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In this first work, commercial steak-like (n= 3) and cured meat (n= 3) analogues with different legume and cereal formulations were studied and compared to their animal-based (n= 3) counterparts. Plant-based products showed lower protein content than meat controls but a good amino acidic profile even though the sum of essential amino acids of plant-cured meats does not fulfill the requirements set by the Food and Agriculture Organization for children. A comparable release of soluble proteins and peptides in the digestates after in vitro digestion was observed in meat analogues as meat products, whereas the digestibility of proteins was lower in plant-based steaks and higher in plant-based cured meats than their counterparts. The overall protein quality and digestibility of products are related to both the use of good blending of protein sources and processes applied to produce them. An adequate substitution of meat with its analogues depends mostly on the quality of raw materials used, which should be communicated to consumers.

Background: In Italy, there is scant evidence on the impact of Community Hospitals (CHs) on clinical outcomes. Aims: To assess the effectiveness of CHs versus long-term care hospital or inpatient rehabilitation facilities on mortality, re-admission, institutionalization, and activation of a home care programme in the Emilia-Romagna Region (ERR-Italy) after acute hospitalisation. Methods: We implemented a cohort study drawing upon the ERR Administrative Healthcare Database System and including hospital episodes of ERR residents subject ≥ 65 years, discharged from a public or private hospital with a medical diagnosis to a CH or to usual care between 2017 and 2019. To control for confounding, we applied a propensity score matching. Results: Patients transferred to CHs had a significantly lower risk of dying but an increased risk of being readmitted to community or acute hospital within 30/90 days from discharge. The hazard of institutionalisation within 30/90 days was significantly lower in the whole population of the CH exposed group but not among patients with cardiac or respiratory chronic diseases or diabetes. The activation of a home care program within 90 days was slightly higher for those who were transferred to a CH. Discussion: The findings of our study show mixed effects on outcomes of patients transferred to CHs compared to those who followed the post-acute usual care and should be taken with cautious as could be affected by the so-called ‘confounding by indication’. Conclusions: The study contributes to the intermediate care available evidence from a region with a well-established care provision through CHs.

A significant quantity of bone-rich poultry by-products must be disposed of by poultry processors. These products still contain a significant amount of nutritionally valuable animal proteins. In the present work, a hydrolysis protocol was optimized to recover the protein fraction of bone-rich poultry by-products while simultaneously minimizing the amount of water required for hydrolysis (thus reducing drying costs) and recycling the hydrolytic broth up to 3 times, to reduce the cost of the proteolytic enzyme. The final hydrolysis conditions involved the use of (protease from B. licheniformis, ≥2.4 U/g; 0.5 V/w of raw material) and a hydrolysis time of 2 h at 65°C. The protein hydrolysate obtained has a high protein content (79–86%), a good amino acid profile (chemical amino acid score equal to 0.7–0.8) and good gastric digestibility (about 30% of peptide bonds are already hydrolyzed before digestion). This supports its use as an ingredient in food, pet food or animal feed formulations.

This chapter aims to address an issue of ancient origins, but more and more topical in a globalized world in which consumers and stakeholders are increasingly aware: the authenticity of food. Foods are systems that can also be very complex, and verifying the correspondence between what is declared and the actual characteristics of the product is often a challenging issue. The complexity of the question we want to answer (is the food authentic?) means that the answer is equally articulated and makes use of many different analytical techniques. This chapter will consider the chemical analyses of foods aimed at guaranteeing their authenticity and will focus on frontier methods that have been developed in recent years to address the need to respond to ever-increasing guarantees of authenticity. Targeted and non-targeted approaches will be considered for verifying the authenticity of foods, through the study of different classes of constituents (proteins, metabolites, lipids, flavors). The numerous approaches available (proteomics, metabolomics, lipidomics) and the related analytical techniques (LC-MS, GC-MS, NMR) are first described from a more general point of view, after which their specific application for the purposes of authentication of food is addressed.

