Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds

Article abstract of DOI:10.1021/jacs.9b02931

Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp²)-H alkylation or cyclopropane products are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active-site geometries, hemoprotein "carbene transferases" could provide an alternative to traditional transition metal catalysts by enabling heterocycle functionalizations with high chemo-, regio-, and stereocontrol. However, carbene transferases accepting heterocyclic substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are characterized by low turnovers or depend on artificially introduced, costly iridium-porphyrin cofactors. We addressed this challenge by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a spectrophotometric high-throughput screening assay based on 1-methylindole C₃-alkylation that enabled rapid analysis of thousands of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with 11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C₃-alkylates indoles with up to 470 turnovers per minute and 18000 total turnovers. We subsequently used this optimized alkylation variant for parallel evolution toward more challenging heterocycle carbene functionalizations, including C₂/C₃ regioselective pyrrole alkylation, enantioselective indole alkylation with ethyl 2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power of directed evolution to enhance the scope of new-to-nature enzyme catalysts.

Full text of DOI:10.1021/jacs.9b02931

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Article
Directed Evolution of a Cytochrome P450 Carbene Transferase
for Selective Functionalization of Cyclic Compounds
Oliver F. Brandenberg, Kai Chen, and Frances H. Arnold
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b02931 • Publication Date (Web): 09 May 2019
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Directed Evolution of a Cytochrome P450 Carbene Transferase for
Selective Functionalization of Cyclic Compounds
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Oliver F. Brandenberg1,†, Kai Chen1, Frances H. Arnold1*
1 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
* Corresponding author
KEYWORDS Biocatalysis, Cytochrome P450, Carbene Transfer, Indole Alkylation, Cyclopropanation.
ABSTRACT: Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp2)‒H alkylation or cyclopropane products
are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active site
geometries, hemoprotein “carbene transferases” could provide an alternative to traditional transition metal catalysts by enabling
heterocycle functionalizations with high chemo-, regio- and stereocontrol. However, carbene transferases accepting heterocyclic
substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are
characterized by low turnovers or depend on artificially introduced, costly iridium–porphyrin cofactors. We addressed this challenge
by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a
spectrophotometric high-throughput screening assay based on 1-methylindole C3-alkylation that enabled rapid analysis of thousands
of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with
11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C3-alkylates
indoles with up to 470 turnovers per minute and 18,000 total turnovers. We subsequently used this optimized alkylation variant for
parallel evolution towards more challenging heterocycle carbene functionalizations, including C2/C3 regioselective pyrrole alkylation,
enantioselective indole alkylation with ethyl-2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of
efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power
of directed evolution to enhance the scope of new-to-nature enzyme catalysts.
Introduction
as indoles, pyrroles and azoles;9 and (mono)oxygenases like cy-
tochrome P450s or flavoenzymes are involved in oxidation of
heterocyclic substrates to induce or lose aromaticity.10–12
Cyclic compounds, particularly heterocycles, are key compo-
nents in biologically active compounds, from the building
blocks of life (e.g. nucleic acids, amino acids and carbohy-
drates) to natural products with pharmacologically relevant ac-
tivities (e.g. vitamins, alkaloids and antibiotics). Accordingly,
heterocycles have been playing an increasingly significant role
in the modern repertoire of medicinal chemistry,1 with mole-
cules containing at least one heterocyclic fragment comprising
nearly 60 % of FDA-approved pharmaceuticals.2 Because het-
erocycles are powerful handles to regulate polarity, lipophilicity
and hydrogen bonding properties, extensive efforts are directed
at tuning these structures to manipulate the pharmacological,
pharmacokinetic and toxicological features of drug candi-
dates.3–5 Therefore, development of selective and efficient
methods for functionalization of heterocycles will streamline
synthesis of medicinal compounds and pharmacophores.
