CN102453067B - A kind of NAD +the preparation method of analogue and application thereof - Google Patents
A kind of NAD +the preparation method of analogue and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of Reduced nicotinamide-adenine dinucleotide (NAD
+) preparation method of analogue and application thereof, its general structure as
nAD
+analogue, be reacted by nmn and corresponding pyrimidine nucleoside acid-like substance to generate, wherein R is
or R is
such NAD
+analogue as the coenzyme of desaturase, also can promote microbial growth.The NAD that the present invention obtains
+analogue can be applicable in the research of biocatalysis, bioanalytical chemistry, metabolic engineering and synthetic biology.
Description
Technical field
The present invention relates to synthesis and the application thereof of the small molecule compound with biologic activity, specifically there is the Reduced nicotinamide-adenine dinucleotide (NAD of various substituent pyrimidine ring substituted adenines ring
+) analogue, and the cofactor that this compound can be used as desaturase reacts for catalytic oxidation-reduction, or as microbial growth promoters, accelerate growth of microorganism.
Background technology
Reduced nicotinamide-adenine dinucleotide (nicotinamide adenine dinucleotide, NAD
+) and its corresponding reduction-state (NADH), being exactly usually said nadide, is the indispensable micromolecular compound of organism, and participate in the redox metabolism in life entity and other a series of important biomolecule chemical process, its structural formula is as follows:
NAD
+chemical structure
NAD
+chemical structure more complicated, character is stable not, expensive.From NAD
+chemical structure can find out that this molecule is formed by connecting by tetra-sodium key by nmn (NMN) fragment and adenylic acid (AMP) fragment.NMN part is the main undertaker of its chemical functional, participate in redox reaction and electron transmission, AMP part then with the relevant domain effect of functional protein and grappling coenzyme, material impact (J.Benach et al.J.Biol.Chem.2003 is had to the selectivity of enzyme specificity identification coenzyme and biochemical reaction, 278,19176-19182).
In cell, NAD
+main plaing a part transmits hydrogen and electronics, and many important oxydo-reductase all depend on NAD
+as its cofactor.Except redox function, in non-oxide reduction vital process, NAD
+play very important effect equally.NAD
+all indispensable micromolecular compound (W.H.Ying.Antioxidants & Redox Signaling 2008,10,179-206) in the vital movements such as cell proliferation, growth, differentiation, apoptosis.NAD
+also can as the substrate of histon deacetylase (HDAC) (sirtuins); enzymatically; ethanoyl on histone is removed; enable histone complete DNA replication dna smoothly, transcribe and critical function (the H.N.Lin et al.Org.Biomol.Chem.2007 such as reparation; 5,2541-2554).
Based on NAD
+structure, people carry out chemical improvement to it, have synthesized a lot of analogue.Nearest a few thing relates to chemical improvement adenylic acid (AMP) partly (C.J.W.Mort et al.Bioorg.Med.Chem.2004,12,475-487), ribose ring part (G.C.Zhou et al.J.Am.Chem.Soc.2004,126,5690-5698), niacinamide part (N.E.Batoux et al.Tetrahedron 2004,60,6609-6617) and tetra-sodium part (L.Chen et al.Bioorg.Med.Chem.Lett.2007,17,3152-3155).H.C.Lo once adopted methyl alcohol be AMP part surrogate and nmn under DCC/DMAP, synthesized NAD
+analogue, and be chiral alcohol (H.C.Lo et al.Angew.Chem.Int.Ed.2002 with above-mentioned analogue as the cofactor reductone of horse liver desaturase (HLADH), 41,478-481), but cofactor structure is too simple, with enzyme in conjunction with undertighten, catalytic efficiency is very low.Above-mentioned work is all be based upon NAD
+original structure carries out on chemically modified basis, is difficult to overcome unstable in its chemical property, the shortcoming such as chemosynthesis, separation difficulty, gained NAD
+analogue can not be dehydrogenated enzyme selectivity identification mostly.
