Research & Development in Fluorous Peptide Synthesis

Fluorous Technologies Inc. (FTI) provides a wide range of fluorous tags and protecting groups, including fluorous versions of BOC, Cbz, PMB, Bn, CDMT, Fmoc and TM groups used in peptide synthesis. We also offer a series of fluorous tagged amino acids. To facilitate the separation and purification of your fluorous compounds, we also have the fluorous sorbents from Fluorous SPE Cartridges to Fluorous HPLC Columns to Fluorous Silica Gel. If you have specific product questions, please contact Kris Mikulan, our technical support specialist by clicking here.

Fluorous Tagged Amino Acids

In a recent publication, Professor Dennis Curran and co-workers at the University of Pittsburgh showed how Fluorous Carbobenzyloxy (F-Cbz) tagged amino acids can be used to create F-Cbz-Phe derivatives. Several reactions were undertaken to show the broad applicability of the F-Cbz-tagged amino acids and to validate that high purity products would result when separations were conducted over FluoroFlash® Silica Gel. Coupling reactions were conducted with four different amines under standard conditions (EDCl, HOBt) as shown. The amines were used in excess (4 equiv). The excess amine and the crude coupled products were isolated in moderate to excellent yields and high levels of purity by either FluoroFlash® SPE or FluoroFlash® HPLC.

Curran, Dennis P., et. al. “Synthesis and Reactions of Fluorous Carbobenzyloxy (F-Cbz) Derivatives of a-Amino Acids.” J. Org. Chem. (2003), 68, 4643-4647.

Enhanced Purification Following Solid-phase Peptide Synthesis

Overkleeft [i] and van Boom [ii] demonstrated how fluorous tags can be used to enhance the purity of a peptide following a conventional solid-phase synthesis, particularly in cases where the target peptide closely elutes with impurities. As a final step before de-linking, the N-terminus was tagged with a fluorous protecting group reagent. Overkleeft used the base-labile fluorous methylsulfonylethoxycarbonyl (F-Msc) with an Fmoc strategy, while van Boom used a fluorous benzyloxycarbonyl (F-Cbz) compound, which is acid labile (but less so than the linker used in the experiment). Following cleavage, the tagged full-length sequences were separated from capped deletion sequences and other impurities using F-SPE cartridges or F-HPLC. Purity of 94-99% was reported for several deliberately-chosen problematic sequences, including the 35-mer LSELDDRADALQAGFSQFESSAAKLKRKYWWKNLK. Overkleeft reported that F-SPE worked well with shorter peptides and that F-HPLC delivered 99% purity on the longer sequence.

Conversely, fluorous techniques can also be used to tag and scavenge unwanted compounds, like solid-phase PS impurities from deletion sequences and unreacted monomers. In relevant work by Seeberger [iii] and co-workers, a fluorous silyl ether cap was used to tag and then extract deletion sequences in the iterative automated solid-phase synthesis of a triglucoside; similar approaches can be applied to peptide synthesis.

[i] Overkleeft, H.S., et al., “A novel, base-labile fluorous amine protecting group: synthesis and use as a tag in the purification of synthetic peptides.” Tetrahedron Letters 2003, 44, 9013-9016.

[ii] Van Boom, J. H., et al., “Use of benzyloxycarbonyl (Z)-based fluorophilic tagging reagents in the purification of synthetic peptides.” Tetrahedron Letters 2002 , 43, 7809.

[iii] Palmacci, E. R.; Hewitt, M. C.; Seeberger, P. H. Angew. Chem. Int. Ed. 2001, 40, 4433.

