Chemistry Reference
In-Depth Information
13.8 A Critical Survey and New Perspectives
Twenty years have passed since the fi rst report by Barton and coworkers of an
artifi cial agent for the hydrolytic cleavage of DNA.
4
Since then, the realization of
such systems has attracted great interest and has led, with Komiyama's ARCUT,
5
to the fi rst examples of DNA manipulation performed with synthetic agents. Still,
several important goals remain unachieved and the recognition of the most promis-
ing guidelines is still not easy.
The ideal features of an artifi cial hydrolytic agent are high activity, high DNA
affi nity and sequence selectivity. Other requirements may vary depending on the
fi nal application: for an artifi cial restriction agent, double-stranded cleavage at the
P - O3
site is a requirement for subsequent religation, while for an antisense drug
the absence of cofactors, activity at very low concentrations and at physiological pH
and temperature are surely important. For all the possible applications, the occur-
rence of an oxidative cleavage, either as main or concurrent pathway, is highly
undesirable.
The last point is of particular relevance with synthetic agents. Metal ions used
by nature in nucleases and phosphatases, e.g. Ca(II), Mg(II), Fe(II), Fe(III) and
Zn(II), do not have, with exception of the Fe(II)/Fe(III) couple, relevant redox
properties. Chemists, however, can pick up the most suitable candidate over the
whole periodic table. The ideal metal ion for the realization of a hydrolytic system
should have a hard character (to bind phosphate oxygen atoms), high Lewis acidity
(to polarize both the phosphate group and the nucleophile) and a fast rate of ligand
exchange (to ensure catalytic turnover).
3o
Several metal ions not used by nature
best fulfi l such requisites, particularly trivalent lanthanide ions, Ce(IV) and Cu(II).
In the case of Cu(II), however, oxidative cleavage pathways have been detected
for most of the hydrolytically active Cu(II)-based systems so far reported, both in
the presence and in the absence of reducing agents and, on some occasions, even of
oxygen. The adventitious presence of reducing agents either in DNA samples
obtained from natural sources or in the
in vivo
environment, makes the use of
Cu(II)-based systems as hydrolytic agents rather discouraging, notwithstanding
their excellent activity.
Moreover, the recent results form Rediijk and coworkers,
9
who discovered
oxidative DNA cleavage with a Zn(II) complex containing a phenol unit, put further
emphasis on the need to assess clearly the reaction mechanism, even in the case of
metal ions without a relevant redox activity.
After the cleavage pathway, the fi rst problem to face is that of the hydrolytic
effi ciency of the artifi cial system. A comparison of the activity of the different agents
reported is rather diffi cult because of the large variety of reaction conditions used.
However, a rough evaluation of the most reactive agents is tentatively shown in
Table 13.1, which reports, where possible, the plasmid DNA degradation rate at a
fi xed agent concentration (5 m M,
′
k
ψ
) and agent to DNA (base pair) ratio (0.125,
′
k
ψ
),
′′
and the Michaelis - Menten parameters.
A fi rst glance at the
k
ψ
data reported, indicates that simple metal ions
and monometallic complexes can hardly achieve relevant reactivity. Such low activ-
ity appears to be related mainly to a low affi nity for DNA. Indeed, when the
k
cat
k
ψ
and
′
′′
