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.

Interactions between biomolecules and structurally disordered calcium phosphate (CaP) surfaces are crucial for the regulation of bone mineralization by noncollagenous proteins, the organization of complexes of casein and amorphous calcium phosphate (ACP) in milk, as well as for structure-function relationships of hybrid organic/inorganic interfaces in biomaterials. By a combination of advanced solid-state NMR experiments and metadynamics simulations, we examine the detailed binding of O-phospho-l-serine (Pser) and l-serine (Ser) with ACP in bone-adhesive CaP cements, whose capacity of gluing fractured bone together stems from the close integration of the organic molecules with ACP over a subnanometer scale. The proximity of each carboxy, aliphatic, and amino group of Pser/Ser to the Ca2+ and phosphate species of ACP observed from the metadynamics-derived models agreed well with results from heteronuclear solid-state NMR experiments that are sensitive to the 13C-31P and 15N-31P distances. The inorganic/organic contacts in Pser-doped cements are also contrasted with experimental and modeled data on the Pser binding at nanocrystalline HA particles grown from a Pser-bearing aqueous solution. The molecular adsorption is driven mainly by electrostatic interactions between the negatively charged carboxy/phosphate groups and Ca2+ cations of ACP, along with H bonds to either protonated or nonprotonated inorganic phosphate groups. The Pser and Ser molecules anchor at their phosphate/amino and carboxy/amino moieties, respectively, leading to an extended molecular conformation across the surface, as opposed to an "upright standing" molecule that would result from the binding of one sole functional group.

Introduction

Bone mineral constitutes nanoparticles of a structurally disordered and carbonate-bearing form of Ca hydroxyapatite [HA; “apatite”; Ca10(PO4)6(OH)2].[1−5] Vast research efforts have been spent to understand the mechanisms of bone mineralization,[5−10] which remain controversial but are believed to be governed by noncollagenous proteins (NCPs) that carry a high density of negatively charged carboxy-bearing (Asp/Glu) and phosphorylated residues.[5,9−11] For decades, mainstream models for the NCP/mineral interactions involved “charge-matching” arguments assuming that the protein adopts a secondary structure with its negatively charged sidechains matching the Ca2+ positions at the apatite mineral surface to provide epitaxial crystal growth.[6,7] However, doubts thereof arose from the subsequently accumulating evidence that (i) the crystal-binding domains of most NCPs lack a well-defined secondary structure[10,11] and (ii) both synthetic and biogenic nanocrystalline apatite particles formed from aqueous solutions—such as body fluids—consist of a “core” of an ordered HA lattice coated by a 1–2 nm thick surface layer (“shell”) of amorphouscalcium phosphate (ACP),[4,12−16] often termed the “hydrated surface layer”.[2,3,17] These recent insights have led to a paradigm shift advocating simpler electrostatic models for which the net charge of the protein and its underlying distribution control the NCP binding at biogenic apatite.[18−21]

An accurate atomistic probing of biomolecular adsorption at structurally disordered inorganic calcium phosphate (CaP) surfaces by experimental techniques is hampered by the particle fragility, coupled with their structural disorder. The most detailed current insight is provided by magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) experiments, often utilizing proteins with isotopic 13C/15N labeling at specific sites in its “crystal”-binding domain. Some MAS NMR reports targeted biomolecular binding at bone mineral,[22−26] but adsorption on model systems of HA nanoparticles is most commonly encountered, encompassing the surface binding of small biomolecules[27−32] and various mineralization-controlling NCPs such as statherin,[33,34] osteopontin,[35] osteonectin,[36,37] and osteocalcin.[38] Here, advanced NMR techniques relying on through-space dipolar interactions, such as 13C{31P} and 15N{31P} rotational-echo double resonance (REDOR)[39] NMR experiments, have been exploited for obtaining (semi-)quantitative 13C–31P and 15N–31P interatomic distance-information along with 2D 13C–13C correlation NMR experiments that offer constraints on the surface-bound molecular conformation.

Considering the difficulties in experimental probing of the surface binding at atomic resolution, computer modeling by atomistic molecular dynamics (MD) and/or metadynamics simulations offers a rich source of structural information on the organic/inorganic interface.[18−20,40−48] They may reveal the precise binding sites of the biomolecule along with its detailed surface-immobilized conformation—henceforth referred to as the binding mode of the molecule. Yet, outstanding problems in computational modeling of biomolecular adsorption at an in vitro- or in vivo-generated nanocrystalline apatite surface concern how its structural disorder and potentially pH-dependent phosphate speciation are accounted for. As discussed further in the Supporting Information, these critical aspects remain essentially ignored in all but a few very recent modeling studies.[44−49]

Interfaces between phosphorylated biomolecules and disordered CaP phases are not only of pivotal importance for understanding bone mineralization but also underpin casein–ACP complex-formation in milk[50−53] and structure–function relationships of hybrid organic/inorganic (bio)materials, such as the bone-tissue-adhesive properties of CaP cements incorporating O-phospho-l-serine (Pser),[54−59] which by their capacity to glue bone tissues together makes them promising for accelerating bone-fracture healing. As unveiled by an array of advanced MAS NMR techniques, we recently demonstrated that the “bone-gluing” strength of such a cement correlates well with its content of an amorphous “ACP/Pser” phase of Pser molecules intimately integrated with ACP across a sub-nm scale.[60] An analogous “ACP/Ser” component forms in l-serine (Ser) doped cements which, however, exhibit poor bone-adhesive properties,[60] as discussed and rationalized further herein.

Herein, we advance the atomic-scale insight into the organic/inorganic interface further by examining cements prepared from uniformly 13C/15N–enriched l-serine and O-phospho-l-serine (the latter synthesized herein for the first time), thereby enabling direct experimental probing of the proximity of each carboxy/aliphatic and amino group to the inorganic phosphate moieties of ACP by 13C{31P} and 15N{31P} REDOR NMR experimentation, respectively. These results are contrasted with information from heteronuclear 13C{31P} and 1H{31P} correlation 2D NMR experiments that reveal the contacts between the organic functional groups of Pser/Ser and the inorganic / phosphate moieties of ACP. We also discuss the similarities and differences between the inorganic/organic contacts in the amorphous ACP/Pser and ACP/Ser biocement components with that of nanocrystalline HA particles grown from a Pser-bearing aqueous solution, henceforth referred to as “Pser@HA”, and constituting a simplified model of a carboxy-bearing or/and phosphorylated protein residue interacting with bone mineral. Moreover, the NMR-derived 13C–31P and 15N–31P interatomic-distance constraints were contrasted with results from state-of-the-art well-tempered metadynamics MD simulations[61,62] employing the INTERFACE force field and the HA-surface preparation protocol of refs (45) and (49) to faithfully model the adsorption of Ser and Pser molecules at the amorphous surface of apatite nanoparticles. These structural models offer a wealth of structural details about the Ser/Pser binding modes (encompassing the surface-bound molecular conformation), as well as a quantification of the relative contribution of each functional group toward the binding via a recently introduced analysis protocol.[48]

Materials and Methods

Preparation of 13C/15N-Enriched Samples

l-serine (Aldrich; 99.5%) and O-phospho-l-serine (Flamma SpA; 95%) with all isotopes at their natural abundance were used as received. [U–13C/15N]-l-serine enriched to 98% with respect to both 13C/15N was purchased from CortecNet (France). It was used to prepare [U–13C/15N]-O-phospho-l-serine, which was obtained as its HCl salt (Figure S1) from the corresponding BocNH-Ser-CO2-tert-Bu analogues via phosphoramidation with tert-butyl N, N′-diisopropylcarbamimidate followed by peroxide oxidation to yield the protected phosphate ester, which was converted into the desired product by global deprotection under acidic conditions; see Section S3 for details. These isotopically labeled Ser and Pser powders are for simplicity abbreviated as Ser* and Pser*, respectively. They were used for producing a nanocrystalline HA powder with surface-immobilized Pser* molecules, as well as cements comprising N mol % of Ser* or Pser*, which are henceforth denoted by SerN and PserN, respectively.

