advanced separation chemistry for life sciences

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1 Fluorous Based Peptide Synthesis and Immobilization in the Formation of a Protease Microarray Marvin S. Yu August 2009 advanced separation chemistry for life sciences

2 Acknowledgements Fluorous Technologies, Inc. Iowa State University Dr. Tadamichi Nagashima Dr. Bru Miriyala Dr. Beatrice Collett Prof. Nicola Pohl Funding NIH STTR Grant

3 Presentation utline I. Introduction II. Fluorous Peptide Synthesis III. Microarray Results IV. Conclusions and Future Directions

4 What is Fluorous Chemistry? Fluorous chemistry is a novel tagging technology that separates desired molecules from complex mixtures. Molecules can be rendered fluorous by the attachment of perfluorocarbon domains. Fluorous tagged molecules can be separated from non-fluorous molecules exploiting fluorophilicity. Non-fluorous Fluorous separation Fluorous Fluorous techniques are marked by high selectivity, low reactivity, and exceptional breadth Fluorous immobilization fluorous-modified surface

5 Carbohydrate Synthesis and Immobilization Ko, K.-S.; Jaipuri, F.A.; Pohl, N. L. J. Am. Chem. Soc. 2005, 127, 13162

6 Histone deacetylases (HDAC) are attractive targets for chemotherapy agents Small molecule libraries of SAHA analogs have been produced and screened for HDAC inhibition Results were validated by comparison with biochemical and SPR based assays Broad Institute researchers are using an integrated approach combining synthesis purification, and immobilization Application of Fluorous Microarrays Vegas, A.J.; Bradner, J.E.; Tang, W.; McPherson,.M.; Greenberg, E.F.; Koehler, A.N.; Schreiber, S.L. Angew. Chemie Intl. Ed. 2007, 46, 4960.

7 Fluorous Microarray Benefits No blocking step Fluorous immobilization Specific display orientation fluorous-modified surface Incubation Excellent spot morphology (40% reduction in spot diameter) Less washing steps High sensitivity High signal-to-noise Low non-specific binding allows use of cell lysates Low and uniform background fluorescence

8 Aminocoumarin Based Protease Microarray Prior to incubation trypsin granzyme B thrombin Salisbury, C.M.; Maly, D.J.; Ellman, J.A. J. Am. Chem. Soc. 2002, 124, 14868

9 Synthesis of Aminocoumarin Peptides I iprmgcl; Ethyl formate 76% yield H tbu Br TBAB, KH 90% yield tbu 4N HCl 94% yield H H-Su EDCI 96% yield Su N H 2 C NH N H C6 NH 2 H-[F26], 66% yield Double chain Rf6 was chosen to ensure good retention on FSPE. ligoethylene glycol linker was used to keep peptide off of the hydrophobic fluorous surface.

10 Synthesis of Aminocoumarin Peptides Fmoc-NH H + H 2 N C N 6 H DIC, HBt Collidine DMF H-[F26] N H N H piperidine Fmoc-NH Fmoc-ACC-[F26] N H linker F Fmoc based peptide synthesis N H linker F N H 2 peptide N H ACC-[F26] (1.36 mmol, 50% yield in 2 steps) Solution phase synthesis throughout with FSPE between couplings 12 peptides synthesized with differing activity to various proteases

11 Solution Phase Experiments Expected reactivity and selectivity for each substrate in good agreement with non-fluorous tagged analog was observed

12 Initial Microarray Results Before incubation After incubation Thrombin Trypsin Plasmin Normalized signal response consistent with known selectivities

13 Incubation in the Absence of Tween-20 Before incubation After incubation Before incubation After incubation Incubation without Tween-20 resulted in little loss of marker signal, HC1. Hydrolysis of the peptides with the appropriate protease observed Larger than anticipated signal upon hydrolysis

14 Fluorous tags are readily incorporated into peptide syntheses. Conclusions Fluorous tagging does not substantially alter peptides for protease activity in solution. Fluorous tagged peptides qualitatively retain appropriate reactivity and specificity in solution based on LC and fluorescence analysis. Fluorous immobilized peptides provide results similar to that obtained in solution, although some anomalies were observed Detergents can adversely effect fluorous immobilization

15 Future work Develop general protocols for fluorous peptide microarray immobilization and detection. Examine different fluorous tags and linkers on peptide microarray performance. Examine other peptide-protein interactions, i.e. kinases, phoaphatases, etc. Expand fluorous microarray concept to include mixed microarrays.

16 Phone: FLURFLASH Fax: Philip E. Yeske, Ph.D. Marvin S. Yu, Ph. D.