracting plasmids, transferring the plasmids into an escherichia coli expression strain BL21-DE3, coating an LB plate containing kanamycin, and culturing for 12 hours. The monoclonal was picked up in 96-well plates, each containing 200. Mu.L of TB medium (containing 50. Mu.g/mL kanamycin, 0.5mM IPTG), incubated at 30℃at 180rpm with shaking for 18h. The individual clones were replicated on LB solid medium plates with a 96-well plate replicator, incubated at 37℃for 12h, and stored in a refrigerator at 4 ℃.
Preparation of pure enzyme solution: the monoclonal was selected and cultured in LB medium (containing 50. Mu.g/mL kanamycin) at 37℃overnight, transferred to TB medium (containing 50. Mu.g/mL kanamycin) at 1% of the inoculum size, cultured at 37℃for 3 hours, induced by adding 0.5mM IPTG, and cultured at 30℃for 18 hours. The cells were collected by centrifugation at 6000rpm at 4 ℃. Resuspended in Buffer A (50 mM sodium phosphate Buffer, pH 8.0, 300mM NaCl,10mM imidazole), and the cell homogenizers were broken down before centrifugation at 13000rpm at 4℃for 20min. The purification was performed by nickel column affinity chromatography (Bio-Rad), the supernatant was added to the column equilibrated with Buffer A, gently mixed for 30min, the hetero protein was rinsed with Buffer A containing 20mM imidazole, the target protein was eluted with Buffer A containing 250mM imidazole, and imidazole was removed by dialysis with sodium phosphate Buffer (0.1M, pH 8.0), and finally the purity was identified by electrophoresis.
2. The construction of mutant M1A (S153L) was identical to that of mutant M1B (Q97L) and serine at position 153 was site-directed mutated to leucine using PCR techniques, except that the primers used were as follows:
S153L-Forward primer: 5'-gttgctgcaccgcttctttcagcctacacttct-3' (SEQ ID NO. 4);
S153L-reverse primer: 3'-agaagtgtaggctgaaagaagcggtgcagcaac-5' (SEQ ID NO. 5).
3. The construction method of the mutant M2 (Q97L/S153L) is the same as that of the mutant M1B (Q97L), except that:
in the step 1, mutant M1B (Q97L) is taken as a DNA template sequence, site-directed mutagenesis is carried out on 153 th amino acid of the modified gene, and specifically serine at 153 th site is mutated into leucine, so that an M2 (Q97L/S153L) mutant strain is obtained; the primers used were: S153L-Forward primer: 5'-gttgctgcaccgcttctttcagcctacacttct-3' (SEQ ID NO. 6),
S153L-reverse primer: 3'-agaagtgtaggctgaaagaagcggtgcagcaac-5' (SEQ ID NO. 7).
4. The construction method of the mutant M3C1 (Q97L/S153L/Y188S) is the same as that of the mutant M1B (Q97L), except that:
in the step 1, mutant M2 is used as a DNA template sequence, site-directed mutagenesis is carried out on amino acid 188 of the modified gene, specifically, tyrosine 188 is mutated into serine, and an M3C1 (Q97L/S153L/Y188S) mutant strain is obtained; the primers used were:
M3C 1-Forward primer: 5'-gcggtggggcctgctagcattgaaaccccgctg-3' (SEQ ID NO. 8),
M3C 1-reverse primer: 3'-cagcggggtttcaatgctagcaggccccaccgc-5' (SEQ ID NO. 9).
Example 3
A synthesis method of chiral lactone compound comprises the following steps:
the synthetic route of the substrate compound a in the step 1 is shown in figure 1:
(1) The preparation of substrate 2a is specifically:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting 4-fluorobenzaldehyde (16 mmol) and methyl acrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 2 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 2a.
(2) The preparation of 5a is specifically as follows:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting 3-bromobenzaldehyde (16 mmol) and methyl acrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 5 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 5a.
(3) The preparation of 10a is specifically as follows:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting 3-methylbenzaldehyde (16 mmol) and methyl acrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 10 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then, 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as an eluent to give substrate compound 10a. (4) the preparation of 12a specifically comprises:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then 2-methylbenzaldehyde (16 mmol) and methyl acrylate (8 mmol) are continuously injected into a round-bottom flask under the nitrogen atmosphere, and then the round-bottom flask is placed in a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 12 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 12a.
