Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

Role of chirality and optical purity in nucleic acid recognition by PNA and PNA analogs

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 PNA(2):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 7 are 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 8 were 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 8 were 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.     

Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays.

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.     

Synthesis of Chiral Heteronucleotide ONA Sequences by a Fmoc/Boc-Based Submonomeric Strategy

Peptide nucleic acid (PNA) is a DNA analog able to form hybridization complexes with complementary DNA or RNA strands. Many PNAs have been described in recent years, particularly chiral PNA analogs. Chiral heteronucleotide ONA (Orn backbone PNA) is an important tool in the antisensing field, but was not been fully explored yet. In the present work, we performed studies toward the synthesis of chiral heteronucleotide ONA sequences by utilizing a Fmoc/Boc-based submonomer approach on solid support. The desired oligomers with different nucleic content and length were obtained in very good yields and high purity. Specific binding to the complimentary ssDNA oligomers was demonstrated.     

Synthesis And Binding Study Of Phosphonate Analogues Of Pnas And Their Hybrids With Pnas

Abstract

The preparation of monomers for the synthesis of phosphonate analogues of peptide nucleic acids containing the four natural nucleobases: thymine, cytosine, adenine and guanine, has been ccomplished. The monomers obtained were used for the automated online solid phase synthesis of pure phosphono-PNA oligomers as well as chimeras consisting of phosphono-PNA and PNA resudies. The hybridization properties of these oligonucleotide mimics to complementary DNA and RNA fragments were studied.     

Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.     

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.     

Foldamers derived from nucleoside beta-amino acids: PNA Or DNA? Can we have both in one place?

The synthesis of a modified thymidine (nucleoside beta-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.     

Foldamers Derived From Nucleoside β-Amino Acids: Pna Or Dna? Can We Have Both In One Place?

The synthesis of a modified thymidine (nucleoside β-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.     

Synthesis and properties of DNA-PNA chimeric oligomers.

Adenine, thymine and cytosine PNA monomers have been prepared using 3-amino-1,2-propanediol as a starting material. The benzoyl group was used to protect the exocyclic amines of the heterocyclic bases of A and C PNA monomers and the backbone primary amine was protected with the monomethoxytrityl group. The thymine and cytosine PNA monomers were used in conjunction with standard DNA synthesis monomers to produce chimeric PNA DNA (PDC) oligomers. Ultraviolet melting studies confirmed that these oligomers form stable hybrids with complementary DNA strands and that mismatches in the DNA but more so in the PNA sections lead to duplex destabilisation.     

肽核酸——一种新型的反义物质

肽核酸是以肽为骨架的一种新型ＤＮＡ模拟物。已经证明肽核酸具有与ＤＮＡ和ＲＮＡ结合的高度亲合性、良好的稳定性及能方便地固相合成等特性。在反义技术和基因治疗中有着很好的前景。本文综述了肽核酸的生物化学特性及其在反义技术方面的应用。     

Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine.

The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the T m of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 degrees C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment.     

dabPNA: design, synthesis, and DNA binding studies

In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs.     

SYNTHESIS AND BINDING AFFINITY OF A CHIRAL PNA ANALOGUE

The synthesis of a chiral peptide nucleic acid (PNA), which is composed of N-aminoethyl-cis-4-nucleobase-L-proline units, was described. The chiral PNA monomers containing all four nucleobases (A, T, C and G) were steroselectively prepared. The x-ray diffraction data from a single crystal confirmed the configuration of a key intermediate. Binding activity of the oligomers with their complementary DNA targets was also investigated.     

Delayed fibril formation of amylin(20-29) by incorporation of alkene dipeptidosulfonamide isosteres obtained by solid phase olefin cross metathesis

The synthesis of a new peptidomimetic structure, the alkene dipeptidosulfonamide isostere, is described. The synthesis is based on a cross metathesis reaction between two allylic building blocks, both in solution and on the solid phase. This method was also applicable to the solid phase synthesis of alkene dipeptide isosteres. Derivatives of amylin(20-29) containing the alkene dipeptidosulfonamide isostere as well as the alkene dipeptide isostere were successfully synthesized using the solid phase cross metathesis method. Investigation of relations between structure and fibril formation of these amylin(20-29) derivatives showed retardation of fibril formation and altered secondary structures, compared to native amylin(20-29).     

Synthesis and binding affinity of a chiral PNA analogue

The synthesis of a chiral peptide nucleic acid (PNA), which is composed of N-aminoethyl-cis-4-nucleobase-L-proline units, was described. The chiral PNA monomers containing all four nucleobases (A. T, C and G) were steroselectively prepared. The x-ray diffraction data from a single crystal confirmed the configuration of a key intermediate. Binding activity of the oligomers with their complementary DNA targets was also investigated.     

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.     

Strand-invading linear probe combined with unmodified PNA

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40 °C at 100 mM NaCl concentration.     

Synthesis and DNA binding properties of dioxime-peptide nucleic acids

Peptide nucleic acids (PNAs) C- or N-modified with dioxime ligands were prepared by solid-phase synthesis using iron(II)-clathrochelates as protected dioxime building blocks. These PNA bind complementary DNA sequence specifically, though with much reduced affinity in comparison with nonmodified PNA. The dioxime-PNA conjugates bind Cu2+ and Ni2+ at microM concentration.     

Composites of peptide nucleic acids with titanium dioxide nanoparticles. III. Kinetics of PNA dissociation from nanocomposites containing DNA/PNA duplexes

When delivering peptide nucleic acids (PNA) into cells in the TiO₂ · PL · DNA/PNA nanocomposites consisting of titanium dioxide nanoparticles coated with polylysine (PL) and immobilized DNA/PNA duplexes, it is important to control the rate of the release of PNA from the carrier due to dissociation of the immobilized DNA/PNA duplex, followed by the desorption of PNA to solution while the DNA remains on the carrier. It was found that the rate constant of dissociation of the DNA/PNA duplex in the TiO₂ · PL · DNA/PNA nanocomposites depended on the number of complementary bases in the duplex. The half-retention time values for PNA in the studied nanocomposites containing the duplexes with 10, 12, 14, and 16 overlapping complementary base pairs were 10, 14, 22, and 70 min, respectively. Thus, it was shown that the rate of the release of PNA from the proposed nanocomposites can be controlled by varying the number of overlapping complementary base pairs in the immobilized DNA/PNA duplex. The method of the PNA immobilization may be used for designing nanocomposites having the optimum time value of the PNA release. The proposed TiO₂ · PL · DNA/PNA nanocomposites can be used to efficiently deliver therapeutically significant PNA drugs for their selective effect on pathogenic nucleic acids in cells.