CN102453067A - Preparation method and application of NAD+(nicotinamide adenine dinucleotide) analogues - Google Patents
Preparation method and application of NAD+(nicotinamide adenine dinucleotide) analogues Download PDFInfo
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- CN102453067A CN102453067A CN2010105247676A CN201010524767A CN102453067A CN 102453067 A CN102453067 A CN 102453067A CN 2010105247676 A CN2010105247676 A CN 2010105247676A CN 201010524767 A CN201010524767 A CN 201010524767A CN 102453067 A CN102453067 A CN 102453067A
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- UORXTGFCFREKJF-GLCBXQDMSA-O C=NC(C(Cl)=CN1[C@@H](C2O)O[C@H](COP(O)(OP(O)(OC[C@H](C(C3O)O)O[C@H]3[n+]3cccc(C(N)=O)c3)=O)=O)C2O)=NC1=O Chemical compound C=NC(C(Cl)=CN1[C@@H](C2O)O[C@H](COP(O)(OP(O)(OC[C@H](C(C3O)O)O[C@H]3[n+]3cccc(C(N)=O)c3)=O)=O)C2O)=NC1=O UORXTGFCFREKJF-GLCBXQDMSA-O 0.000 description 1
- SMADXYGVDMUPOG-LEXLCYFYSA-O CC(C(N)=N1)=CN([C@@H](C2O)OC(COP(O)(OP(O)(OCC(C(C3O)O)O[C@H]3[n+]3cccc(C(N)=O)c3)=O)=O)C2O)C1=O Chemical compound CC(C(N)=N1)=CN([C@@H](C2O)OC(COP(O)(OP(O)(OCC(C(C3O)O)O[C@H]3[n+]3cccc(C(N)=O)c3)=O)=O)C2O)C1=O SMADXYGVDMUPOG-LEXLCYFYSA-O 0.000 description 1
- 0 CCC1=CC11*=CC(CC[*+])C1 Chemical compound CCC1=CC11*=CC(CC[*+])C1 0.000 description 1
- UHLVTZVBYHMCSF-UHFFFAOYSA-O COP(O)(OCCC(C1)(COC1[NH2+]C=CC=C(C=C)C(N)=O)O)=O Chemical compound COP(O)(OCCC(C1)(COC1[NH2+]C=CC=C(C=C)C(N)=O)O)=O UHLVTZVBYHMCSF-UHFFFAOYSA-O 0.000 description 1
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention discloses a preparation method and application of NAD+ (nicotinamide adenine dinucleotide) analogues. The NAD+ analogues of which the structural general formula is disclosed in the specification are generated by reacting nicotinamide mononucleotide with corresponding pyrimidine nucleotide analogues, wherein R is disclosed in the specification. The NAD+ analogues can be used as a coenzyme of dehydrogenase, and can also promote growth of microbes. The NAD+ analogues can be applied to research of biological catalysis, bioanalysis chemistry, metabolism engineering and synthetic biology.
Description
Technical field
The present invention relates to have small molecule compound synthetic of BA and use, specifically have the Reduced nicotinamide-adenine dinucleotide (NAD of various substituent pyrimidine ring substituted adenines rings
+) analogue, and the cofactor that this compound can be used as desaturase is used for the catalytic oxidation-reduction reaction, or as microorganism growth promotor, promotes microorganism growth.
Background technology
Reduced nicotinamide-adenine dinucleotide (nicotinamide adenine dinucleotide, NAD
+) and it go back ortho states (NADH) accordingly, be exactly usually said nicotinamide adenine dinucleotide, be the indispensable micromolecular compound of organism, participate in redox metabolism and other a series of important biomolecule chemical processes in the life entity, its structural formula is following:
NAD
+Chemical structure
NAD
+The chemical structure more complicated, character is stable inadequately, costs an arm and a leg.From NAD
+Chemical structure can find out that this molecule is to be formed by connecting through the tetra-sodium key nmn (NMN) fragment and amp (AMP) fragment.NMN partly is the main undertaker of its chemical functional; Participate in redox reaction and electron transport; The AMP part is the grappling coenzyme with the dependency structure territory effect of functional protein then; To the selectivity of enzyme specificity identification coenzyme and biochemical reaction have material impact (J.Benach et al.J.Biol.Chem.2003,278,19176-19182).
