Cofactors with photocatalytic activity, synthetic method and application

Abstract

The invention provides a cofactor with photocatalytic activity as well as a synthesis method and application thereof, the cofactor comprises a diphosphate group, two ribose are connected to two ends of the diphosphate group respectively, one ribose is connected with adenine, the other ribose is connected with a # imgabs 0 # or # imgabs 1 # functional group, and the cofactor further contains two cations; wherein-R is any one of hydrogen, alkoxy, alkyl, N, N-dialkylamino, halogen, nitryl, cyano, sulfonic acid group, ester group, imino group, carbonyl, keto group, carboxyl, aldehyde group or hydroxyl. The cofactor can be efficiently assembled with natural NAD < + > dependent protein to construct the artificial photoenzyme with a photocatalytic function; in addition, the cofactor can be added with different-R groups and cations according to actual needs, and can be combined with different proteins to realize different catalytic functions.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic chemistry and enzyme catalysis, and in particular to a class of cofactors with photocatalytic activity, a synthesis method and an application thereof. Background Art

[0002] Enzyme catalysis is gaining more and more attention in academia and industry as a green method for constructing chiral molecules. Unlike chemical catalysis, enzymes control reactions by binding to substrate molecules through multiple levels of interactions such as hydrogen bonds, hydrophobicity, and electrostatics. Therefore, enzyme catalysis has the advantages of high efficiency and high selectivity. With the development of the bio-industry, the production cost of enzymes has been greatly reduced, making their large-scale application possible. Innovations in enzyme engineering technology also make it possible to regulate catalytic performance at the amino acid level.

[0003] However, there are only a limited number of natural enzymes, and the chemical reactions they can catalyze are also limited. A large number of chemical reactions of great value cannot be achieved using enzyme catalysts, which greatly limits the application of enzyme catalysts in various fields.

[0004] Among the known natural enzymes, a large proportion of them require the assistance of cofactors when performing catalytic functions. These cofactors include NAD + , NADP + , FAD, FMN, ThPP, PLP, porphyrin, etc. The structure and function of cofactors determine the types of reactions that the enzymes that bind to them can catalyze, while the enzyme protein structure is used to activate substrate molecules, control the selectivity of reactions, etc. We believe that by developing new artificial cofactors with non-natural functions and combining them with known natural enzyme proteins, we can design artificial enzymes with non-natural catalytic functions, thereby expanding the functional boundaries of enzyme catalysis systems.

[0005] There are precedents for the design and synthesis of artificial cofactors. For example, John Hartwig's research group at the University of California, Berkeley, developed an artificial porphyrin cofactor with an Ir metal center. After binding to proteins such as CYP119, this type of artificial cofactor can activate the CH bond to construct a CN bond. Other porphyrin artificial cofactors with metal centers have also been widely synthesized and applied. Therefore, there is a need for an artificial cofactor that can bind to NAD + The combined protein is efficiently assembled and has photocatalytic function, while being able to catalyze artificial cofactors of asymmetric photoreactions.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first object of the present invention is to provide a class of cofactors with photocatalytic activity, which can react with natural NAD +Relying on efficient protein assembly, an artificial photoenzyme with photocatalytic function is constructed; in addition, the cofactor can add different -R groups and cations according to actual needs, combine with different proteins, and realize different catalytic functions.

[0008] The second purpose of the present invention is to provide a method for synthesizing the above-mentioned cofactor with photocatalytic activity. The synthesis method is a new synthesis route that can synthesize the above-mentioned cofactor with photocatalytic activity; at the same time, the synthesis method has a high synthesis yield and has good application prospects.

[0009] The third object of the present invention is to provide the application of the above-mentioned cofactor having photocatalytic activity, wherein the above-mentioned cofactor can be used with NAD + It is protein-dependent and catalyzes asymmetric cycloaddition reactions.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides a cofactor with photocatalytic activity, wherein the cofactor comprises: a diphosphate group, two riboses are connected to both ends of the diphosphate group, one ribose is connected to adenine, and the other ribose is connected to or Functional group, the cofactor also has two cations; Wherein, -R is any one of hydrogen, alkoxy, alkyl, N,N-dialkylamino, halogen, nitro, cyano, sulfonic acid, ester, imino, carbonyl, keto, carboxyl, aldehyde or hydroxyl.

[0011] Its chemical structure is shown below:

[0012] The -R group is not limited to the above groups. Theoretically, the -R group can be any group. The -R group is used to adjust the electrical properties and steric hindrance of the cofactor. Different functional groups can be selected according to the needs of catalysis to change the auxiliary catalytic properties of the cofactor. In the above structure, the -R group can be located at or The sugar ring can be located at the ortho, meta or para position of The ortho, para or meta position of the -R group, the position and type of the -R group and the position of the sugar ring affect the electrical properties and steric hindrance of the cofactor, thereby regulating the catalytic properties of the cofactor.

[0013] M + It is any cation used to balance the electrical properties of the cofactor, ultimately affecting the solubility of the cofactor.

[0014] The present invention also provides a method for synthesizing a cofactor having photocatalytic activity, comprising the following steps: The halogenated benzoyl chloride and the benzene containing a substituent are subjected to Friedel-Crafts acylation reaction to obtain the halogenated benzophenone compounds; or, The bromine-substituted 2-iodobenzoic acid and substituted thiophenol were subjected to Ullmann coupling under the catalysis of copper powder, followed by Friedel-Crafts acylation to obtain halogenated thioxanthones. The halogenated benzophenone / halogenated thioxanthone compound is protected under acid catalysis, and then subjected to a one-pot multi-step glycosylation reaction with 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone to obtain a benzophenone nucleoside / thioxanthone nucleoside; Subsequently, a selective phosphorylation reaction at the 5-position of ribose was carried out under the conditions of phosphorus oxychloride and trimethyl phosphate to obtain benzophenone nucleoside monophosphate / thioxanthone nucleoside monophosphate; The benzophenone nucleoside monophosphate / thioxanthone nucleoside monophosphate is subjected to a condensation reaction with adenosine monophosphate through methylimidazole salt to obtain a benzophenone cofactor / thioxanthone cofactor.

