Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-900, SP, Brazil.
Laboratory of Development and Innovation, Butantan Institute, Av. Vital Brasil, 1500, São Paulo 05503-900, SP, Brazil.
Molecules. 2020 Jul 2;25(13):3027. doi: 10.3390/molecules25133027.
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-MS 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 MS data confirms the presence of the protonated alkaloids, and the MS 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.
术语“柱孢藻毒素”（CYNs）是指一类结构相关的蓝藻代谢产物，由三环胍基基团和羟甲基尿嘧啶部分组成。在环境水生样品中，大多数报告涉及柱孢藻毒素（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，优化了一种从同一样品中完全回收 CYN、7-脱氧柱孢藻毒素（7D-CYN）和 7-脱氧-去磺酸柱孢藻毒素（7D-desulfo-CYN）的分离方案。该分离方案可根据样品的性质（固体生物量、培养液或环境水样）进行调整，可耐受高达 4 L 浓培养液或 400 mg 冻干生物量。为了定量分析 CYN 生物碱，我们验证了一种 LC-DAD-MS 方法，该方法利用了尿嘧啶基团的紫外吸收（λ 262nm）。采用正离子模式的电喷雾电离（ESI），高分辨率 MS 数据证实了质子化生物碱的存在，并且 MS 碎片分配被报告为 CYN 结构的分子结构的补充证明。我们使用相同的冻干样品分离了三种具有不同水溶性的 CYN 生物碱，其纯度范围为 95%至 99%。使用流式细胞术评估体外坏死和凋亡，评估了纯化的 CYN 及其 CYN 的合成降解产物（去磺酸柱孢藻毒素）的生物活性。由于存在四个功能基团：胍基、尿嘧啶、C-7 羟基和硫酸根残基，因此 CYN 在 HepG2 细胞中的致死效力大于其他类似物。
