Anomeric Proton Research Articles

Benzyl Glycosides from the Aerial Parts of Gynostemma laxum and Their NF-κB Inhibitory Activity in HepG2 Cells

Nuclear transcription factor-κB (NF-κB) represents a family of Rel domain-containing proteins including RelA, RelB, c-Rel, NF-κB1, and NF-κB2. The activation of NF-κB has been linked to multiple pathophysiological conditions such as cancer, arthritis, asthma, inflammatory bowel disease, and other inflammatory conditions. NF-κB can be activated by various stimuli, such as microbial and viral products, cytokines, DNA damage and noxious chemicals. The induction of a number of pro-inflammatory mediators occurs as the results of increased inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activities. Therefore, suppression of iNOS and COX-2 activities can be an important approach to preventing inflammation in organs. NF-κB and the signaling pathways that regulate many physiological processes, including the innateand adaptiveimmune responses, cell death, and inflammation have become a focal point for intense drug discovery and development efforts. Indeed, growing evidences has validated NF-κB as a target for anti-inflammatory and anticancer agents. Gynostemma genus (Cucurbitaceae) is a climbing vine, attaching itself to supports using tendrils. Over 30 species of Gynostemma are known to grow throughout China, mostly in the South-west. In Vietnam, there are only two species of the Gynostemma genus recorded to date, Gynostemma pentaphyllum (Thunb.) Makino and Gynostemma laxum (Wall.) Cogn. Of these, G. laxum is distributed throughout Vietnam from the plains to mountainous areas. The aerial parts of G. laxum have been used as a bitter stomachic, a laxative, anti-inflammatory as well as anti-obesity. Up to date, no report has been reported about chemical components from this plant. In our screening project for antiinflammatory agents from natural sources, we found G. laxum to possess anti-inflammatory effects. As a part of our continuing research to elucidate the anti-inflammatory compounds, we report herein the isolation, structural elucidation, and evaluation of the inhibitory effects on NFκB activation and iNOS and COX-2 expression of a new benzyl glycoside, 2,4-dihydroxybenzyl-O-α-L-rhamnopyranoside (1), along with nine known compounds from G. laxum. The dried aerial parts of G. laxum were extracted with MeOH and partitioned with n-hexane and ethyl acetate, and water. From these fractions and subsequently separation, one new and nine known compounds were isolated (see Figure 1). Compound 1 was obtained as a white amorphous powder and its molecular formula, C13H18O7, determined on the basis of ESI MS at m/z 287 [M+H] (positive) and HR ESI MS at m/z 287.1147 [M+H] (calcd C13H19O7 for 287.1131). The H-NMR spectrum of 1 (DMSO-d6) (Table 1) showed the following signals: one secondary methyl group at δH 1.03 (d, J = 6.0 Hz), assigned to H-6 of the rhamnose; one anomeric proton at δH 4.72 (br s); one oxygenated methylene (δH 4.42 and 4.50); and one 1,2,4-trisubstituted aromatic ring with ABX coupling patterns [δH 7.20 (d, J = 8.0 Hz), 7.25 (d, J = 8.0 Hz), and 7.96 (s)]. The C-NMR and DEPT spectra (Table 1) revealed 13 carbon signals, of which, 7 signals were assigned to a benzyl moiety and 6 signals belonged to rhamnose moiety. The Hand C-NMR data of 1 (Table 1) were similar to those of benzyl-O-α-L-rhamnopyranoside except for an appearance of two hydroxyl groups at aromatic ring. In the HMBC spectrum (see Figure 2), two proton signals at δH 4.45 and 4.50 of oxygenated methylene group correlated with carbons C-1 (δC 126.3), C-2 (δC 148.1), and C-6 (δC 126.4); one proton signal at δH 7.25 (H-6) correlated with carbons C-2 (δC 148.1), C-4 (δC 154.0), and C-7 (δC

Biosynthesis and Identification of an N-Oxide/N-Glucuronide Metabolite and First Synthesis of an N-O-Glucuronide Metabolite of Lu AA21004

