phosphoramiditteknologi

Phosphoramidites are key intermediates in the synthesis of oligonucleotides, particularly in automated DNA and RNA sequencing and synthesis. They are organophosphorus compounds characterized by a phosphorus atom bonded to an amidite group (–N(R)–), an alkoxy group, and a chlorine atom. The general structure includes a phosphoramidite moiety attached to a nucleoside or nucleotide base, enabling controlled polymerization reactions.

In oligonucleotide synthesis, phosphoramidites are used in solid-phase synthesis methods, where they react with hydroxyl groups on a growing DNA or RNA strand. The reaction proceeds via activation with a tetrazole catalyst, forming a phosphite triester intermediate, which is then oxidized to a phosphate triester. This process allows for the stepwise addition of nucleotides, facilitating the construction of defined sequences.

Phosphoramidite chemistry was pioneered by Leslie Levene and later refined by Philip S. Anderson and others in the 1980s, leading to the development of automated synthesis instruments. The method offers high efficiency, reproducibility, and scalability, making it indispensable in molecular biology, genomics, and biotechnology. Variations of phosphoramidites, such as those modified with fluorescent dyes or locked nucleic acids (LNAs), expand their applications in research and diagnostics.

The synthesis of phosphoramidites involves coupling a nucleoside with a chlorophosphite reagent, followed by amidation with a secondary amine. Purification and characterization are critical to ensure high-quality intermediates for downstream applications. While phosphoramidites are primarily used in DNA/RNA synthesis, their principles also inform the design of other phosphoramidate-based compounds in medicinal chemistry and materials science.