Continuous Flow Synthesis of A2E Guided by Design of Experiments and High-Throughput Studies

N-Retinylidene-N-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.