An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp 3)–H bonds

Article abstract of DOI:10.1038/s41557-019-0343-5

The ability to selectively functionalize ubiquitous C–H bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mech

Full text of DOI:10.1038/s41557-019-0343-5

Articles
https://doi.org/10.1038/s41557-019-0343-5
An enzymatic platform for the asymmetric
amination of primary, secondary and tertiary
C(sp3)–H bonds
Yang Yangꢀ ꢀ1, Inha Cho1, Xiaotian Qiꢀ ꢀ2, Peng Liuꢀ ꢀ2* and Frances H. Arnoldꢀ ꢀ1*
The ability to selectively functionalize ubiquitous C–H bonds streamlines the construction of complex molecular architec-
tures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene
transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(sp3)–H bonds. These fully geneti-
cally encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad
spectrum of enantioselective C(sp3)–H amination reactions. These catalysts can aminate a variety of benzylic, allylic and
aliphatic C–H bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of
primary C(sp3)–H bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quater-
nary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess
a tertiary C(sp3)–H bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule
catalysts. Further engineering allowed for the enantioselective construction of methyl–ethyl stereocentres, which is notori-
ously challenging in asymmetric catalysis.
he development of general systems for the highly enantiose- venerable example of outstanding stereocontrol10,11. Over the past
lective transformation of C(sp3)–H bonds lies at the forefront six years, our laboratory12,13 and others14–17 have repurposed these
T
of current efforts to advance transition metal-catalysed C–H enzymes and other haem proteins to catalyse synthetically useful
functionalization1–5. In principle, several classes of enantioselective reactions that are not known to nature. In particular, we have engi-
C(sp3)–H functionalization can be developed, depending on the top- neered haem proteins for abiological C–H functionalization leading
icityanddegreeofsubstitution(primary,secondaryortertiary)ofthe to the formation of C–C and C–N bonds through a carbene18 or
sp3-hybridized carbon atom undergoing functionalization (Fig. 1). nitrene19–21 transfer mechanism. These results collectively showcase
As depicted in Fig. 1a, effective enantiodiscrimination of the two enzymes’ potential for performing abiological asymmetric C–H
prochiral secondary C(sp3)–H bonds at a methylene unit leads to functionalization reactions, using earth-abundant iron in a fully
the formation of a trisubstituted stereogenic centre1. On the other genetically encodable protein that can be tuned by evolution. The
hand, performing asymmetric primary C(sp3)–H functionalization capabilities of enzymes to solve key outstanding problems in asym-
by differentiating the two prochiral methyl substituents can serve as metric catalysis, however, have not yet been tested in these C–H
a powerful means to access challenging all-carbon quaternary ste- functionalization processes.
reocentres (Fig. 1b)1,6,7. As a distinct alternative, the enantioselective
Herein we describe the development of general cytochrome
functionalization of a tertiary C(sp3)–H bond will enable the con- P450-based biocatalysts for the asymmetric amination of primary,
version of readily available precursors into valuable products that secondary and tertiary C(sp3)–H bonds in the synthesis of chiral
feature a tetrasubstituted stereocentre (Fig. 1c). Due to the presence diamines, a key pharmacophore in numerous antiviral and anti-
of a pre-existing stereogenic centre at the site of attachment, this bacterial agents (Fig. 1d and Supplementary Fig. 1)22,23. Inspired by
process would require the enantioconvergent functionalization8 of the pioneering work of other groups in the area of transition metal-
a tertiary C(sp3)–H bond to convert both enantiomers of the race- catalysed intramolecular C(sp3)–H amination4,5,24–30, as well as our
mic substrate into the same major enantiomer, a daunting challenge own studies19–21, we envisioned a unified enzymatic strategy for
that so far remains out of the reach of small-molecule transition the catalytic asymmetric assembly of diverse 1,2- and 1,3-diamines
metal catalysts. In this context, identifying a set of structurally from abundant amine precursors. As outlined in Fig. 1d, using a
related catalysts as a unified platform for the asymmetric function- previously established one-step procedure23, the aliphatic amine (I)
alization of all three types of aliphatic C–H bonds will accelerate can be readily converted to the corresponding sulfamoyl azide (II)
further development and the application of C–H functionalization in excellent yield. Our proposed biocatalytic C(sp3)–H amination
technologies.
