Alan Wong

Magnesium incorporation into hydroxyapatite

The incorporation of Mg in hydroxyapatite (HA) was investigated using multinuclear solid state NMR, X-ray absorption spectroscopy (XAS) and computational modeling. High magnetic field (43)Ca solid state NMR and Ca K-edge XAS studies of a ∼10% Mg-substituted HA were performed, bringing direct evidence of the preferential substitution of Mg in the Ca(II) position. (1)H and (31)P solid state NMR show that the environment of the anions is disordered in this substituted apatite phase. Both Density Functional Theory (DFT) and interatomic potential computations of Mg-substituted HA structures are in agreement with these observations. Indeed, the incorporation of low levels of Mg in the Ca(II) site is found to be more favourable energetically, and the NMR parameters calculated from these optimized structures are consistent with the experimental data. Calculations provide direct insight in the structural modifications of the HA lattice, due to the strong contraction of the M⋯O distances around Mg. Finally, extensive interatomic potential calculations also suggest that a local clustering of Mg within the HA lattice is likely to occur. Such structural characterizations of Mg environments in apatites will favour a better understanding of the biological role of this cation.

Probing heteronuclear N-15-O-17 and C-13-O-17 connectivities and proximities by solid-state NMR spectroscopy

Heteronuclear solid-state magic-angle spinning (MAS) NMR experiments for probing (15)N-(17)O dipolar and J couplings are presented for [(2)H(NH(3)),1-(13)C,(15)N,(17)O(2)]glycine.(2)HCl and [(15)N(2),(17)O(2)]uracil. Two-dimensional (15)N-(17)O correlation spectra are obtained using the R(3)-HMQC experiment; for glycine.(2)HCl, the intensity of the resolved peaks for the CO and C-O(2)H (17)O resonances corresponds to the relative magnitude of the respective (15)N-(17)O dipolar couplings. (17)O-(15)N REDOR curves are presented for glycine.(2)HCl; fits of the initial buildup (DeltaS/S < 0.2) yield effective dipolar couplings in agreement with (+/-20%) the root-sum-squared dipolar couplings determined from the crystal structure. Experimental (15)N-(17)O REAPDOR curves for the (15)N resonances in glycine.(2)HCl and uracil fit well to the universal curve presented by Goldbourt et al. (J. Am. Chem. Soc. 2003, 125, 11194). Heteronuclear (13)C-(17)O and (15)N-(17)O J couplings were experimentally determined from fits of the quotient of the integrated intensity obtained in a heteronuclear and a homonuclear spin-echo experiment, S(Q)(tau) = S(HET)(tau)/S(HOM)(tau). For glycine.(2)HCl, (1)J(CO) was determined as 24.7 +/- 0.2 and 25.3 +/- 0.3 Hz for the CO and C-O(2)H resonances, respectively, while for uracil, the average of the two NH...O hydrogen-bond-mediated J couplings was determined as 5.1 +/- 0.6 Hz. In addition, two-bond intramolecular J couplings, (2)J(OO) = 8.8 +/- 0.9 Hz and (2)J(N1,N3) = 2.7 +/- 0.1 Hz, were determined for glycine.(2)HCl and uracil, respectively. Excellent agreement was found with J couplings calculated using the CASTEP code using geometrically optimized crystal structures for glycine.HCl [(1)J(CO)(CO) = 24.9 Hz, (1)J(CO)(COH) = 27.5 Hz, (2)J(OO) = 7.9 Hz] and uracil [(2h)J(N1,O4) = 6.1 Hz, (2h)J(N3,O4) = 4.6 Hz, (2)J(N1,N3) = 2.7 Hz].

Natural abundance 43Ca solid-state NMR characterisation of hydroxyapatite: identification of the two calcium sites

Natural abundance 43Ca solid‐state NMR of hydroxyapatite (Ca10(PO4)6(OH)2) was performed at three different fields (8.45, 14.1 and 18.8 T). The two crystallographically distinct calcium sites of the apatite structure were spectroscopically resolved at 18.8 T. The 43Ca NMR interaction parameters (δiso, CQ and etaQ) of each site were determined by multiple magnetic‐field simulations. The peaks with δiso = 11.2 ± 0.8 and − 1.8 ± 0.8 ppm, both with CQ = 2.6 ± 0.4 MHz, were assigned to the Ca(II) and Ca(I) sites, respectively, on the basis of their relative intensities. Copyright

