Enantioselective, intermolecular benzylic C-H amination catalysed by an engineered iron-haem enzyme

Article abstract of DOI:10.1038/nchem.2783

C-H bonds are ubiquitous structural units of organic molecules. Although 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-haem cofactor.

Full text of DOI:10.1038/nchem.2783

ARTICLES
PUBLISHED ONLINE: 29 MAY 2017 | DOI: 10.1038/NCHEM.2783
Enantioselective, intermolecular benzylic C–H
amination catalysed by an engineered
iron-haem enzyme
Christopher K. Prier†, Ruijie K. Zhang†, Andrew R. Buller, Sabine Brinkmann-Chen
and Frances H. Arnold
*
C–H bonds are ubiquitous structural units of organic molecules. Although 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-haem
cofactor.
–H bonds may be the most common functional groups in rhodium15–17, ruthenium18,19 and manganese19,20 catalysts. Highly
organic molecules but are typically considered inert toward diastereoselective C–H amination using a chiral nitrogen source
C
chemical transformation. By selectively acting on these has also been achieved using a rhodium catalyst21. These systems
bonds and installing new functional groups directly into the hydro- represent major advances in C–H functionalization; none,
carbon framework of organic compounds, C–H functionalization however, offer the combination of high catalytic efficiency (turn-
technology has the potential to streamline synthetic routes, over), high enantioselectivity across diverse substrates, and ready
leading to less wasteful and more sustainable chemical pro- access to the chiral catalyst.
duction1–3. Modern C–H functionalization methods, however, rely
Cytochrome P450 monooxygenases are nature’s catalysts for the
heavily on catalytic complexes of precious transition metals (such direct insertion of oxygen into C–H bonds. These iron-dependent
as rhodium, ruthenium and iridium), which are neither cheap nor enzymes perform hydroxylation with high reaction rates and turn-
green1. Many enzymes, by contrast, catalyse very challenging reac- overs, and do so under mild physiological conditions22. They also
tions using base metals; their activities are often only possible act with excellent selectivity in complex settings, often hydroxylat-
because the protein plays a key role, modifying or enhancing the ing only one C–H bond out of many in diverse secondary metab-
inherent reactivity of the metal cofactor4. We envisioned that olites23. In contrast to this strategy for oxygenation, nature does
enzymes dependent only on base metals could be exploited not introduce nitrogen into organic molecules via C–H functionali-
through reaction design and evolution to perform non-biological zation. Biosynthetic routes to amines instead rely exclusively on
C–H functionalization reactions, potentially with efficiencies and functional group manipulation of pre-oxidized substrates, and bio-
selectivities exceeding those of known chemical catalysts.
Due to the presence of nitrogen in many bioactive synthetic C–H amination using this general approach24,25
and natural compounds, methods for directly transforming In striking contrast to both natural and synthetic amination strat-
catalytic multi-enzyme cascades have been engineered for formal
.
sp3-hybridized C–H bonds into C–N bonds have been pursued egies, it has been shown that haem proteins can catalyse nitrene
intensively5–9. Many of the methods that have been developed are transfer under appropriate reaction conditions26–32. In particular,
either intramolecular (requiring that a nitrogen source already be variants of cytochrome P450BM3 from Bacillus megaterium that
present in the same molecule as the targeted C–H bond) or utilize feature a serine axial ligand to the haem iron in place of the wild-
directing groups (also requiring the presence of specific functional- type cysteine ligand, which we term cytochrome P411s (ref. 33),
ity in the starting material). An elegant yet more elusive transform- are proficient catalysts for nitrene transfer. Based on this manifold,
ation is the intermolecular C–H amination of unfunctionalized we envisioned the catalytic cycle for intermolecular amination
hydrocarbons; such a reaction provides a dramatically simplifying shown in Fig. 2. First, reduction of the ferric state of the haem cofac-
and convergent disconnection for the synthesis of amines from tor, with electrons derived from NADPH, gives the ferrous state 1.