To expand nature’s capacity for functionalization of (hetero)cy-
clic molecules, we wished to use recently developed “carbene
transferase” enzymes to functionalize carbon‒hydrogen and
carbon‒carbon double bonds in heterocycles. Carbene transfer-
ases are engineered heme-dependent proteins capable of trans-
ferring carbene moieties to different target compounds.13 Reac-
tivities developed in recent years include carbene transfer to
olefins,14–16 alkynes,17 and X‒H bonds (X = C, N, S, Si, B)18–23
to generate chemical structures often not found in nature. These
enzymatic transformations grant biocatalytic access to im-
portant pharmaceuticals including levomilnacipran, ticagrelor,
tasimelteon, tranylcypromine and others.24–26
However, enzymatic carbene transfer for heterocycle derivati-
zation faces several challenges. Carbene transfer has been
achieved mostly for sterically unhindered functional groups,
such as terminal olefins. Thus it remains to be determined
whether sterically more demanding, internal carbon‒carbon
double bonds in heterocycles can be efficiently functionalized.
A second challenge for enzymatic carbene transfer to heterocy-
cles is selectivity. Synthetic chemists have used transition metal
catalysts based on Rh, Ru, Pd, Cu and others to selectively
transfer carbenes to heteroarenes, such as indoles and pyrroles,
One major way to derivatize heterocyclic fragments is to di-
rectly functionalize C‒H bonds with new chemical entities.6 Al-
ternatively, one can derivatize ring structures by functionalizing
existing C‒C bonds7. Nature employs different enzymatic ma-
chineries to accomplish these two processes. For instance, me-
thyltransferases install methyl moieties on the C‒H bonds of
DNA bases;8 flavin-dependent halogenases are responsible for
installing halogens on heterocycle-based natural products, such
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yielding either cyclopropanes or C(sp2)‒H alkylation prod-
ucts.27–32 Mimicking this activity would require enzymes to
have exquisite control over chemo-, regio- and stereo-selectiv-
ity. Here, we set out to direct the evolution of enzyme catalysts
with high activity and selectivity in heterocycle carbene func-
tionalization reactions.
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Towards this goal, we used a two-stage engineering approach.
We first evolved a cytochrome P450 variant for C3 alkylation
of indoles, using a high-throughput spectrophotometric screen
that allowed us to evaluate several thousand enzyme variants
per generation. This enabled a mutagenesis strategy targeting
the entire P450 protein sequence, which delivered a variant con-
taining 11 mutations as well as a truncation of a large part of the
non-catalytic reductase domain and which chemoselectively
and efficiently alkylates indoles. Hypothesizing that this en-
zyme, evolved for alkylation of a heterocyclic substrate, would
provide a launch pad for further exploration of challenging het-
erocycle functionalizations, in the second stage we performed
parallel directed evolution to engineer a set of P450 catalysts
capable of regio-divergent pyrrole alkylations, enantioselective
indole alkylations with α-disubstituted carbenes, and stereose-
lective cyclic alkene cyclopropanations.
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Results and Discussion
Leveraging a high-throughput screening assay to evolve a
cytochrome P450 for indole alkylation
We began by testing a range of hemoproteins for carbene trans-
fer (using ethyl diazoacetate (EDA) as carbene precursor) to a
selection of heterocyclic substrates (Supplementary Table 1).
Cytochrome P450-BM3 variant P411-CIS, a serine-ligated var-
iant previously evolved for styrene cyclopropanation,14 stood
out as highly active for C3-alkylation of 1-methylindole, cata-
lyzing this reaction with 14% yield and 690 TTN. To our de-
light, we found that the resulting alkylated product 3a showed
a pronounced red-shift in UV absorbance compared to 1-me-
thylindole. This difference in absorbance properties allowed us
to develop a simple UV-Vis spectrophotometric screening as-
say, in which the catalytic activities of enzyme variants for the
alkylation reaction (performed in whole Escherichia coli (E.
coli) cells) could be measured by UV absorbance at 305 nm in
a 96-well plate reader (Figure 1A, Supplementary Figure 1).
Figure 1: Evolution of P411-CIS for 1-methylindole C3
alkylation. (A) Screening reaction used to evolve P411 variants for
higher indole alkylation activity. (B) Domain structure of P450-
BM3. As a crystal structure of the entire P450-BM3 protein is
lacking, individual domain structures with approximate domain
boundaries are shown (pdb entries: heme domain 4h2314; FMN
domain 1bvy33; FAD domain 4dqk34); co-factors are shown in red.