In cell, NAD
+Main plaing a part transmitted hydrogen and electronics, and many important oxydo-reductase all depend on NAD
+As its cofactor.Except the redox function, in non-oxide reduction vital process, NAD
+Play important effect equally.NAD
+In vital movements such as cell proliferation, growth, differentiation, apoptosis all be indispensable micromolecular compound (W.H.Ying.Antioxidants&Redox Signaling 2008,10,179-206).NAD
+Also can be used as the substrate of histon deacetylase (HDAC) (sirtuins); Under enzyme catalysis, the ethanoyl on the histone is removed, make histone can accomplish dna replication dna smoothly, transcribe and critical function (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 analogues.Recently a few thing relate to chemical improvement amp (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), vitamin PP 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 surrogate and the nmn of AMP part under DCC/DMAP, synthesized NAD
+Analogue, and be chiral alcohol (H.C.Lo et al.Angew.Chem.Int.Ed.2002,41 with above-mentioned analogue as the cofactor reductone of horse liver desaturase (HLADH); 478-481); But the cofactor structure is too simple, and with the undertighten that combines of enzyme, catalytic efficiency (is very low.Above-mentioned work all is to be based upon NAD
+Original structure carries out being difficult to overcome unstable on its chemical property on the chemically modified basis, shortcomings such as chemosynthesis, separation difficulty, gained NAD
+Analogue can not be discerned by the desaturase selectivity mostly.
Applicant's previous work [Zhao Zongbao, Liu Wujun, Wu Siguo, Hou Shuhua, a kind of NAD
+Analogue and synthetic and application thereof, application number: 200810010285.1] the synthetic NAD of institute
+Analogue can promote mikrobe such as intestinal bacteria and yeast saccharomyces cerevisiae growth; Its reduced form also can be used as the desaturase cofactor and is used for the catalytic oxidation-reduction reaction, but these analogue biological activitys are relatively low.
In a word, the NAD of existing bibliographical information
+Analogue mainly is the suppressor factor as biological catalyst, and only a few can be used for biocatalysis or bionic catalysis reaction, but can not be discerned by biological catalyst specifically.Therefore, need design and rational to have the NAD of other constitutional featuress
+Analogue makes them more effectively by enzyme identification, shows biocatalytic Activity.NAD with these biological characteristicses
+Analogue can play a significant role in fields such as bioanalysis chemistry, biocatalysis, metabolic engineering or synthetic biologies.
Summary of the invention
The present invention be in summing up document about NAD chemical improvement achievement and the active basis of associated biomolecule on, to NAD
+Structure is analyzed, and has kept vitamin PP monokaryon glycosides redox functional zone, the VITAMIN B4 loop section is transformed a pyrimidine ring structure with modification substitute the NAD that project organization is novel
+Analogue.
NAD involved in the present invention
+Analogue has following general structure:
Nmn is a beta configuration in the structural formula; Two ribose unit are D-form;
The R unit that links to each other with Nucleotide does
Below be referred to as A, wherein R
1Be halogen, F for example, Cl, Br; C
1-C
5Alkyl, methyl for example, ethyl; C
1-C
5Alkane hydroxyl, for example methylol.
Or R does
Below be referred to as B, wherein R
2Be C
1-C
5Alkyl, methyl for example, ethyl.
NAD among the present invention
+Three steps of synthetic branch of analogue carry out, and summarize as follows:
The first step: reference literature (S.M.Graham et al.Org.Lett.2004,6,233-236) synthetic nmn;
Second step: reference literature (M.Yoshikaw et al.Tetrahedron Lett.1967,5065-5068) synthetic nucleosides list phosphoric acid;
The 3rd step: the nucleosides list phosphoric acid that the nmn that the first step is obtained and second step obtain carries out idol and connects, and obtains target NAD
+Product.The 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-N (DMF) and DMSO 99.8MIN. (DMSO), condensation reagent is triphenylphosphine (Ph
3P), 1, the two piperidines (PyrS) of 1-two mercaptan
2Or phenylbenzene disulfide (PhS)
2Or 2,2 '-bipyridyl disulfide (PyS)
2With the 1-Methylimidazole;
Document (L.Q.Chen et al.J.Med.Chem.2007,50,5743-5751) use N, dinethylformamide (DMF) is a solvent, N, N-carbonyl dimidazoles (CDI) is a condensation reagent;
Document (J.Lee et al.Chem.Commun.1999,729) uses methane amide to be solvent, N, and N '-NSC 57182 (DCC), morpholine are condensation reagent, MnCl
2, MgSO
4Be catalyzer.
