Diels-alder reaction enzyme mutant, preparation method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly provides a Diels-Alder reaction enzyme mutant as well as a preparation method and application thereof. The present application relates to functional modification and strengthening of a Diels-Alder (D-A) reaction enzyme MaDA1 derived from mulberry, so that the enzyme catalyzes intermolecular D-A reaction of a non-natural dienophile containing a ''handle group'' (carboxyl, ester or a precursor thereof) and various dienophiles, so that further modification of the enzymatic D-A product can be realized through decarboxylation functionalization reaction, and diversification and stereoselective synthesis of a cyclohexene skeleton structure are realized.

Description

TECHNICAL FIELD

[0001] The application 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

[0002] It is well known that the functional diversity of drug molecules is determined by their structural diversity. Due to the complexity of their three-dimensional structure, polysubstituted six-membered ring skeleton structures are widely present in a variety of drugs with biological diversity activities, such as the anticonvulsant drug cannabidiol, the antiviral drug oseltamivir, the antitumor drug taxol and the antidepressant drug brexanolone (Allosteride) A). Therefore, chemists are very active in developing stereoselective methods to effectively 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. Figure 1

[0003] Enzymes as biological catalysts play an increasingly important role in the synthesis of drugs and natural products. Therefore, the development and use of enzymes that can stereoselectively catalyze Diels-Alder reactions provide a new idea for solving the problems of poor stereoselectivity and low efficiency faced by traditional chemical catalysis of D-A reactions. However, enzymes often have high substrate specificity, making it difficult for enzyme catalysis to efficiently achieve diversified synthesis. SUMMARY

[0004] The first object of the present application is to provide a preparation method of a Diels-Alder reaction enzyme mutant and its application in the synthesis of unnatural D-A products.

[0005] The second object of the present application is to combine enzymatic D-A reaction and decarboxylation functionalization to achieve the diversified synthesis of cyclohexene type compounds.

[0006] In a first aspect, the present application provides a mutant of D-A reaction enzyme MaDA1, and the amino acid sequence of the mutant is shown in SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0007] MaDA1 mentioned in the present application 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 present application.

[0008] ​The mutant M1 protein of the D-A response enzyme MaDA1 provided in the application does not have a signal peptide sequence, as shown in SEQ ID No. 2.

[0009] The mutant M2 protein of the D-A response enzyme MaDA1 provided in the application does not have a signal peptide sequence, as shown in SEQ ID No. 3.

[0010] The mutant M3 protein of the D-A response enzyme MaDA1 provided in the application does not have a signal peptide sequence, as shown in SEQ ID No. 4.

[0011] In a second aspect, the application provides a gene encoding the mutant, and the nucleotide sequence of the gene is shown in SEQ ID No. 5; or shown in SEQ ID No. 6; or shown in SEQ ID No. 7.

[0012] In a third aspect, the application provides a biological material containing the gene, and the biological material is an expression cassette, a plasmid, a vector, a microorganism, an animal cell, or a plant cell.

[0013] In a fourth aspect, the application provides the use of the mutant, the gene, or the biological material in catalyzing Diels-Alder reaction to synthesize natural or unnatural D-A products; and the reaction substrate of the Diels-Alder reaction is an unnatural dienophile and a diene.

[0014] In the use provided in the application, the unnatural dienophile is an unnatural dienophile containing a carboxyl group, an ester group, or a precursor thereof.

[0015] In a fifth aspect, the application provides the use of the mutant, the gene, or the biological material in the diversification and stereoselective synthesis of a cyclohexene skeleton structure.

[0016] In the use of the diversification and stereoselective synthesis of the cyclohexene skeleton structure provided in the application, after the mutant of the D-A response enzyme MaDA1 is used to generate an enzymatic D-A product, the D-A product is acetylated, the ester group is hydrolyzed, and then an N-hydroxy phthalimide ester is introduced; through a decarboxylation functionalization reaction, different substituents on the cyclohexene skeleton are introduced.

[0017] In the use of the diversification and stereoselective synthesis of the cyclohexene skeleton structure provided in the application, the decarboxylation functionalization reaction includes but is not limited to the following: decarboxylation alkylation, decarboxylation alkenylation, decarboxylation alkynylization, decarboxylation arylation, and Curtis rearrangement reaction.

[0018] In the use provided in the application, the temperature for catalyzing the Diels-Alder reaction is 30-37℃, and the pH is 6.0-6.5.

[0019] The present application has the following advantages:

[0020] (1) The polysubstituted six-membered ring skeleton structure is widely present in various drugs with biological diversity activity (A of FIG. 1), and the present application provides a chemical-enzymatic synthesis strategy for realizing the diversified synthesis of cyclohexene type compounds by combining the respective advantages of enzyme catalysis and chemical catalysis (B of FIG. 1). Figure 1 Figure 1 (2) The present application finds that the known DA reaction enzyme MaDA1 can recognize unnatural dienophile with a handle group (carboxyl and its derivatives) as a substrate; by mutating and screening MaDA1, a MaDA1 mutant (M3) with greatly improved activity and stereoselectivity is obtained, which can realize the construction of a cyclohexene skeleton with medium to excellent yield and good stereoselectivity; the handle group in the enzymatic D-A product can be further derived by decarboxylation functionalization, further enriching the structure of the enzymatic product, and realizing the efficient diversified synthesis of cyclohexene compounds. The present application develops a novel chemical-enzymatic coupling strategy, which provides a new possibility for the synthesis of important chemical precursors or natural products containing a six-membered ring.

[0021] (2) The present application finds that the known DA reaction enzyme MaDA1 can recognize unnatural dienophile with a handle group (carboxyl and its derivatives) as a substrate; by mutating and screening MaDA1, a MaDA1 mutant (M3) with greatly improved activity and stereoselectivity is obtained, which can realize the construction of a cyclohexene skeleton with medium to excellent yield and good stereoselectivity; the handle group in the enzymatic D-A product can be further derived by decarboxylation functionalization, further enriching the structure of the enzymatic product, and realizing the efficient diversified synthesis of cyclohexene compounds. The present application develops a novel chemical-enzymatic coupling strategy, which provides a new possibility for the synthesis of important chemical precursors or natural products containing a six-membered ring. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort based on these drawings.

[0023] Figure 1 A of FIG. 1 is a representative drug molecule containing a six-membered ring skeleton structure; B is the chemical-enzymatic coupling strategy proposed by the present application.

