阅读说明：本技术 一种糖核苷酸及其衍生物的制备方法 (Preparation method of sugar nucleotide and derivatives thereof ) 是由 房俊强 李爽 王鹏 于 2020-07-10 设计创作，主要内容包括：本发明公开了一种糖核苷酸及其衍生物的制备方法。本发明以单糖及其衍生物为原料,利用生物酶催化合成糖核苷酸及其衍生物,未反应完的原料和杂质经钡离子选择性沉淀,离心除去沉淀物,最后得到高纯度的糖核苷酸及其衍生物。本发明公开的生物酶来自原核生物,具有蛋白表达量高、底物适应性宽、催化效率高等优点,并且生物酶催化合成具有高效性、高区域选择性等优势。本发明还优化了生物酶的反应浓度,将价廉、易得的单糖及其衍生物高效地合成昂贵的糖核苷酸及其衍生物。本发明还使用离子沉淀法对产物进行纯化,该纯化步骤操作简单易于放大,并且能够降低成本,提高产物收率。(The invention discloses a preparation method of sugar nucleotide and derivatives thereof. The invention takes monosaccharide and derivatives thereof as raw materials, utilizes biological enzyme to catalyze and synthesize sugar nucleotide and derivatives thereof, the unreacted raw materials and impurities are selectively precipitated by barium ions, and precipitates are removed by centrifugation, thus obtaining the sugar nucleotide and derivatives thereof with high purity. The biological enzyme disclosed by the invention is derived from prokaryotes, has the advantages of high protein expression amount, wide substrate adaptability, high catalytic efficiency and the like, and has the advantages of high efficiency, high regioselectivity and the like in the catalytic synthesis of the biological enzyme. The invention also optimizes the reaction concentration of the biological enzyme, and efficiently synthesizes cheap and easily obtained monosaccharide and derivatives thereof into expensive sugar nucleotide and derivatives thereof. The invention also uses an ion precipitation method to purify the product, the purification step is simple to operate and easy to amplify, the cost can be reduced, and the product yield can be improved.)

1. A method for preparing sugar nucleotide and derivatives thereof is characterized in that the method comprises the following steps:

step (1) synthesizing sugar nucleotide and a derivative crude product thereof by using the biological enzyme to catalyze monosaccharide and the derivative thereof;

purifying by using an ion precipitation method to obtain sugar nucleotide and derivatives thereof;

the sugar nucleotide and the derivative thereof have the structure of a general formula I:

the glycosyl is selected from monosaccharide and a residual group of a derivative thereof after one molecular of hydroxyl is removed;

the nitrogenous base is selected from thymine, cytosine, adenine, guanine or uracil;

n is selected from 1 and 2;

preferably, the ionic precipitation method is selected from barium ion selective precipitation methods.

2. The method for preparing sugar nucleotides and derivatives thereof according to claim 1, wherein the step (2) comprises: and (2) adding the mixed solution containing the crude product of the sugar nucleotide and the derivatives thereof obtained in the step (1) into an organic solvent, performing protein denaturation and precipitation, removing protein, then adding a salt solution, centrifuging, collecting supernatant, adsorbing and removing impurities by using an ion exchange resin medium, and collecting supernatant to obtain the sugar nucleotide and the derivatives thereof.

3. The method for preparing sugar nucleotides and derivatives thereof according to claim 2, wherein the salt solution is a solution containing metal ions; preferably, the solution of metal ions is a solution containing barium ions; more preferably, the barium ion solution is a barium chloride solution; the barium ions can form complex precipitates with nucleoside triphosphate and nucleoside diphosphate.

4. The method for preparing sugar nucleotides and derivatives thereof according to claim 2, wherein the organic solvent in step (2) is selected from the group consisting of: methanol, ethanol, acetone; preferably, the organic solvent is ethanol; more preferably, the organic solvent is glacial ethanol; adding glacial ethanol into the mixed solution, standing at 4 deg.C for 10-60min, precipitating protein, and removing protein.

5. The method for preparing sugar nucleotides and derivatives thereof according to claim 2, wherein the mixture is centrifuged at 4-10 ℃ in step (2) and the supernatant is collected.

6. The method for preparing sugar nucleotides and derivatives thereof according to claim 2, wherein the ion exchange resin medium in step (2) is a strong acid type cation exchange resin medium.

7. The method of claim 1, wherein the sugar nucleotides and derivatives thereof,

the glycosyl is selected from

The nitrogenous base is selected from cytosine, adenine, guanine and uracil.