Pser@HA Specimen

5.0 mL of a 500 mM aqueous solution of CaCl2 in deionized water was pipetted in a 20 mL flask placed in a water bath at 37 ± 2 °C, whereupon Pser*·HCl was added to the solution to yield a concentration of 10 mM. 5 mL of 300 mM (NH4)3PO4(aq) was added at mL/min with continuous magnetic stirring. The pH of the solution was subsequently raised to 7.5 by dropwise addition of 1 M NaOH(aq), leading to precipitation of floccular ACP particles. Deionized water was added to a final volume of 20 mL, and the flask was sealed. The suspension was aged for one week at 37 ± 0.2 °C, leading to a final pH value of 5.3. The as-formed Pser@HA particles were isolated by centrifugation, cleaned twice with deionized water, and then dried in a desiccator at room temperature for 4 days.

The Pser content was estimated as ≈ 5 wt % by contrasting the integrated 15N NMR intensity from the Pser@HA specimen with that of the Pser16 cement with known Pser content. The transmission electron microscopy images of Figure S2 reveal agglomerates of crystalline domains with variable sizes (from a few nm to 10–20 nm) that are fused together by ACP. The surface area was estimated to be 159 m2/g by the Brunauer–Emmett–Teller model[63] (N2 uptake at relative pressures (P/P0) of 0.05–0.15, using a Micrometrics ASAP2020 volumetric adsorption analyzer).

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]·H2O,[64,65] henceforth denoted CaPser.

Ser/Pser-Doped Cements

The SerN and PserN cements with N = {8, 16, 30} were prepared in batches of 0.25 g by first dissolving/suspending each organic Ser* and Pser* additive in 60 μL of deionized water (see Table S1), whereupon a powder of α-Ca3(PO4)2 (α-TCP) (see refs (54) and (60) for preparation details) was added at liquid-to-powder (L/P) ratio of 0.24 mL/g. The powder and 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. For practical reasons, pH measurements of the cement pastes prior to their setting were performed on larger SerN/PserN batches of 3.0 g and a slightly higher L/P = 0.4 mL/g ratio. Table S1 presents the pH value associated with each cement preparation, obtained as an average over three independent preparations.

Solid-State NMR Experiments

The solid-state NMR experimentation was performed with Bruker Avance-III spectrometers operating at the magnetic fields (B0) of 9.4 T or 14.1 T, which provided {1H, 13C, 31P} Larmor frequencies of {−400.1, −100.6, −162.0} MHz and {−600.1, −150.9, −242.9} MHz, respectively, along with 60.4 MHz for 15N at 14.1 T. A fine powder of each specimen was packed in a ZrO2 rotor of outer diameters of either 2.5 mm (thin-wall), 3.2, or 4 mm, as specified along with each MAS rate (νr) in the figure captions and in Section S1, which provides all details of each NMR experiment. Full rotors were employed throughout, except for the REDOR NMR[39] experiments, for which each sample was restricted to the center 1/3 volume of a 4 mm rotor. 1H/13C, 31P, and 15N chemical shifts are quoted relative to neat tetramethylsilane (TMS), 85% H3PO4(aq), and solid 15NH4Cl, respectively.

Metadynamics Simulations

The preparation of the simulated systems and the well-tempered metadynamics simulations were performed as described in ref (48). Here, we only recapitulate the most essential information and refer to Section S2 and ref (48) for details. A box with side-lengths {3.9, 4.3, 7.8} nm (periodic boundary conditions) contained the simulated system of three components (Figure a): one Ser or Pser molecule in a water phase (2600 H2O molecules) that interfaced a “HA slab”, whose interior consisted of an HA lattice of the biologically relevant P63/m modification.[66] The “surface” segment was generated according to ref (45), which mimics a disordered surface at a nanocrystalline HA-particle by introducing acidic protons at randomly selected phosphate groups accompanied by Ca2+-cation removal until a charge-neutral surface is obtained, whose {, , } speciation matches that of phosphate ions in an aqueous solution at a given pH value; see Figure . The HA surface and the Pser/Ser protonation states were implemented for the experimentally relevant pH values of 4.5 for Pser and 7.4 for Ser, as explained further in Section S2.

Figure 1

(a) Simulated three-component system, consisting of a “HA slab” with a lattice of crystalline HA and a disordered surface that interface a water phase that contains one Pser molecule (or Ser; not shown). The surface atoms are enlarged for better visualization. The box lengths along the z and x directions are indicated, along with the (approximate) lengths of the water and lattice/surface domains. (b,c) Dependence of the average number of protons per phosphate group at the as-prepared HA surface (red curve) along with the net number of protons of the molecular forms of (b) Pser and (c) Ser (black curves) plotted against the pH value of the solution. The most stable molecular form of (b) Pser and (c) Ser at a given pH value is shown on top of each graph, where the colored circles enclose the functional group(s) with altering protonation states between the pH domains marked by black vertical lines. The vertical green lines mark the simulated pH values of 4.5 and 7.4. Each number inside the graph specifies the respective pKa values of the organic functional groups, as well as the inorganic {H3PO4, H2PO4−, and } surface species (the phosphate speciation at the surface matches that of the surrounding solution[45]).

The atomistic MD simulations involved NVT ensembles at the temperature T = 37 °C, utilizing the GROMACS v2018.1 platform[67] and the following force-fields: INTERFACE[45,49] for the HA slab, CHARMM36 (July 2017)[68] for the Pser/Ser molecules, and TIP3P[69] (without Lennard-Jones terms for H[70]) for water. Well-tempered metadynamics[61,62] with a bias factor of γ = 5 and 32 independent “walkers”[62,71] were employed to ensure an efficient sampling of the molecular conformations and accelerate the convergence, using the variational enhanced sampling (VES) protocol[72] implemented in the PLUMED2.4 software.[73] Two collective variables were exploited for locating the most stable surface-bound molecular conformation, involving the distance between the center of the HA slab and the COO–/ atom of the respective Ser/Pser molecule along with an interaction-energy dependent function defined in Section S2. The reported modeled parameters and their uncertainties are averages over 4 independent simulations.

Results and Discussion

Local 31P Environments Probed by MAS NMR

Figure displays 31P MAS NMR spectra recorded from the nanocrystalline Pser@HA sample and the PserN/SerN cements with N = {8, 16, 30}. The peak intensities of the single-pulse-acquired NMR spectra of the left panel quantitatively reflect the relative phase constituents of each sample, whereas those of the right panel of Figure were obtained by 1H → 31P CP, thereby only revealing 31P NMR signals from phosphate groups nearby protons. The directly excited 31P MAS NMR peak shapes from the cements are complex due to the contributing signals from unreacted α-TCP (Figure S4). The absence of protons in the α-TCP structure, however, renders the 1H → 31P CPMAS spectra considerably simpler because they only comprise 31P resonances from 1H-bearing phases, which predominantly involve the amorphous ACP/Pser or ACP/Ser components.[60] We henceforth focus on the 1H → 31P CPMAS NMR spectra along with the deconvolutions into their underlying NMR-peak components and the associated best-fit parameters presented in Table S6. The NMR spectra from the cements in Figure overall match those presented in ref (60) from near-identical preparations with 13C/15N isotopes at natural abundance.

