Detoxification of tetrodotoxin by thiol sinthe liver of puffer fish

河豚毒素(TTX，1)是河豚鱼体内的一种强效神经毒素，对河豚和人类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天然TTX类似物4-L-半胱氨酸TTX(4-CysTTX，2)。本文报道了2的结构确认和TTXs与硫醇的反应。用顺序液相色谱从200g河豚的肝脏中分离得到2(约0.4 mg)。根据光谱数据，确定2为1的类似物，C4上的赤道羟基被半胱氨酸的硫原子取代。用Marfey试剂测定了2经I_2氧化得到的半胱氨酸，确定其构型为L。2是由4，9-无氢TTX(4)与大过量(100-500当量)反应生成的。半胱氨酸在磷酸盐缓冲液(pH7.0-8.0)中，40℃，90 mm。这是一种pH依赖性反应。其他硫醇、谷胱甘肽、巯基乙醇和半胱氨酸甘氨酸与4发生类似的反应，在C4=1时生成硫取代化合物。在较长的反应时间下(8小时以上)，ME和GSH也与1反应生成相同的4-谷胱甘肽TTX的衍生物。我们还证实了4-谷胱甘肽TTX可以被谷氨酰转肽酶降解为4-半胱氨酸甘氨酸TTX，然后再被二肽酶降解为2。根据小鼠生物测定结果，口服硫醇。与Fujii等人的结果相反，TTX并不能有效地降低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.