CN102453067B - A kind of NAD +the preparation method of analogue and application thereof - Google Patents

Abstract

The invention discloses a preparation method and application of a nicotinamide adenine dinucleotide (NAD ⁺ ) analogue, the general structural formula of which is as follows NAD ⁺ analogues are produced by reacting nicotinamide mononucleotide with corresponding pyrimidine nucleotide analogues, where R is or R for This type of NAD ⁺ analog can be used as a coenzyme of dehydrogenase, and can also promote the growth of microorganisms. The NAD ⁺ analogue obtained by the invention can be applied to the research of biocatalysis, bioanalytical chemistry, metabolic engineering and synthetic biology.

Description

A kind of preparation method and application of NAD+ analog

technical field

The present invention relates to the synthesis and application of a class of small molecule compounds with biological activity, in particular to the analogue of nicotinamide adenine dinucleotide (NAD ⁺ ) which has a pyrimidine ring replacing an adenine ring with various substituents. substance, and the compound can be used as a cofactor of dehydrogenase to catalyze redox reactions, or as a microorganism growth promoter to promote the growth of microorganisms.

Background technique

Nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NAD ⁺ ) and its corresponding reduced state (NADH), commonly known as coenzyme I, are indispensable small molecular compounds in living organisms, and participate in the oxidation of living organisms Reductive metabolism and a series of other important biochemical processes, its structural formula is as follows:

Chemical structure of NAD ⁺

The chemical structure of NAD ⁺ is relatively complex, its properties are not stable enough, and it is expensive. From the chemical structure of NAD ⁺ , it can be seen that the molecule is formed by linking nicotinamide mononucleotide (NMN) fragments and adenosine monophosphate (AMP) fragments through pyrophosphate bonds. The NMN part is the main bearer of its chemical function, participating in redox reactions and electron transfer, while the AMP part interacts with the relevant domains of functional proteins to anchor the coenzyme, which has an important effect on the selectivity of the enzyme specific recognition of the coenzyme and biochemical reactions. Important impact (J. Benach et al. J. Biol. Chem. 2003, 278, 19176-19182).

In cells, NAD ⁺ mainly plays the role of transferring hydrogen and electrons, and many important redox enzymes depend on NAD ⁺ as their cofactor. In addition to the redox function, NAD ⁺ also plays a very important role in the non-redox life process. NAD ⁺ is an indispensable small molecule compound in life activities such as cell reproduction, growth, differentiation, and apoptosis (WHYing. Antioxidants & Redox Signaling 2008, 10, 179-206). NAD ⁺ can also be used as the substrate of histone deacetylase (sirtuins), under the catalysis of the enzyme, the acetyl group on the histone is removed, so that the histone can successfully complete important functions such as DNA replication, transcription and repair (HNLin et al. Org. Biomol. Chem. 2007, 5, 2541-2554).

Based on the structure of NAD ⁺ , people have chemically modified it and synthesized many analogues. Some recent work involves chemical modification of adenosine monophosphate (AMP) moiety (CJWMort et al. Bioorg. Med. Chem. 2004, 12, 475-487), ribose ring moiety (GCZhou et al. J. Am. Chem. Soc. 2004,126,5690-5698), nicotinamide moiety (NEBatoux et al.Tetrahedron 2004,60,6609-6617) and pyrophosphate moiety (L.Chen et al.Bioorg.Med.Chem.Lett.2007,17,3152 -3155). HCLo once used methanol as a substitute for AMP and nicotinamide mononucleotide under DCC/DMAP to synthesize NAD ⁺ analogues, and used the above analogues as cofactors of horse liver dehydrogenase (HLADH) Sexual alcohol (HCLo et al.Angew.Chem.Int.Ed.2002, 41, 478-481), but the structure of the cofactor is too simple, the combination with the enzyme is not tight enough, and the catalytic efficiency is very low. The above work is based on the chemical modification of the original structure of NAD ⁺ , which is difficult to overcome its chemical instability, chemical synthesis, and separation difficulties. Most of the obtained NAD ⁺ analogs cannot be selectively recognized by dehydrogenases. .