[0015] The synthetic route of benzophenone (BpAD) cofactor is as follows: ; or, The synthetic route of thioxanthone ketone (TXAD) cofactor is as follows: .

[0016] For the synthesis of benzophenone (BpAD) cofactors, the first step is to obtain the halogenated benzophenone intermediate 1 through the Friedel-Crafts acylation reaction of halogen-substituted benzoyl chloride and substituted benzene.

[0017] For the synthesis of thioxanthone ketone (TXAD) cofactors, the first step is to perform Ullmann coupling between 2-iodobenzoic acid containing halogen substituents and thiophenol containing substituents under the catalysis of copper powder, and then perform intramolecular Friedel-Crafts acylation with concentrated sulfuric acid to obtain the halogenated thioxanthone intermediate 3.

[0018] The ketone carbonyl group was subsequently protected with ethylene glycol under acid catalysis to afford intermediates 2 and 4.

[0019] Under the action of n-butyl lithium, it is glycosylated with commercially available 2,3,5-tribenzyloxy-D-ribonoyl-1,4-lactone, and then reduced and dehydroxylated under the action of triethylsilane and boron trifluoride ether, and then the benzyl protecting agent is removed under the action of boron tribromide to obtain benzophenone nucleoside BpN or thioxanthone nucleoside TXN. Under the conditions of phosphorus oxychloride and trimethyl phosphate, the ribose 5 position is selectively phosphorylated to obtain benzophenone nucleoside monophosphate BpMP / thioxanthone nucleoside monophosphate TXMP.

[0020] Finally, benzophenone nucleoside monophosphate BpMP / thioxanthone nucleoside monophosphate TXMP is condensed with adenosine monophosphate (AMP) through a condensation reaction of methyl imidazolium salt to obtain the target product benzophenone (BpAD) cofactor or thioxanthone ketone (TXAD) cofactor.

[0021] During the reaction, or The carbonyl group in the group needs to be protected before the next glycosidation reaction can be carried out. Protecting groups are not limited to ethylene glycol.

[0022] Preferably, as a further specific embodiment, the reaction process of the condensation of the methylimidazole salt comprises the following steps: Dissolving the thioxanthone nucleoside / benzophenone nucleoside in dry dimethylformamide, adding N,N-diisopropylethylamine and then adding methylimidazole salt; After stirring, add 5'-adenosine monophosphate and magnesium chloride dissolved in dimethylformamide, and react at 15 ℃-30 ℃ for 1 h-1.5 h; The aqueous phase was then backwashed with dimethylformamide, eluted with an aqueous solution of triethylamine carbonate containing 20 wt % acetonitrile, and freeze-dried.

[0023] The most important step in the reaction process is the condensation of methylimidazole salt, which can couple two phosphonic acid bonds, so that the thioxanthone nucleoside / benzophenone nucleoside is connected to another ribose and adenine through the phosphonic acid group.

[0024] Preferably, as a further specific embodiment, in the glycosidation reaction step, the protected halogenated thioxanthone / halogenated dibenzophenone needs to be dissolved in an organic solvent, placed at -78°C for reaction for 30 minutes under the protection of an inert gas, and then 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone dissolved in an organic solvent is added.

[0025] Low temperature and inert gas protection are used in the reaction process to protect the generated ketone on the one hand and inhibit unnecessary side reactions on the other hand, thereby increasing the yield of the product.

[0026] Preferably, as a further specific embodiment, during the substitution reaction under the conditions of phosphorus oxychloride and trimethyl phosphate, it is necessary to adjust the pH of the reaction system to pH = 8 before post-treatment.

[0027] Preferably, as a further specific embodiment, during the substitution reaction under the conditions of phosphorus oxychloride and trimethyl phosphate, a 30 wt% methanol aqueous solution system is also required for purification.

[0028] Preferably, as a further specific embodiment, the organic solvent is tetrahydrofuran, which may also be replaced by a non-polar solvent such as ether, n-hexane, 1,4-dioxane, etc.; The inert gas is any one of nitrogen, helium, neon or argon.

[0029] In the present invention, the reaction method used is an existing method, but the reaction path is a new reaction path. Through the synthesis method of the present invention, a cofactor with a completely new structure can be synthesized; at the same time, different -R groups can be installed in the cofactor or the position of the key group can be changed according to the catalytic requirements, thereby achieving a change in the cofactor structure and further achieving a change in the catalytic function.

[0030] The present invention also provides a method for synthesizing a cofactor with photocatalytic activity. The synthesized cofactor with photocatalytic activity is combined with NAD + Dependent on protein binding, application in catalytic asymmetric cycloaddition reactions.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) This cofactor can react with natural NAD + Relying on efficient protein assembly, artificial photoenzymes with photocatalytic function were constructed; (2) The cofactor can add different -R groups and cations according to actual needs, bind to different proteins, and achieve different catalytic functions; (3) The synthesized artificial cofactor can catalyze asymmetric photoreactions, making the resulting product chiral. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Figure 1 For using the molecular structure BpAD-a and binding protein Rr12α HSDH-M3 or Ps3α Different products generated by the photocatalytic "2+2" cycloaddition reaction of the catalyst formed by HSDH-M4; Figure 2 : Figure 2 For using molecular structure TXAD-a and binding protein Sa The catalyst formed by ADH-M3 undergoes photocatalytic 1,5-hydrogen atom transfer to construct β-lactam products. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0034] Example 1 1. Synthesis of compound 1a Dissolve 4-bromobenzoyl chloride (46 mmol) and toluene (5 g) in dichloromethane (100 mL), slowly add anhydrous aluminum chloride (AlCl3, 55 mmol) under ice bath, and stir at room temperature for 2 h. Pour the reaction solution into 300 mL of ice water, extract three times with dichloromethane (DCM, 300 mL), dry with anhydrous sodium sulfate (10 g), and evaporate to dryness. Then, dissolve the crude product in heated n-hexane (300 mL), cool to precipitate a white solid, filter, and dry to obtain compound 1a. Other modified benzophenones can be prepared according to this method. Compound 1a11 g, white solid, yield 90%.

