(13)C CP MAS NMR and crystal structure of methyl glycopyranosides

The X-ray diffraction analysis, (13)C CP MAS NMR spectra and powder X-ray diffraction patterns were obtained for selected methyl glycosides: alpha- and beta-d-lyxopyranosides (1, 2), alpha- and beta-l-arabinopyranosides (3, 4), alpha- and beta-d-xylopyranosides (5, 6) and beta-d-ribopyranoside (7) and the results were confirmed by GIAO DFT calculations of shielding constants. In X-ray diffraction analysis of 1 and 2, a characteristic shortening and lengthening of selected bonds was observed in molecules of 1 due to anomeric effect and, in crystal lattice of 1 and 2, hydrogen bonds of different patterns were present. Also, an additional intramolecular hydrogen bond with the participation of ring oxygen atom was observed in 1. The observed differences in chemical shifts between solid state and solution come from conformational effects and formation of various intermolecular hydrogen bonds. The changes in chemical shifts originating from intermolecular hydrogen bonds were smaller in magnitude than conformational effects. Furthermore, the powder X-ray diffraction (PXRD) performed for 4, 5 and 7 revealed that 7 existed as a mixture of two polymorphs, and one of them probably consisted of two non-equivalent molecules.     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

Thermal, dynamic and structural properties of drug AT1 antagonist olmesartan in lipid bilayers

It is proposed that AT1 antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT1 receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC), 13C magic-angle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles. 13C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as 13C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche:trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile.     

Quantitation of aliphatic suberin in Quercus suber L. cork by FTIR spectroscopy and solid-state (13)C-NMR spectroscopy

This work determined that the percentage of suberin in cork may be found by solid-state (13)C cross polarization/magic angle spinning (CP/MAS) NMR spectroscopy and by FTIR with photoacoustic detection (FTIR-PAS) spectroscopy. A linear relationship is found between the suberin content measured through CP/MAS spectral areas and that measured gravimetrically. Furthermore, application of a partial least squares (PLS1) regression model to the NMR and gravimetric data sets clearly correlates the two sets, enabling suberin quantification with 90% precision. Suberin quantitation by FTIR-PAS spectroscopy is also achieved by a PLS1 regression model, giving 90% accurate estimates of the percentage of suberin in cork. Therefore, (13)C-CP/MAS NMR and FTIR-PAS proved to be useful and accurate noninvasive techniques to quantify suberin in cork, thus avoiding the traditional time consuming and destructive chemical methods.     

Crystal structure and solid state 13C NMR analysis of nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-gluco- and D-galactopyranosides

The X-ray diffraction analysis of o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside (1), m-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, p-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside was performed. It was found that except in the case of 1, all other crystals have one molecule in the independent part of the crystal unit cell. The results support the opinion that the nitro group does not conjugate effectively with the phenyl ring. In the 13C CP MAS spectrum of 1 the signals are split, confirming the presence of two independent molecules. Similarly, the 13C CP MAS NMR spectrum of p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside indicated the presence of two non-equivalent molecules in the crystal unit. One of these molecules has more conformational freedom enabling rotation of the phenyl ring.     

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

(13)C MAS NMR and photo-CIDNP reveal a pronounced asymmetry in the electronic ground state of the special pair of Rhodobacter sphaeroides reaction centers

Reaction centers of wild-type Rhodobacter sphaeroides were selectively (13)C-isotope labeled in bacteriochlorophyll and bacteriopheophytin. (13)C solid-state CP/MAS NMR and photo-CIDNP were used to provide insight into the electronic structure of the primary electron donor and acceptor on the atomic scale. The first 2-dimensional photochemically induced dynamic nuclear polarization (photo-CIDNP) (13)C-(13)C solid-state MAS NMR spectra reveal that negative charging of the two BChl rings of the primary donor is involved in ground-state tuning of the oxidation potential of these cofactors in the protein via local electrostatic interactions. In particular, the (13)C shifts show moderate differences in the electronic structure between the two BChl molecules of the special pair in the electronic ground state, which can be attributed to hydrogen bonding of one of the BChl molecules. The major fraction of the electron spin density is strongly delocalized over the two BChl molecules of the special pair and the photochemically active BPhe. A small fraction of the pi-spin density is distributed over a fourth component, which is assigned to the accessory BChl. Comparison of the photo-CIDNP data with "dark" NMR spectra obtained in ultra high field indicates a rigid special pair environment upon photoreaction and suggests that structural changes of the aromatic macrocycles of the two BChl molecules of the special pair do not significantly contribute to the reorganization energy associated with the charge-transfer process.     