This article describes the biosynthesis and identification of a new class of metabolites, a piperazine N-oxide/N-glucuronide metabolite 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-1-β-D-glucuronic acid-piperazine 1-oxide (4). The metabolite was found in urine and plasma from humans and animals dosed with 1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide (Lu AA21004, 1), as a novel multimodal antidepressant under development for treatment of depression. Human liver microsomes in combination with uridine 5'-diphosphoglucuronic acid were used as an in vitro system to generate enough material of 4 to perform one- and two-dimensional (1)H and (13)C NMR experiments for structure elucidation. Based on rotating frame Overhauser enhancement spectroscopy NMR experiments, the distance correlation between a piperazine proton and the anomeric proton of the glucuronic acid moiety is of a magnitude similar to that of the H-3' and H-5' protons and can only be explained by proximity in space and the postulated structure (4). The structural analog, the N-O-glucuronic acid conjugate 6-{4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-yloxy}-1-β-D-glucuronic acid (3) was also observed in biological samples from humans and animals and the first organic synthesis and structural identification of this metabolite is also reported. Treatment of the glucuronide metabolites 3 and 4 with β-glucuronidase gave mainly the expected hydrolysis product, the hydroxyl amine 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-ol (2).

1,N2-Etheno-2′-deoxyguanosine Adopts the syn Conformation about the Glycosyl Bond When Mismatched with Deoxyadenosine

The oligodeoxynucleotide 5′-CGCATXGAATCC-3′·5′-GGATTCAATGCG-3′ containing 1,N2-etheno-2′-deoxyguanosine (1,N2-εdG) opposite deoxyadenosine (named the 1,N2-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,N2-εdG by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and by Escherichia coli polymerases pol I exo– and pol II exo–. At pH 5.2, the Tm of this duplex was increased by 3 °C as compared to the duplex in which the 1,N2-εdG lesion is opposite dC, and it was increased by 2 °C compared to the duplex in which guanine is opposite dA (the dG·dA duplex). A strong NOE between the 1,N2-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A20 H2 proton and the mismatched A19 H2 proton from the complementary strand, establish that 1,N2-εdG rotated about the glycosyl bond from the anti to the syn conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T5 CH3. A strong NOE between A20 H2 and A19 H2 protons established that A19, opposite to 1,N2-εdG, adopted the anti conformation and was directed toward the helix. The downfield shifts of the A19 amino protons suggested protonation of dA. Thus, the protonated 1,N2-εdG·dA base pair was stabilized by hydrogen bonds between 1,N2-εdG N1 and A19 N1H+ and between 1,N2-εdG O9 and A19N6H. The broad imino proton resonances for the 5′- and 3′-flanking bases suggested that both neighboring base pairs were perturbed. The increased stability of the 1,N2-εdG·dA base pair, compared to that of the 1,N2-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,N2-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,N2-εdG.

Structural Studies of the Capsular Polysaccharide Produced by Leuconostoc mesenteroides ssp. cremoris PIA2

The structure of the capsular polysaccharide (CPS) produced by Leuconostoc mesenteroides ssp. cremoris PIA2 has been determined using component analysis and NMR spectroscopy. (1)H and (13)C resonances were assigned using 2D NMR experiments, and sequential information was obtained by (1)H,(1)H-NOESY and (1)H,(13)C-HMBC experiments. The CPS consists of linear pentasaccharide repeating units with the following structure: →3)-β-D-Galf-(1→6)-β-D-Galf-(1→2)-β-D-Galf-(1→6)-β-D-Galf-(1→3)-β-D-Galp-(1→, in which four out of the five sugar residues have the furanoid ring form, a structural entity found in bacteria but not in mammals. The analysis of the magnitude of the homonuclear three-bond coupling constants of the anomeric protons for the five-membered sugar rings indicates that the sugar residues substituted at a primary carbon atom show one kind of conformational preferences, whereas those substituted at a secondary carbon atom show another kind of conformational preferences.