The applicant's previous work [Zhao Zongbao, Liu Wujun, Wu Siguo, Hou Shuhua, a NAD ⁺ analog and its synthesis and application, application number: 200810010285.1] The synthesized NAD ⁺ analog can promote the growth of microorganisms such as Escherichia coli and Saccharomyces cerevisiae; Its reduced form can also be used as a dehydrogenase cofactor to catalyze redox reactions, but these analogs have relatively low biological activity.

In conclusion, the NAD ⁺ analogues reported in the existing literature are mainly used as inhibitors of biocatalysts, and very few can be used in biocatalytic or biomimetic catalytic reactions, but cannot be specifically recognized by biocatalysts. Therefore, there is a need to rationally design NAD ⁺ analogs with other structural features to allow them to be more efficiently recognized by enzymes and exhibit biocatalytic functions. NAD ⁺ analogues with these biological properties can play an important role in the fields of bioanalytical chemistry, biocatalysis, metabolic engineering or synthetic biology.

Contents of the invention

The present invention analyzes the structure of NAD ⁺ on the basis of summarizing the results of NAD chemical modification and related biological activities in the literature, retains the redox functional region of nicotinamide mononucleoside, and modifies the adenine ring part Substitution of the pyrimidine ring structure to design novel NAD ⁺ analogues.

The NAD ⁺ analogs involved in the present invention have the following general structural formula:

In the structural formula, the nicotinamide mononucleotide is in the β-configuration; the two ribose units are in the D-configuration;

The R unit attached to the nucleotide is Hereinafter referred to as A, wherein R ¹ is halogen, such as F, Cl, Br; C ₁ -C ₅ alkyl, such as methyl, ethyl; C ₁ -C ₅ alkylhydroxy, such as hydroxymethyl.

or R for Hereinafter referred to as B, wherein R ² is C ₁ -C ₅ alkyl, such as methyl, ethyl.

The synthesis of NAD ⁺ analogs in the present invention is carried out in three steps, summarized as follows:

The first step: refer to the literature (S.M.Graham et al.Org.Lett.2004, 6, 233-236) to synthesize nicotinamide mononucleotide;

The second step: refer to the literature (M.Yoshikaw et al.Tetrahedron Lett.1967, 5065-5068) to synthesize nucleoside monophosphate;

The third step: coupling the nicotinamide mononucleotide obtained in the first step with the nucleoside monophosphate obtained in the second step to obtain the target NAD ⁺ product. The specific operation process refers to the literature (TV Abramova et al.Bioorg.Med.Chem.2007,15,6549-6555), the literature (LQChen et al.J.Med.Chem.2007,50,5743-5751) or the literature (J.Lee et al. Chem. Commun. 1999, 729).

Literature (TV Abramova et al.Bioorg.Med.Chem.2007,15,6549-6555) uses a mixed solvent of N,M-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), and the condensation reagent is three Phenylphosphine (Ph ₃ P), 1,1-dithiol bispiperidine (PyrS) ₂ or diphenyl disulfide (PhS) ₂ or 2,2′-dipyridyl disulfide (PyS) ₂ and 1-methylimidazole;

Literature (L.Q.Chen et al.J.Med.Chem.2007,50,5743-5751) uses N,N-dimethylformamide (DMF) as solvent, N,N-carbonyldiimidazole (CDI) as condensation reagent ;

Literature (J.Lee et al.Chem.Commun.1999, 729) uses formamide as solvent, N, N'-dicyclohexylcarbodiimide (DCC), morpholine as condensation reagent, MnCl ₂ , MgSO ₄ as catalyst.

The dehydrogenase used in the present invention is E. coli K12 malic enzyme (UniProt code P26616). The mutant dehydrogenase used in the present invention is to utilize Single point mutation kit or literature (JXWang, et al.J.Microbiol.Methods 2007, 71, 225-230) introduced amino acid mutations into Escherichia coli K12 malic enzyme to obtain mutant malic enzyme (L301R and L301R/Q392C) . The malic enzyme used in the present invention is expressed and purified according to the literature (JXWang, et al. Protein Expr. Purif. 2007, 53, 97-103).