Effects of backbone and side chain on the molecular environments of chiral cavities in polysaccharide-based biopolymers

The effects of the backbone and side chain on the molecular environments in the chiral cavities of three commercially important polysaccharide-based chiral sorbents--cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), amylose tris(3,5-dimethylphenylcarbamate) (ADMPC), and amylose tris[(S)-alpha-methylbenzylcarbamate] (ASMBC)--are studied by attenuated total reflection infrared spectroscopy (ATR-IR), X-ray diffraction (XRD), 13C cross-polarization/magic-angle spinning (CP/MAS) and MAS solid-state NMR, and density functional theory (DFT) modeling. These sorbents are used widely in preparative-scale chiral separations. ATR-IR is used to determine how the H-bonding states of the C=O and NH groups of the polymer depend on the backbone and side chain. The changes in the polymer crystallinity are characterized with XRD. The changes in the polymer helicity and molecular mobility for polymer-coated silica beads (commercially called Chiralcel OD, Chirapak AD, and Chiralpak AS) are probed with 13C CP/MAS and MAS solid-state NMR. The IR wavenumbers and the NMR chemical shifts for the polymer backbone monomers and dimers and the side chains are predicted at the DFT/B3LYP/6-311+g(d,p) level of theory. It is concluded that the molecular environments of the C=O, NH, and phenyl groups show significant differences in intramolecular and intermolecular interactions and in the nanostructures of the chiral cavities of these biopolymers. These results have implications for understanding how the molecular environments of chiral cavities of these polymers affect their molecular recognition mechanisms.     

The use of solid-state NMR and X-ray crystallography as complementary tools for studying molecular recognition

Solid-state NMR is rapidly becoming available as a routine technique for studying the structure of crystalline or noncrystalline solids. This technique has an advantage over crystallography in that single crystals are not necessary, but it has the disadvantage that the information obtained does not produce a direct picture of the molecule and its environment. On the other hand, solid-state NMR can be done on mixtures, and it gives information about phase distribution in a manner similar to that of X-ray powder pattern analysis.Crystallographic effects such as polymorphism, multiple molecules per asymmetric unit, disorder and salvation can frequently be detected using NMR. Sometimes molecular point group symmetry can also be deduced based on the number of independent nuclei that are detected. The NMR method is sensitive to changes in the electronic structure of a molecule as sensed by the nuclei, and the effects are measured as changes in the isotropic chemical shift of individual nuclei.In this paper, we will give examples of the combined use of X-ray crystallography and ¹³CP/MAS (cross polarization/magic angle spinning) NMR for studying hostguest materials and cocrystals. We have learned how to use NMR to tell us about keto/enol composition in the solid state, to detect the presence of trapped solvent molecules, to detect hydrogen-bond formation and to evaluate molecular conformation and unusual packing pattern effects. We will also present a brief background of the ¹³CP/MAS NMR technique and three case studies in which solid-state NMR and X-ray crystallography are used together to understand materials' structures and properties     

Homogeneous modification of cellulose with succinic anhydride in ionic liquid using 4-dimethylaminopyridine as a catalyst

Cellulose, extracted from sugarcane bagasse, was successfully succinylated in ionic liquid 1-buty-3-methylimidazolium (BMIMCl) using 4-dimethylaminopyridine (DMAP) as a catalyst. Parameters investigated included the mass ratio of DMAP/succinic anhydride in a range from 0% to 15%, reaction time (from 30 to 120 min), reaction temperature (from 60 to 110 °C). The succinylated cellulosic derivatives had a degree of substitution (DS) ranging from 0.24 to 2.34. It was found that the DS of succinylated cellulosic derivatives using DMAP as a catalyst was higher than that without any catalyst under the same reaction conditions. The products were characterized by FT-IR, solid-state CP/MAS ¹³C NMR, and thermal analysis. FT-IR and solid-state CP/MAS ¹³C NMR spectra showed that succinoylation occurred at C-6, C-2 and C-3 positions. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Structural aspects in semicrystalline samples of the mannan II family