Tagged Reagents for Solution-Phase Peptide Synthesis

Fluorous analogs of classic protecting group reagents are commercially available, and various research groups have also developed others. These compounds allow easy removal of post-deprotection adducts (which remain fluorous-tagged) from solution. Behavior of protected groups, as well as deprotection conditions, are generally similar to non-fluorous counterparts. Fluorous analogs of the following protecting groups are among those available:

Fluorous compound Used for

F-Boc-ON His, Lys, Trp, amine / N-terminus

F-tBuOH Cys, Thr, Tyr, Ser

F-Trityl-OH Cys, His, Thr, Asn, Gln, Ser

F-Cbz-OSu amine / N-terminus

F-Msc-Cl & -OSu amine / N-terminus

F-SEM-Cl amine / N-terminus

F-Fmoc-OSu ( in development) amine / N-terminus

The Curran group reported on the synthesis of eighteen F-Cbz-protected amino acids [i], and had earlier investigated the use of F-Boc, including cleavage conditions and recovery/reuse of the resulting fluorous isopropyl alcohol [ii] .

Fluorous peptide coupling agents are also available. Dembinski [iii] reported the use of fluorous CDMT as a solution-phase condensation reagent in better yields than conventional CDMT, including when coupling a,a -disubstituted amino acids. The fluorous hydroxytriazine by-product was easily separated onto a fluorous stationary phase.

Other fluorous compounds suitable for amine, alcohol, and carboxylic acid protection are also available, as are F-DCC and other coupling reagents.

[i] Curran, Dennis P., et. al. “Synthesis and Reactions of Fluorous Carbobenzyloxy (F-Cbz) Derivatives of a-Amino Acids.” J. Org. Chem. (2003), 68, 4643-4647.

[ii] Curran, Dennis P., et. al. “Fluorous Boc (F-Boc) Carbamates: New Amine Protecting Groups for use in Fluorous Synthesis.” J. Org. Chem. (2001), 66, 4261-4266.

[iii] Markowicz, Marcin W.; Dembinski, Roman. “Fluorous Coupling Reagents: Application of 2-Chloro-4,6-bis[(heptadecafluorononyl)oxy]-1,3,5-triazine in Peptide Synthesis.” Synthesis 2004, No. 1, 80-86.

Fluorous Universal Support

As described above, fluorous enhancements are compatible with classical peptide synthesis techniques. There is also some interest in fluorous compounds as supports for sequence elaboration. Mizuno and co-workers have investigated the use of heavily-fluorinated amines anchoring C-terminal amide and carboxyl peptides, using an Fmoc strategy followed by a simple fluorous/organic liquid-liquid extraction [i]. As Dr. Mizuno notes, “the strategy of fluorous synthesis is designed to combine the advantages of solid-phase synthesis (facile purification) with those of traditional organic synthesis in the liquid-phase synthesis (purification of intermediates and reaction monitoring; large scale reaction).

[i] Mamoru Mizuno, et al. “Peptide synthesis on Fluorous support.” Tetrahedron Letters (2004), 45, 3425–3428.

Fluorous Capping Reagent

The development of a new capping reagent for use in solid-phase peptide synthesis was completed by Kumar and Montonari[i]. A fluorous trivalent iodonium salt was synthesized to be compatible with short to medium-length peptide synthesis using t-Boc chemistry. This fluorous capping reagent was used only at sites where peptide couplings were incomplete. The capped peptides were removed by fluorous flash silica gel.

[i] Krishna Kumar and Vittorio Montanari. “Just Add Water: A New Fluorous Capping Reagent for Facile Purification of Peptides Synthesized on Solid Phase.” J. Am. Chem. Soc (2004), 126, 9528-9529

For Information about commercially-available fluorous peptide synthesis reagents and pre-tagged amino acids - click here

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Three New Fluorous Patents Issue in 2004

This year has seen three new U.S. patents issue in the area of fluorous chemistry, all of which are exclusively licensed to FTI. The first patent (6673539) covers fluorous techniques used in conjunction with solid supports. Patent 6727390 covers fluorous phosphines and phosphine oxides, two broadly useful classes of materials. Lastly, patent 6734318 describes the fundamental concept of triphasic fluorous synthesis and separation, an exciting new technology of significant potential in process chemistry. FTI is the established leader in fluorous chemistry and intellectual property is a key asset of the company.