of 15
Enantioselective, Intermolecular Benzylic C–H Amination
Catalyzed by an Engineered Iron-Heme Enzyme
Christopher K. Prier
,
Ruijie K. Zhang
,
Andrew R. Buller
,
Sabine Brinkmann-Chen
, and
Frances H. Arnold
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East
California Boulevard, MC 210-41, Pasadena, CA 91125, United States
Abstract
C–H bonds are ubiquitous structural units of organic molecules; while these bonds are generally
considered to be chemically inert, the recent emergence of methods for C–H functionalization
promises to transform the way synthetic chemistry is performed. The intermolecular amination of
C–H bonds represents a particularly desirable and challenging transformation for which no
efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of
an iron-containing enzymatic catalyst, based on a cytochrome P450 monooxygenase, for the
highly enantioselective, intermolecular amination of benzylic C–H bonds. The biocatalyst is
capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to
valuable benzylic amines. Iron complexes are generally poor catalysts for C–H amination: in this
catalyst, the enzyme’s protein framework confers activity on an otherwise unreactive iron-heme
cofactor.
Table of contents summary
The intermolecular amination of C–H bonds is an enabling transformation for the synthesis of
nitrogen-containing molecules, yet a challenging activity for chemical catalysts. Here we engineer
an iron-based enzyme for this reaction, demonstrating that a protein can confer a difficult new
function upon an otherwise unreactive base metal.
C–H bonds may be the most common functional groups in organic molecules but are
typically considered inert toward chemical transformation. By selectively acting on these
bonds and installing new functional groups directly into the hydrocarbon framework of
organic compounds, C–H functionalization technology has the potential to streamline
synthetic routes, leading to less wasteful and more sustainable chemical production.
1
3
Modern C–H functionalization methods, however, rely heavily on catalytic complexes of
precious transition metals (such as rhodium, ruthenium, and iridium), which are neither
cheap nor green.
1
Many enzymes, by contrast, catalyze very challenging reactions using
Correspondence and requests for materials should be addressed to F.H.A. (frances@cheme.caltech.edu).
These authors contributed equally to this work.
Author Contributions:
C.K.P. and R.K.Z. designed, carried out, and analyzed all amination experiments, with F.H.A. providing
guidance. C.K.P., R.K.Z., and S.B.-C. obtained protein crystals. R.K.Z. and A.R.B. solved the crystal structure. C.K.P. and F.H.A.
wrote the manuscript with input from all of the authors.
The authors declare no competing financial interests.
HHS Public Access
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Nat Chem
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base metals; their activities are often only possible because the protein plays a key role,
modifying or enhancing the inherent reactivity of the metal cofactor.
4
We envisioned that
enzymes dependent only on base metals could be exploited through reaction design and
evolution to perform non-biological C–H functionalization reactions, potentially with
efficiencies and selectivities exceeding those of known chemical catalysts.
Due to the presence of nitrogen in many bioactive synthetic and natural compounds,
methods for directly transforming sp
3
-hybridized C–H bonds into C–N bonds have been
pursued intensively.
5
9
Many of the methods that have been developed are either
intramolecular (requiring that a nitrogen source already be present in the same molecule as
the targeted C–H bond) or utilize directing groups (also requiring the presence of specific
functionality in the starting material). An elegant yet more elusive transformation is the
intermolecular C–H amination of unfunctionalized hydrocarbons; such a reaction provides a
dramatically simplifying and convergent disconnection for the synthesis of amines from
alkanes (Fig. 1). Recognizing the potential of this transformation, several groups have
pursued innovative strategies for intermolecular C–H amination. While many of these
methods are racemic,
10
14
enantioselective intermolecular C–H amination via the generation
and transfer of metal nitrenoids has been developed using rhodium,
15
17
ruthenium,
18
,
19
and
manganese
19
,
20
catalysts. Highly diastereoselective C–H amination using a chiral nitrogen
source has also been achieved using a rhodium catalyst.
21
These systems represent major
advances in C–H functionalization; none, however, offers the combination of high catalytic
efficiency (turnover), high enantioselectivity across diverse substrates, and ready access to
the chiral catalyst.
