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Fluorous Peptide, Oligonucleotide and Oligosaccharide Synthesis

Compatible with Solution-phase & SPPS Strategies for Research and Production

Fluorous chemistry represents a novel orthogonal phase which can be used in the synthesis and purification of peptides, oligonucleotides, and carbohydrates.  Fluorous techniques for the synthesis of these molecules can be categorized into four broad classes:

• Solid phase or solution phase supported synthesis with terminal fluorous tagging
• Solid phase or solution phase supported synthesis with fluorous capping
• Solution phase fluorous supported synthesis
• Solution phase synthesis using fluorous tagged reagents

In all cases, the fluorous products can be separated from the non-fluorous products using either fluorous solid phase extraction (F-SPE) or fluorous liquid-liquid extraction (F-LLE).

Methods of Use

Solid phase or solution phase supported synthesis with terminal fluorous tagging

As shown in Figure 1, this strategy uses a terminal fluorous tagging with conventional peptide, oligonucleotide, or carbohydrate synthesis.  In the example shown, traditional iterative solid phase synthesis is used with capping step, generally acetate capping, used between couplings.  At the end of the synthesis, a fluorous tag is added to the finished oligomer resulting in a mixture of non-fluorous deletion sequences and a fluorous tagged desired product.  Cleavage from the resin, followed by a fluorous separation and detagging yields the pure finished peptide, oligonucleotide, or carbohydrate.

The use of fluorous tags in conjunction with solid phase peptide synthesis was initially described by van Boom et al from Leiden University, first using an acid labile fluorous Z tag, then a base labile fluorous Msc tag. [1]  The basic methodology uses Fmoc based solid phase chemistry to build the amino acid chain. After each condensation step, any free amines are capped with an acetate group. After the desired number of iterations, the deprotection of the final Fmoc group is followed by tagging with a fluorous version of a Cbz or Msc group. (The use of a pre-tagged amino acid, such as those available from FTI, in the final coupling step could be envisioned.) The peptides are cleaved from the resin to provide a mixture of the desired fluorous tagged peptide and undesired truncated sequences, which do not contain a fluorous tag. The fluorous tagged material is then easily separated from the non-tagged material by fluorous SPE or HPLC using a FluoroFlash® sorbent.  Van Boom and co-workers quickly and reliably purified peptides from 7-35 amino acids.  Fluorous Technologies, Inc. has developed a fluorous Fmoc tag which possesses better reactivity and stability than the F-Msc tag and has used the same strategy in the purification of peptides up to 41 amino acids in length.

The same terminal tagging strategy has been employed independently by Bannwarth [2] and Pearson [3] in the synthesis of oligonucleotides.  In the work described by Pearson, long oligonucleotides up to 100 nt in length were synthesized and purified using a fluorous DMT tagged phosphoramidite as the final nucleotide.  Cleavage from the solid support was followed by treatment on a Fluoro-Pak cartridge which retained the fluorous tagged oligonucleotide while washing off the non-fluorous capped deletions.  An on-cartridge detritylation then provided the purified oligo with excellent recovery and ODU’s.  Fluorous tools specifically for oligonucleotide chemistry can be obtained from our partners at Berry and Associates.

Solid phase or solution phase supported synthesis with fluorous capping

An alternative to terminal fluorous tagging is fluorous capping.  In this strategy, all the undesired sequences are fluorous tagged while the final desired sequence is non-fluorous.  This strategy was first demonstrated by Peter Seeberger, now at the ETH, and co-workers in the automated synthesis of carbohydrates.[4]  After each glycoside coupling they used a fluorous silane to cap any unreacted materials before proceeding to the next deprotection-coupling iteration.  Krishna Kumar from Tufts University developed a fluorous alkylating agent which was employed as a peptide capping agent.  All deletion sequences were fluorous capped and later separated from the desired sequence either by precipitation or by F-SPE.[5] 

Fluorous Technologies, Inc. has conducted fluorous capping in peptide synthesis using a fluorous carboxylic acid as the capping reagent under amide coupling conditions.  The capping strategy can be used not only for preparative purposes, but also as a process development tool by identifying those couplings which are problematic.  During process development when each coupling is followed by fluorous capping, it can be quickly and precisely determined which couplings result in n-1 sequences and if those sequences are the result of inefficient coupling or inefficient deprotection.  Analysis, detection, and identification of the capped sequences are readily determined using F-HPLC with a MS detector.