[0035] 2. Synthesis of compound 2a Dissolve compound 1a (10 g) in toluene (100 mL), add p-toluenesulfonic acid monohydrate (200 mg) and ethylene glycol (10 mL). React at 130 °C for 24 h. After cooling, pour the reaction solution into 1N NaOH (100 mL) solution, collect the organic phase and dry it with anhydrous sodium sulfate (10 g). After evaporation, it can be directly used in the next reaction.

[0036] 3. Synthesis of BpN-a The protected bromobenzophenone 2a (3.18 g) was dissolved in tetrahydrofuran, and the mixture was moved to -78 °C under nitrogen protection, and 2.5 M tetrahydrofuran solution of n-butyl lithium (5 mL) was slowly added. After reacting at -78 °C for 30 minutes, a tetrahydrofuran solution (10 mL) of 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone (4.18 g) was added. After reacting at -78 °C for 30 minutes, the reaction solution was poured into a saturated aqueous ammonium chloride solution (100 mL), extracted twice with EA (100 mL), backwashed with saturated brine (200 mL), dried with anhydrous sodium sulfate (10 g), and spun dry. The crude product was dissolved in dichloromethane (30 mL), moved to an ice bath, and triethylsilane (2.8 mL) and boron trifluoride ether solution (1.9 mL) were added and reacted for 1 h. The above reaction solution was poured into a saturated sodium bicarbonate solution (100 mL) and extracted with DCM (100 mL). The DCM phase was dried over anhydrous sodium sulfate and then spin-dried. The crude product was dissolved in tetrahydrofuran (50 mL), and a 10% hydrochloric acid solution (10 mL) was added and reacted at room temperature overnight. DCM (100 mL) was added to the reaction solution for extraction, dried over anhydrous sodium sulfate, and spin-dried. The crude product was crudely purified on silica gel (PE / EA=2 / 1) The crude product was redissolved in dichloromethane (20 mL) and 1M BCl3 dichloromethane solution (15 mL) was added at -78°C for 3 h. After the reaction solution was evaporated to dryness, 40 mL of water was added to the paste to precipitate solids, which were filtered to obtain a white solid. The crude product was recrystallized from anhydrous ethanol to obtain the target product. (BpN-a), BpN-a1.1 g, white solid, yield 34%.

[0037] 4. Synthesis of BpMP-a Benzophenone nucleoside (1 g) was dissolved in trimethyl phosphate solution (8 mL), and phosphorus oxychloride (0.5 mL) was added under ice bath. After reacting under ice bath for 2 h, 10 mL of water was added to the reaction solution, and the pH was adjusted to 8 with sodium bicarbonate. Purification was performed using a C18 column (30% methanol aqueous solution system) to obtain BpMP-a , BpMP-a1.2 g, white foam, yield 77%.

[0038] 5. Synthesis of BpAD-a BpMP-a (400 mg) was dissolved in dry DMF (4 mL), and DIPEA (0.6 mL) was added followed by benzenesulfonic acid methyl imidazole salt (400 mg). After stirring at room temperature for 1 min, it was added to a DMF (4 mL) solution of AMP (350 mg) and MgCl2 (80 mg). After reacting at room temperature for 1 h, it was poured into water and the aqueous phase was backwashed with DCM twice. It was separated and purified using a C18 column (eluted with a triethylamine carbonate aqueous solution (50 mM) containing 20 wt% acetonitrile) and freeze-dried to obtain (BpAD-a).

[0039] BpAD-a460 mg, white solid, yield 77%, identification results are shown below.

[0040] 1 H NMR (400 MHz, D2O) δ 8.14 (s, 1H), 7.76 (s, 1H), 7.33 – 7.23 (m,4H), 7.17 – 7.10 (m, 2H), 6.93 (d, J = 8.0 Hz, 2H), 5.75 (d, J = 3.9 Hz, 1H),4.74 (d, J = 6.5 Hz, 1H), 4.28 – 4.22 (m, 2H), 4.15 (dt, J = 18.7, 3.4 Hz, 7H),3.96 (dd, J = 6.7, 4.4 Hz, 1H), 2.13 (s, 3H). 13 C NMR (101 MHz, D2O) δ 215.31, 198.46, 154.88, 152.22, 148.08,144.73, 144.29, 139.06, 136.07, 133.02, 130.14, 128.81, 125.90, 118.17,87.28, 83.35, 83.11, 83.03, 82.90, 82.81, 77.31, 74.57, 71.26, 69.77, 65.89,65.84, 64.97, 46.56. 31 P NMR (162 MHz, D2O) δ -10.77, -10.90, -11.05, -11.18. Example 2 1. The synthesis method of compound 1b is the same as that of Example 1, except that compound 1a is replaced by 1b , 12.2 g, white solid, yield 90%.

[0041] 2. The synthesis method of compound 2b is the same as that of Example 1.

[0042] 3. BpN-b The synthesis method is the same as that of Example 1. Compound BpN-b 0.9 g, white solid, yield 26%.

[0043] 4. BpMP-b The synthesis method is the same as that of Example 1. Compound BpMP-b 1.4 g, white foam, yield 87%.