(C) Evolutionary lineage from P411-CIS to P411-HF T327P
A328S. Data were obtained from two independent assays with six
replicates per variant, run in 96-well plates under aerobic
conditions with 10 mM 1 and 15 mM 2 at room temperature (RT)
for 16h.
and seven (N201S, L215Q, V281L, K472T, N573D, F646S,
Q674*) were introduced by random mutagenesis. A final round
of double site-saturation mutagenesis library screening on
P411-HF revealed mutations T327P and A328S. This enzyme
delivered the alkylated product with up to 2700 TTN in aerobic
96-well plate screening reactions (Figure 1C).
P411-CIS is a 1048-residue protein consisting of a heme do-
main containing the active site (residues 1-470) and a reductase
domain that shuttles electrons from NADPH to the heme do-
main and is essential for P450-BM3’s native oxo-transfer chem-
istry (residues 471-1048, subdivided into FMN and FAD do-
mains; Figure 1B).35 Having a screen that enables analysis of
several thousand enzyme variants, we introduced mutations
throughout the heme and reductase domains using both random
mutagenesis and site-saturation mutagenesis and screened the
resulting libraries for improved 1-methylindole alkylation ac-
tivity (Supplementary Table 2).
P411-HF: A catalyst for chemoselective alkylation of unpro-
tected indoles
P411-HF is a highly active alkylation enzyme: analytical
whole-cell reactions with P411-HF under anaerobic conditions
with 30 mM 1-methylindole and 1.5 equivalents of EDA deliv-
ered alkylation product 3a in 95% yield with almost 12,000
TTN (Figure 2A, entry 2). Under catalyst-limiting conditions,
P411-HF delivered more than 18,000 TTN (Figure 2A, entry 4).
Under aerobic conditions, variant P411-HF T327P A328S
proved superior to P411-HF and delivered the alkylated product
in 74% yield and with 1670 TTN (Figure 2A, entry 6; P411-HF
delivered 3a in 46% yield under these conditions). Thus, even
with four rounds of directed evolution under aerobic conditions,
After six rounds of directed evolution for formation of 3a, of
which the last four rounds were conducted under aerobic con-
ditions, we obtained a P411 variant we refer to as P411-HF
(Heterocycle Functionalization) (Figure 1C). This variant con-
tains eight mutations in the heme domain, three in the FMN do-
main and a stop codon at the end of the FMN domain (Q674*),
which deletes the entire FAD domain (Figure 1B, Supplemen-
tary Figure 2). Of these mutations, five were introduced by site-
saturation mutagenesis (V87T, H92F, L181G, I263M, T438C),
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P411-HF is about an order of magnitude more effective under
anaerobic conditions. Furthermore, P411-HF delivers almost
two orders of magnitude higher TTN than recently reported var-
iants of the hemoproteins Mb or YfeX catalyzing indole alkyl-
ations (170 to 240 TTN),36,37 and up to three orders of magni-
tude higher TTN than Rh, Cu, Pd or Fe-based transition metal
catalysts reported for the same type of reaction.30,31,38–40
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To investigate catalyst lifetime, we tested sequential additions
of 10 mM 1 and 2 substrate equivalents to whole-cell reactions
in 30-min intervals. This did not increase product formation,
which indicates that the enzyme is inactivated during the reac-
tion (Supplementary Figure 3A). Furthermore, a 10-minute
EDA pre-incubation of P411-HF whole-cell catalyst resulted in
a 26% loss in catalytic activity (Supplementary Figure 3B). Pre-
sumably, alkylation of enzyme residues and/or the porphyrin
cofactor limits enzyme lifetime, which may possibly be ad-
dressed by further directed evolution.41
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P411-HF anaerobic whole-cell reactions reached completion af-
ter 2 h. The high initial turnover frequency (TOF) of 470 turn-
overs min-1 represents a 35-fold increase over the parent variant
P411-CIS (Figure 2B). Under aerobic conditions, P411-HF
T327P A328S showed an initial rate of 21 turnovers min-1, com-
pared to 3 turnovers min-1 for P411-CIS (Figure 2C). The initial
rates of P411-HF compare well to previously disclosed Mb var-
iants, which exhibited ~10 turnovers min-1 in the alkylation of
indole with EDA under anaerobic conditions.36
The most striking mutation found during the directed evolution
was the truncation of the FAD domain; introduction of the stop
codon by Q674* reduces the size of the protein catalyst by 35%
(from 1048 to 673 residues) and increases reaction yield by ca.
