A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium

A lambda-carrageenan-degrading Pseudoalteromonas bacterium, strain CL19, was isolated from a deep-sea sediment sample. A lambda-carrageenase from the isolate was purified to homogeneity from cultures containing lambda-carrageenan as a carbon source. This is the first report of the isolation of lambda-carrageenase together with the gene sequence for the enzyme. The molecular mass of the purified enzyme was approximately 100 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that the enzyme is a monomer. The optimal pH and temperature for activity were about 7 and 35 degrees C, respectively. The enzyme had specific activity of 253 U/mg protein. The enzyme required monovalent salts for the activity. Carbohydrates, such as sorbitol, sucrose, trehalose, improved the enzyme stability. The pattern of lambda-carrageenan hydrolysis showed that the enzyme is an endo-type lambda-carrageenase, and the final main product was a tetrasaccharide of the lambda-carrageenan ideal structure with galactose 2,6-disulfate at the reducing end, indicating the enzyme cleaves the beta-1,4 linkages of its backbone structure. Furthermore, the gene (cglA) encoding the enzyme was sequenced. It encoded a mature protein of 103 kDa (917 amino acids). Remarkably, the deduced amino acid sequence showed no similarity to any reported proteins.     

Complete 1H and 13C NMR assignment of digeneaside, a low-molecular-mass carbohydrate produced by red seaweeds

Digeneaside (alpha-D-mannopyranosyl-(1-->2)-D-glycerate) was extracted from the red algae, Bostrychia binderii, and purified by adsorption and gel-filtration chromatography. HPLC and ESI-MS techniques were used to follow purification steps and characterize digeneaside. NMR spectroscopy experiments (1D 1H, 13C, DEPT and 2D HMQC, COSY and TOCSY) were used to fully assign the 1H and 13C spectra.     

Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy

High resolution NMR has been applied for assessment of lipid classes and acyl stereospecific positions of fatty acids in marine phospholipids and triacylglycerols. 1D and 2D NMR techniques in combination with recording of a number of reference standards have been used to interpret the (1)H and (13)C NMR spectra of fish gonads. (13)C NMR spectra gave information regarding the polyunsaturated fatty acids (PUFAs) in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The carbonyl resonances showed that n-3 PUFAs primarily were esterified in the sn-2 position of PC and PE. The glycerol resonances showed that the PC/PE ratio was higher in roe than in milt and that roe comprised more triacylglycerols than milt. Thin layer chromatography showed that milt contained 2.4 times more cholesterol than roe, which was also found by integrating the (1)H NMR spectra. Concentration (mol%) of n-3 fatty acids were calculated from the (1)H NMR data and showed 44.8 and 36.3% in roe and milt, respectively.     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).     

Complete 1H and 13C spectral assignment of floridoside

Floridoside (2-O-alpha-D-galactopyranosylglycerol) was extracted from the red marine alga Rhodymenia palmata, and purified by ion-exchange chromatography: 1D and 2D NMR spectroscopy experiments were used to unambiguously assign the complete 1H and 13C spectra.     

The structure of an iridoid glycoside, 8-deoxyshanzhiside, from Lamiophlomis rotata

8-deoxyshanzhiside was extracted from Lamiophlomis rotata (Benth.) Kudo. Extensive NMR spectroscopy techniques were used to fully assign the (1)H and (13)C spectra. X-ray investigation was used to identify its conformation, and absolute configuration.     

Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [(13)C,(1)H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.     

Synthesis and anticancer activity of novel chiral D-glucose derived bis-imidazoles and their analogs.

A series of novel D-glucose derived bis-imidazoles and their analogs, which possess potential in bioorganic and supramolecular chemistry, were designed and synthesized from methyl alpha-D-glucoside through protection, bis-bromination, N-alkylation and deprotection. All new compounds were characterized by HRMS, 1H, 13C and DEPT NMR spectroscopy as well as elemental analysis. The 1H-(1)H and 1H-(13)C 2D NMR spectra for some compounds were also recorded. Some regular features of 13C and 1H NMR spectra were summarized. The anticancer activity of some compounds was evaluated.     

Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in vitro

Pseudomonas fluorescens GcM5-1A, isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, was cultured in Luria Broth medium (LB). The clarified culture was extracted with ethyl acetate, and two dipeptides were purified from the extract. The chemical structures of 1 and 2 were identified as cyclo(-Pro-Val-)and cyclo(-Pro-Tyr-), respectively, by MS, ¹H NMR, ¹³C NMR,¹H-¹H COSY, 1H -¹³C COSY spectra. Bioassay results showed that the two compounds were toxic to both suspension cells and seedlings of Pinus thunbergii, which may offer some clues to research the mechanism of pine wilt disease caused by PWN.     

Minor betalains in fruits of Hylocereus species

Betacyanins in peel and flesh of fruits of different Hylocereus species were identified by means of GC/MS, electrospray MS/MS, HPLC as well as (1)H and (13)C NMR techniques. As hitherto unknown pigments: betanidin 5-O-(2'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside, betanidin 5-O-(4'-O-malonyl)-beta-D-glucopyranoside and betanidin 5-O-[(5'-O-E-sinapoyl)-2'-O-beta-D-apiofuranosyl]-beta-D-glucopyranoside were elucidated. The sinapoyl moiety attachment position in the structure of betacyanins was established for the first time. The peel contained a more complex pattern of betacyanins with apiofuranosyl moiety. Other recently identified pigments were also present in the samples and their (1)H or (13)C NMR spectra were recorded. In the case of phyllocactin and its 4'-isomer the migration of the malonyl group was noticed.     

NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain PCC 6308

1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy has been used to characterize cyanophycin, a multi-l-arginyl-poly-[l-aspartic acid] polypeptide from the cyanobacterium Synechocystis sp. strain PCC 6308. 1H, 13C and 15N chemical shifts and 1JHN and 1JCN coupling constants were measured in isolated 15N-labeled cyanophycin, and showed chemical shift values and J-couplings consistent with the reported polypeptide structure. 15N enrichment levels were determined from the extent of 1H-15N J-coupling in 1H NMR spectra of cyanophycin. Similar experiments using 13C-15N coupling in 13C NMR spectra were not useful in determining enrichment levels.     

Separation of floridoside and isofloridosides by HPLC and complete (1)H and (13)C NMR spectral assignments for D-isofloridoside

Isofloridosides (1-O-alpha-D-galactopyranosylglycerol) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga Porphyra umbilicalis (Linné) Kützing (Bangiales, Rhodophyta). Their separation was achieved by HPLC (NH(2) P50 column) after successive purification of the crude extract by ion-exchange chromatography and HPLC (Sugar-Pak TM1 column). 1D and 2D NMR spectroscopy experiments allowed to completely assign the (1)H and (13)C spectra of D-isofloridoside.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

Glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose: synthesis and characterization of glycosylamines, and the crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine

A total of nine glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose were synthesized using primary amines having various groups in their ortho- or para-positions. Among these, six are monoglycosylamines, including one primary glycosylamine, and three are bis-glycosylamines. All these compounds were characterized by 1H, 1H-1H COSY, 1H-13C COSY and 13C NMR spectroscopy and FTIR spectra. The FAB mass spectra provided the molecular weights of the products by exhibiting the corresponding molecular ion peaks. The crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine revealed the C-1 glycosylation, the beta-anomeric nature, and the 4C1 chair conformation of the saccharide unit in the product. In the lattice two types of dimers exist. While one type of dimer is formed through O-H...O type of interactions, the other type is formed via C-H...O type of interactions. In the direction of these C-H...O type of interactions, the dimeric units are connected to form a chain.     

