CN116479074A - Method for synthesizing rare saccharide nucleotide by GDP-Mannose - Google Patents

Method for synthesizing rare saccharide nucleotide by GDP-Mannose Download PDF

Info

Publication number
CN116479074A
CN116479074A CN202310060828.5A CN202310060828A CN116479074A CN 116479074 A CN116479074 A CN 116479074A CN 202310060828 A CN202310060828 A CN 202310060828A CN 116479074 A CN116479074 A CN 116479074A
Authority
CN
China
Prior art keywords
gdp
reaction
mannose
enzyme
gmd
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310060828.5A
Other languages
Chinese (zh)
Inventor
文留青
张家彬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Materia Medica of CAS
Original Assignee
Shanghai Institute of Materia Medica of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institute of Materia Medica of CAS filed Critical Shanghai Institute of Materia Medica of CAS
Publication of CN116479074A publication Critical patent/CN116479074A/en
Pending legal-status Critical Current

Links

Classifications

    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to a method for synthesizing rare saccharide nucleotides from GDP-Mannose, wherein the rare saccharide nucleotides are represented by the following formula I. The method comprises the following steps: the rare sugar nucleotide is prepared by an enzyme catalytic reaction at 25-40 ℃ in the presence of an enzyme and a cofactor regeneration system by adopting a one-pot method by taking GDP-Mannose as a starting sugar nucleotide. The method of the present application can reduce the production cost and difficulty in purifying the product by using a cofactor regeneration system to produce cofactors in the synthesis of rare glycosidic acids.

