The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

中文翻译：

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Desulfo 和两性离子 Cylindrospermopsin 生物碱的替代分离方案及其在 HepG2 细胞中的毒性比较

术语圆柱藻素 (CYN) 是指结构相关的一类蓝藻代谢物，由三环胍基团和羟甲基尿嘧啶部分组成。环境水生样品中的大多数报告都提到了圆柱藻素 (CYN)，而关于其他 CYN 生物碱的报告很少，部分原因是缺乏通用的分离方案。因此，我们使用市售的固相萃取 (SPE) 小柱优化了分离方案，以完全回收 CYN、7-脱氧-柱状植物素 (7D-CYN) 和 7-脱氧-脱硫-柱状植物素 (7D-脱硫-CYN)来自同一份。隔离协议可根据样品（固体生物质、培养液或环境水样）的性质进行调整，并且可耐受高达 4 L 的浓培养液或 400 mg 的冻干生物质。为了定量 CYN 生物碱，我们验证了 LC-DAD-MS2 方法，该方法利用了尿嘧啶基团的紫外吸收 (λ 262 nm)。在正离子模式下使用电喷雾电离 (ESI)，高分辨率 MS1 数据证实了质子化生物碱的存在，并且报告了 MS2 碎片分配作为 CYN 分子结构的补充证据。我们使用相同的冻干样品分离出三种不同水溶性的 CYN 生物碱，纯度在 95% 到 99% 之间。通过使用流式细胞术在体外评估坏死和细胞凋亡来评估纯化的 CYN 以及 CYN 的合成降解产物（脱硫圆柱精子素）的生物活性。CYN 在 HepG2 细胞中的致死效力高于其他类似物，