1.7-fold, driven primarily by an increase in protein expression
level (ca. 1.6-fold) with a slight increase in TTN (Figure 1B,
Supplementary Figure 4A). Comparable positive effects of de-
leting the FAD domain have subsequently been observed for
other P411-catalyzed carbene transfer reactions: FAD trunca-
tion discovered in the course of this study proved beneficial for
carbene C(sp3)‒H insertion18 as well as cyclopropanation reac-
tions (Supplementary Figure 4B).42 Notably, FAD domain de-
letion significantly destabilized the protein, as whole-cell alkyl-
ation assays performed with a protein denaturant pre-treatment
revealed up to 33% lower activity of P411-CIS Q674* com-
pared to P411-CIS (Supplementary Figure 4C).43,44 Thus, FAD
domain deletion exerts a destabilizing effect, but contributes to
higher P411 expression levels and carbene transfer activity.
Mounting evidence suggests that native P450-BM3 adopts a ho-
modimeric quaternary structure in solution via FAD domain in-
teractions, and that the dimeric form is required for oxo-transfer
activity.34,45–48 The data presented here suggest that for non-nat-
ural carbene-transfer reactions a different, possibly monomeric,
catalyst structure may be suitable or even beneficial. Attempts
to obtain crystal structures of the FAD-truncated P411-HF var-
iant have unfortunately not been successful.
Figure 2: Characterization of P411-HF. (A) Comparison of
P411-CIS and P411-HF in analytical (400 µL) biocatalytic whole-
cell reactions for formation of 3a under aerobic or anaerobic con-
ditions and different substrate and catalyst loadings. (B,C) Kinetics
of whole cell-reactions for formation of 3a under anaerobic (B) and
aerobic (C) conditions. Reactions were run with cells at OD600=30
with 30 mM 1 and 45 mM 2 (anaerobic, B), or 10 mM 1 and 15
mM 2 (aerobic, C). Initial turnover frequencies were calculated
from samples analyzed 10 minutes after starting the reaction. Data
in (A) to (C) were obtained from two to three independent assays
with two replicates per variant.
state of the heme cofactor even in the absence of reductant, in
stark contrast to P411-CIS. While native P450 oxo-transfer
chemistry requires a stoichiometric supply of electrons, P411
carbene transfer catalysis functions without an electron supply
as long as the iron cofactor is in the ferrous state.14 Therefore,
truncating the reductase domain can be expected to be less con-
sequential for carbene transfer catalysis. The extent to which
FAD domain truncation contributes to maintaining the enzyme
in the catalytically active ferrous state remains to be deter-
mined.
Lastly, we tested the substrate scope of P411-HF and its suita-
bility for synthesis of alkylated indoles. With 1-methylindole,
preparative-scale reactions (0.6 mmol) delivered 3a with 83 %
isolated yield (108 mg) (Figure 3). To our delight, we found that
P411-HF selectively alkylates unprotected indoles at the C3 po-
sition. No N‒H insertion products were observed, and alkylated
products were isolated in good yields across a range of substi-
tuted, unprotected indoles (Figure 3). We observed a clear reac-
tivity trend, with only trace amount of alkylation product ob-
tained for C4-substituted indoles, comparatively low yields for
C5-substituted indoles, and good yields for indoles with substi-
Next, we compared 1-methylindole alkylation activities of
P411-CIS and P411-HF as purified proteins and with different
reducing agents (Supplementary Figure 5). We found that puri-
fied P411-CIS catalyzes 1-methylindole alkylation with 165
TTN with sodium dithionite as reductant, 135 TTN with
NADPH as reductant, but only 8 TTN in the absence of reduct-
ant. To our surprise, P411-HF showed 1890 TTN with NADPH
(corresponding to 94% yield), 1750 TTN with sodium dithio-
nite, and an almost equally high 1670 TTN without any reduct-
ant present. Thus, P411-HF maintains a catalytically competent
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tutions at C6 or C7. Presumably, substitutions at C4 or C5 inter-
C3 position.53 Enzyme catalysis can take advantage of precise
substrate orientation within the active site, and thus potentially
drive site selectivity independent of electronic or steric bias.54
Accordingly, we speculated that the P411-HF active site could
be tuned by directed evolution to favor alkylation at either the
C2 or C3 position.
