Super-stable artificial coenzyme factor CNMN, and synthesis method and application thereof

Abstract

The application discloses an ultra-stable artificial coenzyme factor CNMN and a synthesis method and application thereof, wherein the ultra-stable artificial coenzyme factor CNMN has the structure shown in the following formula 1. The ultra-stable artificial coenzyme factor CNMN has ultra-high thermal stability, far exceeds existing natural coenzyme factors NAD / NADP, and commonly used artificial coenzyme factors NMN and NR, and can be effectively applied to catalytic reactions at room temperature, elevated temperature and high temperature.

Description

Technical Field

[0001] This invention relates to the field of artificial coenzyme technology, specifically to an ultrastable artificial coenzyme factor CNMN, its synthesis method, and its applications. Background Technology

[0002] In nature, all living organisms rely on redox reactions catalyzed by thousands of oxidoreductases, and these oxidoreductases utilize only a few coenzymes to complete the entire catalytic reaction. Among them, nicotinamide adenine dinucleotide (NAD) + NADH and its phosphorylated form (NADP) + As a key coenzyme, NADPH is used by two-thirds of oxidoreductases for the transfer and storage of hydrogen anions, which ultimately participate in the synthesis of a variety of high-value-added biomolecules.

[0003] However, these coenzymes have some significant limitations, such as their demanding storage conditions, requiring storage at -20°C; low thermal stability, making them unsuitable for long-term high-temperature catalytic reactions; and high application costs.

[0004] Currently, artificial coenzyme factors, due to their designable structure, are gaining widespread attention as alternatives to natural coenzyme factors. Therefore, developing artificial coenzyme factors with high stability, low cost, simple structure, and the ability to replace natural coenzymes is particularly important. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the limitation that coenzyme factors have low thermal stability and cannot be applied to high-temperature catalytic reactions; and to provide an ultra-stable artificial coenzyme factor CNMN, its synthesis method and application, which solves the above problem.

[0006] A highly stable synthetic coenzyme factor, CNMN, has the structure shown in Formula 1:

[0007] Formula 1:

[0008] A method for preparing an ultrastable artificial coenzyme factor CNMN includes:

[0009] Compound 2 with the structure shown in Formula 2 is obtained. Formula 2:

[0010] POCl3 was added dropwise to the solution of compound 2, and the reaction was stirred at below 0°C under a protective atmosphere. After the reaction was completed, an acid-binding agent was added to terminate the reaction. Alternatively, compound 2 was used to perform a phosphorylation reaction with nicotinamide ribophosphoryl kinase. After the reaction, the target product was obtained by separation and purification using an amino functional column.

[0011] The process of obtaining compound 2 includes:

[0012] Compound 1 was prepared by mixing 3-carbamoyl-1-(2,4-dinitrophenyl)pyridine chloride with (1R,2S,3R,5R)-3-amino-5-(hydroxymethyl)cyclopentane-1,2-diol hydrochloride, adding solvent, then adding an acid-binding agent and stirring the mixture. Ammonia was then added to the mixture to continue the reaction. After separation and purification, compound 2 was obtained.

[0013] The solvent is methanol;

[0014] And / or, the acid-binding agent is sodium acetate.

[0015] The separation and purification process of compound 2 is as follows:

[0016] The reaction product was subjected to rotary evaporation to remove the solvent, dissolved in water and filtered. The filtrate was then subjected to rotary evaporation to remove the solvent, and then purified and separated using a silica gel column to obtain compound 2.

[0017] The process for obtaining compound 1 is as follows:

[0018] Nicotinamide was reacted with 2,4-dinitrochlorobenzene by heating and stirring to obtain the crude product of the compound. The crude product of the compound was then separated and purified to obtain compound 1.

[0019] The protective atmosphere is a nitrogen atmosphere.

[0020] The process of separating and purifying the target product using an amino functional column includes: first, equilibrating the column environment with a methanol solution containing acetic acid; then, dissolving the reaction solution in methanol, loading the sample, rinsing the column with a methanol solution containing acetic acid, and finally eluting the product with an aqueous solution containing acetic acid and lyophilizing it to obtain the target product.

[0021] The above-mentioned ultrastable artificial coenzyme factor CNMN is used in any of the following (1)-(3);

[0022] (1) Used for catalytic reactions of oxidoreductases at room temperature, elevated temperature and high temperature;

[0023] (2) Application of coenzymes as oxidoreductases in catalytic reactions;

[0024] (3) As an oxidizing agent or reducing agent.

[0025] In the room temperature, heated and high temperature catalytic reactions of the oxidoreductase, the oxidoreductases participating in the reaction include, but are not limited to, one of the following: thermophilic hydrogenase, thermophilic glucose dehydrogenase, thermostable glucose dehydrogenase, myoglobin and cytochrome P450 enzyme.

