Method for industrially synthesizing monosialotetrahexosyl ganglioside

A technology of ganglioside and monosialic acid, which is applied in the field of industrial synthesis of monosialotetrahexosylganglioside, can solve the problems of low conversion rate, long synthesis cycle, and many steps, and achieve short cycle and simple steps , low cost effect

Pending Publication Date: 2020-08-07
武汉糖智药业有限公司
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Problems solved by technology

Since it is derived from pig brain, the current extraction technology is not fine enough, GM1 obtained by biochemical synthesis is an ideal way
At present, the technology for synthesizing GM1 has certain defects, such as long synthesis cycle, many steps, and low conversion rate.
So far, there have been studies on the preparation of tumor vaccine GM3 using fluorinated lactose (Lac-F) as a raw material, but there is no patented technology for preparing GM1 using fluorinated lactose (Lac-F) as a raw material; in addition, there are documents (“Strategies for chemoenzymatic synthesis of carbohydrates” Wanqing Li, John B. McArthur, Xi Che, etc., Carbohydrate research, No. 472, 2019, pages 86-97) use sphingosine to replace fluorine in Lac-F to prepare Lac-Sph, and then prepare GM1 scheme, but this scheme has the disadvantages of low conversion rate and purification in each step of reaction, which increases the time cost and labor cost in product preparation
In addition, there is a literature ("One-pot multi-enzyme (OPME) systems for chemoenzymaticsynthesis of carbohydrates." Hai Yu and Xi Chen et al., Org.Biomol.Chem., 2016 No. 14, pp. 2809-2818) introduced GalA As a substrate, UDP-GalNAc is synthesized by various enzymes, but there is no research on the efficient synthesis of GM1 or its intermediates using this method

Method used

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  • Method for industrially synthesizing monosialotetrahexosyl ganglioside
  • Method for industrially synthesizing monosialotetrahexosyl ganglioside
  • Method for industrially synthesizing monosialotetrahexosyl ganglioside

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Step 1: Add 10.23g of fluorinated lactose, 12.98g of pentasodium sialate, 26.85g of disodium cytidine triphosphate and 6.09g of magnesium chloride hexahydrate in sequence in a 2L fermenter, and adjust the pH to 9 with sodium hydroxide. Then add 100U NmCSS, 172UPmST1 and 8.8mg PmPPA, add water to make up to 800mL. React at 30°C for 4 hours, and detect the consumption and production of Lac-F and GM3-F by TLC. For TLC detection, see figure 2 .

[0090] Step 2: Add 6.63g N-acetylgalactosamine (GalNac), 21.49g adenosine triphosphate disodium, 19.8g uridine triphosphate trisodium and 6.09g magnesium chloride hexahydrate successively in another 2L fermenter, adjust with sodium hydroxide pH to 8. Then add sodium hydroxide to adjust the pH to 8, add 240U NahK, 170U Agx1, 8.8mg PmPPA and add water to make up to 1mL, react at 37°C for 4h, and check the reaction progress by TLC, see image 3 A, HPLC quantification of UDP-GalNac, see image 3 b. Mix the obtained reaction solut...

Embodiment 2

[0093] Embodiment 2: purify GM1-Sph

[0094] The reaction solution in step 4 was adjusted with 1M NaOH, and the pH was adjusted to 7.5; then 800 mL of ethanol was added, settled overnight and then centrifuged. The supernatant was statically adsorbed with 1L ion exchange packing DEAE for 2h. Take out the supernatant, and use a membrane machine to pass through the membrane: the pressure is 8-9kg, and the membrane: 300-500D polyamide composite membrane. Ultrafiltration until the solution is completely dry, add 400mL of absolute ethanol to a 2000mL beaker, and then ultrafiltration until completely dry, repeat twice, add 100mL of water to wash out the product, filter through a 0.22μm membrane, and obtain about 50mL of the solution. Add the aqueous solution obtained in the previous step into a 120g Fresh column with a filler of 50μm tC18, and the elution conditions are: 500mL 100% water—500mL 50% methanol water—1200mL 80% methanol water—500mL 100% methanol. The fraction containing...

Embodiment 3

[0095] Embodiment 3: GM1-Sph high performance liquid phase detection

[0096] Dissolve 1 mg of the white solid obtained in the previous step in 1 mL of aqueous solution, and pass through a 0.22 μm membrane. After treatment, it was detected by high performance liquid chromatography. The elution conditions are shown in Table 1, and the detection conditions are:

[0097] High performance liquid chromatography: Unimicro ChromStation, chromatographic column: Ultimate XB-C18 (Yuexu, 4.6×250mm, 3.5μm), mobile phase A: isopropanol:methanol:acetonitrile=35:40:25; mobile phase B: Water; flow rate: 0.8mL / min; temperature: 40°C, column temperature: 40°C, detector: evaporative light scattering (flow: 2.50L / min).

[0098] HPLC results before and after purification see Figure 8 , The retention time of GM1-Sph is 9.5min, the purity is greater than 99%, and the recovery rate is 90%.

[0099] Table 1: Elution Conditions

[0100]

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Abstract

The invention relates to a method for industrially synthesizing monosialotetrahexosyl ganglioside. According to the method, monosialotetrahexosyl ganglioside is prepared from lactose fluoride (Lac-F)as a raw material through a five-step continuous reaction catalyzed by 10 enzymes, and the preparation scale from gram level to kilogram level is reached, so that the requirement of industrial production of GM1 is met, and a traditional process route for extracting from pig brains is replaced. Besides, the process for preparing monosialotetrahexosyl ganglioside is shortened into five steps, and intermediate products are not purified in the first three steps, so that the product loss caused by purification in the intermediate steps is reduced, and the time cost is saved.

Description

【Technical field】 [0001] The invention relates to a method for industrially synthesizing monosialotetrahexosylganglioside, and belongs to the technical field of biochemical catalysis synthesis. 【Background technique】 [0002] Monosialotetrahexosylganglioside (GM1) is a substance that is extracted from pig brain and has an effect on nerve cell function damage. Its structure includes a hydrophobic ceramide and a hydrophilic oligosaccharide chain with sialic acid, which are linked together by glycosidic bonds. Hydrophobic ceramides mainly contain sphingosine and fatty acids, which are linked by imino groups. Sphingosine is composed of polyhydroxy aliphatic amines with a chain length of 18 or 20 carbon atoms, and its structural formula is: [0003] [0004] Existing technology for the extraction of GM1 is based on improving product purity, yield, reducing product cost, reducing the usage of toxic organic solvents, etc. A large amount of research has been carried out and the...

Claims

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Application Information

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IPC IPC(8): C12P19/44
CPCC12P19/44
Inventor 王鹏曹学峰
Owner 武汉糖智药业有限公司
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