Murbach-Oliveira Giulia, Banerjee Kalpita, Nociari Marcelo M, Thompson David H
Department of Chemistry, Multidisciplinary Cancer Research Facility, Purdue University, West Lafayette, Indiana 47907, United States.
Weil Cornell Medical College, Ophthalmology Department, Dyson Research Institute, 1300 York Avenue, New York, New York 10065, United States.
ACS Bio Med Chem Au. 2022 Feb 18;2(3):297-306. doi: 10.1021/acsbiomedchemau.1c00060. eCollection 2022 Jun 15.
-Retinylidene--retinylethanolamine (A2E) is the most studied lipid bisretinoid. It forms lipofuscin deposits in the retinal pigment epithelium (RPE), causing vision impairment and blindness in eye conditions, such as Stargardt's disease, cone-rod dystrophy, Best's macular dystrophy, and potentially age-related macular degeneration. Synthetic A2E is often used for inducing the accumulation of lipofuscins within the lysosomes of RPE cells in culture as an in vitro surrogate of retinal lipofuscin buildup, providing insights into the mechanisms of these eye conditions. Many reports describing the use of synthetic A2E employ material that has been prepared using a one-pot reaction of all-trans-retinal (ATR) and ethanolamine at room temperature for 48 h. We have revisited this synthesis by performing a design of experiments (DoE) and high-throughput experimentation workflow that was tailored to identify the most productive combination of the variables (temperature, solvent, and reagent equivalences) for optimization of A2E yield. Our DoE findings revealed that the interaction of ethanolamine with acetic acid and ATR was pivotal for the formation of A2E in high yield, indicating that imine formation is the critical step in the reaction. Armed with these results, we were able to optimize the method using a microfluidic reactor system before upscaling those conditions for continuous flow synthesis of A2E. This revised method enabled a more efficient production of material, from a reaction time of 48 h to a residence time of 33 min, with an accompanying yield improvement from 49 to 78%. Furthermore, we implemented a simple method to evaluate the quality of the A2E produced using optical spectroscopy and LC-MS characteristics to assure that the biological properties observed with A2E samples are not confounded by the presence of oxidized impurities that are commonly present in conventional A2E samples.
视黄叉 - 视黄基乙醇胺（A2E）是研究最多的脂质双视黄醛。它在视网膜色素上皮（RPE）中形成脂褐素沉积物，在诸如斯特格黄斑营养不良、锥杆营养不良、贝斯特黄斑营养不良以及可能的年龄相关性黄斑变性等眼部疾病中导致视力损害和失明。合成A2E常用于在培养的RPE细胞溶酶体内诱导脂褐素积累，作为视网膜脂褐素积累的体外替代模型，为了解这些眼部疾病的机制提供线索。许多描述合成A2E使用的报告采用的材料是通过全反式视黄醛（ATR）和乙醇胺在室温下一步反应48小时制备的。我们通过进行实验设计（DoE）和高通量实验工作流程重新审视了这种合成方法，该流程旨在确定变量（温度、溶剂和试剂当量）的最有效组合以优化A2E产量。我们的DoE结果表明，乙醇胺与乙酸和ATR的相互作用对于高产率形成A2E至关重要，这表明亚胺形成是反应的关键步骤。基于这些结果，我们能够在将这些条件扩大用于A2E的连续流合成之前，使用微流控反应器系统优化该方法。这种改进的方法使材料生产更高效，反应时间从48小时缩短至停留时间33分钟，同时产率从49%提高到78%。此外，我们实施了一种简单方法，利用光谱学和LC-MS特性评估所生产A2E的质量，以确保A2E样品所观察到的生物学特性不会因传统A2E样品中常见的氧化杂质的存在而混淆。
