Author ORCID Identifier

https://orcid.org/0000-0002-9868-3289

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Chemistry

First Advisor

Dr. Robert B. Grossman

Abstract

One of the remarkable steps in loline alkaloid biosynthesis is the installation of an ether bridge between two unactivated C atoms in 1-exo-acetamidopyrrolizidine (AcAP). LolO, a 2-oxoglutarate-dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of AcAP and the resulting alcohol's cycloetherification to give N-acetylnornoline (NANL). The mechanism of hydroxylation is well understood, but the mechanism of the oxacyclization is not. I synthesized difluorinated analogs of AcAP in an attempt to further understand the mechanism of the unusual cycloetherification step.

I prepared 6,6-F2-AcAP in eight steps from N,O-protected 4-oxoproline. The key step was a Dieckmann condensation that annulated the A ring onto the B ring. When I subjected 6,6-F2-AcAP to LolO, the enzyme was able to catalyze both the hydroxylation and the cycloetherification to make 6,6-F2-NANL, suggesting that the LolO has a flexible active site, as it did not differentiate between the natural substrate (AcAP) and this difluorinated analog of AcAP. Also, it suggested that the cycloetherification mechanism most likely involves a C(7) radical as opposed to a C(7) carbocation. Then, I prepared 7,7-F2-AcAP from 3-oxoproline in 17 steps where the key step was radical cyclization to make the pyrrolizidine ring in 5-exo-dig fashion. By contrast, when I subjected the difluorinated analog 7,7-F2-AcAP to LolO, the cycloetherification step was shut down completely, giving 2-OH-7,7-F2-AcAP as the sole product. Because 7,7-F2-AcAP completely blocks the cycloetherification step, it may be used in the future to further understand the cycloetherification of 2-hydroxy-AcAP by accumulating and characterizing the LolO intermediates responsible for cycloetherification.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.008

Funding Information

This study was supported by the National Institutes of Health Grant (GM113106) in 2017-2019.

Available for download on Monday, July 04, 2022

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