Stereochemical elucidation of molecules with multiple chiral centers is difficult. Even with VCD spectroscopy, excluding all but one diastereomeric structural candidates is challenging because stereochemical inversion of one chiral center...

Stereochemistry has a substantial impact on the biological activity of various drugs. We investigated the role of stereochemistry of ceramides in inducing the production of exosomes, a type of extracellular vesicle, from neuronal cells, with a potential benefit in improving the clearance of amyloid‐β (Aβ), a causal agent of Alzheimer's disease. A stereochemical library of diverse ceramides with different tail lengths was synthesized with the purpose of varying stereochemistry (D‐erythro: DE, D‐threo: DT, L‐erythro: LE, L‐threo: LT) and hydrophobic tail length (C6, C16, C18, C24). The exosome levels were quantified using TIM4‐based exosome enzyme‐linked immunosorbent assay after concentrating the conditioned medium using centrifugal filter devices. The results revealed a pivotal role of stereochemistry in determining the biological activity of ceramide stereoisomers, with the superiority of those based on DE and DT stereochemistry with C16 and C18 tails, which demonstrated significantly higher exosome production, without a significant change in the particle size of the released exosomes. In transwell experiments with Aβ‐expressed neuronal and microglial cells, DE‐ and DT‐ceramides with C16 and C18 tails significantly decreased extracellular Aβ levels. The results reported here are promising in the design of non‐classic therapies for the treatment of Alzheimer's disease.