Refined Structures of O-Phospho-l-serine and Its Calcium Salt by New Multinuclear Solid-State NMR Crystallography Methods

Abstract

O -phospho- l -serine (Pser) and its Ca salt, Ca[ O -phospho- l -serine]·H 2 O (CaPser), play important roles for bone mineralization and were recently also proposed to account for the markedly improved bone-adhesive properties of Pser-doped calcium phosphate-based cements for biomedical implants. However, the hitherto few proposed structural models of Pser and CaPser were obtained by X-ray diffraction, thereby leaving the proton positions poorly defined. Herein, we refine the Pser and CaPser structures by density functional theory (DFT) calculations and contrast them with direct interatomic-distance constraints from two-dimensional (2D) nuclear magnetic resonance (NMR) correlation experimentation at fast magic-angle spinning (MAS), encompassing double-quantum–single-quantum (2Q–1Q) 1 H NMR along with heteronuclear 13 C{ 1 H} and 31 P{ 1 H} correlation NMR experiments. The Pser and CaPser structures before and after refinements by DFT were validated against sets of NMR-derived effective 1 H– 1 H, 1 H– 31 P, and 1 H– 13 C distances, which confirmed the improved accuracy of the refined structures. Each distance set was derived from one sole 2D NMR experiment applied to a powder without isotopic enrichment. The distances were extracted without invoking numerical spin-dynamics simulations or approximate phenomenological models. We highlight the advantages and limitations of the new distance-extraction procedure. Isotropic 1 H, 13 C, and 31 P chemical shifts obtained by DFT calculations using the gauge including projector augmented wave (GIPAW) method agreed very well with the experimental results. We discuss the isotropic and anisotropic 13 C and 31 P chemical-shift parameters in relation to the previous literature, where most data on CaPser are reported herein for the first time.

Nuclear Magnetic Resonance and Metadynamics Simulations Reveal the Atomistic Binding of l-Serine and O-Phospho-l-Serine at Disordered Calcium Phosphate Surfaces of Biocements

“…The NMR-derived { M 2 (C j –P)} data from the polycrystalline Pser/CaPser powders were validated against those calculated from their crystal structures. ,, As expected − and discussed further in Section S1.3, the NMR-derived dipolar second moments are consistently lower than their theoretical counterparts ( ). Yet, the ratios are essentially constant (Table S5): a very good agreement is observed for all relative M 2 (C j –P) values within each Pser or CaPser structure, which remain well within the experimental uncertainties.…”

“…The Pser@HA synthesis protocol was preceded by extensive testing of optimal preparation conditions by using Pser (Flamma SpA) with concentrations in the 5−200 mM range. As verified by Figure S3, [Pser] ≥ 20 mM yielded products with little/no surface immobilization but predominantly comprised the Ca salt of Pser, Ca[O-phospho-L-serine]•H 2 O, 64,65 liquid were mixed with a spatula for ≈30 s. The cement paste was cured at 21 °C and 30% relative humidity for 15−30 min and then transferred to a sealed plastic bag that was stored at 37 °C for 7 days at 100% humidity. All cement specimens were thereafter stored under dry conditions in a desiccator.…”

“…We onwards focus on the two D-HMQC NMR signals centered at the {δ P , δ C } shift-pairs of {3.0, 184.2} ppm and {2.4, 170.0} ppm, which are tentatively assigned to COO – moieties that are strongly surface bound predominantly/solely via electrostatic C O ··· Ca 2+ interactions and C O ··· H bonds, respectively. While noting that those two contact modes were predicted by modeling (Section ), the NMR-peak assignments were based on the distinctly different 13 C and 31 P chemical shifts involved in each correlation: the high shifts of the {δ P , δ C } = {3.0, 184.2} ppm signal are well-aligned with COO – moieties nearby groups (see Sections and 3.2), whereas the lower chemical shifts of the {2.4, 170.0} ppm correlation are commensurate with H-bond-mediated 13 C OO – ··· contacts, whose 13 C chemical shift incidentally matches that of the 13 C OOH group of polycrystalline Pser that manifests analogous inter molecular COOH··· motifs. ,, Notably, Figure reveals a lower NMR-signal intensity at 184 ppm from the Ser16 cement relative to its Ser8 counterpart, suggesting a concurrently reduced number of C O ··· Ca 2+ contacts for increasing batched Ser content, along with earlier and more qualitative findings by Mathew et al and our inferences in Section .…”

“… a 15 N– 31 P and 13 C– 31 P dipolar second moments obtained for the as-indicated functional groups from 15 N­{ 31 P} and 13 C­{ 31 P} REDOR NMR experiments, respectively, and compared with data calculated either from metadynamics-derived structural models (for Ser16, Pser16, and Pser@HA) or from the crystal structures of Pser and CaPser . The M 2 (C j –P) data uncertainties are ±1.7 kHz 2 (NMR) and ±2.2 kHz 2 (metadynamics model), with kHz 2 ≡ 1000 s –2 . b Net experimental/calculated values; the NMR analysis afforded the extraction of three M 2 (CO–P) values of {23.6, 9.3, 13.3} kHz 2 for the 13 C O shifts at {184, 177, 174.5} ppm, respectively. c Values within parentheses represent the contribution to the net M 2 (N–P) and { M 2 (C j –P)} values from the Pser···HA contacts alone; the contribution from the intra molecular 15 N– 31 P and 13 C j – 31 P dipolar interactions account for the difference. d The { M 2 (C j –P)} and M 2 (N–P) values were obtained from the Pser (Flamma) and isotopically enriched Pser* samples, respectively. …”

“… a 15 N– 31 P and 13 C– 31 P dipolar second moments obtained for the as-indicated functional groups from 15 N­{ 31 P} and 13 C­{ 31 P} REDOR NMR experiments, respectively, and compared with data calculated either from metadynamics-derived structural models (for Ser16, Pser16, and Pser@HA) or from the crystal structures of Pser and CaPser . The M 2 (C j –P) data uncertainties are ±1.7 kHz 2 (NMR) and ±2.2 kHz 2 (metadynamics model), with kHz 2 ≡ 1000 s –2 . …”