Chemical structure of the core oligosaccharide of aerotolerant Campylobacter jejuni O:2 lipopolysaccharide

The structure of the core oligosaccharide of aerotolerant Campylobacter jejuni 0:2 lipopolysaccharide was determined and found to contain 3-deoxy-D-manno-octulosonic acid (Kdo), L-glycero-D-manno-heptose (LD-Hep), D-galactose, D-glucose, and phosphorylethanolamine (PEtn). Based on 1H, 13C and 31P NMR spectroscopic studies including 2D COSY, TOCSY, ROESY and heteronuclear 1H-31P and HMQC experiments it was established that the oligosaccharide has the following structure: [structure: see text].     

Structure-activity study of phosphoramido acid esters as acetylcholinesterase inhibitors

Phosphoramido acid esters (CH(3))(2)NP(O)X(p-OC(6)H(4)-CH(3)) (containing P-Cl (1), P-O (2), P-F (3), P-CN (5), and P-N (4,6) bonds, X for 2, 4 and 6 is OCH(3), (C(2)H(5))(2)N and morpholin) have been synthesized to investigate the structure-activity study of AChE enzyme inhibition, through the parameters logP, delta(31)P and IC(50). After their characterization by (31)P, (31)P{(1)H}, (13)C, (1)H NMR, IR and mass spectroscopy, the parameters logP and delta(31)P ((31)P chemical shift in NMR) were used to evaluated the lipophilicity and electronical properties. The ability of compounds to inhibit human AChE was predicted by PASS software (version 1.193), and experimentally evaluated by a modified Ellman's assay.     

Structure of the O-specific polysaccharide of Proteus mirabilis O16 containing ethanolamine phosphate and ribitol phosphate

The O-specific polysaccharide of Proteus mirabilis O16 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, HMQC-TOCSY, and 1H,31P HMQC experiments, along with chemical methods. The polysaccharide was found to be a ribitol teichoic acid-like polymer having the following structure [structure: see text].     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Structure analyses of DNA oligomers by use of heteronuclear 2D NMR.

DNA oligomer d(CGGAAGACTCTCCTCCG):d(CGGAGGAGAGTCTTCCG) named UASG (17mer M.W. = 11 kDa) was studied by 1H NMR and heteronuclear two dimensional (2D) NMR. All the labile protons and half of the non-exchangeable protons were assigned by use of conventional 1H 2D experiments including NOESY using 1-1 echo excitation for water suppression. Signal degeneracy in the sugar proton region made it difficult to make assignments of the remaining half of the non-exchangeable protons of the oligomer in 1H 2D spectra. Here we report a new strategy using 1H/13C and 1H/31P heteronuclear single-quantum correlation spectroscopy combined with homonuclear three dimensional NOESY-TOCSY. By this strategy, most of the proton resonances of the oligomer have been assigned, and it turned out that the whole conformation of the oligomer is B-form like.     

Structure of the O-specific polysaccharide of Proteus penneri 103 containing ribitol and 2-aminoethanol phosphates

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 103 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,(13)C HMQC, 1H, 31P HMQC, and HMBC experiments. It was found that the polysaccharide is built up of oligosaccharide-ribitol phosphate repeating units and thus resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was established:where Etn and Rib-ol are ethanolamine and ribitol, respectively. This structure is unique among the known structures of Proteus O-antigens and, therefore, we propose classification of the strain studied into a new Proteus serogroup, O73. The molecular basis for cross-reactivity between O-antiserum against P. penneri 103 and O-antigens of P. mirabilis O33 and D52 is discussed.     

Tetrahydroisoquinoline alkaloids and 2-deoxyribonolactones from Aristolochia arcuata

2-Deoxyribonolactones and four tetrahydroisoquinoline alkaloids were isolated from the acetone extract of the leaves of Aristolochia arcuata Mast., together with pinitol, sequoyitol, glycerol, fructose, sucrose, eupomatenoid-7, salsolinol, and 6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline. Their structures were determined on the basis of spectroscopic methods, mainly using 1H, 13C, 15N, and 31P NMR.     

1H, 13C, and 31P nuclear magnetic resonance spectral assignments for tobramycin, 2'-(adenosine-5'-phosphoryl)-tobramycin and 2'-(adenosine-5'-thiophosphoryl)-tobramycin

Two enzymatically modified derivatives of tobramycin have been prepared by gentamicin nucleotidyl transferase-catalyzed adenylylation of tobramycin, using ATP and (Sp)-ATP alpha S as adenylylation substrates. (EC 2.7.7.46). The 1H, 13C, and 31P NMR spectra have been assigned for tobramycin, 2'-(adenosine-5'-phosphoryl)-tobramycin (TbAMP) and 2'-(adenosine-5'-thiophosphoryl)-tobramycin (TbAMPS). Several one- and two-dimensional NMR techniques have been utilized, notably, 1H-1H homonuclear correlation spectroscopy at 470 or 500 MHz and 13C-1H heteronuclear correlation spectroscopy at 50.3 MHz. The 1H assignments for tobramycin are similar to those previously reported for rings I and III of kanamycin A. The 13C assignments for tobramycin were similar to those previously reported, except for reversal of the assignments for anomeric carbons in the glycosyl rings. The 1H and 13C assignments for tobramycin were used to guide the assignments of the spectra for TbAMP and TbAMPS. Nearly complete assignments were obtained for these two derivatives of tobramycin. From the measured proton coupling constants, only small conformational changes were observed upon modification of tobramycin by adenylylation. From the proton and carbon spectra of the adenylylated derivatives the 2' position is shown to be the site of adenylation. Large downfield shifts of the 2'proton and carbon resonances are easily observed and are more pronounced for TbAMPS than for TbAMP.     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.     