Cytochrome P450 monooxygenases are nature’s catalysts for the direct insertion of oxygen
into C–H bonds. These iron-dependent enzymes perform hydroxylation with high reaction
rates and turnovers, and do so under mild physiological conditions.
22
They also act with
excellent selectivity in complex settings, often hydroxylating only one C–H bond out of
many in diverse secondary metabolites.
23
In contrast to this strategy for oxygenation, nature
does not introduce nitrogen into organic molecules via C–H functionalization. Biosynthetic
routes to amines instead rely exclusively on functional group manipulation of pre-oxidized
substrates, and biocatalytic multi-enzyme cascades have been engineered for formal C–H
amination using this general approach.
24
,
25
In striking contrast to both natural and synthetic amination strategies, it has been shown that
heme proteins can catalyze nitrene transfer under appropriate reaction conditions.
26
32
In
particular, variants of cytochrome P450
BM3
from
Bacillus megaterium
that feature a serine
axial ligand to the heme iron in place of the wild-type cysteine ligand, which we term
cytochrome P411s,
33
are proficient catalysts for nitrene transfer. Based on this manifold, we
envisioned the catalytic cycle for intermolecular amination shown in Figure 2. First,
reduction of the ferric state of the heme cofactor, with electrons derived from NADPH, gives
the ferrous state
1
. Reaction with a nitrene source, here tosyl azide (TsN
3
), then provides the
putative iron nitrenoid
2
. Subsequent reaction of this intermediate with an alkane such as 4-
ethylanisole (
3
) would deliver the C–H amination product
4
and regenerate the ferrous state
of the catalyst (
1
). A competing process observed in P411-catalyzed nitrene transfer is the
reduction of the nitrenoid, generating the undesired by-product
p
-toluenesulfonamide
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(TsNH
2
,
5
). Cytochrome P411 catalysts have been identified for
intramolecular C–H
amination reactions;
26
,
27
in these cases, the targeted C–H bond is necessarily present in the
enzyme active site when the reactive nitrenoid
2
is formed, biasing productive C–H
amination over the deleterious nitrenoid reduction pathway. In contrast, a catalyst for the
desired intermolecular amination must bind a separate alkane substrate and promote its
amination faster than the transient nitrenoid species is consumed in the reductive side
reaction.
Results and discussion
Reaction discovery and directed evolution
In initial studies, we found that almost all cytochrome P411s reported to date display no
activity toward intermolecular C–H amination of alkane
3
– including catalysts that were
developed for ostensibly similar reactions such as intramolecular C–H amination
26
,
27
and
intermolecular aziridination
28
– instead they exclusively reduce the azide to the sulfonamide
5
(Supplementary Table 1). Other heme-containing proteins, as well as the heme cofactor
alone (iron protoporphyrin IX), are similarly inactive (Supplementary Table 2). Remarkably,
however, we found that P411
BM3
variant P-4, engineered for the imidation of allylic
sulfides,
29
shows promiscuous activity toward intermolecular C–H amination. Under
anaerobic conditions, 4-ethylanisole (
3
) undergoes amination at the benzylic carbon in 11%
yield and 14% ee by variant P-4, which differs from wild-type P450
BM3
by 17 mutations
(Fig. 3). Even more encouragingly, a single mutation to P-4, A82L (also identified in the
context of the sulfimidation reaction), provides a greater than 4-fold improvement in yield
(to 51%) and delivers the product
4
in 77% ee. Similarities between the nitrene transfer
transition states of the sulfimidation and C–H insertion reactions may account for why
evolution for one reaction engendered activity toward the other, leading to variants with
expanded promiscuous capabilities toward nitrene transfer.
We next performed sequential rounds of site-saturation mutagenesis of selected residues in
P-4 A82L and screening to improve C–H amination activity and enantioselectivity. We
targeted sites in the heme domain that were either previously shown to impact the activity
and/or selectivity of P450s, residues that are highly conserved in P450s, or residues that
were already mutated in P-4 relative to wild-type P450
BM3
(see Supplementary Information
for details).
