Synthesis of 6,6‐ and 7,7‐Difluoro‐1‐acetamidopyrrolizidines and Their Oxidation Catalyzed by the Nonheme Fe Oxygenase LolO

• Published in: Chembiochem : a European journal of chemical biology Vol. 23; no. 13; pp. e202200081 - n/a
• Main Authors: Panth, Nabin, Wenger, Eliott S., Krebs, Carsten, Bollinger, J. Martin, Grossman, Robert B.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 05-07-2022
• Subjects: Biosynthesis; Catalysis; Chemical reactions; Hydroxylation; Iron; Ketoglutaric Acids; Oxidation; Oxidation-Reduction; Oxygenase; Oxygenases
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202200081

LolO, a 2‐oxoglutarate‐dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of 1‐exo‐acetamidopyrrolizidine (AcAP), a pathway intermediate in the biosynthesis of the loline alkaloids, and the cycloetherification of the resulting alcohol. We have prepared fluorinated AcAP analogues to aid in continued mechanistic investigation of the remarkable LolO‐catalyzed cycloetherification step. LolO was able to hydroxylate 6,6‐difluoro‐AcAP (prepared from N,O‐protected 4‐oxoproline) and then cycloetherify the resulting alcohol, forming a difluorinated analogue of N‐acetylnorloline and providing evidence for a cycloetherification mechanism involving a C(7) radical as opposed to a C(7) carbocation. By contrast, LolO was able to hydroxylate 7,7‐difluoro‐AcAP (prepared from 3‐oxoproline) but failed to cycloetherify it, forming (1R,2R,8S)‐7,7‐difluoro‐2‐hydroxy‐AcAP as the sole product. The divergent LolO‐catalyzed reactions of the difluorinated AcAP analogues provide insight into the LolO cycloetherification mechanism and indicate that the 7,7‐difluorinated compound, in particular, may be a useful tool to accumulate and characterize the iron intermediate that initiates the cycloetherification reaction.
Two difluorinated analogues of 1‐exo‐acetamidopyrrolizidine (AcAP), an intermediate in loline alkaloid biosynthesis, were prepared and subjected to LolO, the enzyme that naturally hydroxylates and cycloetherifies AcAP. LolO (2‐oxoglutarate‐dependent nonheme Fe oxygenase) hydroxylates and cycloetherifies 6,6‐difluoro‐AcAP, but it only hydroxylates 7,7‐difluoro‐AcAP. The resulting 7,7‐difluoro‐2‐hydroxy‐AcAP may be useful for studying the LolO‐catalyzed cycloetherification of 2‐hydroxy‐AcAP.