Wei-Chen Chang

Incorporation of fluorine into pharmaceuticals, agrochemicals, and molecular-imaging agents is of growing importance. Multiple synthetic fluorination methods have recently emerged, and metalloenzymes that are potentially capable of C(sp3)–H fluorination have been reported. Nevertheless, direct, regioselective fluorination of aliphatic carbon centers remains an unsolved problem. Here, we show for the iron(II) and 2-oxoglutarate-dependent (Fe/2OG) l-lysine 4-chlorinase, BesD, which might be envisaged to support C(sp3)–H fluorination by the direct cognate of its native chlorination mechanism, that the enzyme can (1) coordinate F– at its Fe(II) cofactor, (2) activate O2 to form a cis-FeIV(O)(F) (fluoroferryl) intermediate, and (3) use the intermediate to abstract hydrogen from its substrate. In what would be the key final step, fluorine (F•) coupling to the substrate radical is unable to compete with the hydroxyl-radical (HO•) “rebound” step characteristic of related hydroxylases. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) data establish that fluorine remains bonded to the iron cofactor through steps 1–3 and therefore available for transfer to the substrate radical. QM/MM calculations suggest that the F•-coupling step is associated with an activation barrier considerably higher than that of HO• rebound, consistent with the observed outcome. The findings experimentally verify prior proposals that the impediment to C(sp3)–H fluorination by the canonical mechanism of an Fe/2OG halogenase lies solely in the final radical-coupling step and set the stage for exploration of whether a potentially surmountable geometric barrier or an insurmountable electronic one is primarily responsible.

The biological formation of three-membered rings catalyzed by nonheme-iron and 2-oxoglutarate dependent (Fe/2OG) enzymes incurs a thermodynamic cost paid by the four-electron reduction of molecular oxygen. Inspection, however, suggests that this chemistry is closely related to the Fenton reaction, where the central ferryl complex serves as a more reactive surrogate for hydrogen peroxide. While this sacrifices some of the oxidative power of O2 to heat, sufficient chemical potential is retained to make small-ring cyclization strongly exergonic while also reducing kinetic barriers to reaction. At the same time, mechanistic features must be introduced that raise the relative activation energies for alternative reaction pathways. Current study of Fe/2OG catalyzed small ring cyclization is helping to identify these features including substrate–cofactor disposition, ferric stabilization and active site preorganization among others that likely work together to define reaction channels and thus determine the reaction outcome.

The prodiginine family constitutes a distinctive class of tripyrrolic natural products notable for their red pigmentation and diverse bioactivities. While the biological activities of the linear prodiginine prodigiosin have been extensively studied, cyclic prodiginines remain comparatively underexplored despite structural features that may confer distinct membrane-perturbing properties and enhanced antibacterial activity. Here, a high-producing colony variant of Streptomyces spectabilis (ISP-5512) is described that yields the cyclic prodiginine metacycloprodigiosin at up to 300 mg L⁻¹ isolated yield, representing the highest reported production to date. This high-yield production enabled a comprehensive biological evaluation of metacycloprodigiosin against a diverse panel of colistin-resistant Gram-negative (CRGN) pathogens as a colistin adjuvant, and demonstrates that 5 µM metacycloprodigiosin reduces the colistin minimum inhibitory concentration (MIC) up to 32 768-fold across A. baumannii, K. pneumoniae, P. aeruginosa, and E. coli isolates harboring both chromosomal and plasmid-mediated (mcr-1) resistance. Initial investigation into the adjuvant action of metacycloprodigiosin reveals that an increase in reactive oxygen species (ROS) could underpin evident colistin potentiation activity in CRGN A. baumannii. These findings establish metacycloprodigiosin as a potent colistin adjuvant, highlight the potential of cyclic prodiginines for combating antimicrobial resistance, and provide insight into structure–activity relationships governing membrane-active antibiotic adjuvants.

The lignan (−)-podophyllotoxin possesses significant antiviral and anticancer activities and thus serves as a precursor to several natural products with therapeutic properties. Biosynthesis of podophyllotoxin involves cyclization of (−)-yatein to yield deoxypodophyllotoxin catalyzed by the iron- and α-ketoglutarate-dependent (Fe/α-KG) oxidase deoxypodophyllotoxin synthase (DPS), which completes the tetracyclic core of podophyllotoxin. Herein, (+)-hydroxy-yatein is also shown to be a substrate for DPS, directly affording (−)-podophyllotoxin as the enzymatic product. Moreover, derivatives of (+)-hydroxy-yatein are also found to be substrates providing synthetic precursors to medicines such as etoposide. Mechanistic analyses utilizing isotopologs and diastereomers of the m-dimethoxy analogue of (+)-hydroxy-yatein indicate antarafacial C–C bond formation during the cyclization reaction and remain consistent with cation-mediated cyclization. A crystallographic study of DPS bound with vanadium(IV) oxide, succinate, and (−)-hydroxy-yatein suggests a subtle interplay of steric interactions between the substrate and the active site that can alter the course of the DPS-catalyzed reaction and thus cyclization versus hydroxylation. Finally, an efficient chemoenzymatic approach to (−)-podophyllotoxin is described that relies only on freeze-dried whole cells after DPS overexpression.

Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) enzymes have garnered strong research interest in past decades due to their ability to catalyze regio- and stereoselective C-H functionalization via a single reactive intermediate, the oxyferryl species. In addition to the hydroxylation reaction that is commonly observed, other reaction outcomes have also been discovered in Fe/2OG enzymes. Among them, halogenation has attracted much research effort with the goal of revealing the molecular determinants to favor halogenation over hydroxylation; however, a full mechanistic picture is still missing. In this study, by investigating a recently identified Fe/2OG halogenase, AdeV, from the biosynthetic pathway of Adechlorin, we show, via biochemical, kinetics, and spectroscopic characterizations, that two oxyferryl intermediates are formed during the AdeV reaction in a sequential manner, which interconvert but only one shows kinetic competency to enable C-H activation and leads to the conversion of 2'-deoxyadenosine monophosphate (2'-dAMP) and 2',3'-dideoxyadenosine monophosphate (ddAMP) to 2'-Cl-dAMP and 2'-Cl-ddAMP, respectively. By applying chemical synthesis and product characterization by detailed NMR analysis, the stereochemical assignment of the AdeV-catalyzed reaction is resolved, whereof the C-H bond cleavage and the C-Cl bond formation occur in a suprafacial manner. Using the experimental observations as a guide, the computational studies reveal that the kinetically competent oxyferryl intermediate structurally exhibits an offline configuration. However, this offline oxyferryl intermediate requires a structural conversion to a metastable inline configuration to perform a regio- and stereospecific C-H activation via a σ reaction channel. The subsequent conversion back to the offline configuration in the hydroxy-ferric state facilitates the final C-Cl bond formation.