page_title: Oligonucleotide synthesis
section_title: Nucleoside phosphoramidites
hierarchical_section_title: Oligonucleotide synthesis / Synthesis by the phosphoramidite method / Building blocks / Nucleoside phosphoramidites
caption_attribution_description: English: Rnaamidite
context_page_description: Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure. The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides, ribonucleosides, or chemically modified nucleosides, e.g. LNA or BNA.
  To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected.
context_section_description: As mentioned above, the naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expeditious synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of internucleosidic linkages is dramatically improved by using 3'-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides (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 (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides. Below, the protecting groups currently used in commercially available and most common nucleoside phosphoramidite building blocks are briefly reviewed:
  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 does 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. By the use of additional steps in the synthetic cycle or alternative coupling agents and solvent systems, the oligonucleotide chain assembly may be carried out using dA and dC phosphoramidites with unprotected amino groups. However, these approaches currently remain in the research stage. In routine oligonucleotide synthesis, exocyclic amino groups in nucleosides 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 used,  strategy is to install a base-labile protection group on the exocyclic amino groups. Most often, two protection schemes are used.
  In the first, the standard and more robust scheme (Figure), Bz (benzoyl) protection is used for A, dA, C, and dC, while G and dG are protected with isobutyryl group. More recently, Ac (acetyl) group is used to protect C and dC as shown in Figure.
  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 (iPr-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 (tri-iso-propylsilyloxymethyl) group, both being removable by treatment with fluoride ion.
  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-boun
caption: 2'-O-protected ribonucleoside phosphoramidites.
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