Novel determination of the total phenolic content in crude plant extracts by the use of 1H NMR of the -OH spectral region.

A novel method for the determination of the total phenolic content using (1)H NMR spectroscopy in the -OH spectral region is presented. The use of DMSO-d(6), which is an aprotic and strongly hydrogen bonding solvent, allows the "appearance" of the relative sharp resonances of phenolic hydroxyl protons in the region of 8-14 ppm. The determination of the total phenolic -OH content requires three steps: (i) a 1D (1)H NMR spectrum is obtained in DMSO-d(6); (ii) a subsequent 1D (1)H NMR spectrum is recorded with irradiation of the residual water signal which results in the elimination or reduction of the phenolic -OH groups, due to proton exchange; and (iii) 1D (1)H NMR spectra are recorded with the addition of a progressively increased amount of salt, NaHCO(3), which results in extensive linebroadening of the COOH resonances thus allowing the discrimination of the phenolic from the carboxylic acid signals. Integration, with respect to the internal standard TSP-d(4), of the signal resonances between 14 and 8 ppm in spectrum (i) which are either eliminated or reduced in intensity in steps (ii) and (iii) allows the quantitation of the total phenolic content. The method was applied to model compounds, a mixture of them and several extracts of natural products. The results of the proposed (1)H NMR method were compared to the Folin-Ciocalteu (FC) reagent method. Additionally, since (1)H NMR refers to the total phenolic hydroxyl protons, a reaction factor, A(e), is proposed that corresponds to the hydroxyl reactivity. The (1)H NMR method is rapid and accurate bearing the inherent advantages of the NMR spectroscopy and can be applied directly in complex extracts. Furthermore, it can be applied in a wide range of matrixes from crude plant extracts and food products to biological samples.

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.

The (17)O-NMR shielding range and shielding time scale and detection of discrete hydrogen-bonded conformational states in peptides.

The (17)O-NMR shielding range and shielding time scale due to hydrogen-bonding interactions in peptides are critically evaluated relative to those of (1)H-NMR. Furthermore, the assumptions and conclusions in previous (17)O-NMR studies on the detection of discrete conformational states in peptides (V. Tsikaris et al., Biopolymers, 2000, Vol. 53, pp. 135-139) are reconsidered. Consistent examination of the method demonstrates that although (17)O shieldings of peptide oxygens are very sensitive to hydrogen bonding interactions, the (17)O-NMR shielding time scale is not advantageous compared to that of (1)H-NMR, and thus it is not suitable for the detection of discrete hydrogen-bonded conformational states in peptides. (17)O-NMR spectroscopy is prone to interpretation errors due to the formation of (17)O-labeled impurities during the synthetic procedures (A. Steinschneider et al., International Journal of Peptide and Protein Research, 1981, Vol. 18, pp. 324-333).

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.

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.

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.

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.

13C- and 57Fe-NMR studies of the Fe-C-O unit of heme proteins and synthetic model compounds in solution: comparison with IR vibrational frequencies and X-ray structural data.

13C- and 57Fe-NMR spectra of several carbon monoxide hemoprotein models with varying polar and steric effects of the distal organic superstructure, constraints of the proximal side, and solvent polarity are reported. The 13C shieldings of heme models cover a 4.0 ppm range that is extended to 7.0 ppm when several hemoglobin CO and myoglobin CO species at different pHs are included. Both heme models and heme proteins obey a similar excellent linear delta(13C) versus nu(C-O) relationship that is primarily due to modulation of pi backbonding from Fe d pi to the CO pi* orbital by the distal pocket polar interactions. There is no direct correlation between delta(13C) and Fe-C-O geometry. The poor monotonic relation between delta(13C) and nu(Fe-C) indicates that the iron-carbon pi bonding is not a primary factor influencing delta(13C) and delta(57Fe). The delta(57Fe) was found to be extremely sensitive to deformation of the porphyrin geometry, and increased shielding by more than 600 ppm with increased ruffling was observed for various heme models of known X-ray structures.

The effects of atropisomerism and porphyrin deformation on 57Fe shieldings in superstructured hemoprotein models.

57Fe NMR chemical shifts of superstructured heme model compounds have been found to be extremely sensitive to atropisomerism and deformation (ruffling) of the porphyrin geometry.

13C CP/MAS NMR Studies of Hemoprotein Models with and without an Axial Hindered Base: (13)C Shielding Tensors and Comparison with Hemoproteins and X-ray Structural Data.

