The C19 position of 25-Hydroxyvitamin D3 faces outward in the vitamin D sterol-Binding pocket of vitamin D-Binding protein

The radiolabeled affinity and photoaffinity analogues of 25-hydroxyvitamin D3 (25-OH-D3) with probes at the C-19 position failed to specifically label the 25-OH-D3-binding pocket of vitamin D-binding protein (DBP). However, a hybrid analogue, with a bromoacetate affinity probe and a photoaffinity probe at C3-OH and C19 positions, respectively, specifically labeled the ligand-binding pocket, suggesting that C3-OH points towards the ‘inside’ of the binding cavity while the C19 position faces away from it.