CN103233051A - Chemical and biological synthesis method for large-scale preparation of nicotinamide adenine dinucleotide - Google Patents
Chemical and biological synthesis method for large-scale preparation of nicotinamide adenine dinucleotide Download PDFInfo
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- CN103233051A CN103233051A CN2012105551266A CN201210555126A CN103233051A CN 103233051 A CN103233051 A CN 103233051A CN 2012105551266 A CN2012105551266 A CN 2012105551266A CN 201210555126 A CN201210555126 A CN 201210555126A CN 103233051 A CN103233051 A CN 103233051A
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Abstract
The invention relates to a chemical and biological synthesis method for large-scale preparation of nicotinamide adenine dinucleotide (NAD). According to the invention, ribofuranose tetraacetate and ethyl nicotinate are adopted as initial raw materials, and the following steps are carried out that: (1) a refluxing reaction is carried out; (2) ammonia ethanol is dropped under a low temperature, such that a reaction is carried out; (3) phosphorus trichloride is dropped under a low temperature, such that a reaction is carried out; and (4) extraction, separation, and purification are carried out, and the material is added into cell fluid and is biologically converted into nicotinamide adenine dinucleotide; and a pure product is obtained by purification and drying.
Description
Technical field
The present invention relates to the method for chemical-biological synthesis method scale preparation nicotinamide adenine dinucleotide.
Background technology
Nicotinamide adenine dinucleotide (being called for short NAD) is the coenzyme of desaturase, also claims nadide, and it is in glycolysis-, and glyconeogenesis is being brought into play irreplaceable effect in tricarboxylic acid cycle and the respiratory chain.Product can be passed NAD with the hydrogen of taking off, and makes it to become NADH.NADH then can by the mode of chemiosmotic coupling, synthesize ATP as the carrier of hydrogen in respiratory chain.
NAD is one of requisite coenzyme of modern biocatalytic reaction, and leucine dehydrogenase, formic acid ammonia dehydrogenase, Hexose phosphate dehydrogenase all need the help of NAD to finish entire reaction.
Because NAD is the coenzyme of the huge oxydo-reductase of quantity in human body and animal body, so it can also be as the direct template of medicinal design, perhaps the activity that changes NAD effect enzyme indirectly according to structure design enzyme inhibitors or the stimulant of NAD perhaps directly suppresses the biosynthesizing of NAD.
NAD simultaneously might be as following very important a kind of antidotal ancillary drug.The deacetylase that the popular Sir2 protein of modern biological study is a kind of NAD of dependence, its effect are the life cycles (anti-ageing) that prolongs cell.
At present NAD mostly is by extracting in the animals and plants, exist yield low, and the cost height extracts the big shortcoming of difficulty, has hindered the development of modern biocatalysis technology greatly.Still do not have chemical-biological method scale preparation NAD at present, if can have important social meaning and economic benefit with chemical-biological synthetic method large-scale production NAD.The chemical-biological synthetic method is compared with traditional extraction process, can greatly reduce cost, and improves yield, reduces the production difficulty, thereby promotes the development of modern biocatalysis technology effectively.
Summary of the invention
The technical problem that the present invention mainly solves is the synthetic nicotinamide adenine dinucleotide of mass-producing chemical-biological, makes that the chemical-biological of nicotinamide adenine dinucleotide is synthetic really to produce economic benefit, social benefit.
To achieve these goals, technical scheme of the present invention is as follows:
A kind of method of chemical-biological method scale preparation nicotinamide adenine dinucleotide, with 1,2,3,5-Tetra-O-Acetyl-D-Ribose, Nikithan is starting raw material, through (1) back flow reaction; (2) low temperature drips the cholamine reaction; (3) low temperature drips the phosphorus trichloride reaction; (4) add behind the extraction separation purification that bio-transformation is nicotinamide adenine dinucleotide in the enchylema, obtain pure product after the purified drying again.
Among the present invention, in the wherein said the first step back flow reaction, the direct disposable adding of catalyzer.
Among the present invention, in the wherein said second step cholamine dropwise reaction, add with the cholamine that is lower than-10 ℃ of temperature.
Among the present invention, the temperature of wherein said second step reaction, control is at-3 ℃～-5 ℃.
Among the present invention, after second step reaction wherein finishes, reclaiming cholamine below 0 ℃ earlier.
Among the present invention, earlier ice cube is added reaction solution during wherein extraction, avoid heat production too fast, destroy product.
Among the present invention, the product after the bio-transformation is wherein used the mobile chromatographic bed of simulation or high speed adverse current chromatogram or column chromatography purification after ceramic membrane micro-filtration or ultrafiltration.
Among the present invention, wherein said catalyzer is the trifluoromethanesulfonic acid methyl esters.
