Nucleic Acids in Chemistry and Biology

160 Chapter 4

• Stereochemistry at a thiophosphate is defined according to the CIP convention with priority SO3
  O5
  O(P).

Oligonucleotide conjugatesare formed by reaction with a complementary reactive group on the biomol-

ecule, such as a peptide, for example to give amide, disulfide or thioether linkages depending on the types

of functionalities involved in the conjugation.

4.4.3 Backbone and Sugar Modifications

Many modifications to the oligonucleotide backbone (the internucleotide linkage and/or sugar moiety) have

found applications in the use of oligonucleotides as antisense agents (see Section 5.7.1) or in synthetic

siRNA (see Section 5.7.2) for the control of gene expression.23–25The most common backbone modi-

fications are described below, noting their advantages and disadvantages for their use.

4.4.3.1 Phosphorothioates. Phosphorothioatelinkages were first prepared by Fritz Eckstein. They

have a non-bridging oxygen atom of a phosphodiester replaced by sulfur.18,19They can be prepared during

solid phase phosphoramidite synthesis by replacement of the oxidation step with a sulfurisation step.

While elemental sulfur (S 8 ) was used originally for this purpose, the sulfurisation step is now carried out

more rapidly and conveniently by use of a reagent such as 3H-1,2-benzodithole-3-one-1,1-dioxide (the

Beaucage reagent)^26 (Figure 4.21).

The replacement of an oxygen atom by sulfur results in a mixture of two diastereoisomers at phosphorus

and these are designated (RP) and (SP).∗For an oligonucleotide containing a single phosphorothioate link-

age, separation of the two diastereoisomers is usually possible by HPLC. For multiple sulfur substitutions,

separation by HPLC is not possible, and the required pure diastereoisomer must be synthesised by stereo-

specific phosphorothioate chemistry developed by Wojciech Stec.^27

Nucleotide phosphorothioates are isopolar and isosteric with phosphates, and generally only one of the

two diastereoisomers is a substrate for native polymerases. (SP)- -Thiotriphosphates (Section 3.3.2) with

a complete DNA polymerase lead to pure (RP) phosphorothioate linkages, since the polymerase extension

reaction proceeds with inversion of configuration (Figure 4.21). However, (RP)- -thiotriphosphate

nucleotides can be accepted as poorer substrates by DNA polymerases using manganese or by the Klenow

fragment of Pol-1 (see Section 5.1.1).

Phosphorothioate modifications in oligonucleotides have been particularly valuable in antisense appli-

cations for clinical use (see Section 5.7.1). They are more resistant to both exo- and endonucleases and are

therefore used to enhance the stability of oligonucleotides in cells and in sera. One disadvantage is that

N

O

O

O (CH 2 ) 9 O-R^2

O

Amino linker R^1 NH(CH 2 ) 6 O-R^2

Thiol linker TrS(CH 2 ) 6 O-R^2

Disulfide linker DMTO(CH 2 ) 6 - S-S-(CH 2 ) 6 O-R^2

Carboxylate linker

R^1 = monomethoxytrityl or trifluoroacetyl

R^2 = oligonucleotide

Figure 4.20 Linkers for oligonucleotide conjugation

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