Previous work [Zhao Zongbao, Liu Wujun, Wu Siguo, Hou Shuhua, a kind of NAD of applicant
+analogue and synthesis thereof and application, application number: 200810010285.1] synthesized by NAD
+analogue can promote that microorganism is as intestinal bacteria and yeast saccharomyces cerevisiae growth; Its reduced form also can be used as desaturase cofactor and reacts for catalytic oxidation-reduction, but these analogue biological activitys are relatively low.
In a word, the NAD of existing bibliographical information
+analogue is mainly as the inhibitor of biological catalyst, and only a few can be used for biocatalysis or bionic catalysis reaction, but can not specifically by biological catalyst identification.Therefore, design and rational is needed to have the NAD of other constitutional featuress
+analogue, makes them more effectively by enzyme identification, shows biocatalytic Activity.There is the NAD of these biological characteristicses
+analogue, can play a significant role in fields such as bioanalytical chemistry, biocatalysis, metabolic engineering or synthetic biologies.
Summary of the invention
The present invention is summing up about on the basis of NAD chemical improvement achievement and relevant biological activity in document, to NAD
+structure dissects, and remains niacinamide monokaryon glycosides redox functional zone, carries out transformation one and substitutes with the pyrimidine ring structure of modifying, the NAD of project organization novelty to VITAMIN B4 loop section
+analogue.
NAD involved in the present invention
+analogue, has following general structure:
In structural formula, nmn is beta configuration; Two ribose unit are D-form;
The R unit be connected with Nucleotide is
hereinafter referred to as A, wherein R
1for halogen, such as F, Cl, Br; C
1-C
5alkyl, such as methyl, ethyl; C
1-C
5alkane hydroxyl, such as methylol.
Or R is
hereinafter referred to as B, wherein R
2for C
1-C
5alkyl, such as methyl, ethyl.
NAD in the present invention
+the synthesis of analogue divides three steps to carry out, and is summarized as follows:
The first step: reference literature (S.M.Graham et al.Org.Lett.2004,6,233-236) synthesizes nmn;
Second step: reference literature (M.Yoshikaw et al.Tetrahedron Lett.1967,5065-5068) synthetic nucleosides monophosphate;
3rd step: the Nucleotide monophosphates that the nmn the first step obtained and second step obtain carries out coupled, obtains target NAD
+product.Specific operation process reference literature (T.V.Abramova etal.Bioorg.Med.Chem.2007,15,6549-6555), document (L.Q.Chen et al.J.Med.Chem.2007,50,5743-5751) or document (J.Lee et al.Chem.Commun.1999,729).
Document (T.V.Abramova et al.Bioorg.Med.Chem.2007,15,6549-6555) use N, the mixed solvent of M-dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) (DMSO), condensation reagent is triphenylphosphine (Ph
3p), the two piperidines (PyrS) of 1,1-bis-mercaptan
2or Diphenyl disulfide ether (PhS)
2or 2,2 '-bipyridyl disulfide (PyS)
2with 1-Methylimidazole;
Document (L.Q.Chen et al.J.Med.Chem.2007,50,5743-5751) uses DMF (DMF) to be solvent, and N, N-carbonyl dimidazoles (CDI) is condensation reagent;
Document (J.Lee et al.Chem.Commun.1999,729) use methane amide is solvent, and N, N '-dicyclohexylcarbodiimide (DCC), morpholine is condensation reagent, MnCl
2, MgSO
4for catalyzer.
Desaturase used in the present invention is e. coli k12 malic enzyme (UniProt codeP26616).Sudden change desaturase used by the present invention utilizes
single-site mutant test kit or document (J.X.Wang, et al.J.Microbiol.Methods 2007,71,225-230) on e. coli k12 malic enzyme, introduce amino acid mutation obtain saltant type malic enzyme (L301R and L301R/Q392C).The present invention's malic enzyme used is all by document (J.X.Wang, et al.Protein Expr.Purif.2007,53,97-103) method expression and purification.
