CN115820576A - Diels-Alder reaction enzyme mutant and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and particularly provides a Diels-Alder reaction enzyme mutant and a preparation method and application thereof. The invention relates to a method for carrying out functional modification and enhancement on Diels-Alder (D-A) reaction enzyme MaDA1 from mulberry, so that the Diels-Alder (D-A) reaction enzyme MaDA1 catalyzes an unnatural dienophile containing a 'handle group' (carboxyl, ester group or precursor thereof) to carry out intermolecular D-A reaction with a plurality of dienes, and further modifies an enzymatic D-A product through decarboxylation functionalization reaction, thereby realizing diversification and stereoselective synthesis of a cyclohexene framework structure.

Description

Diels-Alder reaction enzyme mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a Diels-Alder reaction enzyme mutant and a preparation method and application thereof.

Background

It is well known that the functional diversity of drug molecules is determined by their structural diversity. Due to the complexity of its three-dimensional structure, the multi-substituted six-membered ring skeleton structure is widely present in various drugs with biodiversity activity, such as the anticonvulsant drug canabidiol, the antiviral drug oseltamivir, the antitumor drug taxol, and the antidepressant drug brexanolone (fig. 1 a). Thus, chemists have been very active in developing stereoselective methods to efficiently synthesize substituted cyclohexanes. Among these methods, the Diels-Alder reaction is generally considered to be one of the most powerful transformations for the rapid construction of new six-membered rings, and therefore the development of asymmetric Diels-Alder reactions has received extensive research interest.

Enzymes play an increasingly important role as biocatalysts in the synthesis of pharmaceuticals and natural products. Therefore, the development and utilization of the enzyme capable of stereoselectively catalyzing the Diels-Alder reaction provide a new idea for solving the problems of poor stereoselectivity and low efficiency of the D-A reaction in the traditional chemical catalysis. However, enzymes often have high substrate specificity, making it difficult to efficiently achieve diverse syntheses using enzymatic catalysis.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of a Diels-Alder reaction enzyme mutant and application thereof in synthesizing a non-natural D-A product.

The second purpose of the invention is to combine enzymatic D-A reaction and decarboxylation functionalization to realize the diversified synthesis of the cyclohexene type compound.

In a first aspect, the present invention provides a mutant of the D-A reactive enzyme MaDA1, said mutant being one or more of the following mutations of the amino acid sequence shown in SEQ ID No. 1:

(1) The M mutation at the 434 th site is V; (2) the 434 th M mutation is V, and the 168 th S mutation is F; (3) The M mutation at the 434 th site is V, the S mutation at the 168 th site is F, and the P mutation at the 368 th site is A.

The amino acid sequence of the mutant of the D-A reactive enzyme MaDA1 provided by the invention is shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.

MaDA1 mentioned in the invention is MaDA-2 in CN110951700A, and the protein sequence of MaDA-2 in CN110951700A is SEQ ID No.12, which is the same as SEQ ID No.1 of the invention.

The mutant M1 of the D-A reactive enzyme MaDA1 provided by the invention is characterized in that M at the 434 th site of the SEQ ID No.1 is mutated into V, and the M1 protein has no signal peptide sequence shown in SEQ ID No. 2.

The mutant M2 of the D-A reactive enzyme MaDA1 provided by the invention is characterized in that M at the 434 th site of the SEQ ID No.1 is mutated into V and S at the 168 th site of the SEQ ID No.1 is mutated into F, and the M2 protein has no signal peptide sequence shown in SEQ ID No. 3.

The D-A reactive enzyme MaDA1 mutant M3 provided by the invention is formed by mutating M at 434 th site of SEQ ID No.1 into V, S at 168 th site into F and P at 368 th site into A, and M3 protein has no signal peptide sequence shown in SEQ ID No. 4.

In a second aspect, the invention provides a gene encoding the above mutant, the nucleotide sequence of the gene is shown in SEQ ID No. 5; or as shown in SEQ ID No. 6; or as shown in SEQ ID No. 7.

In a third aspect, the invention provides a biological material containing the gene, wherein the biological material is an expression cassette, a plasmid, a vector, a microorganism, an animal cell or a plant cell.

In a fourth aspect, the present invention provides the use of a mutant as described above or a gene as described above or a biomaterial as described above for the catalytic synthesis of a natural or unnatural D-A product by a Diels-Alder reaction; the reaction substrates for the Diels-Alder reaction are non-natural dienophiles and dienes.

In the applications provided herein, the non-natural dienophile is a non-natural dienophile containing a carboxyl group, an ester group, or a precursor thereof.

In a fifth aspect, the present invention provides the use of the above mutant or the above gene or the above biomaterial in the diversification and stereoselective synthesis of cyclohexene backbone structures.

In the application of the diversification and stereoselective synthesis of the cyclohexene framework structure, provided by the invention, after an enzymatic D-A product is generated by adopting a mutant of the D-A reaction enzyme MaDA1, the acetyl protection is carried out on the D-A product, and N-hydroxyphthalimide ester is introduced after the ester group is hydrolyzed; the introduction of different substituents on the cyclohexene skeleton is realized through decarboxylation functionalization reaction.

In the application of the diversification and stereoselective synthesis of the cyclohexene skeleton structure provided by the invention, the decarboxylation functionalization reaction comprises but is not limited to the following reactions: decarboxylation alkylation, decarboxylation alkenylation, decarboxylation alkynylation, decarboxylation arylation and Curtis rearrangement reaction.

In the application provided by the invention, the temperature for catalyzing Diels-Alder reaction is 30-37 ℃, and the pH value is 6.0-6.5.

The invention has the beneficial effects that:

(1) The invention provides a chemical-enzymatic synthesis strategy (B in figure 1) for realizing diversified synthesis of cyclohexene type compounds by combining respective advantages of enzyme catalysis and chemical catalysis to make up for deficiencies of each other and combining the advantages of enzyme catalysis and chemical catalysis.

(2) The present inventors have found that the known DA-reactive enzyme MaDA1 can recognize a non-natural dienophile having a handle group (carboxyl group and its derivatives) as a substrate; through carrying out mutation and activity screening on the MaDA1, a MaDA1 mutant (M3) with greatly improved activity and stereoselectivity is obtained, and the mutant can realize the construction of a cyclohexene framework with moderate to excellent yield and good stereoselectivity; the handle group in the enzymatic D-A product can be further derived through decarboxylation functionalization, the structure of the enzymatic product is further enriched, and the efficient and diversified synthesis of the cyclohexene compound is realized. The invention develops a novel chemical-enzymatic coupling strategy, and provides a new possibility for the synthesis of important chemical precursors or natural products containing six-membered rings.

