Nucleic Acids in Chemistry and Biology

150 Chapter 4

pivaloyl chloride), which couples the H-phosphonate diester to a nucleoside hydroxyl group (Figure 4.9).
The resultant H-phosphonate diester is relatively inert to further phosphitylation, such that the chain may
be extended without prior oxidation. Oxidation of all the phosphorus centres is carried out simultaneously
at the end of the synthesis. An advantage of this chemistry is that oxidation is subject to general base catal-
ysis and this allows nucleophiles other than water to be substituted during oxidation to give a range of
oligonucleotide analogues.
Unfortunately, a serious side reaction occurs if an H-phosphonate is premixed with activating agent before
coupling. The H-phosphonate rapidly dimerises to form a symmetrical phosphite anhydride. Subsequent reac-
tion of this with a hydroxyl group gives rise to a branched trinucleotide derivative. The complete elimination
of this side reaction, even under optimal conditions, is probably impossible and may account for the lower
yields obtained by this route.

4.1.4 Solid-Phase Synthesis

The essence of solid-phase synthesis is the use of a heterogeneous coupling reaction between a deoxynu-
cleoside derivative in solution and another residue bound to an insoluble support. This has the advantage
that a large amount of the soluble deoxynucleoside derivative can be used to force the reaction to high
yield. The support-bound product dinucleotide is removed from the excess of reactant mononucleoside
derivative simply by filtration and washing. Other reactions are also carried out heterogeneously and
reagents removed similarly. This process is far faster than a conventional separation technique in solution
and easily lends itself to mechanisation. Protocols and full details of the chemistry are available.6,7There
are four essential features of solid-phase synthesis.

4.1.4.1 Attachment of the First Deoxynucleoside to the Support. Of the many types of support that

have been used for solid-phase synthesis of oligonucleotides, only controlled pore glass (CPG) and poly-
styrene have proved to be generally useful. CPG beads are ideal in being rigid and non-swellable. They are
manufactured with different particle sizes and porosities and they are chemically inert to reactions involved
in oligonucleotide synthesis. Currently, 500–1000 Å porosities are favoured, the latter for synthesis of chains
longer than 80 residues. The silylation reactions involved in functionalisation of glass (introduction of reactive

B^1

O

O

DMTO

HOP

O

B^2

O

O

HO

Support

B^1

O

O

DMTO

PO B

2

O

O

H

O

Support

O

PO

C5'

O

O

C3'

O

PO

C5'

RHN

O

C3'

O

PO

C5'

S

O

C3'

S 8

+

I 2 /H 2 O

I 2 /RNH 2

Me 3 CCOCl/pyridine

Figure 4.9 Formation of an internucleotide bond by the solid-phase H-phosphonate method

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