[0024] Figure 2 is the activity test result of the M434 single mutant of the present application.

[0025] Figure 3 is the activity detection result of the double mutant of the present application.

[0026] Figure 4 is the activity detection result of the triple mutant of the present application.

[0027] Figure 5 is the comparison of catalytic effects after three mutations of MaDA1.

[0028] Figure 6 is the reaction condition optimization result of the present application.

[0029] ​Figure 7 is the substrate scope of the MaDA1 mutant of the present application.

[0030] Figure 8 is the decarboxylation coupling reaction of the DA product of the present application. DETAILED DESCRIPTION

[0031] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0032] If not specifically stated, the reagents and raw materials used in the following examples are commercially available. The strains, vectors, culture media and reagents used in the following examples are mainly:

[0033] The competent cells of E. coli DH5a and DH10Bac are both purchased from Zhuangmeng Biotechnology Co., Ltd. The insect cells Sf21 and Hi5 for expression are purchased from Invitrogen Corporation. The insect expression vector pI-secSUMOstar is purchased from LifeSensors Corporation.

[0034] LB solid medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, 1.5% agar.

[0035] LB liquid medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.

[0036] The plasmid miniprep kit and gel recovery kit are both purchased from Tiangen Biochemical Technology Co., Ltd.; PCR primer synthesis and plasmid sequencing are completed by Goldensun Biotechnology Co., Ltd.

[0037] Example 1 Synthesis of dienophile

[0038]

[0039] To a solution of S1 (3.4 g, 14.7 mmol, 1.0 equiv.) in DCM (150 mL) was added DHP (1.61 g, 17.6 mmol, 1.2 equiv.) and PPTS (185 mg, 0.74 mmol, 0.05 equiv.) at room temperature. After 12 h, the reaction solution was rotary evaporated, and the crude product was purified by recrystallization with ether to obtain S2 (3.4 g, 74%) in the form of white solid. 1H NMR (400 MHz, CDC13) δ 12.09 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 6.59 (dd, J = 9.0, 2.4 Hz, 1H), 5.51 (t, J = 3.2 Hz, 1H), 4.37 (s, 2H), 3.96 3.50 (m, 2H), 2.11 1.58 (m, 6H).

[0040]

[0041] To a solution of S3 (1.0 g, 3 mmol, 1.0 equiv.) in tetrahydrofuran (100 mL) was added a solution of 3-methylbut-2-en-l-ol (0.9 mL, 9 mmol, 3.0 equiv.) and PPh3 (2.4 g, 9 mmol, 3.0 equiv.) in THF (50 mL) at -78 °C, followed by the addition of DEAD (2 mL, 12 mmol, 4.0 equiv.) and slowly warmed to room temperature. After stirring for 30 min, the reaction was spun dry to give the crude product, which was used in the next step without further purification.

[0042] To a solution of the crude product from the previous step in DCM (100 mL) was added montmorillonite K10 (2 g) at 0 °C and slowly warmed to room temperature. After stirring overnight, the reaction mixture was filtered to remove the montmorillonite K10 and spun dry, and purified by silica gel column chromatography (petroleum ether / acetone = 15 / 1) to give S5 (248 mg, 28%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 12.57 (s, 1H), 7.55 (d, J = 8.9 Hz, 1H), 6.42 (d, J = 8.9 Hz, 1H), 6.19 (s, 1H), 5.26 (t, J = 7.2 Hz, 2H), 4.37 (s, 2H), 3.45 (d, J = 7.2 Hz, 2H), 1.83 (s, 4H), 1.77 (s, 3H).

[0043]

[0044] To a solution of S5 (190 mg, 0.6 mmol, 1.0 equiv.) in MeCN (20 mL) was added Et3N (20 uL, 0.14 mmol, 0.2 equiv.) and PPh3 (183 mg, 0.70 mmol, 1.1 equiv.) at room temperature. After stirring for 2 h, the residue was dissolved in CH2Cl2:H2O (25.6 mL:38.4 mL) and 2 M NaOH (3.2 mL, 10 equiv.) was added. After stirring at room temperature for 4 h, the solution was partitioned, the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo and purified by recrystallization from diethyl ether / dichloromethane to give S7 (282 mg, 92%, 2 steps) as a white solid. 1 H NMR (400 MHz, CDC13) δ 13.22 (s, 1H), 7.99 (d, J = 15.3 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.45 - 7.32 (m, 5H), 6.98 (d, J = 15.3 Hz, 1H), 6.42 (d, J = 8.9 Hz, 1H), 6.18 (s, 1H), 5.28 (s, 3H), 3.46 (d, J = 7.3 Hz, 2H), 1.83 (s, 3H), 1.77 (s, 2H).

[0045]

[0046] To a solution of S7 (180 mg, 0.375 mmol, 1.0 equiv.) in THF (8 mL) was added a solution of S8 (123 mg, 0.75 mmol, 2.0 equiv.) in THF (4 mL) at -20 °C. After stirring for 1 h, the solution was concentrated in vacuo (<30 °C) and purified by silica gel column chromatography (petroleum ether / dichloromethane = 1 / 3-1 / 15) to give the diene 1a (106 mg, 77%) as a yellow solid. 1 H NMR (400 MHz, CDC13) δ 13.22 (s, 1H), 7.99 (d, J = 15.3 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.45 - 7.32 (m, 5H), 6.98 (d, J = 15.3 Hz, 1H), 6.42 (d, J = 8.9 Hz, 1H), 6.18 (s, 1H), 5.28 (s, 3H), 3.46 (d, J = 7.3 Hz, 2H), 1.83 (s, 3H), 1.77 (s, 2H).

[0047]

[0048] To a solution of S7 (180 mg, 0.375 mmol, 1.0 equiv.) in THF (8 mL) was added a solution of S9 (112 mg, 0.75 mmol, 2.0 equiv.) in THF (4 mL) at room temperature, followed by heating at 55 °C overnight. When TLC showed the disappearance of starting material, the reaction mixture was cooled to room temperature and rotary evaporated, purified by silica gel column chromatography (dichloromethane / acetone = 100 / 1-100 / 3) to give the dienophile 1b (85 mg, 65%) as a yellow solid. 1 H NMR (400 MHz, Acetone-d6) δ 13.69 (s, 1H), 9.39 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.37 (dt, J = 15.1, 2.0 Hz, 1H), 7.20 (dt, J = 15.1, 3.6 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 5.26 (dddt, J = 7.2, 5.7, 2.7, 1.4 Hz, 1H), 4.41 (dq, J = 5.6, 3.6, 2.9 Hz, 2H), 4.28 (t, J = 5.4 Hz, 1H), 3.36 (d, J = 7.2 Hz, 2H), 1.77 (s, 3H), 1.63 (s, 3H).