Among the present invention, the method for wherein said recovery cholamine is to reclaim cholamine in vacuum tightness greater than the high vacuum of-0.099MPa.
Among the present invention, wherein said after reclaiming cholamine, reclaim methyl alcohol, the mode of described recovery methyl alcohol is vacuum recovery, thin film evaporation or molecular distillation, and the mode that wherein said vacuum reclaims reclaims methyl alcohol for adopting Roots vaccum pump and reactor mechanical sealing technology vacuum tightness greater than the high vacuum of-0.099MPa.
Advantage of the present invention is: compare with traditional extraction process, can greatly reduce cost, improve yield, reduce the production difficulty, thereby promote the development of modern biocatalysis technology effectively.
Description of drawings
Fig. 1 is schema of the present invention.
Embodiment
Further understand and understanding for making constitutional features of the present invention and the effect reached had, cooperate detailed explanation in order to preferred embodiment and accompanying drawing, be described as follows:
Referring to Fig. 1,
Embodiment 1:
In a reactor, add 20 kilograms of 1,2,3,5-Tetra-O-Acetyl-D-Riboses, with 200 kilograms of tetrahydrofuran (THF) dissolvings, add 10 kilograms of Nikithans again, directly add 5 kilograms of trifluoromethanesulfonic acid methyl esters.Be warming up to and produce reflux, pick up counting after backflow is arranged, reacted 3 hours.After reaction finished, decompression and solvent recovery after recovery finishes, added 150 kilograms of dissolve with ethanol.
150 kilograms of ethanol are pumped in No. two reactors, open frozen cooling to-5 ℃.Open the liquid ammonia steel bottle pressure loading valve, begin logical ammonia, metering, feeds 10 kilograms of ammonia after, finish to lead to ammonia.Open frozen cooling to below-10 ℃, get the low temperature cholamine.The low temperature cholamine is splashed in reactor, after dropwising, pick up counting, accurately control temperature of reaction-3 ℃～-5 ℃, reacted 48 hours.
Open sieve time vacuum pump, control vacuum tightness is greater than-0.099MPa, at 0 ℃ of following reclaim under reduced pressure cholamine, reclaim finish after, control vacuum tightness is greater than-0.099MPa, at decompression recycling ethanol below 20 ℃.After recovery finishes, in still, add 150 liters of diethyl phosphites, begin to drip 20 kilograms of phosphorus trichlorides, pick up counting after dropwising, kept low-temp reaction 20 hours.Reaction adds 25 kilograms of ice cubes after finishing in reactor, add 150 liters of diethyl phosphites and 200 kilograms of frozen water again, extracts water layer, column chromatography purification.HPLC detects, be that the nicotinamide gland mononucleotide aqueous solution more than 90% joined in the full cell fermentation liquid reaction 24 hours with relative content, the ceramic membrane cross-flow microfiltration is removed solid, going up high speed adverse current chromatogram again separates, nanofiltration is concentrated to 200 liters, and lyophilize obtains 15.1 kilograms of the pure product of nicotinamide adenine dinucleotide.HPLC detects, and content is 97.3%.
Embodiment 2:
In No. three reactors, add the 1,2,3,5-Tetra-O-Acetyl-D-Ribose double centner, with 1000 kilograms of dioxane dissolvings, add 50 kilograms of Nikithans again, directly add 25 kilograms of trifluoromethanesulfonic acid methyl esters.Be warming up to and produce reflux, pick up counting after backflow is arranged, reacted 3 hours.After reaction finished, decompression and solvent recovery after recovery finishes, added 750 kilograms of dissolve with ethanol.
750 kilograms of ethanol are pumped in No. four reactors, open frozen cooling to-5 ℃.Open the liquid ammonia steel bottle pressure loading valve, begin logical ammonia, metering, feeds 50 kilograms of ammonia after, finish to lead to ammonia.Open frozen cooling to below-10 ℃, get the low temperature cholamine.The low temperature cholamine is splashed in No. three reactors, after dropwising, pick up counting, accurately control temperature of reaction-3 ℃～-5 ℃, reacted 48 hours.
Open sieve time vacuum pump, control vacuum tightness is greater than-0.099MPa, at 0 ℃ of following reclaim under reduced pressure cholamine, reclaim finish after, reclaiming ethanol with molecular distillation equipment below 20 ℃.After recovery finishes, in still, add 750 liters of dimethylbenzene, begin to drip the phosphorus trichloride double centner, pick up counting after dropwising, kept low-temp reaction 20 hours.Reaction adds 75 kilograms of ice cubes after finishing in reactor, add 750 liters of dimethylbenzene and 1000 kilograms of frozen water again, extracts water layer, column chromatography purification.HPLC detects, be that the nicotinamide gland mononucleotide aqueous solution more than 90% joined in the full cell fermentation liquid fermentation 24 hours with relative content, the ceramic membrane cross-flow microfiltration is removed solid, separate with the mobile chromatographic bed of simulation again, nanofiltration is concentrated to 1000 liters, and lyophilize obtains 77.6 kilograms of the pure product of nicotinamide adenine dinucleotide.HPLC detects, and content is 98.2%.
