Search

Peptide nucleic acids (PNAs) are a class of artificial oligonucleotide mimics that have garnered much attention as precision biotherapeutics for their efficient hybridization properties and their exceptional biological and chemical stability. However, the poor cellular uptake of PNA is a limiting factor to its more extensive use in biomedicine; encapsulation in nanoparticle carriers has therefore emerged as a strategy for internalization and delivery of PNA in cells. In this study, we demonstrate that PNA can be readily loaded into porous silicon nanoparticles (pSiNPs) following a simple salt-based trapping procedure thus far employed only for negatively charged synthetic oligonucleotides. We show that the ease and versatility of PNA chemistry also allows for producing PNAs with different net charge, from positive to negative, and that the use of differently charged PNAs enables optimization of loading into pSiNPs. Differently charged PNA payloads determine different release kinetics and allow modulation of the temporal profile of the delivery process. In vitrosilencing of a set of specific microRNAs using a pSiNP-PNA delivery platform demonstrates the potential for biomedical applications.

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (KD) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

A novel synthesis of C(2)-modified peptide nucleic acids (PNAs) is proposed, using a submonomeric strategy with minimally protected building blocks, which allowed a reduction in the required synthetic steps. N(3)-unprotected, d-Lys- and d-Arg-based backbones were used to obtain positively charged PNAs with high optical purity, as inferred from chiral GC measurements. “Chiral-box” PNAs targeting the G12D point mutation of the KRASgene were produced using this method, showing improved sequence selectivity for the mutated- vs wild-type DNA strand with respect to unmodified PNAs.

The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-l-lysine (PLL) as an adhesion layer to concomitantly assess the increase in sensitivity with the increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface, exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO) for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitches was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. A comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array.

Biosensors and materials for biomedical applications generally require chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. The use of modified poly(l-lysine) (PLL) polymers with appended oligo(ethylene glycol) (OEG) and thiol-reactive maleimide (Mal) moieties (PLL-OEG-Mal) offers control over the presentation of functional groups. These reactive groups can readily be conjugated to, for example, probes for DNA detection. Here we demonstrate the reliable conjugation of thiol-functionalized peptide nucleic acid (PNA) probes onto predeposited layers of PLL-OEG-Mal and the control over their surface density in the preceding synthetic step of the PLL modification with Mal groups. By monitoring the quartz crystal microbalance (QCM) frequency shifts of the binding of complementary DNA versus the density of Mal moieties grafted to the PLL, a linear relationship between probe density and PLL grafting density was found. Cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed to establish the absolute probe density values at the biosensor surfaces. These data provided a density of 1.2 × 1012probes per cm2per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.

Silicon nanowire chips can function as sensors for cancer DNA detection, whereby selective functionalization of the Si sensing areas over the surrounding silicon oxide would prevent loss of analyte and thus increase the sensitivity. The thermal hydrosilylation of unsaturated carbon–carbon bonds onto H-terminated Si has been studied here to selectively functionalize the Si nanowires with a monolayer of 1,8-nonadiyne. The silicon oxide areas, however, appeared to be functionalized as well. The selectivity toward the Si–H regions was increased by introducing an extra HF treatment after the 1,8-nonadiyne monolayer formation. This step (partly) removed the monolayer from the silicon oxide regions, whereas the Si–C bonds at the Si areas remained intact. The alkyne headgroups of immobilized 1,8-nonadiyne were functionalized with PNA probes by coupling azido-PNA and thiol-PNA by click chemistry and thiol–yne chemistry, respectively. Although both functionalization routes were successful, hybridization could only be detected on the samples with thiol-PNA. No fluorescence was observed when introducing dye-labeled noncomplementary DNA, which indicates specific DNA hybridization. These results open up the possibilities for creating Si nanowire-based DNA sensors with improved selectivity and sensitivity.

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol–gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson–Crick–Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol–gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing.

Peptide synthesis is a fundamental feature of life. However, it still remains unclear how the contemporary translation apparatus evolved from primitive prebiotic systems and at which stage of the evolution peptide synthesis emerged. Using simple molecular architectures, in which aminoacyl transfer of phenylalanine occurs either between two ends of a PNA stem loop structure, between two PNAs in a duplex, or between two PNAs assembled on a PNA template, we show that bona fidetemplate instructed phenylalanine transfer can take place. Thus, we have identified conditions which allow template assisted intermolecular aminoacyl transfer using simple ester aminolysis chemistry primitively analogous to the ribosomal peptidyl transferase reaction in the absence of anchimeric assistance from ribose and ribosome catalysis. These results help define the minimum chemical boundary conditions for the translation process and also give insight into the possibilities for the prebiotic emergence of RNA-independent translation.

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.

The realization of a DNA biosensor based on Double Tilted Fiber Bragg Grating (DTFBG) for label-free detection has been demonstrated. To our knowledge this is the first time that a biosensor has been realized with such kind of device. The surface of the optical fiber has been functionalized with peptide nucleic acid (PNA) in order to capture DNA strands. The changes of the interference fringes visibility of the grating, due to the PNA-DNA binding, proved the occurred fiber hybridization. The re-use of the fiber for multiple measurements and the selectivity of the sensor have been also investigated.

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.

Molecular computing is potentially one of the most powerful tools for the development of massive parallel computing protocols. In the present paper, a first example of the use of PNA:PNA interactions in molecular computing is described. A series of short PNA sequences have been designed with a four base stretch coding for variables and solutions. Hybridization of the components in different combinations was tested both in solution and in a microarray format. A series of PNA representing the solutions were spotted on a microarray surface in order to simulate the hardware. A series of PNA representing the variables, labeled with TAMRA, were used to interrogate the device enabling to solve non-deterministic logic operations. The system was shown to be able to solve a two-variable equation with a high signal to noise ratio. This paper intends to provide a proof of principle that PNA, on account of their stability and specificity of binding, are most suitable for constructing organic-type computers.

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 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.

A duplex PCR method was developed to simultaneously detect the presence of hazelnut and peanut in raw materials and commercial products. It was found to be able to specifically detect traces of the investigated products down to 50 pg of their target DNA.A duplex PCR method was developed to simultaneously detect the presence of hazelnut and peanut in raw materials and commercial products. It was found to be able to specifically detect traces of the investigated products down to 50 pg of their target DNA.

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 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.

Protonation and the Cu^II complexation constants of the dansylated polyamines N-dansylethylenediamine (1), N-dansyldiethylenetriamine (2), N-dansyltriethylenetetramine (3), N'-[2-(dansylamino)ethyl]diethylenetriamine (4), and tris(2-dansylaminoethyl)amine (5) were determined by glass-electrode potentiometry in MeOH/H2O 9 : 1 (v/v) solution. For ligands 3 and 4, the determinations were also performed in aqueous solution. The complexes formed by these ligands in neutral form correspond to those observed for the analogous unsubstituted monoprotonated amines, whereas, when the ligands are deprotonated at the sulfonamide moiety, the species parallel those of the corresponding amines. The molecular structures of the complexes were deduced from the VIS absorption spectra. The crystal structure of the [CuL2H−2] complex 6 of ligand 1 (L) was determined by X-ray diffraction. The study of the photophysical properties of the ligands 3 – 5 showed that they are good fluorescent sensors for copper(II), which induced fluorescence quenching. Time-resolved fluorescence measurements allowed us to clarify the sensing mechanism. The pH dependence of the quenching effect demonstrated that it occurs for all Cu²⁺ complexes, even for species in which the sulfonamide moiety is not deprotonated. Sensing of Cu²⁺ was compared with that of other metal ions (Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺), and selectivity was studied as a function of pH. Ligands 3 and 4 were found to be selective chemosensors for Cu²⁺ in weakly acidic solution (pH ca. 4 – 5).

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.

Three diaminodiamido ligands (S,S)-N,N'-bis(prolyl)ethanediamine (ProNN-2), (S,S)-N,N'-bis(N-methylvalyl)ethanediamine (Me2ValNN-2), and (S,S)-N,N'-bis(N-methylphenylalanyl)-ethanediamine (Me2PheNN-2) were synthesised and their complex formation equilibria with copper(II) investigated in aqueous solution by potentiometry and, for ProNN-2, by electronic spectrophotometry. ProNN-2 forms the species [CuLH]3+, [Cu2L2]4+, [Cu2L2H-2]2+ and [CuLH-2], Me2PheNN-2 forms the complexes [CuLH]3+, [Cu2L2H-2]2+ and [CuLH-2], whereas Me2ValNN-2 forms the monomer [CuLH-1]+ but not the dimer. The dimeric cation [Cu2L2H-2]2+, of Me2PheNN-2 has severe steric requirements, as demonstrated by the X-ray crystal structure of the complex [Cu2L2H-2]Cl2· 12H2O, of the corresponding non-methylated ligand. Since copper(II) complexes of the ligands examined are used as additives to the mobile phase to perform chiral resolution of D,L-amino acids in RP-HPLC, the present results provide valuable clues to an understanding of the mechanism of the enantiomeric separation.

The synthesis and full spectroscopic characterization (IR, 1D and 2D TOCSY and ROESY NMR, MS) of a novel capped β-cyclodextrin bearing a 6 A,6 D-dideoxy-N,N′-3,6,9-trioxa-undecanoyl-(L,L)-bis-alanyl bridge are described. The chiral recognition properties of the product towards Dns-amino acids have been examined by capillary electrophoresis.

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy) was synthesized by C6-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d.. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.

Background: Mushroom poisoning by some species of the C5rhzrius (Agaricales) often lead to irreversible renal failure caused by the nephrotoxin orellanine. In 1994 and 1995, six poisoning outbreaks involving ten individuals in Northern Italy and in Austria were investigated. Methodq: A total of 87 clinical samples (urine and blood samples including renal biopsy material of three patients) were examined for the presence of orellanine by thin layer chromatography. Results: Orellanine can be detected after a relatively long period following poisoning by performing a simple thin layer chromatography technique using small quantities of renal biopsy material. No toxin was found in urine or blood samples. Conclusiorrs: Orellanine is rapidly concentrated in the kidneys in a relatively soluble form and cannot be detected in urine, blood and dialysis fluids at the time when first symptoms appear

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 −.

A modified β‐cyclodextrin bearing a 2‐aminomethylpyridine binding site for copper(II) (6‐deoxy‐6‐[N‐(2‐methylamino)pyridine)]‐β‐cyclodextrin, CDampy was synthesized by C6‐monofunctionalization. The acid‐base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D‐ or L‐Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred ciscoordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D‐Trp, but not of that of L‐Trp. In Trp‐containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D‐Trp and two conformers of L‐Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997.© 1997 Wiley‐Liss, Inc.