Method for large-scale synthesis of tetrodotoxin
The present invention provides a method for biological and chemical synthesis of tetrodotoxin, specifically comprising the following steps: using cheap and easily available benzyl acetate as a raw material, and performing biological fermentation to obtain an optically pure intermediate ((5S, 6R)-5, 6-dihydroxycyclohexa-1, 3-dienyl) methyl acetate (formula I); and performing a series of chemical conversions on the intermediate to finally obtain tetrodotoxin at a purity of 95% or more without purifying the product. The present invention has the potential of large-scale production, and is an excellent alternative to the existing extraction method from a pufferfish.
1 . A method for preparing tetrodotoxin represented by formula TTX, comprising:
(1) subjecting a compound of formula II to an intramolecular cyclization with a base in a solvent to obtain a compound of formula III:
wherein R is C 1-6 alkyl or substituted alkyl, and the base is selected from the group consisting of potassium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate; and the solvent is selected from the group consisting of methanol, ethanol and toluene;
(2) removing Boc protecting group from the compound of formula III to obtain a compound of formula IV:
(3) reacting the compound of formula IV with Cbz methylthiourea to obtain a compound of formula V:
wherein, a metal Lewis acid is used in the reaction;
(4) subjecting the compound of formula V to a molecular cyclization to obtain a compound of formula VI-a and a compound of formula VI-b:
wherein, the compound of formula VI-a and the compound of formula VI-b are separated by a chiral preparative HPLC;
(5) hydrolyzing the compound of formula VI-b to obtain the compound of formula VI-a:
wherein, the compound of formula VI-a and the compound of formula VI-b are separated by a chiral preparative HPLC; and
(6) hydrogenating the compound of formula VI-a in a solvent to remove the protecting group Cbz, and then obtaining the tetrodotoxin after filtration and concentration,
wherein, Cbz represents benzyloxycarbonyl, and the solvent is selected from the group consisting of methanol, ethanol, ethyl acetate and isopropanol.
2 . The method for preparing tetrodotoxin according to claim 1 , further comprising using an aqueous mixed fiber microporous filter membrane for the filtration.
3 . The method for preparing tetrodotoxin according to claim 1 , wherein, the molecular cyclization in step (4) is carried out with an acid.
4 . The method for preparing tetrodotoxin according to claim 3 , wherein, the reaction in step (5) is carried out with an acid.
5 . The method for preparing tetrodotoxin according to claim 1 , wherein, the metal Lewis acid in step (3) is copper chloride.
6 . The method for preparing tetrodotoxin according to claim 5 ,
wherein, in the step (2) reaction, acetonitrile is used as a solvent, trimethyliodosilane is used as a reagent, and the reaction temperature is from −10° C. to 50° C.
7 . The method for preparing tetrodotoxin according to claim 6 , wherein, the reaction temperature is 0° C.
8 . The method for preparing tetrodotoxin according to claim 1 , wherein, in step (1), the base is potassium carbonate and the solvent is methanol.
9 . A method for preparing tetrodotoxin, comprising the following steps:
(1) subjecting a compound of formula II to an intramolecular cyclization with a base in a solvent to obtain a compound of formula III;
wherein R is C 1-6 alkyl or substituted alkyl, and the base is selected from the group consisting of potassium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate; and the solvent is selected from the group consisting of methanol, ethanol and toluene;
(2) removing Boc protecting group from the compound of formula III to obtain a compound of formula IV:
wherein, acetonitrile is used as a solvent, trimethyliodosilane is used as a reagent, and the reaction temperature is from −10° C. to 50° C.;
(3) reacting the compound of formula IV with Cbz methylthiourea to obtain a compound of formula V
wherein, a metal Lewis acid is used in the reaction;
(4) subjecting the compound of formula V to a molecular cyclization to obtain a compound of formula VI-a and a compound of formula VI-b
wherein, the compound of formula VI-a and the compound of formula VI-b are separated by a chiral preparative HPLC;
(5) hydrolyzing the compound of formula VI-b to obtain the compound of formula VI-a
wherein, the compound of formula VI-a and the compound of formula VI-b are separated by a chiral preparative HPLC; and
(6) subjecting the compound of formula VI-a to hydrogenation in a solvent to remove Cbz protecting group to obtain a compound of formula TTX after filtration
wherein, Cbz represents benzyloxycarbonyl, and the solvent is selected from the group consisting of methanol, ethanol, ethyl acetate and isopropanol; and an aqueous mixed fiber microporous filter membrane is used for the filtration.
10 . The method for preparing tetrodotoxin according to claim 1 , wherein, the metal Lewis acid is selected from the group consisting of lead chloride, lead acetate, silver nitrate, copper chloride and cuprous chloride.
11 . The method for preparing tetrodotoxin according to claim 9 , wherein, the metal Lewis acid is selected from the group consisting of lead chloride, lead acetate, silver nitrate, copper chloride and cuprous chloride.