Durum wheat (Triticum turgidumL. subsp. durum(Desf.) Husn) is a major food source in Mediterranean countries since it is utilized for the production of pasta, leavened and unleavened breads, couscous, and other traditional foods. The technological and nutritional properties of durum wheat semolina depend mainly on the type of gluten proteins and on their amount, which is a genotype- and environment-dependent trait. Gluten proteins are also responsible for celiac disease (CD), an autoimmune enteropathy with a prevalence of about 0.7–2% in the human population. At this purpose, two Italian durum wheat cultivars, Saragolla and Cappelli, currently used for monovarietal pasta, were chosen to compare (i) the reserve and embryo proteome, (ii) the free and bound phenolics, antioxidant activity, and amino acid composition, and (iii) the content of immunogenic peptides produced after a simulated gastrointestinal digestion. The results obtained from 2 years of field cultivation on average showed a higher amount of gluten proteins, amino acids, and immunogenic peptides in Cappelli. Saragolla showed a higher abundance in bound phenolics, antioxidant enzymes, and stress response proteins in line with its higher antioxidant activity. However, the impact of the year of cultivation, largely depending on varying rainfall regimes through the wheat growth cycle, was significant for most of the parameters investigated. Differences in technological and nutritional characteristics observed between the two cultivars are discussed in relation to the influence of genetic and environmental factors.

The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz’s trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography–mass spectrometry (LC–MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 μg/g, respectively. The results of the LC–MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaeaL.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.

Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.

Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogues which have been described for the first time almost twenty years ago and were immediately found to be excellent tool in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems and in the so called anti-gene strategy make them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the modification of the PNA backbone, and their possible use in medicinal chemistry with an update of this topics in view of emerging new trends and opening of new possibilities In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several examples of their use in cellular or animal systems are presented, with exciting new fields of research such as microRNA (miR) targeting and gene repair.

The design and the synthesis of a PNA oligomer containing a pyrenyl residue in the backbone were performed. PNA sequence was chosen complementary to a "G rich" target sequence involved in G- quadruplex formation. The pyrenyl unit replaced a nucleobase in the middle of the PNA through covalent linkage to the backbone by a carboxymethyl unit. A systematic study on the binding properties of this probe towards DNA and RNA complementary strands was carried out by UV and fluorescence spectroscopy. UV melting curves indicated that the PNA probe bind more tightly to RNA rather than to DNA. Thermodynamic data obtained by Van't Hoff fitting of the melting curves indicated that, in the case of RNA, a more favorable interaction occurs between the pyrenyl unit and the RNA nucleobases, leading to a very favorable enthalpic contribution. The fluorescence analysis showed specific quenching of the pyrene emission associated to the formation of the full-match PNA-DNA or PNA-RNA duplexes. Again, this behavior was more evident in the case of RNA, consistent with the stronger interaction of the pyrenyl unit with the complementary strand. In order to study the sequence specificity of the pyrenyl-PNA probe (pyr-PNA), recognition experiments on mismatched DNA and RNA sequences were also performed.

In this paper, the synthesis of a short modified peptide nucleic acid PNA, obtained by using several different Lamino acids as synthons, is shown. The synthesis was performed by a submonomeric strategy, obtaining a model trimeric PNA containing embedded amino acid derived side chains in its backbone that mimic the peptide sequence PKKKRKV, which is a nuclear localization signal NLS widely used for translocating cargo molecules into cell nuclei. Fluorescence experiments demonstrated that this modified PNA, and not a standard unmodified PNA having the same nucleobase sequence, was able to penetrate Rhabdomyosarcoma cellnuclei, exactly behaving as the NLS standard peptide.

Two peptide nucleic acids (PNAs) containing three adjacent modified chiral monomers (chiral box) were synthesized. The chiral monomers contained either a C2- or a C5-modified backbone, synthesized starting from D- and L-arginine, respectively (2D- and 5L-PNA). The C2-modified chiral PNA was synthesized using a submonomeric strategy to avoid epimerization during solid-phase synthesis, whereas for the C5-derivative, the monomers were first obtained and then used in solid-phase synthesis. The melting temperature of these PNA duplexes formed with the full-match or with single-mismatch DNA were measured both by UV and by CD spectroscopy and compared with the unmodified PNA. The 5L-chiral-box-PNA showed the highest Tmwith full-match DNA, whereas the 2D-chiral-box-PNA showed the highest sequence selectivity. The PNA were spotted on microarray slides and then hybridized with Cy5-labeled full match and mismatched oligonucleotides. The results obtained showed a signal intensity in the order achiral >2D-chiral box >5L-chiral box, whereas the full-match/mismatch selectivity was higher for the 2D chiral box PNA. Chirality, 2010. © 2010 Wiley-Liss, Inc.

