Large Chemical Shift Research Articles

Structural investigation of amorphous Na2O–P2O5–B2O3 correlated with its ionic conductivity

A series of sodium borophosphate compounds was prepared by twin roller quenching technique. The compounds have the following compositions: (10B2O3–(90−x) P2O5−xNa2O) (BPNax) and (10Na2O–(90−x) P2O5−xB2O3) (NPBox) with x=0, 5, 10, 15, 20mol%. DTA and XRD are used to evidence the crystallization conditions.Change in the local structural environment has been investigated by Raman and by 11B, 31P MAS and QMAS NMR spectroscopies.The large 31P chemical shift of P3 and P2 units with increasing x and the decrease in 11B chemical shift of B4 units with decreasing x indicate that phosphorous is linked to boron through a BO. This feature reveals that the short-range order is amended in the network.Optical absorption was used to determine glassy energy gap Eg, which we used to calculate size polydispersity index and optical index.Electric and dielectric properties were investigated through impedance spectroscopy.For all compositions, variation in the temperature of dc conductivity was found to follow an Arrhenius type relationship. An improvement in the ionic conductivity was reported with the increase of both alkali and borate contents in the glass host.

Multichannel MRI labeling of mammalian cells by switchable nanocarriers for hyperpolarized xenon.

We demonstrate a concept for multichannel MRI cell-labeling using encapsulated laser-polarized xenon. Conceptually different Xe trapping properties of two nanocarriers, namely macrocyclic cages as individual hosts or compartmentalization into nanodroplets, ensure a large chemical shift separation for Xe bound in either of the carriers even after cellular internalization. Two differently labeled mammalian cell populations were imaged by frequency selective saturation transfer resulting in a switchable "two-color" xenon-MRI contrast at micro- to nanomolar Xe carrier concentrations.

Improved volume selective (1) H MR spectroscopic imaging of the prostate with gradient offset independent adiabaticity pulses at 3 tesla.

Volume selection in (1) H MR spectroscopic imaging (MRSI) of the prostate is commonly performed with low-bandwidth refocusing pulses. However, their large chemical shift displacement error (CSDE) causes lipid signal contamination in the spectral range of interest. Application of high-bandwidth adiabatic pulses is limited by radiofrequency (RF) power deposition. In this study, we aimed to provide an MRSI sequence that overcomes these limitations. Measurements were performed at 3 T with an endorectal receive coil. A semi-LASER sequence was equipped with low RF power demanding gradient-modulated offset-independent adiabaticity (GOIA) refocusing pulses with WURST(16,4) modulation, with a 10 kHz bandwidth. Simulations and phantom studies verified that the GOIA pulses select slices with a flat top and sharp edges and minimal CSDE. The sequence timing was tuned to an optimal citrate signal shape at an echo time of 88 ms. Patient studies (n = 10) demonstrated that high quality spectra with reduced lipid artifacts can be obtained from the whole prostate. Compared with PRESS acquisition at 145 ms the signal-to-noise ratio (SNR) of citrate is increased up to 2.6 and choline up to 1.3. An MRSI sequence of the prostate is presented with minimized spectral lipid contamination and improved SNR, to facilitate routine clinical acquisition of metabolic data.

Folding dynamics and pathways of the trp-cage miniproteins.

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9–7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

Understanding API-polymer proximities in amorphous stabilized composite drug products using fluorine-carbon 2D HETCOR solid-state NMR.

A simple and robust method for obtaining fluorine-carbon proximities was established using a (19)F-(13)C heteronuclear correlation (HETCOR) two-dimensional (2D) solid-state nuclear magnetic resonance (ssNMR) experiment under magic-angle spinning (MAS). The method was applied to study a crystalline active pharmaceutical ingredient (API), avagacestat, containing two types of fluorine atoms and its API-polymer composite drug product. These results provide insight into the molecular structure, aid with assigning the carbon resonances, and probe API-polymer proximities in amorphous spray dried dispersions (SDD). This method has an advantage over the commonly used (1)H-(13)C HETCOR because of the large chemical shift dispersion in the fluorine dimension. In the present study, fluorine-carbon distances up to 8 Å were probed, giving insight into the API structure, crystal packing, and assignments. Most importantly, the study demonstrates a method for probing an intimate molecular level contact between an amorphous API and a polymer in an SDD, giving insights into molecular association and understanding of the role of the polymer in API stability (such as recrystallization, degradation, etc.) in such novel composite drug products.

