CPMAS Spectra Research Articles

CPMAS NMR platform for direct compositional analysis of mycobacterial cell-wall complexes and whole cells

Tuberculosis and non-tuberculosis mycobacterial infections are rising each year and often result in chronic incurable disease. Important antibiotics target cell-wall biosynthesis, yet some mycobacteria are alarmingly resistant or tolerant to currently available antibiotics. This resistance is often attributed to assumed differences in composition of the complex cell wall of different mycobacterial strains and species. However, due to the highly crosslinked and insoluble nature of mycobacterial cell walls, direct comparative determinations of cell-wall composition pose a challenge to analysis through conventional biochemical analyses. We introduce an approach to directly observe the chemical composition of mycobacterial cell walls using solid-state NMR spectroscopy. 13C CPMAS spectra are provided of individual components (peptidoglycan, arabinogalactan, and mycolic acids) and of in situ cell-wall complexes. We assigned the spectroscopic contributions of each component in the cell-wall spectrum. We uncovered a higher arabinogalactan-to-peptidoglycan ratio in the cell wall of M. abscessus, an organism noted for its antibiotic resistance, relative to M. smegmatis. Furthermore, differentiating influences of different types of cell-wall targeting antibiotics were observed in spectra of antibiotic-treated whole cells. This platform will be of value in evaluating cell-wall composition and antibiotic activity among different mycobacteria and in considering the most effective combination treatment regimens.

Leucopterin, the white pigment in butterfly wings: structural analysis by PDF fit, FIDEL fit, Rietveld refinement, solid-state NMR and DFT-D.

Leucopterin (C6H5N5O3) is the white pigment in the wings of Pieris brassicae butterflies, and other butterflies; it can also be found in wasps and other insects. Its crystal structure and its tautomeric form in the solid state were hitherto unknown. Leucopterin turned out to be a variable hydrate, with 0.5 to about 0.1 molecules of water per leucopterin molecule. Under ambient conditions, the preferred state is the hemihydrate. Initially, all attempts to grow single crystals suitable for X-ray diffraction were to no avail. Attempts to determine the crystal structure by powder diffraction using the direct-space method failed, because the trials did not include the correct, but rare, space group P2/c. Attempts were made to solve the crystal structure by a global fit to the pair distribution function (PDF-Global-Fit), as described by Prill and co-workers [Schlesinger et al. (2021). J. Appl. Cryst. 54, 776-786]. The approach worked well, but the correct structure was not found, because again the correct space group was not included. Finally, tiny single crystals of the hemihydrate could be obtained, which allowed at least the determination of the crystal symmetry and the positions of the C, N and O atoms. The tautomeric state of the hemihydrate was assessed by multinuclear solid-state NMR spectroscopy. 15N CPMAS spectra showed the presence of one NH2 and three NH groups, and one unprotonated N atom, which agreed with the 1H MAS and 13C CPMAS spectra. Independently, the tautomeric state was investigated by lattice-energy minimizations with dispersion-corrected density functional theory (DFT-D) on 17 different possible tautomers, which also included the prediction of the corresponding 1H, 13C and 15N chemical shifts in the solid. All methods showed the presence of the 2-amino-3,5,8-H tautomer. The DFT-D calculations also confirmed the crystal structure. Heating of the hemihydrate results in a slow release of water between 130 and 250 °C, as shown by differential thermal analysis and thermogravimetry (DTA-TG). Temperature-dependent powder X-ray diffraction (PXRD) showed an irreversible continuous shift of the reflections upon heating, which reveals that leucopterin is a variable hydrate. This observation was also confirmed by PXRD of samples obtained under various synthetic and drying conditions. The crystal structure of a sample with about 0.2 molecules of water per leucopterin was solved by a fit with deviating lattice parameters (FIDEL), as described by Habermehl et al. [Acta Cryst. (2022), B78, 195-213]. A local fit, starting from the structure of the hemihydrate, as well as a global fit, starting from random structures, were performed, followed by Rietveld refinements. Despite dehydration, the space group remains P2/c. In both structures (hemihydrate and variable hydrate), the leucopterin molecules are connected by 2-4 hydrogen bonds into chains, which are connected by further hydrogen bonds to neighbouring chains. The molecular packing is very efficient. The density of leucopterin hemihydrate is as high as 1.909 kg dm-3, which is one of the highest densities for organic compounds consisting of C, H, N and O only. The high density might explain the good light-scattering and opacity properties of the wings of Pieris brassicae and other butterflies.