Flavonoids from Tanacetum vulgare flowers

Flowers of tansy (Tanacetum vulgare L.) are used in various medicinal forms as antihelminthic and cholegogic agents [1–5]. The pharmacological properties of tansy flower preparations are due mainly to the essential oil (thujone and other terpenoids) and flavonoids. According to various literature sources [3–5], apigenin, acacetin, luteolin, cinaroside, eupatilin, jaceidin, and jaceoside are the main flavonoids in tansy flowers. However, reports in the domestic and foreign literature are somewhat contradictory. The goal of the present work was to study the flavonoid composition of tansy flowers growing in Samara Oblast (Russia). We studied tansy flowers collected near Nizhnee Sancheleevo village, Samara Oblast (July, 2008). Flowers (150 g) were extracted exhaustively with EtOH (70%) by combining maceration (24 h) with subsequent thermal extraction at 85–90°C. The aqueous alcohol extracts were evaporated in vacuo to a thick residue (~50 mL). The condensed extract was dried over L 40/100 silica gel. The resulting powder (extract + silica gel) was placed on a layer of silica gel that was formed in CHCl3 and eluted by CHCl3 and CHCl3:EtOH in various ratios (97:3, 95:5, 93:7, 90:10, 88:12, 85:15, 80:20, 70:30). The separation of the compounds was monitored by TLC on Silufol UV 254 and Sorbfil PTSKh-AF-A-UV plates using CHCl3:EtOH (4:1) and CHCl3:MeOH:H2O (26:14:3). Fractions containing the dominant compound 1 were combined. The precipitate that formed in these was separated and recrystallized from EtOH to afford 1 in 0.2% yield of the air-dried raw material mass. Fractions containing compounds 2–4 (each separately) were transferred to a chromatographic column (sorbent height 4.0 cm; diameter, 5 cm). The respective chromatographic column was eluted by H2O and aqueous EtOH (20%, 40, 70, and 96). Purification over polyamide columns afforded 2 (96% EtOH eluent), 3 (40% EtOH eluent), and 4 (70% EtOH eluent). The compounds were purified additionally by recrystallization from aqueous EtOH. The chemical structures of flavonoids 1–4 were elucidated using PMR and UV spectroscopy and mass spectrometry in addition to chemical transformations. Flavonoids 1 and 3 were cleaved by acid hydrolysis (10% HCl, 100°C, 2 h) and -glucosidase (Fluka, Hungary) into glucose and the aglycons, which were identified by TLC as acacetin (5,7-dihydroxy-4 methoxyflavone) (2) and apigenin (5,7,4 -trihydroxyflavone) (4), respectively. Flavonoids 2 and 4 were also isolated from tansy raw material in the free state. The PMR spectrum of 1 showed two 2H doublets at 8.07 and 7.14 ppm with SSCC (J) 9 Hz that were assigned to H-2 ,6 and H-3 ,5 , respectively; two 1H doublets at 6.84 and 6.45 ppm with J = 2.5 Hz that were characteristic of ring A protons (H-8 and H-6), and a singlet for H-3 at 6.95 (flavone compound). A 3H singlet at 3.87 for the C-4 OCH3 group and a singlet for the 5-OH of the flavonoid were observed in the PMR spectrum. The carbohydrate was placed on the 7-OH group on the basis of these results and those from enzymatic hydrolysis, and UV spectroscopy (lack of a bathochromic shift of the short-wavelength absorption band in the presence of NaOAc) [6]. The glucose was bonded as -D-glucopyranosyl (characteristic doublet for the anomeric proton at 5.35 ppm with J = 7.2 Hz). The chemical studies and spectra data suggested that 1 had the structure 5,7-dihydroxy-4 -methoxyflavone 7-O-Dglucopyranoside (tilianin) [7]. The chemical structure of 3 was studied analogously. Based on UV, NMR, and mass spectral data in addition to results from chemical transformations it was identified as 5,7,4 -trihydroxyflavone 7-O-D-glucopyranoside (cosmosiin).

Negative Ion CID Fragmentation of O-linked Oligosaccharide Aldoses—Charge Induced and Charge Remote Fragmentation

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M - H](-) ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M - H](-) ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M - 2H](2-) ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the (0,2)A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable α/β pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form.