8. The process for preparing sugar nucleotides and derivatives thereof according to any one of claims 1 and 7, wherein the process comprises:

the sugar nucleotide and the derivative thereof are selected from:

r in formula IV₁Selected from methyl, azido-substituted methyl, trifluoro-substituted methyl;

r in formula V₂Is selected from methyl, azido-substituted methyl and trifluoro-substituted methyl.

9. The method for preparing sugar nucleotides and derivatives thereof according to any one of claims 1, 7 and 8, wherein the step (1) comprises: preparing solution of monosaccharide or its derivative and nucleoside triphosphate, regulating pH, adding biological enzyme, reacting for 12-24 hr, and terminating reaction to obtain mixed solution containing sugar nucleotide and its derivative.

10. The method for preparing sugar nucleotide and its derivatives as claimed in any one of claims 1, 7 and 8, wherein the biological enzyme in step (1) is one or more of kinase, sugar nucleotide synthetase and pyrophosphorylase; preferably, the kinase is selected from one or more of glucuronic acid kinase (AtGlcAK), galactokinase (BiGalK), N-acetamido hexokinase (NaHK), L-fucokinase/guanosine diphosphate-fucose pyrophosphorylase (FKP); the sugar nucleotide synthetase is selected from one or more of Arabidopsis UDP-sugar pyrophosphorylase (AtUSP), Pasteurella multocida N-acetylglucosamine 1-phosphate uridine transferase (PmGlU), N-acetylglucosamine 1-phosphate uridine transferase (AGX1), guanosine diphosphate-mannose pyrophosphorylase (PfManC), L-fucokinase/guanosine-fucose pyrophosphorylase (FKP) and cytidine monophosphate-sialic acid synthetase (NmCSS); the pyrophosphatase is selected from inorganic pyrophosphatase; more preferably, the concentration of the biological enzyme is 0.01-2 mg/mL.

11. The method of claim 1, 7 or 8, wherein the concentration of monosaccharide or monosaccharide derivative is 50-500 mM.

12. The method for preparing sugar nucleotides and derivatives thereof according to any one of claims 1, 7 and 8, wherein the concentration of the nucleoside triphosphates and derivatives thereof is 100 mM and 700 mM.

13. The method for preparing sugar nucleotides and derivatives thereof according to any one of claims 1, 7 and 8, wherein the pH of the reaction solution in step (1) is 7.0-9.0.

14. The method for preparing sugar nucleotides and derivatives thereof according to any one of claims 1, 7 and 8, wherein the reaction temperature in step (1) is 10-50 ℃ and the reaction time is 12-24 h.

Technical Field

The invention relates to the technical field of biosynthesis and purification, in particular to a preparation method of sugar nucleotide and derivatives thereof.

Background

Sugars are one of the three biomacromolecules essential for cell life activities. Sugars play an extremely important role in the course of life activities, being energy sources for life activities, and also being structural components of organisms. The saccharides can exist in the forms of free sugar chains and glycoconjugates with non-sugar substances such as proteins, lipids and the like, and play a key role in various processes of normal life activities such as intercellular interaction, inflammatory reaction, signal transduction, fertilization, development and the like; in addition, sugars and glycoconjugates are involved in the development of major diseases. The research of the functions and molecular mechanisms of sugar chains in the processes of disease occurrence, development and transfer from a new perspective of sugar science is a hot spot of scientific research. The efficient acquisition of carbohydrate compounds with definite chemical structure is one of the bottlenecks that restrict the scientific development of the whole sugar. The synthesis of the oligosaccharide catalyzed by the glycosyl transferase has the advantages of high efficiency, good selectivity, no need of protecting sugar substrates and the like, is one of important means for synthesizing carbohydrate compounds, but sugar nucleotide donors required by the synthesis are difficult to prepare and expensive, and the application of the sugar nucleotide donors in the synthesis of the oligosaccharide is limited to a certain extent.

Sugar nucleotides (sugar nucleotides) are structurally compounds in which the reducing end of a monosaccharide is linked to the terminal phosphate group of a nucleoside diphosphate or nucleoside monophosphate, and are activated forms of monosaccharides. The sugar nucleotide is a donor substrate required by the synthesis of the oligosaccharide catalyzed by Leloir type glycosyltransferase, is a necessary donor substrate in the in vitro complex oligosaccharide enzymatic synthesis process, and has very important functions in the aspects of researching substrate recognition and other biochemical properties of glycosyltransferase; the analogues and derivatives of the sugar nucleotides also have wide application prospects in the aspects of specific ligand targeted delivery and new drug development.

