Antioxidant activities and phenolic composition of extracts from Greek oregano, Greek sage, and summer savory.

Oregano vulgare L. ssp. hirtum (Greek oregano), Salvia fruticosa (Greek sage), and Satureja hortensis (summer savory) were examined as potential sources of phenolic antioxidant compounds. The antioxidant capacities (antiradical activity by DPPH* test, phosphatidylcholine liposome oxidation, Rancimat test) and total phenol content were determined in the ethanol and acetone extracts of the dried material obtained from the botanically characterized plants. The ground material was also tested by the Rancimat test for its effect on the stability of sunflower oil. The data indicated that ground material and both ethanol and acetone extracts had antioxidant activity. Chromatographic (TLC, RP-HPLC) and NMR procedures were employed to cross-validate the presence of antioxidants in ethanol and acetone extracts. The major component of all ethanol extracts was rosmarinic acid as determined by RP-HPLC and NMR. Chromatography indicated the presence of other phenolic antioxidants, mainly found in the acetone extracts. The presence of the flavones luteolin and apigenin and the flavonol quercetin was confirmed in the majority of the extracts by the use of a novel (1)H NMR procedure, which is based on the strongly deshielded OH protons in the region of 12-13 ppm without previous chromatographic separation. This deshielding may be attributed to the strong intramolecular six-membered ring hydrogen bond of the OH(5)...CO(4) moiety.

Novel lipophilic amidate oxorhenium and oxotechnetium complexes as potential brain agents: synthesis, characterization and biological evaluation.

Novel oxorhenium and oxotechnetium complexes based on the tetradentate 1-(2-hydroxybenzamido)-2-(pyridinecarboxamido)benzene, H3L, ligand have been synthesized and characterized herein. Thus, by reacting equimolar quantities of the triply deprotonated ligand L3- with the suitable MO3+ precursor, the following neutral MOL complexes could be easily produced following similar synthetic routes: M = Re (1), M = 99gTc (2), and M = 99mTc (3). Complexes 1 and 2, prepared in macroscopic amounts, were chemically characterized and their structure determined by single-crystal X-ray analysis. They are isostructural metal chelates, adopting a distorted square pyramidal geometry around the metal. The N3O donor atom set of the tetradentate ligand defines the basal plane and the oxygen atom of the M = O core occupies the apex of the pyramid. Complex 3 forms quantitatively at tracer level by mixing the H3L ligand with Na99mTcO4 generator eluate in aqueous alkaline media and using tin chloride as reductant in the presence of citrate. Its structure was established by chromatographic comparison with prototypic complexes 1 and 2 using high-performance liquid chromatographic techniques. When challenged with excess glutathione in vitro, complex 3 is rapidly converted to hydrophilic unidentified metal species. Tissue distribution data after administration of complex 3 in vivo revealed a significant uptake and retention of this compound in brain tissue.

Identification and quantification of caffeic and rosmarinic acid in complex plant extracts by the use of variable-temperature two-dimensional nuclear magnetic resonance spectroscopy.

A combination of advanced nuclear magnetic resonance (NMR) methodologies for the analysis of complex phenolic mixtures that occur in natural products is described, with particular emphasis on caffeic acid and its ester derivative, rosmarinic acid. The combination of variable-temperature two-dimensional proton-proton double quantum filter correlation spectroscopy (1H-1H DQF COSY) and proton-carbon heteronuclear multiple quantum coherence (1H-13C HMQC) gradient NMR spectroscopy allows the identification and tentative quantification of caffeic and rosmarinic acids at 243 K in extracts from plants of the Lamiaceae family, without resorting to previous chromatographic separation of the components. The use of proton-carbon heteronuclear multiple bond correlation (1H-13C HMBC) gradient NMR spectroscopy leads to the complete assignment of the correlations of the spins of H2a and H3a with the ester and carboxyl carbons of rosmarinic and caffeic acid, even at room temperature, and confirms the results of the above methodology Quantitative results are in reasonable agreement with reverse phase HPLC measurements.

Synthetic analogs for oxovanadium(IV/V)-glutathione interaction: an NMR, EPR, synthetic and structural study of oxovanadium(IV/V) compounds with sulfhydryl-containing pseudopeptides and dipeptides.

