Transition Metal Derivatives of Peptide Nucleic Acid (PNA) OligomersSynthesis, Characterization, and DNA Binding

Abstract

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.

Synthesis, Spectroscopic Properties and Electrochemical Oxidation of RuII‐Polypyridyl Complexes Attached to a Peptide Nucleic Acid Monomer Backbone

“…UV/Vis and emission spectra measurements on these monomers M1 and M2 and their respective intermediates, 1 2, 4 and 5 show similar behaviour to those that have been reported previously by our group and by Metzler-Nolte and co-workers. [23,32,44] Electrochemical measurements showed that all complexes give rise to a reversible one-electron Ru II /Ru III process. The in-sertion of a linker group between the metal center and the PNA unit in M2 relative to M1 only has a small effect on the luminescence and electrochemistry of the ruthenium complex.…”

“…[23] Subsequently, the same group described the attachment of a tris(bipyridine) Ru II complex to a PNA heptamer where the metal centre had been inserted to the amino end of the sequence by automated synthesis. [32] On hybridisation of this Ru II -bearing PNA heptamer with a complementary DNA strand, the CD (circular dichroism) spectra showed a positive Cotton effect by a shift in the maximum from 266 nm (PNA.DNA bioconjugate) to 280 nm (Ru-PNA-DNA bioconjugate). [32] Metal-containing PNAs have also been synthesised by Achim et al for purposes that include investigation of the electron transfer within PNA [33] and the increase in PNA-DNA stability through metal binding.…”

“…[32] On hybridisation of this Ru II -bearing PNA heptamer with a complementary DNA strand, the CD (circular dichroism) spectra showed a positive Cotton effect by a shift in the maximum from 266 nm (PNA.DNA bioconjugate) to 280 nm (Ru-PNA-DNA bioconjugate). [32] Metal-containing PNAs have also been synthesised by Achim et al for purposes that include investigation of the electron transfer within PNA [33] and the increase in PNA-DNA stability through metal binding. [34] In an effort to enhance the versatility of insertion of Ru II complexes into PNAs, our group has envisaged an alternative approach involving the preparation of robust Ru II complexes attached to the PNA backbone, which could be then inserted into a specific site within a PNA sequence.…”

“…The Ru II / Ru III redox centers in 2, 4 and 5 have E°f (reversible potentials) of +857 mV, +884 mV and +901 mV, respectively, vs. the established chemistry, availability, reversible electrochemistry and chemical stability, [19,20] ferrocene has been coupled to PNA monomers [21][22][23][24][25][26][27][28][29] or sequences. [30][31][32] Ferrocene, however, exhibits only electrochemical and no excited-state properties, in comparison to metal complexes, such as polypyridyl-Ru II complexes, which feature both electrochemical and photochemical properties that can be utilised in biosensing. Metzler-Nolte et al reported the synthesis of a Ru II complex attached to a PNA monomer, [23] bis(bipyridyl)(4-methyl-4Ј-carboxylic acid bipyridyl)ruthenium(II) thymine-PNA methyl ester [Ru-(T-PNA)-OMe].…”

Quantifizierung der Zellaufnahme metallierter Peptide und Peptidnucleinsäuren durch Atomabsorptionsspektroskopie

“…[24] Die Resultate zeigen wie erwartet eine Duplexbildung von 5·7 (Schmelztemperatur T m = 29.7 8C) und 6·7 (T m = 36.1 8C, siehe auch Hintergrundinformationen, Abbildung S1). Die Differenz DT m = 6.3 8C schreiben wir einer anziehenden elektrostatischen Wechselwirkung zwischen dem positiv geladenen NLS-Peptid und dem überhängenden, negativ geladenen 5'-Ende der DNA im Duplex 6·7 zu.…”