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Fluorous Affinity Purification - Frequently Asked Questions

1.  Can the Fluoro-Pak™ columns be reused?

The columns are meant to be disposed after each use.  Fluorous trityl by-products are retained on the column, and it is difficult to assure that there will be no contamination in multiple runs. 

2.  Are deletion sequences removed?

Fluorous affinity purification involves the selective retention of any oligonucleotide that has a 5'-FDMT group.  It is ideal for separating these compounds from the failure sequences that result from incomplete coupling throughout the synthesis, i.e., those that are successfully capped by acetic anhydride and thus cannot couple further.  However, it should be recognized that there are still undesired FDMT-on oligonucleotides in the final crude synthesis product, and these will not be discriminated by fluorous affinity purification.  For example, incomplete capping is a common problem in oligonucleotide synthesis.  Uncoupled material that is also uncapped leaves a 5'-hydroxyl group that then is extended in the next coupling cycle to produce a final oligonucleotide with a deleted base.  This n-1 strand (or n-2, etc.) will lead to a final FDMT-on oligonucleotide that differs from the target strand by one or more base.  Nonetheless, failure sequences (which are removed by fluorous affinity purification) are a major contaminant, especially with longer oligonucleotides, where they are the major products of the synthesis.

3.  What sort of yields may I expect from fluorous affinity purification?

The short answer is that this method will recover most or all of the available material that was fluorous-tagged.  The actual amount isolated will depend on the overall synthesis yield, which is subject to the quality of the synthesis as well as the length of the oligomer, since the yield decays as chain length increases. 

Berry & Associates have examined a variety of oligonucleotides in lengths ranging from 30 to 100 nucleotides.  In each case, HPLC was used to estimate the amount of fluorous-tagged oligonucleotide as a percentage of the total oligonucleotide content, and the final yields of purified, detritylated product reflected 70-100% recovery of the oligonucleotide derived from the FDMT-on material in the crude mixture.  For example, from a 0.2 micromole synthesis of a sample mixed-base 100-mer, it was estimated that six A260 units of the crude material could be assigned to FDMT-on material, the rest being failure sequences, which are the major products of a long synthesis.  After Fluoro-Pak™ purification, six A260 units of purified, detritylated oligonucleotide were isolated, reflecting a quantitative recovery of available material.  Other examples on 0.2 micromole scale include 9-16 A260 units (60-100%) of a 50-mer and 9-11 A260 units (nearly quantitative) of a 75-mer.  About 30 A260 units were recovered from a 1 micromole synthesis of a 100-mer, and 44 A260 units from a 75-mer.  Again, the amount of oligonucleotide recovered will depend on the quality of the synthesis, so it important to realize that the fluorous affinity purification method will recover a high level of full-length material, whether that amount is large or small. 

See below for before-and-after RP-HPLC traces a 75-mer. Note that some DMT-bearing materials at around 15-20 minutes may be observed, especially for long oligonucleotides.  These impurities result from the incomplete and/or reversible nature of detritylations during synthesis.  In traditional DMT-on purifications, these impurities are often not noticed, since they appear in the same reagion as the DMT-on strand.  Thus, a further advantage of the fluorous affinity purification method is potential resolution from DMT-on impurities as well as failure sequences.

Before Fluorous Affinity Purification:


After Fluorous Affinity Purification (on-column detritylation):

 


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

 

4. Can I use HPLC to purify the FDMT-on oligonucleotide and remove the trityl group later?  Does the FDMT group behave like a DMT group during detritylation?

If you wish to use HPLC to isolate FDMT-on oligonucleotides and detritylate later, an RP HPLC column may be used, or for maximum selectivity, contact Berry & Associates about a Fluoro-Pak™ HPLC column.  Yields of recovered FDMT-on oligonucleotides will be lower than those obtained by on-column detritylation, since the FDMT group is lost to some degree upon manipulation, just as a DMT group is. 

After purification, the FDMT group may be removed using the same conditions (e.g. aqueous acetic acid) that are used for DMT removal.  The kinetics of removal of the FDMT and DMT groups have been measured and are essentially the same; the DMT group comes off 1.2 faster than the FDMT group in 80% acetic acid/acetonitrile.  The UV absorbance maximum of the FDMT cation (504 nm) is the same as the DMT cation under these conditions.

 

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