de la Iglesia Pablo, Fonollosa Elena, Diogène Jorge
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou, km 5.5. 43540, Sant Carles de la Ràpita, Tarragona, Spain.
Rapid Commun Mass Spectrom. 2014 Dec 15;28(23):2605-16. doi: 10.1002/rcm.7057.
Esterification is one of the most important metabolic routes of lipophilic marine toxins in shellfish. In this work we assessed several chemical acylation reactions aimed at obtaining acyl ester analogues via partial synthesis from the free toxins. The procedures developed including sensitive and selective methods based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be applied to obtain reference materials that may be used as analytical standards (internal/external) for method development and calibration, as well as to perform toxicological in vitro and in vivo studies.
Acylation systems involved both anhydrous and non-anhydrous fatty acid or acid halides as a source of the acyl radical, and several catalysers of the reaction. A series of mass spectrometric experiments involving product ion scans and multiple reaction monitoring (MRM) were used to confirm the identity and to elucidate the fragmentation pathways of the synthesised products.
Reaction yields regarding reaction time and temperature were examined at sub-nmol scale for the acylation system consisting of palmitic anhydride and 4-(dimethylamino)pyridine (DMAP) in anhydrous pyridine, showing the best conditions at 75 °C for 60 min, 75 °C for 120 min and 100 °C for 270 min for cyclic imines, azaspiracid-1 and pectenotoxin-2, respectively. The esterification approach was verified at a larger scale for the esterification of gymnodimine-A (GYM-A), which kept a good yield >90% for the synthesis of 10-O-palmitoyl-GYM-A.
Acyl ester analogues of lipophilic marine toxins have been synthesised and their structure elucidated by LC/MS/MS. For acyl ester analogues identical to natural metabolites, the procedures developed have potential to be applied for the semi-synthesis of metabolites in a sustainable, scalable and controlled way, avoiding extensive and tedious isolation and purification procedures from naturally contaminated shellfish. For the semi-synthetic esters structurally different than those found in shellfish, they may have applicability as internal standards for accurate quantifications of natural metabolites present in complex matrices.
酯化是贝类中亲脂性海洋毒素最重要的代谢途径之一。在本研究中，我们评估了几种化学酰化反应，旨在通过从游离毒素进行部分合成来获得酰基酯类似物。所开发的程序包括基于液相色谱/串联质谱（LC/MS/MS）的灵敏且选择性的方法，可用于获得可作为方法开发和校准的分析标准品（内标/外标）的参考物质，以及进行毒理学体外和体内研究。
酰化体系涉及无水和非无水脂肪酸或酰卤作为酰基源，以及几种反应催化剂。一系列涉及产物离子扫描和多反应监测（MRM）的质谱实验用于确认合成产物的身份并阐明其裂解途径。
在亚纳摩尔规模下，考察了由棕榈酸酐和4-（二甲基氨基）吡啶（DMAP）在无水吡啶中组成的酰化体系关于反应时间和温度的反应产率，结果表明，对于环亚胺、azaspiracid-1和pectenotoxin-2，分别在75℃反应60分钟、75℃反应120分钟和100℃反应270分钟时条件最佳。对裸甲藻毒素-A（GYM-A）的酯化反应进行了更大规模的验证，其在合成10-O-棕榈酰-GYM-A时保持了>90%的良好产率。
已合成亲脂性海洋毒素的酰基酯类似物，并通过LC/MS/MS阐明了其结构。对于与天然代谢产物相同的酰基酯类似物，所开发的程序有潜力以可持续、可扩展和可控的方式应用于代谢产物的半合成，避免从天然污染的贝类中进行广泛且繁琐的分离和纯化程序。对于结构与贝类中发现的不同的半合成酯，它们可能适用于作为内标，用于准确定量复杂基质中存在的天然代谢产物。