Constraints on the incorporation mechanism of chlorine in peralkaline and peraluminous Na2O-CaO-Al2O3-SiO2 glasses

Incorporation mechanisms of Cl in peralkaline and peraluminous Na2O-CaO-Al2O3-SiO2 glasses as a model system for phonolitic melts were investigated using 35Cl, 23Na, 27Al, and 29Si magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. The size and large distribution of electric field gradients for 35Cl causes loss of signal in the MAS NMR experiment and this, in combination with the low concentration of Cl and the large chemical shift dispersion, means that even at the highest available fields we are at the limits of MAS NMR. Nevertheless clear differences in the Cl environment in peralkaline and peraluminous glasses can readily be seen. In both glass types Cl exists in relatively symmetric Na-Ca-Cl environments. The 35Cl chemical shift indicates that the Cl environment is dominated by the presence of Na cations, consistent with the Na/Ca ratio of 5/1 in the glasses. 35Cl MAS NMR spectra of the peraluminous glasses show a larger chemical shift distribution and a more positive isotropic chemical shift, ~-75 ppm, than the peralkaline glasses, ~-100 ppm. They also have a larger quadrupole coupling constant with a larger distribution, indicating greater disorder in the peraluminous glasses. It is likely that there are more Ca cations present in the Cl environments in the peraluminous glasses than in the peralkaline glasses despite their having the same Na/Ca ratio. In the peralkaline glasses the formation of Na-Ca-Cl environments leads to a decrease in the number of network-modifying cations, which causes a polymerization of the glass network. No effect on the glass polymerization was observed in the peraluminous glasses. Some 35Cl signal is also lost in the static spectra indicating that ~20% of Cl for a peralkaline glass and more than ~70% for a peraluminous glass must be in environments where there is a large enough electric field gradient that the resulting very broad line is unobservable. These environments could be simply Na-Ca-Cl with higher electric field gradients than those producing the observed 35Cl signal or non-bridging Cl environments like for example Al-Cl. The Cl environment in the present mixed Na2O-CaO aluminosilicate glasses appears to be more disordered than was to be expected from previous NMR spectroscopic studies on simpler glass compositions.

Design and Characterization of Two Bifunctional Cryptophane A‐Based Host Molecules for Xenon Magnetic Resonance Imaging Applications

AbstractCryptophanes have been shown to be a promising tool in xenon magnetic resonance imaging (MRI) applications. The affinity of xenon for molecular host structures such as cryptophane‐A (CrA) and the large xenon chemical shift that can be detected in the presence of such hosts has motivated the synthesis of a number of cryptophane‐based MRI biosensors. Bifunctional biosensors that incorporate both a fluorophore and a cryptophane moiety are flexible tools for evaluating the intracellular localization and quantifying cellular uptake/binding of such xenon hosts in conjunction with testing their MRI capabilities. In this study, we compare the performance of a new compound that bears tetramethylrhodamine (TAMRA), CrA‐PEG‐TAMRA (PEG=polyethylene glycol), with our previously synthesized compound in which fluorescein (FAM) was the optical reporter. We demonstrate that these biosensors are suitable candidates for MRI‐based cell‐tracking studies; specifically, the new addition of a rhodamine derivative will allow for multiplexing experiments and showed slightly improved NMR spectroscopic behavior. Importantly, both biosensors display equally good cell‐labeling efficiency, which facilitates the acquisition of high‐quality xenon magnetic resonance images in two different cell lines. This study describes a simple synthetic approach for the production of biologically compatible bifunctional compounds. These biosensors can be used as they are for cell labeling but can also serve as building blocks for further functionalization and cryptophane‐based xenon biosensor design with MRI and fluorescence multiplexing options.

ChemInform Abstract: New Frontiers and Developing Applications in 19F NMR

AbstractReview: 395 refs.

