What are Phosphoramidites?

What are Phosphoramidites?

Published by BroadPharm on July 10, 2023

A phosphoramidite, also known as an amidite, is a chemical compound used in the synthesis of oligonucleotides, which are short chains of nucleotides. They consist of a nucleoside base (adenine, guanine, cytosine, thymine, or uracil) attached to a phosphoramidite group, which enables covalent bonding with the growing chain of nucleotides during the synthesis process.

Phosphoramidites in Oligonucleotide Synthesis

Phosphoramidites are primarily used for the process of building an oligonucleotide sequence, which is divided into multiple steps, typically three or four. Each step involves specific reactions and purification steps to add individual nucleotides to the growing oligonucleotide chain and assemble the desired longer sequence. During the synthesis, the amidite is activated and attached to a solid support, such as a resin or a glass bead. Through a series of coupling, deprotection, and washing steps, the oligonucleotide chain is elongated one nucleotide at a time, following a predefined sequence of amidites. After synthesis completion, the oligonucleotide is released from the solid support and purified for further applications (Figure 1).

Figure 1. Multi-Step Solid-Phase Oligonucleotide Synthesis

This process has been automated through high-throughput synthesizers and advancements in solid-phase synthesis techniques, enabling faster and more efficient production of synthetic oligonucleotides.This automated method enables the rapid and efficient synthesis of custom oligonucleotides for various applications, such as DNA sequencing, gene synthesis, and molecular diagnostics.

Recently, the ABMS lab at University of Freiburg presented one-pot-liquid-phase synthesis, a technique involving the entire process taking place within a single reaction vessel or system, without the need for intermediate purification steps between nucleotide additions, streamlining the process and reducing the overall synthesis time. In this approach, the nucleotides, amidites, and reagents required are combined together and subjected to a series of reactions and conditions that facilitate both coupling and deprotection simultaneously (Figure 2).

Figure 2. One-Pot-Liquid-Phase Synthesis

While one-pot synthesis offers the advantage of simplicity and speed, it can be challenging to achieve high purity and yields compared to traditional stepwise synthesis methods. The choice between the two techniques depends on factors such as the desired oligonucleotide length, sequence complexity, time constraints, and the specific requirements of the research or application. Researchers consider factors such as cost, efficiency, scalability, and product purity when deciding which approach to adopt.

Phosphoramidite Supplier

BroadPharm offers a wide array of different phosphoramidites such as DNA, RNA, biotin, fluorescent dye, and Click-Chem phosphoramidites to empower our customers' advanced research worldwide. These compounds feature a broad selection of nucleotide modifications and functional groups to choose from.

Journal References

Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels
Guido Creusen, Cecilia Oluwadunsin Akintayo, Katja Schumann, and Andreas Walther
Journal of the American Chemical Society 2020 142 (39), 16610-16621
DOI: 10.1021/jacs.0c05488