Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Article abstract of DOI:10.1021/acscatal.9b02508

Here we report a cytochrome P450 variant that catalyzes C₂-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis.

Full text of DOI:10.1021/acscatal.9b02508

Letter
pubs.acs.org/acscatalysis
Cite This: ACS Catal. 2019, 9, 8271−8275
Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole
Amidation
Oliver F. Brandenberg,†,‡ David C. Miller,† Ulrich Markel,†,§ Anissa Ouald Chaib,†,⊥
and Frances H. Arnold*,†
†Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
S
* Supporting Information
ABSTRACT: Here we report a cytochrome P450 variant that
catalyzes C2-amidation of 1-methylindoles with tosyl azide via
nitrene transfer. Before evolutionary optimization, the enzyme
exhibited two undesired side reactivities resulting in reduction
of the putative iron-nitrenoid intermediate or cycloaddition
between the two substrates to form triazole products. We
speculated that triazole formation was a promiscuous
cycloaddition activity of the P450 heme domain, while
sulfonamide formation likely arose from surplus electron
transfer from the reductase domain. Directed evolution
involving mutagenesis of both the heme and reductase
domains delivered an enzyme providing the desired indole
amidation products with up to 8400 turnovers, 90% yield, and
a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work
expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450
scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis.
KEYWORDS: biocatalysis, cytochrome P450, nitrene transfer, indole amidation, chemoselectivity
ne of the hallmarks of enzyme catalysis is the ability to
the C2-amidated product in low yields and turnovers. However,
Oprecisely control reaction selectivity. Through finely the enzymatic reactions yielded two additional products: tosyl
sulfonamide, likely originating from over-reduction and
protonation of the iron-nitrenoid intermediate,6,9,14 and a
triazole, likely arising from cycloaddition of the two
substrates.33 Thus, we were confronted with a three-way
chemoselectivity problem (Figure 1A). Building on the
adaptability of the P450 scaffold for selectivity challenges,18,34
we report here a comprehensive directed evolution strategy
targeting both the P450 heme and reductase domains (Figure
1B) to engineer a variant having high indole amidation activity
and chemoselectivity over the undesired reduction and
cyclization pathways. This work highlights the potential of
enzyme engineering to achieve precise control over non-
natural substrates and reaction pathways and expands the
scope of enzymatic nitrene transfer to synthetically valuable
heterocycles.
We started this work by testing a range of P450 variants,
expressed in Escherichia coli and utilized as whole-cell catalysts,
for the amidation of 1-methylindole 2a with tosyl azide 1
(TsN3) as the nitrene precursor (Table S1). The C2 amidation
product 3a was formed in very low yield by several P450
variants, with the serine-ligated P450 variant P411-CIS35
tuned active site geometries, high levels of substrate binding
specificity and preferential stabilization of transition states can
be achieved, often resulting in excellent chemo-, regio-, and
stereoselectivity. Furthermore, enzyme selectivity can be
engineered using methods of directed evolution to access
different products from the same set of substrates.1−5
We and others previously engineered native hemoproteins to
become selective catalysts for synthetically valuable non-
natural carbene and nitrene transfer reactions.6 Hemoprotein
“nitrene transferases” catalyze intra- and intermolecular C−H
amination,7−13 sulfimidation,14,15 aziridination,16 aminohy-
droxylation,17 and most recently highly selective intramolecular
C−H amination to yield β-, γ-, or δ-lactams.18
In our ongoing effort to develop enzymes for new nitrene
transfer reactions and inspired by recent reports of
hemoprotein-catalyzed carbene transfer for C−H functionali-
zation of indoles,19−21 we investigated indole amidation with
sulfonyl azides. Although this reaction can proceed without
catalyst,22,23 in recent years Pd, Ir, Rh, and Co-based catalysts
have been developed for regioselective indole amidations.24−32
Hemoproteins would offer the advantages of functioning with
an earth-abundant iron cofactor in aqueous media at ambient
temperatures.
