Detoxification of tetrodotoxin by thiol sinthe liver of puffer fish

河豚毒素（TTX, 1）是一种强效的神经毒素，其在河豚体内的代谢途径及对人体的代谢途径尚不清楚。我们从2000年春季采集的河豚肝脏中发现了一种新的TTX天然类似物，4-L-cycteineTTX （4-CysTTX,2）。本文描述了2的结构确认，以及TTXs与硫醇的反应（约0.4 mg）从200 g河豚肝脏中分离得到。根据光谱数据，2被确定为1的类似物，其中C4上的赤道羟基被半胱氨酸的硫原子取代。用Marfey试剂对I_2氧化得到的半胱氨酸进行分析，证实半胱氨酸的构型为L。2是由4,9-无氢ttx(4)与大量过量（100-500等量）半胱氨酸在磷酸盐缓冲液（pH 7.0-8.0）中，在40℃下反应90 mm产生的。这是一个pH依赖性反应。其他硫醇，谷胱甘肽（GSH）、巯基乙醇（ME）和半胱氨酸也同样与4反应，在C4(1)上生成硫取代化合物。在硫醇过量（超过20000当量）和较长反应时间（超过8小时）的条件下，ME和GSH也与1反应，从4生成相同的衍生物。我们还证实了4-谷胱甘肽ttx被谷氨酰转肽酶水解为4-半胱氨酸甘氨酸ttx，然后被二肽酶水解为2。根据小鼠生物测定结果，口服硫醇。与Fujii等人的研究结果相反，与TTX联合用药不能有效降低TTX的毒性。

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.