[0044] 5. BpAD-b The synthesis method is the same as that of Example 1.

[0045] Compound BpAD-b420 mg, white solid, yield 68%, identification results are shown below.

[0046] 1 H NMR (500 MHz, D2O) δ 8.14 (s, 1H), 7.78 (s, 1H), 7.40 – 7.27 (m,6H), 6.67 (d, J = 8.3 Hz, 2H), 5.74 (d, J = 4.4 Hz, 1H), 4.75 (d, J = 6.8 Hz, 1H),4.24 (t, J = 4.7 Hz, 2H), 4.22 – 4.07 (m, 8H), 3.99 (t, J = 5.8 Hz, 1H), 3.69 (s,3H). 13 C NMR (126 MHz, D2O) δ 197.56, 163.13, 154.93, 152.27, 148.10,143.96, 139.09, 136.44, 132.68, 130.01, 128.48, 125.99, 118.17, 113.47,87.27, 83.27, 83.09, 83.01, 82.93, 77.35, 74.57, 74.52, 71.34, 69.76, 65.94,65.90, 65.00, 64.96, 55.53. 31 P NMR (202 MHz, D2O) δ -10.95, -11.05, -11.27, -11.37. Example 3 1. The synthesis method of compound 1c is the same as that of Example 1, except that compound 1a is replaced by 1c , 12 g, white solid, yield 90%.

[0047] 2. The synthesis method of compound 2c is the same as that of Example 1.

[0048] 3. BpN-c The synthesis method is the same as that of Example 1. Compound BpN-c 1.0 g, white solid, yield 29%.

[0049] 4. BpMP-c The synthesis method is the same as that of Example 1. Compound BpMP-c 1.3 g, white foam, yield 81%.

[0050] 5. BpAD-c The synthesis method is the same as that of Example 1.

[0051] Compound BpAD-c422 mg, white solid, yield 68%, identification results are shown below.

[0052] 1 H NMR (500 MHz, D2O) δ 8.18 (s, 1H), 7.83 (s, 1H), 7.38 (q, J = 8.1 Hz,4H), 7.22 (t, J = 7.9 Hz, 1H), 7.08 – 7.01 (m, 1H), 6.97 (d, J = 7.7 Hz, 1H),6.93 (d, J = 2.3 Hz, 1H), 5.79 (d, J = 4.6 Hz, 1H), 4.77 (d, J = 6.7 Hz, 1H), 4.29(q, J = 5.0 Hz, 2H), 4.25 – 4.09 (m, 7H), 4.04 – 3.96 (m, 1H), 3.69 (s, 3H). 13C NMR (126 MHz, D2O) δ 198.48, 158.51, 155.02, 152.35, 148.23,144.64, 139.15, 137.40, 135.77, 130.36, 129.53, 126.01, 123.16, 119.13,118.25, 114.49, 87.18, 83.21, 83.20, 83.12, 83.05, 82.98, 77.38, 74.54,71.32, 69.81, 65.84, 65.80, 64.98, 64.94, 55.41. 31 P NMR (202 MHz, D2O) δ -10.86, -10.95, -11.14, -11.24. Example 4 1. The synthesis method of compound 1d is the same as that of Example 1, except that compound 1a is replaced by 1d , 11.8 g, yellow solid, yield 85%.

[0053] 2. The synthesis method of compound 2d is the same as that of Example 1.

[0054] 3. BpN-d The synthesis method is the same as that of Example 1. Compound BpN-d 1.3 g, yellow solid, yield 36%.

[0055] 4. BpMP-d The synthesis method is the same as that of Example 1. Compound BpMP-d 1.4 g, yellow solid, yield 85%.

[0056] 5. BpAD-d The synthesis method is the same as that of Example 1.

[0057] Compound BpAD-d430 mg, white solid, yield 70%, identification results are shown below.

[0058] 1 H NMR (400 MHz, D2O) δ 8.13 (s, 1H), 7.80 (s, 1H), 7.37 (d, J = 8.0 Hz,2H), 7.25 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 6.17 (d, J= 8.8 Hz, 2H),5.73 (d, J = 4.4 Hz, 1H), 4.76 (d, J = 6.8 Hz, 1H), 4.27 – 4.08 (m, 9H), 4.02(dd, J = 6.8, 4.9 Hz, 1H), 2.71 (s, 6H). 13 C NMR (101 MHz, D2O) δ 196.71, 154.92, 153.79, 152.18, 148.05,142.88, 138.98, 137.76, 132.90, 129.54, 126.01, 121.87, 118.23, 110.28,87.41, 83.36, 83.02, 82.93, 77.32, 74.68, 71.42, 69.67, 65.97, 64.92, 39.12. 31 P NMR (162 MHz, D2O) δ -10.95, -11.07, -11.31, -11.43. Example 5 1. Commercially purchased 1e .

[0059] 2. The synthesis method of compound 2e is the same as that of Example 1.

[0060] 3. BpN-e The synthesis method is the same as that of Example 1. Compound BpN-e 1.0 g, white solid, yield 32%.

[0061] 4. BpMP-e The synthesis method is the same as that of Example 1. Compound BpMP-e 1.2 g, white foam, yield 77%.

[0062] 5. BpAD-e The synthesis method is the same as that of Example 1.

[0063] Compound BpAD-e 420 mg, white solid, yield 71%, identification results are shown below.