Figure 2

31P MAS NMR spectra recorded at 14.00 kHz MAS either directly by single pulses (left panel) or by 1H → 31P CP (right panel) from (a,b) Pser@HA, or from the as-indicated (c–f) SerN and (g–l) PserN cements. Note that no NMR peak from remnants of the proton-free α-TCP precursor (Figure S4) appear in the CPMAS-derived spectra; the latter are shown together with the best-fit spectra (orange traces) and the deconvolutions into the as-indicated peak components given in each legend. The narrow NMR peaks at 1.4 ppm and −1.0 ppm stem from minor impurities of brushite (CaHPO4·2H2O) and CaPser (Ca[O-phospho-l-serine]·H2O), respectively, while that marked by an asterisk in (d) derives from an unknown impurity. Table S6 lists the best-fit NMR parameters.

Both the directly excited and CP-derived 31P MAS NMR spectra of the Pser@HA specimen (Figure a,b) are typical for nanocrystalline HA particles, which involve a crystalline HA “core” coated by a surface layer (“shell”) of ACP.[2−4,12−17] The “core” and “shell” components produce a narrow and broad resonance, respectively,[4,12−14] centered at the corresponding 31P chemical shifts δP ≈ 3.0 ppm and δP ≈ 2.2 ppm (Figure a,b and Table S6). The lower shift of the ACP component reflects the acidic solution (pH = 5.3) surrounding the Pser@HA particles before their isolation, which yields a surface layer of ACP enriched in protonated phosphate moieties[4,13,14,16,74] resonating at lower 31P shifts than nonprotonated groups.[75−77] Moreover, the close 1H–31P distances of /H2PO4− groups emphasize the “ACP” contribution in the 1H → 31P CPMAS NMR spectrum (Figure b) relative to the directly excited counterpart (Figure a); also see Table S6.

Cements prepared from α-TCP and water in the absence of Pser consist of disordered HA.[60,78−80] Although ACP and HA particles are unstable in acidic aqueous solutions, they become stabilized by negatively charged organic additives in weakly acidic solutions.[4] The pH value of the precursor paste before the cement setting is reduced for increasing Pser/Ser content, while at a fixed doping level N, Pser yields a markedly lower pH value than Ser (Table S1). Hence, the highly acidic PserN cement pastes preclude HA formation, whereas the Ser8 and Ser16 samples comprise significant HA contributions (Figure d,f and Table S6), as proved unambiguously by the heteronuclear correlation NMR results of Section . The progressively reduced pH values for the Pser-bearing cements also manifest as 31P resonance displacements of the ACP/Pser phase toward lower chemical shifts for increasing N. These acidic conditions also induced a minor brushite (CaHPO4·2H2O) formation, as signified by the narrow resonance at δP = 1.4 ppm[60,81,82] (Figure h,j,l). It is instructive to contrast the 31P shifts among the ACP (≈2.2 ppm; Figure a,b), ACP/Ser (≈2.3 ppm; Figure d,f), and ACP/Pser (0.7–1.8 ppm; Figure h,j,l) phases with that from amorphous tricalcium phosphate of approximate stoichiometry Ca3(PO4)2·nH2O (δP ≈ 3 ppm)[81,83,84] and the H31 environments of amorphous CaHPO4·nH2O (δP ≈ 1.5 ppm).[4,77]

Organic/Inorganic Interface Probed by 1H–31P Correlation NMR

Figure presents the 1H MAS NMR spectra recorded from the Pser@HA sample along with the SerN and PserN cements. The various 1H sites of the present specimens may be of organic {COOH, POH, NH3, CH} or inorganic {OH, H} origin, along with H2O molecules that may either constitute an integral component of the inorganic ACP and HA structures (“structure-bound”), associated with the inorganic/organic ACP/Pser and ACP/Ser components or being mobile physisorbed species. The latter 1H2O molecules typically resonate in the 4.5–5.5 ppm spectral region,[13,14,16,74,85,86] where Figure suggests that they constitute a significant fraction of the entire proton reservoir in the Pser@HA and PserN/SerN samples. Moreover, the characteristic 1H NMR peak at δH ≈ 0 ppm[12−14,16,74,85,86] from OH groups in the HA lattice is observed from several specimens. We refer to Mathew et al.(60) for discussions on the various 1H resonance assignments made from nominally identical PserN and SerN cement compositions.

Figure 3

1H NMR spectra recorded at 9.4 T and 14.00 kHz MAS from the as-indicated SerN and PserN cements, as well as the Pser@HA and Pser samples. The green trace in (g) is a vertical expansion. The sharp peaks at 3.8/1.1 ppm in (f,g) stem from a minor isopropanol impurity, whereas those marked by asterisks in the NMR spectra from Pser@HA and Ser8 derive from HA-associated surface water molecules, as discussed in detail in refs (60) and (74)

The remainder of the article focuses on the probing of the organic/inorganic interface in the Pser@HA, Pser16, and Ser16 specimens, which were examined by dipolar-based MAS NMR experimentation, such as the dipolar-mediated heteronuclear multiple-quantum coherence (D-HMQC) 1H{31P} NMR spectra[60,82,87,88] shown in Figure . They were recorded from the Pser@HA, Pser16, and Ser16 specimens using a short HMQC excitation period (τexc = 176 μs) to ensure detection predominantly of 1H and 31P sites separated by at most a few hundreds of pm. In each D-HMQC spectrum, a 2D NMR correlation peak centered at the chemical-shift pair {δ1, δ2} ≡ {δP, δH} evidences close spatial proximity between 1H and 31P sites resonating at the (average) chemical shifts δH and δP, respectively.

Figure 4

1H{31P} D-HMQC correlation NMR spectra acquired at 34.00 kHz MAS from the (a) Ser16, (b) Pser16, and (c) Pser@HA specimens using a short HMQC excitation period of 176 μs. The 2D NMR spectra are shown together with projections along the 1H (horizontal) and 31P (vertical) dimensions at the top and to the right, respectively; the green trace in (c) is a three-fold vertical expansion of the 1H projection. Directly excited (“single-pulse”) 1H MAS NMR from crystalline Pser and Ser are displayed by red traces in (a,b). Most of the 1H resonances marked along the horizontal dimension are of organic origin, whereas the peaks assigned along the 31P dimension originate either from HA (blue color) or the ACP/Pser or ACP/Ser cement constituents (cyan color). The 2D NMR regions {δP, δH} ≈ {1.6, 5.8} ppm and {δP, δH} ≈ {4.3, 5.0} ppm marked by the respective yellow and blue rectangles in (c) are discussed in Section ).

Figure a offers direct and unambiguous evidence for an intimate ACP/Ser integration across a sub-nm scale, whereas the Pser16 counterpart reveals HMQC correlations stemming from both intramolecular 1H–31P dipolar interactions of Pser and Pser···ACP contacts of the ACP/Pser phase (Figure b). The 1H{31P} D-HMQC spectral signatures observed from a PserN cement in the high-δH region reflect correlations between acidic protons and (in)organic phosphate groups. Hence, it depends strongly on the pH value before cement setting (i.e., on the Pser content; Table S1). Indeed, the higher pH = 5.3 of the solution that immersed the Pser@HA particles renders the 2D NMR spectrum in Figure c closer to that reported previously from a Pser4 specimen[60] than that from Pser16. As expected from the results of Figures and 3, the D-HMQC NMR spectra recorded from the Pser@HA powder (see Figure c) and the Ser16 cement (Figure a) manifest the HA-characteristic correlation at {δP, δH} = {2.9, 0} ppm.[13,14,16,74,86]

Correlations between the organic protons of the {NH3, CH} moieties and / groups of ACP dominate the 1H NMR spectral range of δH ≲ 10 ppm, as becomes evident by contrasting the projection of the 2D NMR spectra of Figure a,b along the 1H dimension with the single-pulse-acquired 1H MAS NMR spectra from the crystalline Pser and Ser samples. The comparatively more intense 2D NMR-peak intensities of the PO4 contacts with 1H shifts ≈9 ppm relative to those of CH···PO4 (4–5 ppm) ppm of either cement suggest much shorter distances between the inorganic phosphate species and the positively charged moiety than the aliphatic groups that do not bond directly to the surface, as indeed predicted by the metadynamics simulations (Section ) and discussed further in Section .