Solution phase fluorous supported synthesis

The third major approach to the fluorous synthesis of peptides, oligonucleotides, and carbohydrates is a solution phase synthesis based on fluorous supported chemistry.  In this approach, the solid phase support is replaced by a fluorous tag and the oligomer is built over iterative cycles from the support.  In this case the reagents are separated from the growing oligomer either by fluorous liquid-liquid extraction (F-LLE) or by fluorous solid phase extraction (F-SPE). 

Researchers at the Nogouchi Institute have synthesize both oligosaccharides and peptides using “heavy” fluorous tags containing multiple fluorous chains with F-LLE purification between coupling iterations.[6]  Recently they have extended their technique using lighter fluorous tags in combination with alternative F-LLE conditions using hydrofluoroethers rather than traditional perfluorocarbons as the fluorous solvent.[7]  In the work described, the desired fluorous tagged oligomer is separated from excess monomer and reagents by partitioning the fluorous material into a fluorous solvent while all other components are contained in the organic or aqueous layer.

In addition to the “heavy” fluorous tags used in F-LLE applications, various groups have also utilized “light” fluorous tags possessing a single fluorous chain in the solution phase fluorous supported synthesis of carbohydrates and peptides.  In these instances, F-SPE is used as the fluorous purification method.  Nicola Pohl at Iowa State University has described the synthesis and purification of carbohydrates using just such an approach.[8]   Between each step of the synthesis, a F-SPE is conducted to separated excess reagents from the desired carbohydrate.  Since all chemistry occurs in solution, there is less excess reagent and monomer used in each step thereby leading to reduced cost and waste of valuable monomer.  Prof. Pohl has also automated the process by which F-SPE is conducted leading to a highly efficient process for the production of oligosaccharides.  For peptides, Prof. Steven Firestine at Wayne State University used a similar strategy for the synthesis of polyamides that are otherwise very difficult to purify.[9]  Prof. Seeberger has also used a light fluorous supported strategy in the synthesis of carbohydrates and peptides in microreactors.[10]

Solution phase synthesis using fluorous reagents

The other major method by which fluorous techniques are used are as reagents in solution phase synthesis of peptides and carbohydrates.  In peptide synthesis in particular, activation of the acid often requires one or more coupling reagents which can sometimes be difficult to remove after the reaction.  Fluorous versions of popular amide coupling reagents provides a facile method for the removal of these reagent by-products.  Fluorous versions of CDMT, Mukaiyama’s salt, and DCC have all been reported in the literature.[11]  Moreover, FTI has conducted internal research on fluorous versions of HOBt.  Depending on the amide coupling reagent used, the fluorous by-products can then be removed using F-SPE, F-LLE, or reverse fluorous solid phase extraction.

Besides coupling reagents, fluorous approaches have been used to prepare stable activated versions of carboxylic acids for peptide synthesis and glycosides for oligosaccharide synthesis.  In each case, an excess of the activated species can be used to ensure complete reaction and unreacted compound along with any fluorous by-products removed by F-SPE.  For carboxylic acids a fluorous version of Marshall resin, FluoMar® has been developed [12], while a fluorous thiol was used for glycosylations.[13]