The dehydrogenase used in the present invention is also lactate dehydrogenase from Lactobacillus helveticus (UniProt code P30901). The mutant lactate dehydrogenase used in the present invention is to utilize The single point mutation kit introduces amino acid mutations into lactate dehydrogenase to obtain mutant lactate dehydrogenase (V152R). The lactate dehydrogenase used in the present invention is expressed and purified according to the literature (JXWang, et al. Protein Expr. Purif. 2007, 53, 97-103).

The microorganism used in the present invention is Escherichia coli DH5α (Beijing Dingguo Biotechnology Company).

The dehydrogenase used in the present invention also has alcohol dehydrogenase, from Saccharomyces cerevisiae (CAS NO.9031-72-5) purchased from Sigma Aldrich.

Table-1 presents the NAD ⁺ analogues of the examples of the present invention.

Table-1. NAD ⁺ Analogue Numbers and Their Corresponding Chemical Structures

Detailed ways

The following examples help to understand this patent, but are not limited to the content of the present invention.

The raw materials used in the examples are respectively referred to the literature (S.M.Graham et al.Org.Lett.2004, 6, 233-236.) to synthesize nicotinamide mononucleotide ammonium salt; refer to the literature (M.Yoshikaw et al.Tetrahedron Lett. 1967, 5065-5068) to synthesize nucleoside monophosphate; refer to literature (T.V.Abramov et al.Bioorg.Med.Chem.2007, 15, 6549-6555.), literature (L.Q.Chen et al.J.Med.Chem.2007, 50,5743-5751) or literature (J.Lee et al.Chem.Commun.1999,729) to synthesize the final product.

Example 1

0.3mmol NMN Dissolve in 2mL DMSO and 2mL DMF mixed solvent, add 1.5mM Ph ₃ P, 1.5mM (PyS) ₂ , 6mM methylimidazole in sequence, react at 26°C for 18min, add 1mM nucleoside monophosphate dissolved in 2mL DMF React at 26°C for 40 minutes. Add 20 mL of acetone to stop the reaction, a large amount of precipitates are formed, the precipitates are collected by centrifugation, and the precipitates are washed three times with 5 mL of acetone. After water-soluble, performic acid-type anion exchange resin was separated and purified to obtain the target product BC-10 with a yield of 35%.

¹ H NMR (D ₂ O, 400MHz): δ9.17(s, 1H), 9.02(d, J=6.1Hz, 1H), 8.72(d, J=7.9Hz, 1H), 8.05(m, 1H) , 7.70(d, J=6.4Hz, 1H), 5.94(d, J=5.5Hz, 1H), 5.55(d, J=3.8Hz, 1H), 4.33(brs, 1H), 4.28(pseudo t, J =5.1Hz, 1H), 4.19(pseudo t, J=4.6Hz, 1H), 4.15-4.12(m, 1H), 4.03-3.93(m, 5H), 3.91-3.89(m, 1H). ¹³ C NMR (D ₂ O, 100MHz): δ165.2, 158.0, 157.8, 155.3, 145.9, 142.4, 139.8, 138.5, 136.0, 133.7, 128.6, 125.5, 125.2, 99.8, 89.5, 86.6, 82.3, 77.4, 74.0, 70.3, 69.0, 64.8, 64.7. ¹⁹ F NMR (D ₂ O, 376MHz): δ-164.9. ³¹ P NMR (D ₂ O, 162 MHz): δ-11.1, -11.2. HRMS: calcd for C ₂₀ H ₂₆ FN ₅ O ₁₅ P ₂ (M+H) ⁺ 658.0963, found 658.0961.

The compound is a white solid that easily absorbs moisture and becomes viscous and dark in color.