Moreover, the HMQC NMR spectrum of Pser@HA reveals two broad but resolved 2D NMR peaks (marked by blue/yellow rectangles in Figure c) not observed from the cements. They are tentatively attributed to either aliphatic protons or water molecules nearby inorganic phosphate groups, where the 2D correlation centered at {δP, δH} ≈ {1.6, 5.8} ppm and extending toward lower 31P and 1H chemical shifts likely reflects CH/H2O··· proximities. Further work is required to reach firmer assignments, particularly for the {δP, δH} ≈ {4.3, 5.0} ppm correlation, whose high 31P shift is atypical of (and let alone ) groups in CaP phases, although 1H–31P correlation NMR peaks at similar shifts have been ascribed to species in the HA lattice.[74] As discussed further in Section S4, the signal at δH ≈ 7 ppm in the 1H{31P} D-HMQC NMR spectra from Pser and Pser@HA is characteristic of structure-bound H2O molecules of “ACP”, encompassing the amorphous surface of nanocrystalline HA.[16,60,89]

Surface Binding of Pser and Ser Modeled by Metadynamics

Overview and General Considerations

Metadynamics simulations were utilized to locate the energetically most favorable molecular adsorption at a structurally disordered HA surface representative for the nanocrystalline Pser@HA particles as well as the ACP component of the Pser16/Ser16 cements. The simulations were performed for a pH-dependent HA surface representative of structurally disordered forms of its crystallographic (100) and (001) faces interfacing a water phase comprising one Ser or Pser molecule with protonation states for each of pH = {4.5, 7.4}; see Figure and Section S2.

Numerous experimental studies on HA nucleation/growth in the presence of both small and large biomolecules suggest a preference for molecular adsorption at the larger (100) or (101) HA surfaces relative to their smaller (001) counterpart,[28,90−93] as also corroborated by previous modeling.[18,40,43−45,94,95] Indeed, the consistently stronger adsorption at the “disordered” (100) surface was recently deduced by metadynamics simulations of the Pser binding at HA across a wide pH range of 4.5–14 (ref (48)): Table S3 reproduces the results for the Pser adsorption at each pH = {4.5, 7.4} value and (100)/(001) surface,[48] along with those obtained herein from Ser. Hence, we focus on the results for (100) at each experimentally relevant pH = 7.4 for Ser16 and pH = 4.5 for Pser16/Pser@HA, whose most probable/representative stable binding modes are exemplified in Figure . No aliphatic CH/CH2 groups of either molecule are discussed because they do not bind directly at the HA surface.

Figure 5

Representative examples of the most stable/probable metadynamics-derived molecular binding modes at a “disordered (100) HA surface” of (a) Ser at pH = 7.4 and (b) Pser at pH = 4.5. All red numbers mark selected distances (in pm) between directly bonded Ser/Pser···HA atoms, while those in black indicate C–P and N–P distances relating to the experimental constraints from NMR. The green numbers in (b) represent intramolecular C–P distances of Pser. For visualization purposes, only a few surface contacts are shown for each organic moiety. Notably, because none of the surface-binding modes of either Ser or Pser involve the sole anchoring of one functional group (Table ), both molecules assume an extended conformation that “caps” the HA surface. We underscore that owing to the distributions of stable binding modes, no single graphical picture can capture all details of the molecular adsorption (Tables , S3, and S4).

Table 1

MD-Derived Surface-Adsorption Dataa

	Pser		Ser
pH	4.5	7.4	4.5	7.4
ΔFads	–40.7	–66.4	–36.6	–37.7
Contributions to Binding Energyb
phosphate	0.72	0.70
carboxy	0.20	0.26	0.57	0.64
amino	0.08	0.04	0.30	0.16
hydroxyl			0.13	0.10
Binding Mode Statisticsc
PN	0.51	0.15
PC	0.23
PCN	0.15	0.74
CN			0.41	0.30
CNO			0.55	0.59

Metadynamics-derived Helmholtz free energy of adsorption (ΔFads) for the Pser and Ser binding at the disordered (100) surface for pH = {4.5, 7.4}; see Section S2. A more negative ΔFads value implies a stronger surface binding. The results representative for the Pser@HA/Pser16 (pH = 4.5) and Ser16 samples (pH = 7.4) are typeset in boldface. The data for Pser are reproduced from ref (48).

Fractional contribution of each functional group to the net adsorption energy, as defined in Section S3. No aliphatic group is surface bound.

Distribution of stable binding modes, where each number reflects the probability (relative fraction/contribution) out of all stable binding modes. The capital letters P, C, N, and O represent the phosphate (Pser), carboxy, amino, and hydroxyl (Ser) group contributions, respectively. Only binding modes with probability ≥ 0.10 are listed.

There are two main groups of A···Bcontact modes (i.e., “bonding types”) between an organic atom site A of Ser/Pser and an inorganic surface-atom B: (i) electrostatic (“ion–ion”) interactions among negatively charged O sites of carboxy/phosphate groups and positive Ca2+ cations of the ACP layer at nanosized HA particles, and (ii) H bonds between an organic (inorganic) proton and an inorganic (organic) O site. Notably, although carboxy/phosphate groups of biomolecules adsorb via both interaction types, each electrostatic bond (e.g., CO···Ca2+) is around 2.5 times stronger than its H-bond counterpart (e.g., CO···).[48] These relative interaction strengths underlie the current consensus that ion–ion interactions dominate the energy landscape of biomolecular adsorption,[18−21] notwithstanding that the H bonds (140–220 pm) are shorter than the O···Ca2+ distances (240–330 pm); see Figure .

Distinct Biomolecular Binding at (100) and (001) HA Faces

Our comments made in Section S2.5 about biomolecules interacting with inorganic species at an essentially amorphous HA surface might seem to preclude any preferential adsorption at a specific HA surface type. This apparent contradiction, however, may be reconciled by noting that the consistently stronger adsorption at the “(100)” surface does not stem primarily from crystallographic/structural features—where the (hkl) notation rather specifies the crystallographic origin of the disordered (100) and (001) surfaces—but merely from its higher molar ratio nCa/nP ≈ 1.55 relative to that of nCa/nP ≈ 1.15 for (001) across the pH-range of 3.8–7.4. Consequently, the comparatively higher Ca2+ abundance (and larger nCa/nP ratio) at the disordered (100) surface relative to its (001) counterpart emphasizes the electrostatic CO/PO···Ca2+ interactions that predominantly govern the net biomolecular adsorption energy,[18−21,48] as confirmed by Table S3.

The above-quoted nCa/nP values were estimated from the chemical speciations of the two outermost layers at each (100) and (001) surface at the end of the metadynamics MD simulation (≈40 ns); see Table of ref (48) for the corresponding precise speciations at the outermost surface layer relevant for the pH values of the present study. Notably, due to minor ion-dissolution processes, the exact Ca2+/phosphate surface speciations vary slightly throughout each metadynamics simulation, thereby not necessarily being identical to those plotted in Figure b,c that represent the initially prepared outermost surface layer; see ref (48) for details.

Ser and Pser Binding Modes at HA/ACP

Figure a exemplifies one of the most probable/representative Ser binding modes at ACP in solutions at physiological pH = 7.4, relevant both for the ACP/Ser component of the SerN cements and the amorphous surface at HA nanoparticles, all of which involve simultaneous {COO–, , OH} anchoring; they account for ≈60% of all stable Ser binding modes, with the remaining constituting a dual carboxy/amino binding (Table ). The absence of any significant binding mode by one functional group alone leads to a near-parallel “capping” of the Ser molecule along the ACP surface, as illustrated in Figure a. Although typically all three {COO–, , OH} moieties bind at the HA surface, Table reveals strikingly different relative contributions of {64, 16, 10}% among the respective groups toward stabilizing/driving the Ser adsorption. The overwhelming carboxy-group contribution stems from its prevalent contact mode of strong electrostatic CO···Ca2+ interactions (Table S3), whereas the modest net contribution of ≈26% to the total adsorption energy from the and OH groups together reflect their primary (for the amino group exclusive) contact mode of weaker H bonds.