Desaturase used in the present invention also has serum lactic dehydrogenase from Lactobacillus helveticus (UniProt code P30901).Sudden change serum lactic dehydrogenase used by the present invention utilizes
single-site mutant test kit is introduced amino acid mutation and is obtained mutant lactate dehydrogenase (V152R) on serum lactic dehydrogenase.The present invention's serum lactic dehydrogenase used is all by document (J.X.Wang, et al.Protein Expr.Purif.2007,53,97-103) method expression and purification.
Microorganism used in the present invention is bacillus coli DH 5 alpha (Beijing Ding Guo biotech company).
Desaturase used in the present invention also has ethanol dehydrogenase, buys in Sigma Aldrich from Saccharomyces cerevisiae (CAS NO.9031-72-5).
Table-1 provides the NAD of the embodiment of the present invention
+analogue.
Table-1.NAD
+the chemical structure of analogue numbering and correspondence thereof
Embodiment
Following examples contribute to understanding this patent, but are not limited to content of the present invention.
Raw material used in embodiment respectively reference literature (S.M.Graham et al.Org.Lett.2004,6,233-236.) synthesizes nmn ammonium salt; Reference literature (M.Yoshikaw et al.Tetrahedron Lett.1967,5065-5068) synthetic nucleosides monophosphate; Reference literature (T.V.Abramova et al.Bioorg.Med.Chem.2007,15,6549-6555.), document (L.Q.Chenet al.J.Med.Chem.2007,50,5743-5751) or document (J.Lee et al.Chem.Commun.1999,729) synthesis final product.
Embodiment 1
By 0.3mmol NMN
be dissolved in 2mL DMSO and 2mL DMF mixed solvent, add 1.5mM Ph successively
3p, 1.5mM (PyS)
2, 6mM Methylimidazole 26 DEG C reaction 18min, adds the 1mM Nucleotide monophosphates that 2mL DMF dissolves
26 DEG C of reaction 40min.Add 20mL acetone termination reaction, have and precipitate generation in a large number, centrifugal collecting precipitation, precipitate three times with 5mL washing with acetone.Water-soluble rear peroxyformic acid type Anion exchange resin separation purifying, obtains target product BC-10, productive rate 35%.
1H NMR(D
2O，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).
13C NMR(D
2O，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.
19F NMR(D
2O，376MHz)：δ-164.9.
31P NMR(D
2O，162MHz)：δ-11.1，-11.2.HRMS：calcd for C
20H
26FN
5O
15P
2(M+H)
+658.0963，found 658.0961.
This compound is white solid, the easy moisture absorption and become sticky thick, and darkens.
Activity of Malic Enzyme measures: by wild-type malic enzyme, saltant type malic enzyme (L301R) and saltant type malic enzyme (L301R/Q392C) are mixed with 1mgmL respectively
-1solution.Preparation 0.2mL reaction mixture (50mM HEPES pH 7.2,3mM L-malate, 5mM MnCl
2, the NAD of 0.2mM
+or BC-10), add 1 μ L enzyme liquid during activation analysis fast, mixing is placed in ultraviolet spectrophotometer, monitors 340nm absorption value continuously, can obtain desired activities data in 1min at 25 DEG C.Enzyme activity unit is defined as: under 25 DEG C of conditions, per minute catalysis produces 1 μm of ol reduced-NAD
+or the enzyme amount needed for BC-10.
Experiment finds, wild-type malic enzyme is to BC-10 and NAD
+enzyme reaction rate be 0.31Umg respectively
-1and 22.5Umg
-1, saltant type malic enzyme (L301R) is to BC-10 and NAD
+enzyme reaction rate be 11.2Umg respectively
-1and 0.25Umg
-1, saltant type malic enzyme (L301R/Q392C) is to BC-10 and NAD
+enzyme reaction rate be 13.6Umg respectively
-1and 0.17Umg
-1.
