Nucleobase‐caged peptide nucleic acids: PNA/PNA duplex destabilization and light‐triggered PNA/PNA recognition

The 2‐(o‐nitrophenyl)‐propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N‐(2‐aminoethyl)glycine peptide nucleic acids (aeg‐PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc‐a^NPP‐aeg‐OH and Fmoc‐c^NPP‐aeg‐OH were synthesized and incorporated into PNA sequences by Fmoc solid phase synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc‐cleavage, coupling, capping, and resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV‐LED irradiation at 365 nm. The nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both strands. PNA/PNA recognition can be completely restored by UV light‐triggered release of the photolabile protecting group. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

β-PNA: peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.     

PNA synthesis using a novel Boc/acyl protecting group strategy.

The synthesis of novel Boc/acyl protected monomers for the synthesis of peptide nucleic acid (PNA) is described. The oligomerization protocol using these new monomers has been optimized with regard to coupling reagents. The use of base-labile acyl protecting groups at the exocyclic amines of the heterocyclic bases (isobutyryl for guanine and benzoyl for adenine and cytosine) and a PAM-linked solid support offers an attractive alternative to the present procedures used in PNA synthesis. This strategy has been applied for the synthesis of a test 17mer PNA on both control pore glass (CPG) and a polystyrene MBHA support and was used in the preparation of PNA-DNA chimeras.     

An efficient, convenient solid-phase synthesis of amino acid-modified peptide nucleic acid monomers and oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.     

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.     

Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers

A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.     

Synthesis and characterization of new chiral peptide nucleic acid (PNA) monomers.

PNAs are DNA analogues in which the nucleic acid's backbone is replaced by a chiral or achiral pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. The easy to modify PNA structure gives the possibility to obtain monomers, and subsequently oligomers, with improved properties. We have synthesised several new PNA monomers, starting from a series of 2'-substituted methyl N-(2-Boc-aminoethyl)glycinates. The pseudodipeptides were obtained using modified Kosynkina's method, based on the reductive amination of N-Boc-protected alpha-amino aldehydes [glycinal, isoleucinal, valinal, tryptophanal, serinal(Bzl), prolinal] with methyl glycinate. The compounds were then acylated with nucleic acid base derivatives by simplified procedure, and the purification was limited to the last step of the synthesis. The applied procedure is useful in synthesis of various chiral PNA monomers.     

New synthesis of PNA-3'DNA linker monomers, useful building blocks to obtain PNA/DNA chimeras

A new synthetic strategy to get the PNA-3'DNA linker with the monomethoxytrityl (Mmt) group as temporary protection of the backbone to be used for the synthesis of PNA/DNA chimeras was employed and a convenient strategy to obtain Mmt PNA monomers was developed. The synthetic strategies take advantage of the introduction of the acid-labile Mmt-protecting group in the first step.     

Backbone amide linker (BAL) strategy for Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of peptide aldehydes.

A rapid and efficient strategy has been developed for the general synthesis of complex peptide aldehydes. N(alpha)-Benzyloxycarbonylamino acids were converted to protected aldehyde building blocks for solid-phase synthesis in four steps and moderate overall yields. The aldehydes were protected as 1,3-dioxolanes except for one case where a dimethyl acetal was used. These protected amino aldehyde monomers were then incorporated onto 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyryl-resin (BAL-PEG-PS) by reductive amination, following which the penultimate residue was introduced by HATU-mediated acylation. The resultant resin-bound dipeptide unit, anchored by a backbone amide linkage (BAL), was extended further by routine Fmoc chemistry procedures. Several model peptide aldehydes were prepared in good yields and purities. Some epimerization of the C-terminal residue occurred (10% to 25%), due to the intrinsic stereolability conferred by the aldehyde functional group, rather than any drawbacks to the synthesis procedure.     

Affinity and selectivity of C2- and C5-substituted "chiral-box" PNA in solution and on microarrays

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 T(m) with 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.     

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.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

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 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.     

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this 'double-duplex invasion', a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the alpha-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G-C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.     

New Fmoc pseudoisocytosine monomer for the synthesis of a bis-PNA molecule by automated solid-phase Fmoc chemistry

The synthesis of N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-(2-N-(benzyloxycarbonyl)isocytosin-5-ylacetyl)glycine monomer and its incorporation into a PNA molecule via automated Fmoc solid-phase chemistry is described.     

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.     

Synthesis of RNA oligomer using 9-fluorenylmethoxycarbonyl (Fmoc) group for 5'-hydroxyl protection

An approach using a new combination of protecting groups in RNA oligomer synthesis is proposed, in which 5'-hydroxyl group of ribose moiety is temporarily protected with the alkaline labile 9-fluorenylmethoxycarbonyl (Fmoc) group and the 2'-hydroxyl group is protected with the acid labile 1-ethoxyethyl (EE) group. The adoption of this method presented great selectivity in removing the 5'-hydroxyl protecting group and facilitated the RNA oligomer synthesis. A RNA pentamer was synthesized by the phosphotriester method in solution.     

PNA technology

Peptide nucleic acids (PNA) are deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or of ribonucleic acid [RNA]), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. Therefore, these molecules are of interest in many areas of chemistry, biology, and medicine, including drug discovery, genetic diagnostics, molecular recognition, and the origin of life. Recent progress in studies of PNA properties and applications is reviewed.     

A convenient route for the preparation of peptide nucleic acid monomers carrying acid-labile groups for the protection of the amino function

A convenient route for the preparation of peptide nucleic acid (PNA) monomers is described. Two different base-labile protecting groups (2-cyanoethyl and 4-nitrophenylethyl) are described for the protection of the carboxylic function of the N-(2-aminoethyl)glycine backbone during the assembly of the monomers. These groups are selectively removed yielding the desired PNA monomers in high yields, the 2-cyanoethyl group being faster and cleaner than the 4-nitrophenylethyl group. The use of PNA monomers for the preparation of DNA–PNA chimeric molecules is also discussed.