[0026] The technical solution of this invention has the following advantages:

[0027] 1. The present invention provides an ultra-stable artificial coenzyme factor CNMN, which has extremely high thermal stability, far exceeding that of existing natural coenzyme factors NAD / NADP, as well as commonly used artificial coenzyme factors NMN and NR, and can be effectively applied to reactions catalyzed at room temperature, elevated temperature and high temperature.

[0028] 2. The present invention provides a method for preparing an ultrastable artificial coenzyme factor CNMN, which has a simple synthetic route, low cost, and easy separation and purification of the product.

[0029] 3. The present invention provides a method for preparing an ultrastable artificial coenzyme factor CNMN, which uses nicotinamide ribokinase instead of chemical synthesis, thereby improving the yield and simplifying the reaction steps.

[0030] 4. This invention also provides a new approach for the ultrastable artificial coenzyme factor CNMN. Its water solubility is higher than that of the natural coenzyme factor NAD, allowing it to be applied in redox reactions catalyzed by oxidoreductases. It can act as a coenzyme for oxidoreductases, transferring protons to them and improving their conversion ability, thus enabling better conversion of the corresponding substrate into the target product. Specifically, CNMN is initially in the form of CNMN. + The oxidant in the CNMNH state is reduced to a reducing agent in the CNMNH state by an oxidoreductase. The CNMNH then undergoes a further oxidoreductase reaction, transferring protons and electrons to the reaction substrate to form the target product. The reducing agent in the CNMNH state is then converted back to CNMN after the reaction. + The oxidant, in a cyclical process, changes state to act as either an oxidant or a reducing agent, thus achieving its purpose as a coenzyme for oxidoreductases. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is a synthetic route diagram of the ultrastable artificial coenzyme factor CNMN in Example 1 of the present invention;

[0033] Figure 2 This is an LCMS result graph of compound 1 in Example 1 of the present invention;

[0034] Figure 3 This is an LCMS result graph of compound 2 in Example 1 of the present invention;

[0035] Figure 4This is an LCMS result diagram of the ultrastable artificial coenzyme factor CNMN in Example 1 of the present invention;

[0036] Figure 5 The above is the 1H NMR spectrum of the ultrastable artificial coenzyme factor CNMN in Example 1 of this invention;

[0037] Figure 6 The image shows the carbon NMR spectrum of CNMN, an ultrastable artificial coenzyme factor, in Example 1 of this invention.

[0038] Figure 7 The image shows the phosphorus NMR spectrum of the ultrastable artificial coenzyme factor CNMN in Example 1 of this invention.

[0039] Figure 8 This is a thermal stability diagram of the ultrastable artificial coenzyme factor CNMN and its analogues at 50°C in Example 3 of the present invention.

[0040] Figure 9 This is a thermal stability diagram of the ultrastable artificial coenzyme factor CNMN and its analogues at 80°C in Example 3 of the present invention.

[0041] Figure 10 The figure shows the hydrogen production results of the ultrastable artificial coenzyme factor CNMN and its analogues in Pf.SHI hydrogenase hydrogen production in Example 4 of the present invention. Detailed Implementation

[0042] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0043] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0044] Example 1

[0045] A highly stable synthetic coenzyme factor, CNMN, has the following synthetic route: Figure 1 As shown, the specific preparation process is as follows:

[0046] (1) Obtain compound 1;

[0047] Nicotinamide (500 mg, 4.0 mmol) was mixed with 2,4-dinitrochlorobenzene (2.5 g, 12 mmol) and heated and stirred at 100 °C for 4 hours. Methanol (3 mL) was added, and then the solution was poured into diethyl ether (40 mL); this process was repeated four times. The solid was filtered and dried under vacuum to give the desired orange powder product (1.11 g, 85.6%), designated as compound 1.

[0048] LCMS identification revealed that compound 1 is 3-carbamoyl-1-(2,4-dinitrophenyl)pyridin-1-ium chloride, and the structure of compound 1 is as follows:

[0049]

[0050] LCMS results (Agilent-Acid): Rt = 0.74 min; m / z [M+H] + =289.1, see Figure 2 .

[0051] (2) Preparation of compound 2;

[0052] Compound 1 (1.7 g, 5.4 mmol) was mixed with (1R,2S,3R,5R)-3-amino-5-(hydroxymethyl)cyclopentane-1,2-diol hydrochloride (900 mg, 4.92 mmol) CAS: 77841-56-6, followed by methanol (30 mL) and then sodium acetate (846 mg, 10.32 mmol). The mixture was stirred at room temperature for 2 h, then ammonia (0.5 mL) was added, and the reaction was continued at room temperature for another 0.5 h to complete the reaction. The solvent was removed by rotary evaporation, and then 2,4-dinitroaniline was dissolved and filtered off in 50 mL of water. The solvent in the filtrate was removed by rotary evaporation, and the filtrate was purified by silica gel chromatography (mobile phase: ethyl acetate: methanol containing 5% acetic acid = 0–100%). The solvent was then removed by rotary evaporation to obtain a brown target solid (1.2 g, 85.7%), designated as compound 2.

