WO2023115798A1 - 一种由UDP-GlcNAc合成稀有糖核苷酸的方法 - Google Patents

一种由UDP-GlcNAc合成稀有糖核苷酸的方法 Download PDF

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WO2023115798A1
WO2023115798A1 PCT/CN2022/093479 CN2022093479W WO2023115798A1 WO 2023115798 A1 WO2023115798 A1 WO 2023115798A1 CN 2022093479 W CN2022093479 W CN 2022093479W WO 2023115798 A1 WO2023115798 A1 WO 2023115798A1
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文留青
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中国科学院上海药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/305—Pyrimidine nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides

Definitions

  • the invention belongs to the field of glycobiology, and in particular relates to a method for synthesizing rare sugar nucleotides by UDP-GlcNAc.
  • glycosylation is one of the most common biological processes in nature for the assembly of complex glycans and glycoconjugated compounds.
  • glycosylation reactions are catalyzed by glycosyltransferases, using sugar nucleotides or lipid-linked sugar phosphates as donors.
  • Sugar nucleotides are composed of a sugar molecule and a nucleoside diphosphate or nucleoside monophosphate.
  • Common sugar nucleotides include: UDP-Glc (uridine diphosphate glucose), UDP-GlcNAc (uridine diphosphate-N-acetylglucosamine), UDP-GlcA (uridine diphosphate gluconic acid), UDP- Gal (uridine galactose diphosphate), UDP-GalNAc (uridine diphosphate-N-acetylgalactosamine), UDP-Xyl (uridine xylose diphosphate), GDP-Fuc (guanosine diphosphate fucose), GDP-Man (guanosine mannose diphosphate) and CMP-Neu5Ac (adenosine sialic acid monophosphate) (their structural formulas are shown below), the sugar moieties of which are commonly found in human polysaccharides in sugar molecules. In addition to these nine sugar nucleotides, other natural sugar nucleotides are called rare sugar nucleotides.
  • Enzymatic reactions based on biosynthetic pathways facilitate the synthesis of common sugar nucleotides.
  • a huge number of glycosyltransferases have been identified and characterized, and the biosynthetic pathways of many important glycans have been revealed.
  • enzymatic glycosylation has become a powerful tool for the synthesis of structurally defined glycans and glycoconjugated compounds for basic research and the development of carbohydrate drugs.
  • rare sugar nucleotides are converted from common sugar nucleotides (such as: D-GlcNAc).
  • D-GlcNAc common sugar nucleotides
  • the entire biological process of transformation occurs on the sugar ring and involves: dehydration, isomerization, epimerization, amination, acetylation, oxidation, decarboxylation, and reduction reactions.
  • researchers generally believe that it is impractical to synthesize rare sugar nucleotides completely following natural biosynthetic pathways due to complex reaction routes, high synthesis costs (expensive cofactors are required), and difficulties in product purification (isomers or separation of epimers).
  • the technical purpose of the present invention is to provide a general method for preparing rare sugar nucleotides without complicated purification operations, which can use UDP-GlcNAc for large-scale, high-yield preparation Rare sugar nucleotides.
  • the invention provides a method for preparing rare sugar nucleotides, the method comprising: making uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) undergo an enzyme-catalyzed reaction in the presence of an enzyme and a cofactor regeneration system get rare sugar nucleotides,
  • UDP-GlcNAc uridine diphosphate-N-acetylglucosamine
  • UDP-GlcNAc is as follows:
  • the rare sugar nucleotides are represented by the following formula:
  • R 1 is AcNH-
  • R 2 and R 3 are each independently OH, NH 2 , AcNH- and OCH(CH 3 )COOH,
  • R 4 is selected from CH 2 OH, COOH, CH 3 and H,
  • R5 is UDP
  • UDP stands for uridine diphosphate.
  • the enzyme is selected from the following groups:
  • the cofactor regeneration system may be one of all regeneration systems selected from renewable NADH, NADPH, NAD + , PMP, and AcCoA.
  • said cofactor regeneration system is selected from:
  • NADH regeneration system consists of NAD + , D-glucose and D-glucose dehydrogenase (BsGH), or NAD + , fatty alcohols (such as ethanol, propanol, isopropanol or isobutanol ) and alcohol dehydrogenase (ADH), or NAD + , formate, and formate dehydrogenase (FDH), or NAD + , phosphite, and phosphite dehydrogenase (PTDH).
  • CRS1 is composed of NAD + , D-glucose and D-glucose dehydrogenase (BsGH), wherein, relative to UDP-GlcNAc, the amount of NAD + used is 0.001-1 equivalent, preferably 0.01 or 0.02 equivalent, D-glucose
  • the dosage is 1 to 10 equivalents, preferably 3 equivalents, and the mass ratio of BsGH:UDP-GlcNAc is 1:5000 to 1:1, preferably 1:2000 or 1:1000;
  • NADPH regeneration system composed of NADP + , D-glucose and BsGH, or composed of NADP + , glucose 6-phosphate and glucose-6-phosphate dehydrogenase (G6PD), or composed of NADP + , fat alcohol (such as ethanol, propanol, isopropanol, or isobutanol) and alcohol dehydrogenase (ADH), or of NADP + , formate, and formate dehydrogenase (FDH), or of NADP + , phosphite and phosphite dehydrogenase (PTDH).
  • CRS2 NADPH regeneration system
  • CRS2 is composed of NADP + , D-glucose and BsGH, wherein, relative to UDP-GlcNAc, the amount of NADP + is 0.001-1 equivalent, preferably 0.01 or 0.02 equivalent, and the amount of D-glucose is 1-10 equivalents, Preferably 3 equivalents, BsGH:UDP-GlcNAc mass ratio is 1:5000 ⁇ 1:1, preferably 1:2000 or 1:1000;
  • NAD + regeneration system composed of NAD + , pyruvate, and D-glucose dehydrogenase (LdhA), or of NAD + , mangalone, and quinone reductase (NfsB), or of NAD + , O 2 and NADH oxidase, or by NAD + , ⁇ -ketoglutarate and glutamate dehydrogenase (GLDH).
  • CRS3 is composed of NAD + , pyruvate and D-glucose dehydrogenase (LdhA), wherein, relative to UDP-GlcNAc, the amount of NAD + is 0.001-1 equivalent, preferably 0.01 or 0.02 equivalent, and the amount of pyruvate It is 1 to 10 equivalents, preferably 3 equivalents, and the mass ratio of LdhA:UDP-GlcNAc is 1:5000 to 1:1, preferably 1:2000 or 1:1000;
  • PMP regeneration system composed of L-glutamic acid and 5'-pyridoxal phosphate (PLP), preferably, the amount of L-glutamic acid is 1 to 10 equivalents relative to UDP-GlcNAc , 5 equivalents, the amount of PLP is 0.01 to 1 equivalent, preferably 0.1 equivalent;
  • CRS5 AcCoA regeneration system: composed of CoA, ATP, sodium acetate, and AcCoA synthetase (ACS), or composed of CoA and S-acetylthiocholine iodide, or composed of CoA, carnitine acetyl Transferase (CAT) and acetylcarnitine, or CoA, phosphotransacetylase (PTA) and acetyl phosphate.
  • CRS5 consists of CoA, ATP, sodium acetate, and ACS, wherein, relative to the starting sugar nucleotide UDP-GlcNAc, CoA is 0.1-1 equivalent, preferably 0.5 equivalent, ATP 2 equivalents, sodium acetate 1-10 Equivalent, preferably 3 equivalents, ACS:UDP-GlcNAc mass ratio is 1:5000-1:1, preferably 1:2000 or 1:1000.
  • CRS1 and CRS2 can be used interchangeably in the reaction to provide reducing power for the reaction.
  • the above-mentioned fatty alcohol refers to a fatty alcohol having 2-8 carbon atoms, preferably 2-6, more preferably 2-4, such as ethanol, propanol, isopropanol or isobutanol.
  • phosphite may be a common phosphite in the art, including but not limited to sodium phosphite, potassium phosphite and the like.
  • UDP-GlcNAc can be a commercially available product, or can be synthesized according to methods known in the prior art, or can be synthesized by the following route 1:
  • D-GlcNAc is phosphorylated into N-acetylglucosamine-1-phosphate under the action of N-acetylhexosamine kinase (NahK, also known as D-GlcNAc kinase), and then N-acetylglucosamine 1-phosphouria D-GlcNAc is obtained under the action of glycosyltransferase (GlmU) and inorganic pyrophosphatase (PPA).
  • ATP adenosine triphosphate
  • PPi means pyrophosphate.
  • Pi represents inorganic phosphate.
  • D-GlcNAc is phosphorylated into N-acetylglucosamine-1-phosphate by D-GlcNAc kinase (NahK) at a pH of about 7.5 and in the presence of Mg 2+ , and then converted from N-acetylglucosamine 1- Phosphouridine acyltransferase (GlmU) catalyzes UDP-GlcNAc, and inorganic pyrophosphatase (PPA) hydrolyzes the by-product pyrophosphate to promote the positive reaction.
  • D-GlcNAc kinase NahK
  • Phosphouridine acyltransferase GlmU
  • PPA inorganic pyrophosphatase
  • the rare sugar nucleotide is selected from one of the following:
  • the method is carried out as follows:
  • UDP-GlcNAc undergoes an enzyme-catalyzed reaction in the presence of Mg534, Mg535 and the CRS1 cofactor regeneration system to obtain UDP-L-RhaNAc; or
  • UDP-GlcNAc undergoes an enzymatic reaction in the presence of WbjB, WbjC and CRS1 cofactor regeneration system to obtain UDP-L-PneNAc;
  • UDP-GlcNAc first undergoes an enzyme-catalyzed reaction in the presence of PglF, and then adds PreQ and CRS1 cofactor regeneration system to undergo an enzyme-catalyzed reaction to obtain UDP-D-QuiNAc; or
  • UDP-GlcNAc undergoes an enzyme-catalyzed reaction in the presence of MurA and PEP, and then adds MurB and CRS2 cofactor regeneration system to undergo an enzyme-catalyzed reaction to obtain UDP-D-MurNAc; or
  • UDP-GlcNAc undergoes an enzymatic reaction in the presence of WbpO and the CRS3 cofactor regeneration system to obtain UDP-D-GlcNAcA;
  • UDP-GlcNAc first undergoes an enzymatic reaction in the presence of WbpO, UXNAcS and CRS3 cofactor regeneration system, and then adds UXNAcS to undergo an enzymatic reaction to obtain UDP-D-XylNAc; or
  • UDP-GlcNAc undergoes an enzymatic reaction in the presence of Cap5P, Cap5O and CRS3 cofactor regeneration system to obtain UDP-D-ManNAcA; or
  • UDP-GlcNAc first undergoes an enzyme-catalyzed reaction in the presence of PglF, and then adds Pat and CRS4 cofactor regeneration system to undergo an enzyme-catalyzed reaction to obtain UDP-D-4n-FucNAc; or
  • UDP-GlcNAc undergoes an enzyme-catalyzed reaction in the presence of PglF first, and then adds PglE and CRS4 cofactor regeneration system to undergo an enzyme-catalyzed reaction to obtain UDP-D-4n-QuiNAc; or
  • UDP-GlcNAc undergoes an enzyme-catalyzed reaction in the presence of PglF first, then adds PglE and a CRS4 cofactor regeneration system to undergo an enzyme-catalyzed reaction, and then adds PglD and a CRS5 cofactor regeneration system to undergo an enzyme-catalyzed reaction to obtain UDP-diNAcBac; or
  • UDP-GlcNAc undergoes an enzymatic reaction in the presence of PseB, PseC, PseH, and CRS4 and CRS5 cofactor regeneration systems to obtain UDP-6-deoxy-AltdiNAc; or
  • UDP-GlcNAc first undergoes an enzymatic reaction in the presence of WbpO and CRS3 cofactor regeneration systems, and then adds WbpB, WbpE, WbpD, and CRS4 and CRS5 cofactor regeneration systems to undergo enzymatic reactions to obtain UDP-D-GlcNAc3NAcA.
  • the synthesis of rare sugar nucleotides may proceed via the following route 2:
  • UDP-D-QuiNAc The synthesis of UDP-D-QuiNAc is catalyzed by enzymes (PglF, PreQ) to achieve C-4, 6 dehydration and C-4 reduction, and CRS1 provides cofactors to promote the reaction to form the target product;
  • UDP-D-MurNAc realizes the transfer and reduction of enolpyruvyl through the enzyme (MurA, MurB) system, and CRS2 provides cofactors to promote the reaction to form the target product.