A new multiresidual LC‐DAD‐MS/MS method for the simultaneous detection of the major flavonoids and glycoalkaloids occurring in tomatoes has been developed and applied to the characterization of seven varieties of raw tomatoes grown in Italy and to their processed products. The new method allowed to determine rapidly the concentration of these secondary metabolites in a single analysis, showing that their content can be quite variable among the different varieties considered. On the contrary, a great depletion in glycoalkaloids and flavonoids is observed after processing, probably due to the harsh thermal treatment, which prevents the recognition of the varieties in the processed products.

The lipid transfer protein LTP, Pru p 3, has been identified as the major allergen present in peach, and its sequence obtained by direct amino acid sequencing has been previously reported. However, several sequences, obtained from cDNA and available in databases, show differences among them and from the originally proposed structure. In this paper, we report the fast and unambiguous determination of the structure of Pru p 3 protein, extracted from three different varieties of peach, by electrospray ionization mass spectrometry ESIMS, both coupled to single stage quadrupole or advanced FTHRMS analyzers. The structure was identical to one of the cDNAderived sequences and different in two positions from the previously reported structure obtained by amino acid sequencing. Moreover, the exclusive localization of the protein in the outer part of the fruits was assessed by MatrixAssisted Laser Desorption Ionization Mass Spectrometry Imaging MALDI MSI. The results reported here demonstrate the full potential of mass spectrometry for rapidly obtaining high quality structural data of relevant food proteins. Copyright © 2009 John Wiley & Sons, Ltd.

A chiral peptide nucleic acid (PNA) beacon containing a C‐5 modified monomer based on L‐lysine was synthesized. The terminal amino group of the lysine side chain was linked to a spacer for future applications on surfaces. The PNA beacon bears a carboxyfluorescein fluorophore and a dabcyl quencher at opposite ends. The DNA binding properties were compared with those of a homologous PNA beacon containing only achiral monomers. Both beacons underwent a fluorescence increase in the presence of complementary DNA, with higher efficiency and higher selectivity (evaluated using single mismatched DNA sequences) observed for the chiral monomer containing PNA. Ion exchange (IE) HPLC with fluorimetric detection was used in combination with the beacon for the selective detection of complementary DNA. A fluorescent peak corresponding to the PNA beacon:DNA duplex was observed at a very low detection limit (1 nM). The discriminating capacity of the chiral PNA beacon for a single mismatch was found to be superior to those observed with the unmodified one, thus confirming the potency of chirality for increasing the affinity and specificity of DNA recognition. Chirality, 2009. © 2008 Wiley‐Liss, Inc.

The interaction with DNA of a peptide nucleic acid (PNA) oligomer (16nt) conjugated with a nuclear localization signal (NLS) peptide, which was previously found to be able to inhibit tumor cell proliferation through block of transcription of the MYCNoncogene, was studied by UV and CD spectroscopy. While data obtained by UV were not conclusive, the use of circular dichroism gave clear‐cut evidence of the formation of a PNA:DNA duplex of exceptionally high stability (Tm≥ 90°C). Using the same approach, the effect of mutations on DNA:PNA stability was evaluated, and was found in accordance with that expected for a Watson–Crick interaction. The role of the NLS peptide was evaluated by using a PNA lacking of this part, which gave rise to less stable PNA:DNA duplexes. Finally, a competition experiment carried out with a 26mer dsDNA, containing the target 16mer sequence in its middle region, in the presence of PNA‐NLS gave evidence for the formation of a ternary complex at 25°, while at higher temperature, the PNA:DNA duplex and the displaced homologous DNA strand were detected. The present results support the possibility of an analogous mechanism of action of this antitumor PNA in vivo. Chirality, 2008. © 2007 Wiley‐Liss, Inc.