Used desaturase is e. coli k12 malic enzyme (UniProt codeP26616) among the present invention.The used sudden change desaturase of the present invention is to utilize
unit point sudden change test kit or document (J.X.Wang; Et al.J.Microbiol.Methods 2007; 71,225-230) on the e. coli k12 malic enzyme, introduce amino acid mutation and obtain mutant malic enzyme (L301R and L301R/Q392C).The used malic enzyme of the present invention all is by document (J.X.Wang, et al.Protein Expr.Purif.2007,53,97-103) method expression and purification.
Used desaturase also has serum lactic dehydrogenase from Lactobacillus helveticus (UniProt code P30901) among the present invention.The used sudden change serum lactic dehydrogenase of the present invention is to utilize
unit point sudden change test kit on serum lactic dehydrogenase, to introduce amino acid mutation to obtain mutant serum lactic dehydrogenase (V152R).The used serum lactic dehydrogenase of the present invention all is by document (J.X.Wang, et al.Protein Expr.Purif.2007,53,97-103) method expression and purification.
Used mikrobe is bacillus coli DH 5 alpha (Beijing ancient cooking vessel state biotech company) among the present invention.
Desaturase used among the present invention also has ethanol dehydrogenase, buys the Aldrich in Sigma from Saccharomyces cerevisiae (CAS NO.9031-72-5).
Table-1 provides the NAD of the embodiment of the invention
+Analogue.
Table-1.NAD
+Analogue numbering and corresponding chemical structure thereof
Embodiment
Following examples help to understand this patent, but are not limited to content of the present invention.
Used raw material is distinguished reference literature (S.M.Graham et al.Org.Lett.2004,6,233-236.) synthetic nmn ammonium salt among the embodiment; Reference literature (M.Yoshikaw et al.Tetrahedron Lett.1967,5065-5068) synthetic nucleosides list phosphoric acid; 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 the synthetic final product of document (J.Lee et al.Chem.Commun.1999,729).
Embodiment 1
With 0.3mmol NMN
Be dissolved in 2mL DMSO and the 2mL DMF mixed solvent, add 1.5mM Ph successively
3P, 1.5mM (PyS)
2, 26 ℃ of reactions of 6mM Methylimidazole 18min adds 2mL DMF dissolved 1mM nucleosides list phosphoric acid
26 ℃ of reaction 40min.Add 20mL acetone termination reaction, have a large amount of depositions to generate, centrifugal collecting precipitation precipitates three times with the 5mL washing with acetone.The peroxyformic acid type anionite-exchange resin separation and purification of water-soluble back gets title 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 a white solid, is prone to the moisture absorption and becomes sticky thickly, and darkens.
The malic enzyme determination of activity: with the wild-type malic enzyme, mutant malic enzyme (L301R) and mutant 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 the ultraviolet spectrophotometer, 25 ℃ of following continuous monitoring 340nm absorption values, can obtain desirable activity data in the 1min.Enzyme activity unit is defined as: PM catalysis produces 1 μ mol reduced-NAD under 25 ℃ of conditions
+Or the required enzyme amount of BC-10.
Experiment finds that the wild-type malic enzyme is to BC-10 and NAD
+Enzyme reaction rate be respectively 0.31Umg
-1And 22.5Umg
-1, mutant malic enzyme (L301R) is to BC-10 and NAD
+Enzyme reaction rate be respectively 11.2Umg
-1And 0.25Umg
-1, mutant malic enzyme (L301R/Q392C) is to BC-10 and NAD
+Enzyme reaction rate be respectively 13.6Umg
-1And 0.17Umg
-1
Explain 1) mutant malic enzyme (L301R) and mutant malic enzyme (L301R/Q392C) can not effectively utilize NAD
+As coenzyme, and BC-10 is fine as the coenzyme activity of sudden change malic enzyme (L301R) and mutant malic enzyme (L301R/Q392C).Therefore, analogue BC-10 has NAD for the activity of aforementioned mutant malic enzyme
+Irreplaceable effect; 2) the wild-type malic enzyme can not effectively utilize BC-10 as coenzyme.Therefore, analogue BC-10 discerns each other as coenzyme and desaturase and has certain specificity.