[0049]

[0050] To a solution of S7 (60 mg, 0.125 mmol, 1.0 equiv.) in THF (1 mL) was added S10 (8.3 mg, 0.069 mmol, 0.55 equiv.) at room temperature, followed by heating at reflux overnight. When TLC showed the disappearance of starting material, the reaction mixture was cooled to room temperature and rotary evaporated, purified by silica gel column chromatography (dichloromethane / acetone = 25 / 1-16 / 1) to give the dienophile 1c (20 mg, 61%) as a white solid. 1 H NMR (400 MHz, Acetone-d6) δ 13.69 (s, 1H), 9.39 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.37 (dt, J = 15.1, 2.0 Hz, 1H), 7.20 (dt, J = 15.1, 3.6 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 5.26 (dddt, J = 7.2, 5.7, 2.7, 1.4 Hz, 1H), 4.41 (dq, J = 5.6, 3.6, 2.9 Hz, 2H), 4.28 (t, J = 5.4 Hz, 1H), 3.36 (d, J = 7.2 Hz, 2H), 1.77 (s, 3H), 1.63 (s, 3H).

[0051]

[0052] To a solution of Sll (2 g, 9.3 mmol, 1.0 equiv.) in CH3CN (60 mL) was added 3-chloro-3-methylbut-l-yne S12 (5.25 mL, 50 mmol, 5.0 equiv.), CuCl2-H2O (12 mg, 0.07 mmol, 0.007 equiv.) and DBU (1.46 mL, 9.8 mmol, 1.05 equiv.) successively at 0 °C. After stirring for 5 h, the reaction mixture was quenched with saturated aqueous NH4C1 solution, extracted with ethyl acetate, the combined organic phase was washed with saturated brine, dried over anhydrous Na2S04, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl ether = 20 / 1) to give S13 (550 mg, 21%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.91 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 8.9 Hz, 2H), 4.66 (s, 2H), 2.65 (s, 1H), 1.71 (s, 6H).

[0053]

[0054] To a solution of aryl propargyl ether S13 (200 mg, 0.72 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added Lindlar catalyst (80 mg, 40 wt%) at room temperature, then the suspension was degassed and backfilled with H2 three times at -78 °C. After stirring at room temperature for 10 h, the reaction mixture was filtered through celite and concentrated under reduced pressure. The residue was redissolved in toluene (20 mL) and heated at reflux overnight. When TLC showed the disappearance of starting material, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 - 1 / 1) gave S14 (70 mg, 35%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.75 (d, J = 2.2 Hz, 2H), 6.86 (d, J = 8.1 Hz, 1H), 5.79 (s, 1H), 5.35 - 5.27 (m, 1H), 4.64 (s, 2H), 3.41 (d, J = 7.2 Hz, 2H), 1.79 (s, 6H).

[0055]

[0056] To a solution of S14 (70 mg, 0.25 mmol, 1.0 equiv.) in MeCN (8 mL) was added Et3N (10 uL, 0.07 mmol, 0.3 equiv.) and PPh3 (68 mg, 0.26 mmol, 1.1 equiv.) at room temperature. The reaction mixture was then heated at 60 °C with stirring for 48 h, cooled to room temperature and spin dried. The residue was dissolved in dichloromethane / water (8 mL: 12 mL) and 2 M NaOH (1 mL) was added. After stirring at room temperature for 4 h, it was extracted with dichloromethane. The organic phases were combined, washed with saturated 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) was added a solution of S8 (46 mg, 5.5 mmol) in THF (7 mL) at 0 °C. It was then stirred overnight and spin dried (<30 °C) to give a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give the dienophile 1d (48 mg, 72%) as a yellow solid. 1 H NMR (400 MHz, CDC13) δ 7.94 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 7.2 Hz, 2H), 7.44 - 7.34 (m, J = 13.0 Hz, 5H), 7.00 - 6.80 (m, 2H), 5.83 (s, 1H), 5.29 (d, J = 11.4 Hz, 3H), 3.41 (d, J = 6.8 Hz, 2H), 1.79 (s, 6H).

[0057] Example 2 Synthesis of different acetyl dienophiles

[0058]

[0059] To a solution of substituted 2-iodobenzene-1,3-diol (0.3 mmol) in DCM (3 mL) was added Et3N (250 μL, 1.8 mmol) and Ac20 (112 μL, 1.2 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched with water and extracted with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to give the crude product, which was used directly in the next step. The crude product from the previous step, boron reagent S18 (97 mg, 0.5 mmol), K3P04 (635 mg, 3 mmol), AsPh3 (12.8 mg, 0.042 mmol) and Pd2(dba)3 (19.2 mg, 0.021 mmol) were dissolved in DMF (5 mL) and stirred at 60 °C overnight. After cooling to room temperature, the reaction mixture was filtered through celite and washed with EtOAc. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness. The resulting mixture was redissolved in DCM (3 mL), and Et3N (250 μL, 1.8 mmol) and Ac20 (112 μL, 1.2 mmol) were added. After stirring at room temperature overnight, the reaction mixture was quenched with water and extracted with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to give the crude product, which was purified by silica gel column chromatography to give the corresponding product.

[0060] Preparation, activity testing and evolution of MaDA1 mutants

[0061] To further improve the catalytic activity and selectivity of MaDA1, the MaDA1 enzyme was evolved in this example, and the mutants obtained were tested for activity.

[0062] 1. Construction and expression of MaDA1 mutants

[0063] MaDA1 mutants were obtained by megaprimer PCR of whole plasmid using different primers in Table 1, i.e. first a large fragment DNA fragment was obtained by PCR amplification using forward primer containing mutation site (MaDA1_M434V_2_F or MaDA1_S168F_2_F or MaDA1_P368A_2_F) and reverse primer pIsec_MaDA1_R as primers, pI-sec-sumostar-tev-MaDA1 as template. Then the PCR product was used as magaprimer, pI-sec-sumostar-tev-MaDA1 plasmid (or plasmid already containing the above site mutation) as template for megaprimer PCR. After DpnI digestion, the product was transformed into E. coli DH5a strain. The clones containing mutant genes were selected from the plate and confirmed by Sanger sequencing. Then the plasmid was transformed into E. coli DH10Bac competent cells to generate recombinant baculovirus. Subsequently, the Bac-to-Bac heterologous expression system was used for expression, and purified by Ni-NTA. All protein concentrations were analyzed using NanoDrop2000 (Thermo). The average protein yield of MaDA1 mutants was from 15 to 25 mg L -1 unequal.