Determination of malic enzyme activity: Wild-type malic enzyme, mutant malic enzyme (L301R) and mutant malic enzyme (L301R/Q392C) were prepared into 1 mg·mL ^-1 solutions respectively. Prepare 0.2mL reaction mixture (50mM HEPES pH 7.2, 3mM L-malate, 5mM MnCl ₂ , 0.2mM NAD ⁺ or BC-10), quickly add 1μL enzyme solution during activity analysis, mix well and place in a UV spectrophotometer , Continuously monitor the 340nm absorption value at 25°C, and the ideal activity data can be obtained within 1min. The enzyme activity unit is defined as: the amount of enzyme required to catalyze the production of 1 μmol reduced NAD ⁺ or BC-10 per minute at 25°C.

It was found that the enzyme reaction speed of wild-type malic enzyme to BC-10 and NAD ⁺ was 0.31U·mg ^-1 and 22.5U·mg ^-1 respectively, and the mutant malic enzyme (L301R) to BC-10 and NAD ⁺ The enzyme reaction rates of the mutant malic enzyme (L301R/Q392C) to BC-10 and NAD ⁺ were 13.6U·mg ^-1 and ^0.25U ·mg ^-1 respectively. 0.17 U·mg ^-1 .

Instructions, 1) mutant malic enzyme (L301R) and mutant malic enzyme (L301R/Q392C) can not effectively use NAD ⁺ as a coenzyme, while BC-10 as mutant malic enzyme (L301R) and mutant malic enzyme ( L301R/Q392C) coenzyme activity is very good. Therefore, the analog BC-10 has an irreplaceable effect of NAD ⁺ on the activity of the aforementioned mutant malic enzyme; 2) the wild-type malic enzyme cannot effectively utilize BC-10 as a coenzyme. Therefore, the analogue BC-10, as a coenzyme, has certain specificity in mutual recognition with dehydrogenase.

Example 2

With embodiment 1 method; Difference with embodiment 1 is that the nucleotide used is The reaction temperature is 26° C., the reaction time is 45 min, and the yield of the target product BC-20 is 32%.

¹ H NMR (D ₂ O, 400MHz): δ9.33(s, 1H), 9.17(d, J=6.2Hz, 1H), 8.86(d, J=7.2Hz, 1H), 8.19(pseudo t, J =6.3Hz, 1H), 7.95(s, 1H), 6.05(d, J=5.2Hz, 1H), 5.72(d, J=3.3Hz, 1H), 4.52-4.44(m, 2H), 4.36-4.28 (m, 2H), 4.18-4.04 (m, 6H). ¹³ C NMR (D ₂ O, 100MHz): δ165.2, 161.9, 155.8, 145.8, 142.4, 139.8, 138.9, 133.7, 128.5, 102.1, 99.8, 89.7, 86.6, 82.3, 77.4, 73.8, 70.3, 68.8, 64.7, 64.6. ³¹ P NMR (D ₂ O, 162 MHz): δ-11.9. HRMS: calcd for C ₂₀ H ₂₆ ClN ₅ O ₁₅ P ₂ (M+H ) ⁺ 674.0667, found 674.0672.

The compound is a white solid that easily absorbs moisture and becomes viscous and dark in color.

Determination of malic enzyme activity: the method is the same as in Example 1, except that the enzyme reaction rate of wild-type malic enzyme to BC-20 is 0.52U mg ^-1 , and that of mutant malic enzyme (L301R) The enzyme reaction rate to BC-20 is 13.7U·mg ^-1 , and the enzyme reaction rate of mutant malic enzyme (L301R/Q392C) to BC-20 is 15.4U·mg ^-1 . It shows that BC-20 can be selectively utilized by mutant malic enzyme (L301R) and malic enzyme (L301R/Q392C), but is hardly utilized by wild-type malic enzyme; meanwhile, wild-type malic enzyme does not utilize BC- 20, while only selectively utilizing NAD ⁺ .

Example 3

With embodiment 1 method; Difference with embodiment 1 is that the nucleotide used is The reaction temperature was 28° C., the reaction time was 30 min, and the yield of the target product BC-30 was 32%.