Drawings

In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.

A of FIG. 1 is a representative drug molecule comprising a six-membered ring backbone structure; b is the chemical-enzyme coupling strategy provided by the invention.

FIG. 2 shows the results of the activity test of the M434 single mutant of the present invention.

FIG. 3 shows the results of activity assays of the double mutants of the present invention.

FIG. 4 shows the results of activity assays of the triple mutants of the present invention.

FIG. 5 is a comparison of catalytic effects of MaDA1 after three mutations.

FIG. 6 shows the results of the reaction condition optimization according to the present invention.

FIG. 7 shows the applicability of the substrate of the MaDA1 mutant of the invention.

FIG. 8 is a decarboxylated coupling reaction of the DA product of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents and raw materials used in the following examples are all commercially available. The strains, carriers, culture media and reagents used in the following examples were mainly:

coli DH 5. Alpha. And DH10Bac competent cells were obtained from Hill Bio Inc. Insect cells Sf21 and Hi5 for expression were purchased from Invitrogen. The insect expression vector pI-secSUMOstar was purchased from LifesSensors.

LB solid Medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and 1.5% of agar.

LB liquid medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl.

Plasmid small-extraction kits and gel recovery kits are purchased from Tiangen biochemistry limited company; PCR primer synthesis and plasmid sequencing were performed by Jin Weizhi Biotechnology, inc.

Example 1 Synthesis of dienophiles

To a solution of S1 (3.4g, 14.7mmol, 1.0equiv.) in DCM (150 mL) at room temperature was added DHP (1.61g, 17.6mmol,1.2equiv.) and PPTS (185mg, 0.74mmol, 0.05equiv.). After 12 hours, the reaction was spin dried and the crude product was purified by recrystallization from ether to yield S2 as a white solid (3.4 g, 74%). ¹ H NMR(400MHz,CDCl ₃ )δ12.09(s,1H),7.66(d,J＝9.0Hz,1H),6.66(d,J＝2.5Hz,1H),6.59(dd,J＝9.0,2.4Hz,1H),5.51(t,J＝3.2Hz,1H),4.37(s,2H),3.96 3.50(m,2H),2.11 1.58(m,6H)。

To a solution of S3 (1.0 g,3mmol,1.0 equiv.) in tetrahydrofuran (100 mL) at-78 deg.C were added 3-methylbut-2-en-1-ol (0.9mL, 9mmol,3.0 equiv.) and PPh ₃ (2.4 g,9mmol,3.0 equiv.) in THF (50 mL), followed by the addition of DEAD (2mL, 12mmol,4.0 equiv.), and slowly warmed to room temperature. After stirring for 30 min, the reaction was spin dried to give the crude product, which was used in the next step without further purification.

Montmorillonite K10 (2 g) was added to a DCM solution of the crude product from the previous step (100 mL) at 0 deg.C and slowly warmed to room temperature. After stirring overnight, the reaction mixture was filtered to remove montmorillonite K10 and spin-dried and purified by silica gel column chromatography (petroleum ether/acetone = 15/1) to give S5 (248mg, 28%) as a white solid. ¹ H NMR(400MHz,CDCl ₃ )δ12.57(s,1H),7.55(d,J＝8.9Hz,1H),6.42(d,J＝8.9Hz,1H),6.19(s,1H),5.26(t,J＝7.2Hz,2H),4.37(s,2H),3.45(d,J＝7.2Hz,2H),1.83(s,4H),1.77(s,3H)。

Et was added to a solution of S5 (190mg, 0.6mmol,1.0 equiv.) in MeCN (20 mL) at room temperature ₃ N (20uL, 0.14mmol, 0.2equiv.) and PPh ₃ (183mg, 0.70mmol, 1.1equiv.). After stirring for 2 hours, the residue was dissolved in CH after spin-drying ₂ Cl ₂ :H ₂ O (25.6 mL. After stirring at room temperature for 4 hours, the solution was separated, extracted with DCM, the organic phases were combined, washed with saturated brine and anhydrous sulfurSodium was dried, filtered, the filtrate was spin dried and purified by recrystallization from ether/dichloromethane to give S7 as a white solid (282mg, 92%,2 steps). ¹ H NMR(600MHz,CDCl ₃ )δ15.48(s,1H),7.70(dd,J＝12.5,7.8Hz,6H),7.58(t,J＝7.3Hz,3H),7.49(q,J＝8.0,7.4Hz,7H),6.26(d,J＝7.5Hz,1H),5.67(s,1H),5.33–5.25(m,1H),4.32(s,1H),3.43(d,J＝6.0Hz,2H),1.81(s,3H),1.73(s,3H)。

To a solution of S7 (180mg, 0.375mmol,1.0 equiv.) in THF (8 mL) at-20 ℃ was added a solution of S8 (123mg, 0.75mmol,2.0 equiv.) in THF (4 mL). Stirring for 1 hour and spin-drying (<At 30 ℃, silica gel column chromatography (petroleum ether/dichloromethane = 1/3-1/15) gave a yellow solid dienophile 1a (106mg, 77%). ¹ H NMR(400MHz,CDCl3)1H NMR(400MHz,CDCl3)δ13.22(s,1H),7.99(d,J＝15.3Hz,1H),7.63(d,J＝8.9Hz,1H),7.45–7.32(m,5H),6.98(d,J＝15.3Hz,1H),6.42(d,J＝8.9Hz,1H),6.18(s,1H),5.28(s,3H),3.46(d,J＝7.3Hz,2H),1.83(s,3H),1.77(s,2H)。

To a solution of S7 (180mg, 0.375mmol,1.0 equiv.) in THF (8 mL) was added a solution of S9 (112mg, 0.75mmol,2.0 equiv.) in THF (4 mL) at room temperature, followed by heating at 55 ℃ and stirring overnight. When TLC showed disappearance of starting material, the reaction mixture was cooled to room temperature and spun dry and purified by silica gel column chromatography (dichloromethane/acetone = 100/1-100/3) to give yellow solid dienophile 1b (85mg, 65%). ¹ H NMR(400MHz,CDCl ₃ )δ13.59(s,1H),7.63(d,J＝8.9Hz,1H),7.38(d,J＝4.4Hz,4H),7.33–7.28(m,1H),7.13(dt,J＝15.2,3.9Hz,1H),6.38(d,J＝8.8Hz,1H),6.14(s,1H),5.34–5.22(m,1H),4.63(s,2H),4.30(dd,J＝3.8,2.0Hz,2H),3.46(d,J＝7.2Hz,2H),1.83(s,3H),1.76(s,3H)。