Getting to know the nitrogen next door: HNMBC measurements of amino sugars

Long-range 1H– 15N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the 1 J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. 15N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6- O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the 15N chemical shifts of the α anomer are downfield of the β form. For the N-acetylated sugars, the β anomer has a unique long-range 15N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the 15N chemical shift of the glucosamine analogs, 6- O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D 2O solution, peaks in the 15N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to 2H– 15N coupling.

1H NMR signal at 2.10 ppm in the spectrum of KMnO4-bleached heparin sodium: identification of the chemical origin using an NMR-only approach

The recently revised European Pharmacopeia and US Pharmacopeia heparin sodium monographs include nuclear magnetic resonance (NMR) tests on both identity and purity. In KMnO(4)-bleached heparin, an unidentified NMR signal is present at 2.10ppm at a level of 15-20% of the mean of signal height of the major glucosamine (GlcNAc/GlcNS,6S) anomeric proton signal at 5.42ppm and of the major iduronic acid (IdoA2S) anomeric proton signal at 5.21ppm. According to the new monographs, no unidentified signals greater than 4% should be detected at that position. Thus, the material did not meet the acceptance criterion. The signal at 2.10ppm has been present at the same level in all released MSD KMnO(4)-bleached heparin sodium batches analyzed over the past 10years. The signal is a result of the KMnO(4) bleaching. No (oversulfated) chondroitin sulfate or dermatan sulfate was detected in this material. A comprehensive NMR study using long-range heteronuclear 2D techniques identifies this signal at 2.10ppm as originating from the acetyl methyl group of (6-sulfated) 2-N-acetyl-2-deoxy-glucono-1,5-lactone. This modified monosaccharide is formed by the KMnO(4) oxidation of the reducing end of a terminal N-acetylglucosamine.

Determination of the primary structure and carboxyl pK As of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional 1H NMR

Determination of the structure of heparin-derived oligosaccharides by 1H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H1 protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional 1H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]3-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆4,5-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift–pD titration curves. Chemical shift–pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pK As of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pK As of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4224-4) contains supplementary material, which is available to authorized users.

Study of octenyl succinic anhydride-modified waxy maize starch by nuclear magnetic resonance spectroscopy

Granular waxy maize starch was reacted with two levels (3 and 15%, based on the weight of starch) of octenyl succinic anhydride (OSA). Structure of the OSA and modified starches was studied by one-dimensional (1D) 1H and 13C and two-dimensional (2D) homonuclear correlation and heteronuclear correlation nuclear magnetic resonance (NMR) spectroscopy. The modified starches were converted to α-limit dextrins prior to NMR analysis. By applying the 1D and 2D NMR techniques, complete assignments of 1H and 13C NMR spectra of the OSA reagent were achieved, and the position of the double bond and ratio of trans to cis isomers were determined. As level of OSA substitution increased, the peak (≈5.38 ppm) for the anomeric proton of internal α-1,4- d-glucosyl units became broader in 1H NMR spectra, suggesting that substitution occurred at the O-2 position. Compared with the 13C NMR spectrum of the native starch, the modified starches gave additional signals at the C-4 peak region and broadening of the C-1, C-2, C-3, and C-4 resonances, but not of the C-6 signal. Those results further suggest that the OS groups were substituted at the O-2 and O-3 positions, but not at the O-6 position.

An alternative scheme for the multiplexed acquisition of 1D and 2D NMR spectra

Spin system selective 1D 1H, 2D DQF-COSY and 2D HSQC NMR spectra were recorded in order to fully assign the 1H and 13C 1D NMR spectra of an asymmetrical β-cyclodextrin derivative. Instead of individually accessing the seven sugar anomeric protons by means of long multiplet selective pulses, only short region selective pulses were used. The simultaneously selected anomeric protons were differentiated by allowing their magnetization to evolve under the sole effect of the chemical shift interaction. In each experiment, the seven recorded spectra were linear combinations of the seven desired ones. The combination coefficients were measured and used to obtain almost perfectly separated sugar unit sub-spectra. This multiplexed acquisition scheme resulted in a time gain factor of about 2.

ChemInform Abstract: A New Sulfated β-Galactan from Clams with Anti-HIV Activity.

A new polysaccharide composed of galactan sulfate with a β-(1→3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing d-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a β-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 μg/mL.