Description

Method for synthesizing rare saccharide nucleotide by GDP-Mannose
Technical Field
The invention belongs to the field of glycobiology, and particularly relates to a method for synthesizing rare saccharide nucleotide.
Background
Glycans, also known as polysaccharides or oligosaccharides, form one of three types of biopolymers with nucleic acids and proteins in nature. It is well known that glycans or glycoconjugates can act as ligands for intercellular interactions or targets for toxins, antibodies and microorganisms. In living cells, monosaccharides are activated by nucleosides via mono-or biphosphoric acids, forming sugar nucleotides as glycosylation donors for glycosyltransferases. Hydrolysis of sugar nucleotides is an energy-generating reaction that provides a driving force for the formation of glycosidic linkages. As substrates for glycosyltransferases, sugar nucleotides have been widely used in the identification of glycosyltransferases and in the study of biosynthesis of glycans and glycoconjugates.
Because of the importance of sugar nucleotides in glycobiology and glycochemistry, chemical and enzymatic methods have been developed to produce sugar nucleotides. Chemical methods can be divided into two ways, including condensation of sugar-1-phosphate with activated nucleoside monophosphates and direct coupling of activated sugar with nucleoside diphosphate. However, 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 typically requires multiple protection/deprotection operations, resulting in low yields. In contrast, enzymatic synthesis only yields products that exhibit only the native anomer configuration. In living cells, sugar nucleotides are produced by salvage pathways or de novo biosynthesis pathways. Including the use of kinases to phosphorylate sugars and pyrophosphorylation by pyrophosphorylase. However, this strategy has only succeeded in producing very few common natural sugar nucleotides. Alternatively, the synthesis of sugar nucleotides from the head of 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 widely recognized that such complex biotransformations are impractical in synthetic applications due to the complex reaction routes, purification difficulties and high manufacturing costs. Of these obstacles, product purification is the most challenging problem, and only high performance liquid chromatography purification has proven to be an effective method of obtaining products from such complex reaction systems on a very small scale (microgram to milligram scale). Thus, most naturally occurring sugar nucleotides have not been successfully prepared in large quantities. Thus, the large number of glycosyltransferases found in gene libraries cannot be biochemically characterized, and the biosynthetic pathways for many important glycans and glycoconjugates remain unclear. This severely hampers the advancement of the sugar science.
Disclosure of Invention
Based on the problems of the prior art described above, the technical object of the present invention is to provide a general method for preparing rare sugar nucleotides without cumbersome purification operations, which is capable of preparing rare sugar nucleotides in large scale and high yield using GDP-Mannose (guanosine diphosphate Mannose).
The present invention provides a method for preparing rare sugar nucleotides, the method comprising: rare sugar nucleotides are prepared by reaction in the presence of an enzyme and cofactor regeneration system (Cofactor regeneration system, CRS) in a one-pot process at 25-40 ℃, preferably 30-37 ℃, using GDP-Mannose as the starting sugar nucleotide.
In particular embodiments, the rare sugar nucleotide is represented by formula I below:
wherein, in the formula I,
R 1 selected from H and hydroxy;
R 2 selected from hydroxyl, amino, and acetamido; and is also provided with
R 3 Selected from methyl and carboxyl.
In a specific embodiment, the enzyme is selected from the following groups of enzyme systems:
enzyme System name Enzymes
OPME2 GMD,GFS
OPME3 GMD,RMD
OPME4 GMD,GTS
OPME5 GMD,ColD,ColC
OPME6 SjGMD
OPME7 GMD,PerA
OPME8 GMD,PerA,PerB
In a specific embodiment, the cofactorRegeneration system selectionFrom the cofactor regeneration system:
cofactor regeneration System name Type(s) Composition of the composition
CRS1 Reduction-guided NADH regeneration system NAD + D-glucose, bsGH
CRS2 Reduction-directed NADPH regeneration System NADP + D-glucose, bsGH
CRS3 Oxidation-directed NAD + Regeneration system NAD + Pyruvic acid, ldhA
CRS4 Amination-directed PMP regeneration system L-glutamic acid, PLP
CRS5 AcCoA regeneration system guided by acetylation CoA, ATP, sodium acetate, ACS
In particular, when CRS1 cofactor regeneration systems are employed, they consist of 0.01 or 0.02 equivalent NAD relative to GDP-Mannose + 3 equivalents of D-glucose and 1 mg or 2 mg of BsGH;
when the CRS2 cofactor regeneration system is employed, it consists of 0.01 or 0.02 equivalents NADP+, 3 equivalents D-glucose and 1 mg or 2 mg BsGH relative to GDP-Mannose;
when the CRS3 cofactor regeneration system is used, it consists of 0.01 or 0.02 equivalent NAD relative to GDP-Mannose + 3 equivalents of pyruvic acid and 1 mg or 2 mg of LdhA;
when the CRS4 cofactor regeneration system was used, it consisted of 5 equivalents of L-glutamic acid and 0.1 equivalent of PLP relative to GDP-Mannose;
when the CRS5 cofactor regeneration system was used, it consisted of 0.05 equivalents of CoA, 2 equivalents of ATP, 3 equivalents of sodium acetate and 5 mg of ACS, relative to GDP-Mannose.
The regeneration process of the cofactor regeneration system is respectively shown in the following reaction formula:
there is no particular limitation on the source of GDP-Mannose. In particular embodiments, GDP-Mannose can be synthesized by scheme 1 as follows:
wherein GDP-Mannose is prepared by reacting Mannose (Mannose) with Adenosine Triphosphate (ATP) under the action of D-Mannose kinase (NahK) to generate Mannose-1-P, and reacting the Mannose with Guanosine Triphosphate (GTP) under the action of Mannose-1-guanyl phosphate transferase (ManC) (the process is synthesized by adopting a one-pot method), PPi represents pyrophosphate, pi represents inorganic phosphate, and PPA represents inorganic pyrophosphatase. The starting sugar nucleotide GDP-Mannose used in the present application is also commercially available.
In specific embodiments, the rare sugar nucleotide is selected from one of the following:
in specific embodiments, for a reduction-directed NADH or NADPH regeneration system, the synthesis of the rare sugar nucleotide proceeds through scheme 2 below:
in scheme 2 above, each rare nucleotide was prepared using a complex enzyme system as shown below in combination with CRS1 or CRS 2:
specifically, in the synthesis of GDP-L-Fucose, GDP-Mannose first undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS1 and GFS are added, and the GDP-L-Fucose product is further prepared by the enzyme catalytic reaction, preferably, the enzyme catalytic reaction is carried out at 37 ℃.
Specifically, in the synthesis of GDP-D-Rha, GDP-Mannose first undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS2 and RMD are added, and the GDP-D-Rha product is further obtained through the enzyme catalytic reaction, preferably, the enzyme catalytic reaction is carried out at 37 ℃.
Specifically, in the synthesis of GDP-6-deoxy-D-Tal, GDP-Mannose first undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS2 and GTS are added, and the product GDP-6-deoxy-D-Tal is further prepared through the enzyme catalytic reaction, preferably, the enzyme catalytic reaction is carried out at 37 ℃.
Specifically, in the synthesis of GDP-L-Colitose, GDP-Mannose is first subjected to an enzyme-catalyzed reaction in the presence of GMD, after the reaction is completed, CRS2,4 and ColC and ColD are added, and the GDP-L-Colitose product is further prepared by the enzyme-catalyzed reaction, preferably, the enzyme-catalyzed reaction is carried out at 37 ℃.
In particular embodiments, for oxidation-directed NAD + A regeneration system, the synthesis of the rare sugar nucleotide is performed by the following scheme 3:
in scheme 3 above, sjGMD (OPME 6) was used in combination with CRS3 to prepare GDP-ManA.
Specifically, in the synthesis of GDP-D-ManA, GDP-Mannose undergoes an enzyme-catalyzed reaction in the presence of SjGMD and CRS3 to yield the product GDP-D-ManA, preferably, the aforementioned enzyme-catalyzed reaction is carried out at 30 ℃.
In specific embodiments, for an amination-directed PMP regeneration system, the synthesis of the rare sugar nucleotide proceeds through scheme 4 below:
in scheme 4 above, GMD, perA Complex enzyme System (OPME 7) was used in combination with CRS4 to prepare GDP-PerNH 2 。
Specifically, in the synthesis of GDP-D-PerNH2, GDP-Man first undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS4 and PerA are added, and the product GDP-D-PerNH is further prepared by the enzyme catalytic reaction 2 Preferably, the aforementioned enzyme-catalyzed reaction is carried out at 37 ℃.
In specific embodiments, for an acetylation-directed AcCoA regeneration system, the synthesis of the rare sugar nucleotide proceeds through scheme 5 below:
in scheme 5 above, GDP-PerNAc was prepared using the GMD, perA, perB complex enzyme system (OPME 8) in combination with CRS4 and CRS 5.
Specifically, in the synthesis of GDP-D-PerNAc, GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD, CRS4 and PerA are added after the reaction is completed, CRS5 and PerB are added after the reaction is completed, and the enzyme-catalyzed reaction is performed to obtain the product GDP-D-PerNAc, preferably, the enzyme-catalyzed reaction is performed at 37 ℃.
In an embodiment, the reaction is carried out at a buffer system at ph=7.3-7.9, preferably 7.5, preferably the buffer system is Tris buffer system.
Although 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, and those skilled in the art can apply the cofactor regeneration system of the present application to any synthesis reaction of other sugar nucleotides with sugar nucleotides as substrates, which require reduction, oxidation, dehydration, isomerization, amination, and acetylation guidance, based on common knowledge and conventional technical means in the art, and these are also included in the scope of the present invention.
In addition to the cofactor regeneration systems listed above, the cofactor regeneration system may also include NAD as shown below + NAD (P) H and AcCoA regeneration system:
the present application selects the cofactor regeneration system described herein as the optimal solution in combination with the complex enzyme system, and a person skilled in the art can apply other cofactor regeneration systems (e.g., as shown above) to the synthesis of related compounds based on common knowledge and conventional technical means in the art, which are also included in the scope of the present invention.
Furthermore, in the present application, the NADH regeneration system can be replaced with the NADPH regeneration system in the reaction, and these are also included in the scope of the present invention.
Although the enzymes involved in each step of reaction are illustrated as specific examples, those skilled in the art can substitute these enzymes for other enzymes having the same function based on common knowledge and conventional technical means in the art so as to be used for the synthesis of the above-described compounds, and these are also included in the scope of the present invention.
Advantageous effects
The greatest advantage of the enzymatic reaction is the high stereoselectivity and regioselectivity. Thus, the multi-step enzyme reaction can be performed at One time, which is called "One-pot multiple enzyme method (One-pot multienzyme reaction, OPME)". Although the biotransformation pathway of rare sugar nucleotides is quite complex, the last step is usually reduction, oxidation, amination, acetylation and isomerization reactions. Other reactions, except isomerisation, are all irreversible processes, requiring co-factors NADH/NADPH, NAD + /NADP + Pyridoxamine 5' -phosphate (PMP) or acetyl coa (AcCoA).
In view of this, the present application contemplates a one-pot approach that avoids stepwise synthesis to synthesize rare sugar nucleotides. The present application can reduce production costs and difficulty in purification of the product by generating cofactors by the cofactor regeneration system (Cofactor regeneration system, CRS) as described above. Through verification, the one-pot multienzyme method is coupled with the cofactor regeneration system, and the reaction can still be smoothly carried out, so that the unstable purification of an intermediate which is difficult to separate is avoided, and the mass synthesis of rare sugar nucleotides by the enzymatic method becomes practical.
Detailed Description
Hereinafter, the technical solutions of the present invention are described in detail through specific embodiments, but these technical solutions are only used to better understand the present application by those skilled in the art, and are not used to limit the scope of the present application.