It has been disclosed in the literature and patents that sugar nucleotides can be prepared by natural extraction, chemical synthesis, biological synthesis, or a combination thereof. Sugar nucleotide exists in the intracellular biological metabolism process, but the concentration level of the sugar nucleotide in the cell is kept low, and a large amount of high-purity sugar nucleotide cannot be obtained by a natural product extraction method for sugar science research. The sugar nucleotides are prepared by chemical synthesis, biological synthesis, or a combination thereof.

Oberth ü r et al report a series of monophosphosphations of 2-deoxysugars in A preparative method for the steric generating of p-2-deoxyglycosylsugars, ([ J ]. Org Lett,2004,6(17):2873-2876) by first obtaining alpha-chlorinated sugars with oxalyl chloride in dichloromethane and then replacing the chlorine atom with phosphoric acid to obtain the more selective products. The method reported by Sawa et al in Glycoproteomic probes for fluorinated synthesis in vivo ([ J ]. Proc Natl Acad Sci,2006,103(33):12371-12376) is to brominate sugars with a hydrobromic acid, titanium tetrachloride system and then to substitute them with tetra-n-butyl ammonium dihydrogen phosphate (TBAP) in toluene to give monophosphorylated products in very high proportions. Wu et al, A retro-evolution of CDP-6-deoxy-D-glycero-L-threo-4-hexose-3-dehydrogenase (E1) from Yersinia pseudofolliculosis: antibiotics for C-3 deoxygenesis in the Biochemistry of 3,6-dideoxyhexoses. ([ J ]. Biochemistry,2007,46(12): 3759) 3767) use trichloroacetonitrile and potassium carbonate in dichloromethane to activate the sugar to form trichloroacetimide, followed by substitution with dibenzyl phosphate to give the configured product, and finally catalytic hydrodebenzylation with palladium on carbon to give sugar 1-phosphate. Muller et al in investigational settings the synthesis of dTDP-2, 6-dioxy-D-erythro-3-hexulose-A porous intermediates in the biosyntheses of rare catalysts, ([ J ]. Tetrahedron Lett.,1997,38(31): 5473. Ampere. 5476) hydrolyses diethyl phosphate with diethyl chlorophosphonate under the alkaline action of Diisopropylethylamine (DIPEA) to give the product. Diphenyl chlorophosphonate was similarly used by Huestis et al in the Lipophilic sugar nitrile synthesis by structure-based design of a nucleotidyltransferase substrates, ([ J ]. Org Biomol Chem,2008,6(3):477-484) to give phosphorylated products. In synthetic of a deoxy heterocyclic derivatives, an exo-difluoro ethylene mole as an amino chemical probe for a growing enzyme in an unused in an unreacted organic sulfide Synthesis, ([ J ]. J. Org Chem,2001,66(20): 6810) butyl lithium is used as a base and dibenzyl chlorophosphonate is used as a reagent to obtain a configuration product after the reaction, and then the benzyl group on the phosphoric acid is removed by hydrogenation with palladium carbon. Chen et al in Expression, purification, and characterization of two N, N-dimethyltransferase catalysts, Ty1M1 and DesVI, included in the biosynthesis of mycamine and desosamine, ([ J ]. Biochemistry,2002,41(29): 9165:. RTM. 9183) and Lazarevi et al in organic N-functionalized UDP-glucosamine catalysts as modified substrates for N-acetylglucosamine catalysts, 2006,341(5): 569:. about. 576) all use phosphoramidites to react directly with sugar under the activation of tetrazole to give sugar benzyl 1-phosphite, followed by oxidation to the product, M-chlorobenzyl phosphate (mC). Moffatt et al, The synthesis and The solvent reactions of nucleotide-5 'phosphorohalides and related compounds, improved methods for The preparation of nucleotide-5' polyphosphates, ([ J ]. J.Am Chem Soc,1961,83(3):649-658) have reported The condensation of Nucleoside Monophosphates (NMP) with morpholine to NMP phosphoromorphine under Dicyclohexylcarbodiimide (DCC). Wittmann et al, 1H-tetrazole as a catalyst in phosphoside interaction reactions, effective synthesis of GDP-fuse, GDP-manose, and UDP-gapase, ([ J ]. J Org Chem 1997,62(7):2144-2147) reported that sugar 1-phosphate and NMP were coupled in anhydrous pyridine by addition of tetrazole and then the sugar nucleoside diphosphate was deprotected to give a sugar phosphorylated product.

Disadvantages of chemically synthesized sugar nucleotides: sugar nucleotide has certain polarity and is not easy to dissolve in organic solvent, so that the yield of chemically synthesized sugar nucleotide is low; in addition, the chemical reaction conditions are severe, and sugar nucleotides are easily decomposed under the conditions of acid and alkali or over-high temperature.

