Mechanistic investigations of SpnF- and SpnL-catalyzed cyclizations in the biosynthesis of spinosyn A

dc.contributor.advisorLiu, Hung-wen, 1952-
dc.creatorKim, Nam Ho, 1975-en
dc.date.accessioned2015-03-03T19:06:17Zen
dc.date.issued2013-12en
dc.date.submittedDecember 2013en
dc.date.updated2015-03-03T19:06:17Zen
dc.descriptiontexten
dc.description.abstractSpinosyn A is a particularly interesting natural product due to its structural complexity and potent insecticidal activity. The biosynthetic pathway of spinosyn A is interesting as it has two unusual features, the SpnF-catalyzed (4+2) cycloaddition and the SpnL-catalyzed cyclization to produce the perhydro-as-indacene core. The work described in this dissertation focuses on elucidating the mechanisms of the SpnF- and SpnL-catalyzed reactions. SpnF has attracted significant interest as a possible Diels-Alderase. To explain how SpnF catalyzes the formation of cyclohexene ring, three plausible mechanisms have been proposed, the Diels-Alder reaction mechanism, the ionic rearrangement mechanism, and the biradical rearrangement mechanism. Kinetic isotope effect studies were performed using four deuterium-labeled mechanistic probes, specially the C4-D, C7-D, C11-D, and C12-D analogs. Currently, the ionic rearrangement mechanism can be excluded, based on the results using the C4-D and C7-D analogs. In addition, how SpnF accelerates the reaction was studied to assess the contribution of an entropic x preorganization compared to enthalpic transition state stabilization. To measure the relative rate enhancements due to structural perturbations, three mechanistic probes were synthesized, the linear analog, the C13-14 Unc analog, and the C2-3 Unc analog. Unfortunately, the linear analog and C13-14 Unc analog didn’t show any turnover activity under either non-enzymatic or enzymatic conditions. Thus, no conclusion could be drawn from incubation with these substrate analogs. Mechanistic studies of SpnL-catalyzed cyclization were devoted to differentiating between the Rauhut-Currier type mechanism and the Michael addition mechanism. Biochemical studies using the C13-F analog as a mechanism-based inhibitor showed the formation of a covalent adduct with SpnL, which is consistent with the Rauhut-Currier type mechanism. Additional experimental data obtained from isotope trace experiments and kinetic isotope effect studies using C12-D analog supports the Rauhut-Currier type mechanism. Biochemical studies concerning the role of SAM in SpnF and SpnL showed that SAM is required for the activity of SpnL, and were inconclusive for SpnF. SpnL mutant studies showed that Cys60 and Glu96 may be important for the catalysis of SpnL. Chemoenzymatic total synthesis of spinosyn A was completed by chemical etherification of 17-pseudoaglycone and D-forosamine.en
dc.description.departmentPharmaceutical Sciencesen
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttp://hdl.handle.net/2152/28735en
dc.subjectSpinosyn Aen
dc.subjectCyclizationen
dc.subjectCycloadditionen
dc.subjectSpnFen
dc.subjectSpnLen
dc.subjectMechanismen
dc.subjectDiels-Alderen
dc.subjectRauhut-Currieren
dc.subjectKinetic isotope effecten
dc.subjectMechanism-based inhibitoren
dc.titleMechanistic investigations of SpnF- and SpnL-catalyzed cyclizations in the biosynthesis of spinosyn Aen
dc.typeThesisen
thesis.degree.departmentPharmacyen
thesis.degree.disciplinePharmacyen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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