|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2003 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 2002 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Metabolic pathway of tetrodotoxin (TTX, 1), a potent neurotoxin in puffer fish, has not been clarified yet, for puffer fish and also for human. We found a new natural analog of TTX, 4-L-cycteineTTX (4-CysTTX,2) from the liver of the puffer fish, Fugu pardalis, collected in spring in 2000. In this report, structural confirmation of 2, and the reaction of TTXs with thiols are described.
2 (ca. 0.4 mg) was isolated from 200 g of liver of the puffer fish by sequential liquid chromatography. Based on the spectroscopic data, 2 was determined as an analog of 1 of which equatorial hydroxyl group at C4 was substituted by sulfur atom of cysteine. Configuration of cysteine was confirmed to be L by using Marfey reagent for cysteine obtained from 2 by I_2 oxidation.
2 was produced by the reaction of 4,9-anhydroTTX (4) and large excess (100-500 equiv.) of cysteine in phosphate buffer (pH 7.0-8.0) at 40℃ for 90 mm. It was pH dependent reaction. Other thiols, glutathione (GSH), mercaptoethanol (ME), and cysteinyl glycine were similarly reacted with 4 to produce sulfur substituted compounds at C4 of 1. On the condition with much more excess of thiols (more than 20000 equiv.) and for longer reaction time (longer than 8 hr), ME and GSH were also reacted with 1 to produce the same derivatives from 4.
We also confirmed that 4-glutathioneTTX was hydrolyzed by glutamyltranspeptidase to 4-cysteinylglysineTTX, and then to 2 by the dipeptidase. According to the results of mouse bioassay, oral administration of thiols. with TTX was not effective to decrease the toxicity of TTX, contrary to the results by Fujii et al.