34
The libraries were screened for enhanced C–H amination activity, and
potential hits were subsequently evaluated for activity across a set of three substrates with
different electronic demands for C–H amination: 4-ethylanisole (
3
), 4-ethyltoluene (
6
), and
ethylbenzene (
7
, Fig. 3). Two mutations, A78V and F263L, improve activity and selectivity
on all three substrates, yielding a catalyst that delivers the benzylic amine products as single
enantiomers (>99% ee). A final mutation, E267D, is neutral with respect to the amination of
4-ethylanisole but provides a two-fold improvement in the amination of ethylbenzene (
7
, to
15% yield), as well as improving the reaction yield and/or selectivity in the C–H amination
of other substrates (Supplementary Table 6). We call this final variant P411
CHA
, for
“cytochrome P411 C–H aminase.” Under the conditions employed for evolution, this
catalyst generates the benzylic amine
4
in 66% yield, with >99% ee and 1,000 turnovers in
whole
Escherichia coli
cells. While 4-ethylanisole is electronically activated toward C–H
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insertion,
15
alkanes
6
and
7
are less activated and display much lower reactivity with the
initial P411 variants. Over the course of evolution from variant P-4 to P411
CHA
, the
amination of 4-ethylanisole was improved by 6-fold, while the effect on the amination of
ethylbenzene was significantly greater (30-fold improvement). Thus, using an electronically
activated substrate (4-ethylanisole) allowed the initial discovery of P411 C–H amination
activity, from which variants that aminate inherently less reactive alkanes were derived.
Subsequent studies revealed that P411
CHA
can support up to 1,300 turnovers (Table 1, entry
3), far exceeding the highest turnover number (TON) reported with
any chiral transition
metal complex for intermolecular, enantioselective C–H amination (the best reported is 85
TON using a chiral manganese porphyrin).
19
The reaction was optimized for yield by
employing two equivalents of tosyl azide; under these conditions, the product
4
is obtained
in 86% yield, with >99% ee and 670 turnovers (Table 1, entry 8). In contrast to the reported
chemical protocols, the reaction with P411
CHA
takes place in water at room temperature; the
reaction can also proceed under aerobic conditions, but the yield is 7-fold higher using the
optimal anaerobic conditions (Supplementary Table 7).
Substrate scope studies
Scope studies, carried out using the yield-optimized conditions, revealed that cytochrome
P411
CHA
aminates a diverse set of arene-containing hydrocarbons. Substitution of the
aromatic ring is tolerated at
para, meta,
and
ortho
positions (Table 2, entries 2–4). While
electron-withdrawing functionality reduces the reactivity of the alkane toward the metal
nitrenoid, several halogenated ethylbenzenes are nevertheless functionalized with >100
turnovers (entries 6–8). The cyclic alkanes indan and tetralin are excellent substrates (entries
9 and 10), while the related 2,3-dihydrobenzofuran displays reduced reactivity (entry 11).
Larger alkylarenes such as ethylnaphthalenes and 4-propylanisole are still accommodated in
the enzyme active site, undergoing amination with varying levels of efficiency (entries 12–
14). Notably, the amination of 1-ethylnaphthalene delivers the nitrogen-containing fragment
of the calcimimetic drug cinacalcet (Sensipar) with the correct absolute configuration (entry
13).
35
Furthermore, although the activity is low, the amination of the methyl group of 4-
methylanisole demonstrates that these catalysts are capable of functionalizing C–H bonds
with bond dissociation energies as high as 90 kcal/mol (entry 15). Benzylic ethers are
suitable substrates for amination, but the products are isolated in racemic form (entries 16–
17). For the amination of isochroman (entry 17), analysis of crude reaction mixtures
demonstrated that the C–H insertion event is moderately enantioselective (65% ee), but the
product undergoes racemization during purification on silica gel (Supplementary Table 10).
