WO2023138545A1 - 一种由GDP-Mannose合成稀有糖类核苷酸的方法 - Google Patents

一种由GDP-Mannose合成稀有糖类核苷酸的方法 Download PDF

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WO2023138545A1
WO2023138545A1 PCT/CN2023/072484 CN2023072484W WO2023138545A1 WO 2023138545 A1 WO2023138545 A1 WO 2023138545A1 CN 2023072484 W CN2023072484 W CN 2023072484W WO 2023138545 A1 WO2023138545 A1 WO 2023138545A1
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gdp
reaction
mannose
enzyme
equivalents
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文留青
张家彬
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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
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of glycobiology, and in particular relates to a method for synthesizing rare sugar nucleotides.
  • Glycans also known as polysaccharides or oligosaccharides, form one of three classes of biopolymers in nature along with nucleic acids and proteins. Glycans or glycoconjugates are well known to serve as ligands for cell-cell interactions or targets for toxins, antibodies and microorganisms. In living cells, monosaccharides are activated by nucleosides via mono- or diphosphates to form sugar nucleotides that serve as glycosylation donors for glycosyltransferases. Hydrolysis of sugar nucleotides is an energy-generating reaction that provides the driving force for glycosidic bond formation. As substrates of glycosyltransferases, sugar nucleotides have been widely used in the identification of glycosyltransferases and in the study of the biosynthesis of glycans and glycoconjugates.
  • sugar nucleotides Due to the importance of sugar nucleotides in glycobiology and glycochemistry, chemical and enzymatic methods have been developed to prepare sugar nucleotides.
  • the chemical method can be divided into two ways, including the condensation of sugar-1-phosphate with activated nucleoside monophosphate and the direct coupling of activated sugar with nucleoside diphosphate.
  • the low solubility of sugar nucleotides in organic solvents, the presence of polar groups, and the instability of glycosidic linkages under severe reaction conditions make chemical synthesis very challenging. Poor regioselectivity often requires multiple protection/deprotection steps, resulting in low yields.
  • enzymatic synthesis yields only products exhibiting only the native-type anomer configuration.
  • sugar nucleotides are produced through salvage or de novo biosynthetic pathways. Includes phosphorylation of sugars by kinases and pyrophosphorylation by pyrophosphorylases. However, this strategy has only successfully produced a very small number of common natural sugar nucleotides. Alternatively, de novo biosynthesis of sugar nucleotides from existing common sugar nucleotides produces most sugar nucleotides in nature. The process involves single or multiple reactions, including dehydration, isomerization, epimerization, oxidation, reduction, amination, and acetylation reactions. It is generally accepted that such complex biotransformations are impractical for synthetic applications due to complex reaction schemes, difficult purification, and high preparation costs.
  • 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 GDP-Mannose (guanosine diphosphate mannose) to prepare rare sugar nucleotides on a large scale and with high yield.
  • GDP-Mannose guanosine diphosphate mannose
  • the invention provides a method for preparing rare sugar nucleotides, the method comprising: using GDP-Mannose as the starting sugar nucleotide at 25-40°C, preferably at 30-37°C, and preparing rare sugar nucleotides by reaction in the presence of enzymes and cofactor regeneration systems (CRS) in a one-pot method.
  • CRS cofactor regeneration systems
  • the rare sugar nucleotide is represented by the following formula I:
  • R is selected from H and hydroxyl
  • R is selected from hydroxyl, amino and acetamido
  • R3 is selected from methyl and carboxyl.
  • the enzyme is selected from the enzyme systems of the following groups:
  • the cofactor regeneration system is selected from the following cofactor regeneration systems:
  • the CRS1 cofactor regeneration system when employed, it consists of 0.01 or 0.02 equivalents of NAD + , 3 equivalents of D-glucose and 1 mg or 2 mg of BsGH relative to GDP-Mannose;
  • the CRS2 cofactor regeneration system When using the CRS2 cofactor regeneration system, it consists of 0.01 or 0.02 equivalents of NADP+, 3 equivalents of D-glucose and 1 mg or 2 mg of BsGH relative to GDP-Mannose;
  • GDP-Mannose can be synthesized by the following route 1:
  • GDP-Mannose was generated by a mannose (NAHK) to generate a mANNOSE-P-P under the action of D-Mannose kinase (NAHK), and then reacted with tricephosphates (GTP) reaction under the action of mannose 1-phosphate gytinase (MANC).
  • GTP mannose 1-phosphate gytinase
  • OSE mannose 1-phosphate gytinase
  • PPI indicates scorposphosphates
  • PI represents inorganic phosphate
  • PPA represents inorganic caramel phosphatase.
  • the starting sugar nucleotide GDP-Mannose used in this application is also commercially available.
  • the rare sugar nucleotide is selected from one of the following:
  • the synthesis of the rare sugar nucleotides is carried out through the following route 2:
  • GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS1 and GFS are added, and the product GDP-L-Fucose is further obtained through an enzyme-catalyzed reaction.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS2 and RMD are added to further obtain the product GDP-D-Rha through an enzyme-catalyzed reaction.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS2 and GTS are added to further obtain the product GDP-6-deoxy-D-Tal through an enzyme-catalyzed reaction.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS2, 4, ColC, and ColD are added to further obtain the product GDP-L-Colitose through an enzyme-catalyzed reaction.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • the synthesis of the rare sugar nucleotides is carried out by the following route 3:
  • GDP-ManA was prepared by combining SjGMD (OPME6) with CRS3.
  • GDP-Mannose undergoes an enzyme-catalyzed reaction in the presence of SjGMD and CRS3 to obtain the product GDP-D-ManA.
  • the aforementioned enzyme-catalyzed reaction is performed at 30°C.
  • the synthesis of the rare sugar nucleotides is carried out through the following route 4:
  • GDP-Man first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS4 and PerA are added to further obtain the product GDP-D- PerNH2 through an enzyme-catalyzed reaction.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • the synthesis of the rare sugar nucleotides is carried out through the following route 5:
  • GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD. After the reaction is completed, CRS4 and PerA are added for the reaction. After the reaction is completed, CRS5 and PerB are added for the enzyme-catalyzed reaction to obtain the product GDP-D-PerNAc.
  • the aforementioned enzyme-catalyzed reaction is carried out at 37°C.
  • the cofactor regeneration system of the present application has been described in detail above using specific embodiments, the cofactor regeneration system of the present application is not limited to the synthesis of the above-mentioned rare sugar nucleotides. Those skilled in the art can apply the cofactor regeneration system of the present application based on common knowledge and conventional technical means in the field to any synthesis reaction of other sugar nucleotides that require reduction, oxidation, dehydration, isomerization, amination, and acetylation-oriented sugar nucleotides as substrates. These are also included within the scope of the present invention.
  • the cofactor regeneration systems may also include NAD + , NAD(P)H and AcCoA regeneration systems as shown below:
  • the present application chooses the combination of the cofactor regeneration system described herein and the complex enzyme system as the optimal solution.
  • Those skilled in the art can apply other cofactor regeneration systems (for example, as shown above) to the synthesis of related compounds based on common knowledge and conventional technical means in this field, and these are also included in the scope of the present invention.
  • NADH regeneration system can be reacted with the NADPH regeneration system Replace each other, 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 performed at one time, which is called "One-pot multienzyme reaction (OPME)".
  • OPME One-pot multienzyme reaction
  • 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, thereby avoiding the purification of unstable and difficult-to-separate intermediates, and making the large-scale synthesis of rare sugar nucleotides by enzymatic method a reality.
  • CRS cofactor regeneration system
  • ManC Mannose-1-phosphate guanylyltransferase
  • PPA pyrophosphatase
  • GMD GDP-D-Mannose-4,6-dehydratase
  • GFS 3,5-epimerase-4-reductase
  • PerA GDP-4-keto-6-deoxy-d-mannose-4-aminotransferase
  • PerB GDP-perosamine N-acetyl transferase
  • 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.
  • lysis buffer 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole; pH 7.5.
  • OPME One-pot multienzyme reaction, a 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;
  • NAD + Nicotinamide adenine dinucleotide
  • NADH reduced nicotinamide adenine dinucleotide
  • NADP + Nicotinamide adenine dinucleotide phosphate
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • 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 D-Mannose kinase from Bifidobacterium longum, D-mannose kinase, derived from Bifidobacterium longum.
  • PPA pyrophosphatase
  • pyrophosphorylase amplified from Escherichia coli O157 by PCR.
  • ManC Mannose-1-phosphate guanylyltransferase, mannose 1-phosphate guanylyltransferase; amplified from E. coli O157 by PCR
  • GMD GDP-D-Mannose-4,6-dehydratase; Guanosine 5′-diphosphate-D-mannose C-4,C-6 dehydratase from Pseudomonas aeruginosa
  • SjGMD GDP-D-Mannose dehydrogenase from Saccharina japonica; Guanosine 5′-diphosphate-D-mannose C-6 dehydrogenase, derived from kelp.
  • RMD GDP-4-keto-6-deoxy-D-mannose reductase; Guanosine 5′-diphosphate-4-keto-6-deoxy-D-mannose C-4 reductase from Pseudomonas aeruginosa
  • GFS GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, guanosine 5′-diphosphate-4-keto-6-deoxy-D-mannose C-3, C-5 isomerase, C-4 reductase bifunctional enzyme; amplified from Escherichia coli O157 by PCR.
  • GTS GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans; Guanosine 5′-diphosphate-6-deoxy-D-talose synthetase, derived from actinomycetes.
  • ColC C-5 epimerase/C-4 reductase, C-5 epimerase/C-4 reductase bifunctional enzyme; amplified from Escherichia coli O55 by PCR method.
  • ColD GDP-4-keto-6-deoxy-D-mannose-3-dehydrase; guanosine 5′-diphosphate-4-keto-6-deoxy-D-mannose C-3 dehydratase; amplified from Escherichia coli O55 by PCR.
  • GDP-Mannose the most common sugar nucleotide in nature, to synthesize rare sugar nucleotides.
  • GDP-Mannose was synthesized in a one-pot procedure starting from D-Mannose by using D-Mannose kinase (NahK), mannose 1-phosphate guanylyltransferase (ManC) and inorganic pyrophosphatase (PPA); the isolated yield was 86% (see Scheme 1 below).
