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.     

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.     

SYNTHESIS AND PROPERTIES OF PNA OLIGOMERS CONTAINING OROTIC ACID DERIVATIVES

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.     

Synthesis and properties of PNA oligomers containing orotic acid derivatives

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.     

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.     

Highly selective single nucleotide polymorphism recognition by a chiral (5S) PNA beacon

A chiral peptide nucleic acid (PNA) beacon containing a C-5 modified monomer based on L-lysine was synthesized. The terminal amino group of the lysine side chain was linked to a spacer for future applications on surfaces. The PNA beacon bears a carboxyfluorescein fluorophore and a dabcyl quencher at opposite ends. The DNA binding properties were compared with those of a homologous PNA beacon containing only achiral monomers. Both beacons underwent a fluorescence increase in the presence of complementary DNA, with higher efficiency and higher selectivity (evaluated using single mismatched DNA sequences) observed for the chiral monomer containing PNA. Ion exchange (IE) HPLC with fluorimetric detection was used in combination with the beacon for the selective detection of complementary DNA. A fluorescent peak corresponding to the PNA beacon:DNA duplex was observed at a very low detection limit (1 nM). The discriminating capacity of the chiral PNA beacon for a single mismatch was found to be superior to those observed with the unmodified one, thus confirming the potency of chirality for increasing the affinity and specificity of DNA recognition.     

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.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Synthesis of New Chiral Peptide Nucleic Acid (PNA) Monomers by a Simplified Reductive Animation Method

Abstract

The aim of this work was the preparation of four new peptide nucleic acid (PNA) monomer backbone by reductive animation of N^α-Boc-protected chiral amino aldehydes, derived from Leu, Phe, Tyr(Bzl), and Thr(Bzl), with methyl glycinate. To the crude 2-substituted methyl N-(2-Boc-aminoethyl)glycinates obtained, thymin-1-ylacetic acid was coupled using TBTU procedure in a one-pot reaction. PNA monomers were isolated and characterized.     

Introduction of chirality into PNA by replacement of the achiral methylene carbonyl linkage to the nucleobase

A novel approach to the introduction of chirality into peptide nucleic acid (PNA) by replacement of the methylene carbonyl linker by an alpha-amino acid derived moiety is described. A monomer compatible with Fmoc-based oligomerization chemistry possessing an L-serine derived linker has been synthesized and incorporated into PNA oligomers. A single, central substitution in a hexathymine PNA strongly destabilized triple helix formation whereas a central substitution in a mixed sequence is much better tolerated. We have investigated the influence of this substitution on the selectivity for strand composition (DNA versus RNA complement) and strand orientation (antiparallel versus parallel) in the context of duplex formation. A PNA 11-mer with a single substitution demonstrates a preference for an antiparallel RNA complement, as judged by thermal denaturation analysis of the complexes.     

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.     

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.     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

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.     

An azo-based PNA monomer: synthesis and spectroscopic study

The full synthetic details and photospectroscopic characterization of a peptide nucleic acid (PNA) monomer suitable for Fmoc-based oligomerization chemistry that bears an azobenzene moiety as a base surrogate are reported. The monomer showed the ability to quench the fluorescence emission of fluorescein and pyrene luminophores and proved to be a competent F?ster resonance energy transfer partner in a PNA-based molecular beacon.     

Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N',N'"-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that (111)In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degrees C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.     

Preparation of ribonuclease S domain-swapped dimers conjugated with DNA and PNA: modulating the activity of ribonucleases

Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.     

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.     

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.