Evaristo Peggion

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.

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.

Phenylalanine oligopeptides: Circular dichroism studies in solution

Abstract The conformations of a series of l -phenylalanine oligomers having the general formula BOC-(Phe) n -OMe ( n = 1–9) were investigated by circular dichroism in a number of solvent systems. These studies indicate that in trifluoroethanol and in hexafluoroisopropanol these oligomers probably form β-associated conformations beginning at the hexamer.

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.

Solution properties of synthetic polypeptides. light scattering and viscosity of poly-γ-ethyl-l-glutamate in dichloroacetic acid and trifluoroethanol

Abstract Poly-γ-ethyl- l -glutamate (PELG) has been studied by viscometry and light scattering in trifluoroethanol (TFE) and dichloroacetic acid (DCA) solutions. In DCA the following Mark-Houwink equation was obtained: [η]=2·02 × 10−4 × M0.73; the exponent is typical of random coiled polymers. In TFE, the equation was [η]=5·01 × 10−7 × M1.30; the exponent in this case indicates a rod-like structure with some flexibility. From the experimental radius of gyration (RG) in TFE, determined by light scattering, the average length per monomeric residue h was calculated. This quantity was found to be strongly dependent on the molecular weight of the polymer. By extrapolation to zero molecular weight, h 0 ∼ 2·0 A .

Conformation and membrane activity of an analogue of the peptaibol antibiotic trichogin GA IV with a lipophilic amino acid at the N-terminus.

We have synthesized by solution‐phase methods two analogues of the 11‐residue lipopeptaibol antibiotic trichogin GA IV in which the N‐terminal n‐octanoyl group is replaced either by an N‐acetylated 2‐amino‐2‐methyl‐l‐undecanoic acid or by an N‐acetylated α‐aminoisobutyric acid. CD, FTIR absorption, and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane‐modifying properties, these results strongly support the view that moving the long acyl moiety from the Nα‐blocking group to the side chain of the N‐terminal extra‐residue does not affect the conformational properties or the membrane activity of trichogin GA IV.

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.

Threonine6‐bradykinin: Conformational study of a flexible peptide in dimethyl sulfoxide by NMR and ensemble calculations

The conformation of the natural peptide threonine6 (Thr6)‐bradykinin, Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Thr6‐Pro7‐Phe8‐Arg9, was investigated in DMSO by nmr spectroscopy and computer simulations. The structural analysis of the Thr6‐peptide is made particularly interesting by the fact that despite the high sequence homology with native bradykinin (only one conservative substitution: Ser6/Thr6) there is a marked and significant difference in the biological profiles of the two peptides.

Investigation of crudes of synthesis of neuropeptide Y by high-performance liquid chromatography-electrospray mass spectrometry

Abstract Neuropeptide Y (NPY) and its modified form, [Leu 31 , Pro 34 ]NPY, are both thirty six amino acids long and they have relative molecular masses of 4250 and 4220. Solid-phase synthesis of both peptides resulted in complex crudes of reaction, which were investigated by means of combines high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS). The combination of these two powerful analytical techniques allowed rapid and reliable identification of the target peptides and furnished comprehensive information on other reaction products, which were mainly peptidic chains containing a smaller number of amino acids compared to those present in the intact peptides. The possible origin of such side-products and the eventual purification and unambiguous identification of both peptides are discussed.