SYNTHESIS OF 6,6- AND 7,7-DIFLUORO-1-ACETAMIDOPYRROLIZIDINES AND THEIR OXIDATION CATALYZED BY THE NONHEME Fe OXYGENASE LolO

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\u27s 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