  • UDP-D-XylNAc is catalyzed by enzymes (WbpO, UXNAcS) to oxidize and decarboxylate the C-6 position, and CRS3 provides the cofactor NAD + to push the reaction towards the formation of the target product;
  • UDP-D-ManNAcA is catalyzed by enzymes (Cap5P, Cap5O) to dehydrogenate the C-2 heterogeneous C-6 position, and CRS3 provides the cofactor NAD + to push the reaction towards the formation of the target product.
  • the synthesis of rare sugar nucleotides may proceed via the following route 4:
  • UDP-D-4n-FucNAc and UDP-D-4n-QuiNAc are catalyzed by PglF to dehydrate C-4, 6, and the C-4 position is aminated by transaminase Pat or PglE, and CRS4 provides the supply of transaminase Body PMP, promote the reaction to the direction of the formation of the target product.
  • the synthesis of rare sugar nucleotides may proceed via the following route 5:
  • UDP-diNAcBac is based on the synthesis of UDP-D-4n-FucNAc system, adding PglD to realize the transfer of acetyl group to the amino group of UDP-D-4n-FucNAc, CRS4 provides the donor PMP of transaminase, CRS5 Provide the cofactor AcCoA to promote the reaction toward the formation of the target product;
  • UDP-6-deoxy-AltdiNAc is catalyzed by enzymes (PseB, PseC, PseH) to achieve C-4, 6 dehydration, C-5 epimerization, and ammoniation , acetylation, CRS4 provides the donor PMP of transaminase, CRS5 provides the cofactor AcCoA, and promotes the reaction to form the target product.
  • UDP-D-GlcNAc3NAcA is catalyzed by enzymes (WbpO, WbpB, WbpE, WbpD) to achieve C-6 oxidation, C-3 dehydrogenation, C-3 ammoniation, aminoacetylation, CRS4 provides transaminase donor PMP, CRS5 provides the cofactor AcCoA to push the reaction toward the formation of the target product.
  • enzymes WbpO, WbpB, WbpE, WbpD
  • CRS4 provides transaminase donor PMP
  • CRS5 provides the cofactor AcCoA to push the reaction toward the formation of the target product.
  • cofactor regeneration system of the present application is not limited to the above specific forms.
  • the technical scheme of the present invention is described above by taking the specifically selected combination of the cofactor regeneration system described herein and the complex enzyme system as an example.
  • those skilled in the art can combine the above-mentioned similar
  • Other cofactor regeneration systems are used in the synthesis of sugar nucleotides, and these are also included in the scope of the present invention.
  • NADH regeneration system CRS1
  • NADPH regeneration system can be used interchangeably. That is, the above-mentioned reaction of the regeneration system using NADH is also applicable if it is replaced by NADPH, and the NADH regeneration system is preferred; the regeneration system using NADPH is also applicable if it is replaced by NADH, and the NADPH regeneration system is preferred, and these are also included in the present invention In the range.
  • the present invention provides the use of a cofactor regeneration system in the preparation of rare sugar nucleotides from UDP-GlcNAc, wherein the cofactor regeneration system can be selected from renewable NADH, NADPH, NAD + , PMP, AcCoA One of a kind among all regenerative systems.
  • said cofactor regeneration system is selected from:
  • NADH regeneration system consisting of D-glucose dehydrogenase (BsGH), NAD+ and D-glucose, or NAD + , fatty alcohol (such as ethanol, propanol, isopropanol, or isobutanol) and alcohol dehydrogenase (ADH), or NAD + , formate and formate dehydrogenase (FDH), or NAD + , phosphite and phosphite dehydrogenase (PTDH); preferably, a NADH regeneration system consisting of D - Glucose dehydrogenase (BsGH), NAD+ and D-glucose, wherein, relative to UDP-GlcNAc, the amount of NAD + is 0.001 to 1 equivalent, preferably 0.01 or 0.02 equivalents, and the amount of D-glucose is 1 to 10 equivalents , preferably 3 equivalents, the mass ratio of BsGH:UDP-GlcNAc is 1
  • the NADPH regeneration system consists of BsGH, NADP + and D-glucose, or consists of NADP + , glucose-6-phosphate and glucose-6-phosphate dehydrogenase (G6PD), or consists of NADP + , fatty alcohols (such as ethanol, propanol, isopropanol, or isobutanol) and alcohol dehydrogenase (ADH), or of NADP + , formate, and formate dehydrogenase (FDH), or of NADP + , phosphite, and phosphorous acid dehydrogenase Enzyme (PTDH); preferably, the NADPH regeneration system is composed of BsGH, NADP + and D-glucose, wherein, relative to UDP-GlcNAc, the amount of NADP + is 0.001 to 1 equivalent, preferably 0.01 or 0.02 equivalent, D-glucose The dosage is 1 to 10 equivalents, preferably 3 equivalents, and the mass ratio of BsGH
  • NAD + regeneration system consisting of NADH, pyruvate and lactate dehydrogenase (LdhA), or NAD + , mangalone and quinone reductase (NfsB), or NAD + , O 2 and NADH oxidase , or consist of NAD + , ⁇ -ketoglutarate and glutamate dehydrogenase (GLDH); preferably, the NAD + regeneration system consists of NADH, pyruvate and lactate dehydrogenase (LdhA), wherein, relative to UDP-GlcNAc, the amount of NAD + is 0.001-1 equivalent, preferably 0.01 or 0.02 equivalent, the amount of pyruvic acid is 1-10 equivalent, preferably 3 equivalents, the mass ratio of LdhA:UDP-GlcNAc is 1:5000-1:1, Preferably 1:2000 or 1:1000;
  • PMP regeneration system which consists of 5'-pyridoxal phosphate (PLP) and L-glutamic acid; preferably, relative to UDP-GlcNAc, the amount of L-glutamic acid is 1 to 10 equivalents, preferably 5 equivalents, The amount of PLP is 0.01 to 1 equivalent, preferably 0.1 equivalent; and
  • AcCoA regeneration system consisting of ATP, sodium acetate, CoA and AcCoA synthase (ACS), or of CoA and S-acetylthiocholine iodide, or of CoA, carnitine acetyltransferase (CAT) and Acetylcarnitine consists of, or consists of CoA, phosphotransacetylase (PTA) and acetyl phosphate, preferably, the AcCoA regeneration system consists of ATP, sodium acetate, CoA and AcCoA synthetase (ACS), wherein, relative to UDP-GlcNAc , CoA 0.1-1 equivalent, preferably 0.5 equivalent, ATP 2 equivalents, sodium acetate 1-10 equivalents, preferably 3 equivalents, ACS:UDP-GlcNAc mass ratio is 1:5000-1:1, preferably 1:2000 or 1:1000 .
  • the cofactor regeneration system of the present application has been described in detail above, the cofactor regeneration system of the present application is not limited to the synthesis of the above-mentioned rare sugar nucleotides. Conventional technical means, the cofactor regeneration system of the present application can be applied to any synthetic reaction that requires reduction, oxidation, amination and acetylation orientation, and these are also included in the scope of the present invention.
  • the biggest advantage of enzymatic reactions is high stereoselectivity and regioselectivity. Therefore, the multi-step enzyme reaction can be carried out at one time, which is called “one-pot multienzyme reaction” (One-pot multienzyme reaction, OPME).
  • One-pot multienzyme reaction OPME
  • the biotransformation pathways of rare sugar nucleotides are complex, the final steps are usually reduction, oxidation, amination, acetylation, and isomerization reactions. Except for the isomerization reaction, other reactions are irreversible processes and require cofactors, such as NADH/NADPH, NAD + /NADP + , 5'-pyridoxamine phosphate (PMP) or acetyl coenzyme A (AcCoA).
  • PMP 5'-pyridoxamine phosphate
  • AcCoA acetyl coenzyme A
  • the present application designs a one-pot method avoiding stepwise synthesis to synthesize rare sugar nucleotides.
  • the present application uses the above-mentioned cofactor regeneration system (Cofactor regeneration system, CRS) to produce cofactors, which can reduce production costs and product purification difficulties. It has been verified that the reaction can still proceed smoothly by coupling the one-pot multi-enzyme method with the above-mentioned cofactor regeneration system, thus avoiding the purification of unstable and difficult-to-separate intermediates, and thus making the large-scale synthesis of rare sugar nucleotides by the enzymatic method become Reality.
  • CRS cofactor regeneration system
  • OPME One-pot multienzyme reaction, one-pot multi-enzyme; OPME is also used to refer to the enzyme system in this article.
  • CRS Cofactor regeneration system, cofactor regeneration system; CRS is also used in this article to refer to five specific cofactor regeneration systems.
  • UDP-GlcNAc Uridine diphosphate-N-acetylglucosamine
  • NAD + Nicotinamide adenine dinucleotide
  • NADH reduced nicotinamide adenine dinucleotide
  • NADP + Nicotinamide adenine dinucleotide phosphate
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • ADH alcohol dehydrogenase, alcohol dehydrogenase
  • FDH formate dehydrogenase, formate dehydrogenase
  • PTDH phosphite dehydrogenase, phosphorous acid dehydrogenase
  • G6PD 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase
  • NfsB oxygen-insensitive NAD(P)H nitrogenreductase, quinone reductase
  • GLDH Glutamate dehydrogenase, glutamate dehydrogenase
  • PTA phosphotransacetylase, phosphotransacetylase
  • CoA coenzyme A, coenzyme A
  • BsGH D-glucose dehydrogenase from Bacillus subtilis, D-glucose dehydrogenase from Bacillus subtilis
  • LdhA lactate dehydrogenase from Leuconostoc mesenteroides, lactose dehydrogenase from Leuconostoc mesenteroides
  • NahK N-acetylhexosamine 1-kinase, N-acetylhexosamine kinase
  • GlmU N-acetylglucosamine 1-phosphate uridylyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase
  • PPA inorganic pyrophosphatase, inorganic pyrophosphatase
  • Mg534 putative dTDP-d-glucose 4,6-dehydratase from Megavirus chiliensis.
  • Mg535 dTDP-4-dehydrorhamnose reductase from Megavirus chiliensis. C-3 epimerization from Chilean giant virus, bifunctional enzyme for C-4 reduction
  • WbjB UDP-GlcNAc C-4,6 dehydratase/C5-epimerase from Pseudomonas aeruginosa Pseudomonas aeruginosa
  • WbjC C-3 epimerase/C-4 reductase from Pseudomonas aeruginosa, C-3 epimerase/C-4 reductase from Pseudomonas aeruginosa
  • MurA UDP-N-acetylglucosamine 1-carboxyvinyltransferase, enolpyruvyltransferase
  • MurB UDP-N-acetylenolpyruvylglucosamine reductase, UDP-N-acetylpentanamide reductase
  • UXNAcS UDP-N-acetylxylosamine synthase Bacillus cereus, UDP-N-acetylpyranamide synthase derived from Bacillus
  • Cap5P UDP-N-acetylglucosamine 2-epimerase from Staphylococcus aureus, UDP-N-acetylglucosamine 2-epimerase from Staphylococcus aureus
  • PglE UDP-N-acetylbacillosamine transaminase from Campylobacter jejuni, UDP-N-acetylbacillosamine transaminase from Campylobacter jejuni
  • PglD UDP-N-acetylbacillosamine N-acetyltransferase from Campylobacter jejuni, UDP-N-acetylglucosamine N-acetyltransferase from Campylobacter jejuni
  • PseB UDP-N-acetylglucosamine 4,6-dehydratase from Helicobacter pylori, UDP-N-acetylglucosamine 4,6-dehydratase from Helicobacter pylori
  • WbpB UDP-N-acetyl-2-amino-2-deoxy-D-glucuronate oxidase from Pseudomonas aeruginosa, UDP-N-acetyl-2-amino-2-deoxy-D-glucuronate from Pseudomonas aeruginosa acid oxidase
  • WbpE UDP-2-acetamido-2-deoxy-3-oxo-D-glucuronate aminotransferase from Pseudomonas aeruginosa, UDP-2-acetamido-2-deoxy-3-oxo-D-glucose from Pseudomonas aeruginosa aldydyl aminotransferase
  • WbpD UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronate N-acetyltransferase from Pseudomonas aeruginosa, UDP-2-acetylamino-3-amino-2,3- from Pseudomonas aeruginosa Dideoxy-D-glucuronide-N-acetyltransferase
  • UDP-GlcNAc was synthesized from D-GlcNAc according to Route 1.