would lead to the formation of the enantioenriched cyclic sulfamide
Enabled by numerous potentially cooperative protein–sub- (III). Subsequent excision of the sulfonyl unit using known proce-
strate interactions in the elaborate chiral scaffold of the active site, dures23 would then furnish the desired chiral diamine (IV, see the
enzymes can exert control over the stereochemical outcome of vari- Supplementary Information for details on converting sulfamides to
ous catalytic reactions, including C–H functionalization9. Among diamines). In addition to its synthetic utility, we postulated that this
naturally occurring enzymatic C–H functionalization processes, chiral diamine synthesis could serve as an ideal platform to iden-
cytochrome P450-catalysed C(sp3)–H hydroxylation represents a tify and evolve haem proteins for the asymmetric amination of all
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA. 2Department of Chemistry, University of
Pittsburgh, Pittsburgh, PA, USA. *e-mail: pengliu@pitt.edu; frances@cheme.caltech.edu
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a
d
H
H
H
FG
Z = CH2 or (CH2)2
H2N
HN
Z
H
N
Z
I
Secondary C(sp3)–H bond
Trisubstituted stereocentre
IV
Chiral diamine
Prochiral
Aliphatic amine
b
Me Me
FG
Me
Primary C(sp3)–H bond
All-carbon
quaternary stereocentre
Prochiral
c
O
O
FG
H
Biocatalytic
S
SO2N3
HN
N
Enantioselective
C(sp3)–H amination
N
Z
Z
II
Tertiary C(sp3)–H bond
Tetrasubstituted stereocentre
III
Racemic
Fig. 1 | the three major types of asymmetric C(sp3)–H functionalization and our envisioned biocatalytic C(sp3)–H amination. a, Enantioselective
functionalization of secondary C(sp3)–H bonds. b, Enantioselective functionalization of primary C(sp3)–H bonds (that is, the desymmetrization of geminal
dimethyl substituents). c, Enantioconvergent functionalization of tertiary C(sp3)–H bonds. d, Enzymatic synthesis of chiral diamines using C(sp3)–H
amination. Red, yellow and blue spheres are generic substituents of the molecule. FG, functional group.
three types of C(sp3)–H bonds (Fig. 1a–c), especially those that have the excellent catalytic efficiency of the engineered P411 enzyme
not succumbed to small molecule-catalysed asymmetric C–H func- relative to previously developed transition metal catalysts.
tionalization.
Using E. coli whole cells that harbour P411Diane2, we surveyed the
substrate scope of this C(sp3)–H amination process for 1,2-diamine
synthesis (Fig. 2b). Electron-donating and -withdrawing sub-
results and discussion
We commenced our study by evaluating a panel of haem proteins— stituents on the aromatic ring were compatible with this process
including variants of cytochromes P450, cytochromes P411 (P450 (2a–2g), affording 1,2-diamines with uniformly high levels of
but with the iron-coordinating cysteine residue replaced by a ser- enantioselectivity. Furthermore, substrates bearing a halogen func-
ine), cytochromes c and globins—in intact Escherichia coli cells tional group handle for further derivatization were accepted by the
for enantioselective diamine synthesis (Fig. 2a). We focused our enzyme (2e and 2f). Steric hindrance at the ortho position was also
initial investigation on the asymmetric synthesis of 1,2-diamine compatible (2h), although lower activity was observed. Structural
derivatives due to the lack of highly enantioselective C–H amina- perturbations such as replacement of the aryl ring by thiophene
tion methods for synthesizing these compounds31. Among the (2i) and variance of the N-substituent (2j) were well tolerated by
haem proteins we tested, a few variants from the cytochrome P450 this biocatalytic C(sp3)–H amination. Moreover, we found that the
superfamily showed low levels of C–H amination activity (see the starting variant P411Diane1 could be employed for the asymmetric
Supplementary Information for details). In particular, a truncated synthesis of 1,3-diamine derivatives using C(sp3)–H amination. In
P411 variant lacking the FAD domain (P411 diamine synthase, also addition to the asymmetric amination of benzylic C(sp3)–H bonds
known as P411Diane1) that we developed during an earlier study on (2k–2n), allylic C(sp3)–H bonds (2o) were also effectively aminated
iron-catalysed carbene insertion into C(sp3)–H bonds18 was over ten with excellent enantioselectivity. In contrast to Rh(OAc)2-based
times more active than other haem proteins, providing a total turn- systems24, competing aziridination product was not observed, high-
over number (TTN) of 450 and an enantiomeric excess (e.e.) of 94% lighting the chemoselectivity of these iron-based biocatalysts.