Embodiment 2
With embodiment 1 method; Be with embodiment 1 difference; Used Nucleotide is 26 ℃ for
temperature of reaction; Reaction times is 45min, and the productive rate of title 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 a white solid, is prone to the moisture absorption and becomes sticky thickly, and darkens.
The malic enzyme determination of activity: method is that with embodiment 1 difference the wild-type malic enzyme is 0.52Umg to the enzyme reaction rate of BC-20 with embodiment 1
-1, mutant malic enzyme (L301R) is respectively 13.7Umg to the enzyme reaction rate of BC-20
-1, mutant malic enzyme (L301R/Q392C) is 15.4Umg to the enzyme reaction rate of BC-20
-1Explain that malic enzyme (L301R) and malic enzyme (L301R/Q392C) that BC-20 can optionally be suddenlyd change utilize, and utilized by the wild-type malic enzyme hardly; Simultaneously, the wild-type malic enzyme does not utilize BC-20, and selective use NAD only
+
Embodiment 3
With embodiment 1 method; Be with embodiment 1 difference; Used Nucleotide is 28 ℃ for
temperature of reaction; Reaction times is 30min, and the productive rate of title 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 a faint yellow solid, is prone to the moisture absorption and becomes sticky thickly, and darkens.
The malic enzyme determination of activity: method is that with embodiment 1 difference the wild-type malic enzyme is 0.64Umg to the enzyme reaction rate of BC-30 with embodiment 1
-1, mutant malic enzyme (L301R/Q392C) is 19.5Umg to the enzyme reaction rate of BC-30
-1Explain that BC-30 can optionally be utilized by mutant malic enzyme (L301R/Q392C), and utilized by the wild-type malic enzyme hardly; Simultaneously, the wild-type malic enzyme does not utilize BC-30, and selective use NAD only
+
Embodiment 4
With embodiment 1 method; Be with embodiment 1 difference; Used Nucleotide is 26 ℃ for
temperature of reaction; Reaction times is 38min, and the productive rate of title 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 a white solid, is prone to the moisture absorption.
The malic enzyme determination of activity: method is that with embodiment 1 difference the wild-type malic enzyme is 0.15Umg to the enzyme reaction rate of BC-40 with embodiment 1
-1, mutant malic enzyme (L301R/Q392C) is 11.3Umg to the enzyme reaction rate of BC-40
-1Explain that BC-40 can optionally be utilized by mutant malic enzyme (L301R/Q392C), and utilized by the wild-type malic enzyme hardly; Simultaneously, the wild-type malic enzyme does not utilize BC-40, and selective use NAD only
+
Embodiment 5
With embodiment 1 method; Be with embodiment 1 difference; Used Nucleotide is 26 ℃ for
temperature of reaction; Reaction times is 40min, and the productive rate of title product BC-50 is 30%.
Or employing is dissolved in 0.3mmol NMN
in the 2mL DMSO solvent; Add 26 ℃ of reactions of 1.5mM CDI 20min, add 26 ℃ of reactions of 2mL DMF dissolved 1mM nucleosides list phosphoric acid
8d.Add 20mL acetone termination reaction, have a large amount of depositions to generate, centrifugal collecting precipitation precipitates three times with the 5mL washing with acetone.The peroxyformic acid type anionite-exchange resin separation and purification of water-soluble back gets title product BC-50, productive rate 24%.
Or adopt 0.2mmol nucleosides list phosphoric acid
The 1mM morpholine is dissolved in the 5mL water, is added dropwise to the 2mM DCC that is dissolved in the 5mL propyl carbinol, 50 ℃ of reaction 5h, and solvent evaporated with 5mL propyl carbinol washing precipitation three times, gets white solid, and oil pump is drained and is thoroughly removed moisture, adds 5mL methane amide dissolved 1mM NMN
0.2mM MnCl
2, 0.2mMMgSO
4, 30 ℃ of reaction 16h.Solvent evaporated, the peroxyformic acid type anionite-exchange resin separation and purification of water-soluble back gets title 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 a white solid, is prone to the moisture absorption.