  • UDP-GlcNAc the most common sugar nucleotide in nature, to synthesize rare sugar nucleotides.
  • UDP-GlcNAc is produced by using D-GlcNAc kinase (NahK, N-acetylhexosamine 1-kinase), N-acetylglucosamine 1-phosphate uridyltransferase (GlmU, N-Acetylglucosamine-1-phosphate uridyltransferase) and inorganic pyrophosphate
  • PPA Inorganic pyrophosphatase
  • the 12 sugar nucleotides synthesized above cover most of the reported rare sugar nucleotides derived from UDP-GlcNAc.
  • the reduction reaction catalyzed by reductases with NADH and NADPH as reducing power is the most common reaction in the biotransformation of rare sugar nucleotides.
  • the biosynthetic pathways from UDP-GlcNAc and UDP-Glc to UDP-L-RhaNAc and UDP-L-PneNAc have been studied clearly. The process requires four steps, including: C-4,6 dehydration, C-5 epimerization, C-3 epimerization, and C-4 reduction, involving two enzymes catalyzing the C-5 epimerization structure (route 2).
  • the intermediates in the synthetic pathway have poor stability and are easily hydrolyzed rapidly to form monosaccharides and UDP.
  • NAD + and NADP + are much cheaper than NADH and NADPH, in order to provide strong reducing power, this application uses D-glucose dehydrogenase (BsGH) from Bacillus subtilis, prepared from NAD + or NADP + NADH or NADPH, thereby greatly reducing costs.
  • BsGH D-glucose dehydrogenase
  • BsGH oxidizes D-glucose to gluconic acid using NAD + or NADP + as a cofactor, while forming NADH (CRS1) or NADPH (CRS2) for the reduction reaction.
  • Mg534 and Mg535 two kinds of enzymes cloned from Chile giant virus (Megavirus chilensis).
  • Mg534 is a bifunctional enzyme that catalyzes C-4,6 dehydration and C-5 epimerization
  • Mg535 is a bifunctional enzyme that catalyzes C-3 epimerization and C-4 reduction.
  • the biosynthesis of UDP-D-QuiNAc is divided into two steps, the C-4,6 dehydratase of UDP-GlcNAc and the C-4,6 reductase catalyzed by C-4, 6 Dehydration and reduction reactions (Scheme 2).
  • the C-4,6 dehydratase (PglF) of UDP-GlcNAc derived from Campylobacter jejuni Campylobacter jejuni
  • no exogenous NAD + /NADP + dehydratase is required.
  • C-4 reductase (PreQ) derived from Bacillus cereus was used.
  • UDP-GlcNAc was incubated with PglF and PreQ enzymes in the presence of CRS1 or CRS2 system.
  • CRS1 (NADH) could obtain higher conversion efficiency. Therefore, the CRS1 (NADH) system was selected in the large-scale synthesis reaction; UDP-GlcNAc was completely converted in the reaction, and UDP-D-QuiNAc was obtained through simple desalination of the P2 column with a yield of 87%.
  • the chemoenzymatic synthesis of UDP-D-QuiNAc requires at least four steps of reaction and severe reaction conditions, compared with which, this method is more efficient and cost-effective.
  • UDP-D-MurNAc was synthesized using E. coli O157-derived enolpyruvyltransferase (MurA) and UDP-N-ethynylpyruvylglucosamine reductase (MurB) (Scheme 2).
  • MurB has intrinsic NADPH oxidase activity and consumes a large amount of NADPH in the reduction reaction.
  • the above-mentioned reduction-oriented CRS system may also include a regenerative system as shown below, but not limited thereto:
  • lactate dehydrogenase (LdhA) from Leuconostoc mesenteroides reduces pyruvate to D-lactate while oxidizing NADH to NAD + .
  • UDP-D-XylNAc was prepared from UDP-GlcNAc by a two-step enzymatic reaction (Route 3), and UXNAcS from Bacillus cereus catalyzed the decarboxylation of UDP-D-GlcNAcA to UDP-D-XylNAc.
  • UDP-GlcNAc was incubated with WbpO in the presence of CRS3, and when UDP-GlcNAc was completely converted, UXNAcS was directly added to the reaction system to generate UDP-D-XylNAc through decarboxylation.
  • the pure product can be obtained through simple desalination of the P2 column, and the yield is 88%.
  • UDP-D-ManNAcA is synthesized from UDP-GlcNAc in the presence of UDP-GlcNAc-2-epimerase and UDP-D-ManNAc dehydrogenase (route 3), the two enzymes used in the reaction (Cap5P and Cap5O) from Staphylococcus aureus.
  • Cap5P catalyzes the interconversion between UDP-GlcNAc and UDP-D-ManNAc, however, this reaction is very unfavorable for the formation of UDP-D-ManNAc, and after equilibrium, less than 10% of UDP-GlcNAc can be converted into UDP-D -ManNAc.
  • Cap5O is a UDP-D-ManNAc dehydrogenase that can specifically recognize UDP-D-ManNAc and push the balance to UDP-D-ManNAc. Therefore, the present application designed a one-pot method, including: Cap5P, Cap5O, CRS3, to synthesize UDP-D-ManNAcA from UDP-GlcNAc. Although the reaction was incomplete, since UDP-D-ManNAcA has one more charge than the raw materials and intermediate products, it can be easily separated from the system by ion exchange resin with a yield of 51%.
  • the above-mentioned oxidation-guided regeneration system may also include the following regeneration system, but not limited thereto:
  • UDP-D-4n-FucNAc and UDP-D-4n-QuiNAc starts from UDP-GlcNAc, firstly the dehydration reaction is catalyzed by UDP-GlcNAc-C4,6 dehydratase, and then the UDP-4-keto-6 C-4 amination of -deoxy-GlcNAc to generate UDP-D-4n-FucNAc, UDP-D-4n-QuiNAc (Scheme 4).
  • the donor of the transaminase is pyridoxamine-5'-phosphate (PMP), which is regenerated from pyridoxal 5'-phosphate (PLP) and L-glutamic acid (shown below as CRS4), without exogenous enzymes.
  • PMP pyridoxamine-5'-phosphate
  • PRP pyridoxal 5'-phosphate
  • CRS4 L-glutamic acid
  • this application uses L-glutamic acid decarboxylase (GadB) from plant Lactobacillus (Lactobacillus plantarum) to hydrolyze the remaining L-glutamic acid into aminobutyric acid, which is easy to purify and remove .
  • GadB L-glutamic acid decarboxylase
  • the reaction intermediate could be easily removed by ion exchange resin to obtain UDP-D-4n-FucNAc with a yield of 70%.
  • UDP-D-4n-QuiNAc was converted from UDP-GlcNAc with a yield of 65%.
  • N-acetylation of rare sugar nucleotides is catalyzed by acetyltransferases, using acetyl-CoA (AcCoA) as the acetyl donor.
  • AcCoA is too expensive to be used for large-scale synthesis.
  • the inventors tried chemical acetylation of amino sugar nucleotides to synthesize UDP-diNAcBac, UDP-6-deoxy-AltdiNAc, UDP-D-GlcNAc3NacA.
  • CoA is much cheaper than AcCoA
  • AcCoA was produced using the regeneration system CRS5 (shown below), where AcCoA is synthesized from sodium acetate, CoA and ATP by AcCoA synthetase (ACS).
  • PglD acetyltransferase
  • Campylobacter jejuni the inventors cloned an acetyltransferase (PglD) involved in the biosynthesis of UDP-diNAcBac from Campylobacter jejuni.
  • PglD can also accept UDP-D-4n-VioNH 2 as a reaction substrate.
  • 10mM UDP-D-4n-QuiNAc incubated with PglD and CRS5 can completely convert UDP-D-4n-QuiNAc to UDP-diNAcBac.
  • UDP-6-deoxy-AltdiNAc was prepared from UDP-GlcNAc with a yield of 85%.
  • UDP-D-GlcNAcA was prepared as described above, and then mixed with 0.1 equivalents of NAD + , CRS4 (10 equivalents of L-glutamic acid, 0.05 equivalents of PLP), CRS5 (0.01 equivalents of CoA, 2 equivalents ATP, 3 equivalents of sodium acetate, and ACS) were reacted together overnight, and GadB and alkaline phosphatase were added to hydrolyze impurities to reduce the difficulty of purification. Due to incomplete reaction, the final yield of UDP-D-GlcNAc3NacA was 41%.
  • acetylation-guided regeneration system can also include the following regeneration system, but not limited thereto:
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the column Prior to purification, the column was equilibrated with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole; pH 7.5). The column was washed with 2 column volumes of lysis buffer and the target protein was eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole; pH 7.5). Protein concentration was determined with BCA protein detection kit.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 grams of UDP-GlcNAc, 0.02 equivalents of NAD + , 3 equivalents of D-glucose, 3 mg of Mg534, 3 mg of Mg535 and 1 mg of BsGH.
  • the reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by a P-2 column, and the product yield was 91%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc, 0.02 equivalent of NAD + , 3 equivalent of D-glucose, 5 mg of WbjB, 5 mg of WbjC and 1 mg of BsGH.
  • the reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction. After the insoluble precipitate was removed by centrifugation, the supernatant was concentrated and purified by P-2 column, and the product yield was 90%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc and 20 mg of PglF. The reaction was carried out at 37°C. After the conversion of UDP-GlcNAc was complete, 0.02 equivalents of NAD + , 3 equivalents of D-glucose, 5 mg of PreQ and 1 mg of BsGH were added. The reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by a P-2 column, and the product yield was 87%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 grams of UDP-GlcNAc, 1.5 equivalents of PEP and 3 mg of MurA. The reaction was carried out at 37°C. After the conversion of UDP-GlcNAc was complete, 0.02 equivalents of NAD + , 3 equivalents of D-glucose, 5 mg of MurB and 1 mg of BsGH were added. The reaction was carried out at 37°C. After the reaction is complete, add an equal volume of cold ethanol to terminate the reaction, centrifuge to remove the insoluble precipitate, then concentrate and purify the supernatant through a P-2 column, then purify with anion exchange resin, and elute with sodium chloride, the product yield is 85% .
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc, 0.02 equivalent of NAD + , 3 equivalent of pyruvate, 5 mg of WbpO and 1 mg of LdhA.
  • the reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction. After the insoluble precipitate was removed by centrifugation, the supernatant was concentrated and purified by P-2 column, and the product yield was 90%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc, 0.02 equivalent of NAD + , 3 equivalent of pyruvate, 5 mg of WbpO and 1 mg of LdhA.
  • the reaction was carried out at 37°C, and after UDP-GlcNAc conversion was complete, 3 mg of UXNAcS was added.
  • the reaction was continued at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction. After the insoluble precipitate was removed by centrifugation, the supernatant was concentrated and purified by a P-2 column, and the product yield was 88%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc, 0.1 equivalent of NAD + , 3 equivalent of pyruvate, 15 mg of Cap5P, 15 mg of Cap5O and 1 mg of LdhA.
  • the reaction was carried out at 37°C. Once the reaction stopped and continued, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by P-2 column, and then purified by anion exchange resin. Elution yielded 51% product.
  • the reaction system contained 20mM Tris-HCl (pH 7.5), 2 grams of UDP-GlcNAc and 20 mg of PglF. The reaction was carried out at 37 °C, and after the conversion of UDP-GlcNAc was complete, 0.05 equivalent of PLP, 10 equivalent of L-glutamic acid and 5 mg of Pat were added. The reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by P-2 column, then purified by anion exchange resin, and eluted by sodium chloride. , The product yield is 70%.
  • the reaction system contained 20mM Tris-HCl (pH 7.5), 2 grams of UDP-GlcNAc and 20 mg of PglF. The reaction was carried out at 37°C, and after the conversion of UDP-GlcNAc was complete, 0.05 equivalent of PLP, 10 equivalent of L-glutamic acid and 10 mg of PglE were added. The reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by P-2 column, then purified by anion exchange resin, and eluted by sodium chloride. , The product yield is 65%.