alkanes (Fig. 1). Recognizing the potential of this transformation, Reaction with a nitrene source, here tosyl azide (TsN3), then
several groups have pursued innovative strategies for intermolecular provides the putative iron nitrenoid 2. Subsequent reaction of this
C–H amination. While many of these methods are racemic10–14
,
intermediate with an alkane such as 4-ethylanisole (3) would
enantioselective intermolecular C–H amination via the generation deliver the C–H amination product 4 and regenerate the ferrous
and transfer of metal nitrenoids has been developed using state of the catalyst (1). A competing process observed in P411-catalysed
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, MC 210-41, Pasadena, California 91125,
USA. †These authors contributed equally to this work. e-mail: frances@cheme.caltech.edu
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2783
ARTICLES
Catalyst
(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% e.e.). A
final mutation, E267D, is neutral with respect to the amination of
4-ethylanisole but provides a twofold 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 P411CHA, for
‘cytochrome P411 C–H aminase’. Under the conditions employed
for evolution, this catalyst generates the benzylic amine 4 in 66%
yield, with >99% e.e. and 1,000 turnovers in whole Escherichia
coli cells. While 4-ethylanisole is electronically activated toward
C–H insertion15, 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 P411CHA, the amination
of 4-ethylanisole was improved by sixfold, 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 amin-
H
H
H
H
N
R3
Nitrogen source
R1
R2
R1
R2
Simple alkanes,
or C–H bonds in
complex molecules
Valuable chiral amines
• Challenging transformation with no highly efficient,
highly enantioselective, Earth-abundant catalysts
• Not a natural function of any known enzyme
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.
nitrene transfer is the reduction of the nitrenoid, generating the ation activity, from which variants that aminate inherently
undesired by-product p-toluenesulfonamide (TsNH2, 5). less-reactive alkanes were derived.
Cytochrome P411 catalysts have been identified for intramolecular
Subsequent studies revealed that P411CHA can support up to 1,300
C–H amination reactions26,27; in these cases, the targeted C–H turnovers (Table 1, entry 3), far exceeding the highest turnover
bond is necessarily present in the enzyme active site when the number (TON) reported with any chiral transition metal complex
reactive nitrenoid 2 is formed, biasing productive C–H amination for intermolecular, enantioselective C–H amination (the best reported
over the deleterious nitrenoid reduction pathway. In contrast, a is 85 TON using a chiral manganese porphyrin)19. The reaction was
catalyst for the desired intermolecular amination must bind a optimized for yield by employing two equivalents of tosyl azide;
separate alkane substrate and promote its amination faster than under these conditions, the product 4 is obtained in 86% yield,
the transient nitrenoid species is consumed in the reductive with >99% e.e. and 670 turnovers (Table 1, entry 8). In contrast to
side reaction.
the reported chemical protocols, the reaction with P411CHA takes
place in water at room temperature; the reaction can also proceed
under aerobic conditions, but the yield is sevenfold higher using the
optimal anaerobic conditions (Supplementary Table 7).
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 amination26,27 and
intermolecular aziridination28—instead they exclusively reduce the
azide to the sulfonamide 5 (Supplementary Table 1). Other haem-
containing proteins, as well as the haem cofactor alone (iron
protoporphyrin IX), are similarly inactive (Supplementary
Table 2). Remarkably, however, we found that P411BM3 variant
P-4, engineered for the imidation of allylic sulfides29, 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% e.e. by variant P-4,
which differs from wild-type P450BM3 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 fourfold improvement in yield (to 51%) and delivers
the product 4 in 77% e.e. 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.
