General Method for the Synthesisof Caged Phosphopeptides: Tools for the Exploration of Signal Transduction Pathways Deborah M. Rothman, M. Eugenio Va ´ zquez, Elizabeth M. Vogel, and Barbara Imperiali* Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 imper@mit.edu Received May 29, 2002 ABSTRACT An interassembly approach for the synthesisof peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has beendeveloped. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford thecorresponding phosphopeptide. The peptidesdescribedherein are based on phosphorylation sitesof kinases involved in cell movement or cell cycle regulation and demonstrate the versatilityof the method and compatibility with the synthesisof polypeptides, including a varietyof encoded amino acids. Kinase-mediated phosphorylation of tyrosine, serine, and threonine residues in polypeptides and proteins represents a central mechanism of cell regulation. As such, it is an area of intense study in which there is considerable potential for the development of new chemical tools. 1 The study of protein kinases involved in cell migration is of particular interest in biological and medicinal studies. The present tools available for the study of phosphorylation-dependent signal transduc- tion pathways are limited when information in real time is desired. For example, chemical inhibition, gene knockout, or point mutation studies can often confirm the significance or essentiality of a protein kinase; however, such methods make it difficult to assess the biochemical role of the protein in real time. A caged compound includes a photocleavable-protecting group that masks an essential functionality; upon removal by photolysis, the functionality is revealed, generating a biologically active molecule. 2 This strategy allows research- ers spatial and temporal control over the release of effector molecules in living systems. 3 There are several characteristics required of a caging group to be used in biological systems. The caged compound, as well as the byproducts of photoly- sis, should be inert in the cell, neither agonizing nor antagonizing the receptor molecule. Only the uncaged effector molecule should alter cell activity. The caged compound must be stable to hydrolysis and enzymatic (1) Woodgett, J. R. Protein Kinase Functions, 2nd ed.; Oxford University Press: New York, 2000. (2) Corrie, J. E. T.; Trentham, D. R. In Biological Applications of Photochemical Switches; Morrison, H., Ed.; Wiley & Sons: New York, 1993; Chapter 5. (3) Park, C.-H.; Givens, R. S. J. Am. Chem. Soc. 1997, 119, 2453- 2463. ORGANIC LETTERS 2002 Vol. 4, No. 17 2865-2868 10.1021/ol0262587 CCC: $22.00 © 2002 American Chemical Soci ety Published on Web 07/30/2002