A systematic study on the combined effects of two lysine-derived stereogenic centres at C2 and C5 of a PNA monomer inserted within an achiral PNA strand was performed by circular dichroism, UV spectroscopy and ESI mass spectrometry by taking into account all four diastereomers. The stereogenic centres induced different preferential helix handednesses in the PNA strands, which in turn affected their ability to bind to the complementary DNA. The helical induction, studied on the PNA–PNA duplexes, evidenced that in the case of “chiral conflict” (induction of opposite helices), the stereogenic centre at C5 was clearly prevalent in determining the preferential helix handedness. The PNA–DNA duplex stability was found to be related to the number and the position of the stereogenic centres inducing a preference for a right-handed helix, as well as with the strength of the helical induction. Thermodynamic parameters evaluated by the Van't Hoff model clearly outlined the different enthalpic and entropic contributions of the substitutions at C2 and C5. These findings expand the knowledge of the role of punctual stereogenic centres in inducing supramolecular chirality in PNAs, demonstrate the ability of right-handed DNA to act as a chiral selector and are essential for the future design of chiral PNAs with improved DNA affinity.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

The understanding of the interaction of chiral species with DNA or RNA is very important for the development of new tools in biology and of new drugs. Several cases in which chirality is a crucial point in determining the DNA binding mode are reviewed and discussed, with the aim of illustrating how chirality can be considered as a tool for improving the understanding of mechanisms and the effectiveness of nucleic acid recognition. The review is divided into two parts: the former describes examples of chiral species interacting with DNA: intercalators, metal complexes, and groove binders; the latter part is dedicated to chirality in DNA analogs, with discussion of phosphate stereochemistry and chirality of ribose substitutes, in particular of peptide nucleic acids (PNAs) for which a number of works have been published recently dealing with the effect of chirality in DNA recognition. The discussion is intended to show how enantiomeric recognition originates at the molecular level, by exploiting the enormous progresses recently achieved in the field of structural characterization of complexes formed by nucleic acid with their ligands by crystallographic and spectroscopic methods. Examples of application of the DNA binding molecules described and the role of chirality in DNA recognition relevant for biotechnology or medicinal chemistry are reported. Chirality, 2007. © 2007 Wiley‐Liss, Inc.

Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogs which have been described for the first time fifteen years ago and were immediately found to be excellent tools in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems, and in the so called antigene strategy, makes them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the recent advancements obtained by the modification of the PNA backbone, and their possible use in medicinal chemistry. In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several recent examples of their use in cellular or animal systems are presented.

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A new DNA extraction method suitable for a wide variety of complex food matrices has been devised and applied in combination with a new polymerase chain reaction (PCR) method for the sensitive detection of a specific DNA sequence univocally identifying the presence of potentially allergenic hazelnut (Corylus avellana). A 156 base pair amplicon corresponding to an internal region of the complementary DNA of the major hazelnut allergen (Cor a 1) was designed, and was found to be highly specific; the method was tested on both pure and complex food matrices and was found to be able to confirm the presence of hazelnut down to 5 pg of its DNA. The sequence of the amplicon was further confirmed by high-performance liquid chromatography (HPLC) analysis with a specific peptide nucleic acid (PNA) probe. A 15-mer PNA probe was expressly designed and synthesized to hybridize an internal sequence of the previously described amplicon. Given its reported sequence specificity and high hybridization efficiency, the PNA probe was used to develop an anion-exchange HPLC method allowing for a fast and reliable confirmation of the identity of the amplified products. The PCR–HPLC method was successfully tested on commercial samples, allowing for the detection of the presence of potentially hidden allergens even in products where the presence of hazelnut as an ingredient or possible contaminant was not reported.