All relevant enzymes used hereinafter were all prepared by E.coli expression system and purified using Ni-NTA. Specifically, mannose-1-phosphate guanylyltransferase (ManC), pyrophosphatase (PPA), GDP-D-Mannose-4,6-dehydratase (GMD), 3, 5-epi-4-reductase (GFS), GDP-4-keto-6-deoxy-D-mannase-4-aminotransferase (PerA) and GDP-perosamine N-acetyltransferase (PerB) were amplified from E.coli O157 by PCR, and GDP-L-colitose synthase (ColC) and GDP-4-keto-6-deoxy-D-mannase-3-dehydrase (ColD) were amplified from E.coli O55. In addition to the enzymes mentioned in the text, other gene sources are listed here: D-Mannose kinase (NahK) from Bifidobacterium longum (Bifidobacterium longum); GDP-4-keyto-6-deoxy-D-Mannose Reductase (RMD) is from Pseudomonas aeruginosa; GDP-6-deoxy-D-talose synthetase (GTS) is from Actinobacillus (Actinobacillus actinomycetemcomitans); GDP-Mannose dehydrogenase (SjGMD) is from kelp (Saccharina japonica); lactate dehydrogenase (LdhA) is derived from Leuconostoc mesenteroides (Leuconostoc mesenteroides), (BsGH) D-glucose dehydrogenase from Bacillus subtilis (Bacillus subtilis). All other genes used in this application are synthetic. The gene synthesis service is provided by golden srey (south Beijing, china) or by a biological organism (Shanghai, china).
All genes were cloned into pET-28a vectors to produce recombinant proteins with a six histidine (His) tag at the N-or C-terminus. The sequenced plasmids were transformed into E.coli BL21 (DE 3) for protein expression. Coli BL21 (DE 3) cells containing recombinant vector pET-28a were cultured in two liters of Luria-Bertani (LB) medium containing 50ug/ml kanamycin at 37℃with a rotating shaker at 200 rpm. When the OD reached 0.8, 0.2mM isopropyl-. Beta. -D-thiogalactoside (IPTG) was added and protein expression was induced overnight at 16 ℃. Cells were collected by centrifugation at 7000rpm for 10 min. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl buffer, 300mM NaCl, 10mM imidazole; pH 7.5). Cells were disrupted with a microfluidizer and the lysate was centrifuged at 12,000g for 30min to remove cell debris. His-tagged proteins were purified using a Ni-NTA agarose column. Prior to purification, the column was equilibrated with lysis buffer (50 mM Tris-HCl, 300mM NaCl, 10mM imidazole; pH 7.5). The column was washed with 2 column volumes of lysis buffer and eluted with elution buffer (50 mM Tris-HCl, 300mM NaCl, 300mM imidazole; pH 7.5). Protein concentration was determined using BCA protein assay kit. Although the sources of the respective enzymes in the present application are described above, the source route thereof is not limited thereto as long as it can realize the functions intended to be realized in the present application.
Terminology
OPME: one-pot multienzyme reaction, one pot multienzyme; OPME is also referred to herein as an enzyme system;
cofactor regeneration system, cofactor regeneration system; CRS is also referred to herein as a specific five cofactor regeneration system;
NAD + nicotinamide adenine dinucleotide;
NADH, reduced nicotinamide adenine dinucleotide;
NADP + nicotinamide adenine dinucleotide phosphate;
NADPH, reduced nicotinamide adenine dinucleotide phosphate;
PLP pyridoxal 5' -phosphate
PMP: pyridoxamine 5' -phosphate
CoA coenzyme A, coenzyme A
ATP adenine nucleoside triphosphate
PPi inorganic pyrophosphorylase
Pi inorganic phosphate
BsGH D-glucose dehydrogenase from Bacillus subtilis, bacillus subtilis-derived D-glucose dehydrogenase
LdhA lactate dehydrogenase from Leuconostoc mesenteroides, leuconostoc mesenteroides-derived lactose dehydrogenase
ACS AcCoA synthase derived from Escherichia coli
NahK, D-Mannose kinase from Bifidobacterium longum, D-mannose kinase, is derived from Bifidobacterium longum.
PPA pyrophosphatase; pyrophosphorylase; PCR was used to amplify from E.coli O157.
And (3) ManC: mannose-1-phosphate guanylyltransferase, mannose 1-guanyl phosphate transferase; amplification from E.coli O157 by PCR
GMD: GDP-D-Mannose-4, 6-dehydroatase; guanosine 5' -diphosphate-D-mannoseDehydration of C-4, C-6 The enzyme(s) present in the sample,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-keyo-6-deoxy-D-mannose reductase; guanosine 5' -diphosphate-4-one-6-deoxy-D-mannose C-4reductase from Pseudomonas aeruginosa
GFS: GDP-4-keyo-6-deoxy-D-mannase-3, 5-epothilase-4-reduction, guanosine 5' -diphosphate-4-one-6-deoxy-D-mannose C-3, C-5 isomerase, C-4reductase bifunctional enzyme; PCR was used to amplify from E.coli O157.
GTS: GDP-6-oxygen-D-talose synthetase from Actinobacillus actinomycetemcomitans; guanosine 5' -diphosphate-6-deoxy-D-taloseThe synthetase is derived from actinomycetes.
ColC: c-5 epi-enzyme/C-4 reduction enzyme, C-5epimerase/C-4reductase bifunctional enzyme; PCR was used to amplify from E.coli O55.
ColD: GDP-4-keyto-6-deoxy-D-mannase-3-dehydrase; guanosine 5' -diphosphate-4-one-6-deoxy-D-mannose C-3 dehydratase; PCR was used to amplify from E.coli O55.
PerA: GDP-4-keyto-6-deoxy-D-mannase-4-aminotransferase; guanosine 5' -diphosphate-4-keto-6-deoxy-D-mannose C-4 aminotransferase was amplified from E.coli O157 by PCR.
PerB: GDP-peramine N-acetyltransferase; GDP-D-PerNH 2N-acetyltransferase, PCR method for amplification from E.coli O157
The synthesis of 7 rare sugar nucleotides (structure as follows) is exemplified below. The coupling and yields of the one-pot multienzyme system and cofactor regeneration system employed are summarized in table 1 below.
TABLE 1 one pot Multi-enzymatic coupling Cofactor Regeneration System (CRS) Synthesis of rare sugar nucleotides
In the above-mentioned table of the contents,
a starting sugar nucleotide. GDP-Mannose was prepared from D-Mannose.
The starting material for the b reaction was 3 g of GDP-Mannose.
The yield of c is calculated relative to GDP-Mannose, based on molar ratio.
The GDP-Mannose, the most common sugar nucleotide in nature, is selected to be used for synthesizing rare sugar nucleotide. GDP-Mannose was synthesized in one pot starting from D-Mannose by using D-Mannose kinase (NahK), mannose 1-guanyl phosphate transferase (ManC) and inorganic pyrophosphatase (PPA); the isolation yield was 86% (see scheme 1 below). The 7 sugar nucleotides synthesized below cover most of the rare sugar nucleotides reported to be derived from GDP-Mannose.
1. Reduction and targeting for the preparation of GDP-L-Fucose, GDP-D-Rha, GDP-6-deoxy-Talose and GDP-L-Colitose (scheme 2).
The reductase-catalyzed reduction reactions with NADH and NADPH as reducing agents are the most common reactions in the bioconversion of rare sugar nucleotides. The biosynthetic pathway from GDP-Mannose to GDP-L-Fucose has been studied. This process requires four steps, including: c-4,6 dehydration, C-5 epimerization, C-3 epimerization and C-4 reduction, involving two enzymatic catalysis (scheme 2). However, intermediates in the synthetic pathway are poorly stable and readily hydrolyse rapidly to monosaccharides and GDP.