[0064] Table 1 Primer sequence information used

[0065]

[0066] 2. Activity test of MaDA1 mutants

[0067] To determine the relative activity of MaDA1 mutants, 200 mM diene 2a, 150 mM dienophile 1a, and 3 pg of MaDA1 mutants were reacted in 100 pL Tris-HCl buffer (pH = 7, 25 mM) at 37 °C for 3 h (2a). The reaction was quenched with 100 pL ice-cold acetonitrile and centrifuged at 15,000 g for 10 min. The supernatant was analyzed by reverse-phase UPLC analysis.

[0068] The conditions for UPLC analysis were as follows: column: ACQUITY UPLC BEH C18 (50 mm x 2.1 mm, 1.7 pm); mobile phase: acetonitrile-water gradient elution, 0-7 min, 30→100% acetonitrile; 7-8 min, 100→30% acetonitrile.

[0069] 3. Evolution of MaDA1

[0070] The conversion of MaDA1 was 8% with 2a as substrate, and endo / exo=5 / 1. M434, S168, R285, N347, P368 in the pocket were selected as mutation hotspots, and focused iterative residue-specific mutation was carried out. First, M434 was mutated to A, V, S, F, and as a supplement, I, L, C, to obtain the first round of best mutant MaDA1_M434V (M1). The activity test results of single mutant are shown in Table 1. Figure 2 The conversion rate was 32%, and endo / exo>15 / 1.

[0071] Second round, MaDA1_M434V as template, S168A, S168V, S168F, R285A, R285F, R285V, R285S, N347A, N347V, N347F, N347S, P368A, P368V, P368F, P368S mutants and as a supplement S168W, S168H, S168Y were constructed, and the second round of best mutant MaDA1_M434V_S168F (M2) was obtained. The activity test results of double mutant are shown in Table 2. Figure 3 The conversion rate was 58%, and endo / exo>15 / 1.

[0072] Third round, MaDA1_M434V_S168F as template, P368A, P368G, P368S, P368T, P368T, P368D, P368N, P368R were constructed, and the third round of best mutant MaDA1_M434V_S168F_P368A (M3) was obtained. The activity test results of triple mutant are shown in Table 3. Figure 4 The conversion rate was 81%, and endo / exo>15 / 1.

[0073] The comparison of catalytic effect of MaDA1 after three mutations is shown in Table 4. Figure 5 In subsequent large number of reactions, 2a was used as diene, and the corresponding yields of MaDA1, M1, M2, M3 were 39%, 62%, 62%, and 92% respectively, TTN was 192, 730, 996, and 2922 respectively, and ee value was 93%, 99%, 99%, and 99% respectively. From the enzyme parameters, compared with wild type MaDa1 (k cat / K M =0.55mM -1 s -1 ), M3 (k cat / K M =19.13mM -1 s -1) 34-fold activity improvement and 12-fold TTN improvement.

[0074] Example 4 Optimization of enzyme reaction conditions

[0075] To determine the optimal reaction pH of MaDA1_M3, 200 mM diene 2a, 150 mM dienophile la, and 3 pg of MaDA1 mutant MaDA1_M3 were reacted in 100 pL of different buffers at 37 °C for 3 h. The reactions were quenched with 100 pL of ice-cold acetonitrile and centrifuged at 15,000 g for 10 min. The supernatants were analyzed by analytical reverse-phase UPLC.

[0076] By comparing the conversion rates and the yields of by-product la', the optimal reaction conditions were determined to be: phosphate buffer, 25 mM, pH = 6.0, 37 °C. The final conversion rate could reach 92%( Figure 6 ).

[0077] Example 5 Substrate spectrum of MaDA1_M3

[0078] To determine the substrate spectrum of MaDA1_M3, 200 pM dienes 2-2k, 150 pM dienophiles la or lb, and 3 pg of MaDA1 mutant MaDA1_M3 were reacted in 100 pL of phosphate buffer (25 mM, pH = 7) for 3 h. The reactions were quenched with 100 pL of ice-cold acetonitrile and centrifuged at 15,000 g for 10 min. The supernatants were analyzed by analytical reverse-phase UPLC. From the experimental results, all aryl-substituted 1,3-dienes reacted with the dienophile to give the corresponding D-A products (see Figure 7 ).

[0079] Example 6 Application of MaDA1_M3 in the synthesis of endo-configured D-A products containing carboxylate handle for derivatization

[0080] To further verify the application of MaDA1 mutant MaDA1_M434V_S168F_P368A (M3) in the synthesis of endo-configured D-A products containing carboxylate handle for derivatization, this example selectively prepared representative substrates enzymatically.

[0081] To a solution of diene precursor (0.026 mmol, 1.2 equiv) in MeOH (0.8 mL) and H2O (0.4 mL) was added K2CO3 (14 mg, 0.104 mmol, 4.0 equiv) at room temperature. The resulting mixture was stirred for 1 h to generate the diene in situ. The resulting solution was then added to 48 mL of reaction solution (0.5-1 mg MaDA1-M3 in 25 mM NaPi, pH = 6.0, 2% DMSO). To this mixture, 440 μL of dienophile (0.022 mmol) DMSO stock (50 mM) was added in four equal portions every 2 h. After incubation at 37 °C for 24 h, the reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (PTLC) to give the corresponding product.

