In this work, we present a new NMR study, coupled with chemometric analysis, on nonvolatile organic honey components. The extraction method is simple and reproducible. The 1H NMR spectra of chloroform extracts acquired with a fast and new pulse sequence were used to characterize and differentiate by chemometric analysis 118 honey samples of four different botanical origins (chestnut, acacia, linden, and polyfloral). The spectra collection, processing, and analysis require only 30 min. The 1H spectrum provides a fingerprint for each honey type, showing many characteristic peaks in all spectral regions. Principal component analysis (PCA) and projection to latent structures by partial least squares-discriminant analysis (PLS-DA) were performed on selected signals of the spectra to discriminate the different botanical types and to identify characteristic metabolites for each honey type. A distinct discrimination among samples was achieved. According to the distance to model criterion, there was no overlap between the four models, which proved to be specific for each honey type. The PLS-DA model obtained has a correlation coefficient R2 of 0.67 and a validation correlation coefficient Q2 of 0.77. The discriminant analysis allowed us to classify correctly 100% of the samples. A classification index can be calculated and used to determine the floral origin of honey as an alternative to the melissopalinology test and possibly to determine the percentage of various botanical species in polyfloral samples. Preliminary data on the identification of marker compounds for each botanical origin are presented.

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially ␣-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.Human cytomegalovirus (HCMV) is a human pathogen responsible for a variety of severe diseases in immunocompromised patients, including pneumonia, gastrointestinal disease, and retinitis in transplant recipients and in patients with AIDS (7). HCMV is also a major cause of congenital defects in newborn children (51). Antiviral agents currently licensed for the treatment of HCMV infections include ganciclovir, foscarnet, and cidofovir, which all inhibit the HCMV DNA polymerase (21). Ganciclovir and cidofovir are nucleoside analogs which function as DNA chain terminators, whereas foscarnet inhibits viral DNA polymerase through binding to its pyrophosphate binding site (12). There is renewed interest in the search for new HCMV inhibitors because of the emergence of drug-resistant viral strains, particularly in immunocompromised patients (42), and because some of these antiviral agents, e.g., ganciclovir and foscarnet, have toxic side effects. Since the HCMV DNA polymerase represents a major target for antiviral chemotherapy, further studies on this enzyme could be important in developing novel drugs.HCMV DNA replication has not been as fully characterized as that of herpes simplex virus (HSV); however, HCMV homologs to six of the seven proteins essential for HSV type 1 (HSV-1) replication (53) have...

The possibility of tracing the botanical and geographical origin of products such as honey has become more important because of market globalization. As a consequence, numerous analytical methods have been applied to the determination of honey authenticity. The scope of the present work is to chromatographically purify and characterize 23 compounds from organic extracts of unifloral (chestnut, linden, orange, acacia, eucalyptus, honeydew) and polyfloral honeys. Of these compounds, 17 were identified as specific markers and were used for botanical discrimination in a previous study based on multivariate statistical analysis of proton nuclear magnetic resonance ((1)H NMR) data. Together with the botanical markers, 6 other substances were isolated and characterized using NMR and mass spectrometry. These phytochemicals belong to several classes, that is, terpenes, organic acids, flavonoids, and others. For the first time, a diacylglyceryl ether and 5 other compounds present in different types of honey were identified and characterized.

NMR can be used in food analysis for origin discrimination and biomarker discovery using a metabolomic approach. Here, we present an example of this strategy to discriminate honey samples of different botanical origins. The NMR spectra of 353 chloroform extracts of selected honey samples were analyzed to detect possible markers of their floral origin. Six monofloral Italian honey types (acacia, linden, orange, eucalyptus, chestnut, and honeydew) were analyzed together with polyfloral samples. Specific markers were identified for each monofloral origin: two markers for acacia (chrysin and pinocembrin), one for chestnut (γ-LACT-3-PKA), two for orange (8-hydroxylinalool and caffeine), one for eucalyptus (dehydrovomifoliol), one for honeydew (a diacylglycerilether) and two for linden (4-(1-hydroxy-1-methylethyl)cyclohexa-1,3-diene-carboxylic acid and 4-(1-methylethenyl)cyclohexa-1,3-diene-carboxylic acid). An NMR-based metabolomic approach that used O2PLS-DA multivariate data analysis allowed us to discriminate the different types of honey. Two different classifiers were built based on different multivariate techniques. The high precision of the classification obtained suggests that this approach could be useful to develop generally applicable metabolomic tools to discriminate the origin of honey samples

