Method for large-scale synthesis of tetrodotoxin

A tetrodotoxin and compound technology, applied in the field of artificial synthesis of tetrodotoxin, can solve problems such as lack of credibility, lack of value for large-scale synthesis and preparation of tetrodotoxin, and inability to produce tetrodotoxin on a large scale

Pending Publication Date: 2022-01-21
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AI-Extracted Technical Summary

Problems solved by technology

However, the steps of the synthetic method reported in related work are lengthy (generally greater than 30 steps), and the synthetic efficiency is very low. Generally, only 1-5 mg of sample can be obtained, and the yield report is not credible in consideration of the measurement method and the systematic error of the measurement equipment. degree, the significance of academic exploration of related work is greater than its practical application value, and it does not have the ...
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Method used

(2) IV to V use non-toxic metal Lewis acid (such as CuCl2 etc.) to replace highly toxic HgCl2 in the original route, which is safer to operate and meets the API production requirements.
(3) The separation of VI-a and by-product VI-b adopts chiral preparative HPLC, and separation effect is good, can prepare in large quantities, has replaced the silica gel thin plate chromatography that is difficult to enlarge in the original route.
(5) VI-a uses a kind of water system mixed fiber microporous membrane to filter to TTX, removes palladium charcoal effect, and filtrate evaporates to dryness and can obtain TT...
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The invention provides a biological and chemical combined method for synthesis of tetrodotoxin, which specifically comprises the following steps: by taking benzyl acetate which is cheap and easy to obtain as a raw material, carrying out biological fermentation to obtain an optically pure intermediate ((5S, 6R)-5, 6-dihydroxycyclohexa-1, 3-dienyl) meta-acetate (formula I), performing a series of chemical conversions on the intermediate, and obtaining tetrodotoxin with the purity of more than 95% without purification of the final product. The method has the potential of large-scale production, and is an excellent substitute for the existing method for extracting from globefish.

Application Domain

Organic chemistryBulk chemical production

Technology Topic

Chemical conversionBiotechnology +5


  • Method for large-scale synthesis of tetrodotoxin
  • Method for large-scale synthesis of tetrodotoxin
  • Method for large-scale synthesis of tetrodotoxin


  • Experimental program(7)

Example Embodiment

[0080] The technical problem to be solved by the present invention is to provide a method for artificially large-scale synthesis of tetrodotoxin that does not rely on natural sources such as puffer fish, that is, a chemical method for gram-scale synthesis of tetrodotoxin. Compared with the method reported in the prior art, the advantage of the chemical synthesis method of the present invention is that the improvement of the synthesis method and the purification method enables the present invention to have the ability to produce tetrodotoxin and its derivatives on a large scale according to the c-GMP standard above the gram level, It is possible to carry out clinical trials of tetrodotoxin as a candidate drug that meets regulatory specifications, and it has laid a solid API foundation for tetrodotoxin to become an important member of the field of human analgesia and detoxification drugs, including:
[0081] (1) Only one step of intramolecular transesterification reaction is needed from II to III, all reagents have only one cheap and easy-to-obtain base, the reaction yield is equivalent to the original route, replacing the 2-step reaction of the original route, and some expensive ones Reagents, operability and scale-up reproducibility of reactions are improved.
[0082] (2) IV to V use non-toxic metal Lewis acid (such as CuCl 2 etc.) to replace the highly toxic HgCl in the original route 2 , Safer operation, in line with API production requirements.
[0083] (3) Chiral preparative HPLC is used for the separation of VI-a and by-product VI-b, which has a good separation effect and can be prepared in large quantities, replacing the silica gel thin plate chromatography that is difficult to scale up in the original route.
[0084] (4) by-product VI-b can be converted into VI-a under dilute acid effect, through once recycling, the total yield of V to VI-a is 68%, compared with the yield of this step reaction 41% of original route. Greater improvement.
[0085] (5) From VI-a to TTX, use a water-based mixed fiber microporous filter membrane to remove palladium carbon, and the filtrate can be evaporated to dryness without further purification to obtain TTX with a purity of more than 95%, which avoids the difficult amplification of the original route. High pressure liquid chromatography separation.
[0086] (6) The present invention is the first to complete the synthesis of tetrodotoxin on a gram-level scale or above in the world, and the characterization data is complete and reliable, which can be used for large-scale preparation of tetrodotoxin API (API) that meets the c-GMP standard, making tetrodotoxin as a regulatory standard It is possible to carry out clinical trials of drug candidates.

Example Embodiment

[0095] Example 1 : the preparation of formula III compound
[0097] At room temperature, 12 g of compound II and 6 g of potassium carbonate were dispersed in 300 ml of methanol, stirred at room temperature for 18 hours, HPLC detected that the raw materials disappeared, and the precipitate was removed by filtration, and most of the methanol was removed by rotary evaporation of the filtrate at room temperature. Then add 300 ml of water, extract the reaction solution three times with 300 ml of ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, filter and spin dry, and use ethyl acetate-petroleum ether (1:3) silica gel column chromatography to obtain white Foamy solid compound III (7.3 g, yield 75%). 1H NMR (CDCl 3 ,400MHz):δ5.16(s,1H),5.02(d,J=6.9Hz,1H),5.01(s,1H),4.42(s,1H),4.41(d,J=6.9Hz,1H) ,4.37(d,J=10.1Hz,1H),4.32(s,1H),4.25(d,J=10.1Hz,1H),3.48(br s,1H),3.44(s,3H),1.46(s ,9H), 1.43(s,3H), 1.38(s,3H), 1.29(s,3H).

Example Embodiment

[0098] Example 2 : the preparation of formula IV compound
[0100] Under ice bath, add 180 ml of freshly prepared TMSI solution (0.32M) to 6.9 g of compound III in 240 ml of anhydrous acetonitrile solution, react at room temperature for 15 minutes, add 450 ml of sodium thiosulfate solution to quench the reaction, and use chloroform- Methanol (9:1) was extracted three times with 450 ml of mixed solvent, the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude compound IV (5.4 g), which was directly put into the next reaction without purification. 1 H NMR (CDCl 3 ,400MHz):δ6.34(br,2H),5.32(d,J=7.2Hz,1H),5.30(s,1H),4.79(d,J=1.2Hz,1H),4.42(d,J= 7.6Hz, 1H), 4.39(s, 1H), 4.38(d, J=10.0Hz, 1H), 4.28(d, J=10.4Hz, 1H), 3.57(s, 3H), 3.36(t, J= 2.0Hz, 1H), 2.03(d, J=8.0Hz, 1H), 1.46(s, 3H), 1.44(s, 6H), 1.38(s, 3H), 1.36(s, 3H).


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