1-Cyanomethyl-2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite

1. High Stability

• Improved stability during purification and storage of the derived phosphoramidite
• Retains reactivity equivalent to conventional amidite reagents

2. Ease of Handling and Physical Properties

• High solubility in organic solvents (soluble in acetone, acetonitrile, and THF)
• Easy-to-handle crystalline solid (melting point: 111°C)

³¹P NMR analysis was used to compare the stability of the phosphorodiamidites; the results are shown below.

Phosphorodiamidite 1

The conventionally used linear phosphorodiamidite 1 (2-cyanoethyl N,N-diisopropylphosphoramidite) is an oily substance. When stored at room temperature, decomposition progressed over 7 days, and ³¹P NMR measurement confirmed the formation of multiple impurities. Furthermore, the phosphoramidite derivative consisting of a primary alcohol obtained using reagent 1 also does not have sufficient stability, resulting in reduced yield.

Phosphorodiamidite 2

In contrast, the newly developed branched phosphoramidite 2 is a solid, and almost no decomposition was observed even after 7 days at room temperature. In addition, the phosphoramidite derivative consisting of a primary alcohol synthesized using reagent 2 is sterically bulky due to its branched structure, giving it high stability and allowing it to be obtained in high yield. Furthermore, phosphoramidite derivatives with a branched structure can be incorporated into oligonucleotides under standard oligonucleiotide synthesis conditions.

Synthesis of phosphoramidite using a primary alcohol

Stability comparison of phosphoramidites synthesized from a primary alcohol (³¹P NMR analysis)

When the conventional linear phosphoramidite was stirred in an AcOEt solution of silica gel for 24 hours, ³¹P NMR measurement showed that it had completely decomposed. In contrast, the branched phosphoramidite showed almost no decomposition, demonstrating its high stability.

Synthesis of DNA using phosphoramidite

As an example, an oligo-DNA bearing cholesterol at the 5'-end was synthesized, and the target oligo-DNA could be obtained under standard DNA synthesis conditions.
Other branched phosphoramidites can likewise be incorporated into oligo-DNA under standard DNA synthesis conditions.

References

1. Y. Hari, Y. Ito: Japanese Patent Application No. 2025-184878, "Phosphoramidite Reagent, Phosphoramidite, and Method for Producing Nucleoside Derivatives"