A series of samples having the mannan II character were prepared by either (i) desincrusting stems of Acetabularia crenulata, or (ii) acetylating these stems, followed by dissolution and recrystallization under deacetylation conditions, or (iii) recrystallizing at low temperature the alkali soluble fraction of ivory nut mannan. The samples were characterized by transmission electron microscopy, X-ray and electron diffraction analysis together with (13)C CP/MAS NMR spectroscopy. Whereas the A. crenulata stems consisted of a mixture of mannan I and mannan II, the recrystallized samples were all of the hydrated mannan II family and occurred in a ribbonlike morphology where the mannan chains were organized with their molecular axis perpendicular to the ribbon long axis. The recrystallized ivory nut mannan samples presented X-ray and electron diffraction diagrams, together with (13)C solid-state NMR spectra recorded at 95% RH, different from those of recrystallized A. crenulata recorded under the same RH conditions. They corresponded therefore to a new allomorph of the mannan II family. Despite this difference, when the recrystallized samples were in an aqueous environment, they revealed an additional well-defined perhydrated phase, which showed the same (13)C solid-state NMR spectrum for both samples. As this phase, which gave 6-band NMR spectra with narrow line-width and low T1, had no counterpart in X-ray diffraction, it was attributed to specific amorphous segments of mannan chains, gaining some mobility when swollen in water. When the samples were totally dried, their NMR spectra lost their resolution, thus indicating the role played by water for the structural organization of the crystalline and amorphous components of mannan II.     

A study of ordered structure in acid-modified tapioca starch by C CP/MAS solid-state NMR

Acid modification of tapioca starch earlier reported to increase the mechanical strength of tablets. The development of ordered structure (double helices) of these starches was monitored after equilibrating at 0.90 a_w (25 °C) using ¹³C CP/MAS NMR and X-ray diffraction. As the hydrolysis time increased, the intensity of the resonance for C1 and C4 amorphous fractions decreased while that for C1 and C4 double helix fractions increased. Relative crystallinity (%) obtained from ¹³C CP/MAS NMR and X-ray diffraction methods both increased sharply initially and then levelled off with hydrolysis time. The initial increase in relative double helix content and crystallinity was due to a hydrolytic destruction in the amorphous domain, retrogradation of the partially hydrolyzed amylose and crystallization of free amylopectin double helices. After 192 h, these two parameters were not significantly different (=0.05) indicating that the double helices that were not arranged into crystalline regions either had been hydrolyzed or crystallized.     

Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli

Physical properties of chiral poly(thioesters), PTEs, prepared by engineered Escherichia coli, were examined by GPC, 13C CP/MAS solid-state NMR, X-ray diffraction, and thermal analysis. Microbial homopolymers of PTEs, poly(3-mercaptopropionate), PMP, and poly(3-mercaptovalerate), PMV, showed different solubility characteristics compared to poly(hydroxyalkanoates), PHAs. Generally, PTEs required higher temperatures for dissolution. Poly(3-mercaptobutyrate), PMB, and PMV dissolve in chloroform, and the molecular weight values were revealed by GPC as 176,000 and 165,000, respectively. The density values for PMP and PMB were 1.42 and 1.27 g/cm3, respectively. These values are similar to those for oxygen analogues. The NMR spectra for PTEs showed that carbonyl carbons are greatly shifted downfield by the sulfur atoms in the chain backbone compared to the PHA family. X-ray powder diffraction data indicated that PTEs are crystalline materials, but they do not crystallize as well as in the PHA family. The melting point, Tm, for PMP was 170 degrees C, which is about 100 degrees C higher than the equivalent oxygen analogue, poly(3-hydroxypropionate), PHP, and almost the same as that of bacterial poly(3-hydroxybutyrate), PHB. According to thermal analysis, only the PMP sample had enhanced heat stability, e.g., the decomposition temperature for PMP was 277 degrees C at 5% weight loss, whereas the values for PHP and PHB were 233 and 260 degrees C at the same weight loss, respectively.     

Structure and dynamics of flour by solid state NMR: effects of hydration and wheat aging

The effects of accelerated aging of wheat seeds on structural and dynamic properties of dry and hydrated (ca 10 wt % H(2)O) flour at a molecular level were investigated by several high and low resolution solid-state NMR techniques. Identification and characterization of domains with different mobility was performed by (13)C direct excitation (DE) and cross-polarization (CP) magic angle spinning (MAS), as well as by (1)H static and MAS experiments. (1)H spin-lattice relaxation times (T(1) and T(1)(rho)) measurements were carried out to investigate molecular motions in different frequency ranges. Experimental data show that the main components of flour (starch and gluten proteins) are in a glassy phase, whereas the mobile fraction is constituted by lipids and, in hydrated samples, absorbed water. A lower proportion of rigid domains, as well as an increased dynamics of all flour components are observed after both seeds aging and flour hydration. Linear average dimensions between 20 and 200 A are estimated for water domains in hydrated samples.     