Received: June 14, 2019
Revised: July 27, 2019
We found that several cytochrome P450 variants catalyze 1-
methylindole C(sp2)−H amidation with tosyl azide, delivering
© XXXX American Chemical Society
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Figure 1. Engineering chemoselectivity in P411-catalyzed indole
amidation. (A) Proposed reaction scenarios: tosyl azide 1 can react
with the P411 iron heme cofactor to form an iron nitrenoid
intermediate (i). The nitrene can react with 1-methylindole 2a to
yield the desired amidation product 3a (ii), or it can be reduced to
give sulfonamide product 4 (iii). Alternatively, 1 and 2a can react to
give cycloaddition product 5a (iv); while this reaction is likely
accelerated by the P411 active site (vide infra), the involvement of the
heme cofactor remains to be determined. (B) P450-BM3 domain
structure; we targeted both the heme and reductase domains for
directed evolution of chemoselective formation of 3a.
Figure 2. Directed evolution for 1-methylindole amidation. Evolu-
tionary lineage from P411-CIS to P411-IA. Reactions were performed
on 400 μL scale with whole-cell catalysts; HPLC yields of 3a, 4, and
5a are given. Data were derived from two independent experiments
performed in duplicate.
versus cycloaddition reaction pathways. We reasoned that
further directed evolution should allow enzymatic discrim-
ination of the two pathways. Indeed, subsequent screening of
site-saturation mutagenesis libraries (encompassing active-site
residues A82, V87, L181, E267, A268, T327, and A328; Table
S2) revealed mutations A328V and A82W, which dramatically
shifted the 3a:5a product ratio from 0.8:1 to over 25:1 for the
desired amidation product. Lastly, mutation A268G further
increased activity by ca. 1.6-fold and resulted in variant P-
YSVWG, delivering 3a in 68% yield, 5730 TTN, and a 97:1
ratio of 3a to 5a (Figure 2, Figure S2). Thus, directed
evolution of the P450 heme domain delivered a variant
showing high catalytic indole amidation activity and excellent
amidation versus cycloaddition discrimination.
Formation of azide reduction product 4 decreased as P411
catalysts gained efficiency in the amidation reaction,
presumably as better binding of the indole acceptor substrate
increased the rate of the desired transformation relative to
nonproductive nitrene reduction.14 However, 4 still repre-
sented 14% of the product (5:1 3a:4) in reactions with variant
P-YSVWG (Figure 2, Table S3), and we asked whether
unproductive nitrene reduction could be suppressed further.
Nitrene reduction is presumably driven by electron transfer
from the P450 reductase domain,9,14 which accepts electrons
from NADPH and relays them to the heme domain, a
prerequisite for P450-BM3’s native oxo-transfer chemistry.36
However, non-natural P411 nitrene transfer catalysis only
requires a single electron to reduce the heme cofactor from the
FeIII to the catalytically competent FeII state, at which point
exhibiting the highest activity (2.1% yield, 100 total turnovers
(TTN)). However, the predominant reaction product was
tosyl sulfonamide 4. This was expected, as nonproductive over-
reduction and protonation of the heme-bound nitrene yielding
the sulfonamide has been a recurring problem for hemopro-
tein-catalyzed nitrene transfer (Figure 1A).6,9,14 More surpris-
ingly, we observed an additional product in whole-cell reaction
mixtures (Figure S1), which we identified as triazole 5a,
presumably arising from cycloaddition of TsN3 and 1-
methylindole.33 Confronted with this three-way chemo-
selectivity problem, we sought to use directed evolution to
increase enzyme activity and selectivity in the P411-catalyzed
formation of 3a.