IDENTIFICATION AND SEQUENCING BY NMR SPECTROSCOPY OF THE CARBOHYDRATE MOIETY IN A SAPONIN FROM Barringtonia asiatica

A trisaccharide chain in a saponin from the seeds of Barringtonia asiatica has been identified and sequenced as {[b-D-galactopyranosyl(1®3)- b-D-glucopyranosyl(1®2)]-b-D-glucuronopyranosyloxy} using a combination of homonuclear and heteronuclear correlation NMR spectroscopy. The 1H and 13C NMR signals of the sugar residues can be determined and distinguished from one other by use of the HMQC-TOCSY technique. Anomeric configurations were unambiguously assigned from the vicinal coupling constants 3JH-1,H-2 of the anomeric protons. Inter-glycosidic linkage assignments were elucidated using HMBC. Keyword: Barringtonia asiatica, carbohydrate, saponin, NMR

Unique Extracellular Matrix Heparan Sulfate from the Bivalve Nodipecten nodosus (Linnaeus, 1758) Safely Inhibits Arterial Thrombosis after Photochemically Induced Endothelial Lesion

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.

Magnetic resonance spectroscopy of the occipital cortex and the cerebellar vermis distinguishes individual cats affected with alpha-mannosidosis from normal cats

A genetic deficiency of lysosomal alpha-mannosidase causes the lysosomal storage disease alpha-mannosidosis (AMD), in which oligosaccharide accumulation occurs in neurons and glia. The purpose of this study was to evaluate the role of magnetic resonance spectroscopy (MRS) in detecting the oligosaccharide accumulation in AMD. Five cats with AMD and eight age-matched normal cats underwent in vivo MRS studies with a single voxel short echo time (20 ms) STEAM spectroscopy sequence on a 4.7T magnet. Two voxels were studied in each cat, from the cerebellar vermis and the occipital cortex. Metabolites of brain samples from these regions were extracted with perchloric acid and analyzed by high resolution NMR spectroscopy. A significantly elevated unresolved resonance signal between 3.4 and 4. ppm was observed in the cerebellar vermis and occipital cortex of all AMD cats, which was absent in normal cats. This resonance was shown to be from carbohydrate moieties by high resolution NMR of tissue extracts. Resonances from the Glc-NAc group (1.8-2.2 ppm) along with anomeric proton signals (4.6-5.4 ppm) from undigested oligosaccharides were also observed in the extract spectra from AMD cats. This MRS spectral pattern may be a useful biomarker for AMD diagnosis as well as for assessing responses to therapy.

New insights into the structure of hybrid κ-/μ-carrageenan and its alkaline conversion

Hybrid κ-/μ-carrageenan, water-extracted from Kappaphycus alvarezzi, was incubated with Pseudoalteromonas carrageenovora κ-carrageenase. The five most abundant hybrid κ-/μ-carrageenan oligosaccharides were purified and their structure was determined by NMR and mass spectrometry. Since the enzyme-resistant fraction was small, these five oligosaccharides were representative of the distribution of the μ-carrabiose moieties along the carrageenan chain. Three main distributions of μ-carrabiose were found: -μ-, -μ-μ- and -μ-μ-μ-. Interestingly, the chemical shifts of the anomeric proton and carbon were strongly influenced by the identity of the adjacent carrabiose, which explains the asymmetrical D6S-H1 NMR signal that we observed. Although μ-carrabiose chemical shifts are usually attributed to blocks of μ-carrabiose as expected in ideal μ-carrageenan, we demonstrate that they are in fact due to μ-carrabiose units positioned between κ-carrabiose units. Using NMR and chromatography to monitor the alkaline conversion of μ- to κ-carrabiose, we found that the μ-carrabiose distribution (-μ-, -μ-μ-, -μ-μ-μ-) did not influence the conversion rate.