S10 (8.3 mg,0.069mmol, 0.55equiv.) was added to a solution of S7 (60mg, 0.125mmol,1.0 equiv.) in THF (1 mL) at room temperature, followed by heating at reflux overnight. When TLC showed disappearance of starting material, the reaction mixture was cooled to room temperature and spin dried and purified by silica gel column chromatography (dichloromethane/acetone = 25/1-16/1) to give the dienophile 1c as a white solid (20mg, 61%). ¹ H NMR(400MHz,Acetone-d ₆ )δ13.69(s,1H),9.39(s,1H),7.75(d,J＝8.9Hz,1H),7.37(dt,J＝15.1,2.0Hz,1H),7.20(dt,J＝15.1,3.6Hz,1H),6.53(d,J＝8.9Hz,1H),5.26(dddt,J＝7.2,5.7,2.7,1.4Hz,1H),4.41(dq,J＝5.6,3.6,2.9Hz,2H),4.28(t,J＝5.4Hz,1H),3.36(d,J＝7.2Hz,2H),1.77(s,3H),1.63(s,3H)。

To a solution of S11 (2g, 9.3mmol,1.0 equiv.) in CH3CN (60 mL) at 0 ℃ were added 3-chloro-3-methylbut-1-yne S12 (5.25mL, 50mmol,5.o equiv.), cuCl in that order ₂ ·H ₂ O (12mg, 0.07mmol, 0.007equ.) and DBU (1.46mL, 9.8mmol, 1.05equ.). After stirring for 5 hours, the mixture was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was spun dry and subjected to silica gel column chromatography (petroleum ether/diethyl ether = 20/1) to give S13 (550mg, 21%) as a white solid. ¹ H NMR(400MHz,CDCl3)δ7.91(d,J＝8.9Hz,2H),7.29(d,J＝8.9Hz,2H),4.66(s,2H),2.65(s,1H),1.71(s,6H)。

Lindlar catalyst (80mg, 40wt%) was added to a solution of arylpropargyl ether S13 (200mg, 0.72mmol,1.0 equiv.) in ethyl acetate (20 mL) at room temperature, and the suspension was degassed at-78 deg.C and then treated with H ₂ Backfilling for 3 times. After stirring at room temperature for 10 hours, the reaction mixture was filtered through celite and spin dried. Will be provided withThe residue was redissolved in toluene (20 mL) and heated at reflux overnight. When TLC showed disappearance of starting material, the reaction mixture was cooled to room temperature and spun dry and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/1-1/1) to give S14 as a white solid (70mg, 35%). ¹ H NMR(400MHz,CDCl ₃ )δ7.75(d,J＝2.2Hz,2H),6.86(d,J＝8.1Hz,1H),5.79(s,1H),5.35–5.27(m,1H),4.64(s,2H),3.41(d,J＝7.2Hz,2H),1.79(s,6H)。

Et was added to a solution of S14 (70mg, 0.25mmol,1.0 equiv.) in MeCN (8 mL) at room temperature ₃ N (10uL, 0.07mmol, 0.3equiv.) and PPh ₃ (68mg, 0.26mmol, 1.1equiv.). After heating at 60 ℃ and stirring for 48 hours, the reaction mixture was cooled to room temperature and spin-dried. The residue was dissolved in dichloromethane/water (8ml: 12ml) and 2M NaOH (1 mL) was added. After stirring at room temperature for 4 hours, the mixture was extracted with dichloromethane. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spin-dried to give a crude solid. To a solution of the crude product from the previous step in tetrahydrofuran (7 mL) at 0 deg.C was added a solution of S8 (46mg, 5.5 mmol) in THF (7 mL). Followed by stirring overnight and spin-drying (<The crude product was obtained at 30 ℃ and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 4/1) to yield the dienophile 1d as a yellow solid (48mg, 72%). ¹ H NMR(400MHz,CDCl ₃ )δ7.94(d,J＝15.5Hz,1H),7.81(d,J＝7.2Hz,2H),7.44-7.34(m,J＝13.0Hz,5H),7.00–6.80(m,2H),5.83(s,1H),5.29(d,J＝11.4Hz,3H),3.41(d,J＝6.8Hz,2H),1.79(s,6H)。

EXAMPLE 2 Synthesis of different acetyldienes

To a solution of substituted 2-iodobenzene-1,3-diol (0.3 mmol) in DCM (3 mL) at room temperature was added Et ₃ N (250. Mu.L, 1.8 mmol) and Ac ₂ After O (112. Mu.L, 1.2 mmol) was stirred overnight, the reaction was mixedThe material was quenched with water and extracted with DCM. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and spun to give the crude product, which was used directly in the next step. The crude product from the previous step, boron reagent S18 (97mg, 0.5 mmol), K ₃ PO ₄ (635mg,3mmol)，AsPh ₃ (12.8 mg, 0.042mmol) and Pd ₂ (dba) ₃ (19.2mg, 0.021mmol) was dissolved in DMF (5 mL) and stirred overnight at 60 ℃. After cooling to room temperature, the reaction mixture was filtered through celite and washed with EtOAc, and the organic layer was dried over anhydrous sodium sulfate and spin dried. The resulting mixture was redissolved in DCM (3 mL) and Et was added ₃ N (250. Mu.L, 1.8 mmol) and Ac ₂ O (112. Mu.L, 1.2 mmol). After stirring overnight at room temperature, it was quenched with water and extracted with DCM. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and spun-dried to give the crude product, which was purified by silica gel column chromatography to give the corresponding product.

Example 3 preparation, activity detection and evolution of MaDA1 mutants

In order to further improve the catalytic activity and selectivity of MaDA1, the MaDA1 enzyme was evolved and the mutants obtained were tested for activity.

1. Construction and expression of MaDA1 mutant

The MaDA1 mutant is obtained by Megawhop PCR amplification (megaprimer PCR of halide plasmid) using the different primers in Table 1 for pI-sec-sumostar-tev-MaDA1 plasmid (the pI-sec-sumostar-tev- -MaDA1 plasmid is disclosed in Chinese patent CN 110951700A), i.e., large fragment DNA fragments are obtained by PCR amplification using first forward primers (MaDA 1_ M434V _2_F or MaDA1_ S168F _2_F or MaDA1_ P368A _ 2_F) containing mutation sites and reverse primers pI sec _ MaDA1_ R as primers and pI-sec-sumostar-tev-MaDA1 as template. Megawhop PCR was performed using this PCR product as a maprimer and pI-sec-sumostar-tev-MaDA1 plasmid (or a plasmid already containing the site mutation) as a template. After DpnI digestion, the product was transformed into E.coli DH 5. Alpha. Strain. Clones containing the mutated gene were selected from the plates and confirmed by Sanger sequencing. The plasmid was then transformed into E.coli DH10Bac competent cells to produce recombinant baculovirus. Followed byAnd then expressed by Bac-to-Bac heterologous expression system and purified by Ni-NTA. All protein concentrations were analyzed using a NanoDrop2000 (Thermo). The average protein yield of the MaDA1 variant was from 15 to 25mg L ^-1 Are not equal.