Jiang et al in A general enzymatic method for the synthesis of natural and 'unorganic' UDP-and TDP-nuclear peptides, ([ J ]. J Am Chem Soc,2000,122(28):6803 and 6804) from Salmonella enterica bacteria in the extraction of thymidine transferase, and then in Escherichia coli over-expression, through in vitro enzymatic reaction of a variety of sugar 1-phosphate into sugar thymidine diphosphate. Products of GDP-mannose derivatives are obtained by Marchesan et al in Chemoenzymatic synthesis GDP-azidodeoxynitriles Non-radioactive probes for biochemical fermentation activity ([ J ]. Chem Commun,2008,36,4321-4323) using guanylyl transferase. Zhang et al, in the demonstration of the conversion of natural produced glycosylated enzymes-catalyzed reactions ([ J ] Science,2006,313(5791):1291-1294), found that glycosyltransferases can also catalyze the occurrence of the reverse reaction, i.e., hydrolysis of the glycosyl group from a product containing the glycosyl group and the resulting glycosylated reactant, the sugar nucleotide.

Biosynthesis method, in vitro simulation of in vivo synthesis pathway of sugar nucleotide and its derivatives, has become the mainstream pathway of sugar nucleotide synthesis at present; because the initial substrate and the product in the reaction system have similar physicochemical properties and approximate molecular weight distribution, the purification of the sugar nucleotide and the derivatives thereof depends on purification means such as molecular sieve chromatography, preparative HPLC and the like at present, the process is complex, the yield is lower, the cost is higher, milligram-level products can be obtained in batches, and the research work in the aspects of the enzymatic synthesis of complex oligosaccharides, the catalytic reaction mechanism of glycosyltransferase and the like is seriously hindered.

Aiming at the defects of the prior art and aiming at solving the bottleneck problem of restricting the synthesis of sugar nucleotide and derivatives thereof, the invention provides a biological enzyme catalytic synthesis method and a universal ion precipitation method independent of column chromatography, aiming at obtaining a large amount of highly purified sugar nucleotide and derivatives thereof on gram level and promoting the enzymatic synthesis of complex oligosaccharides and the research and development of sugar drugs.

Disclosure of Invention

The invention aims to solve the bottleneck problem of restricting the synthesis of sugar nucleotide and derivatives thereof, provide a method for obtaining high-purity sugar nucleotide and derivatives thereof on gram level in large quantity, and promote the synthesis of complex oligosaccharide and the research and development of sugar drugs.

In order to achieve the above objects, the present invention provides a method for preparing sugar nucleotides and derivatives thereof, the method comprising:

step (1) synthesizing sugar nucleotide and a derivative crude product thereof by using the biological enzyme to catalyze monosaccharide and the derivative thereof;

and (2) purifying by using an ion precipitation method to obtain sugar nucleotide and derivatives thereof:

the sugar nucleotide and the derivative thereof have the structure of a general formula I:

the glycosyl is selected from monosaccharide and its derivative, and is residual group after removing one molecule of hydroxyl,

the nitrogenous base is selected from thymine, cytosine, adenine, guanine or uracil,

n is selected from 1 and 2;

preferably, the ionic precipitation method is selected from barium ion precipitation methods.

Further, the step (2) includes: and (2) carrying out protein denaturation removal treatment on the mixed liquid containing the crude product of the sugar nucleotide or the derivative thereof obtained in the step (1), then adding a salt solution for selective precipitation, collecting supernatant, adsorbing impurities by using an ion exchange resin medium, and freeze-drying the treated supernatant to obtain the sugar nucleotide or the derivative thereof.

Further, the salt solution is a solution containing metal ions; preferably, the solution of metal ions is a solution containing barium ions; more preferably, the barium ion solution is a barium chloride solution; more preferably, the barium ion solution is a barium chloride solution; the barium ions can form complex precipitates with nucleoside triphosphate and nucleoside diphosphate.

Still further, the organic solvent in the step (2) is selected from: methanol, ethanol, acetone; preferably, the organic solvent is ethanol; more preferably, the organic solvent is glacial ethanol; adding glacial ethanol into the mixed solution, standing at 4 deg.C for 10-60min, precipitating protein, and removing protein.

Further, the mixed solution in the step (2) is centrifuged at 4-10 ℃, and the supernatant is collected.

Further, the ion exchange resin medium in the step (2) is a strong acid type cation exchange resin medium.

Further, the glycosyl is selected from

The nitrogenous base is selected from cytosine, adenine, guanine and uracil.

