Re-characterization of three conjugated linolenic acid isomers by GC-MS and NMR

Conjugated linolenic acids (CLN) refer to a group of octadecatrienoic acids with three conjugated double bonds. Minor positional and geometrical differences among CLN isomers make their separation and identification difficult. We have used GC-MS and NMR to study three common CLN isomers namely alpha-eleostearic acid, beta-eleostearic acid and punicic acid, finding that some signals of olefinic carbon atoms in NMR spectra were mistakenly assigned in the literature. The present study was therefore undertaken to re-characterize the location of CC double bonds and assign the chemical signals of proton and carbon atoms using (1)H NMR, (13)C NMR, (1)H-(1)H two-dimensional correlation spectra ((1)H-(1)H COSY) and (13)C-(1)H two-dimensional correlation spectra ((13)C-(1)H COSY). The geometrical structure of double bonds in these three CLN isomers was identified using homonuclear decoupling technique.     

360-MHz 1H nuclear-magnetic-resonance spectroscopy of sialyl-oligosaccharides from patients with sialidosis (mucolipidosis I and II)

360-MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl-oligosaccharides isolated from urine of sialidosis patients. Their structures are related to the complex asparagine-linked glycan chains of glycoproteins. By correlation of these spectra and comparison with spectra of reference glycopeptides and sialyl-lactose isomers it was possible to assign all signals belonging to anomeric, mannose H-2, sialic acid H-3 and N-acetyl protons. The number of the consituting monosaccharide residues of the oligomers can be obtained by integration of the above-mentioned signals. The chemical shifts of the anomeric and mannose H-2 protons give information about the type of glycan structure (mono-, bi-, triantennary) and the presence of terminal sialic acid at each of the antennas. The chemical shifts of sialic acid H-3 protons are typical for sialic acid residues in 2 leads to 3 or 2 leads to 6 linkage to galactose.     

Rapid type 2 molybdenum(V) electron-paramagnetic resonance signals from xanthine oxidase and the structure of the active centre of the enzyme.

Rapid type 2 molybdenum(V) e.p.r. signals from reduced functional xanthine oxidase have been further investigated. These signals, which show strong coupling of two protons to molybdenum, have been obtained under a variety of new conditions: specifically either at pH 8.2 in the presence of borate ions, or at pH 10.1--10.7 with or without various other additions. Parameters of the signals were obtained with the help of computer simulations. In at least some of these signals, the coupled protons must be located on the enzyme rather than on bound species. The relationship between type 1 and type 2 Rapid signals is discussed. They may represent geometrical isomers, or alternatively, hydroxyl uptake as a ligand of molybdenum may be involved in formation of type 2 species.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and Characterization of 3,13- and 2,13-Octadecadienyl Compounds for Identification of the Sex Pheromone Secreted by a Clearwing Moth,Nokona pernix

Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M?18]⁺ at m?z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth, Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.     

An unambiguous assignment method by 2D selective-TOCSY-HSQC and selective-TOCSY-DQFCOSY and structural analysis by selective-TOCSY-NOESY experiments of a biantennary undecasaccharide

In this paper we present a newly developed 2D selective-TOCSY-HSQC experiment for unambiguous assignment of individual sugar components of a biantennary undecasaccharide. The correlation signals between the protons and carbons of a selectively excited sugar component in the undecasaccharide were observed in a 2D selective-TOCSY-HSQC experiment. The network of proton signals of this sugar was observed in a 2D selective-TOCSY-DQFCOSY experiment. These two experiments enabled us to perform unambiguous sequential assignment of both protons and carbons of the individual sugar components in the undecasaccharide. In addition, a three-dimensional structural analysis was performed by a 2D selective-TOCSY-NOESY experiment, which clearly distinguishes between inter-sugar and intra-sugar ring NOE signals.     

Chemistry of Heliangine

Novel three-proton signals at δ3.6~2.0 in the 60 and 100Mc NMR spectra of helianginone (II) were interpreted by several analytical methods. These protons were concluded to form an isolated ABC system which contains two chemical shifts, δ_AC= 100.2 and δ_BC= 81.6 cps, and three coupling constants, J_AB=4.9, J_BC= 10.2 and J_CA= -14.8 cps. In this system it is very significant that J_CA is negative. These protons were assigned as in the partial structure (a) of II.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

New Geometric Isomers of Oxooctadecadienoate in Copper-catalyzed Decomposition Products of Linoleate Hydroperoxide

Methyl linoleate hydroperoxide produced by autoxidation was refluxed with 10^-4 M Cu-naphthenate in benzene. Two new geometrical isomers of oxooctadecadienoate (compounds I and II) were found in addition to the four known isomers. They were isolated by a Sephadex LH-20 column chromatography with chloroform-hexane (2:1) and purified by HPLC on Nucleosil ®100-5 and Zorbax ODS columns. UV, IR, MS, and ¹H-NMR spectra were measured. The geometry of conjugated dienes were assigned from the coupling constants of the olefinic protons. Compounds I and II were identified as 13-oxo-trans-9, cis-11- and 9-oxo-cis-10, trans-12-octadecadienoate, respectively. Each of them had a cis double bond adjacent to the oxo group. The hydroperoxides of the same geometry as compounds I and II were also detected in autoxidation products.     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

Isotope-edited 1D and 2D n.m.r. spectroscopy of 13C-substituted carbohydrates.