The application uses two enzymes, namely dehydratase (GMD) from pseudomonas aeruginosa (Pseudomonas aeruginosa) and 3,5-epimerase-4-reductase (GFS) from escherichia coli O157. GMD is a C-4,6 dehydratase and GFS is a bifunctional enzyme of C-5 epimerization and C-4 reductase. However, the use of NADH and NADPH for large amounts of synthesis is too costly. Due to NAD + And NADP + Is much cheaper than NADH and NADPH, and in order to provide a strong reducing power, the present application uses D-glucose dehydrogenase (BsGH) from Bacillus subtilis (Bacillus subtilis), which is composed of NAD + Or NADP + NADH or NADPH is prepared, thereby greatly reducing the production cost.
As shown below, in this CRS system, bsGH uses NAD + Or NADP + D-glucose is oxidized to gluconic acid as a cofactor, and NADH (CRS 1) or NADPH (CRS 2) is formed for the reduction reaction.
Measurement in 50. Mu.l reaction SystemAnalytical reactions containing 10mM GDP-Mannose, 20mM Tris-HCl (pH 7.5), GMD, GFS, CRS1 or CRS2 were tested. The results demonstrate that both NADH (CRS 1) and NADPH (CRS 2) are well accepted by GMD. Surprisingly, when the enzymatic reaction was coupled to a CRS1 or CRS2 reaction system, the GDP-Mannose was completely converted to GDP-L-Fucose in less than two hours, indicating that the CRS system can significantly promote the reaction. Meanwhile, due to instability of intermediates in the reaction, a plurality of hydrolysates were detected in the reaction without CRS system. This phenomenon has proved to be impractical from the side to step through the synthesis of rare sugar nucleotides. Gradient concentration studies showed 0.01 equivalent of NADP + Or 0.02 equivalent of NAD + Complete conversion of GDP-Mannose can be achieved. Thus, in large scale synthesis reactions, the present application selects NAD at low cost + CRS1 system as raw material. A large scale synthesis reaction was performed at 37 ℃, comprising: 3 g GDP-Mannose, 10 mg GMD were reacted, GDP-Mannose was consumed and heated to 60℃for 15 minutes, then 20 mg GFS and CRS1 system (0.02 eq NAD were added to the system + 3 equivalents of D-glucose and BsGH). The reaction was complete, the addition of cold ethanol was terminated, and due to complete conversion of the reaction and small amounts of cofactor NAD + By using the method, the pure product can be obtained through simple desalting of the P2 column, and the yield is 93%.
The GDP-L-Colitose was obtained from GDP-Mannose by coupling with the CRS2 and CRS4 systems using an enzymatic reaction (scheme 2). Because of less hydrolysis of the product, GDP-L-Colitose was obtained in 86% yield by simple desalting with P2 column. The inventors have also attempted to use high concentrations of NADPH/NADH (10 mM) as cofactor in large scale GDP-L-Fucose and GDP-L-Colitose synthesis reactions, the results of which are not ideal due to the strong product inhibition effect. Moreover, the low yield of the reaction product and the high concentration of cofactor in the system make the isolation of the product very difficult.
Whereas for the synthesis of GDP-Mannose to GDP-D-Rha, the biosynthesis of GDP-D-Rha is a two-step, C-4,6 dehydratase and C-4reductase catalyzed C-4,6 dehydration and reduction reaction of GDP-Mannose (scheme 2). The present application uses the C-4,6 dehydratase (GMD) of the GDP-Mannose from Pseudomonas aeruginosa (Pseudomonas aeruginosa) without the need for an exogenous NAD+/NADP+ dehydratase. The reduction reaction also used was C-4Reductase (RMD) from Pseudomonas aeruginosa. In the analytical reaction, GDP-Mannose was incubated with both GMD and RMD enzymes in the presence of CRS1 or CRS2 systems, and the results showed that both NADH and NADPH were accepted by the enzymes, but that CRS2 (NADPH) gave higher conversion efficiencies. Thus, CRS2 (NADPH) systems were selected for large scale synthesis reactions; GDP-Mannose was completely converted in the reaction, and GDP-D-Rha was obtained in 87% yield by simple desalting with P2 column.
GDP-D-6-deoxy-Talose was obtained from GDP-Mannose using a synthesis strategy similar to GDP-D-Rha (enzymatic reaction using OPME2 enzyme system (GMD, GTS), coupled with CRS1 or CRS2 system) (scheme 2). Because the product is less hydrolyzed, GDP-D-6-oxygen-Talose is obtained through simple desalting of the P2 column, and the yield is 91%.
2. Oxidative guidance for the preparation of GDP-ManA (line 3).
In NAD + Or NADP + Biological oxidation catalyzed by dehydrogenases is a major pathway for synthesizing carboxylic acid sugars and their derivatives in nature as cofactors.
In a one-pot reaction newly designed in this application (scheme 3), the biological oxidation of GDP-Mannose with NAD + Is used in combination with the regeneration system (CRS 3).
As shown below, in CRS3, lactate dehydrogenase (LdhA) from Leuconostoc mesenteroides (Leuconostoc mesenteroides) reduced pyruvate to D-lactate while NADH was oxidized to NAD + 。
Used in this application is oxidase (SjGMD) from Saccharina japonica. The inventors found that when CRS3 (0.01 equivalent NAD + 3 equivalent weightPyruvic acid, ldhA), sjGMD may consume GDP-Mannose in less than 3 hours and convert it to GDP-ManA. This is much faster than chemical oxidation reactions requiring toxic catalysts and severe reaction conditions (100 ℃ and 48 hours). The reaction is completely converted, and the pure product can be obtained through simple desalting of the P2 column, and the yield is 77%.
3. Preparation of GDP-PerNH 2 Is directed to the amination reaction (scheme 4).
GDP-PerNH 2 Starting from GDP-Mannose, the dehydration reaction is first catalyzed by GDP-Mannose-C4,6 dehydratase, and then the C-4 position of GDP-4-keyto-6-deoxy-mAN is aminated by a transaminase to form GDP-PerNH 2 (route 4). The donor of the transaminase is pyridoxamine 5'-phosphate (PMP), which is regenerated from pyridoxal 5' -phosphate (PLP) and L-glutamic acid (as shown below for CRS 4), without the need for exogenous enzymes.
For the synthesis of GDP-PerNH 2 C-4 aminotransferase (PerA) from Vibrio cholerae (Vibrio cholera) was used. Since the activity of GMD is always inhibited by transaminase, the reaction is first incubated with GDP-Mannose and GMD, and after complete conversion of GDP-Mannose, CRS4 and PerA are added to the reaction system for synthesis of GDP-PerNH 2 . Since the amino conversion reaction is incomplete, up to 10-fold equivalent of L-glutamic acid is used to promote the amination reaction, but high concentration of L-glutamic acid is difficult to directly remove. In order to simplify the purification of the product, the present application uses L-glutamic acid decarboxylase (GadB) from Lactobacillus plantarum (Lactobacillus plantarum) to hydrolyze the remaining L-glutamic acid to aminobutyric acid, which is easily purified and removed. Although the amination reaction is not complete, the intermediate of the reaction can be easily removed by the ion exchange resin because the product has an additional amino groupObtaining GDP-PerNH 2 The yield thereof was found to be 62%.
4. N-acetylation-directed reactions for the preparation of GDP-PerNAc (scheme 5).
N-acetylation of rare sugar nucleotides is catalyzed by acetyltransferase, using acetyl coa (AcCoA) as an acetyl donor. However, acCoA is too expensive to be used for large scale synthesis. Since CoA is much cheaper than AcCoA, acCoA is produced using regeneration system CRS5 (shown below), where AcCoA is synthesized from sodium acetate, coA and ATP by an AcCoA synthase (ACS).
In an attempt to enzymatically acetylate and synthesize GDP-PerNAc, the inventors cloned an acetyltransferase (PerB) from Vibrio cholerae (Vibrio cholera) involved in the biosynthesis of GDP-PerNAc. 10mM GDP-PerNH 2 Incubation with PerB and CRS5 can completely convert GDP-PerNH2 to GDP-PerNAc. The catalytic amount of CoA used reduces the cost of large-scale synthesis, and it is important that no side reactions occur. To avoid isolation of intermediate GDP-PerNH 2 The present application proceeds directly to a one-pot process, where dual CRSs are used. First, GDP-Mannose was incubated with GMD, and when no GDP-Mannose remained, CRS4 (5 equivalents of L-glutamic acid, 0.05 equivalents of PLP) and PerA were added, followed by CRS5 (0.002 equivalents of CoA, 2 equivalents of ATP, 3 equivalents of sodium acetate, ACS) and PerB. After the reaction was completed, gadB was added to hydrolyze excess L-glutamic acid, and GDP-PerNAc was obtained by purification with P2 and an ion exchange resin in 82% yield.
Examples
Example 1: large scale synthesis and purification of GDP-L-Fuc:
the reaction system contains 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose, 10 mg GMD and the reaction is carried out at 37 ℃. After complete GDP-Mannose conversion, the reaction was followedThe reaction mixture was heated to 60℃for 15 minutes to quench the reaction, and 0.02 equivalent of NADP was added + 3 equivalents of D-glucose, 20 mg of GFS and 1 mg of BsGH. After the reaction is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, eluting by sodium chloride, and obtaining the product with the yield of 93%.
Example 2: large scale synthesis and purification of GDP-D-Rha:
the reaction system contains 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose, 10 mg GMD and the reaction is carried out at 37 ℃. After complete GDP-Mannose conversion, 0.01 equivalent NAD was added + 3 equivalents of D-glucose, 5 mg of RMD and 1 mg of BsGH. After the reaction is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, wherein the product yield is 96%.
Example 3: large scale synthesis and purification of GDP-6-deoxy-D-Talose:
the reaction system contains 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose, 10 mg GMD and the reaction is carried out at 37 ℃. After the GDP-Mannose conversion was complete, the reaction was quenched by heating the reaction solution to 60℃for 15 minutes, and 0.01 equivalent of NADP was added + 3 equivalents of D-glucose, 20 mg of GTS and 1 mg of BsGH. After the reaction is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, eluting by sodium chloride, and obtaining the product with the yield of 91%.
Example 4: large scale synthesis and purification of GDP-L-Colitose:
the reaction system contains 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose, 10 mg GMD and the reaction is carried out at 37 ℃. After the completion of GDP-Mannose conversion, the reaction mixture was heated to 60℃for 15 minutes to quench the reaction, and 0.1 equivalent of PLP, 5 equivalents of L-glutamic acid, and 0.01 equivalent of NADP were added + 3 equivalents of D-glucose, 20 mg of ColC, 20 mg of ColD and 1 mg of BsGH. After the reaction is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, purifying by an anion exchange resin, eluting by sodium chloride, and obtaining the product with the yield of 86%.
Example 5: large scale synthesis and purification of GDP-D-ManA:
the reaction system contained 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose, 0.1 eq NAD+, 3 eq pyruvic acid, 100 mg SjGMD and 5 mg LdhA. The reaction was carried out at 30 ℃. After the reaction is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, purifying by an anion exchange resin, eluting by sodium chloride, and obtaining the product with the yield of 54%.
Example 6: GDP-D-PerNH 2 Large scale synthesis and purification of (c):
the reaction system contained 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose and 10 mg GMD. The reaction was carried out at 37 ℃. After the GDP-Mannose conversion was complete, 0.1 eq PLP, 10 eq L-glutamic acid and 20 mg PerA were added. The reaction is carried out at 37 ℃, after the reaction is completed, the reaction is stopped by adding an equal volume of cold ethanol, after insoluble precipitate is removed by centrifugation, the supernatant is concentrated and purified by a P-2 column, then is purified by anion exchange resin, and is eluted by sodium chloride, and the product yield is 62%.
Example 7: large scale synthesis and purification of GDP-PerNAc:
the reaction system contained 20mM Tris-HCl (pH 7.5), 3 g GDP-Mannose and 10 mg GMD. The reaction was carried out at 37 ℃. After the GDP-Mannose conversion was complete, 0.1 eq PLP, 5 eq L-glutamic acid and 20 mg PerA were added. The reaction was carried out at 37℃and 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 is completed, adding an equal volume of cold ethanol to terminate the reaction, centrifuging to remove insoluble precipitate, concentrating and purifying the supernatant by a P-2 column, purifying by an anion exchange resin, eluting by sodium chloride, and obtaining the product with the yield of 82%.
Sugar nucleotides prepared in the above examples 1 H NMR 13 The C NMR data are shown in Table 2 below.
TABLE 2
/>
In summary, the present application successfully developed a general method for efficiently preparing rare sugar nucleotides. By using sugar nucleotide synthetases of different clonal origins, complex natural synthetic routes are recombined for the synthetic reaction. The addition of cofactor regeneration system provides excessive cofactor to promote the final irreversible reaction to produce target sugar nucleotide. By this method, 7 rare sugar nucleotides which are difficult to obtain are prepared from GDP-Mannose on a large scale and efficiently, and the whole process does not need complicated purification operation. More importantly, the catalytic amount of expensive cofactor is used to reduce the cost of large scale synthesis. Since the last biotransformation step of most reported rare sugar nucleotides involves a reduction, oxidation, amination or acetylation reaction, the method of the invention can be easily extended to other rare sugar nucleotides whose biosynthetic pathway is clear.