[0082]

[0083] To a solution of diene precursor (0.026 mmol) in methanol (0.8 mL) and water (0.4 mL) was added K2CO3 (14 mg, 0.104 mmol) at room temperature. After stirring for 1 h, the diene was generated in situ. The resulting solution was then added to 48 mL of reaction solution (0.5-1 mg MaDA1-M3, 25 mM NaPi, pH = 6.0, 2% DMSO). Subsequently, 440 μL of dienophile (0.022 mmol) DMSO stock (50 mM) was added in four equal portions every 2 h. After incubation at 37 °C for 24 h, the reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was 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, and the NMR and yield data are as follows:

[0084]

[0085] 3a: 1H NMR (600 MHz, Acetone-d6) δ 12.84 (s, 1H), 9.19 (s, 1H), 8.47 (s, 1H), 8.10 (s, 2H), 7.88 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.34 - 7.24 (m, 5H), 6.93 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 0.9 Hz, 1H), 6.78 (dd, J = 8.4, 2.1 Hz, 1H), 6.74 (s, 2H), 6.46 (d, J = 8.8 Hz, 1H), 5.55 - 5.51 (m, 1H), 5.19 (dddd, J = 7.2, 5.9, 2.7, 1.4 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H), 5.04 (d, J = 12.6 Hz, 1H), 4.73 - 4.67 (m, 1H), 4.34 (dd, J = 10.1, 7.1 Hz, 1H), 4.03 (td, J = 10.0, 5.9 Hz, 1H), 3.25 (p, J = 6.7 Hz, 2H), 2.48 (dd, J = 17.5, 5.7 Hz, 1H), 2.26 (dd, J = 17.5, 9.9 Hz, 1H), 1.80 (s, 3H), 1.71 (s, 3H), 1.59 (s, 3H).

[0086]

[0087] 3b: 1 H NMR (600 MHz, Acetone-d6) δ 12.84 (s, 1H), 9.19 (s, 1H), 8.47 (s, 1H), 8.10 (s, 2H), 7.88 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.34 - 7.24 (m, 5H), 6.93 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 0.9 Hz, 1H), 6.78 (dd, J = 8.4, 2.1 Hz, 1H), 6.74 (s, 2H), 6.46 (d, J = 8.8 Hz, 1H), 5.55 - 5.51 (m, 1H), 5.19 (dddd, J = 7.2, 5.9, 2.7, 1.4 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H), 5.04 (d, J = 12.6 Hz, 1H), 4.73 - 4.67 (m, 1H), 4.32 (dd, J = 9.9, 7.1 Hz, 1H), 3.99 (td, J = 9.9, 5.9 Hz, 1H), 3.26 (p, J = 6.7 Hz, 2H), 2.45 (dd, J = 17.4, 5.6 Hz, 1H), 2.24 (dd, J = 17.5, 9.8 Hz, 1H), 1.78 (s, 3H), 1.73 (s, 3H), 1.61 (s, 3H).

[0088]

[0089] 3c: 1 H NMR (400 MHz, Acetone-d6) δ 12.81 (s, 1H), 9.18 (s, 1H), 8.34 (s, 2H), 7.86 (d, J = 9.0 Hz, 1H), 7.37 - 7.17 (m, 5H), 6.44 (d, J = 8.8 Hz, 1H), 6.26 (s, 2H), 5.47 - 5.41 (m, 1H), 5.24 - 5.15 (m, 1H), 5.07 (d, J = 12.6 Hz, 1H), 5.02 (d, J = 12.6 Hz, 1H), 4.64 (s, 1H), 4.27 (dd, J = 10.4, 7.2 Hz, 1H), 4.00 (td, J = 10.4, 5.9 Hz, 1H), 3.27 (h, J = 7.2 Hz, 2H), 2.45 (dd, J = 17.3, 5.7 Hz, 1H), 2.22 (dd, J = 17.0, 10.4 Hz, 1H), 1.77 (s, 3H), 1.73 (s, 3H), 1.62 (s, 3H).

[0090]

[0091] 3d: 1 H NMR (400 MHz, Acetone-d6) δ 12.77 (s, 1H), 8.31 (s, 2H), 7.87 (d, J = 8.9 Hz, 1H), 7.35 - 7.23 (m, 5H), 6.89 (s, 2H), 6.45 (d, J = 8.8 Hz, 1H), 5.50 - 5.41 (m, 1H), 5.23 - 5.15 (m, 1H), 5.08 (d, J = 12.6 Hz, 1H), 5.02 (d, J = 12.6 Hz, 1H), 4.73 (s, 1H), 4.32 (dd, J = 10.4, 7.2 Hz, 1H), 4.05 (td, J = 10.3, 5.9 Hz, 1H), 3.77 (s, 3H), 3.26 (p, J = 7.9 Hz, 2H), 2.47 (dd, J = 17.3, 5.8 Hz, 1H), 2.23 (dd, J = 17.1, 10.1 Hz, 1H), 1.78 (s, 3H), 1.72 (s, 3H), 1.61 (s, 3H).

[0092]

[0093] 3e: 1H NMR (400 MHz, Acetone-d6) δ 12.87 (s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.71 (s, 2H), 7.29 (q, J = 4.1 Hz, 5H), 6.44 (d, J = 8.8 Hz, 1H), 6.04 (s, 2H), 5.50 (s, 1H), 5.20 (t, J = 6.6 Hz, 1H), 4.69 - 4.59 (m, 1H), 4.30 (dd, J = 9.8, 6.9 Hz, 1H), 3.95 (td, J = 9.7, 6.0 Hz, 1H), 3.26 (d, J = 7.1 Hz, 2H), 2.44 (dd, J = 17.3, 5.8 Hz, 1H), 2.23 (dd, J = 17.1, 9.6 Hz, 1H), 2.03 (s, 3H), 1.77 (s, 3H), 1.73 (s, 3H), 1.61 (s, 3H).

[0094]

[0095] 3f: 1 H NMR (600 MHz, Acetone-d6) δ 12.75 (s, 1H), 9.21 (s, 1H), 8.23 (s, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.54 (s, 1H), 7.38 - 7.21 (m, 5H), 6.93 (d, J = 8.7 Hz, 1H), 6.45 (d, J = 8.8 Hz, 1H), 6.26 (d, J = 8.7 Hz, 1H), 5.49 (dt, J = 2.8, 1.4 Hz, 1H), 5.20 (ddt, J = 8.6, 5.9, 1.4 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H), 5.03 (d, J = 12.6 Hz, 1H), 4.73 - 4.64 (m, 1H), 4.35 (dd, J = 9.9, 7.1 Hz, 1H), 3.94 (td, J = 9.8, 6.0 Hz, 1H), 3.26 (p, J = 6.7 Hz, 2H), 2.46 (dd, J = 17.4, 5.7 Hz, 1H), 2.25 (dd, J = 17.5, 9.7 Hz, 1H), 1.79 (s, 3H), 1.73 (s, 3H), 1.61 (s, 3H).