TABLE 1 primer sequence information Table

2. Activity assay of MaDA1 mutants

To determine the relative activity of the MaDA1 mutants, 200 μ M diene 2a, 150 μ M dienophile 1a, and 3 μ g MaDA1 mutant were reacted in 100 μ L Tris-HCl buffer (pH =7,25mm) for 3 hours at 37 ℃ (2 a). The reaction was quenched with 100. Mu.l ice-cold acetonitrile and centrifuged at 15,000g for 10 min. The supernatant was analyzed by analytical reverse phase UPLC.

The conditions for UPLC analysis were: a chromatographic column: ACQUITY UPLC BEH C18 (50 mm. Times.2.1mm, 1.7 μm); mobile phase: gradient eluting with acetonitrile-water, 0-7min, 30 → 100% acetonitrile; 7-8min, 100 → 30% acetonitrile.

3. Evolutionary process of MaDA1

With 2a as substrate, the conversion of MaDA1 was 8%, endo/exo =5/1. The pocket of M434, S168, R285, N347, P368 was selected as the hot spot for mutation and focused iterative residue-specific mutations were performed. Firstly, selecting an M434 site mutation which is critical to the reaction selectivity and activity to be A, V, S, F and a supplementary I, L, C to obtain the first round of optimal mutation MaDA1_ M434V (M1), wherein the activity test result of the single mutant is shown in figure 2, the conversion rate is 32%, and endo/exo is more than 15/1.

In the second round, maDA1_ M434V is used as a template to respectively construct S168A, S168V, S168F, R285A, R285F, R285V, R285S, N347A, N347V, N347F, N347S, P368A, P368V, P368F, P368S mutant and supplementary S168W, S168H, S168Y, so as to obtain the second round of optimal mutant MaDA1_ M434V _ S168F (M2), wherein the results of the double mutant activity test are shown in FIG. 3, the conversion rate is 58%, and endo/exo is more than 15/1.

In the third round, maDA1_ M434V _ S168F is used as a template to respectively construct P368A, P368G, P368S, P368T, P368T, P368D, P368N and P368R to obtain a third round of optimal mutants MaDA1_ M434V _ S168F _ P368A (M3), wherein the results of the activity test of the three mutants are shown in FIG. 4, the conversion rate is 81%, and the endo/exo is more than 15/1.

The catalytic effect of MaDA1 after three mutations is compared in fig. 5. In the subsequent bulk reaction, maDA1, M2, M3 gave yields of 39%,62%,62%,92%, respectively, and TTN of 192, 730, 996, 2922, respectively, with ee values of 93%,99%,99%,99%, respectively, using 2a as the diene. From the enzymological parameters, relative to the wild type MaDa1 (k) _cat /K _M ＝0.55mM ^-1 s ^-1 ),M3(k _cat /K _M ＝19.13mM ^-1 s ^-1 ) There was a 34 fold increase in activity, and a 12 fold increase in TTN.

Example 4 optimization of enzyme reaction conditions

To determine the optimal reaction pH for MaDA1_ M3, 200. Mu.M diene 2a, 150. Mu.M dienophile 1a, and 3. Mu.g of the MaDA1 mutant MaDA1_ M3 were reacted in 100. Mu.L of different buffers for 3 hours at 37 ℃. The reaction was quenched with 100. Mu.l ice-cold acetonitrile and centrifuged at 15,000g for 10 min. The supernatant was analyzed by analytical reverse phase UPLC.

By comparing the conversion and the yield of the byproduct 1a', the optimal reaction conditions were obtained as follows: phosphate buffer, 25mm, ph =6.0,37 ℃. The final conversion reached 92% (fig. 6).

Example 5 substrate spectra of MaDa1 u M3

To determine the substrate profile of MaDA1_ M3, 200 μ M diene 2-2k, 150 μ M dienophile 1a or 1b and 3 μ g MaDA1 mutant MaDA1_ M3 were reacted in 100 μ L phosphate buffer (25mm, ph = 7) for 3 hours. The reaction was quenched with 100. Mu.l ice-cold acetonitrile and centrifuged at 15,000g for 10 min. The supernatant was analyzed by analytical reverse phase UPLC. From the experimental results, it was found that all aryl substituted 1,3 dienes reacted with dienophiles to give the corresponding D-A products (see FIG. 7).

Example 6 application of MaDa1 u M3 to the Synthesis of D-A products containing the endo configuration from which carboxylate handles can be derived

To further validate the utility of the MaDA1 mutant MaDA1_ M434V _ S168F _ P368A (M3) in the synthesis of D-A products containing the endo configuration available for derivatizing carboxylate handles, this example selectively performed enzymatic preparation of representative substrates.

To a diene precursor (0.026mmol, 1.2 equiv.) in MeOH (0.8 mL) and H at room temperature ₂ K was added to O (0.4 mL) solution ₂ CO ₃ (14mg, 0.104mmol,4.0 equiv.). The resulting mixture was stirred for 1 hour to generate the diene in situ. The resulting solution was then added to 48mL of reaction solution (0.5-1mg MaDA1-M3 in 25mM NaPi, pH =6.0,2% DMSO). To this mixture, 440. Mu.L of dienophile (0.022 mmol) DMSO stock (50 mM) was added in four aliquots every 2 hours. After incubation at 37 ℃ for 24 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were washed with brine, over Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (PTLC) to give the corresponding product.

