Biosynthesis of C-labeled branched polysaccharides by pestalotiopsis from C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-¹³C)glucose, D-(2-¹³C)glucose, D-(4-¹³C)glucose, D-(6-¹³C)glucose or a mixture of D-(1-¹³C)glucose and D-(2-¹³C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. ¹³C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of ¹³C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-¹³C)glucose was low, compared to that of cellulose and curdlan.     

Using simple 13C NMR linewidth and relaxation measurements to make detailed chemical shift assignments in triacylglycerols and related compounds

Two simple experiments measuring the ¹³C linewidths ν_1/2 and spin–lattice relaxation times T₁ of each of the signals in the spectrum of trilinolein indicate that the ν_1/2 and T₁ values are consistent with the different degrees of motional freedom expected for the various ¹³C nuclei. However, for each chain, the ν_1/2 and T₁ measurements indicate a small reversal in mobility at C-10 relative to C-9 before motional freedom again steadily increases on each chain starting at C-11. The T₁ experiment allows unambiguous assignments of the C-8 signal and C-14 signal, which differ by only 0.010 ppm. Measurements of ¹³C ν_1/2 and T₁ values on tripalmitin provide secure assignments for the C-5 and C-6 signals, for which conflicting assignments have been reported. The T₁ measurements also show that among the tightly clustered C-8 through C-12 signals, the C-11 signals are the most downfield, while the C-12 signals are the most upfield, again contrary to a previous report. Similar measurements of ¹³C ν_1/2 and T₁ values on other triacylglycerols or related compounds may prove equally useful in making chemical shift assignments and detecting any discontinuities in motional freedom along a chain. The benefits and possible limitations of ultrahigh field NMR for studying triacylglycerols and related compounds are discussed.     

Structure of Co2(CO)6(dppm) and Co2(CO)5(CHCO2Et)(dppm) (dppm = Ph2PCH2PPh2) and exchange reaction with CO: An experimental and computational study

Crystal structures of Co₂(CO)₆(dppm) (1) and Co₂(CO)₅(CHCO₂Et)(dppm) (2) (dppm = Ph₂PCH₂PPh₂) show asymmetry with respect to the orientation of the phenyl groups in 1 and owing to the bridging ethoxycarbonylcarbene ligand in 2. The effect of this asymmetry was recognized in the solid-state ³¹P NMR spectra of 1 and 2 and in the solid-state and solution ¹³C NMR spectra of 2 as well, but not in the solid-state and solution ¹³C NMR spectra of 1. In CH₂Cl₂ solution under an atmosphere of ¹³CO, the CO ligands of both complexes exchange with ¹³CO. The overall rate of ¹³CO exchange at 10 °C was found to be k_obs = 0.107 × 10⁻³ s⁻¹ for 1 and k_obs = 0.243 × 10⁻³ s⁻¹ for 2. Two-layered ONIOM(B3LYP/6-31G(d):LSDA/LANL2MB) studies revealed fluxional behavior of 1 with rather small barriers of activation of the rearrangements. Four possible isomers have been computed for 2, close to each other energetically.     

Comparison of the glycosaminoglycans isolated from the skin and head cartilage of Gould's arrow squid (Nototodarus gouldi)

The glycosaminoglycans from the skin and head cartilage of the squid Nototodarus gouldi have been isolated and characterised by constituent disaccharide and neutral sugar analysis, ¹³C nuclear magnetic resonance (NMR) spectroscopy, anion exchange and size exclusion chromatography. The glycosaminoglycans from both tissues are chondroitin sulphate species. The skin consists principally of unsulphated but relatively highly glycosylated material. The chondroitin sulphate from the head cartilage is more highly sulphated, predominantly C-4,6diS (chondroitin sulphate E), with a higher molecular weight than the skin derived material but somewhat less highly glycosylated. To provide a standard for the assignment of the ¹³C NMR spectrum, C-4,6diS was chemically prepared from bovine tracheal chondroitin sulphate. This showed that it is not possible to distinguish between a mixture of the monosulphates, C-4S and C-6S, and the C-4,6diS by one-dimensional and simple two-dimensional ¹³C NMR techniques.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

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.     

Nuclear magnetic resonance spectroscopy of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid multi-conjugates: unusual bile acid metabolites in human urine

Complete ¹H and ¹³C resonance assignments were made for a new type of 3β,7β-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the ¹H and ¹³C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.     

Sesquiterpene polyol esters from Celastrus rosthornianus

Two new sesquiterpene polyol esters with β-dihydroagarofuran skeleton were isolated from the root bark of Celastrus rosthornianus. Their structures were elucidated, mainly on the basis of spectral analyses, as 1 β-acetoxy-8β,9-dibenzoyloxy-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran and 1β-acetoxy-9-benzoyloxy-8β-(β-furanocarbonyloxy)-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran. The complete assignments of ¹³C NMR chemical shifts for both compounds on the basis of ¹H-¹³C chemical-shift correlation spectrum were also carried out.     