Illustrating, 1) saltant type malic enzyme (L301R) and saltant type malic enzyme (L301R/Q392C) effectively can not utilize NAD
+as coenzyme, and BC-10 is very well active as the coenzyme of sudden change malic enzyme (L301R) and saltant type malic enzyme (L301R/Q392C).Therefore, analogue BC-10 has NAD for the activity of aforementioned saltant type malic enzyme
+irreplaceable effect; 2) wild-type malic enzyme can not effectively utilize BC-10 as coenzyme.Therefore, analogue BC-10 identifies to have certain specificity mutually as coenzyme and desaturase.
Embodiment 2
With embodiment 1 method; Difference from Example 1 is, Nucleotide used is
temperature of reaction is 26 DEG C, and the reaction times is 45min, and the productive rate of target product BC-20 is 32%.
1H NMR(D
2O，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).
13C NMR(D
2O，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.
31P NMR(D
2O，162MHz)：δ-11.9.HRMS：calcd forC
20H
26ClN
5O
15P
2(M+H)
+674.0667，found 674.0672.
This compound is white solid, the easy moisture absorption and become sticky thick, and darkens.
Activity of Malic Enzyme measures: method is with embodiment 1, and difference from Example 1 is, the enzyme reaction rate of wild-type malic enzyme to BC-20 is 0.52Umg
-1, saltant type malic enzyme (L301R) is 13.7Umg to the enzyme reaction rate of BC-20 respectively
-1, saltant type malic enzyme (L301R/Q392C) enzyme reaction rate to BC-20 is 15.4Umg
-1.Illustrate that BC-20 can optionally be utilized by the malic enzyme (L301R) that suddenlys change and malic enzyme (L301R/Q392C), and utilized by wild-type malic enzyme hardly; Meanwhile, wild-type malic enzyme does not utilize BC-20, and only selective use NAD
+.
Embodiment 3
With embodiment 1 method; Difference from Example 1 is, Nucleotide used is
temperature of reaction is 28 DEG C, and the reaction times is 30min, and the productive rate of target product BC-30 is 32%.
1H NMR(D
2O，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).
13C NMR(D
2O，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.
31P NMR(D
2O，162MHz)：δ-11.2.HRMS：calcd for C
20H
26BrN
5O
15P
2(M+H)
+718.0162，found 718.0147.
This compound is faint yellow solid, the easy moisture absorption and become sticky thick, and darkens.
Activity of Malic Enzyme measures: method is with embodiment 1, and difference from Example 1 is, the enzyme reaction rate of wild-type malic enzyme to BC-30 is 0.64Umg
-1, saltant type malic enzyme (L301R/Q392C) enzyme reaction rate to BC-30 is 19.5Umg
-1.Illustrate that BC-30 can optionally be utilized by saltant type malic enzyme (L301R/Q392C), and utilized by wild-type malic enzyme hardly; Meanwhile, wild-type malic enzyme does not utilize BC-30, and only selective use NAD
+.
Embodiment 4
With embodiment 1 method; Difference from Example 1 is, Nucleotide used is
temperature of reaction is 26 DEG C, and the reaction times is 38min, and the productive rate of target product BC-40 is 30%.
1H NMR(D
2O，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).
13C NMR(D
2O，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.
31P NMR(D
2O，162MHz)：δ-11.9.HRMS：calcd for C
21H
29N
5O
15P
2(M+H)
+652.1057，found 652.1072.
This compound is white solid, the easy moisture absorption.
Activity of Malic Enzyme measures: method is with embodiment 1, and difference from Example 1 is, the enzyme reaction rate of wild-type malic enzyme to BC-40 is 0.15Umg
-1, saltant type malic enzyme (L301R/Q392C) enzyme reaction rate to BC-40 is 11.3Umg
-1.Illustrate that BC-40 can optionally be utilized by saltant type malic enzyme (L301R/Q392C), and utilized by wild-type malic enzyme hardly; Meanwhile, wild-type malic enzyme does not utilize BC-40, and only selective use NAD
+.