In this paper we report an innovative and unconventional method based on circular dichroism for the identification of peanut DNA in food, which can be detected after PCR amplification at the nanomolar level by using an achiral PNA probe complementary to a tract of the peanut Ara h 2 gene and an achiral 3,3′‐diethylthiadicarbocyanine dye [DiSC2(5)]. Peanuts are one of the most common causes of severe allergic reactions to foods and are particularly dangerous when they are “hidden” (undeclared) in food. For better protection of consumers, detection methods are required to specifically detect the presence of hidden allergens in a wide variety of food items. Alternative to the detection of the proteins is the determination of species‐specific DNA, which is more resistant to technological treatments. PNAs are very specific probes able to recognize DNA sequences with high affinity and evidence for the binding can be obtained by using the DiSC2(5) dye, which aggregates onto the PNA–DNA duplex giving rise to a characteristic visibile band at 540 nm. Because the PNA–DNA duplex is in a right‐handed helical conformation, the aggregation of the dye to the duplex gives also rise to a strong CD signal in the 500–600 nm region with a strong exciton coupling due to the formation of multimeric species, since the handedness of the helix is transferred to the dye aggregate. The dye does not interact with the free single‐stranded DNA and although aggregating on the achiral PNA, this interaction is obviously not detectable by circular dichroism. Thus, only the formation of the PNA–DNA duplex, which takes place only upon specific Watson–Crick hydrogen binding between the PNA and the DNA bases, is detected, ensuring a very high specificity and sensitivity. The method has been optimized in a model system by using a synthetic oligonucleotide complementary to the PNA probe, showing that the intensity of the signal is linearly related to the amount of the DNA. The optimized method has been applied to the identification and quantitation of DNA extracted and amplified by PCR from peanuts and from peanut‐containing foods, allowing for a very sensitive detection at a very low level (few pmol). © 2005 Wiley‐Liss, Inc. Chirality 17:515–521, 2005.

Modified cyclodextrins bearing a metal binding site and a dansyl fluorophore 6-deoxy-6-N-Nα-5-dimethylamino-1-naphthalenesulfonylaminoethyl-aminocylamino-β-cyclodextrin, containing l-Phe S-1, l-PhGly S-2 or l-Pro S-3 moieties, were used as enantioselective fluorescence sensors for the discrimination of enantiomers of the amino acids valine and proline, according to a ligand exchange mechanism. The best conditions to perform enantiomeric analyses were studied and a fast protocol using fluorescence quenching by the copperiiamino acid complexes in a fluorescence microplate reader was developed, allowing the detection of samples with high enantiomeric excess. Calibration of the fluorescence response as a function of enantiomeric composition was obtained using the Stern–Volmer model; the calibration curves showed good linearity, allowing fast evaluation of enantiomeric excess within 6 error. Two calibration curves and triplicate analyses of six valine samples were performed in two minutes.

Two enantiomeric chiral PNAs bearing three adjacent d‐ or l‐lysine‐based residues in the middle of the strand (“chiral box” PNAs, sequence H‐GTAGALysTLysCLysACT‐NH2) have been used as models in order to comprehensively study the effects of the stereogenic centers on PNA conformation and on PNA binding properties to complementary PNA and DNA strands. The binding properties of the two enantiomeric PNAs and of their homologous achiral PNA have been extensively studied by UV and CD spectroscopy and by mass spectrometry, both in the antiparallel and in the parallel mode with complementary PNA and DNA strands. In the antiparallel PNA:PNA duplexes, l‐Lys PNA were found to form left‐handed, and d‐Lys PNA right handed helices, while in parallel duplexes, the reversed helicities were observed. Correspondingly, the preferred mode of binding and the best mismatch recognition of the d‐Lys containing PNA with (right handed) DNA was found to be in the antiparallel orientation, while that of l‐Lys PNA was found to be in the parallel mode. A rationale which correlates the preferred handedness of the PNA‐PNA duplexes to the directionality of the binding to complementary DNA duplexes has been devised according to structural data and considering the “retro–inverso” concept widely used for peptides. Chirality 17:S196–S204, 2005.© 2005 Wiley‐Liss, Inc.