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fere with productive indole binding in the active site. Also, only
low activity was observed for 1,2-dimethylindole; alkylation of
this substrate was improved by the M263A mutation, which
likely creates more space in the active site above the heme co-
factor and enables productive binding of this sterically hindered
1,2-disubstituted indole.
We found that P411-HF catalyzes carbene transfer to 1-
methylpyrrole with 180 TTN, showing a 3:1 selectivity for the
electronically favored C2-alkylated product (Figure 4A, Supple-
mentary Figure 6, Supplementary Table 3). Subsequent site-sat-
uration mutagenesis and screening revealed a new variant with
mutation M263T, providing a 1:1 ratio of both products and
confirming that active site mutations can indeed shift the selec-
tivity. Further evolution yielded two divergent P411 lineages
that selectively yield the C2 or C3 alkylation products: variant
P411-HF M263T G181E A82T makes the C3-alkylated product
with 705 TTN and 9:91 C2:C3 ratio, whereas variant P411-HF
M263T T87W G181K yields the C2-alkylated product with 485
TTN and 98:2 C2:C3 ratio (Figure 4A). Thus, the P411-HF ac-
tive site can be sculpted with only three rounds of mutagenesis
and screening to exert precise control over pyrrole heterocycle
alkylation regioselectivity. The resulting variants may provide
starting points for evolving enzymes to selectively alkylate
other pyrrole derivatives.
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Next, we asked whether P411-HF could serve as a starting point
to evolve for enantioselective indole alkylation using an α-di-
substituted carbene precursor (e.g., ethyl-2-diazopropanoate,
Me-EDA), thereby delivering chiral alkylated indole core 5
found in bioactive natural products.56 Alkylation of 1-methylin-
dole with Me-EDA represented a difficult biocatalytic reaction:
no activity was found for either free hemin or for P411-CIS, and
variant P411-HF M263Y delivered the product with only 2
TTN. This low activity, however, was sufficient for GC-MS
based screening, and we evolved this variant over seven rounds
of site-saturation mutagenesis and screening to obtain variant
P411-HF T87A Y263E A328Y L437M A268G T327P, which
delivers 5 with 775 TTN (Figure 4B, Supplementary Figure 7,
Supplementary Table 4). While this represents a >300-fold in-
crease in catalytic activity, enantioselectivity remained at ca.
50% over the course of evolution. The limited enantioselectiv-
ity may be grounded in the enzymatic reaction mechanism: nu-
cleophilic attack by the indole C3 carbon at the iron-carbene
center followed by protonation is generally considered the
mechanistic pathway of indole alkylation.36,38,57 This process
would require the enzyme to have perfect stereocontrol over the
protonation step in order to yield high enantioselectivity. We
speculate that further engineering of the P411-HF active site
could possibly fine-tune hydrogen- bonding networks between
the reaction intermediate and the protein, which might allow
better control of the protonation step and lead to higher stere-
oselectivity. The evolved catalyst is nonetheless among the few
P411’s efficiently catalyzing transfer of an α-disubstituted car-
bene.
Figure 3: P411-HF indole alkylation substrate scope. Prepara-
tive-scale reactions were performed at 0.6 mmol scale (1-methyl-
indole) or 0.3 mmol scale (all other substrates) to yield the C3-al-
kylated products. Isolated yields are given; TTN are based on the
isolated yield. aFor 1,2-dimethylindole, reactions were performed
with variant P411-HF M263A and HPLC yield and TTN are given.
Utilizing P411-HF as a starting point for additional cyclic
alkene functionalization reactions
While evolving for new functions, enzymes may gain activities
for additional reactions that were not actively selected for.