A theoretical and spectroscopic (NMR and IR) study of indirubin in solution and in the solid state

AbstractThe infrared spectrum of indirubin in the solid state and the 1H, 13C and 15N NMR spectra in DMSO‐d6 solution as well as the 13C and 15N CPMAS spectra have been recorded. Different types of calculations (B3LYP, GIAO, and GIPAW) have been carried out. Experimental and calculated values were compared yielding commensurate results. E/Z isomerism was explored, as was oxo/hydroxy tautomerism.

Incomplete Electrocyclization of a Sterically Hindered 1,4-Diphosphabutadiene.

Butadiene is the simplest neutral acyclic closed-shell π-conjugated system and is typically sufficiently stable enough to avoid electrocyclization to cyclobutene. In contrast, most congeners of butadiene containing heavier elements are easily converted into the corresponding 4-membered cyclobutene system. Herein, we demonstrate that the gauche 1,4-diphosphabutadiene (P=C-C=P) skeleton in a sterically encumbered 2,3-bis(phosphanylidene)-1,4-disilinane can be remarkably perturbed due to "incomplete electrocyclization" where P=C-C=P partially form the corresponding 1,2-dihydrodiphosphete (3,4-diphosphacyclobutene) by [2+2] electrocyclization. 31 P NMR data obtained in solution indicated that the coexistence of a closed ring substantially reduces the open-ring characteristics of the P=C-C=P moiety. However, the 31 P CP-MAS spectrum of 2,3-bis(phosphanylidene)-1,4-disilinane showed that the P=C-C=P structure is predominant in the solid-state. Single-crystal X-ray analysis revealed that decreasing the temperature promoted the generation of small amounts of incomplete 1,2-dihydrodiphosphete system in the crystalline state. Furthermore, the 1,2-dihydrodiphosphete units disappeared upon warming the single crystal, and this unique solid-state electrocyclization reaction was reversible.

13C NMR of DIPAC and DIPAB organic ferroelectrics

The diisopropylammonium chloride (C6H16ClN, DIPAC) and diisopropylammonium bromide (C6H16BrN, DIPAB) molecular crystals are recently discovered ferroelectrics with sufficiently high spontaneous polarization and Curie temperature. We performed first studies of these crystals by 13C NMR. CP MAS spectra were collected within large temperature ranges covering the Curie points. The reconstructive phase transition from the initial orthorhombic P212121 structure of DIPAB to the monoclinic ferroelectric P21 structure leads to an abrupt alteration in the 13C spectrum. The 13C spectra for DIPAC and DIPAB in the ferroelectric P21 phase are quite similar with four lines at lower frequencies, which correspond to the CH3 groups, and two lines with close chemical shifts, which correspond to two CH groups. The transition into the paraphase leads to gradual reduction of the interline distances in the low-frequency quadruplet and in the doublet. The step-like changes in the interline frequency shifts at this transition indicating its first order. The analysis of the spectrum evolution in the paraphase shows that only a CH group lays in the reflection plane above the P21 → P21/m transition, while the second CH group only moves closer to the reflection plane upon further heating.

Expanding solid-state phosphorus nuclear magnetic resonance insights into Mozzarella cheese ageing

With the addition of reference spectra and 1H31P cross-polarisation kinetics we have increased the analysis available by 31P NMR of the cheese ageing process. A previous study demonstrated that solid-state 31P NMR can be used to monitor the state of phosphorus in ageing cheeses. It is possible to differentiate between the mobile and immobile phosphorus species present in the cheese casein micelles. In this work, proton to phosphorus cross polarisation (CP) kinetic experiments (1H31P CP-MAS) (where MAS = magic angle spinning) enable the observation of the state of the colloidal calcium phosphate (CCP) and phosphoserine (PSer) separately in the casein micelles. Also, 1H31P CP-MAS spectra can be used to indirectly monitor the levels of micellar calcium. From the data gathered, we were able to conclude these points about the ageing process of Mozzarella cheese; in the first 20 days the cheese texture is dependent on the dynamics of free water and immobile calcium and phosphate, the CCP phosphate is mobilised at an exponential rate with the majority mobilised within the first 20 days, the driving force for CCP mobilisation is possibly the migration of water from the cheese serum into the casein proteins and CCP phosphorus is preferentially mobilised while immobile phosphoserine levels remains relatively unchanged during the early (40 days) of Mozzarella cheese ageing.

