144 Chapter 4
4.1.1 Overall Strategy for Chemical Synthesis
Nucleic acids are sensitive to a wide range of chemical reactions (see Chapter 8), and relatively mild reac-
tion conditions are required for their chemical synthesis. The heterocyclic bases are prone to alkylation,
oxidation and phosphorylation and the phosphodiester backbone is susceptible to hydrolysis. In the case of
DNA, acidic hydrolysis occurs more readily than alkaline hydrolysis because of the lability of the glyco-
sylic bond, particularly in the case of purines (depurination, see Section 8.1). Such considerations limit the
range of chemical reactions in oligodeoxyribonucleotide synthesis to (1) mild alkaline hydrolysis; (2) very
mild acidic hydrolysis; (3) mild nucleophilic displacement reactions; (4) base-catalysed elimination reactions;
and (5) certain mild redox reactions (e.g.iodine or Ag(I) oxidations and reductive eliminations using zinc).
The key step in the synthesis of oligodeoxyribonucleotides is the specific and sequential formation of
internucleoside 3→ 5 phosphodiester linkages. The main nucleophilic centres on a 2-deoxyribonucleo-
side are the 5- and 3-hydroxyl groups and, in the case of dC, dG and dA, the exocyclic amino groups.
To form a specific 3–5linkage between two nucleosides, the nucleophilic centres not involved in the
reaction must be protected. The first 5-unit requires a protecting groupon the 5-hydroxyl as well as on
the nucleobase, whereas the second 3-unit requires protection of the 3-hydroxyl as well as the nucleo-
base. In the example of joining a 5-dA unit to a 3-dG unit (Figure 4.1), R^1 and R^2 protect the 5-dA and
R^3 and R^4 protect the 3-dG. One of the two units requires phosphorylation or phosphitylation on the
unprotected hydroxyl group and is then joined to the other nucleoside in a coupling reaction. The resulting
dinucleoside monophosphate is now fully protected. Usually the phosphate group carries a protecting group
R^5 , introduced during the phosphorylation (phosphitylation) step, such that the internucleotide phosphate
is a triester.To extend the chain, one of the two terminal hydroxyl-protecting groups R^1 or R^3 must be
selectively removed to which a new protected nucleoside unit may be attached.
Where R^1 and R^3 are conventional protecting groups, oligonucleotide synthesis is referred to as solution-
phase.Solution-phase synthesis has largely been superseded by a solid-phasemethod, where either R^1 or R^3
is an insoluble polymeric or inorganic support (Section 4.1.4). Whereas extension of the chain in solution-
phase synthesis is possible in either the 3→ 5 or 5→ 3 directions, in solid-phase synthesis the oligonu-
cleotide can be extended only in one direction. The conventional protecting group removed prior to each
coupling step (R^1 or R^3 , whichever is not the solid-support) is a temporaryprotecting group. R^2 , R^4 , R^5 and
the solid support are all permanentprotecting groups, and must remain stable throughout the oligonucleotide
synthesis. They are only removed at the end of the synthesis to generate the final deprotected oligonucleotide.
4.1.2 Protected 2-Deoxyribonucleoside Units
The most convenient way to assemble an oligonucleotide is to utilise preformed deoxynucleoside phosphate
[P(V)] or phosphite [P(III)] derivatives as building blocks, and to couple these sequentially to a terminal
OOR^1 OOR^3 OOR P(^5) O
O
N
N
N
N
NHR^2
N
NH
N
N
O
NHR^4
O
HO
R^1 O
N
N
N
N
NHR^2
O
R^3 O
HO NH
N
N
O
NHR^4
N
- Figure 4.1 Joining of a 5-dA unit to a 3-dG unit
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