Preparation of cellulose derived from corn stalk and its application for cadmium ion adsorption from aqueous solution

Cellulose was isolated from corn stalk and modified by graft copolymerization to produce an absorbent material (AGCS-cell), which was characterized by scanning electron microscope and energy disperse spectroscopy (SEM-EDS), X-ray diffraction (XRD) and solid-state CP/MAS (13)C NMR. The results showed that AGCS-cell had better adsorption potential for cadmium ion than unmodified cellulose because of the addition of functional groups (CN and OH groups) and the lower crystallinity. The Langmuir isotherms gave the best fit to the data and gave an adsorption capacity was 21.37mgg(-1), which was close to unpurified cellulose (AGCS) and reflected the feasibility of using AGCS-cell as an adsorbent to remove cadmium ions.     

Solid-state 13C NMR and X-ray diffraction of dermatan sulfate

Dermatan sulfate in the solid state has been studied by 13C CP/MAS nmr and X-ray diffraction in order to establish the ring conformation of the L-iduronate moiety. The solid state nmr spectrum is similar to the solution spectrum obtained previously, indicating that a ring conformation at least approximating to 1C4 predominates in the solid state. X-ray powder diffraction data from the same sample indicate the presence of the 8-fold helix form previously observed by fiber diffraction, and interpreted in terms of a 4C1 ring form. A likely explanation of the results is that a distorted 1C4 L-iduronate ring conformation, not considered in the initial X-ray analysis, may emerge to provide a satisfactory interpretation of all available physical-chemical data.     

Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52

The Ca2+-ATPase of cardiac muscle cells transports Ca2+ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca2+ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. 13C MAS NMR measurements are also presented on phospholamban that is 1-13C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca2+ regulation that may involve phospholamban as both an inhibitor of the Ca2+ ATPase and as an ion channel.     

Solid-state NMR determination of sugar ring pucker in (13)C-labeled 2'-deoxynucleosides

The H3'-C3'-C4'-H4' torsional angles of two microcrystalline 2'-deoxynucleosides, thymidine and 2'-deoxycytidine.HCl, doubly (13)C-labeled at the C3' and C4' positions of the sugar ring, have been measured by solid-state magic-angle-spinning nuclear magnetic resonance (NMR). A double-quantum heteronuclear local field experiment with frequency-switched Lee-Goldberg homonuclear decoupling was used. The H3'-C3'-C4'-H4' torsional angles were obtained by comparing the experimental curves with numerical simulations, including the two (13)C nuclei, the directly bonded (1)H nuclei, and five remote protons. The H3'-C3'-C4'-H4' angles were converted into sugar pucker angles and compared with crystallographic data. The delta torsional angles determined by solid-state NMR and x-ray crystallography agree within experimental error. Evidence is also obtained that the proton positions may be unreliable in the x-ray structures. This work confirms that double-quantum solid-state NMR is a feasible tool for studying sugar pucker conformations in macromolecular complexes that are unsuitable for solution NMR or crystallography.     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.     

Characterization of Cimetidine–Piroxicam Coprecipitate Interaction Using Experimental Studies and Molecular Dynamic Simulations

The crystalline states of cimetidine and piroxicam, when coprecipitated from solvents containing 1:1 mole ratio, were transformed to amorphous states as observed using powder X-ray diffraction (PXRD). Amorphous forms of drugs generally exhibit higher water solubility than crystalline forms. It is therefore interesting to investigate the interactions that cause the transformation of both the crystalline drugs. Intermolecular interactions between the drugs were determined using Fourier-transform infrared spectroscopy (FTIR) and solid-state ¹³C CP/MAS NMR. Molecular dynamic (MD) simulation was performed for the first time for this type of study to indicate the specific groups involved in the interactions based on radial distribution function (RDF) analyses. RDF is a useful tool to describe the average density of atoms at a distance from a specified atom. FTIR spectra revealed a shift of the C≡N stretching band of cimetidine. The ¹³C CP/MAS NMR spectra indicated downfield shifts of C₁₁, C₁₅ and C₇ of piroxicam. RDF analyses indicated that intermolecular interactions occurred between the amide oxygen atom as well as the pyridyl nitrogen of piroxicam and H-N₃ of cimetidine. The hydrogen atom (O–H) at C₇ interacts with the N₁ of cimetidine. Since the MD simulation results are consistent with, and complementary to the experimental analyses, such simulations could provide a novel strategy for investigating specific interacting groups of drugs in coprecipitates, or in amorphous mixtures.