Iridoid glucosides from roots of Vietnamese Paederia scandens

Four iridoid glucosides, three of which are dimeric were isolated from the methanol extract of roots of Vietnamese Paederia scandens (Lour) Merrill together with the five known glucosides, paederoside, asperuloside, paederosidic acid, asperulosidic acid and geniposide. Seven sulfur-containing iridoid glucosides were also isolated. The structures of the iridoid glucosides were determined by a combination of high-resolution NMR, MS, IR and UV spectra, and chemical reaction such as acetylation.     

Sn and C NMR study of some trivinyltin(IV) compounds and their complexes

The¹¹⁹Sn and ¹³C NMR spectra of ten trivinyltin(IV) compounds in solutions of non-coordinating (deuteriochloroform, trideuterionitromethane) and coordinating (hexadeuteriodimethyl sulphoxide) solvents have been studied. From δ(¹¹⁹Sn) chemical shifts and ¹J(¹¹⁹Sn,¹³C) coupling constants an evaluation of the coordination number of the central tin atom and the shape of coordination polyhedra around the tin atom has been carried out. Various effects on the δ(¹³C) chemical shifts of both carbon atoms of the vinyl group are also discussed.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Iridoid glucosides from Kickxia abhaica D.A. Sutton from Scrophulariaceae

Two iridoid glucosides namely; 6-acetylantirrinoside (1), 6'-O-p-hydroxybenzoylantirrinoside (2) were isolated from the aerial parts of Kickxia abhaica. Beside that, three known iridoid glucosides, antirrinoside (3), antirride (4) and mussaenosidic acid (5), one flavone glycoside (6) and a hexitol, d-mannitol (7) were isolated. The structures of the iridoid glucosides 1-2 were established by 1D and 2D NMR spectral data, including COSY, HMQC and HMBC experiments, as well as HRMS.     

Preparation of 6-deoxy-6-fluorocellulose

6-Deoxy-6-fluorocellulose was prepared from cellulose 2,3-diacetate (1) or cellulose 2,3-dibenzoate (2) in various solvents, and was characterized by ¹⁹F and ¹³C NMR measurements. The best product, having ds of 0.95 at C-6 and 0.04 at C-3, was prepared from cellulose 2,3-dibenzoate in nitrobenzene. Other combinations of starting material and solvent gave a lower (≈ 0.8) ds of fluorine at C-6 and higher (≈ 0.12) at C-2 or C-3. Substitution at C-2 was observed when the combination of 1 and 1,4-dioxane, or 2 and chloroform was used. The products substituted at C-2 by fluorine were relatively resistant to acid hydrolysis.     

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.     

Biosynthetic studies of lactucin derivatives in hairy root cultures of Lactuca floridana

Biosynthetic studies of the guaianolide-type sesquiterpene lactones 11βH,13-dihydrolactucin-8-O-acetate and 8-desoxylactucin were performed in Agrobacterium rhizogenes—transformed hairy root cultures of blue-flowered lettuce, Lactuca floridana. The ¹³C NMR spectra of the two guaianolides labelled by incorporation of [1-¹³C], [2-¹³C], [1,2-¹³C₂]acetate and [2-¹³C]mevalolactone showed patterns of enrichment consistent with a previously proposed biogenetic pathway for guaianolide-type sesquiterpene lactones via the acetate-mevalonate-germacradiene route.     

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.     

Protonation and NMR studies on C-acetate enriched Ru(OAc)2(Binap). Acetate as a source of water in P---C bond splitting

The P---C bond splitting reaction of Ru(OAc)₂(Binap), containing ¹³C=O-enriched acetate, with 2 equiv. of triflic acid at 80 °C, has been studied. NMR spectroscopy (and specifically ¹³C NMR data) reveal that acetic anhydride and water are produced, thus explaining the end product, which may be thought of as developing due to water adding across the P---C bond. An intermediate 10 derived from attack of acetate on a P-atom is recognised. Complex 10 is shown to contain a cyclic five-membered ring, Ru---{(P---OC(Me)(=O)} fragment which develops via acetate attack on a P-atom. Crystal structures for two Ru(OAc)₂(MeO---Biphep) derivatives are reported.     

Detection of glycogen in a glycogen storage disease by C nuclear magnetic resonance

The livers of gsd/gsd rats homozygous for the glycogen storage disease phosphorylase b kinase deficiency were observed by ¹³C NMR using a surface coil. Clear signals were detected from glycogen. The concentration of glycogen as determined by NMR was 3-times that found in normal strains agreeing well with chemical determinations Starvation did not significantly reduce the glycogen content of the livers with glycogen storage disease whereas it reduced the signal below detectability in normal rats. Difference spectra of starved normal rats from fed gsd/gsd rats gave spectra similar in appearance to that of purified glycogen. Glycogen both in vivo and in vitro is fully visible using ¹³C NMR.     

The C NMR spectroscopy of carrageenans: calculation of chemical shifts and computer-aided structural determination

The set of ¹³C NMR absorptions produced by all the carbons of the diads potentially present in carrageenans is reported. They were obtained by calculation for unreported diads plus the compilation of up-to-date chemical shift data. A computer program was developed in order to aid in the matching of experimental data to the chemical shift data bank reported here.