In neutral and alkaline solutions, Pser preferentially anchors at the ACP surface by all three {, COO–, } moieties,[48] leading to a significantly more negative adsorption energy than that for Ser (Table ). The stronger binding originates primarily from the organic phosphate group of Pser, which is the main adsorption promoter and involved in all stable binding modes regardless of the precise pH and (100)/(001) surface type;[48] see Table S3. However, the Pser–ACP binding weakens significantly in the acidic solutions relevant for the Pser16 and Pser@HA sample preparation conditions (3.9 ≤ pH ≤ 5.3). This feature may be traced to a lower amount of Ca2+ cations at the CaP surface and thereby fewer CO···Ca2+ and PO···Ca2+ electrostatic contacts, where the latter are diminished further because they are superseded by weaker POH···HPO4 bonds (Table S4) accompanying the onset of protonation of the organic phosphate group for pH 6.8 (Figure ). These effects combine into a significantly weakened Pser adsorption at pH = 4.5 (Table ), which incidentally nearly matches that of Ser at the comparatively Ca-richer HA surface at pH = 7.4 that promotes electrostatic interactions.

Relative to the Pser surface immobilization at pH = 7.4, the weaker Pser···ACP contacts in acidic solutions are reflected in a larger distribution of distinct binding modes at pH = 4.5 (Table ), along with an overall more modest COO– participation: roughly half of all surface-bound Pser molecules anchor by a dual binding of their phosphate and amino groups, as depicted by Figure b, whereas all other binding constellations occur either by the phosphate/carboxy moieties (≈23%) or by all three groups together (≈15%); see Table . (We remind that all binding modes not listed in Table are insignificant, such as the anchoring of one functional group alone of either molecule). Yet, although the and COO– moieties participate in ≈90% and ≈40% of all binding modes, respectively, Table S3 reveals that their corresponding net energy contributions to stabilizing the adsorption only amount to ≈8% () and ≈20% (COO–) due to the higher CO···Ca2+ interaction-energy per bond relative to that of NH···PO and the overall dominant PO···Ca2+ interactions.

Out of all functional groups of Pser, the simulations predict the strongest binding at the ACP surface by the phosphate group. However, the broad 31P NMR peak from the ACP/Pser phase (Figures and 4) cannot discriminate between the organic and inorganic phosphate contributions, which underscores the very intimate Pser···ACP contacts in the Pser/ACP phase across a sub-nm scale. Indeed, NMR experiments sensitive to the 31P–31P distance distributions revealed essentially equal average distances in synthetic/pristine ACP and the ACP/Pser components in PserN cements.[60]

13C–31P Correlation NMR Reveals Ser/Pser–ACP Contacts

Figure presents 1H → 13C CPMAS spectra observed from the polycrystalline Ser, Pser, and CaPser powders along with that of the nanocrystalline Pser@HA specimen and the PserN/SerN cements with N = {8, 16, 30} prepared from the Pser*/Ser* precursors. The strikingly different 13C NMR peak widths observed from the precursors relative to Pser@HA or any cement reflect the distinctly different local order of the 13C environments. Hence, even routine 13C CPMAS NMR experiments give qualitative evidence for organic/inorganic contacts. Although heteronuclear 17O–43Ca NMR experiments would enable the most direct probing of the electrostatic CO/PO···Ca2+ interactions that dominate the molecular adsorption, such experiments are precluded for NMR-signal sensitivity reasons along with further sample preparation obstacles which require isotopic 17O and 43Ca labeling.[96−98] Consequently, we resorted to less challenging 13C–31P NMR experiments, which merely probe the Pser/Ser···ACP contacts via their 13C–31P distances in 13C–O···Ca2+···O–31P motifs.

Figure 6

13C CPMAS NMR spectra obtained at 9.4 T and 9.00 kHz MAS from (a) polycrystalline Pser (black trace) and CaPser (red trace), (b) Pser@HA, and as-indicated (c–e) PserN cements, as well as (f) polycrystalline Ser along with (g,h) SerN cements. All NMR spectra were recorded from specimens prepared from 13C-enriched precursors, except for those shown in (a,f). The asterisk in (h) marks a minor peak from an unknown impurity.

Figure displays 13C{31P} D-HMQC NMR[87,88] spectra recorded from the Pser16 and Ser16 cements, where each 13C–31P proximity is revealed by a 2D NMR cross peak centered at the shift-pair {δP, δC}. Owing to the phosphate group of Pser, however, the 13C{31P} D-HMQC spectrum observed from Pser16 primarily reflects the intramolecular 13C–31P distances, which also account for the observed NMR-intensity increase along 13CO 13CH 13CH2 in Figure a; this is particularly evident when contrasting the various peak intensities of the HMQC projection with their counterparts of the corresponding 1H → 13C CPMAS NMR spectrum (Figure S5). The 2D NMR-peak intensities reflect semiquantitatively the relative through-space 13C–31P distances. The significantly emphasized intensity and the higher 13C chemical-shift dispersion of the 13COO–···31P correlations from the Ser16 cement relative to the Pser16 counterpart (as is also evident from the 13C CPMAS NMR spectra of Figure ) corroborates the metadynamics predictions (Section ): the carboxy group of Ser anchors directly at the ACP surface, in contrast with (a majority of) the Pser molecules. However, owing to the HMQC-signal buildup across longer distances, cross-peaks associated with all13C sites are detected for the present τexc values (Figure ), despite that none of the CH/CH2 groups of either Pser/Ser molecule bind directly to any species of ACP.

Figure 7

13C{31P} D-HMQC correlation NMR spectra obtained at B0 = 14.1 T and νr = 24 kHz from (a) Pser16 and (b) Ser16 cements by using a HMQC excitation period of (a) 1.75 ms and (b) 2.0 ms. The 2D NMR spectra are shown together with projections along the 13C (horizontal) and 31P (vertical) dimensions at the top and to the right, respectively, along with their 1H → 13C CPMAS NMR spectra acquired under the same conditions. (c) Zoom around the carboxy-resonance region of the 2D NMR spectrum in (b). The rectangles indicate 13C–31P correlations resolved at the 13C chemical shifts of 184 ppm (blue), 177/174 ppm (yellow), and 170 ppm (grey).

The close COO–···ACP contacts of the Ser molecules are reflected in emphasized 2D NMR-signal intensities in the zoom around the 13COO– region of the 2D NMR spectrum of the Ser16 cement (Figure c): four 2D NMR ridges are resolved at the 13C chemical shifts {184.2, 176.8, 174.0, 170.0} ppm. Notably, none of them coincides with that of δC = 175.2 ppm from polycrystalline Ser (Figure f). As is most transparent from Figure S5, the NMR peaks around δC = {184, 170} ppm are markedly emphasized (the latter peak is barely discernible in the CPMAS NMR spectrum), whereas the comparatively reduced intensities between 181 ppm and 172 ppm stem from Ser molecules further away from the inorganic phosphate groups. Its significant 13C shift-dispersion reflects a range of similar yet distinct COO–··· proximities of weakly bound Ser molecular configurations at the ACP surface (along with some resonances from nonbonded molecules; see Section and the comments mentioned above).