Substrate scope studies. Scope studies, carried out using the yield-
optimized conditions, revealed that cytochrome P411CHA aminates
a diverse set of arene-containing hydrocarbons. Substitution of the
aromatic ring is tolerated at para, meta and ortho positions (Table 2,
N
N
Nitrene reduction
FeIII
N
unproductive
N
O
e–
Ser
H
TsNH2 (5)
Cytochrome P411
e–, 2H+
TsN3
– N2
Ts
N
N
N
N
N
FeIV
N
FeII
N
N
N
2
1
O
O
Ser
H
Ser
H
Iron nitrenoid
NHTs
Me
Nitrene transfer
productive
We next performed sequential rounds of site-saturation muta-
genesis of selected residues in P-4 A82L and screening to improve
C–H amination activity and enantioselectivity. We targeted sites
in the haem domain that were either previously shown to impact
the activity and/or selectivity of P450s, residues that are highly con-
Me
MeO
MeO
Benzylic amine (4)
Alkane (3)
served in P450s, or residues that were already mutated in P-4 relative Figure 2 | Proposed mechanism of cytochrome P411-catalysed
to wild-type P450BM3 (see Supplementary Information for details)34. intermolecular C–H amination. Reaction of the aminating reagent, tosyl
The libraries were screened for enhanced C–H amination activity, azide, with the ferrous porphyrin (1) generates an enzyme-bound iron
and potential hits were subsequently evaluated for activity across a nitrenoid intermediate (2). This nitrenoid then inserts into a C–H bond in the
set of three substrates with different electronic demands for C–H alkane, delivering a benzylic amine product. The nitrogen atoms in a plane
amination: 4-ethylanisole (3), 4-ethyltoluene (6), and ethylbenzene represent the enzyme’s haem cofactor. Ts, 4-toluenesulfonyl; Ser, serine.
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2783
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80
70
60
50
40
30
20
10
0
>99%
>99%
80%
NHTs
Me
TsN3
P411 variant
Me
Whole cells
R
R
77%
R = OMe, Me, H
Me
Me
Me
>99%
3
MeO
>99%
>99%
6
7
Me
>99%
98%
14%
>99%
92%
P-4
P-4
A82L
P-4
A82L
A78V
P-4
P411CHA
A82L
A78V
F263L
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-catalysed 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 OD600 = 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, and numbers above bars represent enantiomeric excess (e.e.); both are colour-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.
entries 2–4). While electron-withdrawing functionality reduces the suitable substrates for amination, but the products are isolated in
reactivity of the alkane toward the metal nitrenoid, several racemic form (entries 16–17). For the amination of isochroman
halogenated ethylbenzenes are nevertheless functionalized with (entry 17), analysis of crude reaction mixtures demonstrated that
>100 turnovers (entries 6–8). The cyclic alkanes indan and the C–H insertion event is moderately enantioselective (65% e.e.),
tetralin are excellent substrates (entries 9 and 10), while the but the product undergoes racemization during purification on
related 2,3-dihydrobenzofuran displays reduced reactivity (entry silica gel (Supplementary Table 10).
11). Larger alkylarenes such as ethylnaphthalenes and
Notably, most of the alkanes evaluated undergo functionalization
4-propylanisole are still accommodated in the enzyme active site, with excellent levels of enantioselectivity (>90% e.e.), a key advan-
undergoing amination with varying levels of efficiency (entries tage of this enzymatic approach over most reported metal catalysts.
12–14). Notably, the amination of 1-ethylnaphthalene delivers the However, P411CHA displays several limitations with regard to sub-
nitrogen-containing fragment of the calcimimetic drug cinacalcet strate scope: it does not aminate substrates much larger than
(Sensipar) with the correct absolute configuration (entry 13)35. those shown here, nor does it functionalize benzylic positions adja-
Furthermore, although the activity is low, the amination of the cent to especially electron-deficient arenes (Supplementary Fig. 1).
methyl group of 4-methylanisole demonstrates that these catalysts Interestingly, cumene and 4-methoxycumene are not aminated by
are capable of functionalizing C–H bonds with bond dissociation P411CHA despite the presence of weak benzylic C–H bonds. We
energies as high as 90 kcal mol–1 (entry 15). Benzylic ethers are have also not found P411CHA to be active toward the amination of
allylic or aliphatic C–H bonds, nor is it 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 P411CHA also has improved
thermostability relative to P-4 (T50 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 catalysts36.
Table 1 | Optimization of the P11CHA-catalysed amination of
4-ethylanisole.
Entry 4-Ethylanisole (3; mM) Tosyl azide (mM) Yield (4) TON
1*
2
5.0
7.5
10
5.0
7.5
10
66%
51%
1,000
1,200
1,300
1,200
1,200
430
630
670
640
610
3†
4
41%
26%
20%
56%
80%
86%
83%
78%
15
15
5
6
20
2.5
2.5
2.5
2.5
2.5
20
2.5
3.75
5.0
7.5
10
7
8‡
9
10
The enzymatic amination reaction can be performed on prepara-
tive scale: from a 0.25 mmol-scale biotransformation, the benzylic
amine 4 was isolated in 78% yield (59.5 mg, 610 TON, >99% e.e.).