Using P411-CIS as the parent (P), site-saturation muta-
genesis and screening at active site positions I263 and T438,
previously shown to be important for P411-catalyzed nitrene
transfer,7,14,16 yielded variant P411-CIS I263Y T438S (P-YS)
(Figure 2, Tables S2, S3). This variant delivered almost 3-fold
higher yields of 3a, but it concomitantly delivered over 7-fold
higher yields of cycloaddition product 5a. This strong
influence of P411 active site mutations on the yield of triazole
5a, with all other reaction parameters identical, indicated that
5a formation took place within (or close to) the P411 active
site (vide infra). We assumed that mutations I263Y and T438S
served to better accommodate both substrates 1 and 2a within
the active site, thereby enhancing the yields of both 3a and 5a
without discriminating between nitrene formation/amidation
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catalysis can proceed without additional electron supply. While
surplus reductant is likely required to reduce any cofactor that
is inadvertently oxidized to the FeIII state, excessive electron
supply aggravates undesired reduction of the iron-nitrenoid
intermediate.6,9,14 Thus, we set out to engineer the reductase
domain toward lower rates of electron transfer and presumably
lower rates of nitrene reduction, anticipating that this would
translate into higher amidation yields.
We first attempted a coarse approach of truncating the entire
reductase domain or the FAD subdomain in variant P-YSVWG
(Figure S3). Removal of the reductase domain proved
deleterious for amidation activity, while FAD domain deletion
(a mutation recently shown to be beneficial for P411 carbene
transfer catalysis)21,37 was slightly less active than P-YSVWG,
showing that full domain deletions are insufficient to enhance
P411 nitrene transfer activity. Next, we used site-saturation
mutagenesis of reductase domain residues involved in electron
relay from NADPH to the heme domain36,38,39 in an attempt
to fine-tune electron transfer rates (Table S2). This identified
beneficial mutation W1046P, located close to the C-terminus
of the reductase domain (Figure S4). W1046 stacks over the
isoalloxazine ring of the FAD cofactor, and replacement with
proline likely alters the NADPH-FAD interaction.39 The
W1046P mutation increased amidation yield by 10% while
reducing sulfonamide yield by 6%, representing an increase in
3a:4 product selectivity from 5:1 to 9:1 (Figure S3, Table S3).
Attempts to further improve the 3a:4 ratio by targeting
additional residues in the FAD-FMN-heme electron transfer
chain were unsuccessful, and we refer to variant P-YSVWG
W1046P as P411-IA (Indole Amidation). P411-IA catalyzes 3a
formation in 78% yield, 8400 TTN, and a 3a:4:5a product
ratio of 110:12:1 at 25 mM substrate loading (Figure 2), while
over 90% yield of 3a and even higher selectivity were observed
at 10 mM substrate loading (Figure S3).
To test whether the W1046P mutation in P411-IA affects
rates of electron transfer and nitrene reduction, we measured
both rates in vitro using purified protein. P411-IA delivered
2060 TTN in vitro, demonstrating that the enzyme functions
in purified form (Figure S5). Next, we used a cytochrome c
reduction assay, which utilizes reduction of cytochrome c by
the P450 reductase domain to provide a spectrophotometric
read-out, to estimate rates of electron transfer from NADPH
across the reductase domain.40 Strikingly, in comparison to
P411-CIS (P) and P-YSVWG, P411-IA showed a ca. 10-fold
reduced electron transfer rate (Figure 3A). However, rates of
nitrene reduction, measured by assessing formation of 4 in the
absence of 1-methylindole (Figure S6), were only reduced ca.
2-fold, indicating that large decreases in electron transfer rate
do not translate linearly into lower rates of nitrene reduction.
Nonetheless, the data support the hypothesis that engineering
the reductase domain to reduce the electron transfer rate can
be beneficial for P411-catalyzed nitrene transfer. Underscoring
the generalizability of this concept, we found that introducing
the W1046P mutation into variant P411-CHA, evolved for
intermolecular C−H amination,12 improved its activity in the
amination of ethylbenzene (Figure S7).