  • the seven sugar nucleotides synthesized below cover most of the reported rare sugar nucleotides derived from GDP-Mannose.
  • the present application adopts two enzymes, dehydratase (GMD) derived from Pseudomonas aeruginosa and 3,5-epimerase-4-reductase (GFS) derived from Escherichia coli O157.
  • GMD dehydratase
  • GFS 3,5-epimerase-4-reductase
  • GMD is a C-4,6 dehydratase
  • GFS is a bifunctional enzyme of C-5 epimerization and C-4 reductase.
  • NADH and NADPH in large quantities is too high.
  • 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 to prepare NADH or NADPH from NAD + or NADP + , thereby greatly reducing production 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.
  • GDP-L-Colitose was obtained from GDP-Mannose (route 2). Due to less hydrolysis of the product, GDP-L-Colitose was obtained through simple desalination of the P2 column with a yield of 86%.
  • the inventors also tried to use high concentrations of NADPH/NADH (10mM) as cofactors in the large-scale synthetic reaction of GDP-L-Fucose and GDP-L-Colitose, but the result of the reaction was not ideal due to the strong product inhibition effect. Moreover, the low yield of reaction products and the high concentration of cofactors in the system make the separation of products very difficult.
  • GDP-D-6-deoxy-Talose was obtained from GDP-Mannose (Route 2) using a similar synthesis strategy to GDP-D-Rha (enzymatic reaction using OPME2 enzyme system (GMD, GTS), coupled with CRS1 or CRS2 system). Due to less hydrolysis of the product, GDP-D-6-deoxy-Talose was obtained through simple desalination of the P2 column with a yield of 91%.
  • lactate dehydrogenase (LdhA) from Leuconostoc mesenteroides reduces pyruvate to D-lactate while oxidizing NADH to NAD + .
  • the biosynthesis of GDP-PerNH 2 starts from GDP-Mannose.
  • the dehydration reaction is catalyzed by GDP-Mannose-C4,6 dehydratase, and then the C-4 position of GDP-4-keto-6-deoxy-mAN is aminated by transaminase to generate GDP-PerNH 2 (route 4).
  • the donor for the transaminase is pyridoxamine-5'-phosphate (PMP), which is regenerated from pyridoxal-5'-phosphate (PLP) and L-glutamate (shown below for CRS4), without exogenous enzymes.
  • this application uses L-glutamic acid decarboxylase (GadB) from Lactobacillus plantarum to hydrolyze the remaining L-glutamic acid into aminobutyric acid, which is easy to purify and remove.
  • GadB L-glutamic acid decarboxylase
  • AcCoA acetyl-CoA
  • AcCoA is too expensive to be used for large-scale synthesis. Since 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).
  • GDP-Mannose was incubated with GMD, and when no GDP-Mannose remained, CRS4 (5 equiv of L-glutamic acid, 0.05 equiv of PLP) and PerA were added, then, CRS5 (0.002 equiv of CoA, 2 equiv of ATP, 3 equiv of sodium acetate, ACS) and PerB were added. After the reaction was complete, excess L-glutamic acid was hydrolyzed by adding GadB, and purified by P2 and ion exchange resin to obtain GDP-PerNAc with a yield of 82%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose, and 10 mg of GMD, and the reaction was carried out at 37°C. After the conversion of GDP-Mannose was completed, the reaction solution was heated to 60° C. for 15 minutes to quench the reaction, and 0.02 equivalents of NADP + , 3 equivalents of D-glucose, 20 mg of GFS and 1 mg of BsGH were added. 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 eluted with sodium chloride. The product yield was 93%.
  • Embodiment 2 Large-scale synthesis and purification of GDP-D-Rha:
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose, and 10 mg of GMD, and the reaction was carried out at 37°C. After the GDP-Mannose conversion was complete, 0.01 equivalents of NAD + , 3 equivalents of D-glucose, 5 mg of RMD and 1 mg of BsGH were added. After the reaction is complete, add an equal volume The reaction was terminated by cold ethanol, and the insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified by P-2 column, and the product yield was 96%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose, and 10 mg of GMD, and the reaction was carried out at 37°C. After the conversion of GDP-Mannose was completed, the reaction solution was heated to 60° C. for 15 minutes to quench the reaction, and 0.01 equivalent of NADP + , 3 equivalents of D-glucose, 20 mg of GTS and 1 mg of BsGH were added. 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 eluted by sodium chloride. The product yield was 91%.
  • Embodiment 4 Large-scale synthesis and purification of GDP-L-Colitose:
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose, and 10 mg of GMD, and the reaction was carried out at 37°C. After the GDP-Mannose conversion was complete, the reaction solution was heated to 60°C for 15 minutes to quench the reaction, and 0.1 equivalent of PLP, 5 equivalents of L-glutamic acid, 0.01 equivalent of NADP + , 3 equivalents of D-glucose, 20 mg of ColC, 20 mg of ColD and 1 mg of BsGH were added. 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, and then purified by anion exchange resin, eluted by sodium chloride, and the product yield was 86%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose, 0.1 equivalent of NAD+, 3 equivalent of pyruvate, 100 mg of SjGMD and 5 mg of LdhA.
