NOE Experiments Research Articles

Neurotoxic meroditerpenoids from the tropical marine brown alga Stypopodium flabelliforme.

Brine shrimp toxicity and TLC analysis guided the isolation of five new and biologically active meroditerpenoids [2beta,3alpha-epitaondiol (1), flabellinol (2), flabellinone (3), stypotriolaldehyde (4), and stypohydroperoxide (5)] along with five known compounds from the marine brown alga Stypopodium flabelliforme collected in Papua New Guinea. The planar structures of compounds 1-5 were determined by extensive spectroscopic analysis (1D and 2D NMR, LRMS, HRMS, IR, and UV), while relative configuration was determined by 1D and 2D NOE experiments. X-ray crystallography confirmed the relative configuration of 2beta,3alpha-epitaondiol (1), and the modified Mosher's ester method was used to establish its absolute configuration. All of the new metabolites were moderately toxic to murine neuro-2a cells (LC50 2-25 microM), and three [2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3)] possessed potent sodium channel blocking activity. Stypotriolaldehyde (4) had a biphasic effect on the concentration of intracellular Ca2+ in rat cerebellar granule neurons (CGN). The previously known compound, stypoldione (6), also modulated intracellular calcium concentration and was cytotoxic in CGN. Metabolites 2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3) displayed moderate cytotoxicity to the NCI-H460 human lung cancer cell line.

Synthesis of functionalized polyhedral oligomeric silsesquioxane (POSS) macromers by microwave assisted 1,3-dipolar cycloaddition

In this paper we report the first synthesis of isoxazolidine and isoxazoline-POSS macromers by 1,3-dipolar cycloaddition reactions of vinyl- and styryl-POSS with N-methyl- C-ethoxycarbonylnitrone and ethoxycarbonyl nitrile oxide, promoted by microwave irradiation. The nature of the resulting cycloadducts has been determined by NOE experiments and supported by computational studies at PM3 and DFT B3LYP/6-31G* levels.

The RNA‐Bound Conformation of Neamine as Determined by Transferred NOE Experiments

The tRNA(Phe)-bound conformation of the aminoglycoside neamine, a member of the neomycin B family, has been investigated by transferred NOE experiments in aqueous solution. This is the first time that the bioactive conformation of an RNA-bound aminoglycoside has been determined by this method. In buffers without divalent Mg(2+) ions, a high degree of electrostatically driven unspecific binding of aminoglycosides to the RNA was observed. Careful optimization of experimental conditions yielded buffer conditions optimized for cryo-probe NMR experiments. In particular, addition of Mg(2+) ions to the solutions was necessary to reduce the amount of unspecific binding as monitored by one-dimensional NMR and surface plasmon resonance experiments. CD spectroscopy was used to probe the effect of aminoglycosides and buffer conditions on the double helical content of tRNA(Phe). Finally the tRNA(Phe)-bound conformation of neamine was determined by trNOE build-up curves and compared with the previously reported crystal structure of neomycin B complexed to this RNA. Although the aminoglycoside in the crystal structure contains several configurational errors, the overall shape of the crystallographically determined RNA-bound structure is identical to the RNA-bound conformation defined by the NMR experiments. Therefore, the crystal structure has been refined by trNOE data. This is particularly important in the context of aminoglycosides being discussed as lead structures for the development of new anti-RNA drugs.

Generation and Reaction of Ammonium Ylides in Basic Two‐Phase Systems

AbstractFor Abstract see ChemInform Abstract in Full Text.

Phenolic Esters from the Rhizomes ofCimicifuga racemosado not Cause Proliferation Effects in MCF-7 Cells

Five phenylpropanoid esters, caffeoylglycolic acid, 2-caffeoylpiscidic acid (cimicifugic acid D), 3,4-dihydroxyphenacyl caffeate (petasiphenone), 3,4-dihydroxyphenyl-2-oxopropyl isoferulate (cimiciphenol) and 3,4-dihydroxyphenacyl isoferulate (cimiciphenone) were isolated from a commercially available extract of the rhizomes of Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L.) for the first time; the known cimicifugic acids A, B, E, F, fukinolic acid, fukiic acid and caffeic acid were also obtained. Cimiciphenone and caffeoylglycolic acid are new natural products. The structures were elucidated by means of spectroscopic data (ESI-MS, 1H-, 13C-NMR, COSY, HMQC, HMBC and NOE experiments). Ferulic acid and isoferulic acid were detected by HPLC analysis in comparison to standards. The estrogenic activity of the isolated compounds was tested in an estrogen-dependent MCF-7 mamma carcinoma cell line; 17beta-estradiol (10(-11) M) and the phytoestrogen coumestrol (10(-7) - 10(-5) M) were used as references. The results suggest that, in contrast to an earlier report, the phenolic esters do not exert a proliferative (estrogenic) effect in this test system.

