P.M.R studies on fully methylated aldohexopyranosides and their 6-deoxy analogues using lanthanide shift reagents

The complexes of lanthanide shift reagents (LSR) with permethylated aldo-hexopyranosides and their 6-deoxy analogues having the gluco, galacto, and manno configurations have been studied. On the basis of shift data from Eu(fod)₃ and Pr(fod)₃, and broadening data from Gd(fod)₃, it was found that the LSR bind preferentially to two neighbouring MeO-oxygens having the axial-equatorial relationship. Because of its steric requirements, the C-5 substituent hinders the binding increasingly in the following order: O-2(ax)-O-3(eq)<O-1(ax)-O-2(eq)<O-4(ax)-O-3(eq). Equatorial groups bind the LSR only weakly. Strong binding to O-6 was found when MeO-6 is predominantly “axially” oriented; when this group has the “equatorial” position, O-6 is not favoured over any other equatorial oxygen. In view of the preference of the LSR to bind to an O(ax)-O(eq) site, it is proposed that O-5 is involved in the binding to the axial O-6. Eu(fod)₃ seems to have less tendency to bind to the O-6(ax)-O-5 site than the other two LSR.     

5,7-Dihydroxy-3,6,8-trimethoxyflavone from the flowers of Gnaphalium elegans

5,7-Dihydroxy-3,6,8-trimethoxyflavone was isolated from the flowers of Gnaphalium elegans. The structure elucidation is based mainly on proton resonance studies using Eu(fod)₃ shift reagent.     

Determination of Stereochemistry at C-16 of Dihydrogibberellin A1 Methyl Ester (Methyl Tetrahyclrogibberellate) and Its 16-Epimer

Stereochemistry at C–16 of dihydrogibberellin A₁ methyl ester (methyl tetrahydrogib-berellate, III) and its 16-epimer was elucidated. NMR analysis employing a shift reagent, Eu(thd)₃ or Eu(fod)₃, was found to be very effective for settling this stereochemical problem.     

Mixed-ligand lanthanide complexes. IV. Paramagnetic shift induced by eu(fod)3pz and Yb(fod)3PZ in the NMR spectrum of di-l-butyl ether

Two pyrazole adducts of Ln(fod)₃, Eu(fod)₃pz and Yb(fod)₃pz have been scanned as potential NMR shift reagents using di-l-butyl ether. Di-l-butyl ether is a very weak donor; even then the complexes associate with the base and induce large isotropic hyperfine shifts in the resonance frequencies of the magnetic substrate nuclei. The t-butyl resonances of the complexes are strongly shifted (in the opposite direction) in the presence of the substrate. This observation together with the fact that the spectra of the substrate are unaffected by addition of any amount of the complexes are taken as evidence that the specific coordination of the complexes to the substrate occurs through the agency of oxygen lone-pair electrons.     

Configurational assignments of C-nitromethyl-C-hydroxy branched-chain carbohydrate derivatives by use of a europium shift-reagent in 1 H-N.M.R. spectroscopy

Configurational assignments for the tertiary alcoholic centers of four branched-chain 3-C-nitromethylglycopyranosides, namely, methyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (1), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-glucopyranoside (4), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (5), and methyl 4,6-O-benzylidene-3-C-nitromethyl-2-O-p-tolylsulfonyl-α-D-glucopyranoside (8), were made on the basis of the downfield chemical shifts of their identifiable protons per molar equivalent of added Eu(fod)₃, as compared with those of model compounds, of known configuration, having a close structural relationship. In some cases, the assignments were corroborated by the position of the acetyl resonances in the unshifted 60-MHz p.m.r. spectra of the corresponding O-acetyl derivatives.     

