Structures of chlorosomes and aggregated BChlc inChlorobium tepidum from solid state high resolution CP/MAS13C NMR

Cross polarization/magic angle spinning (CP/MAS)¹³C (solid state high resolution) NMR spectra were observed for chlorosomes and BChlc aggregates. Similarity of both kinds of spectra (except for some signals assignable to proteins and lipids in chlorosomes) indicates that BChlc's in chlorosomes are present just as in synthetic BChlc aggregates. Chemical shifts for C13¹ carbonyl and C3¹ hydroxylethyl carbons indicate hydrogen bonding between them. Comparison of solution and solid state¹³C NMR chemical shifts shows the five coordinated nature of BChlc aggregates. Some chemical shift differences were attributable to ring currents shifts. Their comparisons with calculated ring current shift values predicted structures for the aggregates. Cross polarization dynamics of the CP/MAS¹³C NMR signals explored dynamic and structural nature of the BChlc aggregates.     

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.     

The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy

Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly¹³C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state¹³C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly¹³C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T₁(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T₁(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces.     

(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.     

Conformational study of silk-like peptides containing the calcium-binding sequence from calbindin D9k using 13C CP/MAS NMR spectroscopy

The calcium-binding sites of calbindin D(9k) have a helix-loop-helix motif. In this study, the helix motifs were replaced by several Ala-Gly repeating regions designed on the basis of the primary sequences of several silk fibroins. The synthesized peptides were treated with several organic solvents to modify the secondary structure of the Ala-Gly repeating regions. The local structures of the Ala-Gly repeating regions, as well as the calcium-binding motif, D(9k)-loop (D(9k)L), were determined by (13)C CP/MAS NMR spectroscopy. In the four peptides containing D(9k)L synthesized, the poly(Ala) domains retain the ability to undergo a conformational transition from alpha-helical to beta-sheet in (A)(12)-D(9k)L despite the presence of the D(9k)L domain at the center of the peptide molecule, but the presence of this domain in the other model peptides synthesized has a marked effect on the conformation of the added silk-like domains. The results showed that the structures of the Ala-Gly repeating regions can be controlled by the choice of both the organic solvent and the amino acid sequence of the Ala-Gly repeating regions without disrupting the secondary structure of D(9k)L suggesting that it may retain its ability to bind calcium ions.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

Cultivation effects on the nature of organic matter in soils and water extracts using CP/MAS 13C NMR spectroscopy

The objective of this study was to examine the chemical structure of the organic matter (SOM) of Oxisols soils in slash and burn agriculture, in relation to its biological properties and soil fertility. The CP/MAS ¹³C technique was used to identify the main structural groups in litter and fine roots as SOM precursors; to identify the changes on the nature of the SOM upon cultivation and the proportion of labile and stable components; and to identify the nature of the organics present in water extracts (DOC). Carbohydrates were the main structural components in litter whereas components such as carbonyl C, carboxyl C,O-alkyl C and alkyl C were more common in SOM. Phenolic C and the degree of aromaticity were similar in litter and SOM. Cultivation resulted in a small decrease in the relative proportion of carbohydrates in SOM, little change in the levels of O-alkyl C and carbonyl C, but an increase in carboxyl C, phenolic C and aromaticity of the SOM. The level of alkyl C in soil was higher than the level of O-alkyl C, indicating the importance of long-chain aliphatics along with lignins in the stabilization of the SOM in Oxisols. The SOM of Mollisols from the Canadian Prairies differed from the Oxisol, with a generally stronger expression of aromatic structures, particularly in a cultivated soil in relation to a native equivalent. Carbohydrate components were the predominant structures in the DOC, indicating their importance in nutrient cycling and vertical translocations in the Oxisol.     

Residue specific hydration of primary cell wall potato pectin identified by solid-state 13C single-pulse MAS and CP/MAS NMR spectroscopy

Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by (13)C single-pulse (SP) magic-angle-spinning (MAS) and (13)C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by (2)H SP/MAS NMR experiments. The study shows that the arabinan side chains hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose-galacturonic acid (Rha-GalA) backbone in RG-I. Potential food ingredient uses of RG-I by tailoring of its structure are discussed.     

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.     

13C CP/MAS NMR Spectral Analysis of 6-O-Tosyl, 6-Deoxy-6-iodo,and 6-Deoxy Derivatives of N-Acetylchitosan in a Solid State

6-O-Tosyl (1, d.s. 0.94, 80% yield), 6-deoxy-6-iodo (2, d.s. 0.49, 86% yield) and 6-deoxy (3, d.s. 0.49, 50% yield) derivatives of N-acetylchitosan were prepared, and a ¹³C CP/MAS NMR spectral analysis was performed because no suitable solvent for 3 was found. The ¹³C signal for CH₃ at C-6 in 3 was detected at 18.9 ppm, and that for C-4 in 1–3 appeared at 72.2–72.7 ppm, which is in a higher magnetic field than those (82.5–86.0 ppm) in N-acetylchitosan, 6-O- (ethylthio), 6-O-(benzylthio)- and 6-O-(methylthio)-thiocarbonyl derivatives, chitosan, and chitin. This strongly suggests a different molecular conformation for 1–3.     