Embodiment 5
With embodiment 1 method; Difference from Example 1 is, Nucleotide used is
temperature of reaction is 26 DEG C, and the reaction times is 40min, and the productive rate of target product BC-50 is 30%.
Or adopt 0.3mmol NMN
be dissolved in 2mL DMSO solvent, add 1.5mM CDI 26 DEG C and react 20min, add the 1mM Nucleotide monophosphates that 2mL DMF dissolves
26 DEG C of reaction 8d.Add 20mL acetone termination reaction, have and precipitate generation in a large number, centrifugal collecting precipitation, precipitate three times with 5mL washing with acetone.Water-soluble rear peroxyformic acid type Anion exchange resin separation purifying, obtains target product BC-50, productive rate 24%.
Or adopt 0.2mmol Nucleotide monophosphates
1mM morpholine is dissolved in 5mL water, is added dropwise to the 2mM DCC be dissolved in 5mL propyl carbinol, 50 DEG C of reaction 5h, and solvent evaporated, with 5mL propyl carbinol washing precipitation three times, obtain white solid, oil pump is drained and thoroughly removed moisture, adds the 1mM NMN that 5mL methane amide dissolves
0.2mM MnCl
2, 0.2mMMgSO
4, 30 DEG C of reaction 16h.Solvent evaporated, water-soluble rear peroxyformic acid type Anion exchange resin separation purifying, obtains target product BC-50, productive rate 56%.
1H NMR(D
2O，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).
13C NMR(D
2O，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.
31P NMR(D
2O，162MHz)：δ-11.3.HRMS：calcd for C
21H
28N
4O
16P
2(M+H)
+655.1054，found 655.1035.
This compound is white solid, the easy moisture absorption.
Activity of Malic Enzyme measures: method is with embodiment 1, and difference from Example 1 is, the enzyme reaction rate of wild-type malic enzyme to BC-50 is 0.63Umg
-1, saltant type malic enzyme (L301R/Q392C) enzyme reaction rate to BC-50 is 2.5Umg
-1.Illustrate that BC-50 can optionally be utilized by saltant type malic enzyme (L301R/Q392C), and utilized by wild-type malic enzyme hardly; Meanwhile, wild-type malic enzyme does not utilize BC-50, and only selective use NAD
+.
Promote Escherichia coli Growth experiment: by LB substratum (peptone 10gL
-1, yeast powder 5gL
-1, sodium-chlor 10gL
-1, pH 7.2) and to add BC-50 to final concentration in 10mL be 100 μMs, with 1: 100 inoculation bacillus coli DH 5 alpha (Beijing Ding Guo biotech company) seed liquor (OD
600=2), at 37 DEG C, cultivate under 200rpm condition; (equivalent NAD is added with control group
+) compare, add the colibacillary OD of BC-50 after cultivating 4h
600be worth higher than control group by 0.5.Illustrate that this BC-50 has obvious promoter action to bacillus coli DH 5 alpha growth.
Embodiment 6
Lactate dehydrogenase activity measures: wild-type serum lactic dehydrogenase and mutant lactate dehydrogenase (V152R) are mixed with 0.1mgmL
-1solution.Preparation 0.2mL reaction mixture (50mMHEPES pH 7.2,200mM D-ALPHA-Hydroxypropionic acid sodium, the NAD of 1mM
+or NAD
+analogue), add 1 μ L enzyme liquid during activation analysis fast, mixing is placed in ultraviolet spectrophotometer, monitors the change of 340nm absorption value at 25 DEG C in 1min continuously.Enzyme activity unit is defined as: under 25 DEG C of conditions, per minute catalysis produces 1 μm of ol NADH or reduced-NAD
+enzyme amount needed for analogue.