Cis-avicennol,a new pyranocoumarin from the root bark of Zanthoxylum elephantiasis

A new pyranocoumarin has been isolated from the root bark of Zanthoxylum elephantiasis and identified as cis-avicennol (6-(3-hydroxy-3-methyl-cis-but-1-enyl)-5-methoxy-2,2-dimethyl- 2H-benzo [1,2b:3,4b′] dipyran-8-one) on the basis of comparison of spectral data with that of trans-avicennol and conversion to tetrahydroavicennol. The usefulness of the lanthanide shift reagent Eu(fod)₃ in the assignment of cis configuration to the butenyl sidechain is briefly discussed.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Lewis-active and Lewis-like substances.

High resolution nuclear magnetic resonance spectra were recorded in a chloroform solution of six Lewis-active or Lewis-like glycosphingolipids in permethylated and permethylated-reduced (LiAlH₄) form. The samples were selected to cover the presently known structural variants of α-fucose linked to galactose and N-acetylglucosamine. Fucα1 → 2Gal, Fucα1 → 3GlcNAc, and Fucα1 → 4GlcNAc gave characteristic and well-separated anomeric resonances. Furthermore, upon reduction there was a strong deshielding effect on Fucα1 → 3GlcNAc and Galβ1 → 3GlcNAc (linkage vicinal to reduced amide), which makes it possible to differentiate type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains. This improved method of nuclear magnetic resonance spectroscopy is discussed in relation to sequence analysis by mass spectrometry, two microscale structural methods using the same type of derivatives and needing no degradations before analysis.     

Detalolactone: A novel pyranocoumarin from the root bark of Zanthoxylum dipetalum

A novel dipyranocoumarin, dipetalolactone {2-oxo-6,6,10,10-tetramethylbenzo[1,2-b:3,4-b′:5,6-b″]tripyran}, has been isolated from the root bark of Zanthoxylum dipetalum and its structure proven by the synthesis of tetrahydrodipetalolactone. A second new pyranocoumarin, dipetaline, has been assigned the tentative structure of 6-(3,3-dimethylallyl)-5-methoxy-2,2-dimethyl-2H-benzo[1,2-b:3,4-b′]dipyran-8-one on the basis of PMR analysis using the lanthanide shift reagent Eu(fod)₃.     

Structural elucidation of polymethoxyflavones from shift reagent proton NMR measurements

The quantitative shift reagent behavior of polymethoxylated flavones in the presence of Pr(fod)₃ shows that for structural elucidation of these molecules the degree of substitution in the neighborhood of the carbonyl group can be determined from the number of signals that are strongly shifted and broadened. The induced shifts of the remaining signals are of complementary help and even the resonances of individual methoxyl groups can be ascribed.     

Isolation of dihydroxylupene and dihydroxylupane from the bark of Lawsonia inermis

Two pentacyclic triterpenes isolated from the bark of henna were identified as 3β, 30-dihydroxylup- 20(29)-ene (hennadiol), and (20S)-3β, 30-dihydroxylupane. The assignment of the C-20 configuration in the latter compound was supported by the analysis of Eu(fod)₃-induced ¹H NMR chemical shifts in the two C-20 epimers synthesized from lupeol.     

Lanthanide- and aromatic solvent-induced shift effects on proton resonances in C-4-methylated steroids and tetracyclic triterpenoids

Lanthanide-induced shifts (LIS) with Eu (dpm)₃ and aromatic solvent induced shifts (ASIS) with C₄H₄, C₅H₅N₁ and C₆F₆ of PMR signals were examined for a series of C-4-methylated steroids and tetracyclic triterpenoids having a hydroxyl, carbonyl or acetoxyl group at position C-3. The magnitude and/or direction of the LIS (or ASIS) of corresponding protons were extensively influenced by the nature of the C-3-functional groups. The possible geometries of Eu (dpm)₃-substrate complexes were also discussed on the basis of the LIS data. The above two techniques in the PMR spectroscopy provided the confirmatory evidence for the structural and stereochemical determination of steroids and triterpenoids.     

Determination of enantiomeric purity of glycerides with a chiral PMR shift reagent.