Two selectors based on modified cyclodextrins containing a metal binding site and a dansyl fluorophore—6‐deoxy‐6‐N‐(Nα‐[(5‐dimethylamino‐1‐naphthalenesulfonyl)aminoethyl]phenylalanylamino‐β‐cyclodextrin—containing D‐Phe (3) and L‐Phe (4) moieties were synthesized. The conformations of the two selectors were studied by circular dichroism, two‐dimensional NMR spectroscopy and time‐resolved fluorescence spectroscopy. Cyclodextrin 4was found to have a predominant conformation in which the dansyl group is self‐included in the cyclodextrin cavity, while 3showed a larger proportion of the conformation with the dansyl group outside the cavity. As a consequence, the two cyclodextrins were found to bind copper(II) with different affinities, as revealed by fluorescence quenching in competitive binding measurements. Addition of D‐ or L‐amino acids induced increases in fluorescence intensity, which were dependent on the amino acid used and in some cases on its absolute configuration. The cyclodextrin 4was found to be more enantioselective than 3, suggesting that the self‐inclusion in the cyclodextrin cavity strongly increases the chiral discrimination ability of the copper(II) complex. Accordingly, a linear fluorescent ligand Nα‐[(5‐dimethylamino‐1‐naphthalenesulfonyl)aminoethyl]‐N1‐propyl‐phenylalaninamide, which has the same binding site and absolute configuration as 4, showed very low chiral discrimination ability. The enantioselectivity in fluorescence response was found to be due to the formation of diastereomeric ternary complexes, which were detected by ESI‐MS and by circular dichroism. Time‐resolved fluorescence studies showed that the fluorescence of the dansyl group was completely quenched in the ternary complexes formed, and that the residual fluorescence was due to uncomplexed ligand.

The oligopeptide fractions of Grana Padano and Parmigiano Reggiano cheese samples, at various stages of ageing (6, 12, 18 and 24 months), were analysed by means of high-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS). The main oligopeptides (< 5000 Da) present in the samples were extracted by using an originally developed method which allowed good enrichment of the oligopeptide fractions and identified according to their molecular weights. The casein sequences compatible with the found molecular weights were determined and the exact oligopeptide sequences were identified by using the fragmentation peaks originated by in-source collisionally-induced dissociation (CID). The ESI-MS method reported here allowed the sequence identification of oligopeptides very quickly with a single liquid chromatography-mass spectrometry run. Oligopeptides were also semi-quantified by comparison with a suitable internal standard (the dipeptide phenylalanyl–phenylalanine, Phe–Phe). This methodology demonstrated that, as far as the main oligopeptides are concerned, Grana Padano and Parmigiano Reggiano presented very similar composition. Anyway, the evolution of the fractions during ageing time was characteristically distinct among the two cheeses: in Grana Padano cheeses most of the oligopeptides reached a maximum at 12 months of ageing and then decreased, whereas in Parmigiano Reggiano cheeses the oligopeptide amounts were usually lower and had a less regular trend. The reason for this different behaviour may be ascribed to the different production techniques.

Lipid A is the endotoxic principle of the lipopolysaccharide fraction from Gram-negative bacteria. It is involved in the elicitation of cytokine production that leads to massive inflammation and to septic shock as a lethal consequence. For this reason, the structural elucidation of lipopolysaccharides from toxic Gram-negative bacteria is an important and complicated task, mainly owing to its natural heterogeneity. Here, a new methodology to infer the distribution of the primary and secondary acyl residues is described, based on electrospray ionization mass spectrometry (ESI-MS) of intact lipid A under high cone voltage in order to achieve in-source fragmentation. Under these conditions, acyl fragmentation is induced and a different regioselective cleavage of secondary fatty acids is observed in positive and negative ESI-MS, allowing the rapid identification of the lipid A structure. Copyright © 2004 John Wiley & Sons, Ltd.

