Synthesis of new chiral peptide nucleic acid (PNA) monomers

We have synthesised a series of new chiral type I peptide nucleic acid monomers in total yields of 36-53%, derived from Val, Ile, Ser(Bzl), Pro, and Trp, employing convenient procedure.     

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin® resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2′-isobutyric acid (HPDI). The base acetic acids of thymine 5 , Z-cytosine 9 , Z-adenine 12 , and 6-O-benzyl guanine 17 were prepared and coupled to the immoblized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel® resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

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.     

Design and synthesis of conformationally frozen peptide nucleic acid backbone: chiral piperidine PNA as a hexitol nucleic acid surrogate

The design and facile synthesis of novel chiral piperidine PNA from naturally occurring 4-hydroxy-L-proline is reported. The stereospecific ring-expansion reaction to get six-membered piperidine derivative from 5-membered pyrrolidine derivative is exploited for this synthesis. The resulting conformationally constrained PNA is utilized for the synthesis of PNA mixmers and the concept is substantiated by UV-Tm studies of the resulting PNA(2):DNA complexes.     

Synthesis of new, base-modified PNA monomers

A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and 5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling reactions. Starting from the base-unprotected 5-iodo-uracil PNA monomer, only the Stille couplings were accomplished successfully, while Suzuki couplings with boronic acids containing the same aryl groups failed. During Sonogashira couplings with terminal alkynes, significant amounts of unrequired furano[2,3-d]pyrimidine by-products were formed. Protection of the lactam function by p-methoxybenzylation prevented the opportunity for intramolecular cyclization as well as formation of a negative charge on the 4-O atom, making it possible to reach almost quantitative yields at Sonogashira couplings and acceptable conversions in Suzuki reactions.     

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases.

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.     

Restricted rotation in chiral peptide nucleic acid (PNA) monomers--influence of substituents studied by means of 1H NMR

The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into "aminoalkyl" part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of 1H NMR at 0, 25 and 40 degrees C in CDCl3. The delta G0 values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties.     

Application of Mitsunobu reaction to solid-phase peptide nucleic acid (PNA) monomer synthesis

PNA type I monomer backbone with a reduced peptide bond was synthesized on a Merrifield resin in Mitsunobu reaction of Boc-aminoethanol with resin-bound o-nitrobenzenesulfonylglycine. The pseudodipeptide 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.     

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.     

In vitro membrane penetration of modified peptide nucleic acid (PNA).

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.     

Inhibiting gene expression with peptide nucleic acid (PNA)--peptide conjugates that target chromosomal DNA

Peptide nucleic acids (PNAs) are nonionic DNA/RNA mimics that can recognize complementary sequences by Watson-Crick base pairing. The neutral PNA backbone facilitates the recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use, it will be necessary to develop straightforward strategies for introducing them into cells. Here, we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both 13 and 19 base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moieties yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.     

An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they are known to form P-loops, which consist of a [PNA]2-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. In contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C+T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J+T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J+T)-containing PNA constructions may be advantageous for use in vivo.     

Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.     

Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.