Fluorous Affinity Purification of Oligonucleotides
Fluoro-Pak® Columns and Fluorous Phosphoramidites
Fluorous Affinity Purification of Oligonucleotides is a quick and simple affinity-based method for the purification of oligonucleotides that relies on the strong interaction of fluorous-tagged oligonucleotides with the fluorinated adsorbent present in Fluoro-Pak™ columns. Fluorous affinity purification is operationally similar to DMT-on purification using a reverse-phase (RP) adsorbent, e.g. RP cartridge purification. Jump to fluorous products and pricing.
Detailed information may be found in our booklet "User Guide: Fluorous Affinity Purification of Oligonucleotides", which is included with each order and may also be downloaded. See also Pearson, W. H.; Berry, D. A.; Stoy, P.; Jung, K.-Y.; Sercel, A. D. J. Org. Chem. 2005, 70, 7114-7122.
The fluorous affinity purification method affords the following advantages over traditional DMT-on RP purification:
• One-pass loading without ammonia removal.
• High selectivity for removal of failure sequences, even with long oligonucleotides. Fluorous-tagged oligonucleotides are strongly retained, whereas non-fluorous materials (failure sequences, other by-products) are poorly retained.
• High recoveries (typically 70-100%), free of failure sequences.
• Excellent for longer oligonucleotides (e.g. 50-100+ mers), where recoveries are nearly quantitative.
• Does not require adopting new techniques.
What does "fluorous" mean, and what is the nature of the fluorous affinity interaction? First of all, "fluorous" has nothing to do with "fluorescent", but refers instead to the element fluorine. In particular, highly fluorinated organic compounds are both hydrophobic and lipophobic, preferring instead to associate with other fluorinated substances. For example, perfluorohexane is insoluble in both water and hexane. Organic molecules that have both an organic domain (e.g. an oligonucleotide) and a perfluoroalkyl domain (e.g., a linear perfluoroalkyl "ponytail") are known as fluorous molecules, and may be separated from non-fluorous molecules by interaction with fluorinated separation media such as Fluoro-Pak™ columns. Fluorous-fluorous interactions are strong and selective. Fluorous affinity interactions are well-documented in the literature and are the basis for the company Fluorous Technologies Inc. (FTI), from whom Berry & Associates have licensed the basic fluorous separation technology.
How is the fluorous method used for oligonucleotide purification? Fluorous Affinity Purification of Oligonucleotides is similar to DMT-on RP cartridge purification, and we have developed protocols that are as "plug-and-play" as possible. The first step is to install a single nucleotide at the 5'-terminus of the oligonucleotide using a fluorous-tagged phosphoramidite. As shown below, the fluorous tag can take the form of a fluorous dimethoxytrityl ("FDMT") group or a fluorous monomethoxytrityl ("FMMT") group, where a fluorous ponytail is attached via an ethylene spacer to a normal DMT or MMT group. The fluorous trityl groups are designed so that they behave just like the non-fluorous versions: the rates of detritylation and absorption maxima of the trityl cations are very similar to what is observed for the parent compounds. Please note that only one coupling of a fluorous-trityl phosphoramidite is required; normal DMT amidites are used for the earlier steps in the synthesis. The synthesis is run in DMT-on mode, leaving the FDMT or FMMT group in place. FDMT- and FMMT-bearing amidites are entirely soluble in acetonitrile and couple normally.
The FDMT- and FMMT-on oligonucleotides are cleaved from the solid support as usual and the base protecting groups are removed according to standard methods. If ammonia is used, it is not necessary to evaporate it. The crude deprotection solution is diluted with a salt-containing loading buffer and applied to a Fluoro-Pak™ column. Binding of the fluorous-tagged oligonucleotide occurs in one pass, leaving most of the failure sequences unbound. Washing with 5-10% acetonitrile in 0.1 M TEAA removes the rest of the failures. On-column detritylation with TFA followed by elution of the purified oligonucleotide is then carried out. Alternatively, the fluorous trityl oligonucleotide can be eluted from the column and detritylated in a separate operation.
Example: Fluorous-tagged mixed-base 75-mers (200 nmol scale) were purified by Fluorous Affinity Purification to provide 9-11 A(260) units of the fully deprotected material. This represents a nearly quantitative recovery of the available FDMT-tagged oligonucleotides present in the crude ammonia deblock solutions (estimated by HPLC). The final 75-mers were analyzed by RP-HPLC (see trace below).