We onwards focus on the two D-HMQC NMR signals centered at the {δP, δC} 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 CO···Ca2+ interactions and CO··· 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 13C and 31P chemical shifts involved in each correlation: the high shifts of the {δP, δC} = {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 13COO–··· contacts, whose 13C chemical shift incidentally matches that of the 13COOH group of polycrystalline Pser that manifests analogous inter molecular COOH··· motifs.[65,99,100] 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 CO···Ca2+ contacts for increasing batched Ser content, along with earlier and more qualitative findings by Mathew et al.(60) and our inferences in Section .

To our knowledge, the present study provides the first 1H/31P/13C correlation NMR signatures of surface-bound Pser molecules at nanocrystalline HA, noting that the previous 31P{1H} and 13C{1H} heteronuclear correlation spectra presented by Wang et al.(32) from similar nanocrystalline HA preparations in Pser-bearing solutions gave no convincing evidence for Pser···HA contacts. For instance, the 13C NMR features merely suggested a prevalence of Pser in crystalline environments than surface-bound ones, which likely resulted from using a very high [Pser] = 200 mM in the solution.[32] Likewise, our 13C CPMAS spectra recorded from various Pser@HA syntheses with increasing Pser concentrations revealed a dominance of crystalline CaPser in all preparations with [Pser] ≥ 20 mM (Figure S3).

Quantitative Probing of Ser/Pser–ACP Contacts by 13C{31P} REDOR NMR

NMR-Derived Dipolar Second Moments

For a quantitative probing of the relative proximities among the {13CO, 13CH, 13CH2} sites of Ser/Pser and the inorganic phosphate groups at the HA/ACP surface, we collected the 13C{31P} REDOR NMR dephasing curves presented in Figure . Here, a rapid (slow) dephasing reflects short (long) distances of a given 13C site to the inorganic phosphate groups of ACP (with the caveat of intramolecular 13C–31P interactions in Pser). Table collects the set of REDOR-derived dipolar second moments[101−106] {M2(CO–P), M2(CH–P), M2(CH2–P)}, extracted by fitting the respective dephasing curves of each sample (Section S1.3). The precise M2(C–P) value depends on the underlying set of interatomic distances {r(C–P)} between a given 13C site and its nearby P atoms via a sum over [r(C–P)]−6 terms. This renders the shortest distance contributions most influential for the net M2(C–P) value.

Figure 8

13C{31P} REDOR NMR dephasing curves (ΔS/S0) recorded at B0 = 14.1 T and νr = 10 kHz and plotted for increasing dipolar recoupling/dephasing periods (τrec) for the 13CO, 13CH, and 13CH2 functional groups of the (a,b) polycrystalline Pser and CaPser powders (13C at natural abundance) along with the 13C-enriched (c) Pser@HA, (d) Pser16, and (e) Ser16 specimens. Each curve in (a–e) corresponds to the best fit of the {τrec, ΔS/S0} data to eq S2, which yielded the dipolar second moments {M2(C–P)} listed in Table . The 13COO– NMR spectral region of the Ser16 sample comprises three distinct resonances at δC = {184, 177, 174.5} ppm, whose associated dephasing curves are labeled by their chemical shifts in the legend in (e). Note the different horizontal scale in (e) relative to (a–d).

Table 2

Experimental and Calculated Dipolar Second Momentsa

	15N–31P		13C–31P
	M2(NH3–P)/kHz2		M2(CO–P)/kHz2		M2(CH–P)/kHz2		M2(CH2–P)/kHz2
sample	exp.	calc.	exp.	calc.	exp.	calc.	exp.	calc.
Ser16	2.7 ± 0.4	4.9 ± 0.7	11.1b	14.0b	8.9	13.0	7.9	9.6
Pser16c	2.8 ± 0.4	5.2 (4.0)±0.7	13.1	13.8 (8.3)	18.2	25.8 (8.9)	75.4	96.8 (5.7)
Pser@HAc	4.0 ± 0.4	5.2 (4.0)±0.7	15.7	13.8 (8.3)	20.9	25.8 (8.9)	73.7	96.8 (5.7)
Pserd	3.2 ± 0.4	4.5	18.6	24.5	18.9	24.8	79.9	102.5
CaPser		4.6	15.9	20.8	27.8	38.2	71.7	93.4

15N–31P and 13C–31P dipolar second moments obtained for the as-indicated functional groups from 15N{31P} and 13C{31P} 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.[65] The M2(C–P) data uncertainties are ±1.7 kHz2 (NMR) and ±2.2 kHz2 (metadynamics model), with kHz2 ≡ 1000 s–2.

Net experimental/calculated values; the NMR analysis afforded the extraction of three M2(CO–P) values of {23.6, 9.3, 13.3} kHz2 for the 13CO shifts at {184, 177, 174.5} ppm, respectively.

Values within parentheses represent the contribution to the net M2(N–P) and {M2(C–P)} values from the Pser···HA contacts alone; the contribution from the intramolecular 15N–31P and 13C–31P dipolar interactions account for the difference.

The {M2(C–P)} and M2(N–P) values were obtained from the Pser (Flamma) and isotopically enriched Pser* samples, respectively.

The NMR-derived {M2(C–P)} data from the polycrystalline Pser/CaPser powders were validated against those calculated from their crystal structures.[64,65,99] As expected[103−106] 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 relativeM2(C–P) values within each Pser or CaPser structure, which remain well within the experimental uncertainties. For Pser, the experimental and calculated M2(CO–P):M2(CH–P):M2(CH2–P) ratios relate as 1.0:1.0:4.3 and 1.0:1.0:4.2, respectively, whereas both data sets for CaPser are 1.0:1.8:4.5.

The markedly different relative M2(CO–P):M2(CH–P) values among the Pser and CaPser crystal structures are noteworthy: Despite the long intramolecular 13CO···31PO4 distances in both structures, the comparatively high M2(CO–P) values (particularly for Pser) originate mainly from multiple intermolecular 13COOH···31PO4 contacts via H bonds.[65,99,100] Moreover, the markedly different M2(CH–P) values in Table reflect distinctly different molecular conformations in the CaPser and Pser crystal structures, whose corresponding θ(P–O–Cβ–Cα) dihedral angles of 280° and 153° translate into intramolecular 13CH···P distances of 341 pm and 384 pm, respectively. Notably, such widely differing Pser conformations are readily discriminated by the 13C{31P} REDOR NMR experiments. Hence, notwithstanding that each M2(C–P) value is dominated by the intramolecular 13C–31P distance for all scenarios where intermolecular Pser···Pser contacts are negligible (thereby obscuring the herein targeted C···ACP contacts), the M2(CH–P) parameter offers a valuable constraint on the conformation of the surface-bound Pser molecules, such as in the Pser@HA and Pser16 systems.

Validation of the Metadynamics-Derived Dipolar Second Moments

The dipolar second moment M2(C–P) reflects all {13C–31P} distances of the adsorbed molecule to its adjacent inorganic phosphate groups (Section S1.3), but cannot unveil the precise underlying distance distribution. However, because the metadynamics simulations do offer such atomistic details (Figure ), we first validated the modeled M2(C–P) data against the experimental counterparts.

Considering that all REDOR-derived M2(C–P) values are underestimated by (Table S5 and Section S1.3), a “perfect match” between the metadynamics-generated models and the experiments should result in for each 13C–31P contact. Notwithstanding a somewhat larger scatter in the ratios of Table S5 (which stems from higher data uncertainties of the simulated systems than the very accurate C/P atom coordinates of the Pser/CaPser crystal structures[64,99]), it is gratifying that the data in Tables and S5 confirmed our expectations for all 13C–31P pairs of both Ser/Pser molecules, except for the modeled M2(CO–P) values relative to the experimental counterparts of Pser@HA and Pser16 that yielded . The somewhat stronger COO–···ACP contacts in both Pser@HA and Pser16 specimens than those predicted by the metadynamics simulations imply that the carboxy group contributes more to the Pser binding than that suggested by the binding mode statistics of Table . However, besides noting that the phosphate/amino binding-mode population is presumably slightly overestimated at the expense of (primarily) the phosphate/carboxy/amino counterpart, more quantitative corrections cannot be made. We remind that the 13C{31P} D-HMQC NMR spectra also suggested weaker COO–···ACP interactions of the Pser molecules than those of Ser (Section ).