  • the reaction system contained 20mM Tris-HCl (pH 7.5), 2 grams of UDP-GlcNAc and 20 mg of PglF.
  • the reaction was carried out at 37°C, and after the conversion of UDP-GlcNAc was complete, 0.05 equivalent of PLP, 10 equivalent of L-glutamic acid and 10 mg of PglE were added.
  • the reaction was carried out at 37[deg.] C. for several hours and 2 equivalents of ATP, 3 equivalents of sodium acetate, 0.005 equivalents of CoA, 5 mg of PglD and ACS were added.
  • After the reaction is complete add an equal volume of cold ethanol to terminate the reaction, and centrifuge to remove the insoluble precipitate. The supernatant is concentrated and purified by P-2 column, and then purified by anion exchange resin, eluted by sodium chloride, and the product yield is 84%. .
  • the reaction system contained 20mM Tris-HCl (PH 7.5), 2 grams of UDP-GlcNAc, 0.05 equivalents of PLP, 5 equivalents of L-glutamic acid, 2 equivalents of ATP, 3 equivalents of sodium acetate, 0.005 equivalents of CoA, 50 mg of PseB, 50 mg PseC, 20 mg PseH and ACS.
  • the reaction was carried out at 37°C. After the reaction was complete, an equal volume of cold ethanol was added to terminate the reaction, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by P-2 column, then purified by anion exchange resin, and eluted by sodium chloride. , the product yield was 85%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 2 g of UDP-GlcNAc, 0.02 equivalent of NAD + , 3 equivalent of pyruvate, 5 mg of WbpO and 1 mg of LdhA. The reaction was carried out at 37°C. After the conversion of UDP-GlcNAc was complete, 0.1 equivalents of NAD + , 0.05 equivalents of PLP, 5 equivalents of L-glutamic acid, 3 equivalents of ATP, 3 equivalents of sodium acetate, 0.01 equivalents of CoA, 50 mg of WbpE, 50 mg WbpB10 mg WbpD and ACS. The reaction was carried out at 37°C.
  • the present application has successfully developed a general method for the efficient preparation of rare sugar nucleotides.
  • sugar nucleotide synthesis-related enzymes from different clonal sources, complex natural synthetic routes are recombined for synthetic reactions.
  • the excess cofactor is provided by adding the cofactor regeneration system to promote the final irreversible reaction and generate the target sugar nucleotide.
  • 12 rare and difficult-to-obtain sugar nucleotides were prepared from the common sugar nucleotide UDP-GlcNAc on a large scale and efficiently, without tedious purification operations in the whole process.
  • the use of catalytic amounts of expensive cofactors reduces the cost of large-scale synthesis.
  • this method of the present invention can be easily extended to other rare sugar nuclei with well-defined biosynthetic pathways nucleotides, so that the method of the present invention has the advantage of strong adaptability.

Abstract

提供了一种由UDP-GlcNAc合成稀有糖核苷酸的方法，所述方法包括：在酶和辅因子再生系统共同存在下使尿苷二磷酸-N-乙酰基葡萄糖胺(UDP-GlcNAc)发生酶催化反应得到稀有糖核苷酸。所述方法可以大规模、高效地制备稀有糖核苷酸，而无需繁琐的纯化操作。

Description

一种由UDP-GlcNAc合成稀有糖核苷酸的方法
本申请要求于2021年12月20日向中国专利局提交的申请号为202111562908.8的中国发明专利申请的优先权权益，该申请的全部内容通过引用并入本文，如同记载在本文中一样。
技术领域
本发明属于糖生物学领域，具体涉及一种由UDP-GlcNAc合成稀有糖类核苷酸的方法。
背景技术
糖基化是自然界中最常见的复杂聚糖和糖缀化合物组装的生物过程之一。生物体内，糖基化反应是糖基转移酶催化的，利用糖核苷酸或脂质连接的磷酸糖作为供体。糖核苷酸是由一个糖分子和一个双磷酸核苷或单磷酸核苷组成的。常见的糖核苷酸包括：UDP-Glc(二磷酸尿苷葡萄糖)、UDP-GlcNAc(尿苷二磷酸-N-乙酰基葡萄糖胺)、UDP-GlcA(二磷酸尿苷葡萄糖酸)、UDP-Gal(二磷酸尿苷半乳糖)、UDP-GalNAc(尿苷二磷酸-N-乙酰基半乳糖胺)、UDP-Xyl(二磷酸尿苷木糖)、GDP-Fuc(二磷酸鸟嘌呤核苷岩藻糖)、GDP-Man(二磷酸鸟嘌呤核苷甘露糖)和CMP-Neu5Ac(单磷酸腺嘌呤核苷唾液酸)(它们的结构式如下所示)，它们的糖分子部分常见于人类聚糖分子中。除了这九种糖核苷酸，其他的天然糖核苷酸被称为稀有糖核苷酸。
基于生物合成途径的酶促反应使得常见糖核苷酸的合成变得容易。随着常见糖核苷酸的大量制备，数量巨大的糖基转移酶被鉴定和表征，同时揭示了许多重要聚糖的生物合成途径。在此基础上，酶促糖基化已经成为合成结构确定的聚糖和糖缀化合物的有力工具，用于基础研究和糖类药物的开发。
Figure PCTCN2022093479-appb-000001
但是，不同于人体内的糖类化合物，大量的结构不同的糖类化合物存在于细菌、植物、古细菌或病毒中，包括diNAcBac、RhaNAc、Rha、VioNAc和QuiNAc等。含有稀有糖的糖类化合物尽管在人体内不存在，但在制药，医药与合成化学等领域拥有巨大的应用潜力。
尽管许多稀有糖已经被发现超过半世纪，但是大量制备它们对应的糖核苷酸仍然十分困难。自然界中，稀有糖核苷酸是由常见糖核苷酸(如：D-GlcNAc)转化而来的。整个 转化的生物过程发生在糖环上，涉及：脱水、异构化、差向异构化、胺化、乙酰化、氧化、脱羧和还原反应。然而，研究者普遍认为，完全遵循天然生物合成途径合成稀有糖核苷酸是不切实际的，因为反应路线复杂，合成成本高(需要昂贵的辅因子)，以及产物纯化困难(异构体或差向异构体的分离)。
目前，只有HPLC纯化被证明是一种可以获得少量稀有糖核苷酸(微克到毫克级)的有效办法。另一方面，化学合成几种简单的稀有糖核苷酸(UDP-GlcNAcA等)也有报道。但是，糖核苷酸在有机溶剂中的低溶解度和分子中的极性与带电基团使得化学合成糖核苷酸极具挑战性。多步合成和繁琐的保护/脱保护通常导致整个路线产率低下。因此，自然界中发现的大多数稀有糖核苷酸还未通过化学合成制得。
由于稀有糖核苷酸来源的限制，基因库中大部分的稀有糖糖基转移酶无法进行生化表征，同时大量的含有稀有糖的糖类化合物的生物合成路径仍然未被揭示清楚。这是极大阻碍糖科学进步的主要原因之一。
发明内容
基于上述现有技术中的存在的问题，本发明的技术目的是提供一种无需繁琐纯化操作的制备稀有糖核苷酸的通用方法，其能够使用UDP-GlcNAc用来大规模、高收率制备稀有糖核苷酸。
本发明提供一种制备稀有糖核苷酸的方法，所述方法包括：在酶和辅因子再生系统共同存在下使尿苷二磷酸-N-乙酰基葡萄糖胺(UDP-GlcNAc)发生酶催化反应得到稀有糖核苷酸，
其中，UDP-GlcNAc的结构如下：
Figure PCTCN2022093479-appb-000002
所述稀有糖核苷酸由下式表示：
Figure PCTCN2022093479-appb-000003
其中，R 1为AcNH-，
R 2和R 3各自独立地为OH、NH 2、AcNH-和OCH(CH 3)COOH，
R 4选自CH 2OH、COOH、CH 3以及H，
R 5为UDP，
UDP表示尿苷二磷酸基。
在具体实施方式中，所述酶选自以下各组：
组别 酶
OPME2 Mg534,Mg535
OPME3 WbjB,WbjC
OPME4 PglF,PreQ
OPME5 MurA,MurB
OPME6 WbpO
OPME7 WbpO,UXNAcS
OPME8 Cap5P,Cap5O
OPME9 PglF,Pat
OPME10 PglF,PglE
OPME11 PglF,PglE,PglD
OPME12 PseB,PseC,PseH
OPME13 WbpO,WbpB,WbpE,WbpD
在实施方式中，所述辅因子再生系统可以为选自可再生NADH、NADPH、NAD +、PMP、AcCoA的所有再生系统中的一种。
特别地，所述辅因子再生系统选自：
(1)NADH再生系统(CRS1)：由NAD +、D-葡萄糖和D-葡萄糖脱氢酶(BsGH)组成，或者由NAD +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NAD +、甲酸和甲酸脱氢酶(FDH)组成，或者由NAD +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成。优选地，CRS1由NAD +、D-葡萄糖和D-葡萄糖脱氢酶(BsGH)组成，其中，相对于UDP-GlcNAc，NAD +的用量为0.001～1当量，优选0.01或0.02当量、D-葡萄糖的用量为1～10当量，优选3当量，BsGH:UDP-GlcNAc的质量比为1:5000～1:1，优选1:2000或1:1000；
(2)NADPH再生系统(CRS2)：由NADP +、D-葡萄糖和BsGH组成，或者由NADP +、6-磷酸葡萄糖和葡萄糖-6-磷酸脱氢酶(G6PD)组成，或者由NADP +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NADP +、甲酸和甲酸脱氢酶(FDH)组成，或者由NADP +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成。优选地，CRS2由NADP +、D-葡萄糖和BsGH组成，其中，相对于UDP-GlcNAc，NADP +的用量为0.001～1当量，优选0.01或0.02当量、D-葡萄糖的用量为1～10当量，优选3当量，BsGH:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000；
(3)NAD +再生系统(CRS3)：由NAD +、丙酮酸和D-葡萄糖脱氢酶(LdhA)组成，或者由NAD +、指甲花醌和醌还原酶(NfsB)组成，或者由NAD +、O 2和NADH氧化酶组成，或者由NAD +、α-酮戊二酸和谷氨酸脱氢酶(GLDH)组成。优选地，CRS3由NAD +、丙酮酸和D-葡萄糖脱氢酶(LdhA)组成，其中，相对于UDP-GlcNAc，NAD +的用量为0.001～1当量，优选0.01或0.02当量、丙酮酸的用量为1～10当量，优选3当量，LdhA:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000；
