Solid state NMR studies on the structural and conformational properties of natural maize starches

Natural maize starches having a range of amylose contents have been characterised by CP/MAS NMR spectroscopy. Chemical shifts, relative resonance intensities, line-widths and spectral shapes were compared at different moisture contents. At 10% moisture content, these parameters showed few significant differences across a range of apparent amylose levels from 0 to 84%. After hydration of the granules to ≈30% moisture, it was found that the amylose content significantly affected the relative signal intensities and line-widths especially of C-1 and C-4 resonances. Narrower line-widths after hydration were attributed to (i) an increased degree of crystallinity, and (ii) disappearance of the signals of amorphous material which, on becoming more mobile, became invisible to the CP/MAS experiment. The enhanced resolution at higher moisture levels revealed signals which were assigned to the amylose–lipid complex, i.e. V-type amylose. The amount of V-amylose detected by NMR increased with both amylose content and lipid content of the granule. Prolonged treatment of the granules with iodine vapour significantly increased the amount of V-type amylose in the high amylose samples, but caused a decrease in their degree of crystallinity. Waxy-maize starch was barely affected by iodination. The results provide evidence that amylose tends to disrupt the structural order within amylopectin crystallities. This effect is enhanced by the formation of the amylose–iodine complex, indicating that V-amylose could be a major crystallite-disrupting agent in native starch granules.     

Changes in C NMR chemical shifts of DNA as a tool for monitoring drug interactions

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]₂ to explore the effects of ligand binding on the ¹³C NMR chemical shifts of the DNA base and sugar carbons. The binding mode of netrospin to TA-rich tracts of DNA has been well documented and served as an attractive model system. For the base carbons, four large changes in resonance chemical shifts were observed upon complex formation: −0.64 ppm for carbon 4 of either Ado4 or Ado6, 1.36 ppm for carbon 2 of Thd5, 1.33 ppm for carbon 5 of Thd5 and 0.94 for carbon 6 of Thd5. AdoC4 is covalently bonded to a heteroatom that is hydrogen bonded to netropsin; this relatively large deshielding is consistent with the known hydrogen bond formed at AdoN3. The three large shielding increases are consistent with hydrogen bonds to water in the minor groove being disrupted upon netropsin binding. For the DNA sugar resonances, large changes in chemical shifts were observed upon netropsin complexation. The 2′, 3′ and 5′ ¹³C resonances of Thd3 and Thd5 were shielded whereas those of Ado4 and Ado6 were deshielded; the ¹³C resonances of 1′ and 4′ could not be assigned. These changes are consistent with alteration of the dynamic pseudorotational states occupied by the DNA sugars. A significant alteration in the pseudorotational states of Ado4 or Ado6 must occur as suggested by the large change in chemical shift of −1.65 ppm of the C3′ carbon. In conclusion, ¹³C NMR may serve as a practical tool for analyzing structural changes in DNA-ligand complexes.     

Inclusion/exclusion of fatty acids in amylose complexes as a function of the fatty acid chain length

Structural models are proposed for amylose-fatty acid complexes depending on the respective chain lengths of their constituents. The three studied fatty acids induce the Vh amylose crystalline type. However, in contrast to lauric and palmitic acids, caprylic acid is not present in crystals. On the basis of the relative amounts of amylose and fatty acid determined in complexes and previous results of molecular modelling, inclusion of lauric and palmitic acids inside the amylose helices is proposed; the acyl chains are included in crystalline areas and the car☐ylic groups in amorphous areas. The absence of caprylic acid in crystals could be due to the solubility of this compound in the crystallization medium.     

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.     

Acylation of natural flavonoids using lipase of candida antarctica as biocatalyst

Several flavonoids (quercetin, hesperidin, rutin and esculin) were acylated with fatty acids using an immobilised lipase from Candida antarctica in 2-methyl-2-butanol at 60 °C. It appears that esculin with primary OH on the sugar part is the most reactive substrate. With palmitic acid as acyl donor, the conversion yields were of about 80, 71 and 38%, respectively, for esculin, rutin and hesperidin. No reaction was observed with aglycon flavonoid (quercetin). For a given flavonoid (rutin), the conversion yield increased from 42 to 76% when the carbon number of the fatty acids rose from C6 to C12. For fatty acids with higher carbon-chain length, both conversion yield and initial rate dropped slightly. Furthermore, compared to the saturated fatty acid (C18: 0), the unsaturated one (C18: 1) exhibited a lower reactivity. For all molecules studied ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analyses indicated that only flavonoid monoester was produced.     

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.     

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.     

