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Just in Time for Summer - Hot Topics!
Fluorous Proteomics
In the May Technical Bulletin we reported on a new application for fluorous affinity tags developed by Dr. Eric Peters and colleagues at the Genomics Institute of the Novartis Research Foundation (GNF), whose findings were detailed at the 229th ACS National Meeting in San Diego and in the April 2005 issue of Nature Biotechnology [1]. Since this seminal article on fluorous proteomics appeared, there has been a tremendous response from the scientific community. Published commentaries highlighting the report have appeared in C&EN News [2], J. Proteome Research [3], Nature Methods [4], and Molecular Biosystems [5]. In addition online publications which have recognized the potential of the technique include the Faculty of 1000, ProteoMonitor, California Separation Society Newsletter and numerous other online forums.
The Peters' group showed that fluorous affinity tags could be utilized to enrich peptides containing amino or thiol groups as well as for post-translational modifications such as phosphorylation. The fluorous tagged peptides are easily removed from the non-tagged peptides using a simple solid phase extraction enabling direct MS analysis of the isolated peptides. The fluorous tagging and enrichment has several advantages over traditional approaches such as biotin-streptavidin including selectivity, ease of recovery of tagged substrates, and cost. One interesting application is the separation of multiply tagged species from singly tagged species, a capability that biotin tagging does not possess. The Peters' group, for example, was able to separate and identify doubly tagged branched peptides from the proteolysis of ubiquitinylated proteins using a fluorous tag.
Some comments from the scientific community regarding fluorous based proteomics:
Dr. Dennis Curran, University of Pittsburgh: "I've always felt that there was excellent potential for fluorous chemistry in analysis, and this paper takes a major step in the right direction. The success of Eric's group with peptide digests and proteins suggests that all kinds of other applications are possible."
"People have been searching hard for solutions on how to put robust tags on peptides to enrich them...My judgment is that this [technique] looks pretty interesting and that it should be in the armamentarium for proteomics research" - Brian T. Chait, Rockefeller University, New York City
"This technology, when optimized and applied to various problems in proteomics science, should be a powerful tool for the enrichment of low-level molecules in biological mixtures." - Thomas Kodadek
Another application of fluorous affinity was presented in a poster at ASMS 2005 by W. Uritboonthai working in the Siuzdak group at The Scripps Research Institute. The poster entitled "Fluorous Based Affinity Mass Spectroscopy" described the isolation, enrichment, and selective detection of fluorous alkylated peptides and small molecule analytes on a fluorous modified porous silicon surface with direct MS analysis from that surface using DIOS-MS. The isolation of molecules having different functionalities and implementation of fractionation methods differentiates this technique from traditional bioaffinity methods.
Fluorous Technologies has introduced a series of fluorous affinity tags for use in the proteomics area. The fluorous iodoacetamide (for tagging thiol residues) and fluorous NHS ester (for tagging amines) are two of the compounds recently commercialized and are available in a range of fluorine contents to meet your specific application needs. Also included are the fluorous maleimide, fluorous thiols and fluorous 5 micron silica gel used in the separation of fluorous tagged peptides from non-tagged peptides. Be sure to visit our "Fluorous Proteomics" webpage to learn more about this exciting new area and to place an order.
Literature Highlight - Fluorous Asymmetric Chemistry
One of the hallmarks of fluorous chemistry is the ability to render molecules fluorous with minimal change in the overall chemistry. In asymmetric synthesis this has been manifested in a variety of fluorous versions of chiral ligands, catalysts, and chiral auxiliaries being synthesized. In general, the reactivity and selectivity of these fluorous molecules have been very similar to their non-fluorous counterparts, but have the advantage of easier separation from the desired product. Two recent examples which have been reported include a fluorous version of a chiral diphenyl prolinol 1 used in CBS reductions and a chiral oxazolidinone 2 utilized in radical additions.
Soos et al generated the chiral oxazaborolidine in situ from 1 and borane-THF [6]. The resulting catalyst was used in the reduction of a number of ketones where the yields and ee's obtained were comparable to that of the non-fluorous version. Based on the data reported, it was noted that 0.6 equivalents of the borane-THF was sufficient for complete reduction of ketone in most cases. The excess of borane had only a slight influence on the enantioselectivity in all cases but one, where the %ee was significantly higher. The researchers reported that not only was the separation of 1 from the desired optically active carbinols simplified by the use of fluorous solid phase extraction (F-SPE), but that 1 was recovered in 98% yield and reused without loss of activity.
Extending previous work on the titanium-mediated asymmetric aldol condensations of fluorous oxazolidinone compounds [7], Hultin et al have described the tin-mediated addition of alkyl radicals to alpha, beta unsaturated imides of 2 [8]. The resultant di-beta-substituted imides were obtained in comparable de's (3-6.6:1) and yields (>80%) to the non-fluorous chiral auxiliary. The fluorous chiral auxiliary provided the added benefit of easy separation of the radical addition product by F-SPE from the tin by-products of the radical addition. Products were generally found to contain <0.10 w/w% tin after F-SPE.
Fluorous HPLC and Fluorous SPE Application Notes Updated
In an effort to provide the best possible resources for our customers we continue to update our Fluorous Application Notes. Recently updated was the Fluorous HPLC application note, where users can now see comparative features to traditional reverse phase columns, recommended solvents and suggestions on other uses for your fluorous HPLC column.
The Fluorous SPE application note was also updated to include improved general procedures, a new ‘how to use’ section, answers to frequently asked questions and recommendations on sample loading and solvent volumes. Our goal is to provide a general and robust protocol, so that our users can see for themselves the value of using fluorous chemistry in their research. Chromatograms obtained from the cited reaction and "Insider tips" from our scientists are included for additional reference. As always, please contact FTI for any questions or if you need any additional information.
ACS National Meeting - Aug 28-Sept 1 in Washington, D.C.
A half day symposium on Monday, August 29 on Recent Advances in Fluorous Chemistry is being co-sponsored by the Division of Organic Chemistry and the Division of Fluorine Chemistry. Topics to be covered include small molecule, peptide, and oligonucleotide synthesis, catalysis, and bioorganic chemistry. A distinguished panel of speakers representing a number of disciplines will present their latest research. The symposium should prove to be an exciting and stimulating overview of some of the latest in fluorous based chemistry.
Are you new to fluorous chemistry and want to learn more? Plan to visit booth #2024 to learn more about what Fluorous Technologies has to offer and how fluorous chemistry can help improve your productivity.
Are you a current FTI customer? Stop by and share your success stories with us! We are always interested to hear how our customers are using our products.
Reprints of FTI-authored papers are available on request from FTI
References:
[1] Brittain, S.; Ficarro, S.; Brock, A.; Peters, E.; "Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry", Nature Biotechnology, 2005, 23(4), 463-468.
[2] C&EN News, "ACS Meeting News: Fluorous Proteomics", April 25, 2005, 33.
[3] Journal of Proteome Research, "Fluorous Proteomics', Vol. 4, 2005, 662.
[4] Nature Methods, "Research Highlights, Chemical Tools, Fluorous Proteomics", Vol. 2, 2005, 406-407.
[5] Molecular Biosystems, "Chemical labeling to analyze low-abundance protein", 2005, 1, 12.
[6] Dalicsek, Z.; Pollreisz, F.; Gomory, A.; Soos, T.; Org. Lett. 2005, 7, 3243.
[7] Hein, J.E.; Zimmerman, J.; Sibi, M.P.; Hultin, P.G.; Org. Lett. 2005, 7, 2755.
[8] Hein, J.E.; ,Hultin, P.G.;Synlett, 2003, 5, 635.