To a solution of diene precursor (0.026 mmol) in methanol (0.8 mL) and water (0.4 mL) at room temperature was added K ₂ CO ₃ (14mg, 0.104mmol). After stirring for 1 hour the diene was formed in situ. The resulting solution was then added to 48mL of the reaction solution (0.5-1mg MaDA1-M3, 25mM NaPi, pH =6.0,2% DMSO). Subsequently, 440. Mu.L of dienophile (0.022 mmol) DMSO stock (50 mM) was added in four aliquots every 2 hours. After 24 h incubation at 37 ℃, the reaction mixture was extracted with ethyl acetate, the organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, spun-dried and purified by Preparative TLC (PTLC) to give the corresponding product. Following the above procedure, D-A products, 3a,3b,3c,3d,3e,3f,3g, etc., were obtained, with the following nuclear magnetism and yield data:

3a: ¹ H NMR(600MHz,Acetone-d ₆ )δ12.84(s,1H),9.19(s,1H),8.47(s,1H),8.10(s,2H),7.88(d,J＝9.0Hz,1H),7.37(d,J＝8.4Hz,1H),7.34–7.24(m,5H),6.93(d,J＝2.0Hz,1H),6.89(d,J＝0.9Hz,1H),6.78(dd,J＝8.4,2.1Hz,1H),6.74(s,2H),6.46(d,J＝8.8Hz,1H),5.55–5.51(m,1H),5.19(dddd,J＝7.2,5.9,2.7,1.4Hz,1H),5.09(d,J＝12.6Hz,1H),5.04(d,J＝12.6Hz,1H),4.73–4.67(m,1H),4.34(dd,J＝10.1,7.1Hz,1H),4.03(td,J＝10.0,5.9Hz,1H),3.25(p,J＝6.7Hz,2H),2.48(dd,J＝17.5,5.7Hz,1H),2.26(dd,J＝17.5,9.9Hz,1H),1.80(s,3H),1.71(s,3H),1.59(s,3H)。

3b: ¹ H NMR(600MHz,Acetone-d ₆ )δ12.84(s,1H),9.15(s,1H),7.85(d,J＝9.0Hz,1H),7.83(s,2H),7.39–7.24(m,5H),6.73(d,J＝8.0Hz,1H),6.44(d,J＝8.8Hz,1H),6.20(d,J＝8.0Hz,2H),5.51(dt,J＝2.9,1.4Hz,1H),5.20(tt,J＝7.2,1.4Hz,1H),5.08(d,J＝12.6Hz,1H),5.03(d,J＝12.6Hz,1H),4.73–4.65(m,1H),4.32(dd,J＝9.9,7.1Hz,1H),3.99(td,J＝9.9,5.9Hz,1H),3.26(p,J＝6.7Hz,2H),2.45(dd,J＝17.4,5.6Hz,1H),2.24(dd,J＝17.5,9.8Hz,1H),1.78(s,3H),1.73(s,3H),1.61(s,3H)。

3c: ¹ H NMR(600MHz,Acetone-d ₆ )δ12.81(s,1H),9.18(s,1H),8.34(s,2H),7.86(d,J＝9.0Hz,1H),7.37–7.17(m,5H),6.44(d,J＝8.8Hz,1H),6.26(s,2H),5.47–5.41(m,1H),5.24–5.15(m,1H),5.07(d,J＝12.6Hz,1H),5.02(d,J＝12.6Hz,1H),4.64(s,1H),4.27(dd,J＝10.4,7.2Hz,1H),4.00(td,J＝10.4,5.9Hz,1H),3.27(h,J＝7.2Hz,2H),2.45(dd,J＝17.3,5.7Hz,1H),2.22(dd,J＝17.0,10.4Hz,1H),1.77(s,3H),1.73(s,3H),1.62(s,3H)。

3d: ¹ H NMR(400MHz,Acetone-d ₆ )δ12.77(s,1H),8.31(s,2H),7.87(d,J＝8.9Hz,1H),7.35–7.23(m,5H),6.89(s,2H),6.45(d,J＝8.8Hz,1H),5.50–5.41(m,1H),5.23–5.15(m,1H),5.08(d,J＝12.6Hz,1H),5.02(d,J＝12.6Hz,1H),4.73(s,1H),4.32(dd,J＝10.4,7.2Hz,1H),4.05(td,J＝10.3,5.9Hz,1H),3.77(s,3H),3.26(p,J＝7.9 Hz,2H),2.47(dd,J＝17.3,5.8Hz,1H),2.23(dd,J＝17.1,10.1Hz,1H),1.78(s,3H),1.72(s,3H),1.61(s,3H)。

3e: ¹ H NMR(400MHz,Acetone-d ₆ )δ12.87(s,1H),7.84(d,J＝8.9Hz,1H),7.71(s,2H),7.29(q,J＝4.1Hz,5H),6.44(d,J＝8.8Hz,1H),6.04(s,2H),5.50(s,1H),5.20(t,J＝6.6Hz,1H),4.69–4.59(m,1H),4.30(dd,J＝9.8,6.9Hz,1H),3.95(td,J＝9.7,6.0Hz,1H),3.26(d,J＝7.1Hz,2H),2.44(dd,J＝17.3,5.8Hz,1H),2.23(dd,J＝17.1,9.6Hz,1H),2.03(s,3H),1.77(s,3H),1.73(s,3H),1.61(s,3H)。

3f: ¹ H NMR(600MHz,Acetone-d ₆ )δ12.75(s,1H),9.21(s,1H),8.23(s,1H),7.85(d,J＝9.0Hz,1H),7.54(s,1H),7.38–7.21(m,5H),6.93(d,J＝8.7Hz,1H),6.45(d,J＝8.8Hz,1H),6.26(d,J＝8.7Hz,1H),5.49(dt,J＝2.8,1.4Hz,1H),5.20(ddt,J＝8.6,5.9,1.4Hz,1H),5.09(d,J＝12.6Hz,1H),5.03(d,J＝12.6Hz,1H),4.73–4.64(m,1H),4.35(dd,J＝9.9,7.1Hz,1H),3.94(td,J＝9.8,6.0Hz,1H),3.26(p,J＝6.7Hz,2H),2.46(dd,J＝17.4,5.7Hz,1H),2.25(dd,J＝17.5,9.7Hz,1H),1.79(s,3H),1.73(s,3H),1.61(s,3H)。

3i: ¹ H NMR(600MHz,Acetone-d ₆ )δ12.85(s,1H),7.95(s,1H),7.72(d,J＝9.0Hz,1H),7.42(d,J＝7.2Hz,2H),7.34(t,J＝7.5Hz,2H),7.27(t,J＝7.3Hz,1H),6.80(t,J＝8.0Hz,1H),6.41(d,J＝8.9Hz,1H),6.28(d,J＝8.0Hz,2H),5.64(s,1H),5.19–5.13(m,1H),4.66–4.57(m,3H),4.43–4.37(m,1H),3.67(t,J＝8.8Hz,1H),3.59(dd,J＝9.2,5.9Hz,1H),3.27(d,J＝7.2Hz,2H),2.59(dtt,J＝9.0,6.3,3.4Hz,1H),2.29(s,1H),1.92(d,J＝18.3Hz,1H),1.77(s,3H),1.72(s,3H),1.60(s,3H)。