Three isotope-edited n.m.r. methods have been applied to selectively 13C-substituted monosaccharides and nucleosides to simplify their spectra and/or measure 1H-1H, 13C-1H, or 13H-13C spin-couplings detected via the labeled site. 1D INADEQUATE spectra allowed the selective detection of the natural-abundance carbons that are spin-coupled to the labeled carbon, and adjustment of the mixing time permitted further discrimination between one-bond and longer-range 13C-13C coupling pathways. Geminal and vicinal 13C-1H coupling constants were determined from the analysis of 1H-1H COSY cross-peaks for those protons coupled to the labeled carbon. Long-range 13C-(HETCOR) and 1H-detected (HMBC) 13C-1H chemical-shift correlation spectra permitted the selective observation of those protons coupled to the labeled site, and JH,H values were measured from data projections. The implications of these methods for structural studies of more complex systems is briefly discussed.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example.

Two types of homonuclear proton COSY experiments are shown to be useful in making resonance assignments in cyanide-ligated cytochrome c peroxidase, a 34 kDa paramagnetic heme protein. Both magnitude COSY and phase-sensitive COSY experiments provide spectra useful for making proton assignments to resonances of strongly relaxed hyperfine-shifted protons. This initial investigation demonstrates that COSY experiments combined with NOESY experiments are feasible for hyperfine-shifted protons of paramagnetic proteins larger than metmyoglobins and ferricytochromes c, for which the nuclear spin-lattice relaxation times are in the range 70-300 ms. Taken together, COSY and NOESY experiments, although not yet widely applied to paramagnetic metalloproteins, provide a reliable protocol for accurately assigning hyperfine-shifted resonances that are part of a metalloenzyme's active site. Specific examples of expected proton homonuclear COSY connectivities that were not observed in these experiments are presented, and utilization of COSY with respect to the proton resonance line widths and apparent nuclear relaxation times is discussed. The COSY experiments presented here provide valuable verification of previously proposed hyperfine resonance assignments for cyanide-ligated cytochrome c peroxidase, which were made by using NOESY experiments alone, and in several instances expand these assignments to additional protons in particular amino acid spin systems.     

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.     

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate.

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.     

Proton NMR studies of myohemerythrin from Themiste zostericola

 One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin (Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the differentiation of the signals from the N_δ-H protons of the five N_ε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr, is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α, β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the binding of N₃ ^– to one of the Fe(II) sites in deoxyMHr, the overlapping His N_δ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in the ¹H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24. Received: 20 May 1996 / Accepted: 24 October 1996     

2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum

Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.     

Ab-inito quantum mechanical calculations of NMR chemical shifts in nucleic acids constituents. II. Conformational dependence of the 1H and 13C chemical shifts in the ribose

The magnetic shielding constant of the different 13C and 1H nuclei of a deoxyribose are calculated for the C2' endo and C3' endo puckerings of the furanose ring as a function of the conformation about the C4'C5' bond. For the carbons the calculated variations are of several ppm, the C3' endo puckering corresponding in most cases to a larger shielding than the C2' endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose-3' and 5' phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides. The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

1H NMR spectra of branched-chain cyclomaltohexaoses (alpha-cyclodextrins)

The 1H NMR spectra of seven branched alpha-cyclodextrins (alpha-CDs) were observed and analyzed in detail. They were compared with spectra of alpha-CD and amylose. Although these branched alpha-CDs consist only of alpha-D-glucose with the same alpha-(1-->4) O-glucosyl binding, aside from one exception, differences in chemical shifts of corresponding signals were significantly large. Especially, differences in the chemical shift in anomeric protons were considerably large. Subtle differences in glucosyl binding directly influences chemical shifts of these protons because anomeric protons are located adjacent to the glucosyl binding sites.     

NMR study of thymulin, a lymphocyte differentiating thymic nonapeptide. Conformational states of free peptide in solution

The nonapeptide less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (formerly called serum thymic factor) is a factor produced by the thymic epithelium, which needs a zinc ion to express its immunoregulatory properties. We report here on 1H and 13C NMR investigation of the conformational properties of the free peptide in aqueous medium and in dimethyl sulfoxide-d6 solution by a combination of homo- and heteronuclear one- and two-dimensional experiments. The various resonances have been assigned in a straightforward manner on the basis of 1H,1H COSY spectroscopy for the recognition of the proton spin systems; two-dimensional NOESY spectra with the correlation peaks across amide bonds and for the amino acid sequence assignment; amide bonds and for the amino acid sequence assignment; 13C,1H COSY experiments using selective polarization transfer from 1H- to 13C-nucleus via the 13C,1H long-range couplings for the attribution of the carboxyl and carbonyl groups; and 13C,1H COSY experiments with selective polarization transfer via the 13C,1H direct couplings for the assignment of all the aliphatic carbons. Other experiments such as pH-dependent chemical shifts, combined use of multiple and selective proton-decoupled 1H and 13C NMR spectra, the temperature and the concentration dependence of the proton shifts of the amide resonances, the solvent dependences of peptide carbonyl carbon resonances, and comparison of the spectra with three different analogues were performed. In aqueous solution, the data are compatible with the assumption of a highly mobile dynamic equilibrium among different conformations, whereas in dimethyl sulfoxide-d6, a more rigid structure is found involving three internal hydrogen bonds. These observations provide an insight into the conformational tendencies of this peptidic hormone in two different media.