¹ H NMR (D ₂ O, 400MHz): δ9.32(s, 1H), 9.16(d, J=6.2Hz, 1H), 8.85(d, J=8.0Hz, 1H), 8.17(pseudo t, J =6.4Hz, 1H), 7.97(s, 1H), 6.04(d, J=5.4Hz, 1H), 5.69(d, J=3.3Hz, 1H), 4.47(brs, 1H), 4.43(pseudo t, J=5.1Hz, 1H), 4.35(pseudo t, J=4.7Hz, 1H), 4.31-4.28(m, 1H), 4.19-4.03(m, 6H). ¹³ C NMR (D ₂ O, 100MHz): ^δ165.5 , _162.7 , 156.2, 146.0, 142.6, 141.9, 139.9, 133.9, 128.7, 99.9, 89.8, 89.2, 86.9, 82.4, 77.5, 74.0, 70.5, 68.9, 64.8, 64.7. 162MHz): δ-11.2. HRMS: calcd for C ₂₀ H ₂₆ BrN ₅ O ₁₅ P ₂ (M+H) ⁺ 718.0162, found 718.0147.

The compound is light yellow solid, easy to absorb moisture and become viscous, and the color becomes darker.

Determination of malic enzyme activity: the method is the same as in Example 1, and the difference from Example 1 is that the enzyme reaction rate of wild-type malic enzyme to BC-30 is 0.64U mg ^-1 , and mutant malic enzyme (L301R/ The enzyme reaction rate of Q392C) to BC-30 is 19.5U·mg ^-1 . It shows that BC-30 can be selectively utilized by the mutant malic enzyme (L301R/Q392C), but almost not utilized by the wild-type malic enzyme; at the same time, the wild-type malic enzyme does not utilize BC-30, but only selectively utilizes NAD ⁺ .

Example 4

With embodiment 1 method; Difference with embodiment 1 is that the nucleotide used is The reaction temperature is 26° C., the reaction time is 38 min, and the yield of the target product BC-40 is 30%.

¹ H NMR (D ₂ O, 400MHz): δ9.33(s, 1H), 9.16(d, J=6.1Hz, 1H), 8.85(d, J=8.0Hz, 1H), 8.19(pseudo t, J =7.0Hz, 1H), 7.60(s, 1H), 6.06(d, J=5.4Hz, 1H), 5.79(d, J=4.3Hz, 1H), 4.68(brs, 1H), 4.48-4.06(m , 9H), 1.87(s, 3H). ¹³ C NMR (D ₂ O, 100MHz): δ165.6, 165.3, 157.2, 145.8, 142.4, 139.8, 138.3, 133.7, 128.6, 104.7, 99.8, 89.0, 86.7, 82.3, 77.4, 73.7, 70.3, 69.2, 64.8, 64.7, 12.3. ³¹ P NMR (D ₂ O, 162MHz): δ-11.9. HRMS: calcd for C ₂₁ H ₂₉ N ₅ O ₁₅ P ₂ (M+H) ^+652.1057 , found 652.1072.

The compound is white solid, easy to absorb moisture.

Determination of malic enzyme activity: the method is the same as in Example 1, and the difference from Example 1 is that the enzyme reaction rate of wild-type malic enzyme to BC-40 is 0.15U mg ^-1 , and mutant malic enzyme (L301R/ The enzyme reaction rate of Q392C) to BC-40 is 11.3U·mg ^-1 . It shows that BC-40 can be selectively utilized by the mutant malic enzyme (L301R/Q392C), but almost not utilized by the wild-type malic enzyme; at the same time, the wild-type malic enzyme does not utilize BC-40, but only selectively utilizes NAD ⁺ .

Example 5

With embodiment 1 method; Difference with embodiment 1 is that the nucleotide used is The reaction temperature is 26° C., the reaction time is 40 min, and the yield of the target product BC-50 is 30%.

Or use 0.3mmol NMN Dissolve in 2mL DMSO solvent, add 1.5mM CDI, react at 26°C for 20min, add 1mM nucleoside monophosphate dissolved in 2mL DMF Reaction at 26°C for 8d. Add 20 mL of acetone to stop the reaction, a large amount of precipitates are formed, the precipitates are collected by centrifugation, and the precipitates are washed three times with 5 mL of acetone. After water-soluble, performic acid-type anion-exchange resin was separated and purified to obtain the target product BC-50 with a yield of 24%.