3j: ¹ H NMR(400MHz,Acetone-d ₆ )δ12.87(s,1H),7.90(s,1H),7.74(d,J＝8.9Hz,1H),6.79(t,J＝8.0Hz,1H),6.46(d,J＝8.9Hz,1H),6.27(d,J＝8.1Hz,2H),5.64(s,1H),5.17(td,J＝6.6,5.9,3.6Hz,1H),4.57(dd,J＝5.8,3.3Hz,1H),4.41–4.30(m,1H),4.01(s,1H),3.76(dd,J＝10.1,8.1Hz,1H),3.67(dd,J＝10.3,6.6Hz,1H),3.27(d,J＝7.2Hz,2H),2.39(ddq,J＝9.8,6.7,2.7Hz,1H),2.25(dt,J＝19.7,3.5Hz,1H),1.95(d,J＝17.8Hz,1H),1.78(s,3H),1.72(s,3H),1.59(s,3H)。

3k: ¹ H NMR(400MHz,Acetone-d ₆ )δ7.98(s,2H),7.72(d,J＝2.1Hz,1H),7.71–7.67(m,1H),7.41–7.30(m,5H),6.84(d,J＝8.3Hz,1H),6.75(t,J＝8.0Hz,1H),6.22(d,J＝8.0Hz,2H),5.53(s,1H),5.32–5.25(m,1H),5.16(d,J＝12.7Hz,1H),5.11(d,J＝12.7Hz,1H),4.55(d,J＝5.6Hz,2H),3.65(q,J＝6.1Hz,1H),3.29(d,J＝7.3Hz,2H),2.28(d,J＝5.9Hz,2H),1.76(s,3H),1.68(s,3H),1.67(s,3H)。

example 7 diversified derivatization of D-A products Using decarboxylation functionalization reactions

Starting from the D-A product, a key intermediate is obtained through chemical conversion, and a diverse multi-substituted multi-chiral-center cyclohexane skeleton can be obtained through decarboxylation functionalization reactions including but not limited to decarboxylation alkynylation, decarboxylation arylation, decarboxylation alkylation, decarboxylation amination, decarboxylation hydrogenation and decarboxylation Giese reactions (decarboxylation coupling reaction is shown in figure 8).

To 3b (90mg, 0.166mmol) in CH at room temperature ₂ Cl ₂ Et (18 mL) was added to the solution ₃ N(278μL,2.0mmol),Ac ₂ O (127. Mu.L, 1.3 mmol), DMAP (60mg, 0.49mmol). Stir overnight and then quench excess reagent with water. The layers were separated and the aqueous layer was extracted with DCM, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spin dried to give the crude product which was taken directly to the next step.

Dissolving the above crude product in acetone (10.5 mL), adding Pd/C (99mg, 10% by weight) and HCOONH ₄ (1.6mg, 0.025mmol). The suspension was degassed at-78 ℃ and backfilled 3 times with argon, followed by 1,4-cyclohexadiene (251 μ L,2.5 mmol). After stirring at room temperature for 1 hour, the reaction mixture was filtered through celite, spin dried to give a crude solid which was washed with hexane/DCM to give S21 as a white solid (89mg, 87%). ¹ H NMR(600MHz,CDCl ₃ )δ7.74(s,1H),7.20(t,J＝8.1Hz,1H),7.00(d,J＝6.8Hz,1H),6.80(s,2H),5.24(s,1H),4.99(td,J＝6.3,5.7,3.5Hz,1H),4.10(s,1H),3.96(s,1H),3.81(td,J＝11.0,5.7Hz,1H),3.20(d,J＝12.8Hz,1H),3.04(d,J＝14.2Hz,1H),2.56(dd,J＝17.6,5.6Hz,1H),2.31(s,4H),2.24–2.12(m,3H),1.85(s,6H),1.74(s,3H),1.70(s,6H)。

To a solution of S21 (50mg, 0.08mmol) in DCM (12 mL) was added N-hydroxytetrachlorophthalimide (28mg, 0.093mmol), DMAP (2.8mg, 0.022mol) and DIC (18. Mu.L, 0.129 mmol) at room temperature. After stirring overnight, the reaction mixture was filtered and spun dry to give the crude product, which was purified by column chromatography (dichloromethane/ether = 20/1) to give 5a (60mg, 82%) as a white solid. ¹ H NMR(400MHz,CDCl ₃ )δ7.56(s,1H),7.22(t,J＝8.2Hz,1H),6.96(d,J＝8.4Hz,1H),6.81(d,J＝7.2Hz,2H),5.33(s,1H),4.96(s,0H),4.32–4.15(m,3H),4.09(s,1H),3.18(dd,J＝14.4,6.2Hz,1H),3.05(dd,J＝16.3,5.0Hz,1H),2.82(dd,J＝17.6,4.9Hz,1H),2.51(dd,J＝17.2,10.0Hz,1H),2.29(s,6H),1.96(s,6H),1.81(s,3H),1.69(s,6H)。

To a solution of S21 (38mg, 0.06mmol) in DCM (8 mL) was added N-hydroxyphthalimide (12mg, 0.074mmol), DMAP (2.0mg, 0.016mol.) and DIC (14. Mu.L, 0.099 mmol) at room temperature. After stirring overnight, the reaction mixture was filtered and concentrated in vacuo to give a crude solid which was purified by column chromatography (dichloromethane/ether = 100/1-20/1) to give 5b (38mg, 83%) as a white solid. ¹ H NMR(600MHz,CDCl ₃ )δ7.85(dd,J＝5.4,3.1Hz,2H),7.75(dd,J＝5.4,3.1Hz,2H),7.59(s,1H),7.22(t,J＝8.2Hz,1H),6.95(d,J＝8.3Hz,1H),6.81(d,J＝7.6Hz,2H),5.33(s,1H),4.97(t,J＝6.9Hz,1H),4.24(dd,J＝13.0,7.4Hz,2H),4.08(s,1H),3.23–3.13(m,1H),3.06(s,1H),2.87–2.80(m,1H),2.53(dd,J＝16.6,9.2Hz,1H),2.28(s,6H),1.95(s,6H),1.82(s,3H),1.69(s,6H)。

