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.     

Synthesis and hybridization properties of DNA-PNA chimeras carrying 5-bromouracil and 5-methylcytosine

The preparation of 5-bromouracil and 5-methylcytosine peptide nucleic acid (PNA) monomers is described. These PNA monomers were used for the preparation of several DNA-PNA chimeras and their hybridization properties are described. The substitution of cytosine by 5-methylcytosine in DNA-PNA chimeras increased duplex stability while substitution of thymine by 5-bromouracil maintained it. Moreover, binding of DNA-PNA chimeras to double-stranded DNA to form triple helices was studied. In contrast to DNA, the presence of 5-methylcytosine and 5-bromouracil in DNA-PNA chimeras destabilized triple helix.     

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.     

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.     

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 of cell-permeable peptide nucleic acids and characterization of their hybridization and uptake properties

Guanidine-based peptide nucleic acid (GPNA) monomers and oligomers containing all four natural (adenine (A), cytosine (C), guanine (G), and thymine (T)) and two unnatural (2-thiouracil (sU) and 2,6-diaminopurine (D)) nucleobases have been synthesized. Thermal denaturation study showed that GPNA oligomers containing alternate D-backbone configuration bind sequence-specifically to DNA and, when incubated with mammalian cells, localized specifically to the endoplasmic reticulum (ER).     

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.     

Construction of peptide conjugates with peptide nucleic acids containing an anthracene probe and their interactions with DNA

We designed and synthesized the peptide nucleic acid (PNA)-peptide conjugates having anthracene chromophores and investigated their interactions with calf thymus DNA, [d(AT)(10)](2), [d(GC)(10)](2), and [d(AT)(10)dA(6)](2). Considering the synthesis compatibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of alpha-helix peptides, we attempted to attach thymine PNA oligomers at the C-terminus of a 14 amino acid alpha-helix peptide that contained a pair of artificial intercalators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, fluorescence and circular dichroism properties. The results observed in this study showed that the designed peptide folded in an alpha-helix structure in the presence of calf thymus DNA, [d(AT)(10)](2), and [d(AT)(10)dA(6)](2) with the chromophores at the side-chain being fixed with a left-handed chiral-sense orientation. The alpha-helix and the anthracene signals were not observed for [d(GC)(10)](2). Incorporation of thymine PNA oligomers into the designed alpha-helix peptide increased the DNA binding ability to [d(AT)(10)dA(6)](2) with increasing the length of the PNA without changing the conformations of the peptide backbone and the anthracene side-chains.     

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 labelled PNA oligomers by a post-synthetic modification approach

The preparation of t-butoxycarbonyl (Boc)-protected O(4)-(o-nitrophenyl) thymine peptide nucleic acid (PNA) monomer is described. This PNA monomer was incorporated into PNA oligomer sequences. The post-synthetic modification of the oligomers to yield fluorescently-labelled PNA oligomers was studied before and after the removal of the protecting groups. In both cases, the desired fluorescently-labelled PNA oligomer was obtained in good yields.     

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 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.     

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.     

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.     

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.     

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.     

Inhibition of PNA triplex formation by N4-benzoylated cytosine.

The synthesis of N-((N4-(benzoyl)cytosine-1-yl)acetyl)- N -(2-Boc-aminoethyl)glycine (CBz) and the incorporation of this monomer into PNA oligomers are described. A single CBzresidue within a 10mer homopyrimidine PNA is capable of switching the preferred binding mode from a parallel to an antiparallel orientation when targeting a deoxyribonucleotide sequence at neutral pH. The resulting complex has a thermal stability equal to that of the corresponding PNA-DNA duplex, indicative of a strong destabilization of Hoogsteen strand PNA binding due to steric interference by the benzoyl moieties. Accordingly, incorporation of the CBz residue into linked PNAs (bis-PNAs) results in greatly reduced thermal stability of the formed PNA:DNA complexes. Thus, incorporation of the CBz monomer could eliminate the stability bias of triplex-forming sequences in PNA used in hybridization arrays and combinatorial library formats. Furthermore, it is shown that the benzoyl moiety does not severely interfere with Watson-Crick hydrogen bonding, thereby presenting an interesting route for novel cytosine modifications.     

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.