(4)PMP再生系统(CRS4)：由L-谷氨酸和5'-磷酸吡哆醛(PLP)组成，优选地，相对于UDP-GlcNAc，L-谷氨酸的用量为1～10当量，5当量，PLP的用量为0.01～1当量，优选0.1当量；
(5)AcCoA再生系统(CRS5)：由CoA、ATP、乙酸钠，以及AcCoA合成酶(ACS) 组成，或者由CoA和S-乙酰硫代胆碱碘化物组成，或者由CoA、肉毒碱乙酰转移酶(CAT)和乙酰肉碱组成，或者由CoA、磷酸转乙酰酶(PTA)和乙酰磷酸组成。优选地，CRS5由CoA、ATP、乙酸钠，以及ACS组成，其中，相对于起始糖核苷酸UDP-GlcNAc，CoA0.1～1当量，优选0.5当量，ATP 2当量、乙酸钠1～10当量，优选3当量，ACS:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000。
上述辅因子再生系统中，CRS1和CRS2在反应中可以互换使用以便为反应提供还原力。
上述的脂肪醇指的是碳原子数为2-8，优选2-6，更优选2～4的脂肪醇，例如乙醇、丙醇、异丙醇或异丁醇。
上述的亚磷酸盐可以是本领域中常见亚磷酸盐，包括但不限于亚磷酸钠，亚磷酸钾等。
以上系统的再生过程分别如下所示：
Figure PCTCN2022093479-appb-000004
CRS1:NADH regeneration system
Figure PCTCN2022093479-appb-000005
CRS2:NADPH regeneration system
Figure PCTCN2022093479-appb-000006
CRS1/2: NAD(P)H regeneration system
Figure PCTCN2022093479-appb-000007
CRS3: NAD +regeneration system
Figure PCTCN2022093479-appb-000008
CRS4: PMP regeneration system
Figure PCTCN2022093479-appb-000009
CRS5:AcCoA regeneration system
下文中，以各具体糖核苷酸的合成为例来详细说明本发明的技术方案。
UDP-GlcNAc可以是市售产品，或者按照现有技术中已知的方法合成，或者可以通过如下路线1合成：
Figure PCTCN2022093479-appb-000010
路线1
其中，D-GlcNAc在N-乙酰己糖胺激酶(NahK，也称为D-GlcNAc激酶)作用下磷 酸化为N-乙酰氨基葡萄-1-磷酸，再在N-乙酰氨基葡萄糖1-磷酸尿苷酰转移酶(GlmU)和无机焦磷酸酶(PPA)作用下得到D-GlcNAc。ATP:三磷酸腺苷，在以上反应中，PPi表示焦磷酸盐。Pi表示无机磷酸盐。
在路线1中，D-GlcNAc在pH为7.5左右，Mg 2+存在条件下，经D-GlcNAc激酶(NahK)磷酸化为N-乙酰氨基葡萄-1-磷酸再由N-乙酰氨基葡萄糖1-磷酸尿苷酰转移酶(GlmU)催化得到UDP-GlcNAc，无机焦磷酸酶(PPA)起到将副产物焦磷酸水解，促进反应正向进行的作用。
在具体实施方式中，所述稀有糖核苷酸选自以下各项之一：
Figure PCTCN2022093479-appb-000011
在具体实施方式中，所述方法如下进行：
UDP-GlcNAc在Mg534、Mg535和CRS1辅因子再生系统共同存在下发生酶催化反应得到UDP-L-RhaNAc；或者
UDP-GlcNAc在WbjB、WbjC和CRS1辅因子再生系统共同存在下发生酶催化反应得到UDP-L-PneNAc；或者
UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PreQ和CRS1辅因子再生系统发生酶催化反应得到UDP-D-QuiNAc；或者
UDP-GlcNAc先在MurA与PEP存在下发生酶催化反应，再加入MurB和CRS2辅因子再生系统发生酶催化反应得到UDP-D-MurNAc；或者
UDP-GlcNAc在WbpO和CRS3辅因子再生系统共同存在下发生酶催化反应得到UDP-D-GlcNAcA；或者
UDP-GlcNAc先在WbpO、UXNAcS和CRS3辅因子再生系统存在下发生酶催化反应，再加入UXNAcS发生酶催化反应得到UDP-D-XylNAc；或者
UDP-GlcNAc在Cap5P、Cap5O和CRS3辅因子再生系统共同存在下发生酶催化反应得到UDP-D-ManNAcA；或者
UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入Pat和CRS4辅因子再生系统发生酶催化反应得到UDP-D-4n-FucNAc；或者
UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PglE和CRS4辅因子再生系统发生酶催化反应得到UDP-D-4n-QuiNAc；或者
UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PglE和CRS4辅因子再生系统发生酶催化反应，然后再加入PglD和CRS5辅因子再生系统发生酶催化反应得到UDP-diNAcBac；或者
UDP-GlcNAc在PseB、PseC、PseH和CRS4和CRS5辅因子再生系统共同存在下发生酶催化反应得到UDP-6-deoxy-AltdiNAc；或者
UDP-GlcNAc先在WbpO和CRS3辅因子再生系统存在下发生酶催化反应，再加入WbpB、WbpE、WbpD和CRS4和CRS5辅因子再生系统发生酶催化反应得到UDP-D-GlcNAc3NAcA。
具体地，不局限于任何理论，在还原导向的NADH或NADPH再生系统存在的情况下，稀有糖核苷酸的合成可能通过以下路线2进行：
Figure PCTCN2022093479-appb-000012
路线2
在上述路线2中，UDP-L-RhaNAc和UDP-L-PneNAc的合成经酶(Mg534,Mg535/WbjB,WbjC)催化实现C-4，6脱水、C-5差向异构化、C-3差向异构化和C-4还原，CRS1提供辅因子NADH或NADPH，推动反应向形成目标产物方向进行；
UDP-D-QuiNAc的合成经酶(PglF,PreQ)催化实现C-4，6脱水和C-4还原，CRS1提供辅因子，推动反应向形成目标产物方向进行；
UDP-D-MurNAc的合成经酶(MurA,MurB)系统实现烯醇丙酮酰转移和还原，CRS2提供辅因子，推动反应向形成目标产物方向进行。
下表总结了在还原导向的NADH或NADPH再生系统存在的情况下，所用酶与CRS(CRS1或CRS2)结合来制备部分稀有核苷酸的部分实例：
Figure PCTCN2022093479-appb-000013
Figure PCTCN2022093479-appb-000014
具体地，不局限于任何理论，在氧化导向的NAD +再生系统存在的情况下，稀有糖核苷酸的合成可能通过以下路线3进行：
Figure PCTCN2022093479-appb-000015
路线3
在上述路线3中，UDP-D-GlcNAcA经WbpO催化实现C-6位氧化，CRS3提供辅因子NAD +，推动反应向形成目标产物方向进行；
UDP-D-XylNAc的合成经酶(WbpO,UXNAcS)催化实现C-6位氧化、脱羧，CRS3提供辅因子NAD +，推动反应向形成目标产物方向进行；
UDP-D-ManNAcA的合成经酶(Cap5P,Cap5O)催化实现C-2位差相异构C-6位脱氢，CRS3提供辅因子NAD +，推动反应向形成目标产物方向进行。
下表总结了在氧化导向的NAD +再生系统存在的情况下，所用酶与CRS3结合来制备部分稀有核苷酸的部分实例：
Figure PCTCN2022093479-appb-000016
具体地，不局限于任何理论，在胺化导向的PMP再生系统存在的情况下，稀有糖核苷酸的合成可能通过以下路线4进行：
Figure PCTCN2022093479-appb-000017
路线4
在上述路线4中，UDP-D-4n-FucNAc和UDP-D-4n-QuiNAc经PglF催化实现C-4，6脱水，经转氨酶Pat或PglE将C-4位氨化，CRS4提供转氨酶的供体PMP，推动反应向形成目标产物方向进行。
下表总结了在胺化导向的PMP再生系统存在的情况下，所用酶与CRS4结合来制备部分稀有核苷酸的部分实例：
Figure PCTCN2022093479-appb-000018
具体地，不局限于任何理论，在乙酰化导向的AcCoA再生系统存在的情况下，稀有糖核苷酸的合成可能通过以下路线5进行：
Figure PCTCN2022093479-appb-000019
路线5
在上述路线5中，UDP-diNAcBac在合成UDP-D-4n-FucNAc系统的基础上，加入PglD实现乙酰基向UDP-D-4n-FucNAc的氨基上转移，CRS4提供转氨酶的供体PMP，CRS5提供辅因子AcCoA，推动反应向形成目标产物方向进行；UDP-6-deoxy-AltdiNAc经酶(PseB,PseC,PseH)催化实现C-4，6脱水、C-5差向异构化、氨化、乙酰化，CRS4提供转氨酶的供体PMP，CRS5提供辅因子AcCoA，推动反应向形成目标产物方向进行。UDP-D-GlcNAc3NAcA经酶(WbpO,WbpB、WbpE、WbpD)催化实现C-6位氧化，C-3位脱氢，C-3位氨化、氨基乙酰化，CRS4提供转氨酶的供体PMP，CRS5提供辅因子AcCoA， 推动反应向形成目标产物方向进行。
下表总结了在乙酰化导向的AcCoA再生系统存在的情况下，所用酶与CRS5结合来制备部分稀有核苷酸的部分实例：
Figure PCTCN2022093479-appb-000020
在实施方式中，所述反应可以在pH＝7.0-8.0，例如pH＝7.5的缓冲体系下进行，优选地，所述缓冲体系为Tris缓冲体系；所述反应可以在20-40℃，优选37℃下进行。
上述反应路线中，关于上述辅因子再生系统，需要说明的是，虽然上文列出了用于糖核苷酸合成的各辅因子再生系统的具体实例，然而本申请中偶联使用的辅因子再生系统并不局限于用于上述常见糖核苷酸的合成，本领域的技术人员基于本领域的公知常识及常规技术手段，可以将本申请的辅因子再生系统应用于任何需要还原、氧化、脱水、异构化、胺化及乙酰化导向的以UDP-GlcNAc为底物的糖核苷酸的合成反应中，这些也均包括在本发明的范围内。
此外，本申请的辅因子再生系统并不局限于上述具体形式。上文中以具体选择的本文所述辅因子再生系统与复合酶系统结合为例来说明本发明的技术方案，然而，本领域的技术人员基于本领域的公知常识及常规技术手段可以将与上述类似的其他辅因子再生系统应用于糖核苷酸的合成中，这些也均包括在本发明的范围内。
此外，关于上述再生系统中所涉及的所有酶，本领域的技术人员基于本领域的公知常识及常规技术手段可以将其用其他具有相似或同等功能的酶进行替换以便用于上述化合物的合成，这些也均包括在本发明范围内。
此外，上述反应路线中，NADH再生系统(CRS1)和NADPH再生系统可以互换使用。即，上述采用NADH的再生系统的反应，替换为NADPH也同样适用，优先选择NADH再生系统；采用NADPH的再生系统，替换为NADH也同样适用，优先选择NADPH再生系统，这些也均包括在本发明的范围内。
另一方面，本发明提供辅因子再生系统在由UDP-GlcNAc制备稀有糖核苷酸中的用途，其中所述辅因子再生系统可以为选自可再生NADH、NADPH、NAD +、PMP、AcCoA的所有再生系统中的一种。
特别地，所述辅因子再生系统选自：
NADH再生系统，其由D-葡萄糖脱氢酶(BsGH)、NAD+及D-葡萄糖组成，或者由NAD +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NAD +、甲酸和甲酸脱氢酶(FDH)组成，或者由NAD +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成；优选地，NADH再生系统，其由D-葡萄糖脱氢酶(BsGH)、NAD+及D-葡萄糖组成，其中，相对于UDP-GlcNAc，NAD +的用量为0.001～1当量，优选0.01或0.02当量、D-葡萄糖的用量为1～10当量，优选3当量，BsGH:UDP-GlcNAc的质量比为1:5000～1:1，优选1:2000或1:1000；
NADPH再生系统，其由BsGH、NADP +及D-葡萄糖组成，或者由NADP +、6-磷酸葡萄糖和葡萄糖-6-磷酸脱氢酶(G6PD)组成，或者由NADP +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NADP +、甲酸和甲酸脱氢酶(FDH)组成，或者由NADP +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成；优选地，NADPH再生系统由BsGH、NADP +及D-葡萄糖组成，其中，相对于UDP-GlcNAc，NADP +的用量为0.001～1当量，优选0.01或0.02当量、D-葡萄糖的用量为1～10当量，优选3当量，BsGH:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000；
NAD +再生系统，其由NADH、丙酮酸和乳酸脱氢酶(LdhA)组成，或者由NAD +、指甲花醌和醌还原酶(NfsB)组成，或者由NAD +、O 2和NADH氧化酶组成，或者由NAD +、α-酮戊二酸和谷氨酸脱氢酶(GLDH)组成；优选地，NAD +再生系统由NADH、丙酮酸和乳酸脱氢酶(LdhA)组成，其中，相对于UDP-GlcNAc，NAD +的用量为0.001～1当量，优选0.01或0.02当量、丙酮酸的用量为1～10当量，优选3当量，LdhA:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000；