Two New Nortriterpenes from Ligularia tongolensis

Abstract Two new nortriterpenes having a ursane type structure were isolated from the roots of Chinese Ligularia tongolensis, and their structures were elucidated by interpretation of spectral data. Their relative stereochemistries were also clarified by detailed analysis of proton–proton coupling constants and by NOE experiments.

Structural and spectral assignment of three forskolin‐like diterpenoids isolated from Plectranthus ornatus

Three labdane diterpenoids were isolated from an acetone extract of Plectranthus ornatus. Their structures, closely related to that of forskolin, were determined by NMR studies. Unambiguous and complete assignments of the 1H and 13C NMR chemical shifts for these substances are presented. The assignments are based on 2D shift-correlated [1H, 1H-COSY, 1H, 13C-gHSQC-1J (C,H), 1H, 13C-gHMBC-(n)J (C,H) (n = 2 and 3)] and NOE experiments.

Cyclohexanoid protoflavanones from the stem-bark and roots of Ongokea gore

Phytochemical investigation of root and stem-bark of the West African medicinal plant Ongokea gore resulted in the isolation of four novel flavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B. The structures of the isolated compounds were elucidated by spectroscopic methods, mainly 1D and 2D NMR, and subsequently, the structures were corroborated by chemical conversion to (−)-( S)-sakuranetin. The absolute configurations, and preferred conformations were determined by NOE experiments and CD measurements.

Stereolabile and Configurationally Stable Atropisomers of Hindered Aryl Carbinols

Carbinols of the Ar-C(OH)R(2) type, Ar being o-isopropylphenyl, exist as stereolabile syn-clinal (sc) and anti-periplanar (ap) atropisomers when R = Me, Et, i-Pr. In the latter compound, the major atropisomer also comprises two enantiomeric forms that interchange with a barrier of 6.4 kcal mol(-1). X-ray diffraction, NOE experiments, and ab initio calculations indicate that the sc-atropisomer is the more stable form when R = Me, i-Pr, t-Bu but is the less stable one when R = Et. NMR spectra at variable temperature allowed the determination of the barriers for the interconversion of the sc- into the ap-atropisomers (DeltaG++ = 7.6, 8.8, and 13.5 kcal mol(-1) for Me, Et, i-Pr, respectively). When R is a tert-butyl group, the two atropisomers are configurationally stable: the ap-atropisomer is obtained as the kinetic controlled compound, which can be transformed into the thermodynamically more stable sc-atropisomer with a free energy of activation of 29.3 kcal mol(-1). Both atropisomers exhibit restricted rotation of the tert-butyl moiety, the corresponding DeltaG++ values being 9.4 and 8.8 kcal mol(-1) for the sc- and ap-atropisomer, respectively.

Diastereoselective Addition of Organolithiums to 1,3‐Oxazolidines Complexed with Aluminum Tris(2,6‐diphenylphenoxide) (ATPH).

AbstractFor Abstract see ChemInform Abstract in Full Text.

Taxadiene Synthase-Catalyzed Cyclization of 6-Fluorogeranylgeranyl Diphosphate to 7-Fluoroverticillenes

The mechanism of the taxadiene synthase-catalyzed cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP, 7) to taxadiene (5) is proposed to proceed through a verticillen-12-yl carbocation intermediate (8) that undergoes an 11 --> 7 proton transfer leading to formation of the C ring. The substrate analogue 6-fluoroGGPP (17) was synthesized to elucidate the stereochemistry of the putative verticillenyl intermediate. It was expected that the inductive electron-withdrawing effect of the fluoro substituent would prevent the critical proton transfer to the Delta(7) double bond and thereby derail the cyclization at the bicyclic stage. Incubation of the fluoro analogue with recombinant taxadiene synthase yielded a mixture of three major and two minor fluoro diterpenes according to GC/MS analyses. The three major products were identified as the exocyclic, endocyclic, and 4(20)-methylene 7-fluoroverticillenes, i.e., Delta(3,7,12 (18)), Delta(3,7,12), and Delta(4(20),7,11) isomers (22, 23, and 24) on the basis of (1)H NMR analyses and comparisons with the parent bicyclic diterpenes. The H1beta, H11alpha (1S,11R) configurations at the bridgehead positions of 22 were established by means of NOE experiments and CD spectra. The absolute configuration of (+)-verticillol (4) was revised after the anomalous dispersion X-ray analysis of (+)-verticillol p-iodobenzoate. Of particular note, all absolute configurations of verticillane diterpenes in the literature should be reversed. This work affords compelling evidence supporting the H11alpha (11R) stereochemistry of the verticillen-12-yl(+) ion intermediate in the taxadiene synthase-catalyzed reaction and illustrates the capability of vinyl fluoro analogues to intercept complex cyclization cascades.

Environment‐Friendly Organic Synthesis Using Bismuth Compounds. Bismuth Triflate Catalyzed Synthesis of Substituted 3,4‐Dihydro‐2H‐1‐benzopyrans.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The disordered conformation of κ-carrageenan in solution as determined by NMR experiments and molecular modeling

The conformation of κ-carrageenan in solution was studied combining 1H and 13C NMR with molecular mechanics. The experimental conditions were chosen to characterize the disordered conformation of the polymer. Particular attention has been given to explore a wide range of experimental conditions as to the dependence on solvent (water and Me 2SO), polymer concentration, temperature, pH, presence of a denaturing agent (guanidinium chloride), and of ions otherwise able to induce conformational order of the carrageenan chains, either in solution (I −) or in the gel state (Rb +). Two-dimensional NOE experiments were analyzed to obtain information on internuclear distances, and molecular mechanics provided the range of energetically accessible conformations. Two inter-residue topological constraints were clearly identified: their combination is rather restricting for the chain and suggests that the disordered conformation of κ-carrageenan is characterized by an intrinsic stiffness with high values of persistent length and characteristic ratio. They also rule out any postulated interchain hydrogen bonds. In contrast, experiments on the temperature dependence of the chemical shift in Me 2SO reveal the existence of two inter-residue intramolecular H-bonds which might contribute positively to the rigidity of the polymer chain. The overall picture emerging from the present results is that of a locally elongated ‘loose single helix’.

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.

Generation and Reaction of Ammonium Ylides in Basic Two‐Phase Systems

AbstractReaction of the quaternary ammonium salts 2a–i with electrophilic alkenes 3, active alkylating agents 7 or aromatic aldehydes 11, carried out in basic two‐phase systems A–D, afforded cyclopropanes 4, cyanoalkenes 8 or cyanooxiranes 12 respectively, via the corresponding ammonium ylides 2+ –. The method is very simple, and gives cyclopropanes 4 and cyanoalkenes 8 in high yield. Under similar conditions, 1‐cyanodienes 8aa,ba were cyclopropanated at the γ,δ‐double bond with formation of vinylcyclopropanes 9a,b. The stereochemistry of the prepared cyclopropanes was elucidated from literature, 1H NMR spectroscopic data, NOE experiments or X‐ray single crystal analysis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Structure and Dynamics of Micelle-bound Human α-Synuclein

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded beta-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the beta-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.

A New Spin Probe of Protein Dynamics: Nitrogen Relaxation in15N−2H Amide Groups

(15)N spin relaxation data have provided a wealth of information on protein dynamics in solution. Standard R(1), R(1)(rho), and NOE experiments aimed at (15)N[(1)H] amide moieties are complemented in this work by HA(CACO)N-type experiments allowing the measurement of nitrogen R(1) and R(1)(rho) rates at deuterated (15)N[(2)D] sites. Difference rates obtained using this approach, R(1)((15)N[(1)H]) - R(1)((15)N[(2)D]) and R(2)((15)N[(1)H]) - R(2)((15)N[(2)D]), depend exclusively on dipolar interactions and are insensitive to (15)N CSA and R(ex) relaxation mechanisms. The methodology has been tested on a sample of peptostreptococcal protein L (63 residues) prepared in 50% H(2)O-50% D(2)O solvent. The results from the new and conventional experiments are found to be consistent, with respect to both local backbone dynamics and overall protein tumbling. Combining several data sets permits evaluation of the spectral density J(omega(D) + omega(N)) for each amide site. This spectral density samples a uniquely low frequency (26 MHz at a 500 MHz field) and, therefore, is expected to be highly useful for characterizing nanosecond time scale local motions. The spectral density mapping demonstrates that, in the case of protein L, J(omega(D) + omega(N)) values are compatible with the Lipari-Szabo interpretation of backbone dynamics based on the conventional (15)N relaxation data.

Sesquiterpene constituents of the liverwort Lophozia ventricosa

The essential oil of Lophozia ventricosa was investigated and yielded the two new sesquiterpenoids ventricos-7(13)-ene and 6,7-epoxyeudesm-3-ene. The first has a new skeleton, which is named ventricosane and is numbered after the structurally closest known sesquiterpene skeleton pentalenane. The absolute configuration of the latter compound was established by conversion to known compounds. The isolated compounds and reaction products were investigated using enantioselective GC and extensive NMR measurements ( 1H; 1H– 1H COSY; HSQC; HMBC and NOE experiments).

Diastereoselective addition of organolithiums to 1,3-oxazolidines complexed with aluminum tris(2,6-diphenylphenoxide) (ATPH)

1,3-Oxazolidines were easily obtained by condensation of N-substituted ( R)-phenylglycinol with aldehydes. Addition of organolithium reagents to 1,3-oxazolidines by complexation with the bulky Lewis acid aluminum tris(2,6-diphenylphenoxide) (ATPH) readily produced the corresponding chiral amines with good yield and high diastereoselectivity. The configuration of the new stereogenic center was shown to be opposite to that of adducts obtained for the same 1,3-oxazolidines using Grignard reagents. The best diastereoselectivity was achieved using N-isopropyl-1,3-oxazolidines. The mechanism of addition was deduced by determining the stereochemistry of the iminium–aluminum complex by NOE experiments.