Development of new research tools by derivatization of saxitoxin analogs

由于麻痹性贝毒研究领域对标准材料和新的科学工具的要求很高，我们开发了新的方法，并通过化学转化制备了几种甲藻毒素(STX)的衍生物。对产毒蓝藻圆环鱼腥藻进行大规模培养，得到其起始原料：C_1、C_2、GTX2和GTX3。将天然污染贝类中难以获得的dcGYX2、dcGYX3、dcSTX和GTX5作为高效液相色谱标准品，用简单的方法从C1、C2衍生化。DcSTX被证明是STX的替代品，作为小鼠生物测定的标准。建立了一种高效的乙酰去氨甲酰撒克酮的制备方法，作为放射性标记STX的模型。还制备了两种亲和凝胶，用于研究毒素在双壳类体内积累和转运的大分子，以及用于电压门控钠通道蛋白的纯化。首先在STX骨架的C11和C13位引入了含有羧基的部分，即11-(2-羧乙硫基)石杉毒素和13-O-半琥珀酰脱氨基石杉毒素。它们通过间隔区与琼脂糖凝胶(Sepharose4B)偶联。前一种亲和凝胶对河豚血浆中STX结合蛋白的纯化是有效的，并在4℃下保存6个月以上。

Due to the high demand for the standard material and new scientific tools in the field of research related to paralytic shellfish toxins, we developed the new nethods and prepared several saxitoxin (STX) derivatives by chemical conversions. Mass culture of toxigenic cyanobacteria Anabaena circinalis was used to obtained starting materials, C1, C2, gonyautoxin-2 and 3 (GTX2,3). As HPLC standards, dcGYX2, dcGYX3, dcSTX and GTX5, which were difficult to obtain from naturally contaminated shellfish, were derivatized by simple methods from C1, C2. DcSTX was shown to work as an alternate for STX as standard for the mouse bioassay. Highly effective method to prepare acetyldecarbamoylsaxiton was developed as a model for radio-labeled STX. Two types of affinity gels were also prepared to utilize for the research on macromolecule involved in the accumulation and transportation of toxins in bivalves and for the purification of voltage gated sodium channel protein. First the moieties having carboxyl were introduced at C11 and C13 of STX skeleton, namely 11-(2-carboxyethylthio) saxitoxin and 13-O-hemisuccinyl decarbamoylsaxitoxin. They were coupled with agarose gel (Sepharose 4B) via spacer. The former affinity gel was proven to be effective in purification of STX-binding protein in puffer plasma and to be stable in storage at 4℃ for more than 6 months.