Phosphonato complexes of platinum(II): Kinetics of formation and phosphorus-31 NMR characterization studies

Reactions of cis-diamminedichloroplatinum(II) with phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and methylenediphosphonic acid (MDP) yield various phosphonatoplatinum(II) chelates which were characterized by phosphorus-31 NMR spectroscopy. The P-31 resonances for the chelates appear at 6–12 ppm downfield as compared to the uncomplexed ligands. All complexes exhibit monoprotic acidic behavior in the pH range 2–10. The chemical shift-pH profiles yielded acidity constants, 1.0 × 10⁻⁴, 1.5 × 10⁻⁴, and 1.3 × 10⁻⁶ M⁻¹, for the PFA, PAA, and MDP chelates. In addition to the monomeric chelate, MDP formed a bridged diplatinum(II,II) complex when it reacted with cis-Pt (NH₃)₂(H₂O)₂²⁺. The P-31 resonance for this binuclear complex appears at 22 ppm downfield from the unreacted ligand.

Rate data for the complexation reactions of the phosphonate ligands with the dichloroplatinum complex are consistent with a mechanism in which a monodentate complex is formed initially through rate-limiting aquation process of the platinum complex, followed by a rapid chelation. For the PFA and PAA complexes, initial binding sites are the carboxylato oxygens. Implications of the various binding modes of the phosphonates in relationship to their antiviral activities are discussed.     

NMR study of paper

High quality antique sheets of paper have been characterized by ¹H NMR relaxations and ¹³C CP MAS spectra. Paper can be regarded as a bicomponent material made of cellulose and water plus a small amount of organic and inorganic impurities. Semicrystalline fibrous cellulose, rich in water, is present in the I and I_β forms. The amorphous cellulose, with a low water content, contains a higher amount of paramagnetic impurities and it is characterized by quite short ¹H spin-lattice relaxations and by ¹¹³C resonances with noticeable chemical shifts. Ad hoc tailored pulse sequences are able to produce ¹³C CP MAS spectra in which only the amorphous content of paper is clearly observed. It is shown that water is fully bound to the cellulose lattice. It also seems reasonable to formulate the hypothesis that a larger concentration of paramagnetic ions is located in the amorphous fraction of highly degraded paper compared with paper in good condition.     

The interaction of azacrown ether with fatty acid in nonpolar solvents and at the organic-aqueous interface

In the paper, we show that lipophilic azacrown ether (22DD) in solvents of low to intermediate polarity forms the complexes with fatty acids, like lauric or palmitic acid. Due to the weak acid-base properties of the azacrown ether-fatty acid system, no proton transfer between the two molecules was observed, as shown by IR and 1H NMR studies. The Job plot exhibits double maximum, suggesting the coexistence of two 22DD-fatty acid complexes, of 1:1 and 1:2 stoichiometry, respectively. Their stability constants were calculated by taking into account the dimerization of fatty acid in toluene. The diffusion coefficients for the free molecules and their complexes were measured with diffusion-ordered spectroscopy (DOSY) NMR in order to prove the close spatial proximity of the molecules. Interfacial tension measurements at the water-toluene interface showed that due to the presence of two decyl chains, 22DD adsorbs at the interface much stronger than dodecanoic (lauric) acid does. The shape of the adsorption isotherm for the mixture of 22DD and lauric acid suggests that the two molecules also interact at the interface in a similar manner as in the bulk of low to intermediate polarity solvents. As a result of the affinity of the fatty acid to strongly surface-active azacrown ether, the interface might be enriched with fatty acid molecules, which without 22DD shows little adsorption at the liquid-liquid interface.     

A new process for the esterification of wood by reaction with vinyl esters

A novel route to wood modification by transesterification of vinyl esters is developed in the current study. The reaction between varied saturated and unsaturated vinyl esters and the hydroxyl groups of maritime pine sapwood (Pinus pinaster Soland) was examined using potassium carbonate as a catalyst. The esterification of wood was investigated by weight percent gain calculations (WPG), Fourier-transform infrared spectroscopy (FTIR) and ¹³C cross-polarization with magic-angle spinning nuclear magnetic resonance spectroscopy (¹³C CP–MAS NMR). Differences in the rates of modification were noted, depending on the vinyl ester used, but relatively high yields were obtained in all cases. The infrared and NMR spectra of the different esterified samples were analysed in detail and the assignment of the signals corresponding to the grafted acyl groups confirmed that esterification occurred.     

Utilization of Uniformly Labeled 13C-Polyunsaturated Fatty Acids in the Synthesis of Long-Chain Fatty Acids and Cholesterol Accumulating in the Neonatal Rat Brain

Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18-carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20-and 22-carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo ¹³C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly ¹³C-enriched 16-and 18-carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U-¹³C-polyunsaturated fatty acid mixture, ¹³C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; ¹³C enrichment remained high in the phospholipids and cholesterol for 15 days. ¹³C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in ¹³C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16-and 18-carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long-chain saturated and monounsaturated fatty acids and cholesterol.     

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP⁺) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by ³¹P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP⁺ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.     