Oligopeptides based on protein (C R -trisubstituted) R-amino acids are known to undergo R-helix a unordered conformation transitions under appropriate experimental conditions. 1 On the other hand, oligopeptides rich in C R -tetrasubstituted R-amino acids present a rather peculiar stereochemistry due to significant constraints imposed on their conformational freedom by these residues. 2 Specifically, most of the C R -tetrasubstituted R-amino acids have been extensively documented to possess a very high intrinsic helix-forming capacity. The narrow conformational space accessible includes both the classical R-helix and the 3 10 -helix. 3 It was shown that among the C R -tetrasubstituted chiral R-amino acids the -branched C R -methyl-L-valine [L-(RMe)Val] is the residue with the most pronounced bias toward the right-handed 3 10 -helix. 2d Several initial experimental evidences have indicated that the terminally blocked -[L-(RMe)Val] 8 -sequence adopts a fully developed, right-handed 3 10 -helical conformation both in the crystal state and in structure-supporting solvents. 4 More recently, the interesting property of this peptide to fold in solution both in the 3 10 -and in the R-helix has emerged. 4c The type of helical conformation adopted was found to depend on experimental conditions, as clearly shown by vibrational and electronic CD techniques. Under appropriate conditions, the conformational transition from 3 10 -to R-helix is very slow (the time scale is on the order of several days).Our observations can be rationalized on the basis of a stabilization of the 3 10 -helical structure by interpeptide interactions. This conclusion is supported by experimental evidence that the extent of peptide self-association is enhanced by increasing concentration and decreasing solvent polarity [e.g., from 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) to 2,2,2-trifluoroethanol (TFE)] as well as temperature. If this assumption is correct, then the conformational transition between the two types of helical structures can be slow because the first step of this process requires disruption of the molecular aggregates. Indeed, this structural helix-helix transition was theoretically predicted to take place very rapidly at the monomeric level. 3b Different (3 10 -and R-helical) polymorphic forms have already been found in the crystal state for mixed oligopeptides of protein and C R -tetrasubstituted R-amino acids. 5 However, our -[L-(RMe)Val] 8 -homooligopeptide system represents a unique tool to fully characterize both kinds of helices in solution and to study their conversion by NMR.In the present work, we initially performed a CD study (Jasco model J-715 dichrograph) of Ac-[L-(RMe)Val] 8 -OtBu (Ac, acetyl; OtBu, tert-butoxy) at a peptide concentration (=1 × 10 -2 M) appropriate for an NMR analysis (results not shown). In TFE, the CD spectrum is typical of a right-handed 3 10 -helical peptide, 4b displaying a negative band at 207 nm accompanied by a weak shoulder centered at 222 nm (the ratio R ) [Θ] 222 /[Θ] 207 is =0.3). This CD pat...

Roasted coffee is subject to commercial frauds, because the high-quality Coffea arabica species, described as "100% Arabica" or "Highland coffee", is often mixed with the less expensive Coffea canephora var. Robusta. The quantification of 16-O-methylcafestol (16-OMC) is useful to monitor the authenticity of the products as well as the Robusta content in blends. The German standard method DIN 10779 is used in the determination of 16-OMC in roasted coffee beans to detect C. canephora in blends, but it is laborious and time-consuming. Here, we introduce a new method that provides a quantitative determination of esterified 16-OMC directly in coffee extracts by means of high-resolution proton nuclear magnetic resonance spectroscopy. Limit of detection and limit of quantitation were 5 and 20 mg/kg, respectively, which are adequate to detect the presence of Robusta at percentages lower than 0.9%. The proposed method is much faster, more sensitive, and much more reproducible than the DIN standard method.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.

The use of band-selective excitation with adiabatic pulses to rapidly obtain NMR spectra of trace components in the presence of strong signals is described, along with qualitative and quantitative examples from food matrices like olive oil and honey.