Further, the sugar nucleotides and derivatives thereof are selected from:

r in formula IV₁Selected from methyl, azido-substituted methyl, trifluoro-substituted methyl;

r in formula V₂Is selected from methyl, azido-substituted methyl and trifluoro-substituted methyl.

Still further, the step (1) includes: preparing solution of monosaccharide or its derivative and nucleoside triphosphate, regulating pH, adding biological enzyme, reacting for 12-24 hr, and terminating reaction to obtain mixed solution containing sugar nucleotide or its derivative.

Further, the biological enzyme in the step (1) is a composition consisting of one or more than two of kinase, sugar nucleotide synthetase and pyrophosphorylase; preferably, the kinase is selected from one or more of glucuronic acid kinase (AtGlcAK), galactokinase (BiGalK), N-acetamido hexokinase (NaHK), L-fucokinase/guanosine diphosphate-fucose pyrophosphorylase (FKP); the sugar nucleotide synthetase is selected from one or more of Arabidopsis UDP-sugar pyrophosphorylase (AtUSP), Pasteurella multocida N-acetylglucosamine 1-phosphate uridine transferase (PmGlU), N-acetylglucosamine 1-phosphate uridine transferase (AGX1), guanosine diphosphate-mannose pyrophosphorylase (PfManC), L-fucokinase/guanosine diphosphate-fucose pyrophosphorylase (FKP) and cytidine monophosphate-sialic acid synthetase (NmCSS); the pyrophosphatase is selected from Pasteurella multocida inorganic pyrophosphatase (PmPPA); more preferably, the concentration of the biological enzyme is 0.01-2 mg/mL.

Furthermore, the concentration of the monosaccharide and the monosaccharide derivative thereof is 50-500 mM.

Further, the concentration of the nucleoside triphosphate and its derivatives is 100-700 mM.

Further, the pH of the reaction solution in the step (1) is 7.0-9.0.

Furthermore, the reaction temperature in the step (1) is 10-50 ℃, and the reaction time is 12-24 h.

Further, the reaction is terminated with an organic solvent in the step (1), and preferably, the organic solvent is an alcohol solvent.

Still further, the method comprises: mixing glucuronic acid (GlcA), Adenosine Triphosphate (ATP), Uridine Triphosphate (UTP), and MgCl₂Preparing aqueous solution of GlcA and MgCl in reaction system₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, the pH of the reaction system is adjusted to 7.0-9.0, glucuronic acid kinase (AtGlcAK), Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) and inorganic pyrophosphatase (PmPPA) are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 during the reaction, and after the detection reaction is finished, glacial ethanol is added to stop the reaction; centrifuging the reaction liquid for 10-20min, gradually adding barium chloride solution into the reaction liquid by using a pipettor, centrifuging the reaction system for 10-60min at the temperature of 4-10 ℃, collecting supernatant, gradually adding barium chloride solution into the reaction liquid by using the pipettor again, repeating the steps until no precipitate is generated, collecting supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the uridine diphosphate glucuronate (formula II).

Preferably, the method comprises: mixing glucuronic acid (GlcA), Adenosine Triphosphate (ATP), Uridine Triphosphate (UTP), and MgCl₂Preparing aqueous solution of GlcA and MgCl in reaction system₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, the pH of a reaction system is adjusted to 7.5, AtGlcAK, AtUSP and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.5 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the supernatant by using a pipettor, centrifuging the reaction system at 4 deg.C and 12000rpm for 20min, collecting the supernatant, gradually adding barium chloride solution into the reaction solution again by using the pipettor, and repeating the stepsCollecting supernatant, concentrating, adding appropriate amount of strong acid type cation exchange resin medium, adsorbing to remove positively charged substances, mixing at 4 deg.C under 100rpm for 20min, and lyophilizing the treated solution to obtain uridine diphosphate glucuronate (formula II).

Still further, the method comprises: mixing galacturonic acid (GalA), ATP, UTP, MgCl₂Preparing into aqueous solution, GalA, MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, the pH of the reaction system is adjusted to 7.0-9.0, galactokinase (BiGalK), Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction; centrifuging the reaction liquid to collect supernatant, gradually adding barium chloride solution into the supernatant by using a pipettor, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting the supernatant, gradually adding the barium chloride solution into the reaction liquid by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the uridine diphosphate galacturonic acid (formula III).

Preferably, the method comprises: mixing galacturonic acid (GalA), ATP, UTP, MgCl₂Preparing into aqueous solution, GalA, MgCl₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, the pH of a reaction system is adjusted to 7.5, BiGalK, AtUSP and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.5 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added for stopping the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the supernatant by using a pipettor, centrifuging the system at 4 ℃ and 12000rpm for 20min, collecting the supernatant, gradually adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the uridine diphosphate semi-phosphateLactobionic acid (formula III).