for the 1,2-diamine product (2a). This demonstrates that the reduc-
Directed evolution of P411Diane1 furnished a complementary set
tase domain of cytochrome P450 is not needed for C–H amination. of enzymatic catalysts that allow for the enantiodivergent amina-
Finally, the absolute stereochemistry of 2a was ascertained by single tion of unactivated secondary aliphatic C(sp3)–H bonds (Fig. 3).
crystal X-ray diffraction analysis.
Amino acid residue 87, which is located in a loop in the active site
We used P411Diane1 as the starting template for directed evolution and known for its importance in substrate recognition in P450-
of 1,2-diamine synthase (Fig. 2a). In an effort to further improve catalysed oxidation and carbene transfer reactions32,33, was found
the enzyme’s activity and enantioselectivity for this C–H amination to play a dominant role in determining the sense of absolute ste-
process, we performed iterative rounds of site-saturation mutagen- reochemistry of the C–H amination product. Specifically, a single
esis (SSM) and screening, targeting amino acid residues close to the A87I mutation inverted the absolute configuration of the newly
haem cofactor. For each round of engineering, enzyme libraries were formed stereocentre. With this initial result, further rounds of SSM
expressed and screened in 96-well plates in the form of whole E. coli and screening led to P411Diane1 L82M A87I Y263W I327S, delivering
cells. Beneficial mutations I327P, Y263W and Q437F were intro- the C(sp3)–H amination product in 96% e.e. (as the R enantiomer).
duced in three rounds, furnishing a tenfold improvement in activ- On the other hand, when leaving A87 unchanged, a single mutation
ity as well as further enhancements in enantioselectivity; undesired I327T resulted in a considerable enhancement in enantioselectiv-
nitrenoid reduction was also effectively suppressed. Using final- ity (32% e.e. to 71% e.e.). This is a rare example of enantiodiver-
variant P411Diane2, the C(sp3)–H amination product formed in 3,490 gent C(sp3)–H amination reactions that are catalysed by engineered
TTN and 99.9% e.e., as determined by chiral gas chromatography. haem proteins.
Moreover, by further lowering the cell density in whole-cell biotrans-
Having engineered a set of enzymes for the asymmetric ami-
formations, this enzymatic C–H amination provided the diamine nation of secondary C(sp3)–H bonds, we questioned whether this
product in 72,000 TTN and 99.9% e.e., thereby demonstrating enantioselective amination could be extended to the conversion
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a
O
O
O
O
E. coli cells
N3
H2N
S
Me
S
N
O
S
N
O
that harbour P411Diane
HN
N
+
M9-N buffer (pH = 7.4)
r.t., 12 h
Ph
Me
Ph
Me
Ph
Trisubstituted stereocentre
1a
2a
3a
Secondary C(sp3)–H bond
P411
TTN of 2a
e.e. of 2a
2a:3a
L437
450
1,140
94%
99%
31:69
57:43
98:2
P411Diane1
L263
P411Diane1 I327P
2,540
99.4%
99.9%
(99.9%)a
P411Diane1 I327P Y263W
P411Diane1 I327P
3,490
(72,000)a
98:2
T327
Y263W Q437F
(P411Diane2
)
b
O
O
O
O
N
O
O
N
O
O
N
S
S
S
S
Me
N
Me
Me
Me
HN
HN
HN
HN
F3C
MeO
F
Cl
2b
2c
2d
2e
X-ray structure of 2a
3,920 TTN
98% e.e.
1,900 TTN
98% e.e.
4,190 TTN
97% e.e.
3,120 TTN
97% e.e.