[0096]

[0097] 3i: 1H NMR (400 MHz, Acetone-d6) δ 12.85 (s, 1H), 7.95 (s, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H), 6.80 (t, J = 8.0 Hz, 1H), 6.41 (d, J = 8.9 Hz, 1H), 6.28 (d, J = 8.0 Hz, 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 = 8.8 Hz, 1H), 3.59 (dd, J = 9.2, 5.9 Hz, 1H), 3.27 (d, J = 7.2 Hz, 2H), 2.59 (dtt, J = 9.0, 6.3, 3.4 Hz, 1H), 2.29 (s, 1H), 1.92 (d, J = 18.3 Hz, 1H), 1.77 (s, 3H), 1.72 (s, 3H), 1.60 (s, 3H).

[0098]

[0099] 3j: 1 H NMR (400 MHz, Acetone-d6) δ 12.87 (s, 1H), 7.90 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H), 6.79 (t, J = 8.0 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H), 6.27 (d, J = 8.1 Hz, 2H), 5.64 (s, 1H), 5.17 (td, J = 6.6, 5.9, 3.6 Hz, 1H), 4.57 (dd, J = 5.8, 3.3 Hz, 1H), 4.41 - 4.30 (m, 1H), 4.01 (s, 1H), 3.76 (dd, J = 10.1, 8.1 Hz, 1H), 3.67 (dd, J = 10.3, 6.6 Hz, 1H), 3.27 (d, J = 7.2 Hz, 2H), 2.39 (ddq, J = 9.8, 6.7, 2.7 Hz, 1H), 2.25 (dt, J = 19.7, 3.5 Hz, 1H), 1.95 (d, J = 17.8 Hz, 1H), 1.78 (s, 3H), 1.72 (s, 3H), 1.59 (s, 3H).

[0100]

[0101] 3k: 1H NMR (400 MHz, Acetone-d6) δ 7.98 (s, 2H), 7.72 (d, J = 2.1 Hz, 1H), 7.71 - 7.67 (m, 1H), 7.41 - 7.30 (m, 5H), 6.84 (d, J = 8.3 Hz, 1H), 6.75 (t, J = 8.0 Hz, 1H), 6.22 (d, J = 8.0 Hz, 2H), 5.53 (s, 1H), 5.32 - 5.25 (m, 1H), 5.16 (d, J = 12.7 Hz, 1H), 5.11 (d, J = 12.7 Hz, 1H), 4.55 (d, J = 5.6 Hz, 2H), 3.65 (q, J = 6.1 Hz, 1H), 3.29 (d, J = 7.3 Hz, 2H), 2.28 (d, J = 5.9 Hz, 2H), 1.76 (s, 3H), 1.68 (s, 3H), 1.67 (s, 3H).

[0102] Example 7 Diversification of D-A products using decarboxylative functionalization reactions

[0103] From D-A products, key intermediates are obtained via chemical transformations, and diverse polysubstituted polychiral center cyclohexane scaffolds are obtained via decarboxylative functionalization reactions, including but not limited to decarboxylative alkynylation, decarboxylative arylation, decarboxylative alkylation, decarboxylative amination, decarboxylative hydrogenation, decarboxylative Giese reaction (see Figure 8 ).

[0104]

[0105] To a solution of 3b (90 mg, 0.166 mmol) in CH2Cl2(18 mL) was added Et3N (278 μL, 2.0 mmol), Ac2O (127 μL, 1.3 mmol), DMAP (60 mg, 0.49 mmol) at room temperature. Stirred overnight, then quenched excess reagents with water. Partitioned, aqueous layer extracted with DCM, combined organic phases, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product which was directly subjected to the next step.

[0106] The above crude product was dissolved in acetone (10.5 mL), Pd / C (99 mg, 10% wt) and HCOONH4(1.6 mg, 0.025 mmol) were added. The suspension was degassed and backfilled with argon 3 times at -78 °C, then 1,4-cyclohexadiene (251 μL, 2.5 mmol) was added. After stirring at room temperature for 1 h, the reaction mixture was filtered through celite and concentrated to give the crude solid which was washed with hexane / DCM to give S21 as a white solid (89 mg, 87%). 1H NMR (400 MHz, CDC13) δ 7.56 (s, 1H), 7.22 (t, J = 8.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 7.2 Hz, 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, 6.2 Hz, 1H), 3.05 (dd, J = 16.3, 5.0 Hz, 1H), 2.82 (dd, J = 17.6, 4.9 Hz, 1H), 2.51 (dd, J = 17.2, 10.0 Hz, 1H), 2.29 (s, 6H), 1.96 (s, 6H), 1.81 (s, 3H), 1.69 (s, 6H).

[0107]

[0108] To a solution of S21 (50 mg, 0.08 mmol) in DCM (12 mL) was added N-hydroxyphthalimide (28 mg, 0.093 mmol), DMAP (2.8 mg, 0.022 mol) and DIC (18 μL, 0.129 mmol) at room temperature. After stirring overnight, the reaction mixture was filtered and spin dried to give the crude product, which was purified by column chromatography (dichloromethane / ether = 20 / 1) to give 5a (60 mg, 82%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.56 (s, 1H), 7.22 (t, J = 8.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 7.2 Hz, 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, 6.2 Hz, 1H), 3.05 (dd, J = 16.3, 5.0 Hz, 1H), 2.82 (dd, J = 17.6, 4.9 Hz, 1H), 2.51 (dd, J = 17.2, 10.0 Hz, 1H), 2.29 (s, 6H), 1.96 (s, 6H), 1.81 (s, 3H), 1.69 (s, 6H).

[0109]

[0110] To a solution of S21 (38 mg, 0.06 mmol) in DCM (8 mL) was added N-hydroxyphthalimide (12 mg, 0.074 mmol), DMAP (2.0 mg, 0.016 mol.) and DIC (14 μL, 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 / ethyl ether = 100 / 1-20 / 1) to give 5b (38 mg, 83%) as a white solid. 1 H NMR (600 MHz, CDC13) δ 7.85 (dd, J = 5.4, 3.1 Hz, 2H), 7.75 (dd, J = 5.4, 3.1 Hz, 2H), 7.59 (s, 1H), 7.22 (t, J = 8.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 7.6 Hz, 2H), 5.33 (s, 1H), 4.97 (t, J = 6.9 Hz, 1H), 4.24 (dd, J = 13.0, 7.4 Hz, 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, 9.2 Hz, 1H), 2.28 (s, 6H), 1.95 (s, 6H), 1.82 (s, 3H), 1.69 (s, 6H).

