James E. Roberts

Inorganic–Organic Hybrid Vesicles with Counterion- and pH-Controlled Fluorescent Properties

Two inorganic-organic hybrid clusters with one or two covalently linked pyrene fluorescent probes, [(n-C(4)H(9))(4)N](2)[V(6)O(13){(OCH(2))(3)C(NH(CO)CH(2)CH(2)CH(2)C(16)H(9))}{(OCH(2))(3)C-(NH(2))}] ((TBA(+))(2)1) and [(n-C(4)H(9))(4)N](2)[V(6)O(13){(OCH(2))(3)C(NH(CO)CH(2)CH(2)CH(2)C(16)H(9))}(2)] ((TBA(+))(2)2), respectively, are synthesized from Lindqvist type polyoxometalates (POMs). The incorporation of pyrene into POMs results in amphiphilic hybrid molecules and simultaneously offers a great opportunity to study the interaction between hybrid clusters and their counterions. 2D-NOESY NMR and fluorescence techniques have been used to study the role of counterions such as tetrabutyl ammonium (TBA) in the vesicle formation of the hybrid clusters. The TBA(+) ions not only screen the electrostatic repulsions between the POM head groups but also are involved in the hydrophobic region of the vesicular structure where they interrupt the formation of pyrene excimers that greatly perturbs the luminescence signal from the vesicle solution. By replacing the TBA(+) counterions with protons, the new vesicles demonstrate interesting pH-dependent fluorescence properties.

Homonuclear J-couplings and rotationally induced sideband enhancements in NMR spectra of rotating solids

We demonstrate direct detection of 13C-13C J-couplings in magic angle sample spinning NMR spectra despite the presence of the much larger homogeneous broadening caused by the homonuclear dipolar couplings. Carbon-carbon J-couplings were observed in doubly 13C-enriched samples of sodium acetate, glycine and glucose. The resolved J-coupling permits carbon-carbon connectivities to be established with standard two-dimensional techniques. Interesting spectral features are observed when the rotational sidebands of the coupled spins overlap: when a sideband from a dipolar-coupled pair approaches the centerband of its partner, a significant enhancement in sideband intensity is observed as well as small shifts in the resonance frequency.

Characterization of very young mineral phases of bone by solid state 31Phosphorus magic angle sample spinning nuclear magnetic resonance and X-ray diffraction

SummaryThe properties of bone mineral change with age and maturation. Several investigators have suggested the presence of an initial or “precursor” calcium phosphate phase to help explain these differences. We have used solid state 31P magic angle sample spinning (MASS) nuclear magnetic resonance (NMR) and X-ray radial distribution function (RDF) analyses to characterize 11-and 17-day-old embryonic chick bone and fractions obtained from them by density fractionation. Density fractionation provides samples of bone containing Ca-P solid-phase deposits even younger and more homogeneous with respect to the age of mineral than the calcium phosphate (Ca-P) deposits in the whole bone samples. The analytical techniques yield no evidence for any distinct phase other than the poorly crystal-line hydroxyapatite phase characteristic of mature bone mineral. In particular, there is no detectable crystalline brushite [DCPD, CaHPO4 2H2O< 1%] or amorphous calcium phosphate (< 8–10%) in the most recently formed bone mineral. A sizeable portion of the phosphate groups exist as HPO42− in a brushite (DCPD)-like configuration. These acid phosphate moieties are apparently incorporated into the apatitic lattice. The most likely site for the brushite-like configuration is probably on the surface of the crystals.

Chemical exchange effects in the NMR spectra of rotating solids

We present a theoretical analysis based on Floquet theory describing the effect of intermediate rate exchange on line shapes in magic angle sample spinning (MASS) spectra. As a test case, 13C spectra were obtained of dimethyl sulfone (DMS) from 25–55 °C. DMS exhibits an axially symmetric powder pattern at 25 °C which becomes asymmetric due to 180° flips about the twofold axis bisecting the CH3–S–CH3 angle. As the temperature is increased, the principal effect on the MASS spectrum is a pronounced broadening of both the center and sidebands. Calculated spectra fit the experimental spectra well and provide information on the rate constants and the activation energy for the exchange process. These results extend the line shape methods successfully used to study molecular motion in static samples to include high resolution MASS.