High-field 1H and 31P NMR studies on the binding of the anticancer agent mitoxantrone to d[CpGpApTpCpG]2

A high-field 1H and 31P-NMR study of the oligomer d[CpGpApTpCpG]2 was carried out in H2O and water signal suppression was employed in all 1H NMR acquisitions. Particular attention was given to imino proton and 31P assignments. Two dimensional 31P-1H shift correlation contours were particularly useful in 31P assignments and confirming previous 1H assignments. Titrimetric addition of aliquots of the anticancer agent mitoxantrone resulted in selective and progressive chemical shifts with critical changes at stoichiometries of 1:1 and 2:1 drug to DNA ratios. The results indicate ultimate intercalative binding of the drug at both C.G. termini of the oligomer in accord with the previously determined C.G. preference and with non-nearest neighbor intercalation.     

Secondary phosphine oxides: tautomerism and chiral recognition monitored by multinuclear NMR spectroscopy of their Rh2[(R)-MTPA]4 adducts

Six secondary phosphine oxides and their tautomeric equilibria as free ligands and in the presence of an equimolar amount of the chiral dirhodium complex Rh* are described and discussed. Discrimination of enantiomers is easily possible by inspecting the (31)P NMR resonances; some (1)H and (13)C NMR resonances are useful as well. H/D exchange of the acidic protons in the phosphine oxides takes place with acetone-d(6), the solvent additive, after some hours but does not obscure the chiral recognition experiment. (103)Rh,(31)P coupling constants are discussed briefly. Decomposition of ligand molecules in 1:1-Rh*-adducts occurs slowly but completely.     

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H,31P COSY, 1H,1H NOESY and 1H,13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.     

Nuclear magnetic resonance study of sphingomyelin bilayers

Bilayers of D-erthro-(N-stearoylsphingosyl)-1-phosphocholine (C18-SPM), previously characterized by differential scanning calorimetry [Bruzik, K. S., & Tsai, M.-D. (1987) Biochemistry 26, 5364-5368] in various phases, were studied by means of wide-line 31P, 2H, high-resolution 13C CP-MAS, and 1H MAS NMR. The fully relaxed gel phase of C18-SPM at temperatures below 306 K displayed 31P NMR spectra characteristic of the rigid phase with frozen rotation of the phosphocholine head group. Three other gel phases existing in the temperature range 306-318 K displayed spectra with incompletely averaged axially symmetric powder line shapes and were difficult to differentiate on the basis of their 31P NMR spectra. The gel-to-gel transition at 306 K was found to be fully reversible. The main phase transition at 318 K resulted in the formation of the liquid-crystalline phase for which spectra with axially symmetric line shapes of uniform width were obtained, regardless of the nature of the starting gel phase. 13C CP-MAS NMR spectra revealed significant differences in the molecular dynamics of sphingomyelin in various phases. All carbon atoms of the polar head group in the liquid-crystalline phase gave rise to a separate resonance lines. Numerous carbon atom signals were doubled in the stable phase, demonstrating the existence of two slowly interconverting conformers.     

Applications of nuclear magnetic resonance in parasitology

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical framework with emphasis on magnetic nuclei of 1/2 spin, including 1H, 13C, and 31P. Biological applications of NMR spectroscopy and magnetic resonance imaging (MRI) are outlined briefly. NMR spectroscopic studies on parasitic protozoa, cestodes, nematodes, trematodes, and hymenopterous insect parasites are reviewed. NMR and MRI investigations on the pathophysiology of the host are also discussed, and the potential future of NMR applications in parasitology outlined.     

Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed.     

Interaction of the antitumor Au(I) complex [Au(Ph2P(CH2)2PPh2)2]Cl with human blood plasma, red cells, and lipoproteins: 31P and 1H NMR studies

The bis-chelated tetrahedral gold(I) complex [Au(dppe)2]Cl, where dppe is Ph2P(CH2)2PPh2, is active in several animal tumor models. When added to human blood plasma in vitro it appears to bind to lipoproteins, giving a slightly broadened 31P NMR signal, and 1H NMR resonances which are too broad to detect. Some lipoprotein is denatured. 31P NMR studies suggest that some [Au(dppe)2]+ is transferred from plasma to red cells with a half-life of ca. 2 hr. The complex binds within red cell membranes and the 1H resonances of intracellular glutathione are unaffected. The 31P NMR resonance from [Au(dppe)2]+ in red cell membranes is observable only when the complex is mobilized by addition of sodium dodecyl sulphate, which also mobilizes membrane phospholipids.     

Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.     

Olanzapine interaction with dipalmitoyl phosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayer: a (13)C and (31)P solid-state NMR study

Phospholipid bilayer interaction of olanzapine (OLZ), a thienobenzodiazepine derivative and an antipsychotic agent, has been studied with (13)C and (31)P solid-state NMR. A dipalmitoyl phosphatidylcholine (60%)/1-palmitoyl-2-oleoyl phosphatidylserine (40%) bilayer (DPPC(60%)/POPS(40%)) with 50 wt.% H(2)O, with and without 10 mol% OLZ have been investigated. The results reveal that both the serine and the choline head groups are affected by OLZ interaction with the bilayer. The OLZ interaction with the serine and the choline head groups appears to be caused by electrostatic attraction to the serine head group carboxyl and repulsion of the choline head group positively charged nitrogen. (31)P MAS NMR experiments show the appearance of two new (31)P resonances both for the PS and the PC phosphorous in the presence of OLZ. Static (31)P NMR spectra demonstrate a decrease in chemical shift anisotropy (CSA) of the OLZ containing bilayer when in the liquid-crystalline phase and an increase in CSA when in the gel state.