Development of new research tools by derivatization of saxitoxin analogs

通过石房蛤毒素类似物的衍生化开发新的研究工具

• 批准号: 12460089
• 负责人: OSHIMA Yasukatsu
• 金额: $ 7.42万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460089/
• 关键词: paralytic shellfish toxins; HPLC standard; chemical conversion; affinity gel; saxitoxin; Anabaena circinalis; gonyautoxin; アフィニティークロマトグラフィー; サキシトキシン結合タンパク; デカルバモイルサキシトキシン; デカルバモイルゴニオトキシン-2,3; 13-アセチルデカルバモイルサキシトキシン

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.

由于麻痹性贝类毒素研究领域对标准物质和新的科学工具的需求很高，我们开发了新的方法，并通过化学转化法制备了几种石房蛤毒素（STX）衍生物。采用大规模培养的方法，从螺旋鱼腥藻（Anabaena circinalis）中获得了起始原料C1、C2、gonyautoxin-2和3（GTX 2、3）。以天然污染贝类中难以获得的dcGYX 2、dcGYX 3、dcSTX和GTX 5为HPLC标准品，通过简单的方法从C1、C2中衍生得到。DcSTX显示出作为STX的替代品作为小鼠生物测定的标准品。建立了一种高效制备乙酰基十氨甲酰基-saxiton的方法，作为放射性标记STX的模型化合物。制备了两种亲和凝胶，用于贝类毒素积累和转运相关大分子的研究和电压门控钠通道蛋白的纯化。首先在STX骨架的C11和C13位引入含羧基的部分，即11-（2-羧基乙硫基）石房蛤毒素和13-O-半琥珀酰十氨甲酰基石房蛤毒素。它们通过间隔物与琼脂糖凝胶（Sepharose 4 B）偶联。结果表明，亲和凝胶可有效纯化河豚血浆中的STX结合蛋白，4℃保存6个月以上稳定。