Chemistry Reference
In-Depth Information
ester - like structures. Third, effi cient hydrolytic agents for phosphate diesters could
be employed as artifi cial restriction enzymes for molecular biology. Such systems
would be highly valuable because they could, in principle, be designed to cleave
DNA with sequence selectivity different from that of the natural enzymes. The
latter, in fact, recognize DNA sequences of only 4, 6 or 8 bases, and such selectivity
may be inadequate for the precise control of the manipulation of large DNA of
higher species. Finally, the realization of antiDNA drugs can be envisaged in a more
distant future.
Since the fi rst example of an artifi cial hydrolitic agent for DNA cleavage,
reported by J. K. Barton in 1987,
4
several promising systems have been proposed
up to the description of the fi rst studies on DNA manipulation with artifi cial agents,
reported in very recent years by Komiyama and coworkers.
5
This chapter highlights
the progress reported during these two decades toward obtaining synthetic nucle-
ases. Particular attention will be devoted to the strategies that can be pursued to
increase the effi ciency and the sequence selectivity of the systems.
13.2 DNA Hydrolysis
Information available on the mechanism of spontaneous hydrolysis of DNA is obvi-
ously scarce due to its virtually absent reactivity. The commonly accepted pathway
for the hydrolysis of deoxynucleotide phosphates involves the nucleophilic attack
of a water (or hydroxide) oxygen at the phosphorus to give a fi ve - coordinate phos-
phate intermediate or transition state (Figure 13.1 ).
3o,p,6
Subsequent cleavage of
either the P-O3
cleavage in the enzymatic process) causes
a strand scission, yielding the R-OH and R-O-PO
3
(H
2
) termini. As mentioned
before, such a reaction is exceedingly slow in physiological conditions (i.e, aqueous
solution at 25 °C and pH 7) and reaction rate constants of about 1
′
or P - O5
′
(usually P - O3
′
10
− 15
s
− 1
have
×
Figure 13.1
Reaction pathway for DNA hydrolysis. The enzymatically promoted P-03
′
scis-
sion is shown
