NMR investigations of the static and dynamic structures of bisphosphonates on human bone: A molecular model

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using ²H, ¹³C, ¹⁵N, and ³¹P nuclear magnetic resonance spectroscopy. The ³¹P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of Pamidronate is well described by a Langmuir-like isotherm, from which we deduce an ∼30-38 Ų surface area per Pamidronate molecule and a ΔG = -4.3 kcal mol^-1. TEDOR of [ ¹³C₃, ¹⁵N] Pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of ³¹P as well as ¹⁵N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of ²H NMR measurements of side-chain ²H-labeled Pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In Pamidronate, the motion is well simulated by a gauche⁺/gauche^- hopping motion of the terminal -CH₂-NH₃⁺ group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH₃⁺-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO₄^3- sites.