Nucleic Acids in Chemistry and Biology

148 Chapter 4

5
-unit to yield a dinucleoside phosphodiester. The main drawback is that the product phosphodiester is
also vulnerable to phosphorylation by the activated deoxyribonucleoside phosphomonoester to give a trisub-
stituted pyrophosphate derivative. An aqueous work-up is necessary to regenerate the desired phosphodiester.
Extension of the chain involves removal of the 3
-protecting group with alkali (for Racetyl) or fluoride ion
(for Rtert-butyldiphenylsilyl, TBDPS) and coupling with another deoxyribonucleoside 5
-phosphate
derivative. To prepare oligonucleotides beyond five units, preformed blocks containing two or more deoxyri-
bonucleotide residues must be coupled. Such blocks require significant effort to synthesise and contain unpro-
tected phosphodiesters that undergo considerable side reactions. The synthetic products of coupling reactions
require lengthy purification. Thus, synthesis of an oligonucleotide of 10–15 residues (the effective limit of the
method) took upwards of 3 months. Although in the late 1970s phosphodiester chemistry was successfully
applied to solid-phase synthesis (Section 4.1.4), the low yields intrinsic to phosphodiester chemistry remained.

4.1.3.2 Phosphotriester. Although this chemistry was first applied to solution-phase synthesis,

it proved particularly successful when applied to solid-phase synthesis in the early 1980s.^2 A
5
-O-(chlorophenyl phosphate) is coupled to a deoxynucleoside attached at its 3
-position to a solid support
(Figure 4.7). The coupling agent (mesitylenesulfonyl 3-nitro-1,2,4-triazolide, MSNT) is similar to that used
in phosphodiester synthesis, except that 3-nitrotriazolide replaces chloride. The coupling agent activates
the deoxyribonucleoside 3
-phosphodiester and allows reaction with the hydroxyl group of the support-bound
deoxyribonucleoside. The rate of reaction can be enhanced by addition of a nucleophilic catalyst such as
N-methylimidazole. This participates in the reaction by forming a more activated phosphorylating inter-
mediate (an N-methylimidazolium phosphodiester), since the N-methylimidazole is a better leaving group.
The product is a phosphodiester and accordingly is protected from further reaction with phosphorylating
agents. The yield is therefore much better than in the case of a phosphodiester coupling, but phosphotri-
ester chemistry could only be used satisfactorily after the development of selective reagents for cleavage
of the aryl protecting group. To extend the chain, the DMT group is removed by the treatment with acid
to liberate the hydroxyl group for further coupling. Note the direction of extension is 3
→ 5
, in contrast
to solid-phase phosphodiester chemistry.
Two side reactions give rise to limitations. During coupling there is a competitive reaction (about 1%) of
sulfonylation of the 5
-hydroxyl group by the coupling agent. This limits the efficiency of phosphotriester
coupling to 97–98%, and thus also the length of oligonucleotide attainable to about 40 residues. More ser-
iously, deoxyguanosine residues are subject to both phosphorylation and nitrotriazole substitution at the
O-6-position unless the O-6-position is protected. O^6 -Phosphorylation is particularly serious since this is not
easily reversible (in contrast to phosphitylation) and leads to chain branching and eventually chain degradation.
The phosphotriester method is particularly useful for large-scale (multi-gram) synthesis of short oligonu-
cleotides. Here the solid support is usually replaced by an acetyl or benzoyl group for solution phase stepwise
synthesis, or by a soluble polymeric carrier.

B^1

O

HO

MMTO

B^2

O

RO

P O

O

O

O

B^1

O

O

MMTO

P

O B

2

O

RO

O

O

SO 2 Cl

+

R = COCH 3 or TBDPS

i, pyridine

ii, aqueous work-up

Figure 4.6 Formation of an internucleotide bond by the phosphodiester method. BT, CBz, ABzor GiB. MMT is

monomethoxytriphenylmethyl

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