The reaction of [VO(CH3COO)2(phen)] (phen = 1,10-phenanthroline) with the sulfhydryl-containing pseudopeptides (scp), N-(2-mercaptopropionyl)glycine (H3mpg), N-(2-mercaptopropionyl)cysteine (H4m2pc), N-(3-mercaptopropionyl)cysteine (H4m3pc) and the dipeptides glycylglycine (H2glygly) and glycyl-L-alanine (H2glyala), in the presence of triethylamine, results in the formation of the compounds Et3NH[VO(mpg)(phen)] (1), (Et3NH)2[VO(m2pc)] (4), [(Et3NH)2[VO(m3pc) (5), [VO(glygly)(phen)] x 2CH3OH (2 x 2CH3OH) and [VO(glyala)(phen)] x CH3OH (3 x CH3OH). Evidence for the molecular connectivity in 2 x CH3OH was established by X-ray crystallography, showing the vanadium(IV) atom ligated to a tridentate glygly2- ligand at the N(amine), N(peptide) and O(carboxylato) atoms. Combination of the correlation plot of the EPR parameters gz versus Az, together with the additivity relationship supported the prediction of the equatorial donor atom sets of the V(IV)O2+ center at various pH values for the V(IV)O2+-glutathione system considered in this study. Model NMR studies (interaction of vanadium(V) with the scp H3mpg) showed that there is a possibility of vanadium(V) ligation to glutathione.

Multinuclear (13C, 17O, 57Fe) NMR studies of carbonmonoxy heme proteins and synthetic model compounds.

13C, 17O and 57Fe NMR spectra of several carbonmonoxy hemoprotein models with varying polar and steric effects of the distal organic superstructure, constraints of the proximal side, and porphyrin ruffling are reported. Both heme models and heme proteins obey a similar excellent linear delta(13C) versus nu(C-O) relationship which is primarily due to modulation of pi-back-bonding from the Fe d(pi) to CO pi* orbital by the distal pocket polar interactions. The lack of correlation between delta(13C) and delta(17O) suggests that the two probes do not reflect a similar type of electronic and structural perturbation. delta(17O) is not primarily influenced by the local distal field interactions and does not correlate with any single structural property of the Fe-C-O unit; however, atropisomerism and deformation of the porphyrin geometry appear to play a significant role. 57Fe shieldings vary by nearly 900 ppm among various hemes and an excellent correlation was found between delta(57Fe) and the absolute crystallographic average displacement of the meso carbon atoms, /Cm/, relative to the porphyrin core mean plane. The excellent correlation between iron-57 shieldings and the average shieldings of the meso carbons of the porphyrin skeleton of TPP derivatives suggests that the two probes reflect a similar type of electronic and structural perturbation which is primarily porphyrin ruffling.

Arg side-chain-backbone interactions evidenced in model peptides by 17O-NMR spectroscopy.

The guanidinium group of arginine possesses a variety of biochemical functions, either by participating in direct interactions in recognition processes, or by stabilizing secondary structures. Three model compounds, selectively (17)O enriched, Ac-Arg-Ala-[(17)O]Pro-NH(2) (1), Piv-Arg-Pro-[(17)O]Gly-NH(2) (2) (C-terminal segment of the luteinizing hormone releasing hormone), and Piv-Nle-Pro-[(17)O]Gly-NH(2) (3), were prepared and studied by (17)O-nmr spectroscopy. A direct hydrogen-bonded interaction between the Arg side chain and the carbonyl main chain carboxy-terminus was found, thus confirming the tendency of Arg to participate in proton-acceptor functions.

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study.

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.

Treatment of experimental allergic encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP) epitope 72-85.

In this report the rational design, synthesis and pharmacological properties of an amide-linked cyclic antagonist analogue of the guinea pig myelin basic protein epitope MBP(72-85) are described. Design of the potent cyclic analogue was based on 2D NOESY nuclear magnetic resonance and molecular dynamics studies carried out in the linear antagonist Ala81MBP(72-85). The cyclic antagonist completely prevented the induction of experimental allergic/autoimmune encephalomyelitis when coinjected with linear and cyclic agonist analogues MBP(72-85) and cyclo(2-9)MBP(72-85).

Design and synthesis of a potent cyclic analogue of the myelin basic protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for induction of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP), such as guinea pig sequence MBP72-85. Two linear and one cyclic analogues based on MBP72-85 have been synthesized and evaluated for EAE induction in Lewis rats. The linear peptide Gln1-Lys2-Ser3-Gln4-Arg5-Ser6-Gln7-+ ++Asp8-Glu9-Asn10-Pro11-Val12 (1) was found to induce EAE, while substitution of the Asp residue at position 8 with Ala resulted in an analogue (2) which suppressed the induction of EAE by its parent peptide. Nuclear magnetic resonance studies of analogue 1 in dimethyl sulfoxide (DMSO) using TOCSY/ROESY techniques revealed a head-to-tail intramolecular interaction (ROE connectivity between betaVal12-gammaGln1), indicating a pseudocyclic conformation for the immunogenic peptide 1. A conformational model was developed using NMR constraints and molecular dynamics. Based on this model, a novel amide-linked cyclic analogue has been designed and synthesized by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9, respectively, of linear analogue 1. The cyclic analogue (3) had similar activity to the linear peptide 1, and the EAE effects induced by cyclic analogue 3 were completely suppressed by co-injection with the Ala81-substituted analogue 2 in Lewis rats. The similar potencies of analogues 1 and 3 support the proposed cyclic comformation suggested for analogue 1 from NMR studies and computer modeling and provides the basis for designing more potent molecules with improved properties such as increased resistance to degradation.15 The present findings suggest that a cyclic conformation for the MBP72-85 epitope positions the carboxyl group of Asp81 correctly and presumably other side groups of the peptide such as Arg78 in a manner which enables functional binding of the trimolecular complex MHC-peptide-T cell receptor resulting in EAE.