Notably, most of the alkanes evaluated undergo functionalization with excellent levels of
enantioselectivity (>90% ee), a key advantage of this enzymatic approach over most reported
metal catalysts. However, P411
CHA
displays several limitations with regard to substrate
scope: it does not aminate substrates much larger than those shown here, nor does it
functionalize benzylic positions adjacent to especially electron-deficient arenes
(Supplementary Fig. 1). Interestingly, cumene and 4-methoxycumene are not aminated by
P411
CHA
despite the presence of weak benzylic C–H bonds. We have also not found
P411
CHA
to be active toward the amination of allylic or aliphatic C–H bonds, nor is it
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capable of aminating non-benzylic ethers. The aromatic motif may be required for
productive substrate binding or to sufficiently lower the activation barrier of C–H insertion.
Directed evolution, however, may improve the activities described here and alter the
substrate scope of these enzymes, potentially by modifying the binding site of the alkane.
Just as evolution within the functional space of nitrene transfer engendered expanded
amination activities, it is possible that non-benzylic amination will be accessible to future
generations of P411 enzymes. Cytochrome P411
CHA
also has improved thermostability
relative to P–4 (
T
50
is increased by ~3 °C, to 61 °C, Supplementary Table 11), suggesting it
may serve as a robust starting point for the evolution of specialized enantioselective C–H
amination catalysts.
36
The enzymatic amination reaction can be performed on preparative scale: from a 0.25 mmol-
scale biotransformation, the benzylic amine
4
was isolated in 78% yield (59.5 mg, 610 TON,
>99% ee). The reaction also proceeds with purified protein, albeit less efficiently than in
whole cells: the P411 catalysts are capable of up to 190 TON
in vitro
vs. 1,300 TON
in vivo
(Supplementary Table 8). Furthermore, the tosyl group present in the C–H amination
products may be removed via treatment with samarium diiodide,
37
providing the
corresponding primary amine with no erosion of enantiomeric excess (see Supplementary
Information for details).
Rate experiments
The fact that amination activity throughout evolution is highly dependent on the electronic
nature of the substrate (electron-rich substrates giving higher yields) suggests that C–H
insertion is rate-determining. We questioned, however, whether improvements in yield arose
from improved catalytic efficiency toward C–H amination or suppression of the competing
azide reduction pathway (Fig. 2). Under
in vitro
conditions, initial rates are enhanced by
greater than 8-fold for the amination of 4-ethylanisole by P411
CHA
compared to P-4,
showing that the mutations indeed increase the rate of the productive reaction
(Supplementary Fig. 2). Independent rate measurements conducted with deuterated alkane
8
show a kinetic isotope effect (
k
H
/
k
D
) of 1.6 in the reaction catalyzed by P411
CHA
,
suggesting only partial C–H bond cleavage in the amination transition state (Fig. 4a),
5
while
also providing further evidence for a rate-determining C–H insertion event.
Crystallography
We obtained an X-ray crystal structure of the penultimate variant in our lineage (P-4 A82L
A78V F263L) at 1.70-Å resolution (Fig. 4b). The beneficial mutations identified in this
study are all located in helices that line the active site; two mutations are located in the
enzyme’s B
helix (A82L, A78V) and two are located in the I helix (F263L, E267D).
Residues in both of these helices are known to mediate substrate binding and/or impact
selectivity in P450-catalyzed oxygenation reactions.
38
40
Like two earlier reported P411
structures,
27
,
33
the structure of the evolved aminase adopts the P450 closed state typically
induced by substrate binding. Compared to the structure of P411 variant P-I263F,
27
which
differs by only six mutations yet performs intermolecular C–H amination with trace activity
(<1% yield), there are only minor movements of the protein scaffold, although the active-site
residue L437 surprisingly adopts an unfavored backbone conformation. On average, the
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volume of the heme distal pocket is reduced by approximately 10% in the evolved variant
compared to P-I263F; the smaller active site potentially enforces productive substrate
binding modes. These observations suggest that the mutations introduced on the path to
P411
CHA
exert local effects that modulate interactions with the azide and alkane substrates
in the active site. Docking simulations revealed plausible substrate conformations for nitrene
transfer, in which the substrates are organized via van der Waals interactions with residues
A87, L263, E267, and V328, among others (see Supplementary Information).