Chemical shift calibration of 1H MAS NMR liver tissue spectra exemplified using a study of glycine protection of galactosamine toxicity

High-resolution (1)H magic angle spinning (MAS) NMR spectroscopy is a useful tool for analysing intact tissues as a component of metabonomic studies. The effect of referencing MAS NMR spectra to the chemical shifts of glucose or to that or trimethylsilylpropionic acid on the resultant multivariate statistical models have been investigated. It is shown that referencing to known chemical shifts of either alpha-glucose or beta-glucose in (1)H MAS NMR-based metabolic data of intact liver tissues is preferred. This has been exemplified in studies of galactosamine toxicity in the rat where co-administration of glycine ameliorates the toxic response. This approach leads to better aligned sets of spectra and reduces the inter-sample variability in multivariate statistical models. If glucose is not present in the tissue under study, then a number of alternative internal reference chemical shifts are presented. Finally, the chemical shift difference between that of the anomeric H1 proton of alpha-glucose and residual water is confirmed as a suitable internal temperature calibration method.

Effect of the frequency of intramolecular motions on the NMR relaxation in liquid state temperature regime

Equations for the spectral densities of complex motion of a spin pair undergoing internal motion and isotropic/anisotropic overall rotation have been considered. The fluctuations of the interproton distances, caused by internal motion, have been taken into account in the theoretical equations. A method allowing a distinction between the isotropic and the anisotropic overall rotation of molecules has been proposed. The effect of the activation parameters of internal motions (known from the solid state study) on the measured T 1 relaxation of the 13C and 1H–1H cross-relaxation rates has been analysed for methyl-β-D-galactopyranoside in DMSO-d6 solution. The conformational trans-gauche jumps of the methylene group are not fast enough to affect the T 1 value of carbon C6 in the liquid state temperatures regime. Only the methyl group rotation is a very fast internal motion. This motion influences the carbon C7 relaxation and methyl protons–anomeric proton cross-relaxation. The values of interatomic distances between anomeric H(C1) and H(C5) as well as the three methyl protons H(C7) have been calculated from the cross-relaxation rates. The distance H(C1)–H(C7) fluctuates due to the rotation of methyl group. The application of the ‘model-free approach’ to study molecular dynamics in solutions is discussed.

Acetylation of α-chitin in ionic liquids

Acetylation of α-chitin using acetic anhydride in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), was performed. First, a mixture of chitin and AMIMBr (2% w/w) was heated at 100 °C for 24 h for dissolution. Then, acetic anhydride (5–20 equiv) was added to the solution and the mixture was heated with stirring at desired temperatures for 24 h. The product was precipitated by the addition of the reaction mixture into methanol. The IR spectrum of the product indicated the progress of acetylation. The degrees of substitution (DS), which were determined from the IR spectra, increased with increasing the amounts of acetic anhydride used for the reaction. The highest DS was 1.86, which was obtained by the reaction using 20 equiv of acetic anhydride at 100 °C. The product with this DS value was soluble in DMSO, and thus the structure of the product was further confirmed by 1H NMR spectroscopy in DMSO- d 6. The DS value estimated by the integrated ratio of signals due to acetyl protons to a signal due to anomeric protons was in good agreement with that determined from the IR spectrum.