O
O
N
O
O
N
O
O
N
O
O
N
O
O
N
S
Me
S
S
S
HN
S
Me
Me
Et
Me
HN
HN
HN
HN
Me
S
Me
Br
2g
2j
2f
2h
2i
3,120 TTN
94% e.e.
4,640 TTN
91% e.e.
1,540 TTN
96% e.e.
1,030 TTN
97% e.e.
4,280 TTN
99% e.e.
O
O
O
O
O
O
O
O
S
Me
S
i-Pr
S
Me
S
Me
O
O
HN
N
HN
N
HN
N
HN
N
S
Me
HN
N
MeO
Cl
2o
2k
2l
2m
2,740 TTN
91% e.e.
2n
5,140 TTN (62,000 TTN)a
99% e.e. (99% e.e.)a
3,370 TTN
92% e.e.
4,000 TTN
98% e.e.
1,540 TTN
70% e.e.
Fig. 2 | enantioselective amination of secondary C(sp3)–H bonds. a, The directed evolution of P411ΔFAD for the enantioselective synthesis of
1,2-diamines. The crystal structure of a variant closely related to P411Diane1 is shown (PDB ID: 5UCW). b, The substrate scope of 1,2-diamine and
1,3-diamine synthesis. The experiments were performed using E. coli that expresses cytochrome P411Diane (OD600ꢀ=ꢀ30) with 10ꢀmM substrate at room
temperature (r.t.) under anaerobic conditions for 12–24ꢀh. See the Supplementeary Information for details. aPerformed using E. coli expressing cytochrome
P411Diane2 or P411Diane1 (OD600ꢀ=ꢀ1.9) with 20ꢀmM substrate at room temperature under anaerobic conditions for 24ꢀh.
of unactivated primary aliphatic C(sp3)–H bonds, which is a ther-
O
O
modynamically more challenging process26. Examination of our
P411Diane collection revealed that the P411Diane1 I327P variant already
displayed excellent activity and enantioselectivity for the aminative
desymmetrization of geminal dimethyl substituents, providing the
desired 1,3-diamine that possesses an all-carbon quaternary stereo-
centre at the β position in 99% e.e. (Fig. 4a). In the realm of asym-
metric catalysis, the desymmetrization of geminal dimethyl groups
has been recognized as a promising solution to bypass the long-
standing problem of enantioselective methylation34. In this context,
our biocatalytic desymmetrization represents a valuable example of
C–N bond formation for the construction of methyl-substituted ste-
reocentres. Subsequent examination of the substrate scope revealed
that this desymmetrization is also applicable to other substrates that
bear various aryl groups (4a–4d). The absolute configuration of 4a
was determined by X-ray diffraction analysis.
S
Me
HN
N
P411Diane1
A87I I327S
Y263W L82M
Me
O
O
2p (R)
1,370 TTN
96% e.e.
S
Me
N3
N
Me
O
O
1b
S
Me
Unactivated secondary
C(sp3)–H bond
P411Diane1 I327T
HN
N
Me
2p (S)
1,100 TTN
71% e.e.
Evolutionary trajectory
A87I (R)
44% e.e.
I327S (R)
80% e.e.
Y263W (R)
92% e.e.
L82M (R)
96% e.e.
Although conceptually similar enantioconvergent transfor-
mations of tertiary alkyl halides have recently received consid-
erable attention35–37, enantioconvergent protocols to transform
both antipodes of a racemic C–H substrate into the same major
enantiomer of the product remain elusive. Previous studies indi-
cate that iron nitrene-mediated C–H amination may involve a
radical mechanism38,39, thus suggesting the possibility of achieving
A87 (S)
32% e.e.
I327T (S)
71% e.e.
Fig. 3 | Amination of unactivated secondary C(sp3)–H bonds. The
enantiodivergent amination of unactivated secondary C(sp3)–H bonds to
access either the (R)- or the (S)-product using engineered P411Diane variants.
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a
O
O
O
O
S
Me
S
Me
P411Diane1 I327P
N3
N
HN
N
Me
r.t., 12 h
Desymmetrization
Me
R
Me
Quaternary stereocentre
R
Primary C(sp3)–H bond
O
O
O
O
O
O
O
O
S
Me
S
Me
S
Me
HN
N
HN
N
HN
N
S
Me
HN
N
Me
Me
Me
Me
CF3
Me
OMe
4a
4b
4c
800 TTN
81% e.e.