The reaction also proceeds with purified protein, albeit less
Reactions were performed with whole E. coli cells overexpressing P411CHA, as in Fig. 3. All reactions
generate benzylic amine
4
in >99% e.e. *Conditions employed during evolution. †Turnover-
optimized conditions. ‡Yield-optimized conditions, employed for evaluating the substrate scope.
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a
b
Me
Table 2 | Substrate scope of enzymatic intermolecular C–H
amination.
TsN3
NHTs
Me
D/H
MeO
MeO
Cytochrome P411CHA
or
In vitro
MeO
D
D
TsN3
NHTs
R''
Cytochrome P411CHA
Whole cells
Me
kH/kD = 1.6
R''
8
R'
R'
V78
E267
Entry 1: R = p-OMe 86% yield, 670 TON, >99% e.e.
L263
Entry 2: R = p-Me
Entry 3: R = m-Me
Entry 4: R = o-Me
Entry 5: R = H
53% yield, 420 TON, >99% e.e.
58% yield, 460 TON, >99% e.e.
14% yield, 110 TON, 87% e.e.
15% yield, 120 TON, >99% e.e.
19% yield, 150 TON, >99% e.e.
19% yield, 150 TON, >99% e.e.
22% yield, 170 TON, >99% e.e.
NHTs
Me
L82
A87
R
Entry 6: R = p-Br
Entry 7: R = p-Cl
Entry 8: R = p-F
NHTs
NHTs
NHTs
V328
O
Entry 11: 21% yield†
Entry 9: 80% yield
Entry 10:76% yield
640 TON, 96% e.e.
660 TON, 95% e.e.
180 TON, 92% e.e.
S400
NHTs
NHTs
Me
NHTs
Me
Et
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 haem in white and the iron atom
in orange. Key active site residues are labelled and shown as sticks in blue.
Residue S400 ligates the iron centre; 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 haem.
MeO
Entry 14: 16% yield
Entry 12: 81% yield
Entry 13: 6% yield
710 TON, >99% e.e.
45 TON, >99% e.e.
140 TON, >99% e.e.
NHTs
O
NHTs
O
NHTs
MeO
Entry 16: 70% yield†
Entry 17: 80% yield†
Entry 15: 5% yield
47 TON
with deuterated alkane 8 show a kinetic isotope effect (kH/kD) of
1.6 in the reaction catalysed by P411CHA, 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.
610 TON
730 TON
Reactions were performed in duplicate with whole E. coli cells overexpressing P411CHA at OD600 = 30
(∼3 μM enzyme), with 2.5 mM alkane and 5 mM tosyl azide. †Isolated yield from a reaction
performed on 0.25-mmol scale (see Supplementary Information for details).
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
efficiently than in whole cells: the P411 catalysts are capable of up to
190 TON in vitro versus 1,300 TON in vivo (Supplementary
Table 8). Furthermore, the tosyl group present in the C–H amin-
ation products may be removed via treatment with samarium diio-
dide37, providing the corresponding primary amine with no erosion
of enantiomeric excess (see Supplementary Information for details).
impact selectivity in P450-catalysed oxygenation reactions38–40
.
Rate experiments. The fact that amination activity throughout Like two earlier reported P411 structures27,33, the structure of the
evolution is highly dependent on the electronic nature of the evolved aminase adopts the P450 closed state typically induced by
substrate (electron-rich substrates giving higher yields) suggests substrate binding. Compared to the structure of P411 variant
that C–H insertion is rate-determining. We questioned, however, P-I263F (ref. 27), which differs by only six mutations yet
whether improvements in yield arose from improved catalytic performs intermolecular C–H amination with trace activity (<1%
efficiency toward C–H amination or suppression of the competing yield), there are only minor movements of the protein scaffold,
azide reduction pathway (Fig. 2). Under in vitro conditions, initial although the active-site residue L437 surprisingly adopts an
rates are enhanced by greater than eightfold for the amination of unfavoured backbone conformation. On average, the volume of
4-ethylanisole by P411CHA compared to P-4, showing that the the haem distal pocket is reduced by approximately 10% in the
mutations indeed increase the rate of the productive reaction evolved variant compared to P-I263F; the smaller active site
(Supplementary Fig. 2). Independent rate measurements conducted potentially enforces productive substrate binding modes. These
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2783
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starting material; TON is calculated as mM reaction product divided by mM P411,
as determined by the CO-binding assay following cell lysis. e.e. is calculated as
(major enantiomer–minor enantiomer)/(major enantiomer + minor enantiomer).
observations suggest that the mutations introduced on the path to
P411CHA 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).