The substrate compound 12a was subjected to a hydrogen spectrum test analysis, and the test results are shown in FIG. 11, and it can be seen from FIG. 11 that the substrate compound 12a was successfully synthesized in the present invention.
(5) 15a is specifically prepared by:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting benzaldehyde (16 mmol) and methyl crotonate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 15 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 15a.
(6) The preparation of substrate 16a is specifically:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting benzaldehyde (16 mmol) and methyl methacrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 16 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 16a.
(7) The preparation of the substrate 17a is specifically:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting 4- (tertiary butyl) benzaldehyde (16 mmol) and methyl acrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 17 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 17a.
Wherein, 17a hydrogen spectrum data: 1 H NMR(400MHz,CDCl 3 ) Delta 7.93 (d, j=8.3 hz, 2H), 7.48 (d, j=8.2 hz, 2H), 3.70 (s, 3H), 3.38-3.26 (m, 2H), 2.76 (t, j=6.7 hz, 2H), 1.34 (s, 9H); that is, substrate compound 17a was successfully synthesized in this example.
(8) The preparation of the substrate 26a is specifically:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then 2-naphthaldehyde (16 mmol) and methyl acrylate (8 mmol) are continuously injected into a round-bottom flask under the nitrogen atmosphere, and then the round-bottom flask is placed in a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 26 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 26a.
Wherein, 26a hydrogen spectrum data: 1 H NMR(400MHz,CDCl 3 ):δ8.46(s,1H),8.00(d,J＝8.6Hz,1h) 7.91 (d, j=8.0 hz, 1H), 7.87-7.79 (m, 2H), 7.54 (dt, j=19.8, 7.0hz, 2H), 3.70 (s, 3H), 3.41 (t, j=6.6 hz, 2H), 2.80 (t, j=6.6 hz, 2H); that is, the substrate compound 26a was successfully synthesized in this example.
The synthetic route of the chiral lactone compound e in the step 2 is shown in the following figure 2;
(1) The preparation of chiral lactone compound 2e is specifically: substrate compound 2a (0.5 mM) and ChKRED20-M3C1 (mutant M3C1,1 mol%) obtained in example 2 were each mutated at amino acids 97, 153 and 188, glutamine at 97 was mutated to leucine, serine at 153 was mutated to leucine, and tyrosine at 188 was mutated to serine) were dispersed in isopropanol (i-PrOH 10%) and then reacted at 37℃for 48 hours to obtain chiral lactone compound 2e.
(2) The preparation of chiral lactone compound 5e is specifically: the substrate compound 10a (0.5 mM) and ChKRED20-M3C1 (1 mol%, amino acids at positions 97, 153 and 188 were mutated, glutamine at position 97 was mutated to leucine, serine at position 153 was mutated to leucine, and tyrosine at position 188 was mutated to serine) obtained in example 2 were dispersed in isopropanol (i-PrOH 10%), and then reacted at 37℃for 48 hours to obtain chiral lactone compound 5e.
(3) The preparation of chiral lactone compound 10e is specifically: the substrate compound 10a (0.5 mM) and ChKRED20-M3C1 (1 mol%, amino acids at positions 97, 153 and 188 were mutated, glutamine at position 97 was mutated to leucine, serine at position 153 was mutated to leucine, and tyrosine at position 188 was mutated to serine) obtained in example 2 were dispersed in isopropanol (i-PrOH 10%), and then reacted at 37℃for 48 hours to obtain the chiral lactone compound 10e.
(4) The preparation of chiral lactone compound 17e is specifically: the substrate compound 17a (0.5 mM) and ChKRED20-M3C1 (1 mol%, amino acids at positions 97, 153 and 188 were mutated, glutamine at position 97 was mutated to leucine, serine at position 153 was mutated to leucine, and tyrosine at position 188 was mutated to serine) obtained in example 2 were dispersed in isopropanol (i-PrOH 10%), and then reacted at 37℃for 48 hours to obtain the chiral lactone compound 17e.
(5) The preparation of chiral lactone compound 26e is specifically: the substrate compound 26a (0.5 mM) and ChKRED20-M3C1 (1 mol%, amino acids at positions 97, 153 and 188 were mutated, glutamine at position 97 was mutated to leucine, serine at position 153 was mutated to leucine, and tyrosine at position 188 was mutated to serine) obtained in example 2 were dispersed in isopropanol (i-PrOH 10%), and then reacted at 37℃for 48 hours to obtain the chiral lactone compound 26e.