[0053] The above separation and purification process is as follows: using dry column loading, first pass through 200 mL of 100% ethyl acetate, then pass through 200 mL of 50% ethyl acetate, and finally elute the target compound with 10% ethyl acetate.

[0054] LCMS analysis revealed that compound 2 is 3-carbamoyl-1-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyridine-1-onium chloride, and the structure of compound 2 is shown in Formula 2 below;

[0055] Formula 2:

[0056] LCMS results (Agilent-Acid): Rt = 0.355 min; m / z [M+H] + =253.2, see Figure 3 .

[0057] The NMR data for compound 2 are as follows:

[0058] 1 H NMR (400MHz, D2O): δppm 9.41(s,1H),9.16(d,J=6.4Hz,1H),9.04-8.90(m,1H),8.26(dd,J=8.0,6.4Hz,1H),5.14-5.05(m,1H),4.45(dd,J=9.4,6.0Hz,1H),4. 13(dd,J=6.0,3.2Hz,1H),3.76(d,J=6.0Hz,2H),2.71(dd,J=13.2,8.0Hz,1H),2.37-2.33(m,1H),2.00(ddd,J=13.2,11.2,8.8Hz,1H).

[0059] (3) Prepare the ultrastable artificial coenzyme factor CNMN shown in Formula 1;

[0060] The obtained compound 2 (1.7 g, 5.26 mmol) was dissolved in trimethyl phosphate (10 mL), and then POCl3 (2.4 g, 15.79 mmol) was added dropwise under a nitrogen atmosphere in an ice-water bath. The mixture was stirred below 0 °C for 1 h, and then water (2 mL) and sodium acetate (6 g) were added to stop the reaction. The target product CNMN (330 mg, 17%) was obtained by separation and purification using an amino functional column.

[0061] The above separation and purification process is as follows: First, the column environment is equilibrated with a methanol solution containing 0.1M acetic acid. Then, the reaction solution is dissolved in 50mL of methanol, and the sample is loaded at a rate of 20mL / min. The column is then rinsed with a methanol solution containing 0.1M acetic acid. The product is then eluted with an aqueous solution containing 0.1M acetic acid and lyophilized.

[0062] LCMS identification revealed that the target product CNMN is ((1R,2R,3S,4R)-4-(3-carbamoylpyridin-1-onth-1-yl)-2,3-dihydroxycyclopentyl)methyl hydrogen phosphate, and the target product CNMN is shown in Formula 1 below;

[0063] Formula 1:

[0064] LCMS results (Agilent-Acid) for the target product CNMN: Rt = 0.376 min; m / z [M+H] + =333.1, see Figure 4 .

[0065] HPLC (Waters-Acid): Rt: 0.362min, 214nm: 95.87%; 254nm: 95.87%;

[0066] NMR data of the target product CNMN are as follows Figure 5-7 As shown, specifically:

[0067] 1 H NMR (400MHz, D2O): δppm 9.35(s,1H),9.13(d,J=6.4Hz,1H),8.97-8.85(m,1H),8.22(dd,J=8.0,6.4Hz ,1H),5.07(dd,J=18.8,9.6Hz,1H),4.44(dd,J=9.6,6.0Hz,1H),4.16(dd,J=6. 0,2.8Hz,1H),4.10(dd,J=10.,5.2Hz,1H),4.02(dd,J=7.6,5.2Hz,1H),2.71(d d,J=13.2,8.4Hz,1H),2.53-2.38(m,1H),2.08(ddd,J=13.2,10.8,8.0Hz,1H);

[0068] 31 C NMR (101MHz, D2O): δppm 165.720,145.286,144.871,143.101,134.077,128.643,76.791,75.598,71.729,66.550,43.070,28.742;

[0069] 31 P NMR (162MHz, D2O): δppm-0.04.

[0070] Example 2

[0071] A superstable artificial coenzyme factor CNMN is synthesized via a different route than that in Example 1, in step (3). Different types of nicotinamide ribophosphoryl kinases (HS-NRK 1 from Human; Klm-NRK from Kluyveromyces marxianus) are used for phosphorylation to obtain the superstable artificial coenzyme factor CNMN. The specific preparation process is as follows:

[0072] Compound 2 (100 mM), ATP (100 mM), magnesium chloride (20 mM) and nicotinamide ribophosphokinase 20 μg / mL were added to phosphate buffer solution and stirred at 40 °C for 2 h. The target product CNMN was obtained by separation and purification using the same amino functional column as in Example 1. The yield is shown in Table 1.