Still further, the method comprises: mixing N-acetylglucosamine (GlcNAc), ATP, UTP, MgCl₂Preparing into aqueous solution of N-acetylglucosamine and MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, then the pH of the reaction system is adjusted to 7.0-9.0, then N-acetylhexosamine kinase (NaHK), Pasteurella multocida N-acetylglucosamine 1-uridine phosphate transferase (PmGlmU) and inorganic pyrophosphatase (PmPPA) are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min for the first 2h during the reaction period, and after the detection reaction is finished, glacial ethanol is added to stop the reaction; gradually adding the barium chloride solution into the reaction liquid by using a liquid transfer device, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting supernatant, gradually adding the barium chloride solution into the reaction liquid by using the liquid transfer device again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding an ion exchange resin medium to adsorb impurities, and obtaining the uridine diphosphate N-acetylglucosamine;

mixing N-acetylglucosamine derivative, ATP, UTP, MgCl₂Preparing into aqueous solution, N-acetylglucosamine derivative, MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, then the pH of the reaction system is adjusted to 7.0-9.0, then NaHK, PmGlmU and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, the reaction is stopped by adding glacial ethanol; boiling the reaction liquid for 10-60min, gradually adding the barium chloride solution into the reaction liquid by using a pipettor, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting the supernatant, gradually adding the barium chloride solution into the reaction liquid by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the uridine diphosphate N-acetylglucosamine derivative.

Preferably, the method comprises: mixing N-acetylglucosamine (GlcNAc), ATP, UTP, MgCl₂Preparing into aqueous solution of N-acetylglucosamine and MgCl₂The final concentration was 200mM and the final concentrations of ATP and UTP were 300mM, and then the pH of the reaction system was adjusted to 7.0, followed byAdding NaHK, PmGlmU and PmPPA, reacting for 12-24h, adjusting the pH to 7.0 every 15min in the first 2h of the reaction period, adding equal volume of glacial ethanol to terminate the reaction after detecting the reaction is finished, and standing for 60min at 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, gradually adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the uridine diphosphate N-acetylglucosamine;

mixing N-acetylglucosamine derivative, ATP, UTP, MgCl₂Preparing into aqueous solution, N-acetylglucosamine derivative, MgCl₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, then the pH of a reaction system is adjusted to 7.0, then NaHK, PmGlmU and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, gradually adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the uridine diphosphate N-acetylglucosamine derivative.

Still further, the method comprises: n-acetylgalactosamine (GalNAc), ATP, UTP, MgCl₂Preparing into aqueous solution, N-acetylgalactosamine, MgCl₂The final concentration is 100-400mM, the final concentration of ATP and UTP is 100-400mM, then the pH of the reaction system is adjusted to 7.0-9.0, and NaHK and N-acetylgalactosamine 1-uridine phosphate are addedReacting transferase (AGX1) and PmPPA for 12-24h, adjusting the pH to 7.0-9.0 every 15min for the first 2h during the reaction, and adding ethanol to terminate the reaction after detecting the reaction is finished; gradually adding the barium chloride solution into the reaction liquid by using a liquid transfer device, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting supernatant, gradually adding the barium chloride solution into the reaction liquid by using the liquid transfer device again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding an ion exchange resin medium to adsorb impurities, and obtaining the uridine diphosphate N-acetylgalactosamine;

mixing N-acetylgalactosamine derivative, ATP, UTP, and MgCl₂Preparing into aqueous solution, N-acetylgalactosamine derivative, and MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, the pH of the reaction system is adjusted to 7.0-9.0, then NaHK, AGX1 and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, ethanol is added to terminate the reaction; and (2) adding the barium chloride solution into the reaction liquid by using a liquid transfer device, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting supernatant, adding the barium chloride solution into the reaction liquid by using the liquid transfer device again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the uridine diphosphate N-acetylgalactosamine derivative.

Preferably, the method comprises: n-acetylgalactosamine (GalNAc), ATP, UTP, MgCl₂Preparing into aqueous solution, N-acetylgalactosamine, MgCl₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, then the pH of a reaction system is adjusted to 7.0, then NaHK, AGX1 and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting the supernatant, gradually adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, and collecting the supernatantConcentrating the clear liquid, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain uridine diphosphate N-acetylgalactosamine;

mixing N-acetylgalactosamine derivative, ATP, UTP, and MgCl₂Preparing into aqueous solution, N-acetylgalactosamine derivative, and MgCl₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, then the pH of a reaction system is adjusted to 7.0, then NaHK, AGX1 and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, gradually adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the uridine diphosphate N-acetamino galactose derivative.

Still further, the method comprises: mixing fucose (Fuc), ATP, Guanosine Triphosphate (GTP), MgCl₂Prepared into aqueous solution, fucose (Fuc), MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and GTP are 100-400mM, the pH of the reaction system is adjusted to 7.0-9.0, then L-fucokinase/guanosine diphosphate-fucose pyrophosphorylase (FKP) and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, glacial ethanol is added to stop the reaction; and (2) adding the barium chloride solution into the reaction liquid by using a liquid transfer device, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting supernatant, adding the barium chloride solution into the reaction liquid by using the liquid transfer device again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the guanosine diphosphate rock trehalose (formula VI).

Preferably, the method comprises: mixing fucose (Fuc), ATP, Guanosine Triphosphate (GTP), MgCl₂Prepared into an aqueous solution, Fuc and MgCl₂The final concentration is 200mM, the final concentrations of ATP and GTP are 300mM, the pH of a reaction system is adjusted to 7.5, then L-fucokinase/guanosine diphosphate-fucose pyrophosphorylase (FKP) and PmPPA are added for reaction for 124h, the pH is adjusted to 7.5 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added for stopping the reaction, and the reaction is kept still for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the guanosine diphosphate fucose (formula VI).

Still further, the method comprises: mixing mannose (Man), GTP and MgCl₂Preparing into aqueous solution, Man and MgCl₂The final concentration is 400mM and the GTP final concentration is 700mM, the pH value of the reaction system is adjusted to 7.0-9.0, then NaHK, guanosine diphosphate-mannose pyrophosphorylase (PfManC) and PmPPA are added for reaction for 12-24h, the pH value is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, ethanol is added to stop the reaction; gradually adding barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4-10 deg.C for 10-60min, collecting supernatant, gradually adding barium chloride solution into the reaction solution by using the pipettor again, repeating the above steps until no precipitate is generated, collecting supernatant, concentrating, adding ion exchange resin medium to adsorb impurities, and obtaining guanosine diphosphate mannose (formula VII)

Preferably, the method comprises: mixing mannose (Man), GTP and MgCl₂Preparing into aqueous solution, Man and MgCl₂The final concentration was 200mM, GTP final concentration was 600mM, the reaction pH was then adjusted to 8.0, and NaHK, PfManC and Pm were then addedPPA is reacted for 12-24h, the pH is adjusted to 8.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added to stop the reaction, and the mixture is kept still for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, adding the barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the guanosine diphosphate mannose (formula VII).

Still further, the method comprises: n-acetylneuraminic acid (Neu5Ac), Cytidine Triphosphate (CTP), MgCl₂Preparing into aqueous solution, Neu5Ac, MgCl₂The final concentration is 100-400mM, the CTP final concentration is 100-400mM, then the pH of the reaction system is adjusted to 7.0-9.0, then cytidine monophosphate-sialic acid synthetase (NmCSS) and PmPPA are added for reaction for 2-4 h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, ethanol is added to terminate the reaction; and (2) adding the barium chloride solution into the reaction liquid by using a liquid transfer device, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting the supernatant, adding the barium chloride solution into the reaction liquid by using the liquid transfer device again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the cytidine monophosphate N-acetylneuraminic acid (formula VIII).

Preferably, the method comprises: n-acetylneuraminic acid (Neu5Ac), Cytidine Triphosphate (CTP), MgCl₂Preparing into aqueous solution, Neu5Ac, MgCl₂The final concentration is 200mM, the CTP final concentration is 300mM, the pH of a reaction system is adjusted to 9.0, then NmCSS and PmPPA are added for reaction for 2-4 h, the pH is adjusted to 8.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added for stopping the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; 1M barium chloride Using a pipetteAnd (2) adding the solution into the reaction liquid one by one, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, adding the barium chloride solution into the reaction liquid one by using a pipettor again, repeating the steps until no precipitate is generated, collecting the supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, adsorbing and removing positively charged substances, uniformly mixing at 4 ℃ and 100rpm for 20min, and freeze-drying the treated solution to obtain the cytidine monophosphate N-acetylneuraminic acid (formula VIII).

Still further, the method comprises: mixing galactose (Gal), ATP, UTP, MgCl₂Preparing into aqueous solution, galactosamine and MgCl₂The final concentration is 100-400mM, the final concentrations of ATP and UTP are 100-400mM, then the pH of the reaction system is adjusted to 7.0-9.0, then galactokinase (BiGalK), AtUSP and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0-9.0 every 15min in the first 2h during the reaction period, and after the detection reaction is finished, ethanol is added for stopping the reaction; gradually adding the barium chloride solution into the reaction solution by using a liquid transfer machine, centrifuging the reaction system at 4-10 ℃ for 10-60min, collecting supernatant, gradually adding the barium chloride solution into the reaction solution by using the liquid transfer machine again, repeating the steps until no precipitate is generated, collecting supernatant, concentrating, and adding an ion exchange resin medium to adsorb impurities to obtain the uridine diphosphate galactose (formula IX).

Preferably, the method comprises: mixing galactose, ATP, UTP, MgCl₂Preparing into aqueous solution, galactosamine and MgCl₂The final concentration is 200mM, the final concentrations of ATP and UTP are 300mM, the pH of a reaction system is adjusted to 7.0, BiGalK, AtUSP and PmPPA are added for reaction for 12-24h, the pH is adjusted to 7.0 every 15min in the first 2h during the reaction period, after the detection reaction is finished, equal volume of glacial ethanol is added for stopping the reaction, and the reaction is stopped for 60min at the temperature of 4 ℃; centrifuging the reaction solution at 4 deg.C and 12000rpm for 20min, and collecting supernatant; gradually adding 1M barium chloride solution into the reaction solution by using a pipettor, centrifuging the reaction system at 4 ℃ and 12000rpm for 20min, collecting supernatant, gradually adding barium chloride solution into the reaction solution by using the pipettor again, repeating the steps until no precipitate is generated, collecting supernatant, concentrating, adding a proper amount of strong acid type cation exchange resin medium, and suckingRemoving positively charged substances, mixing at 4 deg.C and 100rpm for 20min, and lyophilizing to obtain uridine diphosphate galactose (formula IX).

The invention provides a preparation method of sugar nucleotide and derivatives thereof, which aims at different preparation targets and needs targeted purification strategies, and simple conversion of monosaccharide and derivatives thereof into sugar nucleotide and derivatives thereof is not simply carried out under catalysis of different enzymes.

The preparation method of the sugar nucleotide and the derivatives thereof provided by the invention improves the catalytic reaction effect of bacterial monosaccharide kinase and sugar nucleotide synthetase, adopts a post-treatment strategy independent of column chromatography, greatly simplifies the purification process, and realizes the large-scale purification preparation of the sugar nucleotide and the derivatives thereof with high added values. The sugar nucleotide synthetase and the sugar kinase related by the invention are all prokaryotic sources, and have the advantages of high protein expression, wide substrate adaptability, high catalytic efficiency and the like, the high-concentration biological enzyme method based on the sugar nucleotide synthetase and the sugar kinase has high synthesis conversion efficiency, cheap monosaccharide is used as a raw material, and the sugar nucleotide is efficiently converted into expensive sugar nucleotide, so that the production cost is greatly reduced; the ion precipitation method is used for the post-purification process of the product, avoids chromatographic purification operations such as column chromatography and the like, reduces the cost of purification steps, is easy to amplify the system and improves the synthesis yield.

The preparation method of the sugar nucleotide and the derivatives thereof provided by the invention can be used for the mass preparation of the sugar nucleotide and the derivatives thereof, and lays a foundation for deeply researching the interaction mechanism and the structure-activity relationship between a sugar chain taking the sugar nucleotide as a donor and an acceptor on a molecular level and the clarification, diagnosis and treatment of disease control.

Drawings

FIG. 1 shows the chemical structural formulas of 12 sugar nucleotides and derivatives thereof.

FIG. 2 is a schematic diagram of the preparation strategy process of high concentration enzymatic synthesis-ion precipitation method.

FIG. 3 is a reaction equation for the synthesis of uridine diphosphate glucuronate.

FIG. 4 is a reaction equation for the synthesis of uridine diphosphate galacturonic acid.

FIG. 5 is a reaction equation for the synthesis of uridine diphosphate N-acetylglucosamine and its derivatives.

FIG. 6 is a reaction equation for the synthesis of uridine diphosphate N-acetylgalactosamine and its derivatives.

FIG. 7 is a reaction equation for the synthesis of guanosine diphosphate fucose.

FIG. 8 is a reaction equation for the synthesis of guanosine diphosphate mannose.

FIG. 9 is a reaction equation for synthesizing N-acetylneuraminic acid cytidine monophosphate.

FIG. 10 is a reaction equation for the synthesis of uridine monophosphate galactose.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.