Discussion

The NMR-derived dipolar second moments of the aliphatic groups of Ser (Table ) suggest M2(C–P) kHz2 as the marker of an absence of direct surface binding of any atom of a given 13C functional group at the ACP surface. We remind that regardless of the (non)adsorption of Pser at ACP, the intramolecular 13C–31P dipolar interactions render all M2(C–P) values of the Pser@HA and Pser16 specimens markedly higher than those from Ser16, as is evident from the dephasing curves of Figure . However, the simulation-derived model enables a separation of the intra/intermolecular contributions to the net M2(C–P) value: indeed, Table confirms the expectation that both CH/CH2 groups of Pser exhibit very low dipolar second moments once their intramolecular contributions are excluded (particularly that of M2(CH2–P), for which the organic phosphate group accounts for ≈95% of the net value).

It is gratifying that each of the three {M2(CO–P), M2(CH–P), M2(CH2–P)} NMR-derived data agrees mutually very well among the Pser@HA and Pser16 samples, which suggests overall (very) similar Pser contacts/distances to the inorganic phosphate moieties. Hence, despite the formally distinct nature of the ACP/Pser phase of the biomedical PserN cements and the Pser adsorption at a nanocrystalline “HA” surface (Pser@HA), the local structure of their organic/inorganic interfaces must be similar in both specimens, thereby consolidating the current consensus that nanocrystalline HA is coated by a layer of “ACP”[4,12−16] (although its precise chemical/structural nature remains unknown). This aspect also justifies that the single metadynamics simulation at pH = 4.5 mimics well the Pser···ACP interactions in both Pser@HA and Pser16 specimens, as well as supporting the physical relevance of the herein employed HA-surface preparation procedure.[45] These important issues are discussed further in Section S2.5.

We now return to the partially overlapping 13C NMR peaks in the carboxy domain of the NMR spectrum from the Ser16 cement (Figure h), which, according to their distinct 13COO–···ACP contacts, were grouped into three regions in the 13C{31P} D-HMQC spectrum of Figure c. As expected from the δC = 184 ppm resonance attributed to CO···Ca2+ motifs (Section ), its very rapid dipolar dephasing is consistent with a sizable dipolar second moment of 23.6 kHz2 (Table ). In contrast, the spectral region marked by a yellow rectangle in Figure c and deconvoluted into two peak components around 177/174.5 ppm reflects more weakly surface-bound COO– groups with lower dipolar second moments of 9.3/13.3 kHz2 (Figure and Table ). Although a large M2(CO–P) value is also anticipated from COO– moieties surface bound via H bonds and giving an NMR peak at 170 ppm, its minor NMR intensity (and thereby very small population) did not permit reliable analyses of the REDOR NMR data (e.g., see Figure S6). Altogether, the varying dipolar second moments of these COO–···ACP contact modes/distances underlie the average value of M2(CO–P) ≈ 11.1 kHz2 (Table ), which qualitatively corroborates the metadynamics predictions of direct COO–···ACP bonds.

We contrasted the D-HMQC NMR-derived and modeled relative populations of the three types of surface-bound COO– sites that produce the three spectral regions marked in Figure c and attributed to COO– sites bound solely by Ca2+ cations (≈184 ppm), sites weakly bound by both electrostatic and H-bond interactions (181–172 ppm), and solely by H bonds (≈170 ppm). Quantitative agreements are not expected because the 2D NMR intensities depend strongly on the precise HMQC excitation/reconversion periods. Deconvolution of the 13C projection of the D-HMQC spectrum (results not shown) yielded fractional populations of {0.27, 0.59, 0.14} for the respective {184, 181–172, 170} ppm resonance regions, whereas the corresponding metadynamics-simulation-derived fractions are {0.68, 0.27, 0.05}. While both experimental and modeled results accorded qualitatively well for the overall most sparse contact mode of solely CO···HPO42− interactions, the main discrepancy concerns the strong dominance of CO–···Ca2+ interactions predicted by the model (68% of all direct surface contacts and consistent with the results of Table S4) compared to the much lower estimate by NMR (27%). The differences presumably stem from the difficulties by NMR to accurately quantify the contributions from the 181–172 ppm resonance-region stemming from “weakly bound” carboxy groups, which may involve non-negligible 13COO– NMR signals from more distant nonbonded molecules that are not accounted for in the simulation analysis.

15N{31P} REDOR NMR Reveals the Amino-Group Binding

Figure a,b displays the 1H → 15N CPMAS NMR spectra of Pser, the 15N-enriched Ser* and Pser* precursor powders, together with those of Pser@HA, Pser16, and Ser16. As for the 13C NMR spectra (Figure ), the Ser/Pser adsorption is mirrored by a significant 15N resonance broadening along with a minor increase in the average chemical shift of ≈2 ppm and ≈5 ppm for the Ser and Pser bearing specimens, respectively. The amino-group binding at ACP was probed by 15N{31P} REDOR NMR experiments on the Pser@HA, Pser16, and Ser16 cements, whose dipolar dephasing curves are displayed in Figure c along with that from the Pser* powder. For the latter, Table S5 reveals a ratio of 0.71 between the NMR-derived M2(N–P) value and that calculated from the Pser crystal structure,[99] which is close to the expected ratio of 0.76 (Section ). Moreover, the ratio of 0.77 between the experimental and modeled M2(P–N) data for the Pser@HA sample suggests a very faithful metadynamics modeling of its ACP/HA contacts (Figure b).

Figure 9

1H → 15N CPMAS NMR spectra recorded from powders of (a) Pser (15N at natural abundance) along with the 13C/15N-enriched Ser* and Pser* precursors, and their (b) Pser@HA, Pser16, and Ser16 products. The NMR peaks at 32.8 ppm and 44.0 ppm observed from the Pser* specimen are attributed to the 15N sites of 13C/15N-enriched O-phospho-l-serine and its HCl salt, respectively. (c) 15N{31P} REDOR NMR dephasing curves recorded from the as-indicated samples. All NMR results were obtained at B0 = 14.1 T and νr = 10 kHz.

Particularly, when recalling the very good mutual match between the {M2(C–P)} sets among the Pser@HA and Pser16 specimens (Section ), the significantly lower M2(N–P) value of the Pser16 cement (as well as for Ser16) relative to Pser@HA is surprising (Table ). It suggests much weaker ACP contacts in both Pser/ACP and Ser/ACP cement components than the counterparts of the Pser@HA particles (also see Section S1.3). Although the NMR-derived dipolar second moments from the cements also suggest longer ACP distances than the metadynamics predictions, the qualitative feature of near-equal M2(N–P) values of the models for both Ser/Pser molecules is supported by the REDOR NMR experiments on the Pser16 and Ser16 samples.

The amino group binds to the ACP surface exclusively via H bonds to the negatively charged O atoms of the phosphate groups. Table S4 reveals that the NH··· contact mode dominates for Pser (pH = 4.5), whereas the amino group of Ser (pH = 7.4) forms an equal number of bonds to and moieties. For the pH conditions of the Pser16 and Ser16 cements, the binding-energy contribution from the amino group is higher for Ser than Pser (Table S3), which also accords with the dipolar second moments of Table : although both the experimental and modeled M2(N–P) values for Pser16 are slightly higher than their Ser counterparts, once excluding the intramolecular 15N–31P Pser contribution, the expectation of a stronger surface binding of the group of Ser is confirmed by its higher modeled M2(N–P) value than that of Pser.