[0073] Table 1:

[0074]

[0075] The LC / MS and NMR results of the ultrastable artificial coenzyme factor CNMN in this embodiment are the same as those of the ultrastable artificial coenzyme factor CNMN in Example 1.

[0076] Example 3

[0077] The thermal stability of an ultrastable synthetic coenzyme factor CNMN and its analogues was determined, and the specific determination process is as follows:

[0078] 1 mM of CNMN, NADP, NAD, NMN, NR-CL, and NCD were placed in different test tubes and incubated at 50℃ or 80℃ for 96 hours. Samples were taken every 12 hours to monitor the remaining amount of coenzymes.

[0079] The structural formula of CNMN:

[0080] The structural formula of NADP:

[0081] The structural formula of NAD:

[0082] The structural formula of NMN:

[0083] The structural formula of NR-CL:

[0084] The structural formula of NCD:

[0085] Thermal stability test structure as follows Figures 8-9 As shown.

[0086] Example 4

[0087] The application of an ultrastable artificial coenzyme factor, CNMN, in hydrogen production by Pf. SHI hydrogenase. Specifically, hydrogen production was tested using coenzymes NADP, NAD, and CNMN in Pf. SHI hydrogenase, and the test methods are as follows:

[0088] 10 mM sodium disulfite (DT), 110 μM PfSHI, and 1 mM coenzyme factor were added to 100 mM HEPES buffer, and a hydrogen production reaction was carried out at 80 °C. The reaction results are as follows. Figure 10 As shown.

[0089] pass Figure 10 It can be seen that CNMN coenzyme, which has higher stability, has a higher hydrogen production efficiency than the hydrogen production reaction involving natural coenzymes NADP and NAD.

[0090] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A superstable artificial coenzyme factor CNMN, characterized in that, It has the structure shown in Equation 1: Formula 1:

2. A method for preparing an ultrastable artificial coenzyme factor CNMN, characterized in that, include: To obtain compound 2 with the structure shown in Formula 2, Formula 2: POCl3 was added dropwise to the solution of compound 2, and the reaction was stirred at below 0°C under a protective atmosphere. After the reaction was completed, an acid-binding agent was added to terminate the reaction. Alternatively, compound 2 was used to perform a phosphorylation reaction with nicotinamide ribophosphoryl kinase. After the reaction, the target product was obtained by separation and purification using an amino functional column.

3. The preparation method according to claim 2, characterized in that, The process of obtaining compound 2 includes: Compound 1 was prepared by mixing 3-carbamoyl-1-(2,4-dinitrophenyl)pyridine chloride with (1R,2S,3R,5R)-3-amino-5-(hydroxymethyl)cyclopentane-1,2-diol hydrochloride, adding solvent, then adding an acid-binding agent and stirring the mixture. Ammonia was then added to the mixture to continue the reaction. After separation and purification, compound 2 was obtained.

4. The preparation method according to claim 3, characterized in that, The solvent is methanol; and / or the acid-binding agent is sodium acetate.

5. The preparation method according to claim 3, characterized in that, The separation and purification process of compound 2 is as follows: The reaction product was subjected to rotary evaporation to remove the solvent, then dissolved in water and filtered. The filtrate was subjected to rotary evaporation to remove the solvent, and then purified and separated using a silica gel column to obtain compound 2.

6. The preparation method according to any one of claims 3-5, characterized in that, The process for obtaining compound 1 is as follows: Nicotinamide was reacted with 2,4-dinitrochlorobenzene by heating and stirring to obtain the crude product of the compound. The crude product of the compound was then separated and purified to obtain compound 1.

7. The preparation method according to any one of claims 2-6, characterized in that, The protective atmosphere is a nitrogen atmosphere.

8. The preparation method according to any one of claims 2-7, characterized in that, The process of separating and purifying the target product using an amino functional column includes: first, equilibrating the column environment with a methanol solution containing acetic acid; then, dissolving the reaction solution in methanol, loading the sample, rinsing the column with a methanol solution containing acetic acid, and finally eluting the product with an aqueous solution containing acetic acid and lyophilizing it to obtain the target product.

9. The use of the ultrastable artificial coenzyme factor CNMN prepared by the preparation method according to claim 1 or any one of claims 2-8 in any one of the following (1)-(3); (1) Used for catalytic reactions of oxidoreductases at room temperature, elevated temperature and high temperature; (2) Application of coenzymes as oxidoreductases in catalytic reactions; (3) As an oxidizing agent or reducing agent.

10. The application according to claim 9, characterized in that, In the room temperature, heated and high temperature catalytic reactions of the oxidoreductase, the oxidoreductases participating in the reaction include, but are not limited to, one of the following: thermophilic hydrogenase, thermophilic glucose dehydrogenase, thermostable glucose dehydrogenase, myoglobin and cytochrome P450 enzyme.

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