Or use 0.2mmol nucleoside monophosphate Dissolve 1mM morpholine in 5mL water, add dropwise 2mM DCC dissolved in 5mL n-butanol, react at 50°C for 5h, evaporate the solvent to dryness, wash the precipitate with 5mL n-butanol three times to obtain a white solid, and pump it dry to remove water completely. Add 5 mL of formamide dissolved 1 mM NMN 0.2mM MnCl ₂ , 0.2mM MgSO ₄ , react at 30°C for 16h. The solvent was evaporated to dryness, dissolved in water, separated and purified by performic acid-type anion exchange resin, and the target product BC-50 was obtained with a yield of 56%.

¹ H NMR (D ₂ O, 400MHz): δ9.34(s, 1H), 9.18(d, J=6.1Hz, 1H), 8.86(d, J=8.1Hz, 1H), 8.20(pseudo t, J =6.8Hz, 1H), 7.58(s, 1H), 6.07(d, J=5.4Hz, 1H), 5.80(d, J=4.5Hz, 1H), 4.48-4.45(m, 2H), 4.37-4.35 (m, 1H), 4.29-4.23(m, 3H), 4.16-4.12(m, 3H), 4.05-4.03(m, 1H), 1.79(s, 3H). ¹³ C NMR (D ₂ O, 100MHz) : δ166.3, 165.6, 151.7, 146.1, 142.6, 139.9, 137.1, 133.9, 128.7, 111.7, 99.9, 88.2, 87.0, 83.0, 77.5, 73.4, 70.6, 69.7, 65.0, 64.9, 11.6. ³¹ P NMR (D ₂ O, 162MHz): δ-11.3. HRMS: calcd for C ₂₁ H ₂₈ N ₄ O ₁₆ P ₂ (M+H) ⁺ 655.1054, found 655.1035.

The compound is white solid, easy to absorb moisture.

Determination of malic enzyme activity: the method is the same as in Example 1, and the difference from Example 1 is that the enzyme reaction rate of wild-type malic enzyme to BC-50 is 0.63U·mg ^-1 , and mutant malic enzyme (L301R/ The enzyme reaction rate of Q392C) to BC-50 is 2.5U·mg ^-1 . It shows that BC-50 can be selectively utilized by the mutant malic enzyme (L301R/Q392C), but almost not utilized by the wild-type malic enzyme; at the same time, the wild-type malic enzyme does not utilize BC-50, but only selectively utilizes NAD ⁺ .

Escherichia coli growth promotion experiment: BC-50 was added to 10 mL of LB medium (peptone 10 g·L ^-1 , yeast powder 5 g·L ^-1 , sodium chloride 10 g·L ^-1 , pH 7.2) to a final concentration of 100 μM, Escherichia coli DH5α (Beijing Dingguo Biotechnology Company) seed solution (OD ₆₀₀ ⁼ 2) was inoculated at a ratio of 1:100, and cultured at 37°C and 200 rpm; The OD ₆₀₀ value of E. coli added with BC-50 was 0.5 higher than that of the control group. It shows that the BC-50 can obviously promote the growth of Escherichia coli DH5α.

Example 6

Determination of lactate dehydrogenase activity: wild-type lactate dehydrogenase and mutant lactate dehydrogenase (V152R) were prepared into a solution of 0.1 mg·mL ^-1 . Prepare 0.2mL reaction mixture (50mM HEPES pH 7.2, 200mM D-sodium lactate, 1mM NAD ⁺ or NAD ⁺ analogue), quickly add 1μL enzyme solution during activity analysis, mix well and place in a UV spectrophotometer, at 25°C Continuously monitor the change of 340nm absorption value within 1min. The enzyme activity unit is defined as: the amount of enzyme required to catalyze the production of 1 μmol NADH or reduced NAD ⁺ analogs per minute at 25°C.

