Elisabetta Schievano

1H nuclear magnetic resonance spectra of chloroform extracts of honey for chemometric determination of its botanical origin.

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.

An NMR-based metabolomic approach to identify the botanical origin of honey

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.

Characterization of markers of botanical origin and other compounds extracted from unifloral honeys.

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.

Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

ABSTRACT 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 α-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.

NMR quantification of trace components in complex matrices by band-selective excitation with adiabatic pulses.

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. Copyright

Green coffee oil analysis by high-resolution nuclear magnetic resonance spectroscopy

In this work, we show how an extensive and fast quantification of the main components in green coffee oil can be achieved by NMR, with minimal sample manipulation and use of organic solvents. The approach is based on the integration of characteristic NMR signals, selected because of their similar relaxation properties and because they fall in similar spectral regions, which minimizes offset effects. Quantification of glycerides, together with their fatty acid components (oleic, linoleic, linolenic and saturated) and minor species (caffeine, cafestol, kahweol and 16-O-methylcafestol), is achieved in less than 1h making use of (1)H and (13)C spectroscopy. The compositional data obtained are in reasonable agreement with classical chromatographic analyses.

Fast Determination of Histamine in Cheese by Nuclear Magnetic Resonance (NMR)

A rapid and quantitative (1)H nuclear magnetic resonance (NMR) method was developed to analyze histamine in cheeses. The procedure is simple because the acid extract is analyzed directly, without any need for further filtration, derivatization, or other manipulation. This NMR method was demonstrated to be specific by 2D total correlation spectroscopy (TOCSY) and heteronuclear multiple-quantum coherence (HMQC) experiments and reliable in terms of linearity, accuracy, recovery, repeatability, and limits of detection (LOD). Good precision, with relative standard deviation (RSD) <4%, recovery of 100%, and a range of 0.6-1 mg/kg for the LOD were obtained. The NMR method was successfully applied to different types of cheese, ranging from soft to hard. No interference from free amino acids, proteins, and other natural components was detected. The NMR method could be transferred to other kinds of food.

Total Synthesis, Characterization, and Conformational Analysis of the Naturally Occurring Hexadecapeptide Integramide A and a Diastereomer

Integramide A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L-Iva(14)-D-Iva(15). Herein, we describe the syntheses of the natural compound and its D-Iva(14)-L-Iva(15) diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy.

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.

Conformation and interactions of bioactive peptides from insect venoms: the bombolitins.

Bombolitins are five structurally related heptadecapeptides originally isolated from the venom of a bumblebee, acting at membrane level and able to enhance the activity of Phospholipase A2. The biological activity of this class of natural peptides seems to be related to the their ability to form amphiphilic helical structures in the presence of phospholipid aggregates or related membrane model systems. We have carried out systematic investigations on a series of bombolitins and their synthetic analogs in order to establish the conditions in which amphipathic helices are formed and to elucidate the details of the interaction with phospholipids and related model systems. We have shown that bombolitins and their analogs interact with phospholipid aggregates and detergent micelles forming amphiphilic helices. By means of the Langmuir film balance technique, coupled with fluorescence microscopy, we have Shown that bombolitins perturbe the structure of phospholipid monolayers, forming phase separated peptide domains. In aqueous solution, in the absence of detergent or phospholipids, bombolitins form oligomeric aggregates with consequent conformational transition from a random coil to an alpha-helical structure. In the aggregate structure, evidence was obtained that helices are oriented in an antiparallel fashion. In this article we summarize the most recent results of conformational studies by CD, NMR and computer simulations on a series of bombolitins and retro-, all-D- and all-D-retro-analogs.