4d
X-ray structure
2,200 TTN
99% e.e.
2,900 TTN
91% e.e.
2,500 TTN
99% e.e.
of 4a
b
O
O
O
O
S
Me
N3
N
S
Me
P411Diane1 L78A A87G I327V Q437G
HN
N
R1
R2
r.t., 12 h
R1
Racemic
Tertiary C(sp3)–H bond
R2
Enantioconvergent transformation
Tetrasubstituted stereocentre
O
O
O
O
O
O
S
Me
N
S
Me
S
Me
HN
HN
N
HN
N
Me
Me
5c
Me
5a
MeO
F
5b
1,910 TTN
76% yield
99% e.e.
770 TTN
30% yield
98% e.e.
1,790 TTN
70% yield
98% e.e.
X-ray structure
of 5a
O
O
O
O
O
O
S
Me
S
Me
S
Me
OMe HN
N
HN
N
HN
N
S
Me
Me
Et
5d
5e
5f
1,810 TTN
72% yield
99% e.e.
1,410 TTN
56% yield
81% e.e.
1,880 TTN
75% yield
99% e.e.
X-ray structure
of 5f
c
O
O
O
O
S
Me
S
Me
N3
N
HN
N
P411Diane1 L82C L181V I327T A330M Q437G
r.t., 12 h
Et
Me
Me
Et
‘Methyl–ethyl’ stereocentre through
enantioconvergent transformation
1c
5g
Racemic
Unactivated tertiary
C(sp3)–H bond
2,120 TTN
82% yield
87% e.e.
Fig. 4 | engineered P411Diane variants for the enantioselective amination of primary and tertiary C(sp3)–H bonds. a, Asymmetric amination of primary
C(sp3)–H bonds (that is, desymmetrization of geminal dimethyl substituents). b, Enantioconvergent amination of tertiary C(sp3)–H bonds. c, The
enantioconvergent construction of a methyl–ethyl stereocentre using tertiary C(sp3)–H amination. Experiments were performed using E. coli that expresses
cytochrome P411Diane1 I327P, P411Diane1 L78A A87G I327V Q437G and/or P411Diane1 L82C L181V I327T A330M Q437G (OD600ꢀ=ꢀ30–40) with 10ꢀmM
substrate at room temperature under anaerobic conditions for 12–24ꢀh.
stereochemical convergence in the enzymatic tertiary C(sp3)–H beneficial mutations L78A, A87G, Q437G and I327V and pro-
functionalization. Nonetheless, such enzymatic enantioconver- vides the diamine product in 76% yield, 1,910 TTN and 99% e.e.
gent transformations would require the same haem protein to (Fig. 4b). More importantly, P411Diane3 was found to be effective
accommodate both antipodes of the racemic substrate, a pro- in the enantioconvergent amination of other substrates (5a–5f).
cess rarely found in nature’s biochemical repertoire. We found Electron-donating (5b), electron-withdrawing (5c) and ortho-
that P411Diane1 could effect the enantioconvergent C(sp3)–H substituents (5d) were effectively tolerated under these condi-
amination to provide diamine product 5a, which features a tet- tions. Substrates that bear heterocycles (5e) and other branching
rasubstituted stereocentre (99% e.e.). Iterative SSM and screen- alkyl groups (5f) could also be transformed with excellent enan-
ing generated an improved variant, P411Diane3, which bears the tioselectivity.