The solid-phase synthesis of chiral peptide nucleic acids (PNAs) usually results in partial epimerization of the products, since the α-nitrogen atom of the amino acid is involved in an amidic bond. It is also time-consuming, since all the chiral monomers bearing different nucleobases have to be independently synthesized. In order to prevent racemization and to speed up the synthetic procedure we adopted a submonomeric approach by using a solid-phase, Boc-based PNA synthesis in which the chiral backbone orthogonally N^α-Fmoc-protected (submonomer) was first linked to the growing chain on the resin, followed by Fmoc-deprotection and derivatization with the carboxymethylnucleobase. The submonomer bearing the D-lysine residue was designed by protecting the N^α-(aminoethyl)amino acid moiety with an Fmoc protecting group, compatible with standard Boc chemistry, and with the use of an MBHA-PS resin, normally employed for PNA synthesis. Different synthetic pathways towards the desired submonomer were studied by using the amino acid D-lysine as a chiral synthon, obtaining a fast method leading to a high yield and an excellent enantiomeric excess of the submonomer. The solid-phase submonomeric reaction conditions were optimized for the synthesis of a thyminyl PNA dimer and then used to synthesize two different chiral PNAs. In this way two advantages were obtained: a lower degree of racemization in the coupling step during the solid-phase synthesis and the possibility of using the same submonomer for every different nucleobase. All the D-lysine-based chiral PNAs were obtained in good yields and, as compared with PNAs synthesized by other coupling methods, showed the highest optical purity reported so far. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Peptide nucleic acids are DNA mimics able to form duplexes with complementary DNA or RNA strands of remarkable affinity and selectivity. Oligopyrimidine PNA can displace one strand of dsDNA by forming PNA2:DNA triplexes of very high stability. Many PNA analogs have been described in recent years, in particular, chiral PNA analogs. In the present article the results obtained recently using PNA derived from N‐aminoethylamino acids 7are illustrated. In particular, the dependence of optical purity on synthetic methodologies and a rationale for the observed effects of chirality on DNA binding ability is proposed. Chirality as a tool for improving sequence selectivity is also described. PNA analogs derived from D‐ or L‐ornithine 8were also found to be subjected to epimerization during solid phase synthesis. Modification of the coupling conditions or the use of a submonomeric strategy greatly reduced epimerization. The optically pure oligothymine PNAs 8were found to bind to RNA by forming triplexes of unusual CD spectra. The melting curves of these adducts presented two transitions, suggesting a conformational change followed by melting at high temperature. Chirality 14:591–598, 2002. © 2002 Wiley‐Liss, Inc.

The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10PNA onin vitrotranscription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is “roadblocked” by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.

Peptide nucleic acids (PNAs) are oligonucleotide analogues with a skeleton made up of N-(2-aminoethyl)glycine units; they bind to complementary DNA and RNA with high stability and specificity. In order to improve the binding specificity, solubility and uptake into cells, many modifications have been introduced, some concerning the introduction of stereogenic centres. With the aim of achieving a selective antiparallel binding with DNA, we report in this paper the synthesis and binding abilities of a chiral PNA decamer (H-GTAGATCACT-NH2) bearing three D-Lys-based monomers (a “chiral box”) in the middle of the strand. Indeed, the antiparallel PNA-DNA duplex showed a melting point of 43 °C (determined both by CD and UV spectroscopy), whereas the parallel PNA-DNA duplex failed to form, as shown by the absence of temperature dependence in the UV and CD spectra. Moreover, hybridization experiments carried out with antiparallel DNA strands bearing single mismatches showed that this PNA was excellent in discriminating between mismatched and matched targets. These results indicate that a high chiral constraint in the middle of a PNA sequence strongly affects the direction selectivity, i.e. the antiparallel/parallel preference in the DNA complexation. In particular, a “D-chiral box” favours a highly specific antiparallel DNA binding, thus allowing possible diagnostic applications for the screening of single-point mutations.

Peptide Nucleic Acids (PNAs) are DNA mimics in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide skeleton composed of N-(2-aminoethyl)glycine units; they bind to complementary DNA strands with high affinity and selectivity. In order to study the effect of stereogenic centers within the backbone on PNA preorganization and DNA binding properties, chiral PNA decamers were synthesized which contained thymine monomers derived from L-Leu and D- or L-Lys inserted either at C-terminus and/or in the middle of an achiral PNA strand. PNAs containing three chiral thymine monomers derived from L-Leu, D- or L-Lys, L-Asp, or D-Glu were also synthesized. CD spectral analyses showed that a charged chiral monomer inserted in the middle of the strand is able to induce a strong preference in the helix handedness of a PNA-PNA duplex. The effect is increased by the presence of three chiral charged monomers. The L-Lys- and L-Asp-PNAs induced a preference for the left-handed and the D-Lys and D-Glu-PNAs for the right-handed conformation. As expected, the PNA-DNA duplexes are dominated by the DNA strand and thus are right-handed with both D- and L-PNAs. However, the D-PNAs, being inherently right-handed, lead to more stable PNA-DNA duplexes than the L-PNAs. The lysine-based PNAs form more stable complexes with the DNA at low ionic strength, due to the electrostatic interactions between the charged lysine side chain and DNA.

The Vilsmeier reagents generated from tertiary and secondary aliphatic and aromatic amides and trifluoromethanesulfonic anhydride (Tf 2O) reacted with different nucleophiles (ROH, RSH, RNH 2) affording the relative iminium and amidinium salts. The former, stable at room temperature, were easily transformed into the corresponding esters or O-alkyl thioesters by treatment with OH −or SH −.

The synthesis of 10 new T-PNA monomers derived from L-amino acids is presented. The monomers were incorporated into decameric PNA oligomers, and the hybridisation with RNA, DNA and PNA complements studied by thermal stability measurements.

The aim of this review is to provide a brief summary of the recent applications of peptide molecules in the food area. Since, in the last few years, there has been growing interest in food authenticity, the role of peptides in this area has assumed greater importance. A fundamental part, as discussed in the article, was played by the growth of innovative analytical techniques, based on mass spectrometry (MS), which allows identifying and quantifying protein fragments in complex mixtures of different components. These procedures have been successfully applied for the identification of peptide markers in food authenticity. Because food‐derived proteins have high variability in chemical and physical properties, it is obvious that an exhaustive proteomic analysis cannot be applied by using a single technique for all objectives. The report shows some examples of various methodologies applied to different food matrices and some trivial aspects are also critically described.

We are happy to publish this special issue dedicated to Prof. Rosangela Marchelli. This issue not only celebrates her long-standing scientific activity on occasion of her significant anniversary, but it is meant to recognize her contribution to Bioorganic Chemistry in the field of Artificial DNA, and in particular of Peptide Nucleic Acids.

Invasion of peptide nucleic acid (PNA) is important to target a specific site in double-stranded DNA. However, it needs improvement in many ways such as sequence versatility and invasion efficiency. It is here reported that one 10-mer strand of -lysine substituted peptide nucleic acid (PNA) efficiently invades double-stranded DNA (dsDNA) in the presence of single-strand binding protein (SSB) via Watson-Crick rule. According to the thermodynamic parameters in this study, highly efficient invasion was achieved by a favorable entropy change. The importance of preorganization of PNAs for efficient strand invasion is indicated.

By using suitably designed chiral, lysine-based PNAs (chPNAs), the stability of complementary chPNAchPNA duplexes can be depressed, maintaining or improving at the same time the stability of the corresponding PNA-DNA duplexes. This approach could be used in order to develop chPNAs able to perform double duplex DNA invasion.

The DNA binding abilities of peptide nucleic acids (PNAs), both achiral and bearing three adjacent d-lysine-based monomers in the middle of the strand (“chiral box” PNA), were studied by means of electrospray mass spectrometry (ESI-MS). In contrast with achiral PNA, “Chiral box” PNA was confirmed to exert high direction control (antiparallel vs. parallel DNA target) in DNA binding.

The DNA binding abilities of peptide nucleic acids PNAs, both achiral and bearing three adjacent d-lysine-based monomers in the middle of the strand “chiral box” PNA, were studied by means of electrospray mass spectrometry ESI-MS. In contrast with achiral PNA, “Chiral box” PNA was confirmed to exert high direction control antiparallel vs. parallel DNA target in DNA binding.

Review: about 80 refs.

Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.

For Abstract see ChemInform Abstract in Full Text.