Cycloartane glycosides from Euphorbia glareosa

We have previously reported the isolation from various species of Euphorbia growing in Georgia of flavonoids [1] and polyphenols [2]. We also found that all studied species contained in addition to the aforementioned classes of compounds triterpene glycosides of the cycloartane series. Herein we communicate results from a study of the structure of cycloartane glycosides isolated from E. glareosa (Euphorbiaceae). Triterpene glycosides were obtained by exhaustive extraction (2 × 5 L) of ground air-dried roots (0.8 kg) at room temperature. The MeOH extract was evaporated and worked up by the literature method [3] to afford triterpenoids (20.5 g total) that were separated over a column of silica gel (L 40/100, Czech Rep.) using CHCl3:CH3OH (1, 15:1), CHCl3:CH3OH:H2O (2, 70:23:4), C6H6:EtOAc (3, 2:1 and 1:1), and CHCl3:C6H14:EtOAc (4, 1:1:1) [3]. We isolated eight compounds designated in order of increasing polarity as 1–8. Compounds 1 and 2 were the genins cyclosiversigenin and asgenin; the others, their glycosides. Compound 1, cyclosiversigenin, C30H50O5 (cycloastragenol, astramembrangenin [4]), yield 0.036% (here and henceforth of the weight of air-dried raw material), mp 238–240°C, [ ] α D 20 +50.4° (c 2.0, MeOH). IR spectrum (KBr, νmax, cm −1): 3500–3350 (OH), 3040, 1760, 1750, 1260–1250. PMR spectrum (Py-d5, δ, ppm, J/Hz, 0 = HMDS): 4.90 (1H, q, J = 7.0, H-16), 3.78 (1H, br.q, H-24), 3.57 (2H, m, H-3, H-6), 0.51 (d, J = 4.0) and 0.23 (br.s, 2H-19); CH3 groups 1.76, 1.46, 1.35, 1.25, 1.21, 1.18, 0.92 [3]. Compound 2, cycloasgenin, C30H48O6, yield 0.011%, mp 234–235°C, [ ] α D 20 +130.4° (c 0.8, MeHO). IR spectrum (KBr, νmax, cm −1): 3450–3350 (OH), 3060 (cyclopropane CH2), 1706–1695 (C=O). PMR spectrum (Py-d5, δ, ppm, J/Hz, 0 = HMDS): 4.92 (1H, q, J = 7.3, H-16), 4.20 (1H, dd, J = 9.8, 2.5, H-11), 3.72 (1H, m, H-6), 1.64 (d, J = 4.0) and 0.46 (br.s, 2H-19), 3.75 (1H, dd, J = 8.8, 5.6, H-24), CH3 groups 1.70, 1.44, 1.41, 1.39. 1.21, 1.16, 0.84 [3]. A study of the acid hydrolysis products of the glycosides [5] showed that they all contained cyclosiversigenin as the genin. The structures of the carbohydrate parts of the glycosides were established using chemical transformations (Hakomori methylation [6] with subsequent methanolysis and GC of the sugars) and IR and PMR spectral data. Compound 3, cyclosiversigenin 3,6-O-β-D-dixylopyranoside, C40H66O13, 0.008%, mp 218–221°C (MeOH), [ ] α D 20 +29.0° (c 0.71, MeOH). IR spectrum (KBr, νmax, cm −1): 3200-3600 (OH), 3040–3060 (cyclopropane CH2). PMR spectrum (Py-d5, δ, ppm, J/Hz): 4.56 and 4.42 (d, J = 7.2, H′ and H′′), 1.70, 1.44, 1.25, 0.95 (eath 3H, s, CH3), 1.16 (9H, s, CH3 × 3), 0.46 (1H, br.s, Ha-19) [7, 8]. Compound 4, cyclosiversigenin 3-O-[β-D-xylopyranoside-(2′-O-acetyl)]-6-O-β-D-xylopyranoside, C42H68O14, 0.03%, mp 253–254°C (MeOH), [ ] α D 20 +31° (c 0.90, MeOH). IR spectrum (KBr, νmax, cm −1): 3360–3500 (OH), 1750, 1260 (ester). PMR spectrum (Py-d5, δ, ppm, J/Hz): 5.32 (1H, br.t, H-2′), 4.68 (1H, d, J = 6.2) and 4.55 (1H, d, J = 7.5); anomeric protons of two xyloses: 1.90 (3H, s, CH3CO), 1.52, 1.41, 1.26, 1.18, 1.15, 1.07, 0.95, (each 3H, s, CH3), 0.45 (1H, d, J = 4.0, Ha-19) [8, 9]. Compound 5, cyclosiversigenin 3-O-β-D-xylopyranoside-6-O-β-D-glucopyranoside, C41H68O14, 0.009%, mp 247– 249°C (MeOH), [ ] α D 20 +37.0° (c 0.5, MeOH). IR spectrum (KBr, νmax, cm −1): 3300–3500 (OH), 3040 (cyclopropane CH2). PMR spectrum (Py-d5, δ, ppm, J/Hz): 4.78 (1H, d, J = 7.8, H-1′′), 4.52 (1H, d, J = 7.4, H-1′), 1.82, 1.40, 1.26, 0.80, (each 3H, s, CH3), 1.16 (9H, s, CH3), 0.44 (1H, d, J = 3.9, Ha-19) [10].