Journal of the American Chemical Society
Article
relative to azide. We therefore tested whether excess sulfide or
slow addition of azide would increase sulfimide formation
relative to sulfonamide. Increasing the sulfide concentration
decreased reduction of azide to sulfonamide and improved the
ratio of sulfimide to sulfonamide, from 0.6 (with 0.5 equiv
sulfide) to 1.8 (with 4 equiv sulfide) (Figure S10, Table S6).
Slow azide addition slightly increased the TTN for sulfimide
and decreased sulfonamide formation in a 2 h reaction (Figure
S11). That higher concentrations of sulfide substrate improve
sulfimide production suggest that protein engineering to
improve the binding of sulfide acceptor substrates could also
produce strong gains in the desired activity. Indeed, the specific
activities of the enzyme catalysts reported here compare
favorably with enantioselective iron-based catalysts, which
routinely require catalyst loadings of 10 mol %.9b Furthermore,
engineering the holoenzyme or reductase domain to favor one-
electron transfers might improve the proportion of desired
product relative to azide reduction, which could allow reaction
with more challenging organic acceptor substrates.
presence of azide substrate as well as sulfide nitrene acceptor.
Calibration curves for all products presented and LC-MS
verification of product formation by enzyme catalysts. This
material is available free of charge via the Internet at http://
AUTHOR INFORMATION
Corresponding Author
■
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the support of the Jacobs Institute
for Molecular Engineering for Medicine at Caltech and the
Department of the Navy, Office of Naval Research (grant
N00014-11-1-0205). C.C.F. is supported by an NSF Graduate
Research Fellowship (DGE-1144469). J.A.M., T.K.H., and
Z.J.W. are supported by NIH Ruth L. Kirschstein National
Research Service Awards (F32GM101792, F32GM108143,
F32EB015846). We thank R. Kelly Zhang and Hans Renata
for helpful comments on the manuscript, and Scott Virgil and
the 3CS catalysis center for HPLC and LC-MS analysis.
CONCLUSIONS
■
This is the first report of intermolecular nitrene transfer
catalyzed by an enzyme, allowing for a mechanistic analysis of
this new enzyme activity. Similar to P450-catalyzed sulfox-
idation, we find that the electronic properties of the sulfide
substrates strongly influence reactivity, though the magnitude
of the substituent effects is greater for nitrene transfer, possibly
owing to the less oxidizing nature of the presumed nitrenoid
intermediate as compared to compound I. The necessity of the
C400S mutation for sulfimidation can be rationalized along
similar lines: the less electron-donating axial serine ligand in
P411 enzymes likely makes the nitrenoid species a more potent
oxidant. The impact of sulfide substituents on sulfimide
formation is also reflected in the generation of sulfonamide
side product, suggesting the nitrenoid undergoes rapid
reduction and can only productively insert into sufficiently
reactive sulfides. Characterization of the redox state of the
heme iron in the presence and absence of nitrene source and
sulfide acceptor supports the proposal that nitrenoid “over-
reduction” competes with productive sulfimide formation and
that the former is a two-electron process resulting in
regeneration of ferric heme. Another interesting aspect of this
enzyme reaction is the strong preference for an aryl
sulfonylazide nitrene source: although monooxygenation
reactions use a small donor substrate (dioxygen), only trace
sulfimidation was observed with small azides such as
ethanesulfonyl azide (Table S4). The ability of the enzyme to
accept larger aryl substrates may be beneficial for development
of enantioselective intermolecular nitrene-transfer catalysts, as
we have observed that a single mutation can dramatically affect
the enantioselectivity of reaction. Intermolecular nitrene
transfer in the form of sulfimidation can now be added to the
impressive array of natural and non-natural chemistry catalyzed
by cytochrome P450 enzymes.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Text, tables, and figures describing methods and results for
reaction set up and quantitation, determination of enzyme
reaction rates, and enzyme mutant generation and composition.
Chromatogram traces demonstrating dependence of enantio-
selectivity on enzyme sequence. Spectroscopic characterization
of the P411BM3-CIS I263A T438S catalyst in the absence and
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dx.doi.org/10.1021/ja503593n | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX