CONSTRAINED FLEXIBILITY IN PNA: DNA BINDING STUDIES WITH BRIDGED AMINOPROPYLGLYCYL PNA

Introduction of methylene bridges in aegPNA and apgPNA molecules give rise to cyclic five and six membered ring structures. Synthesis of a new six membered cyclic PNA monomer, aminopipecolyl PNA (pipPNA) is reported. Incorporation of pipPNA into PNA oligomers and comparative binding with target DNA sequences is studied.     

6-Thioguanine in Peptide Nucleic Acids. Synthesis and Hybridization Properties

Abstract

The synthesis of N-((2-amino-6-benzylthiopurine-9-yl)acetyl)-N-(2-tBoc-aminoethyl)glycine 4 and its incorporation into a peptide nucleic acid (PNA) oligomer are described. Introduction of a single 6-thioguanine residue (6sG) in the PNA of a 10-mer PNA:DNA heteroduplex resulted in a decrease in Tm of 8.5°C. Furthermore, we observed a hypochromic and a bathochromic shift of 6 nm above 346 nm when the 6sG containing PNA was hybridized to its complementary DNA strand.     

Conformationally Restrained Chiral PNA Conjugates: Synthesis and DNA Complementation Studies

Abstract

In recent times, PNA (I), a structural mimic of DNA in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine (aeg) linkage has emerged as a potential antisense therapeutic agent.¹ A major limitation of PNAs from an application perspective is their poor solubility in aqueous medium and being achiral, they bind to cDNA in both parallel (N-PNA/5′-DNA) and antiparallel (N-PNA/3′-DNA) modes. In this connection, we have designed spermine conjugated and conformationally constrained PNA analogues to generate the 4-aminoprolyl backbone (II).² These were synthesised and evaluated for their DNA binding abilities by using UV and CD spectroscopic studies. It is seen that incorporation of one 4-aminoprolyl unit at the N-terminus of a PNA chain not only enhances the inherent binding of PNA to DNA, but also imparts significant bias in parallel and antiparallel binding with cDNA. Conjugation of spermine at C-terminus enhanced the PNA solubility.     

In Vitro Membrane Penetration of Modified Peptide Nucleic Acid (PNA)

Abstract

Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments, showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.     

PNA∗Synthesis and Diagnostic Application

Abstract

An optimized automated PNA synthesis protocol is reported. Under optimal conditions the product yield of a test 17-mer PNA is approximately 90 %. The average coupling yield is 99.4 %. The synthesis strategy is Boc/Z. The protocol is developed in a 5 pmole scale but is easily scaled up to 10–50 μmole scale syntheses on the automated synthesizer (ABI 433A). DNA capture experiments by PNA was used to develop a method for PNA-mediated purification of genomic Chlamydia DNA from urine. This purification removed efficiently substances that impeded DNA amplification.

     

APPLICATION OF MITSUNOBU REACTION TO SOLID-PHASE PEPTIDE NUCLEIC ACID (PNA) MONOMER SYNTHESIS

ABSTRACT

PNA type I monomer backbone with a reduced peptide bond was synthesized on a Merrifield resin in Mitsunobu reaction of Boc-amino ethanol with resin-bound o-nitrobenzenesulfonylglycine. The pseudo dipeptide secondary amine group was deprotected by thiolysis and acylated with thymin-1-ylacetic acid. The monomer was released as a methyl ester. The procedure seems to be of general applicability and allows various modifications of PNA structure by using diverse alcohols and amino acid esters.     

Triplex Formation Between DNA and Mixed Purine-Pyrimidine PNA Analog with Lysines in Backbone

Abstract

Melting UV experiments and mixing curves indicated slow triplex formation between lysine comprising PNA and DNA complement in 100mM Na⁺ solution.     

Different effects of cGMP and cAMP in the intestine of the European eel,Anguilla anguilla

The regulation of salt absorption in the sea water cell intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E ₁ on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increase the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying Cl^– transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine vasoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.Abbreviations AP III rat artriopeptin III - 8 Br cAMP 8 Br cyclic adenosine monophosphate - 8 Br cGMP 8 Br cyclic guanosine monophosphate - g _t transepithelial conductance - I _sc short circuit current - IC ₅₀ half-maximal inhibitory concentration - NaK ATPase Na-K-adenosine monophosphate - NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid - PGE prostaglandin E ₁ - R _t tissue resistance - SITS 4-acetamide-4-isothiocyano-stilbene-2,2-disulfonic acid - V _t transepithelial voltage difference - VIP porcine vasoactive intestinal peptide     

Synthesis of trans-L/D-2-(Tert-butoxycarbonyl- aminomethyl)-4-(thymin-1-yl) Pyrrolidin-1-yl Acetic Acid

Abstract

To delineate the binding preferences of stereochemically divergent pyrrolidine PNAs, synthesis of all four diastreomeric monomers of I and the systematic complexation studies of the resultant PNAs with complementary DNA/RNA is essential. We herein report the synthesis of trans-L/D-2-(tert-butoxycarbonylaminomethyl)-4-(thymin-1-yl) pyrrolidin-1-yl acetic acids I, their incorporation in PNA oligomers and DNA binding studies will be presented.     

Long Range Electron Transfer in PNA/DNA Duplexes

Abstract

The conductive properties of PNA/DNA is examined. The electron donor is covalently linked to a fixed site in PNA which unambiguously places the acceptor in the hybrid duplex. The study shows that PNA/DNA acts like an insulator.     

Recognition of Double-Stranded Dna by Peptide Nucleic Acid

Abstract

Peptide nucleic acid (PNA) is an oligonucleotide mimic in which the backbone of DNA has been replaced by a pseudopeptide. We here show that there are distinct variations as to how PNA oligomers interact with double-stranded DNA depending on choice of nucleobases. Thymine-rich homopyrimidine PNA oligomers recognise double-stranded polynucleotides by forming PNA₂-DNA triplexes with the DNA purine strand. By contrast, cytosine-rich homopyrimidine PNAs add to double-stranded polynucleotides as Hoogsteen strands, forming PNA-DNA₂ triplexes, while homopurine, or alternating thymine-guanine, PNA oligomers invade DNA to form PNA-DNA duplexes.     

SYNTHESIS AND BIOLOGICAL ACTIVITIES OF CYCLIC ADP-CARBOCYCLIC-RIBOSE AND ITS ANALOGS

An efficient synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was achieved. Treatment of N ¹-carbocyclic-ribosyla-denosine bisphosphate derivative 10 with AgNO₃ in the presence of molecular sieves 3A in pyridine gave the desired cyclic product in 93% yield, which was deprotected to give the target cyclic ADP-carbocyclic-ribose (2).     

Synthesis of Peptide Nucleic Acid Monomers

Abstract

The chemical synthesis of peptide nucleic acid (PNA) monomers is described using Fmoc (backbone), anisoyl (cytosine, adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/diphenylcarbamoyl (guanine) protecting group combinations. For the guanine monomer the alkylation was realized both in a Mitsunobu [DIAD, triphenylphosphine or (4-dimethylaminophenyl)diphenylphosphine, tert-butyl glycolate] and in a low-temperature, sodium-hydride mediated alkylation (tert-butyl bromoacetate) to give the N⁹ -substituted derivative.     

ENGINEERING PREFERENCES OF HAIRPIN PNA BINDING TO COMPLEMENTARY DNA: EFFECT OF N7G IN aeg/aep PNA BACKBONE

AegPNA and aepPNA monomeric units bearing the N7-guanine nucleobase as a substitute for C⁺ have been demonstrated to bind to a GC base-pair of a duplex in a pH-independent manner when placed in the third strand. The aepPNA backbone exerts a preference for binding in the antiparallel Hoogsteen mode over the parallel Hoogsteen mode.     

Pna Array Technology in Molecular Diagnostics

Abstract

A comparative study using immobilised DNA and PNA oligomers demonstrates the suitability of PNA molecules as sequence specific capture probes in the detection of single point mutations in a DNA analyte and in the analysis of complex analyte mixtures.     

DNA-PHONA-PNA Chimeric Molecules: Contributions to Binding Against Complementary DNA

Abstract

The synthesis of a DNA-PHONA-PNA chimeric molecule using the Mmt protection strategy is described. The chimeric oligomer shows duplex binding properties that are comparable to PNA. Obviously, PHONA building blocks can be incorporated into PNAs without distortion of the PNA structure

     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

Summary The preparation of the thymine peptide nucleic acid (PNA) monomer carrying a 2-nitrophenyl group in position 4 is described. This monomer is incorporated into PNA oligomers and reacted with amines to yield PNA oligomers carrying 5-methylcytosine derivatives. During the deprotection-modification step two side reactions were detected: degradation of PNA oligomer from theN-terminal residue and modification ofN ⁴-tert-butylbenzoyl cytosine residue. Protection of theN-terminal position and the use ofN ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

The preparation of the thymine peptide nucleicacid (PNA) monomer carrying a 2-nitrophenyl group in position4 is described. This monomer is incorporated into PNAoligomers and reacted with amines to yield PNA oligomerscarrying 5-methylcytosine derivatives. During thedeprotection-modification step two side reactions weredetected: degradation of PNA oligomer from the N-terminal residue and modification of N ⁴-tert-butylbenzoyl cytosine residue. Protection of the N-terminal position and the use of N ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Evidences for complex formation between L-dabPNA and aegPNA

Continuing our research on the development of nucleopeptides as ODN analogs for biomedical and bioengineering applications, here we report the synthesis and the chemical–physical characterization of a homoadenine hexamer based on a l-diaminobutyric acid (l-DABA) backbone (dabPNA), and its binding studies with a complementary aegPNA. We demonstrated by CD and UV experiments that the l-dabPNA binds the aegPNA forming a complex with good thermal stability, that we identified as a left-handed triplex.