4.17.1.5.2. Non-nucleoside Phosphoramidites

Non-nucleoside phosphoramidites are the phosphoramidite reagents designed to introduce various functionalities at the termini of synthetic oligonucleotides or between nucleotide residues in the middle of the sequence. In order to be introduced inside the sequence, a non-nucleosidic modifier has to possess at least two hydroxy groups, one of which is often protected with a DMT group while the other bears the reactive phosphoramidite moiety.
Non-nucleosidic phosphoramidites are used to introduce desired groups that are not available in natural nucleosides or that can be introduced more readily using simpler chemical designs. A very short selection of commercial phosphoramidite reagents is shown in Scheme for the demonstration of the available structural and functional diversity. These reagents serve for the attachment of 5'-terminal phosphate (1), NH2 (2), SH (3), aldehydo (4), and carboxylic groups (5), CC triple bonds (6), non-radioactive labels and quenchers (exemplified by 6-FAM amidite 7 for the attachment of fluorescein and dabcyl amidite 8, respectively), hydrophilic and hydrophobic modifiers (exemplified by hexaethyleneglycol amidite 9 and cholesterol amidite 10, respectively), and biotin amidite 11.

Figure 4.22: Non-nucleoside phosphoramidites for 5'-modification of synthetic oligonucleotides.

4.17.1.5.3. The Synthetic cycle

Scheme 4.6: Synthetic cycle for preparation of oligonucleotides by phosphoramidite method.

Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5'-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle (Scheme 4.6) and consists of four chemical reactions:

• Step 1 – De-blocking (detritylation): The DMT group is removed with a solution of an acid, such as TCA or Dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene) and washed out, resulting in a free 5'-terminal hydroxyl group on the solid support-bound oligonucleotide precursor.
• Step 2 – Coupling: A nucleoside phosphoramidite (or a mixture of several phosphoramidites) is activated by an acidic azole catalyst, 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. This mixture is brought in contact with the starting solid support (first coupling) or a support-bound oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. This reaction is very rapid and requires, on small scale, about 20 s for its completion. The phosphoramidite coupling is also highly sensitive to the presence of water and is commonly carried out in anhydrous acetonitrile. Unbound reagents and by-products are removed by washing.
• Step 3 – Capping: After the completion of the coupling reaction, a small percentage of the solid support-bound 5'-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) short oligomers. This is done by acetylation of the unreacted 5'-hydroxy groups using a mixture of acetic anhydride and 1-methylimidazole or, less often, DMAP as catalysts. Excess reagents are removed by washing.
• Step 4 – Oxidation: The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. This step can be substituted with a sulfurization step to obtain oligonucleotide phosphorothioates. In the latter case, the sulfurization step is carried out prior to capping.