The results are shown in Table-2:

Table-2 Lactate dehydrogenase activity assay results

The above results indicated that 1) the analogues BC-10, BC-20, BC-30 and BC-40 act as coenzymes of mutant lactate dehydrogenase (V152R). The activity of the mutant lactate dehydrogenase (V152R) using these coenzymes was comparable to that of the wild-type lactate dehydrogenase using NAD ⁺ as a coenzyme. However, NAD ⁺ and the analogue BC-50 were less enzymatically active as coenzymes for mutant lactate dehydrogenase (V152R). Therefore, BC-10, BC-20, BC-30 and BC-40 have NAD ⁺ irreplaceable effects on the activity of mutant lactate dehydrogenase (V152R). 2) The wild-type lactate dehydrogenase cannot effectively use analogues BC-10, BC-20, BC-30, BC-40 and BC-50 as coenzymes. Therefore, these analogs have a certain specificity in mutual recognition as coenzymes and dehydrogenases.

Example 7

Activity determination of alcohol dehydrogenase using reduced NAD ⁺ analogues: make NAD ⁺ analogues into a 10mM solution, treat with an equal volume of 10mM NaBH ₄ aqueous solution to obtain the reduced state of the analogues, and set aside; prepare alcohol dehydrogenase 1.0 mg·mL ^-1 solution. Prepare 0.2mL reaction mixture (50mM HEPES pH 7.2, 5mM acetaldehyde, 5mM MnCl ₂ , 0.5mM reduced NAD ⁺ analog or NADH), quickly add 1μL enzyme solution during activity analysis, mix well and place in a UV spectrophotometer In the process, the change of the 340nm absorption value was continuously monitored within 1min at 25°C. The enzyme activity unit is defined as: the amount of enzyme required to catalyze the production of 1 μmol NAD ⁺ or NAD ⁺ analogs per minute at 25 °C.

The results are shown in Table-3:

Table-3 Activities of Alcohol Dehydrogenase Utilizing Reduced NAD ⁺ Analogues

The above results indicated that the reduced forms of NAD ⁺ analogues BC-10, BC-20, BC-30, BC-40 and BC-50 can act as coenzymes of alcohol dehydrogenase to catalyze the reduction of acetaldehyde production tool ethanol. Wherein, the alcohol dehydrogenase uses the reduced form of the analogue BC-50 as a coenzyme, and its activity reaches more than 10% of the activity when using NADH as a coenzyme.

Can find out by above embodiment:

1) The present invention establishes a chemical synthesis method for a class of novel NAD ⁺ analogues, which is simple and effective, and can be used for the preparation of other NAD ⁺ analogues with similar structures;

2) The NAD ⁺ analog prepared by the present invention can be used as a coenzyme of dehydrogenase to catalyze redox reactions;

3) The NAD ⁺ analog prepared by the present invention can be used as a specific coenzyme for dehydrogenase or mutant dehydrogenase, and its effect for catalytic reactions is better than that of using NAD ⁺ as a coenzyme, and can be applied in biocatalysis and biological in analytical chemistry;

4) The highly active combination of mutant dehydrogenase/NAD ⁺ analogs based on the present invention is an artificial new system independent of the original redox catalytic system in nature, and may be used as a unique tool in bioanalytical chemistry, metabolic engineering and It is of great value in synthetic biology research;

5) The NAD ⁺ analogs prepared in the present invention can be used as microbial growth regulators to change the growth behavior of microorganisms and be applied in biochemical engineering research or production.

Claims (4)

1. an application of NAD ⁺ analogs, characterized in that: it has the following general structural formula: In the structural formula, the nicotinamide mononucleotide is in the β-configuration; the two ribose units are in the D-configuration; The R unit attached to the nucleotide is Wherein R ¹ is one of halogen, C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkoxyl; or R for Wherein R ² is one of C ₁ -C ₅ alkyl groups; The NAD ⁺ analog acts as a coenzyme of dehydrogenase for catalyzing redox reactions. 2. According to the application of the NAD ⁺ analogue described in claim 1, it is characterized in that: its structural formula is as follows, 3. According to the application of NAD ⁺ analogs described in claim 1, it is characterized in that: its structural formula is as follows, 4. An application of the NAD ⁺ analogue according to claim 1, characterized in that: the NAD ⁺ analogue according to claim 1 is used as a microorganism accelerator for promoting the growth of microorganisms.