A highly regular fraction of a fucoidan from the brown seaweed Fucus distichus L

A fucoidan fraction consisting of L-fucose, sulfate, and acetate in a molar proportion of 1:1.21:0.08 was isolated from the brown seaweed Fucus distichus collected from the Barents Sea. The 13C NMR spectrum of the fraction was typical of regular polysaccharides containing disaccharide repeating units. According to 1D and 2D 1H and 13C NMR spectra, the fucoidan molecules are built up of alternating 3-linked alpha-L-fucopyranose 2,4-disulfate and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp-(2,4-di-SO3-)-(1-->4)-alpha-L-Fucp-(2SO3-)-(1-->. The regular structure may be only slightly masked by random acetylation and undersulfation of several disaccharide repeating units.     

Two-dimensional 13C-1H heteronuclear correlation NMR spectroscopic studies for the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with a new Helicobacter pylori eradicating agent (TG44) in the amorphous state

The interaction of a newly developed Helicobacter pylori eradicating agent (TG44, 4-methylbenzyl-4'-[trans-4-(guanidinomethyl)cyclohexylcarbonyloxy]-biphenyl-4-carboxlylate monohydrochloride) with cyclomaltoheptaose (beta-cyclodextrin, beta-CyD) in the solid state was studied by high-speed frequency-switched Lee-Goldburg (FSLG) (13)C-(1)H heteronuclear correlation (HETCOR) NMR experiments. The TG44/beta-CyD solid complex in a 1:1 stoichiometry was prepared by the grinding method. Powder X-ray diffractometry confirmed that the complex is in an amorphous state. The solid-state (13)C signals of TG44 and beta-CyD were significantly broadened by the complexation. As the temperature increased, the (13)C signals of the aromatic moieties of TG44 were insignificantly influenced, whereas those of the cyclohexyl moiety became sharper. The T1(rho) H values of the aromatic moieties of TG44 were almost the same as those of the beta-CyD carbons, whereas those of other TG44 carbons gave much smaller values. The (13)C-(1)H HETCOR spectra gave the intermolecular correlation peaks between the aromatic carbons of TG44 and the beta-CyD protons or between the biphenyl protons of TG44 and the beta-CyD carbons, when measured using longer contact times (500 and 1500mus). On the basis of these solid NMR spectroscopic data together with aqueous NMR data, we assume that beta-CyD includes predominantly the biphenyl moiety of TG44 in the solid state. (13)C-(1)H HETCOR spectroscopy is particularly useful for the determination of inclusion modes of the complexes that occurring in an amorphous form.     

An antibacterial hydroxy fusidic acid analogue from Acremonium crotocinigenum

A fusidane triterpene, 16-deacetoxy-7-beta-hydroxy-fusidic acid (1), was isolated from a fermentation of the mitosporic fungus Acremonium crotocinigenum. Full unambiguous assignment of all (1)H and (13)C data of 1 was carried out by extensive one- and two-dimensional NMR studies employing HMQC and HMBC spectra. Compound 1 was tested against a panel of multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA) strains and showed minimum inhibitory concentration values of 16 microg/ml.     

Diastereomeric stilbene glucoside dimers from the bark of Norway spruce (Picea abies)

As part of a long-term study of the chemical defenses of Norway spruce (Picea abies) against herbivores and pathogens, a phytochemical survey of the phenolics in the bark was carried out. Eight stilbene glucoside dimers, designated as piceasides A-H (1a-4b), were isolated as four 1:1 mixtures of inseparable diastereomers. Their structures were determined by extensive spectroscopic means including 1D (1H and 13C) and 2D NMR (1H-1H COSY, HSQC, HMBC, ROESY) spectra, and were supported by enzymatic hydrolysis and computational analysis.     

Structure elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 62D1.

The O-antigen polysaccharide of the lipopolysaccharide from the enteroaggregative Escherichia coli strain 62D1 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy revealed the components of the repeating unit. Two-dimensional NOESY and heteronuclear multiple-bond correlation experiments were used to deduce the sequence. 1H and 13C NMR spectra indicate heterogeneity in the polysaccharide. Methylation analysis and 1H NMR spectra of native and Smith-degraded material show that the majority (65%) of the repeating units has the following structure: Minor resonances in the NMR spectra are consistent with the presence of repeating units which lack the alpha-d-Galp terminal residue (35%).