Kamal H. Mroue

Proton-Detected Solid-State NMR Spectroscopy of Bone with Ultrafast Magic Angle Spinning

While obtaining high-resolution structural details from bone is highly important to better understand its mechanical strength and the effects of aging and disease on bone ultrastructure, it has been a major challenge to do so with existing biophysical techniques. Though solid-state NMR spectroscopy has the potential to reveal the structural details of bone, it suffers from poor spectral resolution and sensitivity. Nonetheless, recent developments in magic angle spinning (MAS) NMR technology have made it possible to spin solid samples up to 110 kHz frequency. With such remarkable capabilities, 1H-detected NMR experiments that have traditionally been challenging on rigid solids can now be implemented. Here, we report the first application of multidimensional 1H-detected NMR measurements on bone under ultrafast MAS conditions to provide atomistic-level elucidation of the complex heterogeneous structure of bone. Our investigations demonstrate that two-dimensional 1H/1H chemical shift correlation spectra for bone are obtainable using fp-RFDR (finite-pulse radio-frequency-driven dipolar recoupling) pulse sequence under ultrafast MAS. Our results infer that water exhibits distinct 1H−1H dipolar coupling networks with the backbone and side-chain regions in collagen. These results show the promising potential of proton-detected ultrafast MAS NMR for monitoring structural and dynamic changes caused by mechanical loading and disease in bone.

Crystallinity and compositional changes in carbonated apatites: Evidence from 31P solid-state NMR, Raman, and AFM analysis

Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and 31P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse 31P NMR linewidth and inverse Raman PO43- ν1 bandwidth were both correlated with powder XRD c-axis crystallinity over the 0.3-10.3 wt% CO32- range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the 31P NMR chemical shift frequency and the Raman phosphate ν1 band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.

Proton-Based Ultrafast Magic Angle Spinning Solid-State NMR Spectroscopy

Protons are vastly abundant in a wide range of exciting macromolecules and thus can be a powerful probe to investigate the structure and dynamics at atomic resolution using solid-state NMR (ssNMR) spectroscopy. Unfortunately, the high signal sensitivity, afforded by the high natural-abundance and high gyromagnetic ratio of protons, is greatly compromised by severe line broadening due to the very strong 1H-1H dipolar couplings. As a result, protons are rarely used, in spite of the desperate need for enhancing the sensitivity of ssNMR to study a variety of systems that are not amenable for high resolution investigation using other techniques including X-ray crystallography, cryo-electron microscopy, and solution NMR spectroscopy. Thanks to the remarkable improvement in proton spectral resolution afforded by the significant advances in magic-angle-spinning (MAS) probe technology, 1H ssNMR spectroscopy has recently attracted considerable attention in the structural and dynamics studies of various molecular systems. However, it still remains a challenge to obtain narrow 1H spectral lines, especially from proteins, without resorting to deuteration. In this Account, we review recent proton-based ssNMR strategies that have been developed in our laboratory to further improve proton spectral resolution without resorting to chemical deuteration for the purposes of gaining atomistic-level insights into molecular structures of various crystalline solid systems, using small molecules and peptides as illustrative examples. The proton spectral resolution enhancement afforded by the ultrafast MAS frequencies up to 120 kHz is initially discussed, followed by a description of an ensemble of multidimensional NMR pulse sequences, all based on proton detection, that have been developed to obtain in-depth information from dipolar couplings and chemical shift anisotropy (CSA). Simple single channel multidimensional proton NMR experiments could be performed to probe the proximity of protons for structure determination using 1H-1H dipolar couplings and to evaluate the changes in chemical environments as well as the relative orientation to the external magnetic field using proton CSA. Due to the boost in signal sensitivity enabled by proton detection under ultrafast MAS, by virtue of high proton natural abundance and gyromagnetic ratio, proton-detected multidimensional experiments involving low-γ nuclei can now be accomplished within a reasonable time, while the higher dimension also offers additional resolution enhancement. In addition, the application of proton-based ssNMR spectroscopy under ultrafast MAS in various challenging and crystalline systems is also presented. Finally, we briefly discuss the limitations and challenges pertaining to proton-based ssNMR spectroscopy under ultrafast MAS conditions, such as the presence of high-order dipolar couplings, friction-induced sample heating, and limited sample volume. Although there are still a number of challenges that must be circumvented by further developments in radio frequency pulse sequences, MAS probe technology and approaches to prepare NMR-friendly samples, proton-based ssNMR has already gained much popularity in various research domains, especially in proteins where uniform or site-selective deuteration can be relatively easily achieved. In addition, implementation of the recently developed fast data acquisition approaches would also enable further developments in the design and applications of proton-based ultrafast MAS multidimensional ssNMR techniques.

Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Investigating Albendazole Desmotropes by Solid-State NMR Spectroscopy

Characterization of the molecular structure and physicochemical solid-state properties of the solid forms of pharmaceutical compounds is a key requirement for successful commercialization as potential active ingredients in drug products. These properties can ultimately have a critical effect on the solubility and bioavailability of the final drug product. Here, the desmotropy of Albendazole forms I and II was investigated at the atomic level. Ultrafast magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy, together with powder X-ray diffraction, thermal analysis, and Fourier transform infrared spectroscopy, were performed on polycrystalline samples of the two solids in order to fully characterize and distinguish the two forms. High-resolution one-dimensional (1)H, (13)C, and (15)N together with two-dimensional (1)H/(1)H single quantum-single quantum, (1)H/(1)H single quantum-double quantum, and (1)H/(13)C chemical shift correlation solid-state NMR experiments under MAS conditions were extensively used to decipher the intramolecular and intermolecular hydrogen bonding interactions present in both solid forms. These experiments enabled the unequivocal identification of the tautomers of each desmotrope. Our results also revealed that both solid forms may be described as dimeric structures, with different intermolecular hydrogen bonds connecting the tautomers in each dimer.

Insights into atomic-level interaction between mefenamic acid and Eudragit EPO in a supersaturated solution by high-resolution magic-angle spinning NMR spectroscopy

The intermolecular interaction between mefenamic acid (MFA), a poorly water-soluble nonsteroidal anti-inflammatory drug, and Eudragit EPO (EPO), a water-soluble polymer, is investigated in their supersaturated solution using high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy. The stable supersaturated solution with a high MFA concentration of 3.0 mg/mL is prepared by dispersing the amorphous solid dispersion into a d-acetate buffer at pH 5.5 and 37 °C. By virtue of MAS at 2.7 kHz, the extremely broad and unresolved (1)H resonances of MFA in one-dimensional (1)H NMR spectrum of the supersaturated solution are well-resolved, thus enabling the complete assignment of MFA (1)H resonances in the aqueous solution. Two-dimensional (2D) (1)H/(1)H nuclear Overhauser effect spectroscopy (NOESY) and radio frequency-driven recoupling (RFDR) under MAS conditions reveal the interaction of MFA with EPO in the supersaturated solution at an atomic level. The strong cross-correlations observed in the 2D (1)H/(1)H NMR spectra indicate a hydrophobic interaction between the aromatic group of MFA and the backbone of EPO. Furthermore, the aminoalkyl group in the side chain of EPO forms a hydrophilic interaction, which can be either electrostatic or hydrogen bonding, with the carboxyl group of MFA. We believe these hydrophobic and hydrophilic interactions between MFA and EPO molecules play a key role in the formation of this extremely stable supersaturated solution. In addition, 2D (1)H/(1)H RFDR demonstrates that the molecular MFA-EPO interaction is quite flexible and dynamic.

Insights into Novel Supramolecular Complexes of Two Solid Forms of Norfloxacin and β-Cyclodextrin

The solid-state properties of novel complexes of β-cyclodextrin and two different solid forms of norfloxacin were investigated at the molecular level, in an attempt to obtain promising candidates for the preparation of alternative matrices used in pharmaceutical oral formulations. In order to evaluate the physical properties inherited from the different polymorphs, these supramolecular systems were characterized using a variety of spectroscopic techniques including natural-abundance (13) C cross-polarization magic-angle-spinning (CP-MAS) nuclear magnetic resonance (NMR), powder X-ray diffraction, and Fourier transform infrared spectroscopy. The intrinsic proton spin-lattice relaxation times detected in (13) C CP-MAS NMR spectra are used to confirm and distinguish the complex formation, as well as to provide better insights into the molecular fragments that are involved in the interaction with β-cyclodextrin.

Proton chemical shift tensors determined by 3D ultrafast MAS double-quantum NMR spectroscopy