[0064] 1 H NMR (400 MHz, Deuterium Oxide) δ 8.20 (d, J = 1.3 Hz, 1H), 7.87 (d, J= 1.4 Hz, 1H), 7.62 – 7.45 (m, 6H), 7.42 – 7.18 (m, 4H), 5.83 (dd, J = 5.2, 1.4Hz, 1H), 4.67 (d, J = 7.0 Hz, 1H), 4.38 (t, J = 5.1 Hz, 1H), 4.31 (t, J = 4.6 Hz,1H), 4.23 – 4.05 (m, 8H). 13 C NMR (101 MHz, D2O) δ 199.47, 155.07, 152.40, 148.39, 139.30,139.23, 136.30, 136.17, 133.39, 130.86, 130.06, 128.73, 128.38, 128.09,118.29, 87.00, 83.32, 83.23, 83.08, 82.99, 77.00, 74.36, 71.33, 69.93, 65.94,65.89, 65.00. 31 P NMR (162 MHz, D2O) δ -10.93, -11.06, -11.18, -11.30. Example 6 1. The synthesis method of compound 1f is the same as that of Example 1, except that compound 1a is replaced by 1f , 12 g, white solid, yield 91%.

[0065] 2. The synthesis method of compound 2f is the same as that of Example 1.

[0066] 3. BpN-f The synthesis method is the same as that of Example 1. Compound BpN-f 0.8 g, white solid, yield 23%, identification results are shown below.

[0067] 4. BpMP-f The synthesis method is the same as that of Example 1. Compound BpMP-f 1.3 g, white foam, yield 81%.

[0068] 5. BpAD-f The synthesis method is the same as that of Example 1.

[0069] Compound BpAD-f416 mg, white solid, yield 67%, identification results are shown below.

[0070] 1 H NMR (400 MHz, Deuterium Oxide) δ 8.17 (s, 1H), 7.87 (s, 1H), 7.57(dt, J = 7.7, 1.6 Hz, 1H), 7.47 – 7.39 (m, 3H), 7.32 – 7.22 (m, 2H), 6.82 –6.75 (m, 2H), 5.80 (d, J = 5.1 Hz, 1H), 4.68 (s, 1H), 4.35 (t, J = 5.1 Hz, 1H),4.30 (t, J = 4.7 Hz, 1H), 4.17 (dp, J = 10.9, 3.7 Hz, 3H), 4.08 (q, J = 4.4, 3.6Hz, 4H), 3.99 (dd, J = 7.0, 5.2 Hz, 1H), 3.75 (s, 3H). 13 C NMR (101 MHz, D2O) δ 197.99, 163.26, 155.07, 152.36, 148.38,139.24, 139.15, 136.94, 132.83, 130.40, 129.69, 128.66, 127.72, 118.29,113.60, 86.97, 83.30, 83.21, 83.10, 83.03, 76.96, 74.40, 71.39, 69.94, 65.95,64.98, 55.58. 31 P NMR (162 MHz, D2O) δ -10.90, -11.02, -11.17, -11.29. Example 7 1. The synthesis method of compound 1g is the same as that of Example 1, except that compound 1a is replaced by 1g , 13 g, white solid, yield 99%.

[0071] 2. The synthesis method of compound 2g is the same as that of Example 1.

[0072] 3. BpN-g The synthesis method is the same as that of Example 1. Compound BpN-g 0.95 g, white solid, yield 28%, identification results are shown below.

[0073] 4. BpMP-g The synthesis method is the same as that of Example 1.

[0074] Compound BpMP-g1.2 g, white foamy substance, yield 75%.

[0075] 5. BpAD-g The synthesis method is the same as that of Example 1.

[0076] Compound BpAD-g415 mg, white solid, yield 67%, identification results are shown below.

[0077] 1 H NMR (400 MHz, Deuterium Oxide) δ 8.19 (s, 1H), 7.87 (s, 1H), 7.58(td, J = 4.6, 3.6, 2.0 Hz, 1H), 7.48 (s, 1H), 7.32 – 7.27 (m, 2H), 7.24 (t, J =8.0 Hz, 1H), 7.07 – 6.95 (m, 3H), 5.82 (d, J = 5.1 Hz, 1H), 4.68 (d, J = 7.1 Hz,1H), 4.38 (t, J = 5.1 Hz, 1H), 4.31 (t, J = 4.7 Hz, 1H), 4.21 – 4.12 (m, 4H), 4.09 (dq, J = 7.0, 4.4, 3.8 Hz, 4H), 3.97 (dd, J = 7.0, 5.2 Hz, 1H), 3.69 (s,3H). 13C NMR (101 MHz, D2O) δ 198.74, 158.55, 155.06, 152.37, 148.38,139.24, 137.44, 136.26, 130.91, 130.00, 129.57, 128.71, 128.00, 123.24,119.22, 118.27, 114.57, 86.98, 83.30, 83.21, 83.07, 83.01, 76.99, 74.37,71.35, 69.94, 65.93, 65.00, 55.42. 31 P NMR (162 MHz, D2O) δ -10.92, -11.04, -11.17, -11.30. Example 8 1. Synthesis of compound 3a Dissolve 5-bromo-2-iodobenzoic acid (10 g), sodium benzenethiol (5 g), copper powder (200 mg), and KOH (8.5 g) in water (100 mL) and stir at 110°C for 12 h. After filtering out the copper powder while hot, cool the solution and adjust the pH to 1 with concentrated hydrochloric acid. Filter the precipitated solid to obtain a white solid, which is then dried. Add concentrated sulfuric acid (50 mL) to the solid and react at 80°C for 2 h. Pour the solid into ice water and filter the precipitated solid using a Buchner funnel to obtain a yellow solid. Wash with anhydrous ethanol, grind, and filter to obtain 3a. , other different modifications can be prepared according to this method.

[0078] Compound 3a7g, light yellow solid, two-step yield 80%.

[0079] 2. Synthesis of compound 4a Compound 3a (10 g) was dissolved in toluene (70 mL), and p-toluenesulfonic acid (200 mg) and ethylene glycol (10 mL) were added. The reaction was carried out at 130 °C for 24 h. After the reaction solution was cooled, it was poured into a 1N NaOH solution (100 mL). 10 g of anhydrous sodium sulfate was added to the organic phase in toluene and dried. The yellow solid obtained by spin drying for 2 h can be directly used in the next reaction. Other different modifications can be prepared according to this method.