Selected References

1. a) Filippov, D. V.; van Zoelen, D. J.; Oldfield, S. P.; van der Marel, G. A.; Overkleeft, H. S.; Drijfhout, J. W.; van Boom, J. H., Use of benzyloxycarbonyl (Z)-based fluorophilic tagging reagents in the purification of synthetic peptides. Tetrahedron Letters 2002, 43, (43), 7809-7812.  b) de Visser, P. C.; van Helden, M.; Filippov, D. V.; van der Marel, G. A.; Drijfhout, J. W.; van Boom, J. H.; Noort, D.; Overkleeft, H. S., A novel, base-labile fluorous amine protecting group: synthesis and use as a tag in the purification of synthetic peptides. Tetrahedron Letters 2003, 44, (50), 9013-9016.
2. Beller, C.; Bannwarth, W., Noncovalent attachment of nucleotides by fluorous-fluorous interactions: application to a simple purification principle for synthetic DNA fragments. Helvetica Chimica Acta 2005, 88, (1), 171-179.
3. Pearson, W. H.; Berry, D. A.; Stoy, P.; Jung, K.-Y.; Sercel, A. D., Fluorous Affinity Purification of Oligonucleotides. Journal of Organic Chemistry 2005, 70, (18), 7114-7122.
4. Palmacci, E. R.; Hewitt, M. C.; Seeberger, P. H., \"Cap-Tag\" - Novel methods fo the rapid purification of oligosaccharides prepared by automated solid-phase synthesis. Angewandte Chemie, International Edition 2001, 40, (23), 4433-4437.
5. Montanari, V.; Kumar, K., Just Add Water: A New Fluorous Capping Reagent for Facile Purification of Peptides Synthesized on the Solid Phase. Journal of the American Chemical Society 2004, 126, (31), 9528-9529.
6. a) Mizuno, M.; Goto, K.; Miura, T.; Hosaka, D.; Inazu, T., A novel peptide synthesis using fluorous chemistry. Chemical Communications (Cambridge, United Kingdom) 2003, (8), 972-973. b) Goto, K.; Miura, T.; Hosaka, D.; Matsumoto, H.; Mizuno, M.; Ishida, H.-k.; Inazu, T., Rapid oligosaccharide synthesis on a fluorous support. Tetrahedron 2004, 60, (40), 8845-8854.  c) Goto, K.; Miura, T.; Mizuno, M., Synthesis of peptides and oligosaccharides by using a recyclable fluorous tag. Tetrahedron Letters 2005, 46, (48), 8293-8297.
7. a) Goto, K.; Mizuno, M., Synthesis of monosaccharide units using fluorous method. Tetrahedron Lett. 2007, 48, (32), 5605-5608.  b) Chu, Q.; Yu, M. S.; Curran, D. P., New fluorous/organic biphasic systems achieved by solvent tuning. Tetrahedron 2007, 63, (39), 9890-9895.
8. Ko, K.-S.; Jaipuri, F. A.; Pohl, N. L., Fluorous-Based Carbohydrate Microarrays. Journal of the American Chemical Society 2005, 127, (38), 13162-13163.
9. Mamidyala, S. K.; Firestine, S. M., Fluorous synthesis of minor groove binding agents related to distamycin. Tetrahedron Letters 2006, 47, (42), 7431-7434.
10. a) Carrel, F. R.; Geyer, K.; Codee, J. D. C.; Seeberger, P. H., Oligosaccharide Synthesis in Microreactors. Org. Lett. 2007, 9, (12), 2285-2288. b) Floegel, O.; Codee, J. D. C.; Seebach, D.; Seeberger, P. H., Microreactor synthesis of b-peptides. Angewandte Chemie, International Edition 2006, 45, (42), 7000-7003.
11.  a) Markowicz, M. W.; Dembinski, R., Fluorous coupling reagents: Application of 2-chloro-4,6-bis[(heptadecafluorononyl)oxy]-1,3,5-triazine in peptide synthesis. Synthesis 2004, (1), 80-86. b) Matsugi, M.; Hasegawa, M.; Sadachika, D.; Okamoto, S.; Tomioka, M.; Ikeya, Y.; Masuyama, A.; Mori, Y., Preparation and condensation reactions of a new light-fluorous Mukaiyama reagent: reliable purification with fluorous solid phase extraction for esters and amides. Tetrahedron Lett. 2007, 48, (23), 4147-4150.
12. Chen, C. H.-T.; Zhang, W., FluoMar, a Fluorous Version of the Marshall Resin for Solution-Phase Library Synthesis. Organic Letters 2003, 5, (7), 1015-1017.
13. Jing, Y.; Huang, X., Fluorous thiols in oligosaccharide synthesis. Tetrahedron Letters 2004, 45, (24), 4615-4618.