  • the reaction was carried out at 30°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, and then purified by anion exchange resin, eluted by sodium chloride, and the product yield was 54%.
  • Embodiment 6 Large-scale synthesis and purification of GDP-D-PerNH 2 :
  • the reaction system contained 20mM Tris-HCl (PH 7.5), 3 grams of GDP-Mannose and 10 mg of GMD. The reaction was carried out at 37°C. When the GDP-Mannose conversion was complete, 0.1 equivalent of PLP, 10 equivalent of L-glutamic acid and 20 mg of PerA 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. After the insoluble precipitate was removed by centrifugation, the supernatant was concentrated and purified by a P-2 column, and then purified by an anion exchange resin and eluted by sodium chloride. The product yield was 62%.
  • the reaction system contained 20 mM Tris-HCl (pH 7.5), 3 g of GDP-Mannose and 10 mg of GMD. The reaction was carried out at 37°C. When the GDP-Mannose transformation is complete, add 0.1 when amount of PLP, 5 equivalents of L-glutamic acid and 20 mg of PerA. The reaction was carried out at 37°C, then 2 equivalents of ATP, 3 equivalents of sodium acetate, 0.05 equivalents of CoA, 20 mg of PerB and 5 mg of ACS were added. 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, and then purified by anion exchange resin, eluted by sodium chloride, and the product yield was 82%.
  • the present application has successfully developed a general method for the efficient preparation of rare sugar nucleotides.
  • sugar nucleotide synthetases 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.
  • seven rare sugar nucleotides that are difficult to obtain were prepared in a large-scale and efficient manner starting from GDP-Mannose, and the whole process did not require tedious purification operations. More importantly, 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 nucleotides with well-defined biosynthetic pathways.

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Abstract

本发明涉及一种由GDP-Mannose合成稀有糖类核苷酸的方法，其中所述稀有糖类核苷酸由下式I表示。所述方法包括：在25-40℃下，以GDP-Mannose为起始糖核苷酸，采用一锅法在酶和辅因子再生系统存在下，通过酶催化反应制备所述稀有糖核苷酸。本申请的方法通过在稀有糖苷酸的合成中采用辅因子再生系统来产生辅因子，可以降低生产成本和产物的纯化难度。

Description

一种由GDP-Mannose合成稀有糖类核苷酸的方法 技术领域
本发明属于糖生物学领域，具体涉及一种合成稀有糖类核苷酸的方法。
背景技术
聚糖，也称为多糖或寡糖，在自然界中与核酸和蛋白质一起形成三类生物聚合物之一。众所周知，聚糖或糖缀合物可作为细胞间相互作用的配体或毒素、抗体和微生物的靶标。在活细胞中，单糖被核苷通过单磷酸或二磷酸激活，形成糖核苷酸作为糖基转移酶的糖基化供体。糖核苷酸的水解是一种产生能量的反应，为糖苷键的形成提供驱动力。作为糖基转移酶的底物，糖核苷酸已被广泛用于糖基转移酶的鉴定以及聚糖和糖缀合物的生物合成研究。
由于糖核苷酸在糖生物学和糖化学中的重要性，已经开发出化学和酶促方法来制备糖核苷酸。化学方法可分为两种方式，包括糖-1-磷酸与活化的单磷酸核苷的缩合和活化的糖与核苷二磷酸的直接偶联。然而，糖核苷酸在有机溶剂中的低溶解度、极性基团的存在以及糖苷键在剧烈反应条件下的不稳定性使得化学合成非常具有挑战性。较差的区域选择性通常需要多步保护/脱保护操作，从而导致收率低。相比之下，酶促合成仅产生仅表现出天然型端基异构体构型的产品。在活细胞中，糖核苷酸是通过补救途径或从头生物合成途径产生的。包括使用激酶对糖进行磷酸化和通过焦磷酸化酶进行焦磷酸化。然而，该策略仅成功制备了极少数常见的天然糖核苷酸。或者，从现有的常见糖核苷酸从头生物合成糖核苷酸在自然界中产生大部分糖核苷酸。该过程涉及单个或多个反应，包括脱水、异构化、差向异构化、氧化、还原、胺化和乙酰化反应。人们普遍认为，由于反应路线复杂、纯化困难和制备成本高，这种复杂的生物转化在合成应用中是不切实际的。在这些障碍中，产物纯化是最具挑战性的问题，只有高效液相色谱纯化被证明是一种从这种复杂反应系统中以极小规模(微克到毫克级)获得产物的有效方法。因此，大多数天然存在的糖核苷酸还没有成功地大量制备。因此，基因库中发现的大量糖基转移酶无法进行生化表征，许多重要聚糖和糖缀合物的生物合成途径仍不清楚。这严重阻碍了糖科学的进步。
