# Regio- and Stereoselective Halogenation by an Iron(II)- and 2‑Oxoglutarate-Dependent Halogenase in the Biosynthesis of Halogenated Nucleosides

## TL;DR

This paper investigates how an iron-dependent enzyme selectively adds chlorine atoms to specific positions in nucleosides, revealing the mechanism behind its regio- and stereoselectivity.

## Contribution

The study identifies two oxyferryl intermediates and their structural transformations that enable selective halogenation over hydroxylation in an Fe/2OG halogenase.

## Key findings

- Two oxyferryl intermediates are formed sequentially during the AdeV reaction.
- The stereochemistry of the C–Cl bond formation is suprafacial.
- Structural conversion between offline and inline oxyferryl configurations is necessary for C–H activation and bond formation.

## Abstract

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.