Nucleic Acids in Chemistry and Biology

oxygen atoms are non-equivalent (i.e.diastereotopic) and the phosphorus atom is a pro-chiral centre. By
substituting one of these oxygen atoms by sulfur (Figure 3.42c) or by distinguishing them isotopically, the
phosphorus can be made into a stereogenic centre for the stereochemical analyses of substitution reactions.

3.2.1.3 Phosphate Monoesters. Monoesters have a single alkyl or aryl group and two ionisable OH

groups. These have pKa 1
1.6 and pKa 2
6.6, so there is an equilibrium in neutral solution (effectively from
pH 5 to pH 8) involving significant concentrations of both the monoanion and dianion. The equivalent oxygen
atoms share the negative charge in both monoanions and dianions and there is partial double bonding to each.
In the monoanion, the hydrogen atom translocates rapidly between the three oxygen atoms making them all
equivalent in solution. These three oxygen atoms are pro-pro-chiral. Thus the use of the three isotopes of oxy-
gen,^16 O,^17 O (Ø) and^18 O (O) is required for stereodifferentiation and has been widely used in stereo-
chemical synthesis and analysis of substitution reactions of phosphate monoesters (Figure 3.43).

3.2.2 Hydrolysis of Phosphate Esters^84

The great stability of phosphate diesters and monoesters during hydrolysis under physiological conditions
is an essential feature of the chemistry of nucleosides and nucleic acids and is intrinsic to life itself. Studies
of mechanisms for their hydrolysis have had to be carried out at elevated temperatures (up to 250°C) and
often at extremes of pH and the data is then extrapolated to ambient temperature and pH 7. Reactivity can
also be enhanced by use of aryl esters, and 4-nitrophenyl esters have been used frequently because of their
enhanced reactivity and convenient chromophoric properties.

Nucleosides and Nucleotides 101

O

P

HOHO OH

O

P

HOHO O

O

P

HOO O

O

P

OO O

pKa 2.12 pKa 7.21 pKa 12.32

Figure 3.41 Orthophosphoric acid and its conjugate bases

P

EtO

O

MeO

S

P

O

EtO

O

P MeO

OPh

EtO

O

MeO

aa b c

RRPP

Figure 3.42 (a) Chiral phosphate triester; (b) pro-chiral oxygens in a phosphate diester; and (c) chiral
phosphothioate diester

P

O

O

O OH

H

H

a b

P O

Figure 3.40 (a) Orthophosphoric acid; and (b) P O dp–ppbonding