Phosphoramidites

Phosphoramidites are the cornerstone reagents in modern oligonucleotide synthesis, enabling the rapid and efficient assembly of DNA and RNA sequences. These compounds are central to the solid-phase synthesis method, which revolutionized the production of oligonucleotides for research, diagnostics, and therapeutics. Their significance extends across a range of applications, including antisense therapies, small interfering RNA (siRNA), and mRNA vaccine development. Alfa Chemistry offers a comprehensive portfolio of high-purity phosphoramidites, tailored to meet the stringent requirements of oligonucleotide synthesis in both laboratory and industrial settings.

Chemical Structure of Phosphoramidite

Each phosphoramidite consists of a 5'-O-DMT sugar moiety (2'-deoxyribose for DNA or 2'-O-protected ribose for RNA), a protected nitrogenous base (adenine, guanine, cytosine, and thymine in DNA; adenine, guanine, cytosine, and uracil in RNA), and, most crucially, a phosphoramidite group at the 3' position of the sugar moiety. The phosphoramidite group includes a 2-cyanoethyl group that safeguards the second hydroxyl on the phosphorus (III) center, and a diisopropylamino group acts as a leaving group, facilitating its removal during synthesis and enabling the phosphoramidite to attach to the growing oligonucleotide chain.

Figure 1. The structure of a standard 2'-deoxyadenosine phosphoramidite.

The Role of Phosphoramidite in Oligonucleotide Synthesis

The phosphoramidite method is the gold standard for synthesizing oligonucleotides. The methodology involves a four-step cycle:

• Coupling: The 5'-hydroxyl group of a growing oligonucleotide chain reacts with an activated phosphoramidite monomer, forming a phosphite linkage.
• Capping: Unreacted hydroxyl groups are acetylated to prevent the formation of truncated sequences.
• Oxidation: The phosphorus (III) center is oxidized to a stable phosphorus (V) phosphotriester using iodine-based reagents.
• Deblocking: Acidic cleavage of the dimethoxytrityl (DMT) group exposes the next reactive hydroxyl for sequential elongation.

This automated, high-yield process enables the synthesis of custom oligonucleotides for applications such as:

• Antisense and siRNA therapeutics
• qPCR probes and primers
• DNA sequencing adapters

Alfa Chemistry offers high-quality phosphoramidites designed to achieve optimal coupling efficiency and minimize side reactions, thus ensuring excellent oligonucleotide purity and functional performance.

Handling and Storage of Phosphoramidite

Phosphoramidites are highly sensitive to moisture and oxygen, requiring careful handling and storage to maintain their stability and reactivity. They should be stored under an inert atmosphere, such as argon or nitrogen, in tightly sealed containers to prevent hydrolysis and oxidation. In addition, ideally, they should be stored at low temperatures, typically at -20°C. When handling, anhydrous solvents and dry glassware are essential to prevent premature degradation. Proper storage and handling help ensure their effectiveness in oligonucleotide synthesis and other phosphoramidite-based applications.

Why Choose Us

• High Purity and Batch Consistency

Alfa Chemistry ensures that all phosphoramidites meet stringent quality standards. This maximizes purity, reduces side product formation, and ensures batch-to-batch consistency, which is crucial for high-fidelity oligonucleotide synthesis in both research and industrial applications.

• Scalability for Research and Industrial Needs

From small-scale laboratory research to large-scale industrial production, Alfa Chemistry provides phosphoramidites in a range of quantities. This scalability ensures that clients receive consistent product quality, whether for early-stage research or commercial-scale oligonucleotide manufacturing.

• Expert Technical Support and Consultation

During the partnership, Alfa Chemistry's team of experts will also provide you with technical assistance. This support helps researchers and manufacturers maximize synthesis efficiency, reduce errors, and streamline the oligonucleotide production process.