Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 149

4.1.3.3 Phosphite Triester. The development of phosphite triester (or phosphoramidite) chemistry

by Caruthers and co-workers in the early 1980s transformed oligonucleotide synthesis into an efficient and

automated process.4,5The crux of this chemistry is a highly efficient coupling reaction between a 5
-hydroxyl

group of a support-bound deoxyribonucleoside and a 5
-DMT-(N-acetylated)-deoxyribonucleoside 3
-O-

(N,N-diisopropyl O-alkyl phosphoramidite (the alkyl group being methyl or 2-cyanoethyl) (Figure 4.8). In

early development of this chemistry, a chlorophosphite was used in place of the N,N-diisopropylphospho-

ramidite, but was found to be unstable on storage. By contrast, a phosphoramidite is considerably less

reactive and requires protonation on nitrogen to make the phosphoramidite into a highly reactive phos-

phitylating agent. A weak acid (such as tetrazole or 4,5-dicyanoimidazole) can do this without causing loss

of the DMT group. The product of coupling is a dinucleoside phosphite, which must be oxidised with

iodine to the phosphotriester before proceeding with chain extension.

The efficiency of coupling is extremely high (98%) and the only major side reaction is phosphityla-

tion of the O^6 -position of guanosine. Fortunately, after coupling, treatment with acetic anhydride and

N-methylimidazole (introduced to cap off any unreacted hydroxyl groups) completely reverses this side

reaction. Solid-phase phosphoramidite chemistry may be used for synthesis of oligodeoxynucleotides up

to 150 residues in length and to prepare products on a scale from micrograms to many grams.

4.1.3.4 H-Phosphonate. Although the origins of this chemistry lie with Todd and co-workers in

the 1950s, the potential in oligonucleotide synthesis emerged more recently.^2 A deoxyribonucleoside

3 
-O-(H-phosphonate) is essentially a tetra-coordinated P(III) species, preferring this structure to the tau-

tomeric tri-coordinated phosphate monoester. Activation is achieved with a hindered acyl chloride (e.g.

B^1

O

O

DMTO

ROPN

B^2

O

O

HO

Support

B^1

O

O

DMTO

PO B 2

O

O

RO

Support

B^1

O

O

DMTO

P

O B

2

O

O

RO

O

Support

N

N

H

NC

NC

N N

NH

+ N

I 2 /H 2 O/pyridine

or

Figure 4.8 Formation of an internucleotide bond by the solid-phase phosphoramidite method. Rmethyl or
2-cyanoethyl

B^1

O

O

DMTO

P

O

O

Cl

O

+

B^2

O

O

HO

Support

B^1

O

O

DMTO

P

O B

2

O

O

O

O

Cl

Support

SO (^2) N
N
N
NO 2
pyridine/N-methylimidazole
Figure 4.7 Formation of an internucleotide bond by the solid-phase phosphotriester method