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Phosphotriester Approach - Both phosphotriester and the phosphite triester methods utilize deoxyribonucleosides as starting materials, and involve the stepwise addition of mononucleotides and oligonucleotides. The amino groups of the nucleosides deoxyadenosine and deoxcytidine are usually benzoylated, and that on deoxyguanosine is protected by an isobutyryl group thymidine requires no protection.

The 5/-OH group is protected by dimethoxytrityl commonly abbreviated as DMTr or (MeO)2 Tr. The amino groups of the bases are freed by mild alkaline hydrolysis, while (MeO)2 Tr is removed by gentle acid hydrolysis.

In the phosphotriester approach, the 3'-OH is coupled with a suitable phosphorylating agent, say, p-chlorophenyl phosphorodichloridate; the phosphate residue has a free OH group which accepts any nucleotide or oligonucleotide with a free 5'-OH group. Therefore, such protected nucleotides have phosphodiesters and serve as the 5'-terminus residue in oligonucleotide synthesis.
Such protected and phosphorylated nucleotides are further modified to make them suitable for joining to the -OH group of the 3' -phosphate residue.
The OH group of the 3'-phosphate residue of such nucleotides is blocked by a suitable agent, e.g., β-cyanoethanol, following which the 5/-OH is freed by mild acid hydrolysis (this removes the (MeO)2Tr). This yields phosphotriester nucleotides, which have, a free 5/-OH.

A desired diester nuecleotide (free -OH at 3'-phosphate residue) is now mixed with the desired triester nucleotide, and agents that promote their coupling are added to the mixture. Coupling is promoted by arylsulfonyl compounds, e.g., tri-iso-propyl- benzene sulfonyl chloride (TPS).
This reaction yields a fully protected dinucleotide, which can be either fully unprotected, or may be selectively unprotected to be used as the starting material for construction of larger molecules. The DNA chains can be constructed either in 3' --> 5' or 5' --> 3’ direction. Tedius purifications are essential after every addition to the growing chain to remove the uncoupled mononucleotides/ oligonucleotides.
This and some other problems are eliminated by using a solid support to which the first nucleotide is fixed.
Somehow fixing the 3'-OH is better than fixing the 5/-0R; generally, this is done by forming on ester between the 3'-OH and a carboxyl group on a solid support, e.g., controlled pore glass beads. This procedure has been adopted for automated stepwise synthesis; a 10-20 nucleotide long chain is synthesized in a few days.

 
 

 
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