Conclusion
Cytochrome P411
CHA
displays the ability to aminate benzylic C–H bonds intermolecularly
in diverse structures with high selectivity, demonstrating that a renewable protein catalyst
based on iron (the most abundant transition metal in the earth’s crust) can solve a long-
standing challenge in synthetic chemistry. The protein does more than simply control the
reactivity of a reactive metal complex – as in the native P450 monooxygenation reaction, the
protein enables a function that the iron cofactor cannot perform on its own. Biocatalysts for
non-natural reactions have alternatively been created by introducing precious metals (such as
iridium and rhodium) into proteins.
41
43
An artificial iridium metalloenzyme has been
shown to perform nitrene transfer; this system is capable of up to ~300 turnovers in
intramolecular
C–H amination reactions.
44
That P411
CHA
achieves intermolecular C–H
amination with the native iron cofactor suggests that costly precious metals – and strategies
for introduction of the non-native metal – are not necessary to achieve highly active
biocatalysts for challenging non-natural reactions. The current work also describes an
evolutionary pathway in which P411s evolved for a more readily accessible nitrene transfer
reaction (sulfimidation) picked up promiscuous activity toward a more challenging reaction
(intermolecular C–H amination). This strategy of stepwise evolution through increasingly
challenging reactivities may be generally useful for engineering enzymes for new activities
not readily found by testing wild-type proteins. Finally, the ability to accelerate C–H
insertion via mutation suggests that cytochrome P411
CHA
may be a platform for evolving
catalysts for diverse C–H functionalization reactions currently inaccessible to chemical
catalysis.
Methods
Expression of P411
BM3
variants
E. coli
BL21
E. cloni
cells carrying a plasmid encoding a P411 variant were grown
overnight in 5 mL Luria-Bertani medium with 0.1 mg/mL ampicillin (LB
amp
, 37 °C, 250
rpm). The preculture was used to inoculate 45 mL of Hyperbroth (HB) medium (prepared
from AthenaES© powder, 0.1 mg/mL ampicillin) in a 125-mL Erlenmeyer flask; this culture
was incubated at 37 °C, 230 rpm for 2 h. Cultures were then cooled on ice (20 min), and
expression was induced with 0.5 mM IPTG and 1.0 mM 5-aminolevulinic acid (final
concentrations). Expression was conducted at room temperature (23 °C), at 130 rpm, for 16–
18 h. Cultures were then centrifuged (2,600 x g, 10 min, 4 °C), and the pellets were
resuspended to an OD
600
of 30 in M9-N minimal media (no nitrogen). Aliquots of the cell
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suspension (4 mL) were used to determine the P411 expression level by CO-binding assay
after lysis by sonication.
Intermolecular C–H amination in whole
E. coli
cells
For amination bioconversions, the cells containing the P411 variant, at OD
600
of 30 in M9-N
media (grown as described above), were degassed by sparging with argon in sealed 6-mL
crimp vials for at least 40 minutes. Separately, a glucose solution (250 mM in M9-N) was
degassed by sparging with argon for at least 10 minutes. An oxygen depletion system (20 μL
of a stock solution containing 14,000 U/mL catalase and 1,000 U/mL glucose oxidase in 0.1
M KPi, pH 8.0) was added to 2-mL crimp vials. All solutions were uncapped and transferred
into an anaerobic chamber. Resuspended cells (320 μL) were added to the vials, followed by
glucose (40 μL, 250 mM in M9-N), alkane (10 μL of a DMSO stock), and tosyl azide (10 μL
of a DMSO stock). Final concentrations were typically 2.5–5.0 mM alkane, 5.0 mM tosyl
azide, and 25 mM glucose; final reaction volume was 400 μL. The vials were sealed,
removed from the anaerobic chamber, and shaken at room temperature and 40 rpm for 16–
20 h. The reactions were quenched by addition of acetonitrile (400 μL) and internal standard
(10 μL of a DMSO stock). This mixture was then transferred to a microcentrifuge tube and
centrifuged at 20,000 x g for 10 minutes. The supernatant was transferred to a vial and
analyzed by HPLC for yield. Reaction samples were extracted with cyclohexane and
analyzed by chiral SFC (supercritical fluid chromatography) for enantiomeric excess (ee).