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 P411BM3
P-4 A82L A78V F263L has been deposited in the Protein Data Bank (PDB) under
accession code 5UCW.
Conclusion
Received 14 March 2017; accepted 21 April 2017;
published online 29 May 2017
Cytochrome P411CHA 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 References
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long-standing challenge in synthetic chemistry. The protein does
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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
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Methods
Expression of P411BM3 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–1 ampicillin (LBamp, 37 °C, 250 rpm). The preculture was used to
inoculate 45 ml of Hyperbroth (HB) medium (prepared from AthenaES powder,
0.1 mg ml–1ampicillin) 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 (2,600g, 10 min, 4 °C), and the
pellets were resuspended to an OD600 of 30 in M9-N minimal media (no nitrogen).
Aliquots of the cell suspension (4 ml) were used to determine the P411 expression
level by CO-binding assay after lysis by sonication.
19. Zhou, X.-G., Yu, X.-Q., Huang, J.-S. & Che, C.-M. Asymmetric amidation of
saturated C–H bonds catalysed by chiral ruthenium and manganese porphyrins.
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Intermolecular C–H amination in whole E. coli cells. For amination
20. Kohmura, Y. & Katsuki, T. Mn(salen)-catalyzed enantioselective C–H
amination. Tetrahedron Lett. 42, 3339–3342 (2001).
bioconversions, the cells containing the P411 variant, at OD600 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 min. Separately, a glucose solution (250 mM in M9-N) was
degassed by sparging with argon for at least 10 min. An oxygen depletion system
(20 µl of a stock solution containing 14,000 U ml–1 catalase and 1,000 U ml–1
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
21. Liang, C. et al. Efficient diastereoselective intermolecular rhodium-catalyzed
C–H amination. Angew. Chem. Int. Ed. 45, 4641–4644 (2006).
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perspectives for synthetic application. Trends Biotechnol. 30, 26–36 (2012).
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natural products. Nat. Prod. Rep. 29, 1251–1266 (2012).
24. Both, P. et al. Whole-cell biocatalysts for stereoselective C–H amination
reactions. Angew. Chem. Int. Ed. 55, 1511–1513 (2016).
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 20,000g for 10 min. The supernatant was transferred to a vial
and analysed by HPLC for yield. Reaction samples were extracted with cyclohexane
and analysed by chiral SFC (supercritical fluid chromatography) for enantiomeric
excess (e.e.). Yield is calculated as mM reaction product divided by mM alkane
25. Schrewe, M., Ladkau, N., Bühler, B. & Schmid, A. Direct terminal alkylamino-
functionalization via multistep biocatalysis in one recombinant whole-cell
catalyst. Adv. Synth. Catal. 355, 1693–1697 (2013).
26. McIntosh, J. A. et al. Enantioselective intramolecular C–H amination
catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew.
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27. Hyster, T. K., Farwell, C. C., Buller, A. R., McIntosh, J. A. & Arnold, F. H.
Enzyme-controlled nitrogen-atom transfer enables regiodivergent C–H
amination. J. Am. Chem. Soc. 136, 15505–15508 (2014).
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2783
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Acknowledgements
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 S. Virgil, J. Kaiser, and
R. D. Lewis for experimental assistance, and O. F. Brandenberg, S. C. Hammer, and S. B. J.
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Author contributions
C.K.P. and R.K.Z. designed, carried out, and analysed 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.
Additional information
Supplementary information and chemical compound information are available in the
online version of the paper. Reprints and permissions information is available online at
www.nature.com/reprints. Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Correspondence and
requests for materials should be addressed to F.H.A.
42. Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a
dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat.
Commun. 6, 7789 (2015).
Competing financial interests
The authors declare no competing financial interests.
6
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