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Preface Caged compounds are biomolecules whose activity or function is inactivated by chemical modification with a photolabile group. Excitation of a caged compound with (near-ultraviolet) light results in specific cleavage of a bond within the molecule that rapidly liberates the functional biomolecule of interest. One main advantage of this light-directed perturbation technique centers around its ability to overcome the mixing and diffusion time limits of fast-flow methods. This is achieved by precharging the biological sample with a caged substrate, ligand, or protein conjugate, followed by light-directed activation of the caged compound which triggers the specific reaction under investigation within the excitation volume. In addition, concentration jumps mediated by light-directed activation of caged compounds can be conducted without physically disturbing the preparation. Caged groups described in this volume generate active biomolecules within nanoseconds of absorbing light and, furthermore, by using two-photon excitation of the caged compound, the concentration jump can be confined to a volume having a diameter of approximately 250 nm with little or no irradiation-based damage to the preparation. Light-directed activation of caged compounds and rapid monitoring of the ensuing reaction using photomultiplier or imaging-based techniques have been used to understand the molecular mechanism of several biochemical reactions and processes in vitro and in vivo. This volume also describes the application of caged compounds to studies that aim to understand the molecular basis of neurotransmission, muscle contraction, and ion channel function. Topics covered in this book include (a) an up-to-date account of the synthetic chemistry and photochemistry of traditional and newer generations of caged groups, (b) a description of new reagents and methods to prepare monofunctional and bifunctional complexes of a diverse array of caged substrates, ligands, and polypeptides, (c) a state-of-the-art practical and theoretical description of lamp- and laser-based excitation sources for light-directed activation of caged compounds in macro- and microscopic samples, (d) the measurement and interpretation of spectroscopic signals derived from light-directed activation of caged compounds to monitor the kinetics of the reaction being studied, and (e) the application of lightdirected activation of caged compounds and caged polypeptides to chemical relaxation studies that aim to understand the molecular mechanism of protein-catalyzed reactions in a diverse array of complex molecular environments. xiii
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PREFACE
The primary objective of this volume is to provide newcomers to the field with a central source of valuable practical and theoretical information on caged compounds that will allow them to use light-directed activation of caged compounds to address molecular mechanisms of biochemical reactions or processes within biological samples. It provides the investigator with the most up-to-date and comprehensive account of caged compounds that should aid in the design of experiments to compare and interpret data obtained from kinetic measurements of specific reactions triggered by lightdirected activation of caged biomolecules. This volume is dedicated to the memory of Professor Fredric S. Fay. GERARD MARRIOTT
Fredric Stewart Fay
1943-1997 The scientific community lost one of its most gifted and productive members on March 18, 1997 when Fred Fay suffered a fatal heart attack. Fred was born in New York City and was educated in its public school system. On graduation from the Bronx High School of Science in 1961, he enrolled at Cornell University where he majored in chemistry and was elected to Phi Beta Kappa. His love and talent for science were evident early, not only in the schoolboy experiments conducted in the family apartment, but