Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 147

with 2-chlorophenyl phosphoro-bis(triazolide) (Figure 4.5, Route a). Despite this being a bifunctional
phosphorylating agent it acts as a monofunctional one in the absence of any stronger catalyst.
In phosphate-triesterchemistry [P(III)] both aryl and alkyl phosphates are highly reactive species. Here,
a methyl group or 2-cyanoethyl group is the preferred protecting group because they can be removed conve-
niently and selectively at the end of the synthesis (Section 4.1.4). Again a bifunctional reagent is used in a
monofunctional manner, but to obtain a sufficiently stable product a phosphoramidite is prepared (Route b).
The monofunctional chlorophosphoramidite can also be used.
H-Phosphonatechemistry does not require protection of the phosphate group, since the internucleoside
H-phosphonate linkage in an oligonucleotide is stable to the conditions used in the assembly of the
oligonucleotide. In a sense, a proton is the protecting group. A 2
-deoxyribonucleoside 3
-H-phosphonate
is prepared by the reaction of a deoxynucleoside with phosphorus trichloride and imidazole or triazole in
the presence of a basic catalyst, such as N-methylmorpholine, followed by an aqueous work-up (Route c).

4.1.3 Ways of Making an Internucleotide Bond

The development of an efficient method for forming an internucleotide bond was for many years the most
central issue in oligonucleotide synthesis.^2 The problem was solved by the development of phosphite tri-
ester chemistry (phosphoramidite) and, to some extent, H-phosphonate chemistry. However, an under-
standing of earlier phosphodiester and phosphotriester chemistry is important (see Section 3.2.3).

4.1.3.1 Phosphodiester. In the pioneering gene syntheses by Khorana and colleagues in the 1960s

and 1970s (see Section 5.4.1),^3 oligonucleotide synthesis involved coupling a 5
-protected deoxynucleo-
side derivative with a 3
-protected deoxyribonucleoside-5
-phosphomonoester (Figure 4.6). The coupling
agent (triisopropylbenzenesulfonyl chloride, TPS) activates the phosphomonoester by a complex reaction
mechanism that gives a powerful phosphorylating agent, which reacts with the 3
-hydroxyl group of the

N

N

N

N NHBz

O

HO

HO

N

N

N

N NHBz

O

HO

DMTO

N

N

N

N NHBz

O

O

DMTO

P

R^4 ONiPr 2

N

N

N

N NHBz

O

O

DMTO

OP O

O

R 3

R 2

R 1

N

N

N

N NHBz

O

O

DMTO

P

O

H

O

R^3

R^2

R^1

OP

O

N

N

N

pyridineDMTCl H

b

R^4 O-P(NiPr 2 ) 2

tetrazole

or

R^4 O-P(NiPr 2 )Cl

EtNiPr 2

a c i, PClii, aqueous work-up^3 /imidazole/Et^3 N

2

i,

ii, aqueous work-up

Figure 4.5 Introduction of a 3
-phosphate by (a) phosphorylation, (b) phosphitylation, and (c) H-phosphonylation.
R^1 , R^3 H, R^2 Cl, 4-chlorophenyl; R^2 , R^3 H, R^1 Cl, 2-chlorophenyl; R^2 H, R^1 , R^3 Cl,
2,5-dichlorophenyl; R^4 methyl or 2-cyanoethyl