Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 161

phosphorothioate-modified oligodeoxynucleotides with mixed phosphorothioate stereochemistry bind
more weakly to complementary RNA targets than do regular phosphate oligomers. Also, in the case of uni-
form phosphorothiate oligonucleotides, there can be a loss of specificity of binding to nucleic acids while
some non-specific binding to proteins may be observed. The challenge of the chemical synthesis of homochi-
ral all-(RP) and all-(SP) oligomers has been accomplished by Wojciech Stec28,29leading to the conclusive
result that oligomers having all-(RP) phosphorothioate linkages bind more tightly to RNA than do their
phosphate counterparts. Single-site stereospecific modifications have been used in mechanistic studies
involving oligonucleotides, for example in studies of the cleavage reaction of ribozymes (see Section 7.6.2).
Replacement of a bridgingoxygen atom by sulfur is more difficult to achieve synthetically. Internucleotide
coupling reactions involving sulfur are more difficult since the sulfur atom is less nucleophilic for phos-
phorus. Nevertheless, oligonucleotides in which the 3
- or 5
-bridging oxygen has been replaced by sulfur
have been prepared and used in mechanistic studies.

4.4.3.2 Phosphorodithioates. Phosphorodithioatelinkages have both the non-bridging oxygen atoms

replaced by sulfur. Such linkages are non-chiral and are completely resistant to cleavage by all known
nucleases. Caruthers has developed a synthesis of phosphorodithioate oligonucleotides that couples a
2
-deoxyribonucleoside 3
-phosphorothioamidite to a support-bound nucleoside 5
-hydroxyl group and is
followed by a sulfurisation step (Figure 4.22). The 2-benzoylthioethyl group is removed by ammonia
deprotection at the end of the synthesis.^30 Although this method can incorporate a phosphorodithioate link-
age at any position in an oligonucleotide, phosphorodithioates are used infrequently because they bind to
complementary oligonucleotides with reduced discrimination and also bind to various proteins.

4.4.3.3 Methylphosphonates. Methylphosphonatesare uncharged analogues of phosphodiester anions

in which a non-bridging oxygen atom of the phosphate group has been replaced by a methyl group
(Figure 4.23a) (several other alkyl or aryl groups attached to phosphorus have also been used).
Oligonucleotides containing methylphosphonate modifications are prepared from 3
-O-methylphospho-
namidite nucleoside building blocks using conditions similar to standard phosphoramidite synthesis.^31 The
methylphosphonate is chiral at phosphorus, so a mixture of isomers occurs and the synthesis of defined
stereoisomers has been accomplished. Methylphosphonate diester linkages have enhanced stability to exo-
and endonucleases and duplexes containing them have elevated Tms. However, as this modification results
in a loss of the phosphate anionic charge, poor aqueous solubility and aggregation of oligonucleotides can
result from multiple methylphosphonate substitutions.

P

O

R3'

NCCH 2 CH 2 O OR5'

P

O

O

R3'

H

R5' O

P

R3'O

O

S

OR5'

P

R3'O

O

S

OR5'

S

S

O O

O

+

polymerase

phosphite triester

H-phosphonate triester

ii, NH 4 OH

S 8

pyridine

[Sp]-dNTPαS[Rp]-dNTPαS

[Rp][Sp]

polymerase

(slow with Mn2+)

i. 3H-1,2-benzodithiole-

3-one-1,1-dioxide

3H-1,2-benzodithiole-3-one-1,1-dioxide

Figure 4.21 Synthesis of oligonucleotide phosphorothioates