Figure 3. Rates of nitrene reduction and indole amidation. (A) Rates
of P411 nitrene reduction to TsNH2 and electron transfer (measured
with the cytochrome c reduction assay) obtained with purified
enzyme in vitro. (B) Amidation kinetics of P411-IA and P411-CIS
whole-cell reactions.
did not require the oxygen depletion system used in previous
hemoprotein nitrene transfer studies.7,12,14−17
In addition to the screening substrate 2a, P411-IA efficiently
amidated N-methylindoles with substitutions at C5 or C6,
delivering the C2-amidated products in 64% to 91% yield and
2300 to 3300 TTN (Figure 4, 3c−3f). In contrast, P411-IA
showed only low amidation activity on unprotected indole;
curiously, P411-IA predominantly delivered the indole cyclo-
addition product 5b (Figure S9). However, one additional
round of site-saturation mutagenesis and screening yielded
P411-IA W82C, showing 6-fold higher indole amidation
activity and delivering the C2-amidated product 3b in 45%
yield with 1220 TTN (Figure 4, Figure S9).
Lastly, we were curious to further investigate the apparent
indole-azide cycloaddition reactions leading to products 5a and
5b. We hypothesized that these reactions take place in the
P411 active site, which may serve as a hydrophobic pocket for
orienting the substrates in the proper orientation for
cycloaddition. We therefore screened site-saturation muta-
genesis libraries of variants P-YS and P411-IA for enhanced
triazole formation with 1-methylindole and indole, respec-
tively. This resulted in variants P-YS W82S and P411-IA W82F
that shifted the amidation:cycloaddition ratio toward triazole
products 5a and 5b (Figure 4, Figure S10, Table S2). This
further illustrates how P411 active-site engineering allows
routing of substrates along different reaction trajectories.
Furthermore, the data point to a critical role for residue 82 in
controlling indole amidation versus cycloaddition selectivity;
residue 82 may act to orient substrate in the active site and
steer reaction specificity. While additional work is required to
elucidate the enzymatic cycloaddition reaction mechanism, the
variants obtained here may serve as starting points to evolve
indole-azide cycloaddition activity.
In small-scale biocatalytic whole-cell reactions, P411-IA
catalyzed 1-methylindole amidation with an initial turnover
frequency of 176 min−1 (Figure 3B) and total turnovers
(>8000) that rank among the highest achieved for
hemoprotein-catalyzed intermolecular nitrene transfer. P411-
IA showed ca. 7-fold lower activity in the presence of oxygen
(Figure S8). Notably, however, P411-IA whole-cell reactions
In summary, we have demonstrated directed evolution of a
cytochrome P411 variant to catalyze indole C2-amidation with
greater than 8000 TTNs, 90% yield, and excellent selectivity
for one out of three possible reaction products. Targeting both
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Ulrich Markel: 0000-0002-5655-5342
Frances H. Arnold: 0000-0002-4027-364X
Present Addresses
‡O.F.B.: Department of Human Genetics, University of
California Los Angeles, Los Angeles, California 90095, USA.
§U.M.: Institute of Biotechnology, RWTH Aachen University,
52074 Aachen, Germany.
⊥A.O.C.: Laboratory of Organic Chemistry, ETH Zu
̈
rich, 8093
̈
Zurich, Switzerland.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
O.F.B. acknowledges support from the Deutsche Forschungs-
gemeinschaft (DFG grant BR 5238/1-1) and the Swiss
National Science Foundation (SNF grant P300PA-171225).
D.C.M. was supported by a Ruth Kirschstein NIH Post-
doctoral Fellowship (F32GM128247). The authors thank Dr.
Christopher K. Prier for help with initial experiments and
comments on the manuscript. We also thank Dr. David K.
Romney, Dr. Xiongyi Huang, and Kai Chen for helpful
comments and discussions.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscatal.9b02508.
■
S
Experimental procedures, supplementary data, calibra-
tion curves, and characterization of amidation and
cycloaddition products (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: frances@cheme.caltech.edu.
ORCID
■
Oliver F. Brandenberg: 0000-0001-5662-1234
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DOI: 10.1021/acscatal.9b02508
ACS Catal. 2019, 9, 8271−8275