Example 4
A synthesis method of chiral lactone compound comprises the following steps:
the synthetic route of the substrate compound 1a in step 1 is as follows:
the method comprises the following steps:
acetonitrile (10 mL) and TBADT prepared in example 1 (800 mg) were added to a round bottom flask and nitrogen was vented; then, under the nitrogen atmosphere, continuously injecting benzaldehyde (16 mmol) and methyl acrylate (8 mmol) into a round-bottomed flask, and then placing the round-bottomed flask into a self-made photoreactor (composed of two 390nm LED light sources, the structure of which is shown in FIG. 7) to react for 12 hours at 20 ℃ to prepare a mixture containing a substrate compound 1 a;
after the reaction is finished, the mixture is put in air to carry out quenching reaction until the color of the reactant is changed from blue to transparent liquid; then 30mL of ethyl acetate was added to the mixture to precipitate TBADT and filtered, and then the filtrate was evaporated under reduced pressure, and purified by column chromatography using petroleum ether/ethyl acetate as eluent to give substrate compound 1a.
The synthetic route of step 2, sex lactone compound 1e is shown below:
the method comprises the following steps: substrate compound 1a (0.5 mM) and ChKRED20-M3C1 (1 mol%) obtained in example 2 were dispersed in isopropanol (10%), followed by reaction at 37℃for 48 hours to obtain chiral lactone compound 1e.
Example 5
The method for synthesizing the chiral lactone compound in this example is the same as in example 4, except that:
ChKRED20-M3C1 in step 2 was replaced with mutant M2 (i.e., the amino acids at positions 97 and 153 were mutated simultaneously, with glutamine at position 97 being mutated to leucine and serine at position 153 being mutated to leucine).
Comparative example 1
The method for synthesizing the chiral lactone compound in this example is the same as in example 4, except that:
the ChKRED20-M3C1 substitution in step 2 was a mutant M1B (the amino acid at position 97 was mutated, and the glutamine at position 97 was mutated to leucine).
Comparative example 2
The method for synthesizing the chiral lactone compound in this example is the same as in example 4, except that:
ChKRED20-M3C1 in step 2 was replaced with mutant M1A (amino acid 153 was mutated and serine 153 was mutated to leucine).
Comparative example 3
The method for synthesizing the chiral lactone compound in this example is the same as in example 4, except that:
the ChKRED20-M3C1 in step 2 was replaced with WT (wild-type ChKRED 20).
Test analysis:
1. the substrate compound 2a of example 3 was subjected to hydrogen spectrum and carbon spectrum test analysis, and the test results are shown in FIGS. 2 and 3, and it can be seen from FIGS. 3 and 4 that the substrate compound 2a was successfully synthesized in the present invention.
The chiral lactone compound 2e prepared in example 3 was subjected to High Performance Liquid Chromatography (HPLC), and the measurement results are shown in fig. 5 and 6, wherein fig. 5 is a liquid phase spectrum of a racemate 2e standard sample, fig. 6 is a liquid phase spectrum of the chiral lactone compound 2e, and it can be seen in combination with fig. 5 and 6 that the chiral lactone compound 2e prepared in this embodiment is an R-configuration lactone compound.
The substrate compound 1a was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 8, and it can be seen from FIG. 8 that the substrate compound 1a was successfully synthesized in the present invention.
The substrate compound 5a was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 9, and it can be seen from FIG. 9 that the substrate compound 5a was successfully synthesized in the present invention.
The substrate compound 10a was subjected to a hydrogen spectrum test analysis, and the test results are shown in FIG. 10, and it can be seen from FIG. 10 that the substrate compound 10a was successfully synthesized in the present invention.
The substrate compound 12a was subjected to a hydrogen spectrum test analysis, and the test results are shown in FIG. 11, and it can be seen from FIG. 11 that the substrate compound 12a was successfully synthesized in the present invention.
The substrate compound 15a was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 11, and it can be seen from FIG. 11 that the substrate compound 15a was successfully synthesized in the present invention.
The substrate compound 16a was subjected to a hydrogen spectrum test analysis, and the test results are shown in FIG. 12, and it can be seen from FIG. 12 that the substrate compound 16a was successfully synthesized in the present invention.