Tris(3-heptafluorobutyryl-d-camphorato)europium(III), Eu(hfbc)₃ was used to determine the optical purities of enantiomeric mixtures of tri-, di- and monoglycerides with various fatty acid chain lengths by proton magnetic resonance (PMR). Synthesized model enantiomers were used to assign PMR signals. Enantiomeric signal separation becomes more difficult if the chain length difference between the fatty acids in the 1- and 3-positions of glycerol becomes smaller. The sign of the enantiomeric shift difference (ΔΔδ) of the terminal acyl CH₃ group of 1-acyl-2,3-distearoyl-sn-glycerol vs its enantiometer remains the same in the series acyl is hexanoyl, butyryl, propionyl, but is reversed for acetyl.The absolute configuration of the main triglyceride of the seed oil of Euonymus alatus was determined to be 3-acetyl-1,2-distearoyl-sn-glycerol and that of a monobutyryl triglyceride fraction from hydrogenated bovine butterfat was confirmed to be mainly 1,2-diacyl-3-butyryl-sn-glycerol. The enantiotopic behaviour of the glycerol CH₂ groups in (nearly) symmetric di- and triglycerides is discussed.     

Conformational analysis of peracetylated hexononitriles

The conformations of six peracetylated hexononitriles in solution have been investigated by Fourier-transform, proton n.m.r. spectroscopy at 90 MHz, with iterative analysis and simulation of many of the spectra. The conformation of tetra-O-acetyl-L-arabinononitrile has been re-examined by the same methods. A shift reagent [Eu(fod)₃-d₃₀] and spectra at 220 MHz were used to improve spectral dispersion, where necessary. For practically all of the derivatives studied, the vicinal, proton-proton coupling-constants are consistent with a zigzag conformation in which the cyano group lies in the plane of the other carbon atoms of the chain, unless this conformation contains a parallel 1,3-interaction of substituents. Other conformers that are also consistent with the coupling constants observed are discussed, including rotamers about chain-terminal, carbon-carbon bonds.     

Transport of Pr3+ across phospholipid membranes by lipophilic β-diketones

Several β-diketones (R.CO.CH₂.CO.R.) with R groups similar to those used in NMR shift reagents have been investigated as carriers for the transport of Pr³⁺ ions across phospholipid vesicular membranes. Only the flourinated diketone fod (1,1,1,2,2,3,3-heptafluoro-7.7-dimethyloctane-4,6 dione) gave a transport rate which approached that obtained using the calcium ionophore A23187. It was established that the NMR method used to follow the kinetics gave an experimental stoichiometry of Pr(fod)_2.8 for an expected transported complex Pr(fod)₃. The advantages of using an NMR method for studying facilitated transport in vesicular membrane systems are discussed, in particular their use in determining stoichiometries of transported species.     

Stereochemistry of the decarboxylation of l-ornithine with ornithine decarboxylase from mouse kidney

Using highly purified ornithine decarboxylase isolated from androgen-treated mice, [1R-²H]putrescine was generated by the decarboxylation of l-ornithine in D₂O, and [1S-²H]putrescine was generated from [2-²H]ornithine by carrying out the decarboxylation in H₂O. Chirality of the putrescines was then determined from the 200-MHz ¹H NMR spectra of their bis-camphanamides in the presence of Eu(fod)₃. These results demonstrated that decarboxylation had taken place with retention of configuration.     

13C- and 1H-N.M.R. spectroscopy of permethylated gluco-, galacto-, and manno-pyranoses and their 6-deoxy analogues

The ¹³C- (25.16 MHz) and ¹H-n.m.r (220, 300 MHz) spectra of permethylated mannopyranoses, their 6-deoxy analogues, and permethylated 6-deoxy-gluco- and -galacto-pyranoses have been analysed with the aid of specific trideuteriomethylation, heteronuclear spin-decoupling, and spectrum simulation. Comparison of spectral data for the aldohexose derivatives and their 6-deoxy analogues shows that the ring conformation is not significantly affected by the presence or absence of MeO-6; all compounds are present in the ⁴C₁ (D) or ¹C₄ (L) conformation. Changes in orientation of the MeO groups have distinct effects on the chemical shifts of carbons and protons of the pyranoid rings and of the MeO groups. The possible origins of these effects are discussed.     