5a (9.0 mg,0.01mmol,1.0 equiv.) and magneton were added to the reaction tube at room temperature. Subsequently, the tube was evacuated and backfilled with argon. Followed by the addition of NiCl ₂ ·6H ₂ O/4,4 '-dimethoxy-2,2' -bipyridine in DMF (100. Mu.L, 0.01mmol,0.1M in DMF) and ethynylzinc chloride S22 (100. Mu.L, 0.033mmol,0.33M in DMF). After stirring for 18 h, quench with half-saturated aqueous ammonium chloride and extract with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2.5/1) to give 6a (5.1mg, 85%). ¹ H NMR(600MHz,CDCl ₃ )δ7.44(s,1H),7.11(t,J＝8.2Hz,1H),6.81(d,J＝8.6Hz,1H),6.70(s,2H),5.18(s,1H),4.93–4.86(m,1H),4.06(s,1H),3.83(t,J＝7.8Hz,1H),3.52–3.42(m,1H),3.12(dd,J＝14.4,5.8Hz,1H),3.04–2.93(m,1H),2.65(dd,J＝17.9,5.8Hz,1H),2.22(s,4H),2.08–1.90(m,10H),1.67(s,3H),1.64(s,3H),1.62(s,3H)。

To a solution of 5a (9.0mg, 0.01mmol) in DMF (0.3 mL) at room temperature was added Ni (acac) ₂ And 2,2' -bipyridine in DMF (0.1mL, 0.01mmol, 0.1M). A THF solution of S23 (0.1mL, 0.033mmol,0.33M in THF) was then added and stirred at room temperature overnight. After this time, it was diluted with EtOAc, quenched with half-saturated aqueous ammonium chloride solution and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 4/1) to give 6b (3.1mg, 50%). ¹ H NMR(600MHz,CDCl ₃ )δ7.56(d,J＝8.4Hz,1H),7.16(t,J＝8.0Hz,1H),6.88(d,J＝8.4Hz,1H),6.77(s,2H),5.20(s,1H),4.97(t,J＝6.6Hz,1H),4.83(s,1H),4.77(s,1H),3.97(s,1H),3.86(t,J＝7.8Hz,1H),3.21–3.16(m,2H),3.06(dd,J＝14.2,5.9Hz,1H),2.51(dd,J＝18.1,5.8Hz,1H),2.29(s,4H),2.15–1.98(m,9H),1.79(s,3H),1.74(s,3H),1.70(s,3H),1.69(s,3H)。

TCNHPI ester 5a (9.1mg, 0.01mmol) and magneton were added to the reaction tube at room temperature. The tube was evacuated and backfilled with argon. NiCl2 glyme and a solution of ditBuBipy in DMF (0.1L, 0.01mmol) were then added. After stirring for 2 minutes, a solution of the arylzinc reagent S24 in THF (0.15mL, 0.0375mmol) was added in one portion. After stirring at room temperature for 12 h, the mixture was diluted with EtOAc, quenched with half-saturated aqueous ammonium chloride solution, and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2/1) to give 6c (5.9mg, 90%). ¹ HNMR(600MHz,CDCl ₃ )δδ7.31(t,J＝7.5Hz,3H),7.24(d,J＝7.4Hz,3H),7.20(t,J＝7.3Hz,1H),7.10(t,J＝8.1Hz,1H),6.68(d,J＝8.6Hz,3H),5.28(s,1H),4.97–4.91(m,1H),3.89–3.84(m,1H),3.81(t,J＝6.2Hz,1H),3.68–3.62(m,1H),3.15(dd,J＝14.7,6.3Hz,1H),3.05(dd,J＝14.8,6.8Hz,1H),2.92(dd,J＝18.4,6.4Hz,1H),2.25(s,4H),2.15(s,6H),1.82(s,3H),1.70(s,3H),1.68(s,3H)。

TCNHPI ester 5a (9.0 mg, 0.01mmol) and magnetons were added to the reaction tube at room temperature. The tube was evacuated and backfilled with argon, followed by NiCl2 glyme and a solution of ditBuBipy in DMF (0.1mL, 0.01mmol). After stirring for 5 minutes, a solution of S25 in THF (0.1mL, 0.033mmol) was added in one portion. After stirring at room temperature for 12 h, the mixture was diluted with EtOAc, quenched with half-saturated aqueous ammonium chloride, and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spin-dried to give a crude oil. It was redissolved in DCM (1 mL) and Et was added ₃ N(6μL,0.06mmol)、Ac ₂ O (14. Mu.L, 0.1 mmol) and DMAP (1.2mg, 0.01mmol). Stir at rt overnight, quench the reaction mixture with water and extract with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2/1) to give a white 6d solid (4.0 mg, 67%). ¹ H NMR(600MHz,CDCl ₃ )δ7.65(d,J＝8.3Hz,1H),7.20(t,J＝8.1Hz,1H),7.01(d,J＝8.5Hz,1H),6.82(d,J＝8.1Hz,2H),5.26(s,1H),5.02–4.96(m,1H),3.90(s,1H),3.59(dd,J＝10.4,7.7Hz,1H),3.21(dd,J＝14.2,6.1Hz,1H),3.08(d,J＝5.0Hz,1H),2.83–2.70(m,1H),2.31(s,4H),2.20–1.82(m,10H),1.72(s,3H),1.71(s,3H),1.69(s,3H),0.97(d,J＝6.4Hz,3H)。

At room temperature, to the reaction tubeTo this was added TCNHPI ester 5a (9.0 mg, 0.01mmol) and magneton. The tube was evacuated and backfilled with argon. Followed by the addition of NiCl ₂ Glyme and ditBuBipy in DMF (0.5mL, 0.1mmol). After stirring for 5 minutes, a solution of S26 in THF (0.5 mL, 0.1mmol) was added in one portion. After stirring at room temperature for 12 h, the mixture was diluted with EtOAc, quenched with half-saturated aqueous ammonium chloride solution and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil. The resulting mixture was redissolved in DCM (1 mL) and Et was added ₃ N(6μL,0.06mmol)、Ac ₂ O (14. Mu.L, 0.1 mmol) and DMAP (1.2mg, 0.01mmol). After stirring at room temperature overnight, quenched with water and extracted with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2/1) to give 6e as an oil (3.3mg, 59%). ¹ H NMR(600MHz,CDCl ₃ )δ7.19(d,J＝8.1Hz,1H),7.14(t,J＝7.9Hz,1H),7.00(d,J＝8.1Hz,1H),6.76(t,J＝8.4Hz,2H),5.59(d,J＝21.7Hz,1H),5.04(t,J＝5.9Hz,1H),4.47(d,J＝17.8Hz,1H),3.17(s,2H),2.35(s,4H),2.29(s,3H),2.19(s,1H),2.08(s,3H),1.91(d,J＝17.6Hz,1H),1.70(s,3H),1.67(s,6H),1.04(dd,J＝24.3,9.7Hz,1H),0.41(dd,J＝42.3,10.6Hz,2H),0.06(d,J＝20.5Hz,2H)。

