Nucleic Acids in Chemistry and Biology

Early syntheses of nucleoside phosphate esters worked mainly with mild condensing agents such as dicy-
clohexylcarbodiimide (DCC). More powerful reagents, such as arenesulfonyl chlorides, were introduced
next and were improved by building in steric factors. Some valuable references to the early phosphorylation
chemistry may be found in a number of reviews.34,88–90After 1980, the demand for faster reactions for oligonu-
cleotide synthesis has switched attention to P(III) chemistry.^91 In a more recent variation, the use of
H-phosphonates as a 4-coordinate P(III) species^92 has been built upon pioneering studies of Todd in the 1950s.

3.2.3.2 Syntheses via Phosphate Diesters.88,89,93 In the diester route to oligonucleotides (Figure 3.54),

the key step is the condensation of a phosphate monoester with an alcohol using DCC. The reactions are
slow, but at room temperature there is no formation of triesters. The mechanism is complex: an initial imidoyl
phosphate adduct of DCC and the 5-nucleotide is probably converted into the cyclic trimetaphosphate
species before reaction with the 3-hydroxyl component and subsequent final formation of the phosphate
diester (Figure 3.54). Since the reaction of trimetaphosphate with alcohols is relatively slow, DCC was
superseded by mesitylenesulphonyl chloride as a faster and more efficient condensing agent.

3.2.3.3 Syntheses via Phosphate Triesters.88,89 The greater reactivity for phosphorylation using

arenesulfonyl chloride as activating agent enables the syntheses of triesters from dialkyl phosphates and
an alcohol, and so it forms the basis of the first triester syntheses of oligonucleotides. The key step here is the
condensation of a suitable nucleotide diester as (RO) 2 POX, with the 3-hydroxyl group of a second nucleo-
side to give a phosphate triester, (RO) 3 PO. To avoid problems arising from the nucleophilicity of chloride
anion, the condensing agents now used are mesitylenesulfonyl tetrazolide (MST)or nitrotriazolide

108 Chapter 3

O

O

RO P

ArO

O

O

RO P

R'O

O

OR'

ArO P

RO

HO

N C N

C 6 H 11

C 6 H 11

O

O

RO P

O C

NC 6 H 11

NHC 6 H 11

C 6 H 11 NH C HNC 6 H 11

O

O

O

RO P

DCC R'O

HO

Cl

Me

Me

S Me

O

O

N

N

N

O 2 N

O

O

RO P

ArO

Me

Me

S Me

O

O

N N

O 2 N N

O

N

RO P

ArO

N N

NO 2

O

H

O

O

P O

OR

H

NC N

C 6 H 11

C 6 H 11

H

O

O

P O

OR

R'OH

H+

DCU

R'

3'-phosphate

diester

phosphodiester chemistry using DCC

phosphate

triester

R = 3'

R'OH

5'-phosphate

monoester

phosphate

diester

R' =5'

phosphotriester chemistry using MSNT

Ar =

2-NO 2 C 6 H 4 C=NOH

& Et 3 N phosphatediester

R'OH deprotection

Figure 3.54 Mechanisms and reagents of phosphodiester (upper) and phosphotriester (lower) chemistry

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