The malic enzyme determination of activity: method is that with embodiment 1 difference the wild-type malic enzyme is 0.63Umg to the enzyme reaction rate of BC-50 with embodiment 1
-1, mutant malic enzyme (L301R/Q392C) is 2.5Umg to the enzyme reaction rate of BC-50
-1Explain that BC-50 can optionally be utilized by mutant malic enzyme (L301R/Q392C), and utilized by the wild-type malic enzyme hardly; Simultaneously, the wild-type malic enzyme does not utilize BC-50, and selective use NAD only
+
Promote the intestinal bacteria growth experiment: with LB substratum (peptone 10gL
-1, yeast powder 5gL
-1, sodium-chlor 10gL
-1, pH 7.2) add among the 10mL BC-50 to final concentration be 100 μ M, with 1: 100 inoculation bacillus coli DH 5 alpha (Beijing ancient cooking vessel state biotech company) seed liquor (OD
600=2),, cultivate under the 200rpm condition at 37 ℃; (add equivalent NAD with control group
+) compare, add the colibacillary OD of BC-50 behind the cultivation 4h
600Value is higher by 0.5 than control group.This BC-50 is described, and growth has obvious facilitation to bacillus coli DH 5 alpha.
Embodiment 6
Lactate dehydrogenase activity is measured: wild-type serum lactic dehydrogenase and mutant serum lactic dehydrogenase (V152R) are mixed with 0.1mgmL
-1Solution.Preparation 0.2mL reaction mixture (50mMHEPES pH 7.2,200mM D-Sodium.alpha.-hydroxypropionate, the NAD of 1mM
+Or NAD
+Analogue), add 1 μ L enzyme liquid during activation analysis fast, mixing is placed in the ultraviolet spectrophotometer, the variation of continuous monitoring 340nm absorption value in 25 ℃ of following 1min.Enzyme activity unit is defined as: PM catalysis produces 1 μ mol NADH or reduced-NAD under 25 ℃ of conditions
+The enzyme amount that analogue is required.
The result is as showing-2:
Table-2 lactate dehydrogenase activities are measured the result
The above results explanation, 1) analogue BC-10, BC-20, BC-30 and BC-40 are as the coenzyme of mutant serum lactic dehydrogenase (V152R).Mutant serum lactic 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 as the coenzyme of mutant serum lactic dehydrogenase (V152R), activity of enzyme reaction is lower with analogue BC-50.Therefore, BC-10, BC-20, BC-30 and BC-40 have NAD for the activity of mutant serum lactic dehydrogenase (V152R)
+Irreplaceable effect.2) the 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 are discerned each other as coenzyme and desaturase and are had certain specificity.
Embodiment 7
Ethanol dehydrogenase utilizes reduced-NAD
+The determination of activity of analogue: with NAD
+Analogue is made into 10mM solution, with equal-volume 10mM NaBH
4The aqueous solution is handled, and obtains the ortho states of going back of analogue, and is 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 the ultraviolet spectrophotometer, the variation of continuous monitoring 340nm absorption value in 25 ℃ of following 1min.Enzyme activity unit is defined as: PM catalysis produces 1 μ mol NAD under 25 ℃ of conditions
+Or NAD
+The enzyme amount that analogue is required.
The result is as showing-3:
Table-3 ethanol dehydrogenases utilize reduced-NAD
+The activity of analogue
The The above results explanation, NAD
+The pairing reduced form of analogue BC-10, BC-20, BC-30, BC-40 and BC-50 can be used 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 it is active more than 10% when utilizing NADH as coenzyme that activity reaches.