Synthesis of Fluorinated Pyridines as 19F NMR pH Indicators

This research was undertaken to synthesizes and evaluate 3,5-dihydroxymethyl-4-(2-fluorophenyl)-2,6-dimethylpyridine and 4-(fluoromethyl)-2,6-dimethylpyridine as indicators of 19F NMR pH. A comparison of 3,5-dihydroxymethyl-4-(2-fluorophenyl)-2,6-dimethylpyridine to 4-(fluoromethyl)-2,6-dimethylpyridine showed that 4-(fluoromethyl)-2,6-dimethylpyridine has a large 19F chemical shift to pH, a suitable pKa value and a good water solubility.

Asymmetric simultaneous phase-inversion cross-polarization in solid-state MAS NMR: Relaxing selective polarization transfer condition between two dilute spins

Double cross polarization (DCP) has been widely used for heteronuclear polarization transfer between 13C and 15N in solid-state magic-angle spinning (MAS) NMR. However, DCP is such sensitive to experimental settings that small variations or deviations in RF fields would deteriorate its efficiency. Here, we report on asymmetric simultaneous phase-inversion cross polarization (referred as aSPICP) for selective polarization transfer between low-γ 13C and 15N spins. We have demonstrated through simulations and experiments using biological solids that the asymmetric duration in the simultaneous phase-inversion cross polarization scheme leads to efficient polarization transfer between 13C and 15N even with large chemical shift anisotropies in the presence of B1 field variations or mismatch of the Hartmann–Hahn conditions. This could be very useful in the aspect of long-duration experiments for membrane protein studies at high fields.

Dismutational and Global‐Minimum Isomers of Heavier 1,4‐Dimetallatetrasilabenzenes of Group 14

Aromatic species with heavier Group 14 elements show remarkable differences in terms of stability, structure, and reactivity. Herein we report our experimental and theoretical investigations regarding isomers of germanium- and tin-containing benzene analogues E2Si4R6 (E=Ge, Sn). The germanium-substituted dismutational isomer with a tricyclic six-membered scaffold is isolable, but unlike the homonuclear Si6 analogue slowly rearranges even at room temperature to give the propellane-type global minimum isomer. In case of E=Sn the dismutational isomer may be an intermediate on the pathway to the propellane-type species obtained, but cannot be detected even at low temperature. Unprecedentedly large chemical shift anisotropies in the (29)Si NMR spectra that increase from the Si6 species through Ge2Si4 to Sn2Si4 are rationalized by progressively larger paramagnetic-term contributions to the chemical shift tensor as a result of diminishing HOMO-LUMO gaps, which are also reflected in the absorption spectra, as well as by appearance and symmetry of these frontier orbitals.

Solid-state NMR/NQR and first-principles study of two niobium halide cluster compounds

Two hexanuclear niobium halide cluster compounds with a [Nb6X12]2+ (X=Cl, Br) diamagnetic cluster core, have been studied by a combination of experimental solid-state NMR/NQR techniques and PAW/GIPAW calculations. For niobium sites the NMR parameters were determined by using variable Bo field static broadband NMR measurements and additional NQR measurements. It was found that they possess large positive chemical shifts, contrary to majority of niobium compounds studied so far by solid-state NMR, but in accordance with chemical shifts of 95Mo nuclei in structurally related compounds containing [Mo6Br8]4+ cluster cores. Experimentally determined δiso(93Nb) values are in the range from 2400 to 3000ppm. A detailed analysis of geometrical relations between computed electric field gradient (EFG) and chemical shift (CS) tensors with respect to structural features of cluster units was carried out. These tensors on niobium sites are almost axially symmetric with parallel orientation of the largest EFG and the smallest CS principal axes (Vzz and δ33) coinciding with the molecular four-fold axis of the [Nb6X12]2+ unit. Bridging halogen sites are characterized by large asymmetry of EFG and CS tensors, the largest EFG principal axis (Vzz) is perpendicular to the X-Nb bonds, while intermediate EFG principal axis (Vyy) and the largest CS principal axis (δ11) are oriented in the radial direction with respect to the center of the cluster unit. For more symmetrical bromide compound the PAW predictions for EFG parameters are in better correspondence with the NMR/NQR measurements than in the less symmetrical chlorine compound. Theoretically predicted NMR parameters of bridging halogen sites were checked by 79/81Br NQR and 35Cl solid-state NMR measurements.