Libration of phenyl groups detected by VT-SSNMR: Comparison with X-ray crystallography.

The X-ray crystal structure of 2-benzyl-1H-benzimidazole, 2BnBzIm, was determined at 293K showing no dynamic phenomena (disorder) of any class. On the other hand, some 13 C NMR signals were absent in the CPMAS spectrum (100MHz, 300K). We decided to carry out variable-temperature SSNMR and discovered that the missing signals are ortho and meta carbons of the phenyl ring of the benzyl group. Line-shape analysis and the Eyring equation were used to determine the barrier, which was compared with the calculated DFT for the gas phase that it is much lower.

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells.

Solid-state NMR is a powerful tool for quantifying chemical composition and structure in complex assemblies and even whole cells. We employed N{P} REDOR NMR to obtain atomic-level distance propensities in intact 15N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorus was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen contributions to the REDOR spectra, the strongest dephasing emerged from the dipolar couplings to phosphorus involving nitrogen peaks ascribed primarily to rRNA, and the weakest dephasing arose from protein amide nitrogens. This approach is applicable to any macromolecular system and provides quantitative comparisons of distance proximities between shift-resolved nuclei of one type and heteronuclear dephasing spins. Enhanced molecular specificity could be achieved through the use of spectroscopic filters or specific labeling. Furthermore, ribosome 13C and 15N CPMAS spectra were compared with those of whole cells from which the ribosomes were isolated. Whole-cell signatures of ribosomes were identified and should be of value in comparing overall cellular ribosome content in whole-cell samples.

Structural, vibrational and DFT studies of di-(pipecolinium acid) squarate

A racemic mixture of (R)- and (S)-pipecolic acid (piperidine-2-carboxylic acid, P2C) forms a stable crystalline complex with squaric acid (3,4-dihydroxy-3-cyclobutene-1,2-dione, H2SQ), at the 2:1 ratio (1). The crystals are built of two pipecolic acid cations (P2C+H) and squarate dianion (SQ2−). Complex 1 has been characterized by single-crystal X-ray analysis, Raman, FTIR and 13C CP MAS spectroscopy. The title complex crystallizes in monoclinic C2/c space group with Z=4 and Z’=0.5. In the crystal structure two types of C2-symmetric complexes with different chirality are identified as [(R)-P2C+H]2SQ2− and [(S)-P2C+H]2SQ2−. The molecules are linked through the COOH⋯O–C of 2.587(1) Å and two N+H⋯O–C hydrogen bonds of 2.756(1) and 2.759(1) Å. In the optimized structure, at the B3LYP/6–311++G(d,p) level of theory, pipecolic acid zwitterion and cation interact with the hydrogen squarate anion. Charge delocalization has been analysed using the Natural Bond Orbital (NBO) method. Solid-state Raman, FTIR and 13C CP MAS spectra have been measured. The potential energy distributions (PED) have been used to assign the vibrational spectra. The crystal and optimized structures have been compared.

Disproportional proton tautomers of pipecolic acid and 2,6-dichloro-4-nitrophenol in a 2:3 complex

A racemic mixture of (R/S)-pipecolic acid (piperidine-2-carboxylic acid, P2C) forms a stable crystalline aggregate with 2,6-dichloro-4-nitrophenol (DCNP) at the unusual 2:3 ratio (1). Complex 1 has been characterized by X-ray analysis, Raman, FTIR and NMR spectra, and its structure (2) has been optimized by the B3LYP/6-31G(d,p) approach. The crystals are monoclinic, P21/n space group. The piperidine rings adopt chair conformations with the equatorial positions of the carboxylic groups. A P2C zwitterion is linked to two 2,6-dichloro-4-nitrophenol molecules through two non-equivalent short COO⋯HO–C hydrogen bonds and a cation (P2C+H) is hydrogen bonded with the DCNP− anion. These P2C molecules are NH⋯O hydrogen bonded. The FTIR spectrum is in agreement with the X-ray studies. The 13C CP MAS spectrum confirms the presence of two different P2C and DCNP units in the complex. In the optimized structure of the isolate aggregate, three DCNP molecules are bonded to two pipecolic acid zwitterions. Charge delocalization has been analyzed using the Natural Bond Orbital (NBO) method.

