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Fluorous Affinity Purification of a Mixed-Base 100-mer

A 100-mer was synthesized using standard phosphoramidites, except that the last coupling was performed with FDMT-T CEP. Cleavage from the support and nucleobase deprotection was carried out with ammonium hydroxide as usual. The crude ammonia solution was subjected to the Fluorous Affinity Purification protocol. The figure below illustrates what is happening at each step.


Trace (a): HPLC of the crude ammonia solution, showing a large separation of the fluorous tagged FDMT-on 100-mer and non-fluorous materials (e.g. failures).

Trace (b): After diluting the crude solution with loading buffer and passing it through a Fluoro-Pak™ column one time, the eluate showed complete binding of the FDMT 100-mer, while most of the non-fluorous material (failure sequences) failed to bind. DMT-on purifications cannot achieve this level of selectivity on long oligonucleotides.

Trace (c): Washing the column with 10% acetonitrile in 0.1 M TEAA removed the rest of the failure sequences without removing the bound FDMT 100-mer.

Trace (d): Another wash with 10% acetonitrile in 0.1 M TEAA showed that all of the failures had been removed.

Trace (e): After on-column detritylation to remove the FDMT group, the fully deprotected 100-mer is eluted. Another trace of the final material is shown below:


C18 RP silica column (4.6 x 150 mm), 1 mL/min, Mobile A = acetonitrile, Mobile B = 0.1 M TEAA

 

 

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