Implications for Bone-Adhesive Properties

We recently reported a strong correlation between the amount of the amorphous ACP/Pser component and the measured shear strength of PserN cements used for gluing two cubes of either cortical bone[54] or steel[60] together across a wide composition range up to N = 87 mol %.[60] The shear strength reflects the bone-adhesive properties.[54] Notably, both parameters exhibit a nonmonotonic trend against the batched Pser content, with an initial increase up to a plateau of near-constant shear-strengths and ACP/Pser contents in cements incorporating 40–60 mol % Pser, followed by their concurrent decrease for increasing N because the bone-adhesive-promoting ACP/Pser component becomes gradually replaced by crystalline CaPser and unreacted Pser.[60] In contrast, SerN cements form an amorphous ACP/Ser phase that only develops with the batched Ser content up to N ≲ 16 mol % (which is insufficient for giving a high shear strength), whereas all remaining Ser remains unreacted.[60]

Consequently, the formation of an amorphous component featuring both significant organic–organic and organic–inorganic interactions appears to be a prerequisite for favorable bone-adhesive properties of an α-TCP-derived cement. Hence, the results herein combined with those of ref (60) suggest that a significant bone-adhesion cannot stem from the adsorption strength of a given biomolecule at ACP alone, because the Pser···ACP binding energy at pH = 4.5 essentially matches with that of Ser···ACP at pH = 7.4 (Table ), implying that both Pser16/Ser16 cements feature similar Pser···ACP and Ser···ACP net interaction strengths. Nonetheless, while both Pser and Ser molecules may enter ACP/Pser and ACP/Ser phases in the respective PserN and SerN specimens with N ≲ 16 mol %, their amounts develop very differently upon increasing organic content (vide supra). Consequently, the main distinction between the PserN and SerN cements concerns their respective degrees of Pser···Pser and Ser···Ser contacts.

Due to its low Pser content ( wt %), the Pser@HA sample is expected to involve essentially “isolated” surface-bound Pser molecules with long Pser···Pser distances. Likewise, the metadynamics modeling involved one sole Pser (or Ser) molecule. Hence, the good agreement between the M2(C–P) NMR results of the Pser@HA and Pser16 samples, as well as with the modeled data, suggest that 16 mol % of either Pser or Ser may be sufficiently low to be dispersed within the ACP matrix without any significant Pser···Pser or Ser···Ser aggregation (i.e., analogously with a “monolayer” adsorption scenario). However, the substantial Pser contribution to the ACP/Pser cement component in all Pser-rich PserN cements—for which the shear strength is maximal—cannot be reconciled with a monolayer Pser adsorption at ACP, but must involve a significant Pser···Pser aggregation, yet with the molecules remaining intimately integrated also with the inorganic species of ACP (see comments in Section 3.3.3 and ref (60)). Although further work is required for a definite proof, we propose that the high bone-adhesion/shear-strength of the PserN cements with 40 ≲ N ≲ 60 mol % stems from a “stickiness” accompanying the high organic content of their ACP/Pser component, in conjunction with its dominance of the entire cement constitution. Naturally, the “stickiness” is low in cements with batched Pser contents ≲ 20 mol %, as well as for all SerN specimens[60] due to their insignificant tendency of Ser···Ser aggregation.

Conclusions

We have presented the first atomistic probing of the Pser and Ser binding at structurally disordered CaP surfaces by a synergistic combination of advanced solid-state NMR experimentation and metadynamics MD simulations, revealing the relative proximities of each molecular functional group and their respective underlying types of bonds, as well as the conformation of the adsorbed molecules. Our study encompassed the organic/inorganic interactions in a sample of surface-bound Pser at nanocrystalline HA particles (Pser@HA) along with two biocements prepared from α-Ca3(PO4)2 doped with 16 mol % of either Pser or Ser. Notably, the very close sets of 13C–31P dipolar second moments observed from the cement and the Pser@HA sample highlight the similarities of the Pser binding at ACP and the structurally disordered surface layer of nanocrystalline HA, thereby corroborating the current consensus that it is faithfully described as “ACP”.[4,13−16]

The Pser and Ser adsorption is primarily mediated by electrostatic interactions between Ca2+ cations and the negatively charged organic COO–/ groups and, to a lesser extent, by H bonds to the inorganic phosphate groups, which involves NH···PO4 and CO/PO···HPO4 contacts for the amino and carboxy/phosphate groups, respectively. The dominance of electrostatic interactions for driving the adsorption implies that the phosphate group of Pser and the carboxy group of Ser are mainly responsible for stabilizing their surface binding, fully consistent with earlier inferences that biomolecules bind at bone minerals mainly via ion–ion interactions.[18−21]

All detailed information about the number of electrostatic/H-bond interactions and the accompanying interatomic distances were extracted from the metadynamics models. They were validated against the NMR-derived interatomic-distance constraints encoded by the dipolar second moments {M2(CO–P), M2(CH–P), M2(CH2–P)} that convey the relative proximities between the 13C atoms of the respective {COO–, CH, CH2} group and the inorganic phosphate moieties of ACP, as well as the M2(N–P) counterpart informing about the ACP contacts. The overall good agreement confirmed the accuracy of our metadynamics simulations, notably the validity of the herein employed HA-surface preparation protocol of ref (45), which produces a disordered apatite surface with pH-dependent phosphate speciation. For our experimental conditions of 3.8 ≤ pH ≤ 5.3 (Pser@HA and Pser16) and pH = 7.4 (Ser16), both Pser and Ser molecules anchor at their amino groups, together with the HPO4− group of Pser and the COO– group of Ser, which leads to an extended conformation of both surface-immobilized molecules. The OH group of Ser also participates in the binding at ACP via both electrostatic OH···Ca2+ interactions and H bonds to the inorganic phosphate groups, but they contribute overall little to the net adsorption energy (≈10%). The only discrepancy between the experiments and models concerned an underestimation of the metadynamics-derived COO–···ACP proximities relative to those deduced by NMR on the Pser@HA and Pser16 specimens.

Besides rationalizing the distinction in bone-adhesive properties of Pser and Ser doped α-Ca3(PO4)2-based cements (Section ), our findings settle some earlier suggestions/speculations about which functional groups are involved in the molecular binding of Ser and Pser at nanocrystalline HA.[90,107−109] We stress that the binding modes discussed herein (Table and Figure ) are the most probable ones from an energetic viewpoint over a distribution of several distinct but stable modes. The nature of the comparatively weak adsorption of small biomolecules (such as amino acids and oligopeptides) at structurally disordered CaP surfaces must be analyzed/discussed in terms of “distributions” and/or “effective contacts”, as those encoded by dipolar second moments. The existence of a distribution of very similar binding modes is indeed mirrored in the broad and typically asymmetric 13C/15N MAS NMR peak shapes observed from each functional group which, except for the COO– moiety of Ser16, remained unresolved. The 13C–31P correlation NMR spectrum of the latter revealed four 13COO– resonances from groups with different proximities to ACP: the two COO– environments closest to the inorganic phosphates were tentatively attributed to those solely involved in electrostatic COO–···Ca2+ interactions (≈184 ppm) and H-bonded COO–··· moieties (≈170 ppm). However, most of the COO– groups bind by both interaction types, as mirrored in a resonance-continuum across the 181–172 ppm spectral region.

We conclude by highlighting the power of the herein-implemented combination of advanced solid-state NMR experiments with metadynamics simulations for an enhanced probing of the detailed biomolecular binding at structurally disordered CaP surfaces, which remains essentially untapped but is potentially very rewarding. Another ubiquitous tool exploited herein concerns the recently introduced Debye–Hückel-based analysis[48] reviewed in the Supporting Information, which offers a straightforward decomposition of the net modeled biomolecular binding energy into its contributions from the various functional groups of the surface-immobilized molecule, as well as for quantifying each individual electrostatic/H-bond interaction energy.