The applications of laser spectroscopy in analytical chemistry and biochemistry: investigating the photophysics of hypericin and hypocrellin, as well as the structure of porcine fructose-1,6-bisphosphatase

Wen, Jin
Major Professor
Jacob W. Petrich
Committee Member
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This dissertation addresses the applications of ultrafast laser spectroscopy in investigating the photophysical properties of hypericin, hypocrellin and their analogs, as well as the structure of protein.;Hypericin and hypocrellin are naturally occurring photosensitizers. The attractions of those compounds are their light induced antiviral and antitumor activities. A variety of techniques, i.e. time-resolved single photon counting, pump-probe experiment and fluorescence upconversion, are utilized to reveal the photophysical properties of hypericin, hypocrellin and their analogs. The primary photoprocess is excited-state H-atom transfer that has been partially attributed to the virucidal activity of hypericin.;Comparison of the excited-state H-atom transfer in hypocrellin A and B, by using time-resolved absorption and fluorescence upconversion techniques, as well as ab initio quantum mechanical calculations, suggests that excited-state conformation change are coupled to the H-atom transfer reaction. A simple model is employed to describe the photophysics of hypocrellin B.;The synthesis of a molecule containing hypericin and luciferin moieties joined by a tether is reported. This hypericin-luciferin tethered molecule exhibits excited-state behavior that is very similar to that of its parent compounds and antiviral activity that is identical, within experimental error, to that of its most closely related parent compound, pseudohypericin. The implications of the chemiluminescent reaction with luciferin and luciferase as an alterative light source for photodynamic therapy are discussed.;Time-resolved fluorescence and direct mutation are employed to study the environment of Trp57 in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase). The studies indicate that Trp 57 in R-state conformer is in the hydrophobic pocket and Trp 57 in T-state conformer is exposed to the solvent, which is consistent with the predictions made on the basis of X-ray crystal structures of Trp 57 FBPase. The unexpected red shift in steady-state fluorescence of Trp57 in R-state relative to that in T-state is attributed in part to the electrostatic perturbation of the Asp127.