The chiral lactone compound 5e was subjected to hydrogen spectrum test analysis, and the test results are shown in fig. 14, and it can be seen from fig. 14 that the substrate compound 5e was successfully synthesized in the present invention.
The chiral lactone compound 10e was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 15, and it can be seen from FIG. 15 that the substrate compound 10e was successfully synthesized in the present invention.
The chiral lactone compound 17e was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 16, and it can be seen from FIG. 16 that the substrate compound 17e was successfully synthesized in the present invention.
The chiral lactone compound 26e was subjected to hydrogen spectrum test analysis, and the test results are shown in FIG. 17, and it can be seen from FIG. 17 that the substrate compound 26e was successfully synthesized in the present invention.
2 chiral value and yield detection
The chiral values and yields of the chiral lactone compounds in examples 4 to 7 and comparative example 1 were measured by High Performance Liquid Chromatography (HPLC), and the results are shown in table 1.
TABLE 1 chiral values and yield measurements
As can be seen from Table 1, wild-type ChKRED20 was unable to catalyze the reduction of gamma-keto ester, mutant M2 (Q97L-S153L), and M2-based mutant M3C1 (Q97L-S153L-Y188S) was able to synthesize chiral lactone compounds with high enantioselectivity with high efficiency. Namely, the invention successfully expands the substrate spectrum, and the mutant strain M3C1 has better substrate universality.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (8)

1. A carbonyl reductase ChKRED20 mutant for chiral lactone compound synthesis, characterized in that amino acids at least two of positions 97, 153 and 188 are mutated using the amino acid sequence of wild type carbonyl reductase ChKRED20 as a starting sequence;
wherein, taking the amino acid sequence of wild carbonyl reductase ChKRED20 as a starting sequence, mutating the 97 th glutamine into leucine;
taking the amino acid sequence of wild carbonyl reductase ChKRED20 as a starting sequence, and mutating 153 th serine into leucine;
the amino acid sequence of wild carbonyl reductase ChKRED20 is taken as a starting sequence, and the 188 th tyrosine is mutated into serine.
2. Use of a carbonyl reductase ChKRED20 mutant as described in claim 1 in the synthesis of chiral lactone compounds.
3. A method for synthesizing chiral lactone compounds using the carbonyl reductase ChKRED20 mutant of claim 1, comprising the steps of:
step 1, under the atmosphere of protective gas, dissolving an aldehyde compound and a compound with a structural formula shown as II in a solvent, adding a photocatalyst tetrabutylammonium decatungstate, and then reacting for 10-14 hours in a photoreactor to obtain a substrate compound a;
step 2, dispersing a substrate compound a and a carbonyl reductase ChKRED20 mutant in a solvent for enzyme catalytic reaction to prepare a chiral lactone compound;
wherein, formula II's structural formula is:wherein R is 1 Is-> Any one of the following.
4. A synthetic method according to claim 3, characterized in that the aldehyde compound
The structural formula is shown as formula I, and the structural formula of the formula I is as follows:wherein R is Any one of them; the R is 2 H, F, NC, cl, br, me, meO, CF of a shape of H, F, NC, cl, br, me, meO, CF 3 、Et、tBu、CF 3 O、CF 3 Any one of S and EtO;
the R is 3 F, me, CN, br and Cl;
the R is 4 Is Me or CF 3 。
5. The synthesis method according to claim 4, wherein the molar ratio of the aldehyde compound to the compound with the structural formula shown as II in the step 1 is 1:1-5:4; the protective gas is nitrogen; the mass ratio of the photocatalyst to the compound with the structural formula shown as II is 3:2-2:1.
6. A synthetic method according to claim 3, characterized in that the conditions of the enzyme-catalyzed reaction in step 2 are: reacting for 42-54 h at 35-38 ℃.
7. The method for synthesizing according to claim 3, wherein the preparation method of the photocatalyst tetrabutylammonium decatungstate is as follows:
the tungstate and tetrabutylammonium bromide are dissolved in a solvent and heated, and the pH value is regulated to be 2-3, and then the reaction is continued for 20-40 min to prepare the photocatalyst.
8. The method of claim 7, wherein the molar ratio of tungstate to tetrabutylammonium bromide is 25-32:10-17; the tungstate is sodium tungstate; the solvent is deionized water; the temperature after heating is 85-95 ℃.
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