Synthesis of a novel C46 ceramide and its skin barrier repair effect verified by molecular dynamics and cellular assays

Objective: Ceramides play a critical role in skin barrier function. Here, we synthesized a novel long-chain ceramide, C46 EOP, via a sustainable catalyst-free route and elucidated its molecular mechanisms and barrier repair efficacy using an integrated computational-experimental strategy. Methods: A novel C46 long-chain ceramide EOP was synthesized via a sustainable catalyst-free two-step cascade route, achieving purity exceeding 98.5%. To elucidate its mechanism of action, molecular docking and molecular dynamics simulations were employed to predict the binding modes and stability of EOP with key skin barrier targets (IL-6, AQP3, ELANE), supported by thermodynamic free energy calculations. The functional outcomes of EOP were experimentally verified by assessing cell viability (MTT), migration, and gene/protein expression (RT-qPCR, ELISA). Results: Molecular docking and molecular dynamic simulation identified stable EOP binding to key targets of skin barrier: IL-6 (ΔG = −5.393 kcal/mol), AQP3 (ΔG = −5.300 kcal/mol), and ELANE (ΔG = −5.543 kcal/mol). In vitro assays demonstrated concentration-dependent inhibition of elastase activity, reaching 34.78% at 772 mg/L. EOP treatment significantly suppressed IL-6 mRNA expression by 89% at 772 mg/L and reduced IL-6 protein levels by 56.83% at 123.52 mg/L. Furthermore, EOP markedly upregulated AQP3 mRNA expression by 42.34% at 1235.2 mg/L and increased AQP3 protein expression by 129% at 123.52 mg/L. Conclusion: This integrated computational-experimental approach validates the multi-target efficacy of the novel synthetic C46 ceramide EOP in skin barrier repair at both molecular and cellular levels. The work deepens the mechanistic understanding of long-chain ceramides and provides a rational strategy for the development of novel dermatological actives.