Implementation of High Resolution 43Ca Solid State NMR Spectroscopy: Toward the Elucidation of Calcium Sites in Biological Materials

Calcium is one of the most abundant cations in living organisms. It is found in the mineral phase of bone and in proteins like calmodulin. However, its exact environment beyond the first coordination sphere is often unknown, thus hampering the understanding of many biological processes. Here, calcium benzoate trihydrate (Ca(C(6)H(5)COO)(2) x 3 H(2)O) was used as a model for the NMR analysis of calcium sites in biological materials, because of the similarity of its calcium coordination, to water and carboxylate ligands, to that in several calcium-proteins. First, calcium-43 magic angle spinning (MAS) and static NMR spectra of a (43)Ca enriched sample were recorded at different magnetic fields, to investigate the electronic environment of calcium. Complex static lineshapes were obtained because of the presence of anisotropic NMR interactions of similar magnitude (chemical shift anisotropy and quadrupolar interaction), and the full interpretation of the spectra required simulations and gauge-including projector augmented wave (GIPAW) DFT calculations. An NMR investigation of the coordination environment of Ca(2+) was carried out, using high resolution (13)C-(43)Ca MAS NMR experiments such as TRAPDOR (transfer of population double resonance) and heteronuclear J-spin-echoes. It was shown that despite the weakness of (13)C-(43)Ca interactions, it is possible to discriminate carbon atoms according to their calcium environment. Long-range calcium-carbon correlations were even evidenced by TRAPDOR, reaching distances >5.6 A. This work demonstrates that by combining solid state NMR experiments, DFT calculations, and simulations, it will be possible to elucidate the electronic and coordination environment of calcium in many important and complex materials.

Natural Abundance 25Mg Solid-State NMR of Mg Oxyanion Systems: A Combined Experimental and Computational Study

Solid-state (25)Mg magic angle spinning nuclear magnetic resonance (MAS NMR) data are reported from a range of organic and inorganic magnesium-oxyanion compounds at natural abundance. To constrain the determination of the NMR interaction parameters (delta(iso), chi(Q), eta(Q)) data have been collected at three external magnetic fields (11.7, 14.1 and 18.8 T). Corresponding NMR parameters have also been calculated by using density functional theory (DFT) methods using the GIPAW approach, with good correlations being established between experimental and calculated values of both chi(Q) and delta(iso). These correlations demonstrate that the (25)Mg NMR parameters are very sensitive to the structure, with small changes in the local Mg(2+) environment and the overall hydration state profoundly affecting the observed spectra. The observations suggest that (25)Mg NMR spectroscopy is a potentially potent probe for addressing some key problems in inorganic materials and of metal centres in biologically relevant molecules.

A High-Resolution 43Ca Solid-State NMR Study of the Calcium Sites of Hydroxyapatite

High resolution Ca-43 solid-state NMR studies of hydroxyapatite (Ca-10(PO4)(6)(OH2)) were performed at 14.1 T. The two crystallographically distinct calcium sites were unequivocally resolved by a triple-quantum magic angle spinning experiment, and the unambiguous assignment of the signals was possible using H-1-Ca-43 rotational echo double resonance and H-1-Ca-43 cross polarization magic angle spinning experiments.

A multinuclear MAS NMR study of calcium-containing aluminosilicate inorganic polymers

Natural abundance 43Ca, 27Al and 29Si MAS NMR spectra were acquired for a series of aluminosilicate inorganic polymers (geopolymers) containing up to 50 wt% of Ca(OH)2, CaCO3, Ca3(PO4)2 and both amorphous and crystalline CaSiO3. The 27Al and 29Si MAS NMR spectra of these geopolymers were generally consistent with those of conventional materials, with their XRD diffractograms showing the presence of the added crystalline phases. Comparison of the 43Ca MAS NMR spectra with those of the calcium starting materials suggests that both the calcium silicates and Ca(OH)2 enter into the geopolymer network, but the spectra of geopolymers containing CaCO3 and Ca3(PO4)2 are identical to those of the original compound, suggesting that the role of these materials is simply as a filler. At the highest concentration of CaCO3, the 43Ca resonance of the aragonite polymorph appears in the geopolymer but disappears on heating at 400 °C for 36 h, suggesting that this is a metastable phase formed in situ under the highly alkaline conditions of geopolymer formation and in the presence of calcite nuclei.

