In the most abbreviated nomenclature currently employed, pNstands for 5-nucleotide, Npfor a
3 -nucleotide and dNpfor a 3-deoxynucleotide (to be precise, a 2-deoxyribonucleoside 3-phosphate). This
shorthand notation is based on the convention that an oligonucleotide chain is drawn horizontally with its
5 -hydroxyl group at the left- and its 3-hydroxyl group at the right-hand end. Thus, pppGpp is the short-
hand representation of the ‘magic spot’ nucleotide, guanosine 3-diphosphate 5-triphosphate, whereas
ApG is short for adenylyl-(3→ 5 )-guanosine, whose 3→ 5 internucleotide linkage runs from the nucleo-
side on the left to that on the right of the phosphate.
2.1.2 Physical Properties of Nucleosides and Nucleotides
Owing to their polyionic character, nucleic acids are soluble in water up to about 1% w/v according to size and
are precipitated by the addition of alcohol. Their solutions are quite viscous, and the long nucleic acid molecules
are easily sheared by stirring or by passage through a fine nozzle such as a hypodermic needle or a fine pipette.
2.1.2.1 Ionisation. The acid–base behaviour of a nucleotide is its most important physical character-
istic. It determines its charge, its tautomeric structure, and thus its ability to donate and accept hydrogen
bonds, which is the key feature of the base:base recognition. The pKavalues for the five bases in the major
nucleosides and nucleotides are listed in Table 2.1.
It is clear that all of the bases are uncharged in the physiological range 5pH9. The same is true for
the pentoses, where the ribose 2,3-diol only loses a proton above pH 12 while isolated hydroxyl groups
ionise only above pH 15. The nucleotide phosphates lose one proton at pH 1 and a second proton (in the
case of monoesters) at pH 7. This pattern of proton equilibria is shown for AMP across the whole pH range
(Figure 2.5).
The three amino bases, A, C and G, each becomes protonated on one of the ring nitrogens rather than
on the exocyclic amino group since this does not interfere with de-localisation of the NH 2 electron lone
pair into the aromatic system. The CNH 2 bonds of A, C and G are about 1.34 Å long, which means that
they have 40–50% double bond order, while the CO bonds of C, G, T and U have some 85–90%
double bond order. It is also noteworthy that the proximity of negative charge of the phosphate residues
has a secondary effect, making the ring nitrogens more basic (pKa0.4) and the amine protons less
acidic (pKa0.6).
2.1.2.2 Tautomerism. A tautomeric equilibrium involves alternative structures that differ only in the
location of hydrogen atoms. The choices available to nucleic acid bases are illustrated by the keto–enol
equilibrium between 2-pyridone and 2-hydroxypyridine and the amine–imineequilibrium for 2-aminopy-
ridine (Figure 2.6). Ultraviolet, NMR and IR spectroscopies have established that the five major bases
exist overwhelmingly ( 99.99%) in the amino-and keto-tautomeric forms at physiological pH (Figure
2.1) and not in the benzene-like enoltautomers, in common use before 1950 (Figure 1.3).
16 Chapter 2
Table 2.1 pKavalues for bases in nucleosides and nucleotides
Bases (site of protonation) Nucleoside 3 -Nucleotide 5 -Nucleotide
Adenine (N-1) 3.63 3.74 3.74
Cytosine (N-3) 4.11 4.30 4.56
Guanine (N-7) 2.20 2.30 2.40
Guanine (N-1) 9.50 9.3 69.40
Thymine (N-3) 9.80 — 10.00
Uracil (N-3) 9.25 9.43 9.50
Note: These data approximate to 20°C and zero salt concentration. They correspond to lossof a
proton for pKa 9 and captureof a proton for pKa 5.