However, such promiscuous activities may in turn serve as evo-
lutionary starting points to evolve novel functions. 49–51 For ex-
ample, we found that variant P411-CIS, previously evolved
from P450-BM3 for styrene cyclopropanation,14,15 showed
more than 30-fold higher activity for 1-methylindole alkylation
than P450-BM3 (Supplementary Table 1). Thus, P411-CIS was
chosen as starting point for evolving indole alkylation activity,
ultimately yielding variant P411-HF (Figure 4A). In turn, we
speculated that P411-HF, engineered to accommodate a hetero-
cyclic structure near the catalytic iron center, may provide a
good starting point for further evolution to unlock additional
carbene transfer reactions to (hetero)cyclic alkenes. To test this
hypothesis, we performed three evolutionary “case studies”, fo-
cusing on regioselective pyrrole alkylation, enantioselective in-
dole alkylation with an α-disubstituted carbene precursor, and
cyclic alkene cyclopropanation.
Finally, we reasoned that P411-HF may provide a good starting
point to unlock cyclopropanation reactions of (hetero)cyclic in-
ternal alkenes. We first tested cyclopropanation using indene
and EDA and found that P411-HF yielded the cis-configured
cyclopropane 6a with 1880 TTN, 95:5 dr and 94% ee (Figure
4C). This was significantly higher than P411-CIS, showing 305
TTN, 69:31 dr and 78% ee (Supplementary Figure 8, Supple-
mentary Table 5), supporting the hypothesis that evolution for
First, we attempted functionalizing pyrroles at either the C2 or
C3 position, a considerable challenge for small-molecule cata-
lysts.27 Transition metal catalysis typically relies on electronic
bias between the two positions and predominantly leads to func-
tionalization of C2‒H bonds.52 Alternatively, bulky substituents
on the nitrogen were shown to shift regioselectivity towards the
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Figure 4: Evolving P411-HF for selective (hetero)cycle functionalization reactions. Top: Schematic representation of the relation be-
tween protein sequence and catalytic function. A given enzyme sequence may exhibit additional ‘promiscuous’ functions that can be opti-
mized by directed evolution.55 We set out to test whether promiscuous activities of P411-HF, gained during evolution for 1-methylindole
alkylation, could serve as starting points to evolve further (hetero)cycle functionalization reactions. (A) Evolution of P411-HF for regiose-
lective 1-methylpyrrole alkylation. (B) Evolution of P411-HF for enantioselective 1-methylindole alkylation with ethyl-2-diazopropanoate.
(C) Evolution of P411-HF for stereoselective cyclic alkene cyclopropanation. Data in (A) to (C) were derived from two independent exper-
iments performed in duplicate; see Supplementary Tables 3 to 6 and Supplementary Figures 6 to 8 for evolutionary lineages and experimental
details.
indole alkylation in parallel raised activity for cyclopropanation
of sterically similar substrates. One additional round of site-sat-
uration mutagenesis and screening delivered P411-HF M263Y,
providing 6a with almost 4000 TTN, 97:3 dr and 96% ee (Fig-
ure 4C). Comparable hemoprotein cyclopropanation activity on
internal alkene substrates was achieved previously only with
iridium porphyrin-substituted hemoprotein variants,58 and this
enzymatic reaction delivers a potentially useful cyclopropane
core recently explored as pharmacophore.59
for the current P411-HF carbene transferase lineage. Nonethe-
less, the results presented here demonstrate that enzymes con-
taining the native iron-heme cofactor are capable of internal al-
kene cyclopropanation, with activity and selectivity tunable by
directed evolution.