[0080] 3. Synthesis of TXN-a The protected bromothioxanthone 4a (3.4 g) was dissolved in tetrahydrofuran and protected with nitrogen, and then moved to -78 °C, and 2.5 M tetrahydrofuran solution of n-butyl lithium (4 mL) was slowly added. After reacting at -78 °C for 30 minutes, a tetrahydrofuran solution (10 mL) of 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone (4.2 g) was added. After reacting at -78 °C for 30 minutes, the reaction solution was poured into a saturated aqueous ammonium chloride solution, extracted twice with EA (40 mL), backwashed with saturated brine (100 mL), dried with 10 g of anhydrous sodium sulfate, and evaporated to dryness. The crude product was dissolved in dichloromethane (30 mL), moved to an ice bath, and triethylsilane (3.3 mL) and boron trifluoride ether solution (2.2 mL) were added and reacted for 1 h. The above reaction solution was poured into a saturated sodium bicarbonate solution (100 mL) and extracted with DCM (50 mL). The DCM phase was dried over anhydrous sodium sulfate and then spun dry. The crude product was dissolved in tetrahydrofuran (80 mL), and a 10% hydrochloric acid solution (10 mL) was added to react at room temperature overnight. DCM (100 mL) was added to the reaction solution for extraction, and the DCM was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was redissolved in dichloromethane (30 mL) again, and a 1M BBr3 dichloromethane solution (20 mL) was added at -78°C, and the reaction was allowed to proceed for 1 h. After the reaction solution was evaporated to dryness, 40 mL of water was added to the black paste, and the solid was precipitated and filtered to obtain an off-white solid. The crude product was recrystallized from anhydrous ethanol to obtain the target product. Other differently modified thioxanthone nucleosides were prepared according to this method.

[0081] Compound TXN-a 1.1 g, yellow solid, four-step yield 32%.

[0082] 4. TXMP-a Synthesis Dissolve thioxanthone nucleoside TXN-a (1 g) in trimethyl phosphate solution (8 mL), transfer to an ice bath and add phosphorus oxychloride (0.34 mL). After reacting for 2 h in an ice bath, add 10 mL of water to the reaction solution and adjust the pH to 8 with sodium bicarbonate. Purify using a C18 column (eluted with triethylamine carbonate aqueous solution (50 mM) containing 20 wt% acetonitrile) and freeze-dry. Other differently modified benzophenone nucleoside monophosphates can be prepared according to this method.

[0083] Compound TXMP-a1.3 g, yellow solid, yield 83%.

[0084] 5. TXAD-a Synthesis TXMP-a (400 mg) was dissolved in dry DMF (4 mL), and DIPEA (0.52 mL) was added followed by benzenesulfonic acid methylimidazolium salt (400 mg). After stirring at room temperature for 1 min, it was added to a DMF solution of 5'-adenosine monophosphate (300 mg) and MgCl2 (70 mg). After reacting at room temperature for 1 h, it was poured into water and backwashed with DCM twice. It was separated and purified using a C18 column (eluted with triethylamine carbonate aqueous solution (50 mM) containing 20 wt% acetonitrile) and freeze-dried.

[0085] Compound TXAD-a 450 mg, yellow solid, yield 75%, identification results are shown below.

[0086] 1 H NMR (400 MHz, D2O) δ 7.84 – 7.77 (m, 2H), 7.69 (s, 1H), 7.47 (dd, J =8.4, 1.9 Hz, 1H), 7.26 (ddd, J = 8.3, 7.1, 1.5 Hz, 1H), 7.22 (s, 1H), 7.11(ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.07 – 6.97 (m, 2H), 5.61 (d, J = 4.6 Hz, 1H),4.74 (d, J = 6.7 Hz, 1H), 4.30 – 4.03 (m, 11H). 13 C NMR (101 MHz, D2O) δ 180.00, 154.23, 151.65, 147.51, 138.60,137.27, 136.93, 132.63, 130.05, 128.37, 126.84, 126.78, 126.70, 126.47,126.38, 125.67, 117.49, 87.04, 83.23, 83.04, 82.95, 82.81, 77.04, 74.68,71.37, 69.78, 65.87, 65.02. 31 P NMR (162 MHz, D2O) δ -10.73, -10.85, -11.04, -11.16. Example 9 1. Compounds The synthesis method of 3b is consistent with that of Example 8; Compound 3b 8.0 g, yellow solid, two-step yield 82%.

[0087] 2. The synthesis of compound 4b was consistent with that of Example 8; 3. Compound TXN-b The synthesis method is consistent with that of Example 8 Compound TXN-b1.0 g, yellow solid, four-step yield 27%.

[0088] 4. TXMP-b The synthesis method is consistent with that of Example 8; Compound TXMP-b1.2 g, yellow solid, yield 73%.

[0089] 5. TXAD-b The synthesis method is consistent with that of Example 8; Compound TXAD-b1.0 g, yellow solid, four-step yield 27%, identification results are shown below.

[0090] 1 H NMR (400 MHz, DMSO- d 6) δ 8.48 (dd, J = 1.8, 0.9 Hz, 1H), 7.93 (d, J =2.9 Hz, 1H), 7.87 – 7.80 (m, 2H), 7.78 (d, J = 8.8 Hz, 1H), 7.43 (dd, J = 8.9,2.9 Hz, 1H), 5.12 (d, J = 7.1 Hz, 1H), 5.01 (d, J = 4.7 Hz, 1H), 4.90 (t, J = 5.4Hz, 1H), 4.74 (d, J = 7.3 Hz, 1H), 3.96 (td, J = 5.0, 3.2 Hz, 1H), 3.90 (s, 4H), 3.76 (td, J = 7.3, 5.3 Hz, 1H), 3.68 – 3.59 (m, 2H). 13C NMR (101 MHz, DMSO) δ 178.39, 158.07, 140.07, 135.62, 130.64,129.48, 128.22, 128.05, 127.37, 126.42, 122.33, 110.32, 85.55, 82.26, 77.69,71.49, 62.04, 55.48. Example 10 1. Compound 3c The synthesis method is consistent with that of Example 8; Compound 3c (6.6 g), pale yellow solid, two-step yield 75%).