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GDP-Mannose(鸟苷二磷酸甘露糖)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25-40℃下，优选在30-37℃下，以GDP-Mannose为起始糖核苷酸，采用一锅法在酶和辅因子再生系统(Cofactor regeneration system,CRS)存在下通过反应制备稀有糖核苷酸。
在具体实施方式中，所述稀有糖核苷酸由下式I表示：
其中，在式I中，
R1选自H和羟基；
R2选自羟基、氨基以及乙酰氨基；并且
R3选自甲基和羧基。
在具体实施方式中，所述酶选自以下各组的酶系统：
在具体实施方式中，所述辅因子再生系统选自如下的辅因子再生系统：
特别地，在采用CRS1辅因子再生系统时，相对于GDP-Mannose，其由0.01或0.02当量NAD+、3当量D-葡萄糖和1毫克或2毫克BsGH组成；
在采用CRS2辅因子再生系统时，相对于GDP-Mannose，其由0.01或0.02当量NADP+、3当量D-葡萄糖和1毫克或2毫克BsGH组成；
在采用CRS3辅因子再生系统时，相对于GDP-Mannose，其由0.01或0.02当量NAD+、3当量丙酮酸和1毫克或2毫克LdhA组成；
在采用CRS4辅因子再生系统时，相对于GDP-Mannose，其由5当量L-谷氨酸和0.1当量PLP组成；
在采用CRS5辅因子再生系统时，相对于GDP-Mannose，其由0.05当量的CoA、2当量ATP、3当量乙酸钠和5毫克ACS组成。
所述辅因子再生系统的再生过程分别如以下反应式所示：
对于GDP-Mannose的来源没有特别限制。在具体实施方式中，GDP-Mannose可以通过如下路线1合成：
其中，GDP-Mannose由Mannose(甘露糖)在D-Mannose激酶(NahK)的作用下与三磷酸腺苷(ATP)生成Mannose-1-P、再在甘露糖1-磷酸鸟苷酰转移酶(ManC)的作用下与三磷酸鸟苷(GTP)反应获得GDP-Mannose(该过程采用一锅法合成),在以上反应中，PPi表示焦磷酸盐，Pi表示无机磷酸盐，PPA表示无机焦磷酸酶。在本申请中使用的起始糖核苷酸GDP-Mannose也可通过商购获得。
在具体实施方式中，所述稀有糖核苷酸选自以下各项之一：
在具体实施方式中，对于还原导向的NADH或NADPH再生系统，所述稀有糖核苷酸的合成通过以下路线2进行：
在上述路线2中，采用如下所示的复合酶系统与CRS1或CRS2结合来制备各稀有核苷酸：
具体地，在GDP-L-Fucose的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS1及GFS，进一步通过酶催化反应制得产物GDP-L-Fucose，优选地，前述酶催化反应在37℃下进行。
具体地，在GDP-D-Rha的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2及RMD，进一步通过酶催化反应制得产物GDP-D-Rha，优选地，前述酶催化反应在37℃下进行。
具体地，在GDP-6-deoxy-D-Tal的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2及GTS，进一步通过酶催化反应制得产物GDP-6-deoxy-D-Tal，优选地，前述酶催化反应在37℃下进行。
具体地，在GDP-L-Colitose的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2，4及ColC、ColD，进一步通过酶催化反应制得产物GDP-L-Colitose，优选地，前述酶催化反应在37℃下进行。
在具体实施方式中，对于氧化导向的NAD+再生系统，所述稀有糖核苷酸的合成通过以下路线3进行：
在上述路线3中，采用SjGMD(OPME6)与CRS3结合来制备GDP-ManA。
具体地，在GDP-D-ManA的合成中，GDP-Mannose在SjGMD和CRS3的存在下发生酶催化反应，得到产物GDP-D-ManA，优选地，前述酶催化反应在30℃下进行。
在具体实施方式中，对于胺化导向的PMP再生系统，所述稀有糖核苷酸的合成通过以下路线4进行：
在上述路线4中，采用GMD,PerA复合酶系统(OPME7)与CRS4结合来制备GDP-PerNH2。
具体地，在GDP-D-PerNH2的合成中，GDP-Man首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS4及PerA，进一步通过酶催化反应制得产物GDP-D-PerNH2，优选地，前述酶催化反应在37℃下进行。
在具体实施方式中，对于乙酰化导向的AcCoA再生系统，所述稀有糖核苷酸的合成通过以下路线5进行：
在上述路线5中，采用GMD,PerA，PerB复合酶系统(OPME8)与CRS4和CRS5结合来制备GDP-PerNAc。
具体地，在GDP-D-PerNAc的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS4及PerA，进行反应，反应完成后，加入CRS5及PerB进行酶催化反应，制得产物GDP-D-PerNAc，优选地，前述酶催化反应在37℃下进行。
在实施方式中，所述反应在pH＝7.3-7.9，优选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。
虽然上文采用具体实施方式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、脱水、异构化、胺化及乙酰化导向的以糖核苷酸为底物的其他糖核苷酸的合成反应中，这些也均包括在本发明的范围内。
除了上述所列举的辅因子再生系统之外，辅因子再生系统还可包括如下所示的NAD+、NAD(P)H以及AcCoA再生系统：
本申请选择本文所述辅因子再生系统与复合酶系统结合为最优方案，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他辅因子再生系统(例如，如上所示)应用于相关化合物的合成中，这些也均包括在本发明的范围内。
此外，在本申请中，NADH再生系统可与NADPH再生系统在反应中 互相替换，这些也均包括在本发明的范围内。
上文虽然以具体实例例示出各步反应所涉及的酶，但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这些酶替换为其他具有相同功能的酶以便用于上述化合物的合成，这些也包括在本发明范围内。
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。