Rational design of hyperpolarized xenon NMR molecular sensor for the selective and sensitive determination of zinc ions

Although Zn(2+) ions are involved in large numbers of physiopathological processes, non-invasive detection of Zn(2+) ions in opaque biological samples remains a huge challenge. Here, we developed a novel zinc-responsive hyperpolarized (HP) (129)Xe-based NMR molecular sensor. This HP (129)Xe-based NMR molecular sensor was synthesized by attaching 2-(diphenylphosphino) benzenamine as ligand for zinc ions to the xenon-binding supramolecular cage, cryptophane. The (129)Xe NMR spectroscopy of such molecular sensor was shifted up to 6.4 ppm in the presence of Zn(2+) ions, which was nearly four times larger than that of the reported similar sensor. The application of the sensor would benefit low concentration detection by using indirect NMR/MRI method. The response exhibited high sensitivity and selectivity as discriminated from other six potentially competing metal ions. The application of this sensor in the analysis of zinc ions in the rat serum samples was also evaluated. The strategy is generally applicable in developing sensitive and selective sensors for quantitative determination of zinc ions.

N1s and O1s double ionization of the NO and N2O molecules.

Single-site N1s and O1s double core ionisation of the NO and N2O molecules has been studied using a magnetic bottle many-electron coincidence time-of-flight spectrometer at photon energies of 1100 eV and 1300 eV. The double core hole energies obtained for NO are 904.8 eV (N1s(-2)) and 1179.4 eV (O1s(-2)). The corresponding energies obtained for N2O are 896.9 eV (terminal N1s(-2)), 906.5 eV (central N1s(-2)), and 1174.1 eV (O1s(-2)). The ratio between the double and single ionisation energies are in all cases close or equal to 2.20. Large chemical shifts are observed in some cases which suggest that reorganisation of the electrons upon the double ionization is significant. Δ-self-consistent field and complete active space self-consistent field (CASSCF) calculations were performed for both molecules and they are in good agreement with these results. Auger spectra of N2O, associated with the decay of the terminal and central N1s(-2) as well as with the O1s(-2) dicationic states, were extracted showing the two electrons emitted as a result of filling the double core holes. The spectra, which are interpreted using CASSCF and complete active space configuration interaction calculations, show atomic-like character. The cross section ratio between double and single core hole creation was estimated as 1.6 × 10(-3) for nitrogen at 1100 eV and as 1.3 × 10(-3) for oxygen at 1300 eV.

Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.

Modeling H-Bonding and Protonation States of Membrane Buried ARG-TYR Pairs

Previous X-ray crystallographic studies have shown that in the activated states of G-protein coupled receptors (GPCRs), or in those of microbial rhodopsins, the side chains in conserved R+Y dyads transiently move within several A of each other. In the M state of bR, substantial side-chain perturbation occurs, as has been shown previously, e.g. by FTIR and solid-state (SS) NMR studies of bR with 15N-labeled R82. No simpler system has been able to model the transient spectral perturbations of R82 in M. Here we report the synthesis of crystalline ω-(4-anisolyl)-dodecylguanidine (ADG) and ω-(4-phenolyl)-dodecylguanidine (PDG), with either natural isotope abundance or 98% 15N-enrichment at the 2 terminal nitrogens. The ADG free base, with a deprotonated guanidine group, is a good spectroscopic model for both IR and 15N-SS-NMR signals that were previously seen from R82 in the M photointermediate, particularly for the unusually large 15N chemical shift splitting. In the crystalline PDG free base, however, this chemical shift splitting is reduced to nearly the same value seen for the HBr salts of ADG, PDG, and arginine itself, all of which have protonated guanidinium groups. The high-resolution X-ray structure of the free-base form of PDG, as well as the IR spectrum of these crystals in KBr disk, show clearly that what crystallizes is actually a zwitterion, with guanidinium and phenolate groups; additionally, 2 H-bonding solvent (methanol) molecules co-crystallize with each PDG. These PDG free-base crystals are surprisingly insoluble in most aprotic solvents. However, they are sufficiently soluble in DMSO to observe IR, UV-vis and 1H NMR spectra. These spectra are all consistent with a ∼50/50 mix of rapidly-interchanging zwitterionic and neutral forms. The high proton polarizability of PDG makes this an interesting model system for buried H-bonded uncharged R+Y dyads in proteins.

NMR assignments for the cis and trans forms of the hemolysin II C-terminal domain

Pathogenic bacteria secrete pore-forming toxins (PFTs) to selectively defend against immune cells and to break through cellular barriers in the host. Understanding how PFTs attack cell membranes is not only essential for therapeutic intervention but for designing agents to deliver drugs to specific human cell subtypes, for example in anti-cancer or anti-viral therapies. Many toxins contain accessory domains that help recognize specific molecular epitopes on the membranes of target cells, including proteins, carbohydrates, and lipids. Here we report NMR assignments for the 94-residue 10 kDa C-terminal accessory domain of Bacillus cereus hemolysin II, HlyIIC, that has no known structural or functional homologues. The HlyIIC domain exists in a dynamic equilibrium due to cis/trans isomerization of its Gly86-Pro87 peptide bond. The cis and trans forms are about equally populated and are in slow exchange on the NMR timescale, giving rise to separate signals for approximately half of the residues in the domain. Assignments for the cis and trans forms were achieved with the aid of a P87M mutant that stabilizes the trans form, and separate sequential walks for the two forms in 3D NMR spectra of the wild-type HlyIIC. Based on backbone chemical shifts, the domain has a α1-α2-β1-β2-β3-β4-α3-β5 order of secondary structure elements. The last strand in the trans form and in the P87M mutant is shortened near Pro87 compared to the cis form. Both cis/trans isomerization and the P87M mutation cause large chemical shift changes throughout HlyIIC, suggesting that the proline is important in stabilizing the structure of the domain. The NMR assignments pave the way for solving the structures of the multiple conformational forms of HlyIIC and establishing their mechanism of interconversion.

Structural Insights for Activation of Retinal Guanylate Cyclase by GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca2+-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca2+ binding in EF-hand 4 and models the Ca2+-free/Mg2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca2+-free activator and Ca2+-bound inhibitor states. This contrasts with large Ca2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.

Interaction between a Transition-Metal Fluoride and a Transition-Metal Hydride: Water-Mediated Hydrofluoric Acid Evolution Following Fluoride Solvation

The reaction between the nickel(II) PCP pincer fluoride complex ((tBu)PCP)Ni(F) [(tBu)PCP = 2,6-C6H3(CH2P(t)Bu2)2] and the tungsten(II) carbonyl hydride CpW(H)(CO)3 (Cp = η(5)-C5H5(-)) leads to hydrofluoric acid evolution and formation of the bimetallic isocarbonylic species [CpW(CO)2(μ-κ,C:κ,O-CO)···Ni((tBu)PCP)]. The process has been monitored through multinuclear ((19)F, (31)P{(1)H}, (1)H) variable-temperature NMR spectroscopy, collecting (19)F T1 data values for a fluoride ligand bound to a transition metal. The extremely short relaxation time (minimum value of 13 ms at 193 K) is ascribed to the large chemical shift anisotropy of the Ni-F bond (688 ppm). The in-depth NMR analysis has revealed that the fluoride-hydride interaction is not direct but water-mediated, at odds with what was previously observed for the "hydride-hydride" case ((tBu)PCP)Ni(H)/CpW(H)(CO)3. Kinetic measurements have unveiled that the first step of the overall mechanism is thought to be solvation of the fluoride ligand (as a result of Ni-F···H2O hydrogen bonding), while further reaction of the solvated fluoride with CpW(H)(CO)3 is extremely slow and competes with the side reaction of fluoride replacement by a water molecule on the nickel center to form the [((tBu)PCP)Ni(H2O)](+) aquo species. Finally, density functional theory analysis of the solvation process through a discrete + continuum model has been accomplished, at the M06//6-31+G(d,p) level of theory, to support the mechanistic hypothesis.

Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs

Fluorine-19 is a spin-½ NMR isotope with high sensitivity and large chemical shift dispersion, which makes it attractive for high resolution NMR spectroscopy in solution. For studies of membrane proteins it is further of interest that (19)F is rarely found in biological materials, which enables observation of extrinsic (19)F labels with minimal interference from background signals. Today, after a period with rather limited use of (19)F NMR in structural biology, we witness renewed interest in this technology for studies of complex supramolecular systems. Here we report on recent (19)F NMR studies with the G protein-coupled receptor family of membrane proteins.