Universal Phosphoramidite Reagent
The advantage of a universal phosphoramidite is that inexpensive solid phase material bearing hydroxy or
amino groups can be used in oligonucleotide synthesis without any additional treatment.
The universal phosphoramidite is coupled to the solid phase in the first cycle of oligonucleotide synthesis
under the standard conditions. Once the solid phase is oxidized and capped, the 1-methoxy -1-ethylidene
(MED) group is cleaved by treating the solid support for at least 20 s with 3% dichloroacetic acid or 2%
trichloroacetic acid in methylene chloride. The standard detritylation subroutine is more than sufficient for
the deprotection of the solid support. The treatment with an acid results in the hydrolysis of the orthoester
to deprotect one of the hydroxy groups while the other one is left protected as an acetate ester. In contrast
to the conventional DMT group, no colorful product is formed in the course of deprotection. Upon washing
with acetonitrile, the solid support is ready to be used for an oligonucleotide assembly.
Final Deprotection. The solid phase should be kept in contact with a deprotecting agent over a period of
time sufficient to complete the 3'-dephosphorylation. The dephosphorylation of P=O 2’-
deoxyoligonucleotides is carried out using one of the following conditions:
1. Conc. aqueous ammonium hydroxide for 2 h at 60°C;
2. Conc. aqueous ammonium hydroxide for 5 h at 25°C;
3. Ammonia-methylamine mixture (AMA) for 1 h at room temperature;
4. Gaseous ammonia: for 2 h at 25°C, 10 bar.
With 2'-O-substituted nucleosides, the 3'-dephosphorylation is more rapid so that the deprotection time can
be safely reduced by 30%.
Please note that the conditions above are the minimal conditions to complete 3'-dephosphorylation. The
deprotection of nucleic bases may require a longer exposure to a deprotecting agent.
To read and download the User Guide for Universal Phosphoramidite Reagent, use this link.