Yield is calculated as mM reaction product divided by mM alkane starting material; TON is
calculated as mM reaction product divided by mM P411, as determined by the CO-binding
assay following cell lysis. Enantiomeric excess (ee) is calculated as (major enantiomer –
minor enantiomer)/(major enantiomer + minor enantiomer).
Data availability
Complete experimental procedures, including synthesis methods for all compounds,
characterization data, and details of bioconversion experiments are described in the
Supplementary Information. The crystal structure of P411
BM3
P-4 A82L A78V F263L has
been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data
Bank (PDB) under accession code 5UCW.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Our research is supported by the National Science Foundation, Division of Molecular and Cellular Biosciences
(grant MCB-1513007) and by funds from the American Recovery and Reinvestment Act (ARRA) through the
National Institutes of Health Shared Instrumentation Grant Program (S10RR027203). C.K.P. thanks the Resnick
Sustainability Institute for a postdoctoral fellowship. R.K.Z. was supported by a National Science Foundation
Graduate Research Fellowship (NSF GRFP; DGE-1144469), is a trainee in the Caltech Biotechnology Leadership
Program, and has received financial support from the Donna and Benjamin M. Rosen Bioengineering Center.
A.R.B. is funded by a Ruth Kirschstein NIH Postdoctoral Fellowship F32G110851. We thank Dr. Scott Virgil, Dr.
Jens Kaiser, and Russell D. Lewis for experimental assistance, and Drs. Oliver F. Brandenberg, Stephan C.
Hammer, and S. B. Jennifer Kan for helpful discussion and comments on the manuscript.
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Figure 1. Intermolecular C–H amination, a simplifying transformation for chiral amine synthesis
Intermolecular C–H amination enables direct and convergent functionalization in which a
simple alkane and a nitrogen atom source are brought together in a single step. In principle,
any C–H bond in the substrate is a potential site of functionalization.
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Figure 2. Proposed mechanism of cytochrome P411-catalyzed intermolecular C–H amination
Reaction of the aminating reagent, tosyl azide, with the ferrous porphyrin (
1
) generates an
enzyme-bound iron nitrenoid intermediate (
2
). This nitrenoid then inserts into a C–H bond
in the alkane, delivering a benzylic amine product. The nitrogen atoms in a plane represent
the enzyme’s heme cofactor. Ts = 4-toluenesulfonyl; Ser = serine.
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Figure 3. Evolution of a cytochrome P411 catalyst for enantioselective C–H amination on
increasingly challenging substrates
Directed evolution, via sequential rounds of site-saturation mutagenesis and screening,
improved both the conversion and enantioselectivity of P411-catalyzed C–H amination. The
initial variant P-4 shows significant activity only on the electronically-activated 4-
ethylanisole (
3
); evolved variants display activity on inherently less activated substrates.
Reactions were performed using whole
E. coli
cells overexpressing the P411 variant,
resuspended to OD
600
= 30, with 5 mM alkane and 5 mM tosyl azide, under anaerobic
conditions. Results are the average of experiments performed with duplicate cell cultures,
each used to perform duplicate chemical reactions (four reactions total). Bars represent
yield; numbers above bars represent enantiomeric excess (ee); both are color-coded to match
the substrate (blue = 4-ethylanisole; red = 4-ethyltoluene; purple = ethylbenzene). Error bars
correspond to one standard deviation. P-4 gives predominantly the
S
enantiomer in the
amination of 4-ethylanisole (
3
); all other variant/substrate combinations give predominantly
the
R
enantiomer.
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Figure 4. Kinetic isotope effect and enzyme structural studies
a,
The kinetic isotope effect in enzymatic C–H amination was determined from independent
in vitro
rate experiments.
b,
Active site view of the P-4 A82L A78V F263L crystal structure,
showing the heme in white and the iron atom in orange. Key active site residues are labeled
and shown as sticks in blue. Residue S400 ligates the iron center; mutations at positions 78,
82, 263, and 267 enhance C–H amination activity and/or selectivity. All beneficial mutations
identified in this study lie in the P411 active site on the distal face of the heme.
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