Sideband intensities in two‐dimensional NMR spectra of rotating solids

In the presence of magic angle sample spinning (MASS), two‐dimensional solid‐state NMR spectra of magnetically dilute I=1/2 nuclei split into rotational sidebands spaced at the spinning frequency in both dimensions. The intensities of these sidebands contain information on the sizes and relative orientations of the interactions present in the two experimental time domains. Here we extend an earlier analysis of sideband intensities in one‐dimensional MASS spectra to two‐dimensional MASS spectra. The resulting equations are applicable to heteronuclear and homonuclear correlation spectra, dipolar/chemical shift spectra, and slow chemical exchange spectra. We use the equations to deduce the orientation of the 15N chemical shift tensor in the amide group of L‐asparagine⋅H2O from the sideband intensities in the 15N–1H dipolar/chemical shift spectrum. This application to an AX2 system enlarges on previous work with AX systems and may be generalized to AXn systems.

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.

Solid state31NMR studies of the conversion of amorphous tricalcium phosphate to apatitic tricalcium phosphate

SummaryThe hydrolytic conversion of a solid amorphous calcium phosphate of empirical formula Ca9(PO4)6 to a poorly crystalline apatitic phase, under conditions where Ca2+ and PO43− were conserved, was studied by means of solid-state magic-angle sample spinning31P-NMR (nuclear magnetic resonance). Results showed a gradual decrease in hydrated amorphous calcium phosphate and the formation of two new PO43−-containing components: an apatitic component similar to poorly crystalline hydroxyapatite and a protonated PO43−, probably HPO42− in a dicalcium phosphate dihydrate (DCPD) brushite-like configuration. This latter component resembles the brushite-like HPO42− component previously observed by31P-NMR in apatitic calcium phosphates of biological origin. Results were consistent with previous studies by Heughebaert and Montel [18] of the kinetics of the conversion of amorphous calcium phosphate to hydroxyapatite under the same conditions.

Electron-nuclear double resonance of copper complexes of human transferrin

Electron-nuclear double resonance (ENDOR) spectroscopy has been used to study ligand and copper hyperfine interactions in Cu(II) complexes of human transferrin. A nearly isotropic superhyperfine interaction of the Cu(II) spin with a single 14N nucleus was identified, and the principal values of its tensor were estimated. All principal values of the copper hyperfine tensor were also directly measured for the first time. Resonances from at least two exchangeable protons were observed, but their origin could not be ascertained. At physiological pH, and in the presence of bicarbonate, ENDOR spectra of the two metal-binding sites were virtually indistinguishable.

Two-Dimensional Dipolar-Chemical Shift NMR in Rotating Solids

An overview of dipolar-chemical shift spectroscopy is presented with emphasis on recent developments to improve sensitivity, enhance information content, and simplify the spectra. Three new experiments are examined. First, a simplified experiment which is not synchronized with the sample rotation is shown to yield the same 2D spectra as previous methods, but with higher signal intensities. Second, an experiment is demonstrated to enhance dipolar sideband intensities, allowing the measurement of weaker couplings at spinning speeds which would otherwise yield vanishingly small sidebands in the dipolar dimension. Lastly, a mechanical technique involving the changing of the spinning speed between the evolution and detection periods of the dipolar chemical shift experiment is shown to yield a dramatic compression of the information in the two-dimensional landscape. The signals obtained are calculated using a versatile formalism which comprises a basis for comparison of the results of different experiments.

Solid State 29Si NMR determination of crystalline silica in natural iron oxide pigments

Synthetic iron oxide pigments have well characterized compositions. In natural iron oxide pigments, commercial products contain various amounts of talc, kaolin, and crystalline silica in addition to the iron oxide. The analysis for crystalline silica requires removing the iron oxide by an acid concentration step followed by x-ray diffraction (XRD) evaluation. However some materials not removed by the acid wash may interfere with quantitative XRD measurements. Another potential drawback of the x-ray analysis is the requirement for the particle size to be on the order of a few micrometers. In principle, nuclear magnetic resonance (NMR) is sensitive to domain sizes down to the order of a few nanometers. We have utilized solid state NMR techniques to obtain 29Si spectra from a number of materials to assess its viability as a quantitative method for measuring crystalline silica in iron oxide pigments. The method employs single pulse excitation with magic angle sample spinning (MASS). A protocol was established for quantitative silica analysis utilizing a reduced pulse angle of 10° with a 20−min relaxation delay between acquisitions. The reduced pulse angle was used to circumvent the long silicon relaxation times (T1) for the crystalline silica species. The spectra were recorded at a measurement frequency of 59.6 MHz with high power proton decoupling (300.1 MHz). A blind test of silica standards was used for comparison with x-ray diffraction analyses as a test of the protocol. Quantitative determination of crystalline silica employed tetrakis(trimethylsilyl)silane as a spin-counting standard.