5b (8mg, 0.01mmol), zn powder (1.3mg, 0.02mmol) and magnetons were charged into a culture tube at room temperature. The tube was then evacuated and backfilled with argon, THF (0.2 mL, anhydrous) and i-PrOH (0.02 mL) were added followed by rapid addition of NiCl ₂ ·6H ₂ O/ditBuBipy in DMF (0.25M, 0.04mL) and PhSiH ₃ (5. Mu.L). The culture tubes were then placed in a pre-warmed 40 ℃ oil bath and stirred overnight, cooled to ambient temperature, quenched with half-saturated ammonium chloride solution, extracted with EtOAc, the organic phases combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun dry to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2/1) to give 6f (4.0)mg,70％)。 ¹ H NMR(600MHz,CDCl ₃ )7.57(d,J＝8.6Hz,1H),7.21–7.14(m,1H),6.98(d,J＝8.6Hz,1H),6.78(d,J＝8.2Hz,2H),5.27(s,1H),4.97(t,J＝7.4Hz,1H),3.92(d,J＝5.0Hz,1H),3.85–3.75(m,1H),3.20(dd,J＝16.1,7.0Hz,1H),3.07(dd,J＝16.1,7.3Hz,1H),2.51(dd,J＝11.0,7.2Hz,1H),2.37–2.31(m,1H),2.30(s,1H),2.27–2.12(m,2H),2.10-2.00(m,9H),1.74(s,3H),1.70(s,3H),1.69(s,3H)。

LiCl (2.5mg, 0.06mmol), 5b (8mg, 0.01mmol), zn powder (2.6mg, 0.04mmol) and Ni (ClO) were charged into a culture tube at room temperature ₄ ) ₂ ·6H ₂ O (1.8mg, 0.005mmol) and magnetons. The tube was evacuated and backfilled with argon and Michael acceptor S27 (9. Mu.L, 0.06 mmol) and MeCN (0.1 mL) were added. After stirring overnight at room temperature, it was quenched with half-saturated ammonium chloride solution and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and spun-dried to give a crude oil which was purified by Preparative TLC (PTLC) (petroleum ether/ethyl acetate = 2/1) to give 6g (5.1mg, 69%). ¹ H NMR(600MHz,CDCl ₃ )7.57(d,J＝8.4Hz,1H),7.36–7.29(m,5H),7.18(t,J＝8.1Hz,1H),6.95(d,J＝8.5Hz,1H),6.79(d,J＝8.1Hz,2H),5.24(s,1H),5.12(d,J＝12.3Hz,1H),5.08(d,J＝12.3Hz,1H),4.97(t,J＝6.9Hz,1H),3.90(s,1H),3.71–3.64(m,1H),3.20(dd,J＝14.5,6.5Hz,1H),3.07(dd,J＝14.6,6.1Hz,1H),2.63(d,J＝4.8Hz,1H),2.49(ddd,J＝15.4,9.6,5.7Hz,1H),2.44–2.36(m,2H),2.30(s,3H),1.97(s,9H),1.90-1.88(m,1H),1.79(dd,J＝17.6,8.5Hz,2H),1.70(s,6H),1.69(s,3H)。

To a solution of compound S21 (6 mg, 0.01mmol) in dry toluene (0.1 mL) at room temperature were added DPPA (2.7. Mu.L, 0.012 mmol) and Et ₃ N (2. Mu.L, 0.015 mmol). After the reaction mixture was stirred at 80 ℃ for 3 hours, bnOH (1.2. Mu.L, 0.012 mmol) was addedThe reaction mixture was added and heating was continued at 80 ℃ with stirring overnight. After cooling to room temperature, the mixture was directly purified by Preparative TLC (PTLC) (petroleum ether/acetone = 2/1) to give 6h (3.0 mg, 43%). ¹ H NMR(600MHz,CDCl ₃ )δ12.64(s,1H),7.56(d,J＝7.9Hz,1H),7.36–7.30(m,5H),7.15(t,J＝8.2Hz,1H),6.75(d,J＝7.4Hz,2H),6.40(d,J＝7.7Hz,1H),5.36(s,1H),5.16–5.01(m,3H),4.90(s,1H),4.50(s,1H),4.31(s,1H),4.14(s,1H),3.23(qd,J＝14.6,7.0Hz,2H),2.86(d,J＝16.9Hz,1H),2.28(s,3H),2.22–1.90(m,10H),1.76(s,3H),1.72(s,3H),1.66(s,3H)。

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (10)

1.D-a reactive enzyme MaDA1 mutant, which is one or more of the following mutations in the amino acid sequence shown in SEQ ID No. 1:

(1) The M mutation at the 434 th site is V; (2) the 434 th M mutation is V, and the 168 th S mutation is F; (3) The M mutation at the 434 th site is V, the S mutation at the 168 th site is F, and the P mutation at the 368 th site is A.

2. Mutant of the D-a reactive enzyme MaDA1 according to claim 1, characterized in that the amino acid sequence of the mutant is shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.

3. A gene encoding the mutant of any one of claims 1-2, the nucleotide sequence of which is shown in SEQ ID No. 5; or as shown in SEQ ID No. 6; or as shown in SEQ ID No. 7.

4. The biomaterial containing the gene of claim 3, wherein the biomaterial is an expression cassette, a plasmid, a vector, a microorganism, an animal cell, or a plant cell.

5. Use of a mutant according to any one of claims 1-2 or a gene according to claim 3 or a biomaterial according to claim 4 for the catalytic synthesis of a natural or non-natural D-a product by Diels-Alder reaction; the reaction substrates for the Diels-Alder reaction are non-natural dienophiles and dienes.

6. The use according to claim 5, wherein the non-natural dienophile is a non-natural dienophile containing a carboxyl group, an ester group or a precursor thereof.

7. Use of a mutant according to any one of claims 1 to 2 or a gene according to claim 3 or a biomaterial according to claim 4 for the diversification and stereoselective synthesis of cyclohexene backbone structures.

8. The use according to claim 7, characterized in that after the enzymatic D-A product has been produced using the mutant of D-A-reactive enzyme MaDA1, the D-A product is protected with acetyl groups and after hydrolysis of the ester groups, N-hydroxyphthalimide esters are introduced; the introduction of different substituents on the cyclohexene skeleton is realized through decarboxylation functionalization reaction.

9. The use according to claim 8, wherein the decarboxylating functionalization reaction comprises but is not limited to the following: decarboxylation alkylation, decarboxylation alkenylation, decarboxylation alkynylation, decarboxylation arylation and Curtis rearrangement reaction.

10. Use according to any one of claims 5 to 9, wherein the temperature at which the Diels-Alder reaction is catalysed is from 30 to 37 ℃ and the pH is from 6.0 to 6.5.

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