[0091] 2. The synthesis method of compound 4c is consistent with that of Example 8; 3. TXN-c The synthesis method is consistent with that of Example 8; TXN-c1.2 g, yellow solid, four-step yield 35%.

[0092] 4. TXMP-c The synthesis method is consistent with that of Example 8; Compound TXMP-c, 1.3 g, yellow solid, yield 83%.

[0093] 5. TXAD-c The synthesis method is consistent with that of Example 8; Compound TXAD-c411 mg, yellow solid, yield 68%, identification results are shown below.

[0094] 1 H NMR (400 MHz, D2O) δ 7.81 – 7.73 (m, 3H), 7.29 – 7.19 (m, 2H), 7.16(dd, J = 8.5, 1.6 Hz, 1H), 7.12 – 7.05 (m, 2H), 7.01 (d, J = 8.0 Hz, 1H), 5.52(d, J = 4.1 Hz, 1H), 4.68 (d, J = 6.3 Hz, 1H), 4.25 – 4.16 (m, 6H), 4.15 – 4.07(m, 4H), 4.02 (dd, J = 6.3, 4.6 Hz, 1H). 13C NMR (101 MHz, D2O) δ 179.92, 154.25, 151.59, 147.38, 144.24,138.59, 137.61, 137.14, 132.54, 128.91, 128.24, 126.88, 126.50, 126.21,125.71, 123.55, 122.86, 117.65, 87.28, 82.91, 82.79, 82.70, 76.99, 74.57,71.18, 69.64, 65.71, 64.92. 31 P NMR (162 MHz, D2O) δ -10.84, -10.96, -11.13, -11.25. Embodiment 11 1. Compound 3d The synthesis method is consistent with that of Example 8; Compound 3d8.3 g, yellow solid, two-step yield 85%.

[0095] 2. The synthesis method of compound 4d is consistent with that of Example 8; 3. TXN-d The synthesis method is consistent with that of Example 8; Compound TXN-d1.1 g, yellow solid, four-step yield 29%.

[0096] 4. TXMP-d The synthesis method is consistent with that of Example 8; Compound TXMP-d1.3 g, yellow solid, yield 79%.

[0097] 5. TXAD-d The synthesis method is consistent with that of Example 8; Compound TXAD-d422 mg, yellow solid, yield 68%, identification results are shown below.

[0098] 1 H NMR (400 MHz, D2O) δ 7.77 (t, J = 4.2 Hz, 2H), 7.37 (s, 1H), 7.22 –7.14 (m, 2H), 6.98 (d, J = 2.9 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.58 (dd, J=8.9, 2.9 Hz, 1H), 5.50 (d, J = 4.3 Hz, 1H), 4.72 (s, 1H), 4.28 – 4.11 (m, 7H), 4.05 (dd, J = 6.1, 4.4 Hz, 3H), 3.99 (dt, J = 7.9, 4.4 Hz, 1H), 3.55 (s, 3H). 13 C NMR (101 MHz, D2O) δ 179.05, 156.65, 154.40, 151.63, 147.46,144.08, 138.59, 137.79, 129.49, 128.83, 127.71, 127.07, 125.88, 123.33,122.72, 121.63, 117.72, 108.75, 87.24, 82.92, 82.86, 82.77, 82.68, 77.01,74.74, 71.22, 69.71, 65.63, 64.86, 55.15. 31 P NMR (162 MHz, D2O) δ -10.74, -10.86, -11.12, -11.24. Application Example 1 like Figure 1 As shown, benzophenone cofactors catalyze the "2+2" cycloaddition reaction Using the molecular structure BpAD-a synthesized in Example 1, the binding protein Rr12α HSDH-M3 or Ps3α HSDH-M4 can realize various intramolecular and intermolecular photocatalytic "2+2" cycloaddition reactions as shown in the figure below.

[0099] Figure 1 The catalyst formed by combining the cofactor in Example 1 of the present invention with the protein is used to catalyze different cycloaddition reactions to finally form products.

[0100] The protein sequence of 12αHSDH-M3 (F184L / V190A / Q150M) is as follows: MKLRGKTAVVTGGAGGIGRAVTRVFVREGARVLFVDVDDDRGRALESELTGAGGEAKFLQADISRRESADQIRDAAVAAFGGIDILVNNAHASRQALLVEHTPEMFELSFGTGFYPTVHLMQACY PQLKQARGSVVNFGSGSALDGMPTMTSYAAAKEAIRAVSRVAANEWAADGIRVNVVCPLAATEGAQAWQQAFPDRAAAAAAKVPLQRIGDPETDIAPVVVFLASDDSKYMTGQTLMADGGSIKLR The protein sequence of Ps3αHSDH-M4 (V74W / L190T / Y200W / I115Y) is as follows: MSVIAITGSASGIGAALKELLARAGHTVIGIDRGQADIEADLSTPGGRETAVAAVLDRCGGVLDGLVCCAGVGWTAANSGLVVAVNYFGVSALLDGLAEALSRGQQPAAVIVGSYAATQPGAAELPM VEAMLAGDEARAIELAEQQGQTHLAYAGSKYAVTCLARRNVVDWAGRGVRLNVVAPGAVETPTLQASKADPRWGESTRRFVAPLGRGSEPREVAEAIAFLLGPQASFIHGSVLFVDGGMDALMRAKTF Application Example 2 Using the structure TXAD-c synthesized in Example 10 and Sa ADH2-M3 protein binding can achieve the following Figure 2 Shown is the intramolecular photocatalytic 1,5-hydrogen atom transfer reaction to construct β-lactam.