[0111]

[0112] To a reaction tube was added 5a (9.0 mg, 0.01 mmol, 1.0 equiv.) and a magnetic stir bar at room temperature. The tube was then evacuated and backfilled with argon. A solution of NiCl2-6H2O / 4,4'-dimethoxy-2,2'-bipyridine in DMF (100 μL, 0.01 mmol, 0.1 M in DMF) and ethynylzinc chloride S22 (100 μL, 0.033 mmol, 0.33 M in DMF) were then added. After stirring for 18 h, the reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give a crude oil, which was purified by preparative TLC (PTLC) (petroleum ether / ethyl acetate = 2.5 / 1) to give 6a (5.1 mg, 85%). 1H NMR (600 MHz, CDC13) δ 7.44 (s, 1H), 7.11 (t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.70 (s, 2H), 5.18 (s, 1H), 4.93 - 4.86 (m, 1H), 4.06 (s, 1H), 3.83 (t, J = 7.8 Hz, 1H), 3.52 - 3.42 (m, 1H), 3.12 (dd, J = 14.4, 5.8 Hz, 1H), 3.04 - 2.93 (m, 1H), 2.65 (dd, J = 17.9, 5.8 Hz, 1H), 2.22 (s, 4H), 2.08 - 1.90 (m, 10H), 1.67 (s, 3H), 1.64 (s, 3H), 1.62 (s, 3H).

[0113]

[0114] To a solution of 5a (9.0 mg, 0.01 mmol) in DMF (0.3 mL) was added Ni(acac)2 and a DMF solution of 2,2’-bipyridine (0.1 mL, 0.01 mmol, 0.1 M) at room temperature. Then a THF solution of S23 (0.1 mL, 0.033 mmol, 0.33 M in THF) was added and stirred at room temperature overnight. After that 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 concentrated to dryness to give a crude oil, which was purified by preparative TLC (PTLC) (petroleum ether / ethyl acetate = 4 / 1) to give 6b (3.1 mg, 50%). 1 H NMR (600 MHz, CDC13) δ 7.44 (s, 1H), 7.11 (t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.70 (s, 2H), 5.18 (s, 1H), 4.93 - 4.86 (m, 1H), 4.06 (s, 1H), 3.83 (t, J = 7.8 Hz, 1H), 3.52 - 3.42 (m, 1H), 3.12 (dd, J = 14.4, 5.8 Hz, 1H), 3.04 - 2.93 (m, 1H), 2.65 (dd, J = 17.9, 5.8 Hz, 1H), 2.22 (s, 4H), 2.08 - 1.90 (m, 10H), 1.67 (s, 3H), 1.64 (s, 3H), 1.62 (s, 3H).

[0115]

[0116] A reaction tube was charged with TCNHPI ester 5a (9.1 mg, 0.01 mmol) and a magnetic stir bar at room temperature. The tube was evacuated and backfilled with argon. NiCl2·glycol dimethyl ether and ditBuBipy in DMF (0.1 L, 0.01 mmol) were then added. After stirring for 2 min, a THF solution of aryl zinc reagent S24 (0.15 mL, 0.0375 mmol) 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 concentrated to dryness to give a crude oil, which was purified by preparative TLC (PTLC) (petroleum ether / ethyl acetate = 2 / 1) to give 6c (5.9 mg, 90%). 1 HNMR (600 MHz, CDC13) δ 7.31 (t, J = 7.5 Hz, 3H), 7.24 (d, J = 7.4 Hz, 3H), 7.20 (t, J = 7.3 Hz, 1H), 7.10 (t, J = 8.1 Hz, 1H), 6.68 (d, J = 8.6 Hz, 3H), 5.28 (s, 1H), 4.97 - 4.91 (m, 1H), 3.89 - 3.84 (m, 1H), 3.81 (t, J = 6.2 Hz, 1H), 3.68 - 3.62 (m, 1H), 3.15 (dd, J = 14.7, 6.3 Hz, 1H), 3.05 (dd, J = 14.8, 6.8 Hz, 1H), 2.92 (dd, J = 18.4, 6.4 Hz, 1H), 2.25 (s, 4H), 2.15 (s, 6H), 1.82 (s, 3H), 1.70 (s, 3H), 1.68 (s, 3H).

[0117]

[0118] A reaction tube was charged with TCNHPI ester 5a (9.0 mg, 0.01 mmol) and a magnetic stir bar at room temperature. The tube was evacuated and backfilled with argon, followed by the addition of a solution of NiCl2·glycol dimethyl ether and ditBuBipy in DMF (0.1 mL, 0.01 mmol). After stirring for 5 min, a solution of S25 in THF (0.1 mL, 0.033 mmol) was added in one portion. After stirring at room temperature for 12 h, the mixture was diluted with EtOAc, quenched with a saturated aqueous solution of ammonium chloride, and extracted with EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude oil. This was re-dissolved in DCM (1 mL), and Et3N (6 μL, 0.06 mmol), Ac2O (14 μL, 0.1 mmol), and DMAP (1.2 mg, 0.01 mmol) were added. After stirring at room temperature overnight, the reaction mixture was quenched with water and extracted with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude oil, which was purified by preparative TLC (PTLC) (petroleum ether / ethyl acetate = 2 / 1) to give 6d as a white solid (4.0 mg, 67%). 1 H NMR (600 MHz, CDC13) δ 7.65 (d, J = 8.3 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.1 Hz, 2H), 5.26 (s, 1H), 5.02 - 4.96 (m, 1H), 3.90 (s, 1H), 3.59 (dd, J = 10.4, 7.7 Hz, 1H), 3.21 (dd, J = 14.2, 6.1 Hz, 1H), 3.08 (d, J = 5.0 Hz, 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 = 6.4 Hz, 3H).