PMP再生系统，其由5'-磷酸吡哆醛(PLP)和L-谷氨酸组成；优选地，相对于UDP-GlcNAc，L-谷氨酸的用量为1～10当量，优选5当量，PLP的用量为0.01～1当量，优选0.1当量；以及
AcCoA再生系统，其由ATP、乙酸钠、CoA和AcCoA合成酶(ACS)组成，或者由CoA和S-乙酰硫代胆碱碘化物组成，或者由CoA、肉毒碱乙酰转移酶(CAT)和乙酰肉碱组成，或者由CoA、磷酸转乙酰酶(PTA)和乙酰磷酸组成，优选地，AcCoA再生系统由ATP、乙酸钠、CoA和AcCoA合成酶(ACS)组成，其中，相对于UDP-GlcNAc，CoA 0.1～1当量，优选0.5当量，ATP 2当量、乙酸钠1～10当量，优选3当量，ACS:UDP-GlcNAc质量比为1:5000～1:1，优选1:2000或1:1000。
虽然上文已经就本申请的辅因子再生系统进行了详细描述，然而本申请的辅因子再生系统并不局限于上述稀有糖核苷酸的合成，本领域的技术人员基于本领域的公知常识及常规技术手段，可以将本申请的辅因子再生系统应用于任何需要还原、氧化、胺化及乙酰化导向的合成反应中，这些也均包括在本发明的范围内。
有益效果
酶反应的最大优点是立体选择性和区域选择性高。因此，多步酶反应可以一次性进行，被称作“一锅多酶法”(One-pot multienzyme reaction,OPME)。尽管稀有糖核苷酸的生物转化途径十分复杂，但是最后一步通常为还原、氧化、胺化、乙酰化和异构化反应。除了异构化反应，其他的反应均为不可逆过程，同时需要辅因子，NADH/NADPH、NAD +/NADP +、5'-磷酸吡哆胺(PMP)或乙酰辅酶A(AcCoA)。
鉴于此，本申请设计避免逐步合成的一锅法来合成稀有糖核苷酸。本申请通过如上文所述的辅因子再生系统(Cofactor regeneration system,CRS)来产生辅因子，可以降低生产成本和产物的纯化难度。经验证，将一锅多酶法与上述辅因子再生系统偶联，反应仍可以顺利进行，从而避免了纯化不稳定、难分离的中间体，进而使酶法大量合成稀有糖核苷酸变成现实。
具体实施方式
下文中，通过具体实施方式来详细本发明的技术方案，然而这些技术方案仅用于使本领域的技术人员更好地了解本申请，而不用于限制本申请的范围。
术语
OPME:One-pot multienzyme reaction,一锅多酶；本文中也用OPME指代酶体系。
CRS:Cofactor regeneration system,辅因子再生系统；本文中也用CRS指代具体的五种辅因子再生系统。
UDP-GlcNAc:尿苷二磷酸-N-乙酰基葡萄糖胺
UDP:尿苷二磷酸
NAD +:烟酰胺腺嘌呤二核苷酸
NADH:还原型烟酰胺腺嘌呤二核苷酸
NADP +:烟酰胺腺嘌呤二核苷酸磷酸
NADPH:还原型烟酰胺腺嘌呤二核苷酸磷酸
PMP：5'-磷酸吡哆胺
AcCoA：乙酰辅酶A
ADH：alcohol dehydrogenase,醇脱氢酶
FDH：formate dehydrogenase,甲酸脱氢酶
PTDH：phosphite dehydrogenase,亚磷酸脱氢酶
G6PD：6-phosphogluconate dehydrogenase,葡萄糖-6-磷酸脱氢酶
NfsB：oxygen-insensitive NAD(P)H nitroreductase,醌还原酶
GLDH：Glutamate dehydrogenase,谷氨酸脱氢酶
PLP:5'-磷酸吡哆醛
CAT：Carnitine acetyltransferase,肉毒碱乙酰转移酶
PTA：phosphotransacetylase,磷酸转乙酰酶
CoA:coenzyme A,辅酶A
ATP:腺嘌呤核苷三磷酸
PPi:无机焦磷酸化酶
Pi:无机磷酸盐
BsGH:D-glucose dehydrogenase from Bacillus subtilis，枯草芽孢杆菌来源的D-葡萄糖脱氢酶
LdhA:lactate dehydrogenase from Leuconostoc mesenteroides,肠膜明串珠菌来源乳糖脱氢酶
ACS:AcCoA synthetase,乙酰辅酶A合成酶
NahK:N-acetylhexosamine 1-kinase,N-乙酰己糖胺激酶
GlmU:N-acetylglucosamine 1-phosphate uridylyltransferase,N-乙酰氨基葡萄糖-1-磷酸尿苷酰转移酶
PPA:inorganic pyrophosphatase,无机焦磷酸化酶
Mg534:putative dTDP-d-glucose 4,6-dehydratase from Megavirus chiliensis.来源于智利巨病毒的C-4,6脱水，C-5差向异构化双功能酶
Mg535:dTDP-4-dehydrorhamnose reductase from Megavirus chiliensis.来自于智利巨 病毒的C-3差向异构化，C-4还原的双功能酶
WbjB:UDP-GlcNAc C-4,6 dehydratase/C5-epimerase from Pseudomonas aeruginosa铜绿假单胞菌来源的UDP-GlcNAc C-4,6脱水酶/C5-差向异构酶
WbjC:C-3 epimerase/C-4 reductase from Pseudomonas aeruginosa,铜绿假单胞菌来源的C-3差向异构酶/C-4还原酶
PglF:UDP-GlcNAc C-4,6 dehydratase from Campylobacter jejuni，空肠弯曲杆菌来源的UDP-GlcNAc的C-4,6脱水酶
PreQ:UDP-GlcNAc C4-reductase from Bacillus cereus，蜡状芽孢杆菌来源的UDP-GlcNAc C-4还原酶(PreQ)
MurA:UDP-N-acetylglucosamine 1-carboxyvinyltransferase,烯醇丙酮酰转移酶
MurB:UDP-N-acetylenolpyruvylglucosamine reductase,UDP-N-乙酰戊戊酰胺还原酶
WbpO:UDP-GlcNAc dehydrogenase from Pseudomonas aeruginosa,铜绿假单胞菌来源的UDP-GlcNAc脱氢酶
UXNAcS:UDP-N-acetylxylosamine synthase Bacillus cereus,芽孢杆菌来源的UDP-N-乙酰吡喃胺合成酶
Cap5P:UDP-N-acetylglucosamine 2-epimerase from Staphylococcus aureus,金黄色葡萄球菌来源的UDP-N-乙酰葡糖胺2-差相异构酶
Cap5O:UDP-N-acetyl-D-mannosaminuronate dehydrogenasefrom Staphylococcus aureus
Pat:C4-aminotransferase from Bacillus cereus,蜡样芽孢杆菌来源的C-4氨基转移酶
PglE:UDP-N-acetylbacillosamine transaminase from Campylobacter jejuni,空肠弯曲杆菌来源的UDP-N-乙酰杆菌胺转氨酶
PglD:UDP-N-acetylbacillosamine N-acetyltransferase from Campylobacter jejuni，空肠弯曲杆菌来源的UDP-N-乙酰氨基葡萄糖N-乙酰转移酶
PseB:UDP-N-acetylglucosamine 4,6-dehydratase from Helicobacter pylori,幽门螺杆菌来源的UDP-N-乙酰氨基葡萄糖4,6-脱水酶
PseC:UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminase from Helicobacter pylori，幽门螺杆菌来源的UDP-4-氨基-4,6-双脱氧-N-乙酰氨基-β-L-阿卓糖胺转氨酶
PseH:UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine N-acetyltransferase from Helicobacter pylori，幽门螺杆菌来源的UDP-4-氨基-4,6-双脱氧-N-乙酰氨基-β-L-阿卓糖胺-N-乙酰转移酶
WbpB:UDP-N-acetyl-2-amino-2-deoxy-D-glucuronate oxidase from Pseudomonas aeruginosa,铜绿假单胞菌来源的UDP-N-乙酰基-2-氨基-2-脱氧-D-葡萄糖醛酸氧化酶
WbpE:UDP-2-acetamido-2-deoxy-3-oxo-D-glucuronate aminotransferase from Pseudomonas aeruginosa,铜绿假单胞菌来源的UDP-2-乙酰氨基-2-脱氧-3-氧代-D-葡萄糖醛酸氨基转移酶
WbpD:UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronate N-acetyltransferase from Pseudomonas aeruginosa，铜绿假单胞菌来源的UDP-2-乙酰氨基-3-氨基-2,3-双脱氧-D-葡萄糖醛酸-N-乙酰转移酶
在下文中例示了12种稀有糖核苷酸(结构如下)的合成。所采用的酶与辅因子再生系统的结合及收率总结在下表1中。
Figure PCTCN2022093479-appb-000021
表1一锅多酶法结合辅因子再生系统(CRS)合成稀有糖核苷酸
Figure PCTCN2022093479-appb-000022
在上表中，
a起始糖核苷酸。UDP-GlcNAc由D-GlcNAc按照路线1合成。
b反应起始原料为2克的UDP-GlcNAc。
c收率的计算是相对于UDP-GlcNAc，基于摩尔比计算。
本申请选取自然界中最为常见的糖核苷酸UDP-GlcNAc用来合成稀有糖核苷酸。UDP-GlcNAc是通过使用D-GlcNAc激酶(NahK，N-acetylhexosamine 1-kinase)、N-乙酰氨基葡萄糖1-磷酸尿苷酰转移酶(GlmU，N-Acetylglucosamine-1-phosphate  uridyltransferase)和无机焦磷酸酶(PPA，Inorganic pyrophosphatase)从D-GlcNAc一锅法合成的，分离产率为86％(参见以下路线1)。
Figure PCTCN2022093479-appb-000023
线路1
上文合成的12种糖核苷酸涵盖了大多数的已报道的源自UDP-GlcNAc的稀有糖核苷酸。
下面对于稀有糖核苷酸的制备进行更详细的介绍。
一、用于制备UDP-L-RhaNAc、UDP-L-PneNAc、UDP-D-QuiNAc和UDP-D-MurNAc的还原导向路线(线路2)。
Figure PCTCN2022093479-appb-000024
线路2
以NADH和NADPH为还原力的还原酶催化的还原反应是稀有糖核苷酸生物转化中最普遍的反应。从UDP-GlcNAc和UDP-Glc到UDP-L-RhaNAc、UDP-L-PneNAc的生物合成途径已经研究清楚。这一过程需要四步，包括：C-4,6脱水、C-5差向异构化、C-3差向异构化和C-4还原，涉及两个酶催化C-5差向异构(路线2)。但是，合成途径中的中间体稳定性差，容易快速水解生成单糖和UDP。
大量合成使用NADH和NADPH成本太高。由于NAD +和NADP +比NADH和NADPH价格便宜很多，为了提供强大的还原力，本申请使用来自枯草芽孢杆菌(Bacillus subtilis)的D-葡萄糖脱氢酶(BsGH)，由NAD +或NADP +制备NADH或NADPH，从而大大降低成本。
如下所示，在还原导向CRS系统中，BsGH使用NAD +或NADP +作为辅因子将D-葡萄糖氧化为葡萄糖酸，而形成NADH(CRS1)或NADPH(CRS2)用于还原反应。
Figure PCTCN2022093479-appb-000025
本申请采用从智利巨病毒(Megavirus chilensis)中克隆的Mg534和Mg535两种酶。Mg534是催化C-4,6脱水和C-5差向异构化的双功能酶，同时Mg535是催化C-3差向异构化和C-4还原的双功能酶。
在50微升的反应体系中测试了包含10mM UDP-GlcNAc、20mM Tris-HCl(PH 7.5)、Mg534、Mg535、CRS1或CRS2的分析反应。结果证实，NADH(CRS1)和NADPH(CRS2)都可以被Mg535很好地接受。令人惊奇的是，当酶促反应与CRS1或CRS2反应体系偶联时，在不到两小时的时间内，UDP-GlcNAc完全转化为UDP-L-RhaNAc，表明CRS系统可以显著促进反应的进行。同时，由于反应中中间体不稳定，在没有CRS体系的反应中检测到多个水解产物。这个现象从侧面证明了分步进行稀有糖核苷酸的合成是不切实际的。梯度浓度研究表明，0.01当量的NADP +或者0.02当量的NAD +就可以实现UDP-GlcNAc的完全转化。因此，在大规模的合成反应中，本申请选择了以低成本NAD +为原料的CRS1体系。在37℃下进行了大规模的合成反应，包含：2克UDP-GlcNAc、Mg534、Mg535和CRS1体系(0.02当量的NAD +、3当量的D-葡萄糖和BsGH)。反应完全，加入冷乙醇终止，并且由于反应的完全转化和少量辅因子NAD +的使用，经过P2柱的简单除盐即可得到纯的产品，收率为91％。