13C cross-polarization magic-angle-spinning (CP/MAS) NMR spectra of several carbonmonoxide (93-99% (13)C enriched) hemoprotein models with 1,2-dimethylimidazole (1,2-diMeIm) and 1-methylimidazole (1-MeIm) as axial ligands are reported. This enables the (13)CO spinning sideband manifold to be measured and hence the principal components of the (13)CO chemical shift tensor to be obtained. Negative polar interactions in the binding pocket of the cap porphyrin model and inhibition of Fe-->CO back-donation result in a reduction in shielding anisotropy; on the contrary, positive distal polar interactions result in an increase in the shielding anisotropy and asymmetry parameter in some models. It appears that the axial hindered base 1,2-dimethylimidazole has little direct effect on the local geometry at the CO site, despite higher rates of CO desorption being observed for such complexes. This suggests that the mechanism by which steric interactions are released for the 1,2-diMeIm complexes compared to 1-MeIm complexes does not involve a significant increase in bending of the Fe-C-O unit. The asymmetry of the shielding tensor of all the heme model compounds studied is smaller than that found for horse myoglobin and rabbit hemoglobin.

31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration.

31P-NMR spectra of NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross-polarization, magic-angle spinning and high-power proton-decoupling on both lyophilized and hydrated samples. Previous studies on the lyophilized complex of L. casei dihydrofolate reductase with NADPH and methotrexate, measuring the isotropic shifts and principal components of the chemical shift tensors, have shown that the 2'-phosphate group of bound NADPH exists as a mixture of the dianionic and monoanionic states [Gerothanassis, I. P, Barrie, P. J., Birdsall, B. & Feeney, J. (1994) Eur J. Biochem. 226, 211-218]. In the present study on hydrated samples, the characterization of the isotropic shift and chemical shift tensors of the 2'-phosphate signal indicates that the 2'-phosphate is almost exclusively in the dianionic state. This is in agreement with earlier 31P-NMR studies in solution [Feeney, J., Birdsall, B., Roberts, G. C. K. & Burgen, A. S. V. (1975) Nature 257, 564-566]. In experiments examining progressively hydrated (6%, 12%, 15%, by mass) samples, the observed signals become increasingly narrower probably because the microenvironments of the 31P nuclei become more homogeneous upon sample hydration. Chemical exchange between mobile water molecules and bound protons close to individual sites on NADPH has been indirectly monitored on a hydrated sample (15% water, by mass) using a pulse sequence proposed by Harbison and coworkers [Harbison, G. S., Roberts, J. E., Herzfeld, J. & Griffin, R. G. (1988) J. Am. Chem. Soc. 110, 7221-7223]. In this experiment, the two diphosphate signals are totally suppressed while the 2'-phosphate phosphorus signal remains: this indicates a significant polarization of the 2'-phosphate nuclei from protons in exchange with those of mobile water molecules.

31P-NMR studies of NADPH, NADP+ and the complex of NADPH and methotrexate with Lactobacillus casei dihydrofolate reductase in the solid state.

31P-NMR spectra on solid samples of NADP+, NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross polarisation, magic angle spinning and high power proton decoupling. The isotropic chemical shifts, the principal components of the shielding tensors and the asymmetry parameters for the 31P nuclei in the 2'-phosphate and pyrophosphate groups have been measured. The isotropic shifts show similar trends to the chemical shifts measured in solution. The isotropic shifts and the shielding tensors for the dianionic and monoanionic states of the 2'-phosphate group have been determined and the presence of both ionisation states has been detected in a solid sample of the lyophilised complex of L. casei dihydrofolate reductase with NADPH and methotrexate. This contrasts with the behaviour in solution, where only the dianionic form is bound to the enzyme. The signals from the two pyrophosphates 31P nuclei in bound NADPH were resolved and identified. The asymmetry parameters in the different ionisation states and the orientations of the shielding tensors within the molecular framework are considered in the context of previous 31P studies on phosphate-containing compounds.

Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]-enkephalin in aqueous solution as revealed by the combined use of 17O-NMR and Fourier-transform infrared spectroscopy.

Solvent-induced and temperature-induced 17O chemical shifts of [17O-Gly2, Leu5]-enkephalin and [17O-Gly3, Leu5]-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu5]-enkephalin and [1-13C-Gly2, Leu5]-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 17O-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.

Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution.

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.

17O NMR and FT-IR study of the ionization state of peptides in aprotic solvents. Application to Leu-enkephalin.

The ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions.

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.

Does a 2----5 beta-turn structure exist in enkephalins? Study of a fully protected Leu-enkephalin in organic solution by 17O-NMR.

The 17O chemical shifts of the Gly-2 and Gly-3 oxygens of a fully protected Leu-enkephalin were measured to be identical in acetone solution. This allows the conclusion that neither of these peptide oxygens is hydrogen bonded and that no specific 2----5 beta-turn structure exists to an appreciable extent.