NMR and parity violation: low-temperature dependence in 1H CRAMPS and 13C CP/MAS ssNMR spectra of alanine enantiomer

Life is based on L-amino acids and D-sugars rather than the enantiomeric D-amino acids and L-sugars. This broken symmetry is now believed to be a feature of fundamental physics-a result of symmetry-breaking induced by the weak force, which makes one enantiomer slightly more stable than the other. An amplification mechanism based on quantum mechanical tunneling could give rise to a second-order phase transition. In order to understand the transition mechanism, we measured the temperature dependence of 1H CRAMPS solid state NMR and 13C CP/MAS spectra of D- and L-alanine crystals from 295 K through to 220 K. Obvious difference of NMR behaviors between two enantiomers was observed in the phase transition which may be related to one suggested by Salam, caused biochirality among twenty amino acids.     

Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy

The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.     

Assessment of the biochemical composition of oilseed rape (Brassica napus L.) 13C-labelled residues by global methods,FTIR and 13C NMR CP/MAS

The biochemical composition of stems, pod walls and roots of oilseed rape (Brassica napus L.) plants, grown in a growth chamber with two levels of N fertiliser, was assessed by two global methods, i.e., serial extraction with the Van Soest's technique and temperature-programmed pyroanalysis (TP-Py). Statistical analysis of the effect of various parameters on the proportion of soluble components, hemicellulose, cellulose and lignin-like components in oilseed rape organs showed that the composition of plant materials depended on the N nutrition conditions during plant growth. Contents of soluble and hemicellulose fractions were affected by the technique used. Elsewhere, both global techniques resulted in similar proportions of skeletal cellulose (respectively 41 and 36% in low and high N stems, 37 and 30% in low and high N pod walls, 32 and 29% in low and high N roots) and of lignin-like components which ranged from about 7% in high N stems and pod walls to 16% in low N roots. Spectroscopy by FTIR showed a significant band at 1650 cm^–1 (amide I in proteins) in the root material (organ with the lowest C/N ratio) and the absence of lignin-specific bands. Carbon distribution by ¹³C NMR CP/MAS of labelled plants indicated that 60–64% was (cellulose + hemicellulose)-C, close to the values obtained by global methods. The proportion of aromatic-C (110–160 ppm) and phenolic ether was higher in roots than in stems and pod walls. Organs from oilseed rape plants with higher N contents exhibited a larger proportion of C in the 171 ppm chemical shift attributed to the peptide bond. The concomitance of a high level of aromatic and proteinaceous components in roots would reveal the presence of tannin–protein complexes in addition with true lignin.     

A C CP/MAS NMR evaluation of the structural changes in wheat straw subjected to different chemical and biological pulping conditions

Wheat straw pulps prepared by chemical (soda) and biological (enzymatic or fungal) treatments were analyzed by ¹³C CP/MAS NMR spectrometry under quantitative acquisition conditions. The most significant changes reflected in the spectra as a result of soda cooking correspond to: (i) decrease of methoxyl content of the residual lignin (56, 153, 147 and 135 ppm), and (ii) deacetylation of hemicellulose fractions and saponification of cinnamyl esters concomitant to the release of alkali-soluble fractions (21 and 172 ppm). Reaction time was the factor with the greatest bearing on the former process, whereas soda concentration and temperature play an additional role in the latter. The decrease of the methoxyl/aryl ratio both after chemical and biological pulping suggests preferential removal of S-type lignin units. The comparison between quality parameters of the pulps and the ¹³C NMR integration data suggests that the linkage breakdown between straw macromolecules has a greater influence on paperboard properties than the neat extent of the chemical and biological removal of lignin fractions.     

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully¹³ C/¹⁵N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue¹³ C-¹⁵N correlation experiments with¹³ C-¹³C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using ¹⁵N instead of¹³ C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the ¹⁵N and ¹³C resonances in human ubiquitin.     

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.     

A method for estimating the nature and relative proportions of amorphous, single, and double-helical components in starch granules by (13)C CP/MAS NMR

An improved method to analyze the (13)C NMR spectra of native starches, which considers the contribution of the V-type conformation and the nature of the amorphous component, has been developed. Starch spectra are separated into amorphous and ordered subspectra, using intensity at 84 ppm as a reference point. The ordered subspectra of high amylose starches show the presence of both V-type single helices and B-type double helices. Relative proportions of amorphous, single, and double-helical conformations are estimated by apportioning intensity of C1 peak areas between conformational types on the basis of ordered and amorphous subspectra of the native starch. Quantitative analysis shows that the V-type single-helical component increases with amylose content of starches. Different amorphous subspectra are needed to provide a consistent analysis of granular starches from diverse sources. The method of preparation was found to be more important than the starch botanical origin in determining (13)C NMR spectral features of amorphous samples.     

The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state 2H NMR

Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using (2)H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ(9)-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2' and 16' positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer. Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-(2)H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime ((2)H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type (2)H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.