Novel poly(imide dioxime) sorbents: Development and testing for enhanced extraction of uranium from natural seawater

A new series of amidoxime-based polymer adsorbents were synthesized at the Oak Ridge National Laboratory (ORNL) by electron beam induced grafting of acrylonitrile and itaconic acid onto polyethylene fiber. Hydroxylamine derivatives of poly(acrylonitrile) (PAN) moiety are demonstrated to possess two kinds of functional groups: open-chain amidoxime and cyclic imide dioxime. The open-chain amidoxime is shown to convert to imide dioxime on heat treatment in the presence of an aprotic solvent, like dimethylsulfoxide (DMSO). The formation of amidoxime and imide dioxime was confirmed by 13C CP-MAS spectra. The adsorbents were evaluated for uranium adsorption efficiency at ORNL with simulated seawater spiked with 8ppm uranium and 5gallon seawater in a batch reactor, and in flow-through columns with natural seawater at the Marine Science Laboratory (MSL) of Pacific Northwest National Laboratory (PNNL) at Sequim Bay, WA. The DMSO-heat-treated sorbents adsorbed uranium as high as 4.48g-U/kg-ads. from seawater. Experimental evidence is presented that the poly(imide dioxime) is primarily responsible for enhanced uranium adsorption capacity from natural seawater. The conjugated system in the imide dioxime ligand possesses increased electron donation ability, which is believed to significantly enhance the uranyl coordination in seawater.

Structure and Dynamics in Functionalized Graphene Oxides through Solid-State NMR

Graphene oxide (GO), a derivative of the supermaterial graphene, has intrinsic proton conductivity, which is similar to Nafion, the most popular proton exchange membrane material currently used in fuel cells. Research into acid-functionalized GOs and determining the role of acidic groups in increasing proton conductivity will help to improve polymer electrolyte membrane performance in fuel cell systems. Multinuclear solid-state NMR (ssNMR) spectroscopy was used to analyze the structure and dynamics of GO and a number of sulfonic acid derivatives of GO, both novel and previously reported. 13C CP-MAS spectra showed the disappearance of surface-based oxygen groups upon GO functionalization and can be used to identify linker group carbon sites in previously synthesized and novel functionalized GO samples with high specificity. Dehydration of these samples allows the collection of 1H spectra with resolved acidic proton and water peaks. The effect of dehydration on the proton spectrum is partially reversible th...

Optimizing Sample Preparation Methods for Dynamic Nuclear Polarization Solid-state NMR of Synthetic Polymers

This work compares the overall sensitivity enhancements provided by dynamic nuclear polarization (DNP) for the solid-state NMR characterization of polymer samples doped with biradicals and prepared either by film casting (FC), or by glass forming (GF) using 1,1,2,2-tetrachloroethane as the solvent. Analysis of amorphous and semicrystalline polymers (polystyrene, poly(ethylene oxide), polylactide, poly(methyl methacrylate)) of varying molecular weights showed that GF provided larger sensitivity enhancements than FC but yielded DNP-enhanced 13C CPMAS spectra of lower resolution for semicrystalline polymers, owing to line-broadening due to conformational distribution of the polymer chains in frozen solution. Moreover, use of deuterated solvents significantly reduced the intensity of the solvent signals in the DNP-enhanced 13C CPMAS spectra of polymers prepared by GF, while preserving the sensitivity enhancement observed for the polymer signals. For the polymers investigated here, both FC and GF performed bet...