Concluding Remarks
Enzyme catalysis represents a potentially sustainable alterna-
tive to chemical synthesis. Unlocking its promise, however, re-
quires that enzymes perform equal or better than state-of-the-
art synthetic methods. In this study, we engineered a set of cy-
tochrome P411 variants for carbene transfer to heterocycles and
internal alkenes, synthetically important substrate classes
largely absent from the repertoire of carbene transferases de-
scribed to date. We tackled this problem with a two-tiered en-
gineering strategy, first subjecting a cytochrome P411 variant
to a comprehensive directed evolution effort, which delivered
the highly active indole C3-alkylation variant P411-HF. Further
engineering of P411-HF towards more challenging heterocycle
and internal alkene functionalizations then delivered a suite of
P411 variants for a diverse set of heterocycle functionalization
Similar results were obtained on a less-activated cyclic alkene,
1,3-cyclohexadiene (Figure 4C, Supplementary Figure 8, Sup-
plementary Table 6). Again, P411-HF showed enhanced cyclo-
propanation activity over its parent P411-CIS, and two addi-
tional rounds of site-saturation mutagenesis and screening
yielded P411-HF M263W G181L, delivering the cis-configured
cyclopropane 6b with 100 TTN, 91:1 dr and 73% ee. Lower
TTNs (< 100) were observed on heterocyclic substrates, includ-
ing 2,3-benzofuran, 2,3-dihydrofuran and benzothiophene,
while no activity was observed with aliphatic internal alkenes
2,4-hexadiene, 2-octene, cyclopentene, cyclohexene or cyclo-
heptene, indicating that these substrates still present a challenge
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ation, enantioselective indole alkylation, and stereoselective cy-
clic alkene cyclopropanation. The evolved enzymes catalyze
hundreds to thousands of turnovers, are fully genetically en-
coded, and function with an earth-abundant iron cofactor. These
variants complement existing transition metal catalysts for se-
lective heterocycle functionalizations, provide potential starting
points for future directed evolution endeavors, and expand the
catalytic repertoire of the carbene transferase family of en-
zymes.
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ASSOCIATED CONTENT
Org.
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Supporting
Information.
Experimental
procedures,
https://doi.org/10.1021/acs.joc.5b01255.
supplementary data, calibration curves, and characterization of
alkylation and cyclopropanation products. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* Frances H. Arnold
frances@cheme.caltech.edu
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Li, Q.-S.; Schwaneberg, U.; Fischer, P.; Schmid, R. D. Di-
rected Evolution of the Fatty‐Acid Hydroxylase P450 BM‐
3 into an Indole‐Hydroxylating Catalyst. Chemistry – A
Present Addresses
†Present address: Howard Hughes Medical Institute, Department
of Human Genetics, University of California Los Angeles, Los
Angeles, California 90095, USA.
European Journal 2000,
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https://doi.org/10.1002/(SICI)1521-
3765(20000502)6:9<1531::AID-CHEM1531>3.0.CO;2-
D.
Kim, D.; Guengerich, F. P. Cytochrome P450 Activation
of Arylamines and Heterocyclic Amines. Annu. Rev. Phar-
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
macol.
https://doi.org/10.1146/an-
nurev.pharmtox.45.120403.100010.
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Funding Sources
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Zheng, Q.; Fang, H.; Liu, W. Post-Translational Modifica-
tions Involved in the Biosynthesis of Thiopeptide Antibi-
otics. Org. Biomol. Chem. 2017, 15 (16), 3376–3390.
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and Engineering Hemoproteins for Abiological Carbene
and Nitrene Transfer Reactions. Curr. Opin. Biotech.
2017, 47, 102–111. https://doi.org/10.1016/j.cop-
bio.2017.06.005.
Coelho, P. S.; Wang, Z. J.; Ener, M. E.; Baril, S. A.; Kan-
nan, A.; Arnold, F. H.; Brustad, E. M. A Serine-Substi-
tuted P450 Catalyzes Highly Efficient Carbene Transfer to
Olefins in Vivo. Nat. Chem. Biol. 2013, 9 (8), 485–487.
https://doi.org/10.1038/nchembio.1278.
Coelho, P. S.; Brustad, E. M.; Kannan, A.; Arnold, F. H.
Olefin Cyclopropanation via Carbene Transfer Catalyzed
by Engineered Cytochrome P450 Enzymes. Science 2013,
339 (6117), 307–310. https://doi.org/10.1126/sci-
ence.1231434.
This work was supported by a grant from the National Science
Foundation, Division of Molecular and Cellular Biosciences to
F.H.A. (NSF MCB-1513007). O.F.B. acknowledges support from
the Deutsche Forschungsgemeinschaft (DFG grant BR 5238/1-1)
and the Swiss National Science Foundation (SNF grant P300PA-
171225). K.C. thanks the Resnick Sustainability Institute at Caltech
for fellowship support.
ACKNOWLEDGMENTS
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(15)
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The authors are truly grateful to Dr. Christopher Prier for help with
initial experimental design and comments on the manuscript. We
also thank Dr. David Romney and Anders Knight for helpful
comments and discussions.
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