4.17.1. 3. Phosphotriester Synthesis

In the 1960s, groups led by R. Letsinger and C. Reese developed a phosphotriester approach. The defining difference from the phosphodiester approach was the protection of the phosphate moiety in the building block 1 (Scheme 4.4) and in the product 3 with 2-cyanoethyl group. This precluded the formation of oligonucleotides branched at the internucleosidic phosphate. The higher selectivity of the method allowed the use of more efficient coupling agents and catalysts, which dramatically reduced the length of the synthesis. The method, led to the automation of the oligonucleotide chain assembly.

Scheme 4.4: Oligonucleotide coupling by phosphotriester method.

4.17.1.4. Phosphite Triester Synthesis

In the 1970s, substantially more reactive P(III) derivatives of nucleosides, 3'-O-chlorophosphites, were successfully used for the formation of inter-nucleosidic linkages. This led to the discovery of the phosphite triester methodology. The group led by M. Caruthers took the advantage of less aggressive and more selective 1H-tetrazolidophosphites and implemented the method on solid phase. The use of 2-cyanoethyl phosphite-protecting group in place of a less user-friendly methyl group led to the nucleoside phosphoramidites currently used in oligonucleotide synthesis.  

4.17.1. 5. Synthesis by the Phosphoramidite Method

4.17.1. 5.1. Building Blocks: Nucleoside phosphoramidites

The naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford expedite synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of inter-nucleosidic linkages is dramatically improved by using 3'-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides called nucleoside phosphoramidites that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides have to be rendered unreactive by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides.

Figure 4.20: Protected 2'-deoxynucleoside phosphoramidites.

Below, the protecting groups currently used in commercially available and most common nucleoside phosphoramidite building blocks are briefly described.

• The 5'-hydroxyl group is protected by an acid-labile DMT (4,4'-dimethoxytrityl) group.'
• Thymine and uracil, nucleic bases of thymidine and uridine, respectively, do not have exocyclic amino groups and hence do not require any protection. Although the nucleic base of guanosine and 2'-deoxyguanosine have an exocyclic amino group, its basicity is low to an extent that it does not react with phosphoramidites under the conditions of the coupling reaction. However, a phosphoramidite derived from the N2-unprotected 5'-O-DMT-2'-deoxyguanosine is poorly soluble in acetonitrile, the solvent commonly used in oligonucleotide synthesis. In contrast, the N2-protected versions of the same compound dissolve in acetonitrile well and hence are widely used. Nucleic bases adenine and cytosine bear the exocyclic amino groups reactive with the activated phosphoramidites under the conditions of the coupling reaction. Although, at the expense of additional steps in the synthetic cycle, the oligonucleotide chain assembly may be carried out using dA and dC phosphoramidites with unprotected amino groups, most often these are kept permanently protected over the entire length of the oligonucleotide chain assembly. The protection of the exocyclic amino groups has to be orthogonal to that of the 5'-hydroxy group because the latter is removed at the end of each synthetic cycle. The simplest to implement and hence the most widely accepted is the strategy where the exocyclic amino groups bear a base-labile protection. Most often, two protection schemes are used.
• In the first, the standard and more robust scheme, Bz (benzoyl) protection is used for A, dA, C, dC, G, and dG are protected with isobutyryl group. More recently, acetyl group is often used to protect C and dC.
• In the second, mild protection scheme, A and dA are protected with isobutyryl or phenoxyacetyl groups (PAC). C and dC bear acetyl protection, and G and dG are protected with 4-isopropylphenoxyacetyl (i-Pr-PAC) or dimethylformamidino (dmf) groups. Mild protecting groups are removed more readily than the standard protecting groups. However, the phosphoramidites bearing these groups are less stable when stored in solution.
• The phosphite group is protected by a base-labile 2-cyanoethyl group.  Once a phosphoramidite has been coupled to the solid support-bound oligonucleotide and the phosphite moieties have been converted to the P(V) species, the presence of the phosphate protection is not mandatory for the successful conducting of further coupling reactions.
• In RNA synthesis, the 2'-hydroxy group is protected with TBDMS (t-butyldimethylsilyl) group or with TOM (t-butyldimethylsilyloxymethyl) group, both being removable by treatment with fluoride ion.

Figure 4.21: 2'-O-Protected ribonucleoside phosphoramidites.

• The phosphite moiety also bears a diisopropylamino (iPr2N) group reactive under acidic conditions. Upon activation, the diisopropylamino group leaves to be substituted by the 5'-hydroxy group of the support-bound oligonucleotide.