Claims (10)

1. A method of preparing rare sugar nucleotides, the method comprising: rare sugar nucleotides were prepared by reaction in the presence of an enzyme and cofactor regeneration system (Cofactor regeneration system, CRS) in a one-pot process at 25-40 ℃ using GDP-Mannose as starting sugar nucleotide.
2. The method of claim 1, wherein the rare sugar nucleotide is represented by formula I:
wherein, in the formula I,
R 1 selected from H and hydroxy;
R 2 selected from hydroxyl, amino, and acetamido; and is also provided with
R 3 Selected from methyl and carboxyl.
3. The method of claim 2, wherein the rare sugar nucleotide is selected from one of:
4. a method according to claim 3, wherein the enzyme is selected from the group of enzyme systems consisting of:
enzyme System name Enzymes OPME2 GMD,GFS OPME3 GMD,RMD OPME4 GMD,GTS OPME5 GMD,ColD,ColC OPME6 SjGMD OPME7 GMD,PerA OPME8 GMD,PerA,PerB
。
5. The method of claim 4, wherein the cofactor regeneration system is selected from the group consisting of:
cofactor regeneration System name Type(s) Composition of the composition CRS1 Reduction-guided NADH regeneration system NAD + D-glucose, bsGH CRS2 Reduction-directed NADPH regeneration System NADP + D-glucose, bsGH CRS3 Oxidation-directed NAD + Regeneration system NAD + Pyruvic acid, ldhA CRS4 Amination-directed PMP regeneration system L-glutamic acid, PLP CRS5 AcCoA regeneration system guided by acetylation CoA, ATP, sodium acetate, ACS
。
6. The method of claim 5, wherein,
when CRS1 cofactor regeneration system is used, CRS1 is regenerated with 0.01 or 0.02 equivalent NAD to GDP-Mannose + 3 equivalents of D-glucose and 1 mg or 2 mg of BsGH;
when using the CRS2 cofactor regeneration system, CRS2 consists of 0.01 or 0.02 equivalents NADP+, 3 equivalents D-glucose, and 1 mg or 2 mg BsGH relative to GDP-Mannose;
when CRS3 cofactor regeneration system is used, CRS3 consists of 0.01 or 0.02 equivalent NAD to GDP-Mannose + 3 equivalents of pyruvic acid and 1 mg or 2 mg of LdhA;
when the CRS4 cofactor regeneration system is employed, CRS4 consists of 5 equivalents of L-glutamic acid and 0.1 equivalent of PLP relative to GDP-Mannose;
when using the CRS5 cofactor regeneration system, CRS5 consisted of 0.05 equivalents of CoA, 2 equivalents of ATP, 3 equivalents of sodium acetate, and 5 mg of ACS, relative to GDP-Mannose.
7. The method according to claim 5 or 6, wherein,
in the synthesis of GDP-L-Fucose, GDP-Mannose firstly undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS1 and GFS are added, and the GDP-L-Fucose is further prepared by the enzyme catalytic reaction;
in the synthesis of GDP-D-Rha, GDP-Mannose firstly undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS2 and RMD are added, and the GDP-D-Rha product is further prepared through the enzyme catalytic reaction;
in the synthesis of GDP-6-oxygen-D-Tal, GDP-Mannose firstly undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS2 and GTS are added, and the GDP-6-oxygen-D-Tal product is further prepared through the enzyme catalytic reaction;
in the synthesis of GDP-L-Colitose, GDP-Mannose firstly undergoes an enzyme catalytic reaction in the presence of GMD, after the reaction is completed, CRS2, CRS4, colC and ColD are added, and the GDP-L-Colitose product is further prepared through the enzyme catalytic reaction;
in the synthesis of GDP-D-ManA, GDP-Mannose undergoes an enzyme catalytic reaction in the presence of SjGMD and CRS3 to obtain a product GDP-D-ManA;
in GDP-D-PerNH 2 In the synthesis of (2), GDP-Man is subjected to enzyme catalytic reaction in the presence of GMD, CRS4 and PerA are added after the reaction is completed, and the GDP-D-PerNH product is prepared by the enzyme catalytic reaction 2 ；
In the synthesis of GDP-D-PerNAc, GDP-Mannose first undergoes an enzyme-catalyzed reaction in the presence of GMD, after the reaction is completed, CRS4 and PerA are added for reaction, after the reaction is completed, CRS5 and PerB are added for enzyme-catalyzed reaction, and the GDP-D-PerNAc product is obtained.
8. The method according to claim 7, wherein for GDP-L-Fucose, GDP-D-Rha, GDP-6-oxygen-D-Tal, GDP-L-Colitose, GDP-D-PerNH 2 And GDP-D-PerNAc, the enzyme-catalyzed reaction is carried out at 37 ℃; for the synthesis of GDP-D-ManA, the enzyme-catalyzed reaction was carried out at 30 ℃.
9. The method according to claim 1, wherein the reaction is carried out at a ph=7.3-7.9, preferably at a buffer system of ph=7.5.
10. The method of claim 9, wherein the buffer system is a Tris buffer system.
CN202310060828.5A 2022-01-21 2023-01-17 Method for synthesizing rare saccharide nucleotide by GDP-Mannose Pending CN116479074A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2022100723043 2022-01-21
CN202210072304 2022-01-21