Embodiment 3:
In No. five reactors, add 200 kilograms of 1,2,3,5-Tetra-O-Acetyl-D-Riboses, with 2000 kilograms of tetrahydrofuran (THF) dissolvings, add the Nikithan double centner again, directly add 50 kilograms of trifluoromethanesulfonic acid methyl esters.Be warming up to and produce reflux, pick up counting after backflow is arranged, reacted 3 hours.After reaction finished, decompression and solvent recovery after recovery finishes, added 1500 kilograms of dissolve with ethanol.
1500 kilograms of ethanol are pumped in No. six reactors, open frozen cooling to-5 ℃.Open the liquid ammonia steel bottle pressure loading valve, begin logical ammonia, metering behind the feeding double centner ammonia, finishes logical ammonia.Open frozen cooling to below-10 ℃, get the low temperature cholamine.The low temperature cholamine is splashed in No. five reactors, after dropwising, pick up counting, accurately control temperature of reaction-3 ℃～-5 ℃, reacted 48 hours.
Open sieve time vacuum pump, control vacuum tightness is greater than-0.099MPa, at 0 ℃ of following reclaim under reduced pressure cholamine, reclaim finish after, with thin-film evaporator at decompression recycling ethanol below 20 ℃.After recovery finishes, in still, add 1500 liters of ethyl acetate, begin to drip 200 kilograms of phosphorus trichlorides, pick up counting after dropwising, kept low-temp reaction 20 hours.Reaction adds 250 kilograms of ice cubes after finishing in reactor, add 1500 liters of ethyl acetate and 2000 kilograms of frozen water again, extracts water layer, column chromatography purification.HPLC detects, be that the nicotinamide gland mononucleotide aqueous solution more than 90% joined in the full cell fermentation liquid reaction 24 hours with relative content, solid is removed in ultrafiltration, go up the resin column chromatography for separation again and obtain nicotinamide adenine dinucleotide, nanofiltration is concentrated to 2000 liters, and lyophilize obtains 155.3 kilograms of the pure product of nicotinamide adenine dinucleotide.HPLC detects, and content is 98.1%.
Advantage of the present invention is: compare with traditional extraction process, can greatly reduce cost, improve yield, reduce the production difficulty, thereby promote the development of modern biocatalysis technology effectively, create 500,000,000 yuans of incomes (to produce NAD5000 kilogram, every kilogram of 100,000 yuan of meters per year) year, 100,000,000 yuans of contribution taxes.The present invention replaces traditional extraction method with the chemical-biological synthesis method, has greatly improved the throughput of important biological catalytic enzyme NAD, and the propelling of world's Biochemical Research manufacturing is had very important meaning.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and the specification sheets is principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the present invention.The protection domain that the present invention requires is defined by appending claims and equivalent thereof.
Claims (10)
1. the method for a chemical-biological method scale preparation nicotinamide adenine dinucleotide, with 1,2,3,5-Tetra-O-Acetyl-D-Ribose, Nikithan is starting raw material, through (1) back flow reaction; (2) low temperature drips the cholamine reaction; (3) low temperature drips the phosphorus trichloride reaction; (4) add behind the extraction separation purification that bio-transformation is nicotinamide adenine dinucleotide in the enchylema, obtain pure product after the purified drying again.
2. the method for nicotinamide adenine dinucleotide is synthesized in mass-producing according to claim 1, in the wherein said the first step back flow reaction, and the direct disposable adding of catalyzer.
3. the method for the synthetic nicotinamide adenine dinucleotide of mass-producing according to claim 1 in the wherein said second step cholamine dropwise reaction, adds with the cholamine that is lower than-10 ℃ of temperature.
4. the method for nicotinamide mononucleotide is synthesized in mass-producing according to claim 1, the temperature of wherein said second step reaction, and control is at-3 ℃～-5 ℃.
5. the method for the synthetic nicotinamide adenine dinucleotide of mass-producing according to claim 1 after second step reaction wherein finishes, is reclaiming cholamine below 0 ℃ earlier.
6. earlier ice cube is added reaction solution when the method for the synthetic nicotinamide adenine dinucleotide of mass-producing according to claim 1, wherein extraction, avoid heat production too fast, destroy product.
7. the method for nicotinamide adenine dinucleotide is synthesized in mass-producing according to claim 1, and the product after the bio-transformation is wherein used the mobile chromatographic bed of simulation or high speed adverse current chromatogram or column chromatography purification after ceramic membrane micro-filtration or ultrafiltration.
