Method for synthesizing sialylated oligosaccharide and analogue thereof

A technology of sialic acid oligosaccharides and analogues, which is applied in the fields of sugar derivatives, organic chemistry, fermentation, etc., can solve the problems of high price and restriction of the synthesis scale of sialic acid oligosaccharides, and achieve the reduction of synthesis costs, good industrial production potential, The effect of the simple synthesis process

Inactive Publication Date: 2014-05-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the yield of this method is high, the starting material of this method - sialic acid molecules are expensive, among which the most common N-acetylneuraminic acid (Neu5Ac), its market price is as high a

Method used

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  • Method for synthesizing sialylated oligosaccharide and analogue thereof
  • Method for synthesizing sialylated oligosaccharide and analogue thereof
  • Method for synthesizing sialylated oligosaccharide and analogue thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1, "One-pot method" synthesis of sialic acid oligosaccharides and their analogs

[0060] Using N-acetylneuraminic acid and its analogues (Formula Ⅰ) as starting materials, through the "whole-cell catalysis" and two-step "enzymatic synthesis" methods, without any separation and purification in the middle, the "one-pot method" synthesizes saliva Acid oligosaccharides and their analogs (formula IV or V).

[0061] The specific experimental steps are divided into two steps:

[0062] 1. The donor compound (N-acetylglucosamine, 1 mmol) shown in VI, MgCl 2 (0.025mmol) and pyruvic acid (2mmol) were added to 10ml of engineering bacteria E.coliΔnanTEK / pNA suspension, mixed evenly, reacted in a shaker at 37°C and 200rpm for 16 hours, and centrifuged at 3000g to obtain the supernatant.

[0063] Among them, the bacterial suspension is a solution obtained by suspending the engineering bacteria E.coliΔnanTEK / pNA in 100mM, pH=7.5 Tris HCl buffer, and the OD of E.coliΔnanTEK / pN...

Embodiment 2

[0073] Example 2, "One-pot method" synthesis of sialic acid oligosaccharides and their analogs

[0074] Carry out according to the method for embodiment 1, difference is as follows, all the other are identical:

[0075] Replace α-2,6-sialyltransferase with α-2,3-sialyltransferase (purchased from Tianjin Suntech Pharmaceutical Science and Technology Co., Ltd., Cat.No.:B-03002);

[0076] In step 1), the donor compound, MgCl 2 , Pyruvate and E.coliΔnanTEK / pNA bacterial suspension, the feeding ratio is 0.8mmol:0.02mmol:1.8mmol:10ml;

[0077] In step 2), the feeding ratio of supernatant, receptor compound, cytidine triphosphate, CMP-sialic acid synthase and sialyltransferase is 10ml:0.75mmol:1.45mmol:1.8U:1.8U;

[0078] The compound of formula XVI was obtained in 55% yield.

[0079] The characterization results of the compound shown in product XVI are as follows:

[0080] 1 H NMR (400MHz,D 2 O): δ4.89(1H,d,J2.8Hz),4.35(1H,d,J8.0Hz),4.27(2H,d,J2.4Hz),4.09–3.91(2H,m),3.90–3.72 ...

Embodiment 3

[0082] Example 3, "One-pot method" synthesis of sialic acid oligosaccharides and their analogs

[0083] Carry out according to the method for embodiment 1, difference is as follows, all the other are identical:

[0084] Substituting the acceptor for compound XI as shown,

[0085] In step 1), the donor compound, MgCl 2 , Pyruvate and E.coliΔnanTEK / pNA bacterial suspension, the feeding ratio is 1.2mmol:0.03mmol:2.2mmol:10ml;

[0086] In step 2), the feeding ratio of supernatant, receptor compound, cytidine triphosphate, CMP-sialic acid synthase and sialyltransferase is 10ml:0.85mmol:1.55mmol:2.2U:2.2U.

[0087] The yield of the compound represented by formula XVII was obtained in 52%.

[0088] The characterization results of the compound shown in product XVII are as follows:

[0089] 1 H NMR (400MHz,D 2 O): δ4.63(1H,d,J7.8Hz),4.25(1H,d,J8.0Hz),4.21(2H,d,J2.4Hz),4.01–3.95(2H,m),3.93–3.82 (5H,m),3.76–3.54(9H,m),3.39(1H,t,J8.0Hz),3.29–3.18(2H,m),2.73(1H,dd,J4.5,12.4Hz),2.84 ...

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Abstract

The invention discloses a method for synthesizing sialylated oligosaccharide and an analogue. The invention provides the method for synthesizing sialylated oligosaccharide and the analogue, the method comprises the followings steps: 1)uniformly mixing a donor compound, MgC12, pyruvate and an engineering bacteria E.coli delta nanTEK/pNA bacterium suspension, reacting, collecting a supernatant; 2)adding an acceptor compound, cytidine triphosphate, CMP-sialic acid synthetase and sialytransferase in the supernatant, uniformly mixing, reacting, and collecting the reaction product which is sialyloligosaccharide or its analogue. The experiment of the present invention proves that the method has the advantages that N-acetylneuraminic acid with low price is taken as a raw material, a method of whole-cell catalysis and a method of two-step enzymatic synthesis are combined, the sialyloligosaccharide and its analogue can be synthesized by an one-pot synthesis method, the synthesis cost of the compounds is greatly reduced, the synthesis process is simple, and the separation method is simple.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing sialylated oligosaccharides and analogs. Background technique [0002] Sialic acid (Sia) is a naturally occurring, negatively charged, pyran monosaccharide containing nine carbon atoms. It is a collective noun with more than 50 members. Neu5Ac and its many derivatives are the main members of the sialic acid family, accounting for 99% of the 43 known species. Oligosaccharides, also known as oligosaccharides, are sugar molecules formed by two or more (generally 2-10) monosaccharide units connected by glycosidic bonds, and are widely distributed in nature. Sialylated oligosaccharides are a class of oligosaccharide compounds that contain one or several sialic acid molecules in their molecules. Sialic acid oligosaccharide compounds have important functions such as biological information transmission. The rapid development of modern biotechnology and analytical ...

Claims

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Application Information

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IPC IPC(8): C12P19/26C07H15/10C07H15/203
Inventor 李学兵吕迅陶勇林白雪
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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