Towards a new assay to investigate electron transfer in DNA

The aim of this work was to develop a new system to investigate electron transfer in DNA using
UV active entities acting as charge acceptor in DNA. The long-range charge transfer in DNA can
be viewed as a series of short range hops between the energetically appropriate guanine bases,
which have the lowest oxidations potential of all the nucleobases. The total charge transport is
considered to be a sequence of single, reverible transfer steps between guanines bases, and these
steps are highly distance dependent since the charge is tunelling between donor and acceptor. It is
characterized as a super exchange mechanism.
Our project is based on the use of redox-indicators (RI) like ferrocene or phenol as charge
acceptor/detector in DNA (Scheme B). The UV transient absorption spectroscopy is used to
measure the oxidation of the charge acceptor during electron-transfer. Both compounds ferrocene
and phenol have lower oxidation potentials than the guanine (E°ox = 1.29 V vs NHE) and possess
distinct UV-absorption spectras which shlould allows us to measure the electron transfer using UV
transient absorption spectroscopy. Ferrocenium, the oxidized form of ferrocene, has a
characteristic absorption at λmax = 615 nm such as the phenoxyl radical which absorbs at λmax =
410 nm.59,100
Nanosecond flash-photolysis has been employed to induce the electron transfer in DNA, using the
4’- pivaloyl modified thymidine T* developed in the Giese group as charge injector. This charge
injector has the advantage to initiate a localized charge transport from a fixed starting point within
the DNA backbone.
Ferrocene was first investigate in a simple D-A system (Scheme C) in order to show that ferrocene
can be used as a charge acceptor in electron transfer processes. The first results based on a RPHPLC
analysis of the irradiated products were very promising. They proved that electron transfer
occurs from the ferrocene to the radical cation because the ketone, the product of electron transfer
was clearly identified on the HPLC chromatogram. However, despite our first hopes, the second
series of experiment based on laser flash photolysis and transient absorption spectroscopy
measurement shows that ferrocenium could not be detected by UV because of its too low
extinction coefficient. The spectroscopic properties of ferrocene can not be used to measure
electron transfer using laser flash photolysis and transient absorption spectroscopy in such
systems.
Electron transfer was then investigate in DNA using phenol as charge acceptor. A phenol
modified nucleoside was synthesized and incorporated into DNA using fully automated solidphase
synthesis.
The choice of the phenol protecting group was a key point of the synthesis of the phenol modified
nucleoside. The acetyl protecting group appeared to be ideal because it withstanded the nucleoside
synthesis conditions, it was compatible with the standard procedures for DNA synthesis and
finally it was easily removed during ammonia treatment used to cleave the DNA strand from the
solid support (Scheme D). The synthesis of the acetyl protected building block was achieved
successfully in 10% yield over 10 steps and its incorporation within oligonucleotides was
performed with efficient coupling using standard automated DNA synthesis.
Photolysis of single and double strand phenol-labeled oligonucleotides followed by HPLC
analysis of the irradiated products demonstrated that phenol is an excellent electron donor. The
electron-transfer rates measured in single and double strand experiments are in agreement with the
low oxidation potential of the phenol.