A Reliable Phosphorylation Reagent——Dibenzyl N,N'-Diisopropylphosphoramidite
Protein phosphorylation plays a very important role in biochemical processes. Enzymes in which phosphate groups are introduced are called kinases, and enzymes in which phosphate groups are removed are called phosphatases. Protein phosphorylation can occur on many kinds of amino acids (the main unit of protein), among which serine, threonine, and tyrosine are the main ones. In addition, functional groups containing phosphoric acid are very common functional groups in some small molecule drugs, especially nucleotide drugs, such as the anti-hepatitis C star drug Sofosbuvir launched in 2013, and the earlier nucleoside drug Tenofo Wei, Adefovir, etc. In addition, with the rise of genetic drugs, some oligonucleotide drugs have been approved by the FDA, including: antisense oligonucleotide drugs (ASOs), nucleic acid aptamer drugs (apatmers), small molecule interference RNA drugs (siRNAs), etc. Some time ago, Nosinagen sodium, a rare disease drug with a price of 700,000 yuan per injection, was directly reduced to less than 33,000 yuan per injection after being "soul bargained" by the national medical insurance negotiator. As soon as the news was broadcast, the Internet was swamped. Everyone was cheering for the negotiators of the national medical insurance, and also felt relieved for the families of the patients. However, what is little known is that this currently the only drug for the treatment of spinal muscular atrophy (SMA) in the world is also an antisense oligonucleotide (ASO) drug (Figure 1). It can be seen that the phosphate functional group endows some drugs with very important and critical pharmacological activities.
Figure 1 Sofosbuvir (left) and Nosinagen Sodium (right)
There are many ways to introduce phosphoric acid functional groups in drug synthesis. Common phosphorylation reagents include: phosphorus oxychloride, pyrophosphoryl chloride, dimethyl chlorophosphate, diphenyl chlorophosphate, etc. (Figure 2). Each of these phosphorylation reagents has advantages and disadvantages. After phosphorus oxychloride and pyrophosphoryl chloride react with alcohol under alkaline conditions, they can be hydrolyzed to obtain phosphoric acid groups. The advantages of these two reagents are low price and easy operation, but the disadvantage is that the product often contains a small amount of similar polar impurities, which makes the purification of the product difficult. Alcohol can also be reacted with dimethyl chlorophosphate or diphenyl ester to generate a phosphate intermediate and then remove the protecting group to obtain a phosphoric acid functional group. The phosphate intermediates generated by these two phosphorylation reagents can be isolated and purified. Dimethyl phosphate intermediates generally require the use of trimethylbromosilane to remove methyl esters, which is not suitable for some acid-sensitive substrates; and diphenyl phosphate intermediates require stoichiometric platinum dioxide as a catalyst to remove Phenyl groups—catalytic amounts of platinum dioxide or palladium on carbon cannot remove phenyl groups, so this synthesis method is too expensive from the perspective of production costs.
Figure 2 Common phosphorylation reagents
This article introduces a reliable phosphorylation reagent - dibenzyl N,N'-diisopropylphosphoramidite, a colorless or light yellow oily substance. Dibenzyl N,N'-diisopropylphosphoramidite is very acid sensitive. Due to the high boiling point of this compound (130°C/0.55mmHg), it is quite difficult to purify by vacuum distillation, so the purity of dibenzyl N,N'-diisopropylphosphoramidite sold on the market is usually not high. Greater than 98%.
Figure 3 Dibenzyl N,N'-diisopropylphosphoramidite and its application
The usual method for introducing phosphoric acid groups with dibenzyl N, N'-diisopropyl phosphoramidite is divided into three steps: (1) dibenzyl N, N'-diisopropyl phosphoramidite and alcohol in four Under the catalysis of nitrogen azole or 4,5-dicyanoimidazole and other weak acids, trivalent phosphorous acid intermediates are generated; (2) phosphorous acid intermediates are oxidized to pentavalent dibenzyl phosphate intermediates, which can be separated and purified (3) two benzyl groups are removed by palladium-carbon catalytic hydrogenation to obtain a phosphoric acid functional group. Compared with the phosphorylation reagents mentioned above, dibenzyl N,N'-diisopropylphosphoramidite has the advantages of mild reaction conditions, simple operation, easy purification of intermediates, and low production cost.
After a lot of technological innovations, our company is now able to produce dibenzyl N,N'-diisopropylphosphoramidite with gas phase purity and NMR purity above 98% on a kilogram scale! At the same time, the company also produces and sells high-quality tetrazole, 4,5-dicyanoimidazole and other related products. Welcome friends in need to call us for inquiries!