Thermodynamic and Structural Behavior of α‐Galactosylceramide and C6‐Functionalized α‐GalCer in 2D Layers at the Air–Liquid Interface

α‐Galactosylceramide (α‐GalCer; KRN7000) is a ligand for the glycoprotein CD1d that presents lipid antigens to natural killer T cells. Therefore, KRN7000 as well as some modified versions thereof have been widely investigated as part of novel immunotherapies. To examine the impact of structural modification, we investigated KRN7000 and C6‐modified KRN7000 at the air–liquid interface using monolayer isotherms, BAM, IRRAS, GIXD, and TRXF. The amino group has no influence on the highly ordered sub‐gel structures found at lateral pressures relevant for biological membranes. Neither lateral compression nor the protonation state of the amino group has a measurable effect on the lattice structure, which is defined by strong and rigid intermolecular hydrogen bonds. However, the first‐order phase transition found for the C6‐functionalized α‐GalCer is connected with an extraordinary surface‐inhibited nucleation. Our study demonstrates that KRN7000 can be functionalized at C6 without significantly changing the structural properties.