[0101] The protein sequence of SaADH2-M3 (Y158I / T190S / S217Q) is as follows: MSYQSLKNKVVIVTGAGSGIGRAIAKKFALNDSIVVAVELLEDRLNQIVQELRGMGKEVLGVKADVSKKKDVEEFVRRTFETYSRIDVLCNNAGIMDGVTPVAEVSDELWERVLAVNLYSAFYSSRA VIPIMLKQGKGVIVNTASIAGIRGGFAGAPITVAKHGLIGLTRSIAAHYGDQGIRAVAVLPGSVKTNIGLGSSKPSELGMRTLTKLMSLQSRLAEPEDIANFVLASDEASFVNGDAVVVDGGLTVL Based on the above data, we can draw the following conclusions: Examples 1-7 are examples of the synthesis of benzophenone cofactors (BpAD); Examples 8-11 are examples of the synthesis of thioxanthone cofactors (TXAD). It can be seen from the examples that the present invention can eventually synthesize cofactors of different types and functions by introducing different -R groups. Taking Example 1 as an example, after the cofactor in Example 1 is combined with the protein, photocatalysis can be achieved for different types of molecules, and asymmetric addition can be achieved to generate chiral products. Taking Example 10 as an example, after the cofactor in Example 10 is combined with the protein, it can catalyze 1,5 hydrogen atom transfer under light conditions to construct chiral β-lactam products.

[0102] In summary, the cofactor synthesized by the present invention is structurally similar to natural NAD + The cofactor is close to that of natural NAD + Combined with efficient protein assembly, artificial enzymes with different catalytic functions are constructed, so that different types of artificial enzymes can be constructed according to actual experimental needs to catalyze asymmetric photoreactions.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of cofactor with photocatalytic activity, characterized in that: The cofactor includes a diphosphate group, two riboses are connected to both ends of the diphosphate group, one ribose is connected to adenine, and the other ribose is connected to or Functional group, the cofactor also has two cations; Wherein, R is any one of hydrogen, alkoxy, alkyl, N,N-dialkylamino, halogen, nitro, cyano, sulfonic acid, ester, imino, carbonyl, keto, carboxyl, aldehyde or hydroxyl.

2. A method for synthesizing a cofactor having photocatalytic activity as claimed in claim 1, characterized in that: The steps include: The halogenated benzoyl chloride and the benzene containing a substituent are subjected to Friedel-Crafts acylation reaction to obtain the halogenated benzophenone compounds; or, Halogen-substituted 2-iodobenzoic acid and substituted thiophenol were subjected to Ullmann coupling under the catalysis of copper powder, followed by Friedel-Crafts acylation to obtain halogenated thioxanthones. The halogenated benzophenone / halogenated thioxanthone compound is protected under acid catalysis, and then subjected to a one-pot multi-step glycosylation reaction with 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone to obtain a benzophenone nucleoside / thioxanthone nucleoside; Subsequently, a selective phosphorylation reaction at the 5-position of ribose was carried out under the conditions of phosphorus oxychloride and trimethyl phosphate to obtain benzophenone nucleoside monophosphate / thioxanthone nucleoside monophosphate; The benzophenone nucleoside monophosphate / thioxanthone nucleoside monophosphate is subjected to a condensation reaction with adenosine monophosphate through methylimidazole salt to obtain a benzophenone cofactor / thioxanthone cofactor.

3. The method for synthesizing a cofactor having photocatalytic activity according to claim 2, characterized in that: The reaction process of the methylimidazole salt condensation comprises the following steps: The benzophenone nucleoside monophosphate / thioxanthone nucleoside monophosphate is dissolved in dry dimethylformamide, and N,N-diisopropylethylamine is added, followed by methylimidazole salt; After stirring, add 5'-adenosine monophosphate and magnesium chloride dissolved in dimethylformamide, and react at 15 ℃-30 ℃ for 1 h-1.5 h; The aqueous phase was then backwashed with dimethylformamide, eluted with an aqueous solution of triethylamine carbonate containing 20 wt % acetonitrile, and freeze-dried.

4. The method for synthesizing a cofactor having photocatalytic activity according to claim 2, characterized in that: In the glycosidation reaction step, the protected halogenated thioxanthone / halogenated dibenzophenone needs to be dissolved in an organic solvent, reacted at -78°C for 30 min under the protection of an inert gas, and then 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone dissolved in an organic solvent is added.

5. The method for synthesizing a cofactor having photocatalytic activity according to claim 2, characterized in that: During the substitution reaction under the conditions of phosphorus oxychloride and trimethyl phosphate, it is necessary to adjust the pH of the reaction system to pH=8 before post-treatment.

6. The method for synthesizing a cofactor having photocatalytic activity according to claim 2, characterized in that: During the substitution reaction under the conditions of phosphorus oxychloride and trimethyl phosphate, a 30 wt% methanol aqueous solution system is also required for purification.

7. The method for synthesizing a cofactor having photocatalytic activity according to claim 2, characterized in that: The organic solvent is a non-polar solvent such as tetrahydrofuran, ether, n-hexane, 1,4-dioxane, etc. The inert gas is any one of nitrogen, helium, neon or argon.

8. A method for synthesizing a cofactor with photocatalytic activity as described in any one of claims 2 to 7, wherein the cofactor with photocatalytic activity as described in claim 1 is synthesized by combining the cofactor with NAD + Dependent on protein binding, application in catalytic asymmetric photoreactions.

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