Background /objective

The phospholipid 1,2-dipalmitoyl-rac-glycero-3-phosphatidylethanolamine (PE) comprises two fatty acid chains: glycerol, phosphate, and ethanolamine. PE participates in critical cellular processes such as apoptosis and autophagy, which places it as a target for designing new therapeutic alternatives in diseases such as pulmonary fibrosis. Therefore, this study aimed obtain PE through a six-step organic synthesis pathway and determine its biological effect on apoptosis induction in normal human lung fibroblasts (NHLF).

Methodology

The first step of the organic synthesis route began with protected glycerol that was benzylated at sn-3; later, it was deprotected to react with palmitic acid at sn-1, sn-2. To remove the benzyl group, hydrogenation was performed with palladium on carbon (Pd/C); subsequently, the molecule was phosphorylated in sn-3 with phosphorus oxychloride and triethylamine, and the intermediate was hydrolyzed in an acid medium to obtain the final compound. After PE synthesis, apoptosis assessment was performed: apoptosis was induced using exposure to annexin V-FITC/propidium iodide-ECD (PI) and quantified using flow cytometry. The experiments were performed in three NHLF cell lines with different concentrations of PE 10, 100 and 1000 µg/mL for 24 and 48 h.

Results

The PE obtained by organic synthesis presented a melting point of 190–192 °C, a purity of 95%, and a global yield of 8%. The evaluation of apoptosis with flow cytometry showed that at 24 h, exposure to PE 10, 100, and 1000 µg/mL induces early apoptosis in 19.42%− 25.54%, while late apoptosis was only significant P < 0.05 in cells challenged with 100 µg/mL PE. At 48 h, NHLF exposed to PE 10, 100, and 1000 µg/mL showed decreasing early apoptosis: 28.69–32.16%, 12.59–18.84%, and 10.91–12.61%, respectively. The rest of the NHLF exposed to PE showed late apoptosis: 12.03–16–42%, 11.04–15.94%, and 49.23–51.28%. Statistical analysis showed a significance P < 0.05 compared to the control.

Conclusion

The organic synthesis route of PE allows obtaining rac-1,2-O-Dipalmitoyl-glycero-3-phosphoethanolamine (1), which showed an apoptotic effect on NHLF.

中文翻译：

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1,2-二棕榈酰-rac-甘油-3-磷脂酰乙醇胺的有机合成及其诱导正常人肺成纤维细胞凋亡的作用

 背景/目标

磷脂1,2-二棕榈酰-外消旋-甘油-3-磷脂酰乙醇胺(PE)包含两条脂肪酸链：甘油、磷酸盐和乙醇胺。 PE 参与细胞凋亡和自噬等关键细胞过程，这使其成为设计肺纤维化等疾病的新治疗替代方案的目标。因此，本研究旨在通过六步有机合成途径获得PE，并确定其对正常人肺成纤维细胞（NHLF）诱导凋亡的生物学作用。

 方法

有机合成路线的第一步从受保护的甘油开始，在sn -3 处进行苄基化；然后脱保护，在sn -1、 sn -2 处与棕榈酸反应。为了除去苄基，用钯碳(Pd/C)进行氢化；随后，用三氯氧磷和三乙胺将分子在sn -3 中磷酸化，中间体在酸性介质中水解得到最终化合物。 PE 合成后，进行细胞凋亡评估：使用膜联蛋白 V-FITC/碘化丙啶-ECD (PI) 诱导细胞凋亡，并使用流式细胞术进行定量。实验在三种 NHLF 细胞系中进行，分别使用不同浓度的 PE 10、100 和 1000 µg/mL，持续 24 和 48 小时。

 结果

有机合成得到的PE熔点为190-192℃，纯度为95%，总收率为8%。流式细胞仪对细胞凋亡的评估显示，24小时时，暴露于PE 10、100和1000 µg/mL会诱导19.42%− 25.54%的早期细胞凋亡，而在用100μg/mL攻击的细胞中，晚期细胞凋亡仅显着P< 0.05。微克/毫升PE。 48 小时时，暴露于 PE 10、100 和 1000 µg/mL 的 NHLF 显示早期细胞凋亡减少：分别为 28.69–32.16%、12.59–18.84% 和 10.91–12.61%。暴露于 PE 的其余 NHLF 显示晚期凋亡：12.03-16-42%、11.04-15.94% 和 49.23-51.28%。统计分析显示与对照相比显着性P< 0.05。

 结论

PE的有机合成路线可以获得外消旋-1,2- O-二棕榈酰基-甘油-3-磷酸乙醇胺( 1 )，其对NHLF表现出凋亡作用。