Evaluation of High Resolution Magic-Angle Coil Spinning NMR Spectroscopy for Metabolic Profiling of Nanoliter Tissue Biopsies

High-resolution magic-angle sample spinning (HR-MAS) (1)H NMR spectroscopy of tissue biopsies combined with chemometric techniques has emerged as a valuable methodology for disease diagnosis and environmental assessments. However, the tissue mass required for such experiments is of the order of 10 mg, and this can compromise the metabolic evaluation because of tissue heterogeneity. Tissue availability is often a limitation for clinical studies due to histopathological requirements, which are currently the gold standard for diagnosis, for example, in the case of tumors. Here, we introduce the use of a rotating micro-NMR detector that optimizes the coil filling factor such that mass-limited samples can be measured. We show the results for measuring nanoliter volume tissue biopsies using a commercial HR-MAS probe for the first time. The method has been tested with bovine muscle and human gastric mucosal tumor tissue samples. The gain in mass sensitivity is approximate up to 17-fold, and the adequate spectral resolution (3 Hz) allows the measurement of the metabolite profiles in nanoliter volume samples, thereby limiting the ambiguity resulting from heterogeneous tissues; thus, the approach presents diagnostic potential for studies by metabonomics of mass-limited biopsies.

Two-dimensional 43Ca–1H correlation solid-state NMR spectroscopy

Calcium-43 (nuclear spin, S=7/2) is an NMR insensitive low-gamma quadrupolar nucleus and up until recently only one-dimensional solid-state (43)Ca NMR spectra have been reported. Through-space correlation experiments are challenging between spin-12 and low-gamma quadrupolar nuclei because of the intrinsically weak dipolar interaction and the often-low natural abundance of the quadrupolar nucleus. Rotary-resonance recoupling (R(3)) has recently been used to re-introduce hetero-nuclear dipolar interactions for sensitive high-gamma quadrupolar nuclei, but has not yet been applied in the case of low-gamma half-integer quadrupolar nuclei. Here an effective and robust 2D (1)H-(43)Ca NMR correlation experiment combining the R(3) dipole-recoupling scheme with 2D HMQC is presented. It is demonstrated that the weak (43)Ca-(1)H dipolar coupling in hydroxyapatite and oxy-hydroxyapatite can be readily re-introduced and that this recoupling scheme is more efficient than conventional cross-polarization transfer. Moreover, three (43)Ca-(1)H dipolar coupled calcium environments are clearly resolved in the structurally unknown oxy-hydroxyapatite. This local information is not readily available from other techniques such as powder XRD and high resolution electron microscopy. R(3)-HMQC is also a desirable experiment because the set-up is simple and it can be applied using conventional multi-resonance probes.

Microfabricated Inserts for Magic Angle Coil Spinning (MACS) Wireless NMR Spectroscopy

This article describes the development and testing of the first automatically microfabricated probes to be used in conjunction with the magic angle coil spinning (MACS) NMR technique. NMR spectroscopy is a versatile technique for a large range of applications, but its intrinsically low sensitivity poses significant difficulties in analyzing mass- and volume-limited samples. The combination of microfabrication technology and MACS addresses several well-known NMR issues in a concerted manner for the first time: (i) reproducible wafer-scale fabrication of the first-in-kind on-chip LC microresonator for inductive coupling of the NMR signal and reliable exploitation of MACS capabilities; (ii) improving the sensitivity and the spectral resolution by simultaneous spinning the detection microcoil together with the sample at the “magic angle” of 54.74° with respect to the direction of the magnetic field (magic angle spinning – MAS), accompanied by the wireless signal transmission between the microcoil and the primary circuit of the NMR spectrometer; (iii) given the high spinning rates (tens of kHz) involved in the MAS methodology, the microfabricated inserts exhibit a clear kinematic advantage over their previously demonstrated counterparts due to the inherent capability to produce small radius cylindrical geometries, thus tremendously reducing the mechanical stress and tearing forces on the sample. In order to demonstrate the versatility of the microfabrication technology, we have designed MACS probes for various Larmor frequencies (194, 500 and 700 MHz) testing several samples such as water, Drosophila pupae, adamantane solid and LiCl at different magic angle spinning speeds.