1H NMR studies of drugs with achiral and chiral lanthanide shift reagents: Applications to the anticonvulsant pheneturide

The anticonvulsant pheneturide, PNT, has been studied by 300 MHz ¹H NMR in CDCl₃ at ambient temperatures with the achiral lanthanide shift reagent (LSR) Eu(FOD)₃, and with the chiral LSR, Eu(HFC)₃. Both LSRs produced spectral simplification of the aryl proton signal region, and substantial lanthanide‐induced shifts (LIS). With added Eu(HFC)₃, enantiomeric shift differences (ΔΔδ) were induced for most nuclei of PNT, indicating substantial potential for direct determination of enantiomeric excess. Valley heights between corresponding signals in the PNT enantiomers as low as 3.6% were achieved for the meta resonance. Least squares line‐fitting was applied to the variation of chemical shift vs. [LSR]/[PNT] molar ratios for both LSRs. Tentative assignments were made for the NH absorptions based on two‐dimensional NMR (COSY45), as well as their relative magnitudes of LIS, ΔΔδ, and lanthanide‐induced line broadening. The PNT conformation reported in the crystal is believed to be retained in solution with added LSR. The relative senses of magnetic nonequivalence were found to be the same among the three sets of aryl protons, and among the three kinds of protons in the ethyl moiety, with high levels of added chiral LSR, using 2D NMR. Chirality 11:529–535, 1999. © 1999 Wiley‐Liss, Inc.     

Scope and limitations of the simple 13C-n.m.r. method of structural analysis of carbohydrates: glucodisaccharides

The scope and limitations of the SIMPLE n.m.r. method (secondary isotope multiplet n.m.r. spectroscopy of partially labelled entities) has been investigated for a series of glucodisaccharies. ¹³C-SIMPLE n.m.r. measurements have been made on solutions of (1→1)- (α,α-trehalose), (1→2)- (sophorose and kojibiose), (1→3)- (laminaribiose), and (1→6)-linked (gentiobiose and isomaltose) glucodisaccharides in (CD₃)₂SO and the results combined with those previously published for (1→4)-linked analogues (maltose and cellobiose). Each linkage (and substitution) type gives rise to a unique pattern of ¹³C isotopomers which, in principle, may be used for complete assignment of the spectra and structural analysis of the molecule. The glucodisaccharides are difficult to analyse, compared with other disaccharides, because the presence of two glucose moieties leads to degeneracies of a few isotopic multiplets which cannot be differentiated by the magnitudes of the isotope effects. Assignments in aqueous solutions were obtained by using the DIS (differential isotope shift) n.m.r. method in conjunction with the results from SIMPLE n.m.r. In practice, nearly all of the signals can be assigned unequivocally and the remaining signals are choices between two possible assignments.     

Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH₄) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.     

Evidence for the electrochromic identification of the change of charges in the four oxidation steps of the photoinduced water cleavage in photosynthesis

After dark adaptation of oxygen-evolving photosystem (PS) II complexes, oscillatory absorption changes (stable >0.5 s) with a period of 4 were previously detected at 514 nm, probably due to electrochromic band shifts of carotenoids [(1984) FEBS Lett. 176, 83-87]. To support this interpretation, we looked for corresponding phenomena in the 650–714 nm range. Here, oscillations were also observed with a difference spectrum giving evidence for an electrochromic band shift of the chlorophyll a absorption band. The oscillation pattern 0:0:+:+ corresponds to those at 514 nm, supporting the former conclusion that the states S₂ and S₃ have a surplus charge compared to S₀ and S₁. This is possible if the electron release pattern 1:1:1:1 is accompanied by an intrinsic proton release pattern 1:0:1:2 for the transition S₀ → S₁ → S₂ → S₃ → S₀.