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Enzymatic modules and Sda carbohydrate antigen synthesis method

A synthetic method and technology for carbohydrate antigens, applied in the field of carbohydrate antigen preparation, can solve the problems of low overall yield, low yield, and narrow substrate applicability, and achieve the effects of strong catalytic specificity, high catalytic efficiency, and wide sources

Active Publication Date: 2020-11-10
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2018, some researchers used the B4GalNTII enzyme obtained from human ovaries to synthesize Sda in vitro, but the yield was extremely low and it was difficult to reach the level of preparation
For chemical synthesis, Sda sugar chains need repeated protection and deprotection operations to ensure regio and stereoselectivity during the synthesis process, resulting in many reaction steps and low overall yield.
[0005] Synthesis of complex oligosaccharides using an enzymatic modular assembly strategy can overcome the shortcomings of chemical synthesis methods, but the synthesis of Sda sugar antigens with mammalian-derived enzymes faces two difficulties: 1. Most of the mammalian-derived enzymes are transmembrane proteins. It is difficult to recombine and express in vitro, and it is difficult to obtain a sufficient amount of active soluble protein; 2. Mammalian-derived enzymes often have strong substrate specificity and narrow substrate applicability

Method used

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  • Enzymatic modules and Sda carbohydrate antigen synthesis method
  • Enzymatic modules and Sda carbohydrate antigen synthesis method
  • Enzymatic modules and Sda carbohydrate antigen synthesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] 1. Chemical synthesis of α-configuration disaccharide compound 3 (GlcNAcβ1,4GalNAcαproN 3 )

[0090] In a 50mL double-necked round bottom flask, the glycosyl acceptor (compound 1) (455mg, 1.16mmol), the glycosyl donor (compound a) (1.08g, 1.74mmol) and dry Molecular sieves were mixed in dry dichloromethane, sealed, filled with argon protection and stirred at room temperature for 30 minutes. Subsequently, the reaction system was cooled to 0°C in a low-temperature reactor, and the accelerator trifluoromethanesulfonic acid (TfOH, 30 μL, 0.35 mmol) was added. After two hours, thin-layer chromatography detected (petroleum ether: ethyl acetate=1:3 , v / v) The receptors basically responded completely. The reaction system was diluted with dichloromethane and filtered with diatomaceous earth. The obtained organic phase was extracted with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and then separated and purifi...

Embodiment 2

[0104] Enzymatic module A method for synthesizing sialylated products 11, 12, 14, 15:

[0105] Add acceptor compound (compound 10 or compound 13) (1.0 equiv), sialic acid (Neu5Ac or Neu5Gc, 1.1 equiv), cytidine triphosphate (CTP, 1.2 equiv), Tris-HCl buffer ( 100mM, pH=8.0) and magnesium chloride (20mM), add double distilled water to adjust the volume to 10mL, shake and mix, add enzyme NmCSS, PmST1M144D. The reaction was carried out at 37° C. and 110 rpm for 12 hours. Thin-layer chromatography detection (ethyl acetate: methanol: water: acetic acid = 4:2:1:0.2, v / v) After the reaction was completed, an equal volume of ice ethanol 10 mL was added to terminate the reaction, and the reaction was left to stand at 4°C for 30 minutes. The reaction system was centrifuged at 4° C. and 10,000 rpm for 10 minutes, the supernatant was concentrated by spinning to dryness, and separated and purified by Bio-Gel P2 gel molecular exclusion chromatography to obtain the product.

[0106] The sy...

Embodiment 3

[0117] Enzymatic Module B method for sialylation of products 17–22, 24, 25:

[0118] Add acceptor compound (compound 4, compound 9, compound 16 or compound 22) (1.0 equivalent), sialic acid (Neu5Ac or Neu5Gc, 1.1 equivalent), cytidine triphosphate (CTP, 1.2 equivalent) to a 50 mL centrifuge tube, Tris-HCl buffer (100mM, pH=8.0) and magnesium chloride (20mM), add double distilled water to adjust the volume to 10mL, shake and mix, add enzymes NmCSS and PmST3. The reaction was carried out at 37° C. and 110 rpm for 12 hours. TLC detection (ethyl acetate: methanol: water: acetic acid = 4:2:1:0.2, v / v). After the reaction was completed, an equal volume of 10 mL of ice ethanol was added to terminate the reaction, and the mixture was left to stand at 4° C. for 30 minutes. The reaction system was centrifuged at 4° C. and 10,000 rpm for 10 minutes, the supernatant was concentrated by spinning to dryness, and separated and purified by Bio-Gel P2 gel molecular exclusion chromatography t...

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Abstract

The invention relates to enzymatic modules and an Sda carbohydrate antigen synthesis method, and relates to six enzymatic modules. One of the six enzymatic modules comprises bifidobacterium longum N-acetylglucosamine kinase, escherichia coli glyconucleoside generating enzyme and campylobacter jejuni [beta]1-4-N-acetylglucosamine transferase. According to the Sda carbohydrate antigen synthesis method, the campylobacter jejuni [beta]1-4-N-acetylglucosamine transferase is utilized to replace human-derived [beta]1-4-N-acetylglucosamine transferase to introduce GalNAc, and Sda carbohydrate antigenepitope is constructed; and Sda carbohydrate antigen and ABH antigen correlated hybridSda carbohydrate antigen is rapidly and efficiently synthesized by helicobacter pylori [alpha]1-2 fucosyltransferase,helicobacter pylori [alpha]1-3-N-acetylglucosamine transferase and human-derived [alpha]1-3-galactotransferase.

Description

technical field [0001] The invention belongs to the technical field of sugar antigen preparation, and in particular relates to an enzymatic module and a Sda sugar antigen synthesis method. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] As early as 1967, scientists discovered a new tissue blood group antigen - Sda sugar antigen. Sda carbohydrate antigen is widely distributed in human red blood cells, body fluids and tissues and organs. The epitope of the Sda sugar antigen is a trisaccharide structure, that is, Siaα2,3(GalNAcβ1,4)Gal (such as figure 1 shown). Sda carbohydrate antigen plays an important role in physiology and pathology. As a blood group antigen, Sda not...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N9/12C12P19/26C12P19/18
CPCC12N9/1051C12N9/1081C12N9/1205C12N9/1029C12N9/1241C12P19/26C12P19/18C12Y204/01051C12Y204/99004C12Y204/01069C12Y207/01006C12Y204/01087C12Y207/01059C12Y203/01004C12Y207/07009Y02A50/30
Inventor 曹鸿志仲侃刘长城
Owner SHANDONG UNIV
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