采用与UDP-L-RhaNAc相同的合成策略(酶促反应与CRS1或CRS2体系偶联)，同时实现UDP-GlcNAc到UDP-L-PneNAc的转化(路线2)。由于产物水解少，经过P2柱的简单除盐得到UDP-L-PneNAc，收率为90％。发明人还尝试在大规模的UDP-L-RhaNAc和UDP-L-PneNAc合成反应中使用高浓度的NADPH/NADH(10mM)作为辅因子，由于很强的产物抑制效应存在，反应的结果并不理想。而且，反应产物的收率低与体系中的高浓度辅因子使得产物的分离十分困难。
而对于UDP-GlcNAc到UDP-D-QuiNAc的合成，UDP-D-QuiNAc的生物合成分为两步，UDP-GlcNAc的C-4,6脱水酶和C-4还原酶催化的C-4,6脱水和还原反应(路线2)。本申请中使用空肠弯曲杆菌(Campylobacter jejuni)来源的UDP-GlcNAc的C-4,6脱水酶(PglF)，不需要外源NAD +/NADP +的脱水酶。还原反应使用的是来自蜡状芽孢杆菌(Bacillus cereus)的C-4还原酶(PreQ)。在分析反应中，UDP-GlcNAc在CRS1或CRS2体系存在下与PglF和PreQ两种酶一起孵育，结果显示，NADH和NADPH都能被酶接受，但是CRS1(NADH)可以获得更高的转化效率。因此，大规模合成反应中选择了CRS1(NADH)体系；UDP-GlcNAc在反应中完全转化，经过P2柱的简单除盐得到UDP-D-QuiNAc，收率为87％。UDP-D-QuiNAc的化学酶法合成需要至少四步反应和剧烈的反应条件，与之相比，本方法更有效率和成本优势。
UDP-D-MurNAc的合成采用大肠杆菌O157来源的烯醇丙酮酰转移酶(MurA)和UDP-N-乙炔丙酮酰葡糖胺还原酶(MurB)(路线2)。然而，MurB具有内在的NADPH氧化酶活性，在还原反应中会消耗大量的NADPH。在本申请中，设计了一锅两步的策略， 即，UDP-GlcNAc同烯醇式丙酮酸磷酸(PEP)和MurA一同孵育，UDP-GlcNAc消耗完全后，将MurB和CRS2(0.01当量NADP +、3当量D-葡萄糖、BsGH)加入反应体系中。过夜反应后，直接使用P2柱与离子交换柱纯化得到UDP-D-MurNAc，收率为85％。经过NMR分析，没有中间体混于产物中，说明CRS2体系可以推动反应进行，使得UDP-GlcNAc完全转化为UDP-D-MurNAc。
上述还原导向的CRS系统还可包括如下所示的再生系统，但不以此为限：
Figure PCTCN2022093479-appb-000026
二、用于制备UDP-D-GlcNAcA、UDP-D-XylNAc和UDP-D-ManNAcA的氧化导向线路(线路3)。
Figure PCTCN2022093479-appb-000027
线路3
以NAD +或NADP +为辅因子，由脱氢酶催化的生物氧化是自然界中合成羧酸糖及其衍生物的主要途径。
在比较研究中，发明人尝试使用来自铜绿假单胞菌(Pseudomonas aeruginosa)来源的UDP-GlcNAc脱氢酶(WbpO)合成UDP-D-GlcNAcA。但是，高浓度的NAD +(10mM)对反应有强烈的抑制作用，反应仅达到20％的转化率，延长反应时间或者添加更多的酶不能明显的提升反应收率。
在本申请中新设计的一锅法反应中(路线3)，UDP-GlcNAc的生物氧化与NAD +的再生体系(CRS3)结合使用。
如下所示，在CRS3中，来自肠系膜明串珠菌(Leuconostoc mesenteroides)的乳酸脱氢酶(LdhA)将丙酮酸还原为D-乳酸，同时将NADH氧化为NAD +。
Figure PCTCN2022093479-appb-000028
发明人发现，当反应体系中加入CRS3(0.01当量NAD +、3当量丙酮酸、LdhA)时，WbpO可以在不到3小时将UDP-GlcNAc消耗完，转化为UDP-D-GlcNAcA。这个相比需要有毒催化剂和剧烈反应条件(100℃和48小时)的化学氧化反应要快很多。由于反应完全转化，经过P2柱的简单除盐即可得到纯的产品，收率为90％。
接下来，由UDP-GlcNAc通过两步酶促反应制备UDP-D-XylNAc(路线3)，来自蜡状芽孢杆菌的UXNAcS催化UDP-D-GlcNAcA脱羧生成UDP-D-XylNAc。发明人发现，UDP-D-GlcNAcA可以在催化量的NAD +存在的条件下脱羧完全转化生成UDP-D-XylNAc。因此，在大规模合成中，UDP-GlcNAc在CRS3存在下与WbpO一起孵育，当UDP-GlcNAc 转化完全后，直接将UXNAcS加入到反应体系中，脱羧生成UDP-D-XylNAc。经过P2柱的简单除盐即可得到纯的产品，收率为88％。
UDP-D-ManNAcA是由UDP-GlcNAc出发，在UDP-GlcNAc-2-差向异构酶和UDP-D-ManNAc脱氢酶的存在下合成的(路线3)，反应中使用的两种酶(Cap5P和Cap5O)来自金黄色葡萄球菌。Cap5P催化UDP-GlcNAc和UDP-D-ManNAc之间的相互转化，然而，该反应对UDP-D-ManNAc的形成非常不利，平衡后，只有不到10％的UDP-GlcNAc可以转化为UDP-D-ManNAc。Cap5O是一种UDP-D-ManNAc脱氢酶，可以特异性的识别UDP-D-ManNAc，推动平衡向UDP-D-ManNAc移动。因此，本申请设计了一锅法，包含：Cap5P、Cap5O、CRS3，用来从UDP-GlcNAc合成UDP-D-ManNAcA。尽管该反应进行不完全，由于UDP-D-ManNAcA较原料与中间产物拥有多一个电荷，可以使用离子交换树脂很容易的将它从体系中分离出来，收率为51％。
上述氧化导向的再生系统还可包括如下所示的再生系统，但不以此为限：
Figure PCTCN2022093479-appb-000029
三、制备UDP-D-4n-FucNAc和UDP-D-4n-QuiNAc的胺化导向路线(路线4)。
Figure PCTCN2022093479-appb-000030
路线4
UDP-D-4n-FucNAc、UDP-D-4n-QuiNAc的生物合成从UDP-GlcNAc开始，首先由UDP-GlcNAc-C4,6脱水酶催化脱水反应，然后通过转氨酶将UDP-4-keto-6-deoxy-GlcNAc的C-4位胺化生成UDP-D-4n-FucNAc、UDP-D-4n-QuiNAc(路线4)。转氨酶的供体是5'-磷酸吡哆胺(pyridoxamine-5'-phosphate,PMP)，它由5'-磷酸吡哆醛(PLP)和L-谷氨酸再生(如下CRS4所示)，无需外源酶。
Figure PCTCN2022093479-appb-000031
为了合成UDP-D-4n-FucNAc，使用了来自蜡状芽孢杆菌(Bacillus cereus)的C-4氨基转移酶(Pat)。由于PglF的活性总是被转氨酶抑制，反应首先将UDP-GlcNAc和PglF一起孵育，当UDP-GlcNAc完全转化后，将CRS4和Pat添加到反应体系中用来合成UDP-D-4n-FucNAc。由于氨基转化反应不完全，因此使用多达10倍当量的L-谷氨酸来推动胺化反应，但是，高浓度的L-谷氨酸很难直接移除。为了简化产物的纯化，本申请使用来自植物乳杆菌(Lactobacillus plantarum)的L-谷氨酸脱羧酶(GadB)将剩余的L-谷氨酸水解为氨基丁酸，氨基丁酸很容易纯化移除。虽然胺化反应进行不完全，但是由于产物具有额外的氨基，可以通过离子交换树脂可以轻松的除去反应的中间体，得到UDP-D-4n-FucNAc，收率为70％。同样的方式，UDP-D-4n-QuiNAc由UDP-GlcNAc转化获得，收率为65％。
四、用于制备UDP-diNAcBac、UDP-6-deoxy-AltdiNAc和UDP-D-GlcNAc3NacA的N-乙酰化导向路线(路线5)。
Figure PCTCN2022093479-appb-000032
路线5
稀有糖核苷酸的N-乙酰化由乙酰转移酶催化，使用乙酰辅酶A(AcCoA)作为乙酰供体。然而，AcCoA太昂贵而无法用来进行大规模合成。最初，发明人尝试对氨基糖核苷酸进行化学乙酰化来合成UDP-diNAcBac、UDP-6-deoxy-AltdiNAc、UDP-D-GlcNAc3NacA。然而，发现不少副产物生成，因此，本申请中采用酶促乙酰化反应。由于CoA比AcCoA便宜很多，因此采用再生系统CRS5(如下所示)产生AcCoA，其中AcCoA是通过AcCoA合成酶(ACS)由乙酸钠、CoA和ATP合成的。
Figure PCTCN2022093479-appb-000033
为了尝试酶促乙酰化合成UDP-diNAcBac，发明人克隆了来自空肠弯曲杆菌(Campylobacter jejuni)的参与UDP-diNAcBac生物合成的乙酰转移酶(PglD)。除了天然底物，发明人发现PglD同样可以接受UDP-D-4n-VioNH 2作为反应底物。10mM的UDP-D-4n-QuiNAc与PglD和CRS5一起孵育，可以将UDP-D-4n-QuiNAc完全转化为UDP-diNAcBac。CoA的催化量使用降低大规模合成的成本，重要的是没有副反应的进行。为了避免分离中间体UDP-D-4n-QuiNAc，本申请直接进行一锅法，其中使用了双CRS。首先，UDP-GlcNAc与PglF一起孵育，当没有UDP-GlcNAc剩余时，添加CRS4(5当量的L-谷氨酸、0.05当量的PLP)和PglE，然后，加入CRS5(0.002当量的CoA、2当量ATP、3当量乙酸钠、ACS)和PglD。反应完全后，加入GadB水解过量的L-谷氨酸，经过P2和离子交换树脂纯化得到UDP-diNAcBac，产率为84％。
同样的方式，UDP-6-deoxy-AltdiNAc由UDP-GlcNAc制备，产率为85％。
为了制备UDP-D-GlcNAc3NacA，从铜绿假单胞菌中克隆了四种酶(WbpO、WbpB、WbpE、WbpD)(路线5)。这四种酶的催化分四步(氧化、氧化、胺化和乙酰化)，每个反应都需要辅因子。WbpB使用NAD +作为辅因子，但它可以在反应过程中再生。大规模合成中，UDP-D-GlcNAcA的制备如前文所述，然后与0.1当量NAD +、CRS4(10当量的L-谷氨酸、0.05当量的PLP)、CRS5(0.01当量的CoA、2当量ATP、3当量乙酸钠、ACS)一起反应过夜，加入GadB和碱性磷酸酶水解杂质，降低纯化难度。由于反应不完全，UDP-D-GlcNAc3NacA的最终收率为41％。
上述乙酰化导向的再生系统还可以包括如下所示的再生系统，但不以此为限：
Figure PCTCN2022093479-appb-000034
实施例
在下文中使用的所有相关的酶，全部通过大肠杆菌表达系统制备，并使用Ni-NTA纯化。具体地，GlmU、PPA、MurA和MurB从大肠杆菌O157通过PCR扩增得到的。除了前文中提到的酶外，这里还列出了其他基因来源：UDP-GlcNAc C-4,6脱水酶/C5-差向异构酶(WbjB)和C-3差向异构酶/C-4还原酶(WbjC)来自铜绿假单胞菌(Pseudomonas aeruginosa)；PseB、PseC和PseH来自幽门螺杆菌(Helicobacter pylori)。除从E.coli中PCR得到的基因，本申请中使用的其他基因都是人工合成的。基因合成服务由金斯瑞(中国南京)或生工生物(中国上海)提供。
所有基因都被克隆到pET-28a载体中，以产生在N端或C端具有六个组氨酸(His)标签的重组蛋白。将经鉴定正确的质粒转化到大肠杆菌BL21(DE3)中以进行蛋白质表达。将含有重组质粒的大肠杆菌BL21(DE3)细胞在两升含有50ug/ml卡那霉素的Luria-Bertani(LB)培养基中在37℃、200rpm的旋转振荡器中培养。当OD值达到0.8时加入0.2mM的异丙基-β-D-硫代半乳糖苷(IPTG)，16℃下过夜诱导蛋白表达。通过在7000rpm下离心10min收集细胞。将细胞沉淀重新悬浮在裂解缓冲液(50mM Tris-HCl缓冲液、300mM NaCl、10mM咪唑；pH 7.5)中。用微流化器破碎细胞并将裂解液12,000g离心30min以去除细胞碎片。使用Ni-NTA琼脂糖柱纯化带His标签的蛋白。在纯化之前，柱子用裂解缓冲液(50mM Tris-HCl、300mM NaCl、10mM咪唑；pH 7.5)平衡。 用2倍柱体积的裂解缓冲液洗涤柱子并用洗脱缓冲液(50mM Tris-HCl、300mM NaCl、300mM咪唑；pH 7.5)洗脱目标蛋白。蛋白质浓度用BCA蛋白质检测试剂盒测定。以上虽然描述了本申请中各酶的来源，然而其来源途径并不限于此，只要其可以在本申请中所意图实现的功能即可。
实施例1：UDP-L-RhaNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.02当量NAD +、3当量D-葡萄糖、3毫克Mg534、3毫克Mg535和1毫克BsGH。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，产物收率为91％。
实施例2：UDP-L-PneNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.02当量NAD +、3当量D-葡萄糖、5毫克WbjB、5毫克WbjC和1毫克BsGH。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，产物收率为90％。
实施例3：UDP-D-QuiNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc和20毫克PglF。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.02当量NAD +、3当量D-葡萄糖、5毫克PreQ和1毫克BsGH。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，产物收率为87％。
实施例4：UDP-D-MurNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、1.5当量PEP和3毫克MurA。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.02当量NAD +、3当量D-葡萄糖、5毫克MurB和1毫克BsGH。反应在37℃下进行。反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为85％。
实施例5：UDP-D-GlcNAcA的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.02当量NAD +、3当量丙酮酸、5毫克WbpO和1毫克LdhA。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，产物收率为90％。
实施例6：UDP-XylNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.02当量NAD +、3当量丙酮酸、5毫克WbpO和1毫克LdhA。反应在37℃下进行，UDP-GlcNAc转化完全后，加入3毫克UXNAcS。反应继续在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，产物收率为88％。
实施例7：UDP-D-ManNAcA的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.1当量NAD +、3当量丙酮酸、15毫克Cap5P、15毫克Cap5O和1毫克LdhA。反应在37℃下进行，一旦反应停止继续进行，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为51％。
实施例8：UDP-D-4n-FucNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc和20毫克PglF。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.05当量的PLP、10当量的L-谷氨酸和5毫克Pat。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为70％。
实施例9：UDP-D-4n-QuiNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc和20毫克PglF。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.05当量的PLP、10当量的L-谷氨酸和10毫克PglE。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为65％。
实施例10：UDP-diNAcBAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc和20毫克PglF。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.05当量的PLP、10当量的L-谷氨酸和10毫克PglE。反应在37℃下进行数小时，加入2当量ATP、3当量乙酸钠、0.005当量CoA、5毫克PglD和ACS。反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为84％。
实施例11：UDP-6-deoxy-AltdiNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.05当量的PLP、5当量的L-谷氨酸、2当量ATP、3当量乙酸钠、0.005当量CoA、50毫克PseB、50毫克PseC、20毫克PseH和ACS。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为85％。
实施例12：UDP-D-GlcNAc3NAcA的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、2克UDP-GlcNAc、0.02当量NAD +、3当量丙酮酸、5毫克WbpO和1毫克LdhA。反应在37℃下进行，UDP-GlcNAc转化完全后，加入0.1当量NAD +、0.05当量PLP、5当量L-谷氨酸、3当量ATP、3当量乙酸钠、0.01当量CoA、50毫克WbpE、50毫克WbpB10毫克WbpD和ACS。反应在37℃下进行，反应完全后，加入等体积的冷乙醇终止反应，离心除去不溶性沉淀后，上清液经P-2柱浓缩纯化，再用阴离子交换树脂纯化，由氯化钠洗脱，产物收率为41％。
以上制备得到的糖核苷酸的 1H NMR数据列于下表2中。
表2
Figure PCTCN2022093479-appb-000035
Figure PCTCN2022093479-appb-000036
Figure PCTCN2022093479-appb-000037
综上，本申请成功地开发了高效制备稀有糖核苷酸的通用方法。通过使用不同克隆来源的糖核苷酸合成相关酶，复杂的天然合成路线被重新组合进行合成反应。通过添加辅因子再生体系提供过量的辅因子，推动最后的不可逆反应，生成目标糖核苷酸。通过这种方法，由常见的糖核苷酸UDP-GlcNAc大规模、高效地制备了12个难以获得的稀有糖核苷酸，整个过程无需繁琐的纯化操作。更为重要的是，昂贵辅因子的催化量使用降低了大规模合成的成本。由于大多数报道的稀有糖核苷酸的最后一个生物转化步骤涉及还原、氧化、胺化或者乙酰化反应，因此本发明的这种方法可以很容易的扩展到其他生物合成途径明了的稀有糖核苷酸，从而使得本发明的方法具有适应性强的优势。

Claims (10)

  1. 一种制备稀有糖核苷酸的方法，所述方法包括：在酶和辅因子再生系统共同存在下使尿苷二磷酸-N-乙酰基葡萄糖胺(UDP-GlcNAc)发生酶催化反应得到稀有糖核苷酸，
    其中，UDP-GlcNAc的结构如下：
    Figure PCTCN2022093479-appb-100001
    所述稀有糖核苷酸由下式表示：
    Figure PCTCN2022093479-appb-100002
    其中，R 1为AcNH-，
    R 2和R 3各自独立地为OH、NH 2、AcNH-和OCH(CH 3)COOH，
    R 4选自CH 2OH、COOH、CH 3以及H，
    R 5为UDP，
    UDP表示尿苷二磷酸基。
  2. 根据权利要求1所述的方法，其中，所述酶为选自以下各组中的一组或多组：
    组别 酶 OPME2 Mg534,Mg535 OPME3 WbjB,WbjC OPME4 PglF,PreQ OPME5 MurA,MurB OPME6 WbpO OPME7 WbpO,UXNAcS OPME8 Cap5P,Cap5O OPME9 PglF,Pat OPME10 PglF,PglE OPME11 PglF,PglE,PglD OPME12 PseB,PseC,PseH OPME13 WbpO,WbpB,WbpE,WbpD
    。
  3. 根据权利要求1或2所述的方法，其中，所述辅因子再生系统选自如下系统之一：
    (1)NADH再生系统(CRS1)：由NAD +、D-葡萄糖和D-葡萄糖脱氢酶(BsGH)组成，或者由NAD +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NAD +、甲酸和甲酸脱氢酶(FDH)组成，或者由NAD +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成；
    (2)NADPH再生系统(CRS2)：由NADP +、D-葡萄糖和BsGH组成，或者由NADP +、6-磷酸葡萄糖和葡萄糖-6-磷酸脱氢酶(G6PD)组成，或者由NADP +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NADP +、甲酸和甲酸脱氢酶(FDH)组成，或者由NADP +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成；
    (3)NAD +再生系统(CRS3)：由NAD +、丙酮酸和D-葡萄糖脱氢酶(LdhA)组成，或者由NAD +、指甲花醌和醌还原酶(NfsB)组成，或者由NAD +、O 2和NADH氧化酶组成，或者由NAD +、α-酮戊二酸和谷氨酸脱氢酶(GLDH)组成；
    (4)PMP再生系统(CRS4)：由L-谷氨酸和5'-磷酸吡哆醛(PLP)组成；
    (5)AcCoA再生系统(CRS5)：由CoA、ATP、乙酸钠，以及AcCoA合成酶(ACS)组成，或者由CoA和S-乙酰硫代胆碱碘化物组成，或者由CoA、肉毒碱乙酰转移酶(CAT)和乙酰肉碱组成，或者由CoA、磷酸转乙酰酶(PTA)和乙酰磷酸组成，
    其中，上述辅因子再生系统中，CRS1和CRS2在反应中可以互换使用。
  4. 根据权利要求1至3中任一项所述的方法，所述稀有糖核苷酸选自以下各项之一：
    Figure PCTCN2022093479-appb-100003
  5. 根据权利要求3所述的方法，其中，所述方法如下进行：
    UDP-GlcNAc在Mg534、Mg535和CRS1辅因子再生系统共同存在下发生酶催化反应得到UDP-L-RhaNAc；或者
    UDP-GlcNAc在WbjB、WbjC和CRS1辅因子再生系统共同存在下发生酶催化反应得到UDP-L-PneNAc；或者
    UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PreQ和CRS1辅因子再生系统发生酶催化反应得到UDP-D-QuiNAc；或者
    UDP-GlcNAc先在MurA与PEP存在下发生酶催化反应，再加入MurB和CRS2辅因子再生系统发生酶催化反应得到UDP-D-MurNAc；或者
    UDP-GlcNAc在WbpO和CRS3辅因子再生系统共同存在下发生酶催化反应得到UDP-D-GlcNAcA；或者
    UDP-GlcNAc先在WbpO、UXNAcS和CRS3辅因子再生系统共同存在下发生酶催化反应，再加入UXNAcS发生酶催化反应得到UDP-D-XylNAc；或者
    UDP-GlcNAc在Cap5P、Cap5O和CRS3辅因子再生系统共同存在下发生酶催化反应得到UDP-D-ManNAcA；或者
    UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入Pat和CRS4辅因子再生系统发生酶催化反应得到UDP-D-4n-FucNAc；或者
    UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PglE和CRS4辅因子再生系统发生酶催化反应得到UDP-D-4n-QuiNAc；或者
    UDP-GlcNAc先在PglF存在下发生酶催化反应，再加入PglE和CRS4辅因子再生系统发生酶催化反应，然后再加入PglD和CRS5辅因子再生系统发生酶催化反应得到UDP-diNAcBac；或者
    UDP-GlcNAc在PseB、PseC、PseH和CRS4和CRS5辅因子再生系统共同存在下发生酶催化反应得到UDP-6-deoxy-AltdiNAc；或者
    UDP-GlcNAc先在WbpO和CRS3辅因子再生系统共同存在下发生酶催化反应，再加入WbpB、WbpE、WbpD和CRS4和CRS5辅因子再生系统发生酶催化反应得到UDP-D-GlcNAc3NAcA。
  6. 根据权利要求1至3中任一项所述的方法，其中，所述反应在pH＝7.0-8.0的缓冲体系下进行。
  7. 根据权利要求6所述的方法，其中，所述缓冲体系为Tris缓冲体系。
  8. 根据权利要求1至3中任一项所述的方法，其中，所述反应在20-40℃下进行。
  9. 根据权利要求8所述的方法，其中，所述反应在37℃下进行。
  10. 辅因子再生系统在由UDP-GlcNAc制备稀有糖核苷酸中的用途，其中所述辅因子再生系统选自：
    NADH再生系统，其由D-葡萄糖脱氢酶(BsGH)、NAD +及D-葡萄糖组成，或者由NAD +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NAD +、甲酸和甲酸脱氢酶(FDH)组成，或者由NAD +、亚磷酸盐和亚磷酸脱氢酶(PTDH) 组成；
    NADPH再生系统，其由BsGH、NADP +及D-葡萄糖组成，或者由NADP +、6-磷酸葡萄糖和葡萄糖-6-磷酸脱氢酶(G6PD)组成，或者由NADP +、脂肪醇(例如乙醇、丙醇、异丙醇或异丁醇)和醇脱氢酶(ADH)组成，或者由NADP +、甲酸和甲酸脱氢酶(FDH)组成，或者由NADP +、亚磷酸盐和亚磷酸脱氢酶(PTDH)组成；
    NAD +再生系统，其由NADH、丙酮酸和乳酸脱氢酶(LdhA)组成，或者由NAD +、指甲花醌和醌还原酶(NfsB)组成，或者由NAD +、O 2和NADH氧化酶组成，或者由NAD +、α-酮戊二酸和谷氨酸脱氢酶(GLDH)组成；
    PMP再生系统，其由5'-磷酸吡哆醛(PLP)和L-谷氨酸组成；以及
    AcCoA再生系统，其由ATP、乙酸钠、CoA和AcCoA合成酶(ACS)组成，或者由CoA和S-乙酰硫代胆碱碘化物组成，或者由CoA、肉毒碱乙酰转移酶(CAT)和乙酰肉碱组成，或者由CoA、磷酸转乙酰酶(PTA)和乙酰磷酸组成。
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