Result is as table-2:
Table-2 lactate dehydrogenase activity measurement results
The above results illustrates, 1) analogue BC-10, BC-20, BC-30 and BC-40 are as the coenzyme of mutant lactate dehydrogenase (V152R).Mutant lactate dehydrogenase (V152R) utilizes the activity of these coenzyme and wild-type serum lactic dehydrogenase to utilize NAD
+activity as coenzyme is suitable.But, NAD
+during with the coenzyme of analogue BC-50 as mutant lactate dehydrogenase (V152R), activity of enzyme reaction is lower.Therefore, BC-10, BC-20, BC-30 and BC-40 have NAD for the activity of mutant lactate dehydrogenase (V152R)
+irreplaceable effect.2) wild-type serum lactic dehydrogenase can not effectively utilize analogue BC-10, BC-20, BC-30, BC-40 and BC-50 as coenzyme.Therefore, these analogues identify to have certain specificity mutually as coenzyme and desaturase.
Embodiment 7
Ethanol dehydrogenase utilizes reduced-NAD
+the determination of activity of analogue: by NAD
+analogue is made into 10mM solution, with equal-volume 10mM NaBH
4aqueous solution process, obtains the reduction-state of analogue, for subsequent use; Ethanol dehydrogenase is mixed with 1.0mgmL
-1solution.Preparation 0.2mL reaction mixture (50mM HEPES pH 7.2,5mM acetaldehyde, 5mM MnCl
2, the reduced-NAD of 0.5mM
+analogue or NADH), add 1 μ L enzyme liquid during activation analysis fast, mixing is placed in ultraviolet spectrophotometer, monitors the change of 340nm absorption value at 25 DEG C in 1min continuously.Enzyme activity unit is defined as: under 25 DEG C of conditions, per minute catalysis produces 1 μm of ol NAD
+or NAD
+enzyme amount needed for analogue.
Result is as table-3:
Table-3 ethanol dehydrogenases utilize reduced-NAD
+the activity of analogue
The above results illustrates, NAD
+reduced form corresponding to analogue BC-10, BC-20, BC-30, BC-40 and BC-50 can as the coenzyme of ethanol dehydrogenase, catalytic reduction acetaldehyde production instrument ethanol.Wherein, ethanol dehydrogenase utilizes the reduced form of analogue BC-50 as coenzyme, and activity reaches and utilizes NADH as more than 10% of activity during coenzyme.
Can be found out by above embodiment:
1) the present invention establishes the NAD of a class formation novelty
+the chemical synthesis process of analogue, the method is simply effective, can be used for the NAD synthesizing other structural similitude
+the preparation of analogue;
2) NAD prepared by the present invention
+the coenzyme that analogue can be used as desaturase reacts for catalytic oxidation-reduction;
3) NAD prepared by the present invention
+analogue can be used as the specificity coenzyme of desaturase or saltant type desaturase, and its effect for catalyzed reaction is better than with NAD
+as the situation of coenzyme, can be applicable in biocatalysis and bioanalytical chemistry;
4) based on saltant type desaturase/NAD of the present invention
+analogue high reactivity combines, and is the artificial new system independent of the original redox-catalyst system of nature, as distinctive tool applications in the research of bioanalytical chemistry, metabolic engineering and synthetic biology, may have important value;
5) NAD prepared by the present invention
+analogue can be used as microbial growth regulator, changes microorganism growth behavior, is applied in biochemical engineering research or production.
Claims (4)
1. a NAD
+the application of analogue, is characterized in that: it has following general structure:
In structural formula, nmn is beta configuration; Two ribose unit are D-form;
The R unit be connected with Nucleotide is
wherein R
1for halogen, C
1-C
5alkyl or C
1-C
5one of in alkane hydroxyl;
Or R is
wherein R
2for C
1-C
5one of in alkyl;
Described NAD
+analogue, as the coenzyme of desaturase, reacts for catalytic oxidation-reduction.
2. according to NAD described in claim 1
+the application of analogue, is characterized in that: its structural formula is as follows,
3. according to NAD described in claim 1
+the application of analogue, is characterized in that: its structural formula is as follows,
4. NAD described in a claim 1
+the application of analogue, is characterized in that: NAD described in claim 1
+analogue as microorganism promotor, for promoting microbial growth.
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