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，Mannose-1-phosphate guanylyltransferase(ManC)、pyrophosphatase(PPA)、GDP-D-Mannose-4,6-dehydratase(GMD)、3,5-epimerase-4-reductase(GFS)、GDP-4-keto-6-deoxy-d-mannose-4-aminotransferase(PerA)和GDP-perosamine N-acetyltransferase(PerB)从大肠杆菌O157通过PCR扩增得到的，GDP-L-colitose synthase(ColC)和GDP-4-keto-6-deoxy-D-mannose-3-dehydrase(ColD)是从E.coli O55中扩增得到。除了正文中提到的酶外，这里还列出了其他基因来源：D-Mannose kinase(NahK)来自长双歧杆菌(Bifidobacterium longum)；GDP-4-keto-6-deoxy-D-Mannose reductase(RMD)来自铜绿假单胞菌；GDP-6-deoxy-D-talose synthetase(GTS)来自放线杆菌(Actinobacillus  actinomycetemcomitans)；GDP-Mannose dehydrogenase(SjGMD)来自海带(Saccharina japonica)；lactate dehydrogenase(LdhA)来自肠系膜明串珠菌(Leuconostoc mesenteroides)、(BsGH)D-glucose dehydrogenase from Bacillus subtilis(枯草芽孢杆菌)。本申请中使用的所有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。将测序确认的质粒转化到大肠杆菌BL21(DE3)中以进行蛋白质表达。将含有重组载体pET-28a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，只要其可以实现在本申请中所意图实现的功能即可。
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；
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；
PLP:5'-磷酸吡哆醛
PMP：5'-磷酸吡哆胺
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,来源于大肠杆菌的乙酰辅酶A合成酶
NahK:D-Mannose kinase from Bifidobacterium longum，D-甘露糖激酶,来源于长双歧杆菌。
PPA:pyrophosphatase；焦磷酸化酶；采用PCR方法从大肠杆菌O157中扩增。
ManC：Mannose-1-phosphate guanylyltransferase，甘露糖1-磷酸鸟苷酰转移酶；采用PCR方法从大肠杆菌O157中扩增
GMD：GDP-D-Mannose-4,6-dehydratase；鸟苷5′-二磷酸-D-甘露糖C-4,C-6脱水酶，来自铜绿假单胞菌
SjGMD：GDP-D-Mannose dehydrogenase from Saccharina japonica；鸟苷5′-二磷酸-D-甘露糖C-6脱氢酶，来源于海带。
RMD：GDP-4-keto-6-deoxy-D-mannose reductase；鸟苷5′-二磷酸-4-酮-6-脱氧-D-甘露糖C-4还原酶，来自铜绿假单胞菌
GFS：GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase，鸟苷5′-二磷酸-4-酮-6-脱氧-D-甘露糖C-3、C-5异构酶，C-4还原酶双功能酶；采用PCR方法从大肠杆菌O157中扩增。
GTS：GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans；鸟苷5′-二磷酸-6-脱氧-D-塔罗糖合成酶,来源于放 线菌。
ColC：C-5epimerase/C-4reductase，C-5差向异构酶/C-4还原酶双功能酶；采用PCR方法从大肠杆菌O55中扩增。
ColD：GDP-4-keto-6-deoxy-D-mannose-3-dehydrase；鸟苷5′-二磷酸-4-酮-6-脱氧-D-甘露糖C-3脱水酶；采用PCR方法从大肠杆菌O55中扩增。
PerA：GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase；鸟苷5′-二磷酸-4-酮-6-脱氧-D-甘露糖C-4转氨酶，采用PCR方法从大肠杆菌O157中扩增。
PerB：GDP-perosamine N-acetyltransferase；GDP-D-PerNH2 N-乙酰转移酶，采用PCR方法从大肠杆菌O157中扩增
在下文中例示了7种稀有糖核苷酸(结构如下)的合成。所采用的一锅多酶系统与辅因子再生系统的偶联及收率总结在下表1中。
表1一锅多酶法偶联辅因子再生系统(CRS)合成稀有糖核苷酸

在上表中，
a起始糖核苷酸。GDP-Mannose由D-Mannose制备。
b反应起始原料为3克的GDP-Mannose。
c收率的计算是相对于GDP-Mannose，基于摩尔比率。
本申请选取自然界中最为常见的糖核苷酸GDP-Mannose用来合成稀有糖核苷酸。GDP-Mannose通过使用D-Mannose激酶(NahK)、甘露糖1-磷酸鸟苷酰转移酶(ManC)和无机焦磷酸酶(PPA)从D-Mannose开始按照一锅法合成；分离收率为86％(参见以下路线1)。下文合成的7种糖核苷酸涵盖了大多数的已报道的源自GDP-Mannose的稀有糖核苷酸。
一、用于制备GDP-L-Fucose、GDP-D-Rha、GDP-6-deoxy-Talose和GDP-L-Colitose的还原导向(线路2)。
以NADH和NADPH为还原剂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GDP-Mannose到GDP-L-Fucose、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(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GDP。
本申请采用铜绿假单胞菌来源(Pseudomonas aeruginosa)的脱水酶(GMD)与大肠杆菌O157来源的3,5-差向异构酶-4-还原酶(GFS)两种酶。GMD是C-4,6脱水酶，GFS是C-5差向异构化和C-4还原酶的双功能酶。但是，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，从而大大降低了生产成本。
如下所示，在这个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。
