Convergent Strategies for the Attachment of Fluorescing Reporter Groups to Peptide Nucleic Acids in Solution and on Solid Phase

Abstract

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.

PNA-Directed Triple-Helix Formation byN 7-Xanthine

“…Thus, we changed to the allyl ester (4, Scheme 1), which affords orthogonal removal condition to Fmoc-deprotection and also increases the solubility of the backbone submonomer in organic solvents. While this compound has been previously synthesized by transesterification from tert-butyl ester, 17 we have employed a convenient 2-step method starting from 2-Naminoethylglycine 18 (3) followed by carbamylation with Fmoc-N-hydroxysuccimide (Fmoc-NHS). We have found compound 3 to be a very useful precursor that may be produced and esterified easily on large scale (ca.…”

Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye

“…10 While conjugation of fluorescent dyes to PNAs that have been removed from solid support and deprotected is a viable alternative, in practice such approach is more time-consuming and requires either changes in PNA structure (e.g., replacement of lysine with glutamic acid) or custom made monomers. 11,12 Labeling of the PNA N-terminus can be done either directly with an activated carboxylic acid derivative of the dye 10 or using custom made monomers, such as lysine conjugated with fluorescein at the ε-amino group. 13,14 Custom made monomers can be also used in labeling of the C-terminus; for example, loading the solid support with S - t -butylmercapto- l -cysteine allowed conjugation of the thiol group with maleimido functionalized rhodamine dye directly on solid support.…”

Nucleobase Modifications in Peptide Nucleic Acids

“…However, these studies indicated that the presumed helical distortion was not so dramatic as to completely prevent base pairing [23,24]. …”