Effect of nixtamalization on the modification of the crystalline structure of maize starch

Endosperm of nixtamalized corn was analyzed using X-ray diffraction. Relative crystallinity changed with lime concentration and steeping. Diffractograms showed peaks corresponding to V-type crystalline structures, indicating formation of complexes during cooking and steeping. Diffraction patterns of the soluble fraction showed that complexed amylose can be leached out during solubilization. While diffraction patterns of the insoluble fraction suggested that some of the formed complexes remain in this fraction. During alkali steeping, release of amylose is strongly inhibited as indicated by the pronounced decrease in the starch–I₂ absorbance of the lime treated samples compared to the lime-free treated sample. This decrease is interpreted as evidence of starch cross-linking during the nixtamalization process. Differences in starch–I₂ absorbance and in X-ray diffraction patterns of the soluble fractions suggested that lime treatment could also modified formation of amylose complexes with lipids.     

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.     

Formation and crystallization of amylose–monoglyceride complex in a starch matrix

The formation of amylose–lipid complexes in a gelatinized potato starch matrix was investigated using potato starch and glycerol monopalmitin. These complexes exist in two forms, with the amounts of each of the forms being dependent on the temperatures and durations of the pre-treatments.

Differential scanning calorimetry (DSC) was used to analyze transition temperatures and melting enthalpies, and thereby determine the amount of the complexes in the samples. X-ray diffraction analysis was used to investigate their crystallinity.

In measurements with DSC, form I started to melt at 88.5°C, and form II at 112.9°C. When complex form II was preheated at 100 or 110°C, its melting point rose to 116.3 and 119.7°C, respectively, because of an annealing effect. The same phenomenon occurred with complex form I: when preheated at 90°C, its melting point rose to 96.8°C. The crystal formation of form II appeared to be slower when treated at 110°C than at 100°C. Their maximum melting enthalpies were reached after about 24 h and 4 h of preheating, respectively. In X-ray diffraction analyses, form II showed a V-pattern, but form I did not. This indicates that form II is more crystalline than form I. It was possible to transform form I into form II when it was heat treated, because form I was then partially or totally melted.

As a comparison, the charged substance cetyltrimethylammonium bromide created complex form I with amylose in the starch matrix, but not form II.     

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and ω-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on ω-substituted fatty acid chain were unfavourable to these reactions. The ¹H NMR and ¹³C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4′′′-OH of the rhamnose residue of rutin.     

Existence of lipid microdomains in bilayer of dipalmitoyl phosphatidylcholine (DPPC) and 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) and their perturbation by chlorpromazine: a 13C and 31P solid-state NMR study

The polyunsaturated fatty acid docosahexaenoic acid (DHA, 22 : 6, n-3) is found at a level of about 50% in the phospholipids of neuronal tissue membranes and appears to be crucial to human health. Dipalmitoyl phosphatidylcholine (DPPC, 16 : 0/16 : 0 PC) and the DHA containing 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) were used to make DPPC (60%)/SDPS (40%) bilayers with and without 10 mol% chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine.

Resonances that are present in ¹³C NMR spectrum of the DPPC (60%)/SDPS (40%) sample and that disappear in presence of 10% CPZ most probably are due to the special interface environment, e.g. the hydrophobic mismatch, at the interface of DPPC and SDPS microdomains in the DPPC/SDPS bilayer. In itself the appearance of resonances at novel chemical shift values is a clear demonstration of a unique chemical environment in the DPPC (60%)/SDPS (40%) bilayer. The findings of the study presented here suggest CPZ bound to the phosphate of SDPS will slow down and partially inhibit such a DHA acyl chain movement in the DPPC/SDPS bilayer. This would affect the area occupied by a SDPS molecule (in the bilayer) and probably the thickness of the bilayer where SDPS molecules reside as well. It is quite likely that such CPZ caused changes can affect the function of proteins embedded in the bilayer.     

Application of CP-MAS and liquid-like solid-state NMR experiments for the study of the ripening-associated cell wall changes in tomato

¹³C and ¹H NMR spectra of an ethanol insoluble material (EIM) prepared from the pericarp of mature-green (MG) and red-ripe (RR) tomato fruits were acquired in ‘liquid-like’ and cross-polarisation with dipolar decoupling and magic angle spinning (CPMAS) conditions using the same triple resonance probe. Such a strategy allowed acquisitions of various NMR experiments aimed at detecting compositional differences as well as distinguishing differences in molecular mobility for various constituent polysaccharides related with the two ripening stages. Increase of the proton dipolar decoupling power levels from 3 to 50–55 kHz during single pulse ¹³C acquisition, led to more intense signals for pectic and hemicellulosic polysaccharides. This behaviour was interpreted as reflecting motional restrictions of these polysaccharides inside the porous cell wall network. Measurements of the proton rotating frame relaxation times T_1ρ in the ‘liquid-like’ conditions and of the proton transverse relaxation times T₂ from CPMAS spectra, revealed changes in mobilities for some pectic polysaccharides in relation with ripening, particularly for the H1 and H5 protons of -1,5 arabinan (Ara) side chains of rhamnogalacturonans. These data are discussed in relation with known pectic modifications occurring during ripening and associated with the tomato fruit softening.