在50微升的反应体系中测试了包含10mM GDP-Mannose、20mM Tris-HCl(PH 7.5)、GMD、GFS、CRS1或CRS2的分析反应。结果证实，NADH(CRS1)和NADPH(CRS2)都可以被GMD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GDP-Mannose完全转化为GDP-L-Fucose，表明CRS系统可以显著促进反应的进行。同时，由于反应中中间体的不稳定，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GDP-Mannose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：3克GDP-Mannose、10毫克GMD进行反应，GDP-Mannose消耗完全加热至60℃，持续15分钟，然后体系中加入20毫克GFS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93％。
采用酶促反应与CRS2和CRS4体系偶联，由GDP-Mannose得到GDP-L-Colitose(路线2)。由于产物水解少，经过P2柱的简单除盐得到GDP-L-Colitose，收率为86％。发明人还尝试在大规模的GDP-L-Fucose和GDP-L-Colitose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GDP-Mannose到GDP-D-Rha的合成，GDP-D-Rha的生物合成分为两步，GDP-Mannose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)。本申请采用铜绿假单胞菌来源(Pseudomonas aeruginosa)的GDP-Mannose的C-4,6脱水酶(GMD)，不需要外源NAD+/NADP+的脱水酶。还原反应使用的同样是来自铜绿假单胞菌的C-4还原酶(RMD)。在分析反应中，GDP-Mannose在CRS1或CRS2体系存在下与GMD和RMD两种酶一起孵育，结果显示，NADH和NADPH都能被酶接受，但是CRS2(NADPH)可以获得更高的转化效率。因此，大规模合成反应中选择了 CRS2(NADPH)体系；GDP-Mannose在反应中完全转化，经过P2柱的简单除盐得到GDP-D-Rha，收率为87％。
采用与GDP-D-Rha相似的合成策略(酶促反应采用OPME2酶系统(GMD,GTS)，与CRS1或CRS2体系偶联)，由GDP-Mannose得到GDP-D-6-deoxy-Talose(路线2)。由于产物水解少，经过P2柱的简单除盐得到GDP-D-6-deoxy-Talose，收率为91％。
二、用于制备GDP-ManA的氧化导向(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。
在本申请中新设计的一锅法反应中(路线3)，GDP-Mannose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+。
在本申请中使用的是来自Saccharina japonica的氧化酶(SjGMD)。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，SjGMD可以在不到3小时将GDP-Mannose消耗完，转化为GDP-ManA。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77％。
三、制备GDP-PerNH2的胺化导向反应(路线4)。
GDP-PerNH2的生物合成从GDP-Mannose开始，首先由GDP-Mannose-C4,6脱水酶催化脱水反应，然后通过转氨酶将GDP-4-keto-6-deoxy-mAN的C-4位胺化生成GDP-PerNH2(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。
为了合成GDP-PerNH2，使用了来自霍乱弧菌(Vibrio cholera)的C-4氨基转移酶(PerA)。由于GMD的活性总是被转氨酶抑制，反应首先将GDP-Mannose和GMD一起孵育，当GDP-Mannose完全转化后，将CRS4和PerA添加到反应体系中用来合成GDP-PerNH2。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GDP-PerNH2，收率为62％。
四、用于制备GDP-PerNAc的N-乙酰化导向反应(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。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。
为了尝试酶促乙酰化合成GDP-PerNAc，发明人克隆了来自霍乱弧菌(Vibrio cholera)的参与GDP-PerNAc生物合成的乙酰转移酶(PerB)。10mM的GDP-PerNH2与PerB和CRS5一起孵育，可以将GDP-PerNH2完全转化为GDP-PerNAc。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GDP-PerNH2，本申请直接进行一锅法，其中使用了双CRS。首先，GDP-Mannose与GMD一起孵育，当没有GDP-Mannose剩余时，添加CRS4(5当量的L-谷氨酸、0.05当量的PLP)和PerA，然后，加入CRS5(0.002当量的CoA、2当量ATP、3当量乙酸钠、ACS)和PerB。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GDP-PerNAc，收率为82％。
实施例
实施例1：GDP-L-Fuc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose、10毫克GMD、反应在37℃下进行。GDP-Mannose转化完全后，将反应液加热到60℃持续15分钟淬灭反应，加入0.02当量NADP+、3当量D-葡萄糖、20毫克GFS和1毫克BsGH。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，由氯化钠洗脱，产物收率为93％。
实施例2：GDP-D-Rha的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose、10毫克GMD、反应在37℃下进行。GDP-Mannose转化完全后，加入0.01当量NAD+、3当量D-葡萄糖、5毫克RMD和1毫克BsGH。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96％。