Experimental Evidence of Specific Solute-Solvent Interactions of Fullerenes C60 and C70 in Solution: One-Dimensional Steady-State Intermolecular 13C, 1H Overhauser Effect Study

Steady-state one-dimensional selective intermolecular carbon-13, proton-1 NOE experiments provide the first experimental evidence of specific solute-solvent interactions of fullerenes C60 and C70 in solution. Copyright 1998 Academic Press. Copyright 1998 Academic Press

Involvement of phospholipids in apolipoprotein B modification during low density lipoprotein oxidation.

An increased amount of phospholipids remained attached on delipidated apolipoprotein B originated from oxidized low density lipoprotein (LDL). 31P nuclear magnetic resonance analysis of such apolipoprotein showed an organic phosphorus peak at -0.55 ppm, which suggests the formation of adducts (most probably Schiff bases) of oxidized phospholipids with apolipoprotein B. The above reaction occurs in parallel with the hydrolysis of oxidized phospholipids, catalyzed by the LDL-attached platelet-activating factor acetylhydrolase, and may contribute to the proatherogenic effect of oxidatively modified low density lipoprotein.

17O NMR Studies of the Solvation State of cissolidustrans Isomers of Amides and Model Protected Peptides

17O shielding constants have been utilized to investigate solvation differences of the cissolidustrans isomers of N-methylformamide (NMF), N-ethylformamide (NEF), and tert-butylformamide (TBF) in a variety of solvents with particular emphasis on aqueous solution. Comparisons are also made with protected peptides of the formulas CH3CO-YOH, CH3CO-Y-NHR (Y = Pro, Sar), and CH3CO-Y-Z-NHR (Y = Pro; Z = D-Ala) selectively enriched in 17O at the acetyl oxygen atom. Hydration at the amide oxygen induces large and specific modifications of the 17O shielding constants, which are practically the same for the cis and trans isomers of NMF, NEF, and the protected peptides. For tert-butylformamide, the strong deshielding of the trans isomer compared to that of the cis isomer may be attributed to an out-of-plane (torsion-angle) deformation of the amide bond andsolidusor a significant reduction of solvation of the trans isomer due to steric inhibition of the bulky tert-butyl group. Good linear correlation between delta(17O) of amides and delta(17O) of acetone was found for different solvents which have varying dielectric constants and solvation abilities. Sum-over-states calculations, within the solvaton model, underestimate effects of the dielectric constant of the medium on 17O shielding, while finite-perturbation-theory calculations give good agreement with the experiment.

Modes of association of concanavalin A with alpha-D-glycosides.

Complexes of Con A with alpha-D-glycosides were studied using 1H-NMR, ESR and fluorescence methods. Correlation times, tau c, for the interaction of the aglycon protons with the manganese ion, present at the S1 site of the protein, were calculated from T1 measurements at two frequencies. The protons of aromatic aglycons have tau c values comparable to the rotational correlation time of the protein molecule, whereas those of non-aromatic aglycons have tau cs 10 to 100 times lower. The correlation times were combined with the experimentally acquired paramagnetic contributions to proton relaxation due to the presence of the manganese ion to yield manganese-proton distances. These distances show that aromatic aglycons have additional favorable contacts with the protein which stabilize the lectin-saccharide interaction. The results are compared to the crystal structure of the methyl alpha-D-glycopyranoside complex with Con A and to models earlier proposed for the binding of monosaccharides to Con A.

Models of binding of 4'-nitrophenyl alpha-D-mannopyranoside to the lectin concanavalin A.

Molecular models for the complex formed between the lectin concanavalin A (Con A) and the saccharide derivative 4'-nitrophenyl-alpha-D-mannopyranoside (alpha-PNM) are presented, combining evidence from 1H-n.m.r. measurements, semi-empirical energy calculations and interactive graphics modelling. The models are in good agreement with the experimental data. Close examination of the models suggests that hydrophobic interactions together with van der Waals interactions and hydrogen bonds contribute to the stability of the complexes. It appears that there is a limited number of possible modes of binding of alpha-PNM to Con A.