Proton NMR spectroscopy in the solid state has recently attracted much attention owing to the significant enhancement in spectral resolution afforded by the remarkable advances in ultrafast magic angle spinning (MAS) capabilities. In particular, proton chemical shift anisotropy (CSA) has become an important tool for obtaining specific insights into inter/intra-molecular hydrogen bonding. However, even at the highest currently feasible spinning frequencies (110-120 kHz), (1)H MAS NMR spectra of rigid solids still suffer from poor resolution and severe peak overlap caused by the strong (1)H-(1)H homonuclear dipolar couplings and narrow (1)H chemical shift (CS) ranges, which render it difficult to determine the CSA of specific proton sites in the standard CSA/single-quantum (SQ) chemical shift correlation experiment. Herein, we propose a three-dimensional (3D) (1)H double-quantum (DQ) chemical shift/CSA/SQ chemical shift correlation experiment to extract the CS tensors of proton sites whose signals are not well resolved along the single-quantum chemical shift dimension. As extracted from the 3D spectrum, the F1/F3 (DQ/SQ) projection provides valuable information about (1)H-(1)H proximities, which might also reveal the hydrogen-bonding connectivities. In addition, the F2/F3 (CSA/SQ) correlation spectrum, which is similar to the regular 2D CSA/SQ correlation experiment, yields chemical shift anisotropic line shapes at different isotropic chemical shifts. More importantly, since the F2/F1 (CSA/DQ) spectrum correlates the CSA with the DQ signal induced by two neighboring proton sites, the CSA spectrum sliced at a specific DQ chemical shift position contains the CSA information of two neighboring spins indicated by the DQ chemical shift. If these two spins have different CS tensors, both tensors can be extracted by numerical fitting. We believe that this robust and elegant single-channel proton-based 3D experiment provides useful atomistic-level structural and dynamical information for a variety of solid systems that possess high proton density.

Acceleration of natural-abundance solid-state MAS NMR measurements on bone by paramagnetic relaxation from gadolinium-DTPA.

Reducing the data collection time without affecting the signal intensity and spectral resolution is one of the major challenges for the widespread application of multidimensional nuclear magnetic resonance (NMR) spectroscopy, especially in experiments conducted on complex heterogeneous biological systems such as bone. In most of these experiments, the NMR data collection time is ultimately governed by the proton spin-lattice relaxation times (T1). For over two decades, gadolinium(III)-DTPA (Gd-DTPA, DTPA=Diethylene triamine pentaacetic acid) has been one of the most widely used contrast-enhancement agents in magnetic resonance imaging (MRI). In this study, we demonstrate that Gd-DTPA can also be effectively used to enhance the longitudinal relaxation rates of protons in solid-state NMR experiments conducted on bone without significant line-broadening and chemical-shift-perturbation side effects. Using bovine cortical bone samples incubated in different concentrations of Gd-DTPA complex, the (1)H T1 values were calculated from data collected by (1)H spin-inversion recovery method detected in natural-abundance (13)C cross-polarization magic angle spinning (CPMAS) NMR experiments. Our results reveal that the (1)H T1 values can be successfully reduced by a factor of 3.5 using as low as 10mM Gd-DTPA without reducing the spectral resolution and thus enabling faster data acquisition of the (13)C CPMAS spectra. These results obtained from (13)C-detected CPMAS experiments were further confirmed using (1)H-detected ultrafast MAS experiments on Gd-DTPA doped bone samples. This approach considerably improves the signal-to-noise ratio per unit time of NMR experiments applied to bone samples by reducing the experimental time required to acquire the same number of scans.

Hybridizing cross-polarization with NOE or refocused-INEPT enhances the sensitivity of MAS NMR spectroscopy

Heteronuclear cross polarization (CP) has been commonly used to enhance the sensitivity of dilute low-γ nuclei in almost all solid-state NMR experiments. However, CP relies on heteronuclear dipolar couplings, and therefore the magnetization transfer efficiency becomes inefficient when the dipolar couplings are weak, as is often the case for mobile components in solids. Here, we demonstrate methods that combine CP with heteronuclear Overhauser effect (referred to as CP-NOE) or with refocused-INEPT (referred to as CP-RINEPT) to overcome the efficiency limitation of CP and enhance the signal-to-noise ratio (S/N) for mobile components. Our experimental results reveal that, compared to the conventional CP, significant S/N ratio enhancement can be achieved for resonances originating from mobile components, whereas the resonance signals associated with rigid groups are not significantly affected due to their long spin-lattice relaxation times. In fact, the S/N enhancement factor is also dependent on the temperature, CP contact time as well as on the system under investigation. Furthermore, we also demonstrate that CP-RINEPT experiment can be successfully employed to independently detect mobile and rigid signals in a single experiment without affecting the data collection time. However, the resolution of CP spectrum obtained from the CP-RINEPT experiment could be slightly compromised by the mandatory use of continuous wave (CW) decoupling during the acquisition of signals from rigid components. In addition, CP-RINEPT experiment can be used for spectral editing utilizing the difference in dynamics of different regions of a molecule and/or different components present in the sample, and could also be useful for the assignment of resonances from mobile components in poorly resolved spectra. Therefore, we believe that the proposed approaches are beneficial for the structural characterization of multiphase and heterogeneous systems, and could be used as a building block in multidimensional solid-state NMR experiments.