Can find out through above embodiment:
1) the present invention has set up the novel NAD of a class formation
+The chemical synthesis process of analogue, this method is simply effective, can be used for the NAD of synthetic other structural similitude
+The preparation of analogue;
2) the prepared NAD of the present invention
+The coenzyme that analogue can be used as desaturase is used for the catalytic oxidation-reduction reaction;
3) the prepared NAD of the present invention
+Analogue can be used as the specificity coenzyme of desaturase or mutant desaturase, and its effect that is used for catalyzed reaction is superior to NAD
+As the situation of coenzyme, can be applicable in biocatalysis and the bioanalysis chemistry;
4) based on mutant desaturase/NAD of the present invention
+The combination of analogue high reactivity is the artificial new system that is independent of the original redox-catalyst system of nature, possibly as distinctive tool applications in bioanalysis chemistry, metabolic engineering and synthetic biology research, have important value;
5) the prepared NAD of the present invention
+Analogue can be used as microbial growth regulator, changes the microorganism growth behavior, is applied in biochemical engineering research or the production.
Claims (5)
1. Reduced nicotinamide-adenine dinucleotide (NAD
+) analogue, it is characterized in that: it has following general structure:
Nmn is a beta configuration in the structural formula; Two ribose unit are D-form;
The R unit that links to each other with Nucleotide does
Below be referred to as A, wherein R
1Be halogen, C
1-C
5Alkyl or C
1-C
5One of in the alkane hydroxyl;
Or R does
Below be referred to as B, wherein R
2Be C
1-C
5One of in the alkyl.
2. according to the said NAD of claim 1
+Analogue is characterized in that: its structural formula is following,
3. according to the said NAD of claim 1
+Analogue is characterized in that: its structural formula is following,
4. said NAD of claim 1
+The application of analogue is characterized in that: the said NAD of claim 1
+Analogue is used for the catalytic oxidation-reduction reaction as the coenzyme of desaturase.
5. said NAD of claim 1
+The application of analogue is characterized in that: the said NAD of claim 1
+Analogue is used to promote microbial growth as mikrobe promotor.
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Cited By (5)
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| CN103463119A (en) * | 2013-08-19 | 2013-12-25 | 渤海大学 | Sir2 inhibitor |
| CN104946706A (en) * | 2014-03-26 | 2015-09-30 | 中国科学院大连化学物理研究所 | Reduction method of NAD (nicotinamide adenine dinucleotide) analogue |
| CN105168236A (en) * | 2015-10-16 | 2015-12-23 | 上海市胸科医院 | Application of nicotinamide adenine dinucleotide in preparation of drug for preventing and treating heart ischemic injuries |
| WO2019222368A1 (en) * | 2018-05-15 | 2019-11-21 | Jumpstart Fertility Pty Ltd | Amino acid salts of nicotinic acid mononucleotide and nicotinamide mononucleotide as anti-ageing agents |
| CN113395995A (en) * | 2018-08-01 | 2021-09-14 | 江普斯塔特生育有限公司 | Niacin and nicotinamide mononucleotide and nucleoside quaternary ammonium salts as anti-aging agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103463119A (en) * | 2013-08-19 | 2013-12-25 | 渤海大学 | Sir2 inhibitor |
| CN103463119B (en) * | 2013-08-19 | 2015-10-28 | 渤海大学 | Sir2 inhibitor |
| CN104946706A (en) * | 2014-03-26 | 2015-09-30 | 中国科学院大连化学物理研究所 | Reduction method of NAD (nicotinamide adenine dinucleotide) analogue |
| CN104946706B (en) * | 2014-03-26 | 2019-07-05 | 中国科学院大连化学物理研究所 | A kind of restoring method of NAD analog |
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| WO2019222368A1 (en) * | 2018-05-15 | 2019-11-21 | Jumpstart Fertility Pty Ltd | Amino acid salts of nicotinic acid mononucleotide and nicotinamide mononucleotide as anti-ageing agents |
| CN113396153A (en) * | 2018-05-15 | 2021-09-14 | 江普斯塔特生育有限公司 | Amino acid salts of nicotinic acid mononucleotide and nicotinamide mononucleotide as anti-aging agents |
| JP7432585B2 (en) | 2018-05-15 | 2024-02-16 | ジャンプスタート ファーティリティ ピーティーワイ リミテッド | Amino acid salts of nicotinic acid mononucleotide and nicotinamide mononucleotide as anti-aging agents |
| CN113395995A (en) * | 2018-08-01 | 2021-09-14 | 江普斯塔特生育有限公司 | Niacin and nicotinamide mononucleotide and nucleoside quaternary ammonium salts as anti-aging agents |
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