New! Install a permanent fluorous tag at any position - enable spotting on fluorous-coated glass slides. While many of our fluorous products focus on the purification of oligonucleotides, fluorous tags have other potential applications in nucleic acid chemistry. We now introduce two new fluorous monomers for the permanent fluorous-tagging of oligonucleotides. Fluorous Modifier CEP is useful for placing a fluorous tag internally or at the 5'-terminus of an oligonucleotide, whereas 3'-Fluorous Modifier CPG is effective for attaching a 3'-fluorous tag. In addition to providing a purification handle, fluorous modifications enable applications where fluorophilicity or high hydrophobicity is desired. For example, the presence of a fluorous tag in an oligonucleotide may allow its immobilization onto fluorous-coated glass slides (Fluorous Technologies, Inc.). Alternatively, placing fluorous monomers at strategic sites in an oligonucleotide may allow intra- or intermolecular fluorous-fluorous interactions, enhancing the attraction between various regions of an oligonucleotide.
New! Simultaneous 5'-phosphorylation and Fluorous Affinity Purification with F-CPR-II CEP. Fluorous Chemical Phosphorylation Reagent II (F-CPR-II CEP, FL 1360) allows fluorous affinity purification with concomitant 5'-phosphorylation, which is especially attractive when synthesizing longmers that will be used in ligase reactions. Further, if a 5'-phosphate is tolerated in a particular application, F-CPR-II may be used as a common handle for fluorous purification, obviating the need to choose a different fluorous phosphoramidite for each different 5'-terminal nucleotide. F-CPR-II CEP is used in the same way as CPR-II (Guzaev, A., et al., Tetrahedron1995, 51, 9375-9384; US Patent 5,959,090, assignee Glen Research; http://www.glenresearch.com//ProductFiles/10-1901.html), i.e., a 6 minute coupling with no capping. The final retro-aldol cleavage to afford the free 5'-phosphorylated oligonucleotide is achieved in the same fashion as found for CPR-II.
New! Enable FDMT-on fluorous affinity purification without having to purchase individual FDMT-bearing nucleoside phosphoramidites. The fluorous affinity purification of oligonucleotides is a powerful technique, but if oligonucleotides with different 5'-nucleobases are being made, it is necessary to have bottles of the four FDMT-tagged nucleoside phosphoramidites installed. However, if a short hydroxyalkyl group is tolerated at the 5'-terminus, FDMT-On Purification Modifier is an effective way to enable fluorous purification in a variety of applications. This phosphoramidite installs a fluorous dimethoxytrityl group via a short tether. After fluorous affinity purification and detritylation, a 3-hydroxy-1-methylpropyl group remains.
Learn more:
Fluoro-Pak® and Fluoro-Pak® II columns
HPLC progression showing the Fluorous Affinity Purification of a mixed-base 100-mer
Fluorous Affinity Purification Products
You will need to order:
(i) one or more fluorous phosphoramidites
(ii) Fluoro-Pak® columns
(iii) Loading Buffer
|
Item
|
Catalog No.
|
Size/pack |
Price (USD)
|
Phosphoramidites |
|||
FL-1000 |
|||
100 µmol |
47.50 |
||
250 µmol |
107.50 |
||
500 µmol |
197.50 |
||
FL-1100 |
|||
100 µmol |
47.50 |
||
250 µmol |
107.50 |
||
500 µmol |
197.50 |
||
FL-1200 |
|||
100 µmol |
47.50 |
||
250 µmol |
107.50 |
||
500 µmol |
197.50 |
||
FL-1220 |
inquire for pricing |
||
FL-1300 |
|||
100 µmol |
47.50 |
||
250 µmol |
97.50 |
||
500 µmol |
177.50 |
||
FL-1360 |
|||
100 µmol |
75.00 |
||
250 µmol |
175.00 |
||
FL-1400 |
|||
100 µmol |
75.00 |
||
250 µmol |
175.00 |
||
FL-1500 |
inquire for pricing |
||
FL-1600 |
|||
100 µmol |
75.00 |
||
250 µmol |
175.00 |
||
FL-1600 |
|||
100 mg |
85.00 |
||
1 g |
475.00 |
||
Columns* |
|||
Fluoro-Pak® columns |
Pack of 10 |
69.50 |
|
| FP-7210-A |
Pack of 10 |
69.50 |
|
Fluoro-Pak® II columns |
Pack of 10 |
95.50 |
|
FP-7220-A (with Luer adaptor) |
Pack of 10 |
95.50 |
|
| Loading Buffer | 200 mL |
24.50 |
|
| User Guide (download) |
*Fluoro-Pak® columns are for 200 nmol of oligonucleotide or less. Fluoro-Pak® II columns are for 1 micromole of oligonucleotide or less. Inquire about bulk Fluoro-Pak™ adsorbent.
Products for Fluorous Affinity Purification of Oligonucleotides: Patent pending, Berry & Associates, Inc.
The use of these products is licensed under U.S. Patents 6,673,539, 6,156,896; 5,859,247; and 5,777,121 and one or more pending patents owned or controlled by Fluorous Technologies, Inc.
"Fluoro-Pak" is a registered trademark of Berry & Associates, Inc.
©2007 Berry & Associates, Inc. All rights reserved.