Publications (1)

Publication Number Publication Date
CN116479074A true CN116479074A (en) 2023-07-25

Family

ID=87223863

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310060828.5A Pending CN116479074A (en) 2022-01-21 2023-01-17 Method for synthesizing rare saccharide nucleotide by GDP-Mannose

Country Status (2)

Country Link
CN (1) CN116479074A (en)
WO (1) WO2023138545A1 (en)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3231791B2 (en) * 1996-09-11 2001-11-26 ヤマサ醤油株式会社 Production method of sugar nucleotide
US6875591B1 (en) * 1999-08-10 2005-04-05 Kyowa, Hakko Kogyo Co., Ltd. Process for producing GDP-fucose
CN102443597A (en) * 2010-10-15 2012-05-09 天津赛科瑞德生物科技有限公司 Enzymatic preparation of sugar nucleotide
CN102409070A (en) * 2011-09-15 2012-04-11 山东大学 Preparation method of rare sugar nucleotides
CN107502615B (en) * 2017-10-16 2020-05-05 中国海洋大学 Gene encoding GDP-mannose-4,6-dehydratase in kelp, and protein and use thereof
CN111676259B (en) * 2020-07-10 2021-11-26 山东大学 Preparation method of sugar nucleotide and derivatives thereof

Also Published As

Publication number Publication date
WO2023138545A1 (en) 2023-07-27

Similar Documents

Publication Publication Date Title
TWI695888B (en) A composition for producing tagatose and methods for producing tagatose using the same
JP2001518783A (en) Oligosaccharide synthesis method
US9193958B2 (en) Method of enzymatically synthesizing 3′-phosphoadenosine-5′-phosphosulfate
EP1816206B1 (en) Method of producing uridine 5 -diphospho-n-acetylgalactosamine
JP3545424B2 (en) Method for producing nucleoside 5'-triphosphate and its application
Frohnmeyer et al. Enzyme cascades for the synthesis of nucleotide sugars: Updates to recent production strategies
Woodyer et al. Regeneration of cofactors for enzyme biocatalysis
Teng et al. Cell-free regeneration of ATP based on polyphosphate kinase 2 facilitates cytidine 5'-monophosphate production
CN114507649B (en) Thermophilic enzyme and method for efficiently synthesizing UDP-glucose and UDP-glucuronic acid by one-pot method
JPWO2004009830A1 (en) Process for producing CMP-N-acetylneuraminic acid
CN116479074A (en) Method for synthesizing rare saccharide nucleotide by GDP-Mannose
JP3833584B2 (en) Process for producing CMP-N-acetylneuraminic acid
US8450088B2 (en) Process for producing CMP-N-acetylneuraminic acid
CN116287064A (en) Method for synthesizing rare sugar nucleotide from UDP-GlcNAc
JP3545425B2 (en) Method for producing uridine diphosphate-N-acetylglucosamine
WO1998048031A1 (en) Process for producing adenosine 5'-triphosphate and use thereof
CN116926150A (en) Method for synthesizing sugar nucleotide from UDP-D-Glc
US6890739B1 (en) Use of uridine diphosphate glucose 4-epimerase
KR101818561B1 (en) Method for Preparing 3'-Amino-2',3'-dideoxyadenosine by Using Nucleoside Phosphorylase Derived from Bacillus
US20050170470A1 (en) Process for producing 2'-deoxyguanosine
CN116355874A (en) Glycosyltransferase mutant and application thereof in preparation of quercetin-3-O rhamnoside
CN116790540A (en) Sucrose synthase mutant with improved catalytic property
JP2001169797A (en) Method for enzymatically producing s-adenosyl-l- methionine
Glaze Mechanistic studies of the enzymes involved in the biosynthesis of CMP-N, N'-diacetyllegionaminic acid and UDP-D-apipose
Bassiri Synthesis of UDP-6-deoxy-6, 6-difluoro-D-GlcNAc: a potential mechanism-based inhibitor of pseB

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination