Nucleic Acids in Chemistry and Biology

group may be achieved using bulky phosphorylating agents such as bis-(2,2,2-trichloro-1,1-dimethyl) phos-
phorochloridate (Figure 3.58a).
For a wide range of sugars and bases, direct phosphorylation at the 5-position of unprotected nucleo-
sides and 2-deoxyribonucleosides using POCl 3 in a trialkyl phosphate solvent system (the Yoshikawa
method^98 ) is probably the simplest and most convenient method. Hydrolysis of the highly reactive phos-
phorodichloridate intermediate with aqueous buffer gives the monophosphate (Figure 3.58b). No protec-
tion of the base or sugar is required, although by-products arising from phosphorylation of the secondary
hydroxyl groups of the sugar vary, depending on the nature of the nucleoside. In the same way, isotopic
oxygen can be readily introduced by the generation of P^18 OCl 3 in situ, while the use of thiophosphoryl
chloride (PSCl 3 ) leads to phosphorothioates.^99 In a modification to the procedure, the use of aqueous pyri-
dine in acetonitrile as solvent (the Sowa–Ouchi method)^100 can provide yields greater than 80% with over
90% regioselectivity.
Synthesis of nucleoside 3-phosphate monoesters requires protection of the 5-hydroxyl group and base
and can be achieved, for example, by DCC-mediated condensation with 2-cyanoethyl phosphate. The
method also allows the preparation of phosphorothioate monoesters (Figure 3.58c).

3.3 Nucleoside Esters of Polyphosphates

3.3.1 Structures of Nucleoside Polyphosphates and Co-Enzymes

Phosphoric acid can form chains of alternating oxygen–phosphorus linkages, which are relatively stable
in neutral aqueous solution (Figure 3.59). The major condensed phosphates of biological importance are
pyrophosphoric acid, (HO) 2 P(O)OP(O)(OH) 2 , its esters and esters of tripolyphosphoric acid. The stability
of such species can be related to that of the corresponding phosphates after due allowance for (1) the
changed stability of the anionic charge and (2) improved leaving group characteristics.
Thus, tetraethyl pyrophosphate is an ethylating agent towards hard nucleophiles and also it is a phosphor-
ylating agent. Tetraphenyl pyrophosphate is exclusively a phosphorylating agent. P^1 ,P^2 -Dialkyl pyrophosphates
have considerable stability towards hydrolysis at ambient pHs where they exist exclusively in the form of a
dianion. This feature is very important for the stability of the pyrophosphate link as a structural feature in many
co-enzymes, including NADH, FAD and CoA. Similarly, P^1 ,P^3 -diesters of tripolyphosphoric acid are stable
components of the ‘cap’ structure of eukaryotic mRNA (Figure 3.60) and P^1 ,P^4 -diadenosyl tetraphosphate

Nucleosides and Nucleotides 111

O

P

O

P

O

P

O

OOOOO O

n

Figure 3.59 Structure of polyphosphates

N

HN

N

N

O

H 2 N

O

HO OH

Me

O

P

O

P

O

P

O

OO OOO O

O

OH

O

P

OO

O

base

O

OH

OP

OO

O

base

O

OH

O P

OO

O

base

Figure 3.60 P^1 , P^3 -Dinucleosidyl triphosphate in the ‘cap’ structure at the 5-end of eukaryotic mRNA