[0119]

[0120] To a reaction tube was added TCNHPI ester 5a (9.0 mg, 0.01 mmol) and a magnetic stir bar at room temperature. The tube was evacuated and backfilled with argon. Subsequently, a solution of NiCl2·glycol dimethyl ether and ditBuBipy in DMF (0.5 mL, 0.1 mmol) was added. After stirring for 5 min, a solution of S26 in THF (0.5 mL, 0.1 mmol) 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 concentrated to dryness to give a crude oil. The resulting mixture was re-dissolved in DCM (1 mL) and Et3N (6 μL, 0.06 mmol), Ac2O (14 μL, 0.1 mmol) and DMAP (1.2 mg, 0.01 mmol) were added. After stirring at room temperature overnight, it was quenched with water and extracted with DCM. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to give a crude oil, which was purified by preparative TLC (PTLC) (petroleum ether / ethyl acetate = 2 / 1) to give 6e as an oil (3.3 mg, 59%). 1 H NMR (600 MHz, CDC13) δ 7.19 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 7.9 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.76 (t, J = 8.4 Hz, 2H), 5.59 (d, J = 21.7 Hz, 1H), 5.04 (t, J = 5.9 Hz, 1H), 4.47 (d, J = 17.8 Hz, 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 = 17.6 Hz, 1H), 1.70 (s, 3H), 1.67 (s, 6H), 1.04 (dd, J = 24.3, 9.7 Hz, 1H), 0.41 (dd, J = 42.3, 10.6 Hz, 2H), 0.06 (d, J = 20.5 Hz, 2H).

[0121]

[0122] A culture tube was charged with 5b (8 mg, 0.01 mmol), Zn dust (1.3 mg, 0.02 mmol) and a magnetic stir bar 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 a rapid addition of a DMF solution of NiCl2-6H2O / di-tBuBipy (0.25 M, 0.04 mL) and PhSiH3(5 μL). The culture tube was then placed in a preheated 40 °C oil bath and stirred overnight, cooled to ambient temperature, quenched with a saturated solution of ammonium chloride, extracted with EtOAc, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness 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 (600 MHz, CDC13) 7.57 (d, J = 8.6 Hz, 1H), 7.21 - 7.14 (m, 1H), 6.98 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.2 Hz, 2H), 5.27 (s, 1H), 4.97 (t, J = 7.4 Hz, 1H), 3.92 (d, J = 5.0 Hz, 1H), 3.85 - 3.75 (m, 1H), 3.20 (dd, J = 16.1, 7.0 Hz, 1H), 3.07 (dd, J = 16.1, 7.3 Hz, 1H), 2.51 (dd, J = 11.0, 7.2 Hz, 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).

[0123]

[0124] A culture tube was charged with 5b (8 mg, 0.01 mmol), Zn dust (1.3 mg, 0.02 mmol) and a magnetic stir bar 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 a rapid addition of a DMF solution of NiCl2-6H2O / di-tBuBipy (0.25 M, 0.04 mL) and PhSiH3(5 μL). The culture tube was then placed in a preheated 40 °C oil bath and stirred overnight, cooled to ambient temperature, quenched with a saturated solution of ammonium chloride, extracted with EtOAc, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness 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%). 1H NMR (600 MHz, CDC13) δ 7.57 (d, J = 8.4 Hz, 1H), 7.36 - 7.29 (m, 5H), 7.18 (t, J = 8.1 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.1 Hz, 2H), 5.24 (s, 1H), 5.12 (d, J = 12.3 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 4.97 (t, J = 6.9 Hz, 1H), 3.90 (s, 1H), 3.71 - 3.64 (m, 1H), 3.20 (dd, J = 14.5, 6.5 Hz, 1H), 3.07 (dd, J = 14.6, 6.1 Hz, 1H), 2.63 (d, J = 4.8 Hz, 1H), 2.49 (ddd, J = 15.4, 9.6, 5.7 Hz, 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, 8.5 Hz, 2H), 1.70 (s, 6H), 1.69 (s, 3H).

[0125]

[0126] To a solution of compound S21 (6 mg, 0.01 mmol) in dry toluene (0.1 mL) was added DPPA (2.7 μL, 0.012 mmol) and Et3N (2 μL, 0.015 mmol) at room temperature. After the reaction mixture was stirred at 80 °C for 3 h, BnOH (1.2 μL, 0.012 mmol) was added to the reaction mixture, and the heating stirring was continued at 80 °C 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 (600 MHz, CDC13) δ 7.57 (d, J = 8.4 Hz, 1H), 7.36 - 7.29 (m, 5H), 7.18 (t, J = 8.1 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.1 Hz, 2H), 5.24 (s, 1H), 5.12 (d, J = 12.3 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 4.97 (t, J = 6.9 Hz, 1H), 3.90 (s, 1H), 3.71 - 3.64 (m, 1H), 3.20 (dd, J = 14.5, 6.5 Hz, 1H), 3.07 (dd, J = 14.6, 6.1 Hz, 1H), 2.63 (d, J = 4.8 Hz, 1H), 2.49 (ddd, J = 15.4, 9.6, 5.7 Hz, 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, 8.5 Hz, 2H), 1.70 (s, 6H), 1.69 (s, 3H).

[0127] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A mutant of D-A reaction enzyme MaDA1, wherein the amino acid sequence of the mutant is shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No.

4. 2.A gene encoding the mutant of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No.5; or shown in SEQ ID No.6; or shown in SEQ ID No.

7. 3.A biological material containing the gene of claim 2, wherein the biological material is an expression cassette, a plasmid, a vector, a microorganism, an animal cell or a plant cell. 4.Use of the mutant of claim 1 or the gene of claim 2 or the biological material of claim 3 in catalyzing Diels-Alder reaction to synthesize natural or unnatural D-A products; wherein the reaction substrates of Diels-Alder reaction are unnatural dienophiles and dienes.

5. Use according to claim 4, characterized in that, The unnatural dienophiles are unnatural dienophiles containing carboxyl, ester or precursors thereof. 6.Use of the mutant of claim 1 or the gene of claim 2 or the biological material of claim 3 in diversification and stereoselective synthesis of cyclohexene skeleton structure.

7. Use according to claim 6, characterized in that, After generating enzymatic D-A products by using the mutant of D-A reaction enzyme MaDA1, the D-A products are acetylated, the ester groups are hydrolyzed and N-hydroxy phthalimide ester is introduced; by decarboxylation functionalization reaction, different substituents on the cyclohexene skeleton are introduced. 8.The use of claim 7, wherein the decarboxylation functionalization reaction comprises decarboxylation alkylation, decarboxylation alkenylation, decarboxylation alkynylization, decarboxylation arylation and Curtis rearrangement.

9. Use according to any one of claims 4 to 8, characterized in that, The temperature for catalyzing Diels-Alder reaction is 30-37℃, and the pH is 6.0-6.5.

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