New-to-nature radical biocatalysis has recently emerged
as a powerful
strategy to tame fleeting open-shell intermediates for stereoselective
transformations. In 2021, we introduced a novel metalloredox biocatalysis
strategy that leverages the innate redox properties of the heme cofactor
of P450 enzymes, furnishing new-to-nature atom-transfer radical cyclases
(ATRCases) with excellent activity and stereoselectivity. Herein,
we report a combined computational and experimental study to shed
light on the mechanism and origins of enantioselectivity for this
system. Molecular dynamics and quantum mechanics/molecular mechanics
(QM/MM) calculations revealed an unexpected role of the key beneficial
mutation I263Q. The glutamine residue serves as an essential hydrogen
bond donor that engages with the carbonyl moiety of the substrate
to promote bromine atom abstraction and enhance the enantioselectivity
of radical cyclization. Therefore, the evolved ATRCase is a bifunctional
biocatalyst, wherein the heme cofactor enables atom-transfer radical
biocatalysis, while the hydrogen bond donor residue further enhances
the activity and enantioselectivity. Unlike many enzymatic stereocontrol
rationales based on a rigid substrate binding model, our computations
demonstrate a high degree of rotational flexibility of the allyl moiety
in an enzyme–substrate complex and succeeding intermediates.
Therefore, the enantioselectivity is controlled by the radical cyclization
transition states rather than the substrate orientation in ground-state
complexes in the preceding steps. During radical cyclization, anchoring
effects of the Q263 residue and steric interactions with the heme
cofactor concurrently control the π-facial selectivity, allowing
for highly enantioselective C–C bond formation. Our computational
findings are corroborated by experiments with ATRCase mutants generated
from site-directed mutagenesis.
Three-rung molecular ladder 8 was prepared in one pot via tandem imine condensation and alkyne metathesis. Catalyst VI is demonstrated to successfully engender the metathesis of imine-bearing substrate 7, while catalyst III does not. The susceptibility of catalyst VI to deactivation by hydrolysis and ligand exchange is demonstrated. Assembly and disassembly of ladder 8 in one pot were demonstrated in the presence and absence of a Lewis acid catalyst.
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