8. the method for nicotinamide adenine dinucleotide is synthesized in mass-producing according to claim 2, and it is characterized in that: wherein said catalyzer is the trifluoromethanesulfonic acid methyl esters.
9. the method for the synthetic nicotinamide adenine dinucleotide of mass-producing according to claim 5 is characterized in that: the method for wherein said recovery cholamine is for reclaiming cholamine in vacuum tightness greater than the high vacuum of-0.099MPa.
10. the method for the synthetic nicotinamide adenine dinucleotide of mass-producing according to claim 1 or 5; it is characterized in that: wherein said after reclaiming cholamine; reclaim methyl alcohol; the mode of described recovery methyl alcohol is vacuum recovery, thin film evaporation or molecular distillation, and the mode that wherein said vacuum reclaims reclaims methyl alcohol for adopting Roots vaccum pump and reactor mechanical sealing technology vacuum tightness greater than the high vacuum of-0.099MPa.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104876994A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide |
| CN104876993A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide phosphate |
| CN104892710A (en) * | 2015-05-19 | 2015-09-09 | 邦泰生物工程(深圳)有限公司 | A method of purifying reduced form beta-nicotinamide adenine dinucleotide |
| WO2018107377A1 (en) * | 2016-12-14 | 2018-06-21 | 邦泰生物工程(深圳)有限公司 | Purification process of nadph |
| CN109053838A (en) * | 2018-07-26 | 2018-12-21 | 四川大学 | Prepare β-nicotinamide mononucleotide or β-niacinamide ribose method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101360421A (en) * | 2005-11-18 | 2009-02-04 | 康乃尔研究基金会有限公司 | Nicotinoyl riboside compositions and methods of use |
| CN102471793A (en) * | 2009-07-27 | 2012-05-23 | 霍夫曼-拉罗奇有限公司 | Enzymatic synthesis of CARBA-NAD |
| CN102477057A (en) * | 2010-11-26 | 2012-05-30 | 北京利德曼生化股份有限公司研发中心 | Thio-nicotinamide adenine dinucleotide, intermediates thereof and preparation methods of thio-nicotinamide adenine dinucleotide and intermediates |
-
2012
- 2012-12-19 CN CN201210555126.6A patent/CN103233051B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101360421A (en) * | 2005-11-18 | 2009-02-04 | 康乃尔研究基金会有限公司 | Nicotinoyl riboside compositions and methods of use |
| CN102471793A (en) * | 2009-07-27 | 2012-05-23 | 霍夫曼-拉罗奇有限公司 | Enzymatic synthesis of CARBA-NAD |
| CN102477057A (en) * | 2010-11-26 | 2012-05-30 | 北京利德曼生化股份有限公司研发中心 | Thio-nicotinamide adenine dinucleotide, intermediates thereof and preparation methods of thio-nicotinamide adenine dinucleotide and intermediates |
Non-Patent Citations (1)
| Title |
|---|
| YANG T.等: "Synthese of Nicotinamide Riboside and Derivatives: Effective Agents for Increasing Nictinamide Adenine Dinucleotide Concentration in Mammalian Cells", 《JOURNAL OF MEDICINAL CHEMISTRY》, vol. 50, no. 26, 6 December 2007 (2007-12-06), pages 6458 - 6461 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104876994A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide |
| CN104876993A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide phosphate |
| CN104892710A (en) * | 2015-05-19 | 2015-09-09 | 邦泰生物工程(深圳)有限公司 | A method of purifying reduced form beta-nicotinamide adenine dinucleotide |
| CN104892710B (en) * | 2015-05-19 | 2017-09-29 | 邦泰生物工程(深圳)有限公司 | A kind of method for purifying reduced form β NADHs |
| WO2018107377A1 (en) * | 2016-12-14 | 2018-06-21 | 邦泰生物工程(深圳)有限公司 | Purification process of nadph |
| CN108431015A (en) * | 2016-12-14 | 2018-08-21 | 邦泰生物工程(深圳)有限公司 | A kind of purifying process of NADPH |
| JP2019503334A (en) * | 2016-12-14 | 2019-02-07 | 邦泰生物工程（深▲セン▼）有限公司 | NADPH purification process |
| US10765967B2 (en) | 2016-12-14 | 2020-09-08 | Bontac Bio-Engineering (Shenzhen) Co., Ltd | Process for purifying NADPH |
| CN108431015B (en) * | 2016-12-14 | 2021-03-23 | 邦泰生物工程(深圳)有限公司 | NADPH purification process |
| CN109053838A (en) * | 2018-07-26 | 2018-12-21 | 四川大学 | Prepare β-nicotinamide mononucleotide or β-niacinamide ribose method |
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