Synthesis, Characterization, and Catalytic Properties of Cationic Hydrogels Containing Copper(II) and Cobalt(II) Ions

Here, we report the synthesis and characterization of a hydrogel based on ethylene glycol diglycidyl ether (EGDE) and 1,8-diamino-3,6-dioxaoctane (DA). Chemically stable Co(II) and Cu(II) coordination complexes were prepared with this nonsoluble polyelectrolyte, poly(EGDE-DA), and studied by ss-NMR, FT-IR, thermogravimetry, and microscopy. Mesopores were found in all the samples, the thermal stability of the polymer matrix was highly affected by the presence of metal ions, and the (13)C CP-MAS spectrum for the Cu(II)-complex evidenced a significant increase in the reticulation degree by Cu(II) ions. The catalytic activity of these materials on H2O2 activation was studied by electron spin resonance (ESR). The Co(II)-poly(EGDE-DA)/H2O2 heterogeneous system produced O2, an anion superoxide (O2(•)¯), and a hydroxyl radical (OH(•)), which diffused into the solution at the time that a decrease in pH was detected. In the same way, the Cu(II)-poly(EGDE-DA)/H2O2 heterogeneous system produced O2 and OH(•). H2O2 activation by the poly(EGDE-DA) complexes with Co(II) and Cu(II) were applied on the decolorization of solutions of the azo-dye methyl orange (MO). In the presence of 63 mM H2O2, 87% of MO was removed in 10 min with Cu(II)-poly(EGDE-DA) and in 110 min with Co(II)-poly(EGDE-DA). In addition, the pharmaceutical product epinephrine was partially oxidized to adrenochrome by the O2(•)¯ released from the Co(II)-poly(EGDE-DA)/H2O2 heterogeneous system.

Experimental study of kerogen maturation by solid-state 13C NMR spectroscopy

Chemical changes in the structure of kerogen during simulated thermal maturation were investigated by solid-state 13С NMR spectroscopy. The spin counting technique was used to evaluate the share of “observable” carbon atoms with increasing hydrous pyrolysis temperature. The obtained one-dimensional CP–MAS spectra showed that up to 50% of ether bonds were destroyed during the initial stages of sample heating. In spite of the loss of a significant part of alkyl chains, kerogen remained aliphatic. The aromatic structure of kerogen underwent considerable changes: two-dimensional 1H–13C heteronuclear solid-state NMR (HETCOR) spectra showed that in the process of hydrous pyrolysis, the redistribution of carbon atoms took place – branched aromatic carbons transform into bridgehead aromatic carbons.

A tris(pyrazol-1-yl)methane bearing carboxylic acid groups at position 4: {1-[bis(4-carboxy-3,5-dimethyl-1H-pyrazol-1-yl)methyl]-3,5-dimethyl-1H-pyrazole-4-carboxylato}sodium dihydrate

A low-resolution X-ray molecular structure of the title hydrated salt, [Na(C19H21N6O6)]·2H2O, displays scorpionate character and resolves apparent ambiguities between solution and solid-state NMR spectroscopies. The (13)CNMR CPMAS spectrum is consistent with this structure showing some splittings, which have been rationalized using GIAO/B3LYP/6-311++G(d,p) theoretical calculations.

Sum of the Parts: Bacterial Biofilms by Solid-State NMR

Bacteria harbor dedicated machinery to assemble amyloid fibers at their cell surface. Curli, produced by E. coli, are among the most well-studied bacterial amyloids and play a significant role in adhesion and in the assembly of bacterial biofilms. Curli interact with secreted polysaccharides to form a biofilm and play a crucial role in determining biofilm morphology, yet the exact composition of curli-integrated biofilms is poorly understood. The determination of biofilm composition by routine methods, such as amino acid hydrolysis and mass spectrometry, is complicated by incomplete dissolution, as well as the perturbative nature of degradative and enzymatic changes. Solid-state NMR can provide valuable information about intact biofilms and is uniquely suited to provide a quantitative perspective on biofilm composition and structure. To dissect the contributions and interactions of curli and polysaccharides in the biofilm framework, we have purified and obtained 13C CPMAS spectra of: (i) the wild-type UTI89 extracellular matrix (ECM); (ii) the curli-free ECM produced by the curli mutant strain UTI89ΔcsgA; and (iii) purified curli. These spectra indicate that the UTI89 ECM has two major components, curli and cellulose, each in a quantifiable amount. The curli and polysaccharide regions of the spectrum also harbor unique chemical-shift contributions that allow us to examine curli-polysaccharide interactions and the modes of action of biofilm inhibitors. Our data define quantitatively the composition of the intact extracellular matrix, including cellulose and amyloid fibers, which impacts bacterial physiology. We are also working to transform cartoon representations of the curli amyloid fiber into a molecular model using selective labeling and REDOR NMR.

CPMAS 13C NMR characterization of humic acids from composted agricultural Saudi waste

CPMAS 13C NMR spectroscopy was used to characterize humic acid (HA) extracted from three types of composted agricultural Saudi waste of different origins, including agricultural crop plants (P), date palm fronds (D), and animal waste (A).The intensity of each region was estimated by the CPMAS spectrum indicating that HA (P) has a lower carbohydrate content than the other two samples. There was a greater presence of aromatic carbon substituted by oxygen or nitrogen in HA (A) than in HA (D), while HA (P) contained the highest content of aliphatic compounds. All samples exhibited strong peaks for a carbonyl of the carboxyl group. However, there was no absorbance for the carbonyl carbon in any of them. The lowest carboxyl group content among the samples was in HA (D) while HA (P), and HA (A) had very similar carboxyl contents. For all samples, the total aliphaticity was higher than the total aromaticity, with HA (P) having the highest total aliphaticity, and HA (A) having the lowest total aliphaticity. The composition and functional groups of the investigated samples did not exactly match those of any previously reported HA models, however, some similarity was noted between the Dragunov's model and HAs extracted from plant PEMs.

MgCl2·6C6H11OH: A High Mileage Porous Support for Ziegler–Natta Catalyst

A new and single phase molecular adduct of MgCl2 with six cyclohexanol molecules MgCl2·6C6H11OH (MgCyOH) has been synthesized. Structural insight to this adduct was obtained by a variety of physicochemical methods. 13C CPMAS spectrum and single pulse MAS spectra with high power proton decoupling recorded at different relaxation time showed the presence of two different sets of magnetically unequal cylcohexanol molecules present around Mg2+. A Raman feature of MgCyOH at 712 cm–1 confirms that cylcohexanol molecules are present around Mg2+ in an octahedral environment. MgCyOH has been used as support material to prepare Ziegler–Natta (Z–N) active catalyst. Textural property of above Z–N catalyst exhibits high surface area (236 m2/g) with high porosity. Above active catalyst has been screened for ethylene polymerization. Depending on the cocatalyst employed (Me3Al, Et3Al, and iso-Bu3Al) and ethylene pressure, polyethylene yield varies an order of magnitude, from 378 to 3570 g/g catalyst, indicating a possibl...

A Slowly Relaxing Rigid Biradical for Efficient Dynamic Nuclear Polarization Surface-Enhanced NMR Spectroscopy: Expeditious Characterization of Functional Group Manipulation in Hybrid Materials

A new nitroxide-based biradical having a long electron spin-lattice relaxation time (T(1e)) has been developed as an exogenous polarization source for DNP solid-state NMR experiments. The performance of this new biradical is demonstrated on hybrid silica-based mesostructured materials impregnated with 1,1,2,2-tetrachloroethane radical containing solutions, as well as in frozen bulk solutions, yielding DNP enhancement factors (ε) of over 100 at a magnetic field of 9.4 T and sample temperatures of ~100 K. The effects of radical concentration on the DNP enhancement factors and on the overall sensitivity enhancements (Σ(†)) are reported. The relatively high DNP efficiency of the biradical is attributed to an increased T(1e), which enables more effective saturation of the electron resonance. This new biradical is shown to outperform the polarizing agents used so far in DNP surface-enhanced NMR spectroscopy of materials, yielding a 113-fold increase in overall sensitivity for silicon-29 CPMAS spectra as compared to conventional NMR experiments at room temperature. This results in a reduction in experimental times by a factor >12,700, making the acquisition of (13)C and (15)N one- and two-dimensional NMR spectra at natural isotopic abundance rapid (hours). It has been used here to monitor a series of chemical reactions carried out on the surface functionalities of a hybrid organic-silica material.