实施例3：GDP-6-deoxy-D-Talose的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose、10毫克GMD、反应在37℃下进行。GDP-Mannose转化完全后，将反应液加热到60℃持续15分钟淬灭反应，加入0.01当量NADP+、3当量D-葡萄糖、20毫克GTS和1毫克BsGH。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，由氯化钠洗脱，产物收率为91％。
实施例4：GDP-L-Colitose的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose、10毫克GMD、反应在37℃下进行。GDP-Mannose转化完全后，将反应液加热到60℃持续15分钟淬灭反应，加入0.1当量PLP、5当量L-谷氨酸、0.01当量NADP+、3当量D-葡萄糖、20毫克ColC、20毫克ColD和1毫克BsGH。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86％。
实施例5：GDP-D-ManA的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose、0.1当量NAD+、3当量丙酮酸、100毫克SjGMD和5毫克LdhA。反应在30℃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54％。
实施例6：GDP-D-PerNH2的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose和10毫克GMD。反应在37℃下进行。当GDP-Mannose转化完全后，加入0.1当量的PLP、10当量的L-谷氨酸和20毫克PerA。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62％。
实施例7：GDP-PerNAc的大规模合成与纯化：
反应体系包含20mM Tris-HCl(PH 7.5)、3克GDP-Mannose和10毫克GMD。反应在37℃下进行。当GDP-Mannose转化完全后，加入0.1当 量的PLP、5当量的L-谷氨酸和20毫克PerA。反应在37℃下进行，然后加入2当量ATP、3当量乙酸钠、0.05当量CoA、20毫克PerB和5毫克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82％。
以上实施例中制备的糖核苷酸的1H NMR和13C NMR数据如下表2中所示。
表2

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GDP-Mannose起始大规模、高效的制备了7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。

Claims (10)

  1. 一种制备稀有糖核苷酸的方法，所述方法包括：在25-40℃下，以GDP-Mannose为起始糖核苷酸，采用一锅法在酶和辅因子再生系统(Cofactor regeneration system,CRS)存在下通过反应制备稀有糖核苷酸。
  2. 根据权利要求1所述的方法，其中，所述稀有糖核苷酸由下式I表示：
    其中，在式I中，
    R1选自H和羟基；
    R2选自羟基、氨基以及乙酰氨基；并且
    R3选自甲基和羧基。
  3. 根据权利要求2所述的方法，其中，所述稀有糖核苷酸选自以下各项之一：
  4. 根据权利要求3所述的方法，其中，所述酶选自以下各组的酶系统：

  5. 根据权利要求4所述的方法，其中，所述辅因子再生系统选自如下的辅因子再生系统：
  6. 根据权利要求5所述的方法，其中，
    在采用CRS1辅因子再生系统时，相对于GDP-Mannose，CRS1由0.01或0.02当量NAD+、3当量D-葡萄糖和1毫克或2毫克BsGH组成；
    在采用CRS2辅因子再生系统时，相对于GDP-Mannose，CRS2由0.01或0.02当量NADP+、3当量D-葡萄糖和1毫克或2毫克BsGH组成；
    在采用CRS3辅因子再生系统时，相对于GDP-Mannose，CRS3由0.01或0.02当量NAD+、3当量丙酮酸和1毫克或2毫克LdhA组成；
    在采用CRS4辅因子再生系统时，相对于GDP-Mannose，CRS4由5当量L-谷氨酸和0.1当量PLP组成；
    在采用CRS5辅因子再生系统时，相对于GDP-Mannose，CRS5由0.05当量的CoA、2当量ATP、3当量乙酸钠和5毫克ACS组成。
  7. 根据权利要求5或6所述的方法，其中，
    在GDP-L-Fucose的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS1及GFS，进一步通过酶催化反应制得产物GDP-L-Fucose；
    在GDP-D-Rha的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2及RMD，进一步通过酶催化反应制得产物GDP-D-Rha；
    在GDP-6-deoxy-D-Tal的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2及GTS，进一步通过酶催化反应制得产物GDP-6-deoxy-D-Tal；
    在GDP-L-Colitose的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS2、CRS4及ColC、ColD，进一步通过酶催化反应制得产物GDP-L-Colitose；
    在GDP-D-ManA的合成中，GDP-Mannose在SjGMD和CRS3的存在下发生酶催化反应，得到产物GDP-D-ManA；
    在GDP-D-PerNH2的合成中，GDP-Man首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS4及PerA，进一步通过酶催化反应制得产物GDP-D-PerNH2；
    在GDP-D-PerNAc的合成中，GDP-Mannose首先在GMD的存在下发生酶催化反应，反应完成后，加入CRS4及PerA，进行反应，反应完成后，加入CRS5及PerB进行酶催化反应，制得产物GDP-D-PerNAc。
  8. 根据权利要求7所述的方法，其中，对于GDP-L-Fucose、GDP-D-Rha、GDP-6-deoxy-D-Tal、GDP-L-Colitose、GDP-D-PerNH2和GDP-D-PerNAc的合成，酶催化反应在37℃下进行；对于GDP-D-ManA的合成，酶催化反应在30℃下进行。
  9. 根据权利要求1所述的方法，其中，所述反应在pH＝7.3-7.9，优选在pH＝7.5的缓冲体系下进行。
  10. 根据权利要求9所述的方法，其中，所述缓冲体系为Tris缓冲体系。
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