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a
hydrogen atom transfer results in the formation of a carbon-cen-
tred radical, which then undergoes the final radical rebound step
with high levels of enantioselectivity. This mechanistic proposal
is further corroborated by our density functional theory calcula-
tions (Fig. 6). Computational studies on an iron porphyrin model
system showed that the triplet state of the key iron nitrenoid inter-
mediate 7 is 3.0kcalmol–1 more stable than the open-shell singlet
state and 11.6kcalmol–1 more stable than the quintet state (see the
Supplementary Information for comparisons between open-shell
singlet, triplet and quintet free energy profiles). The calculated
Mulliken spin densities of 7 revealed a value of 0.84 on the nitrogen
atom and 1.16 on the iron atom, demonstrating substantial radical
characters on both the nitrogen and iron centres. Furthermore, our
computations suggest that the hydrogen atom transfer is irrevers-
ible and occurs through triplet transition state TS1 with an energy
barrier of 15.7kcalmol–1; the subsequent radical rebound step that
proceeds through quintet transition state TS2 requires an activa-
tion energy of 18.4kcalmol–1, suggesting sufficient lifetime of the
carbon-centred radical to allow for stereoablation and the subse-
quent enantioselective C–N bond formation observed in the enzy-
matic reaction.
In conclusion, we have developed a biocatalytic platform for the
asymmetric amination of a variety of C(sp3)–H bonds, permitting
a diverse range of synthetically useful chiral diamines to be pre-
pared with excellent enantioselectivity. These biocatalysts are fully
genetically encoded and thus can be easily tuned and reconfigured
through DNA manipulation. Empowered by directed evolution, this
genetically encoded platform allowed for the rapid development of
highly active biocatalysts for the enantioselective amination of pri-
mary, secondary and tertiary aliphatic C–H bonds. Notably, some of
these processes—such as the enantioconvergent tertiary C(sp3)–H
amination—have not been successfully implemented with small-
molecule catalysts. We anticipate that this biocatalytic platform can
be further leveraged to tackle other challenges in enantioselective
C–H functionalization and asymmetric catalysis in general.
O
O
O
O
O
O
S
Me
S
Me
+
S
Me
P411Diane
r.t., 12 h
N3
N
HN
N
Me HN
N
Me
Me
6a
6b
2,700 TTN (86% yield)
E/Z = 97:3
80 TTN (2.6% yield)
E/Z = 97:3
(E )-1d
80% e.e.
89% e.e.
O
O
O
S
O
O
O
S
Me
+
Me
S
Me
P411Diane
r.t., 12 h
HN
N
Me N3
N
Me HN
N
Me
6a
6b
540 TTN (17% yield)
E/Z = 30:70
1,300 TTN (40% yield)
E/Z = 30:70
(Z )-1d
76% e.e.
82% e.e.
b
O
O
O
O
S
Me
N3
N
S
Me
HN
N
Ph
Me
Ph
Me
Racemic
Enantioenriched
[Fe]II
Biocatalytic
[Fe]II
Enantioconvergent
C(sp3)–H amination
O
O
O
O
S
Me
S
Me
[Fe]III
[Fe]III
N
N
N
N
H
Ph
Me
Ph
Me
Carbon-centred
radical
Nitrogen-centred
radical
Fig. 5 | mechanistic insight. a, Scrambling of olefin stereochemistry during
the allylic C(sp3)–H amination of (E)- and (Z)-1d using P411 biocatalyst
(P411Diane1 I327P). b, The proposed mechanism for the enantioconvergent
amination of tertiary C(sp3)–H bonds.
The effective discrimination between two minimally differenti- methods
Expression of P411 variants. E. coli (E. cloni BL21(DE3)) cells carrying plasmid
ated methyl and ethyl groups to construct methyl–ethyl stereocen-
encoding the appropriate P411 variant were grown overnight in 4ml of lysogeny
broth with ampicillin. A preculture (3ml) was used to inoculate 27ml of hyper
broth with ampicillin in a 125ml Erlenmeyer fask. Tis culture was incubated at
37°C and 230r.p.m. for 2.5h. Te culture was then cooled on ice for 20min and
induced with 0.5mM isopropyl-β-d-thiogalactoside and 1.0mM 5-aminolevulinic
acid (fnal concentrations). Te expression was conducted at 20°C and 130r.p.m.
for 16–18h. E. coli cells were then pelleted by centrifugation (4,500g, 3min,
4°C). Te media was removed and the resulting cell pellet was resuspended in
M9-N bufer to OD600 =30–40. An aliquot of this cell suspension (2ml) was
taken to determine P411 concentration using the hemochrome assay afer lysis
by sonication. When applicable, the remaining cell suspension was further
diluted with M9-N bufer to the OD600 used for the biotransformation and the
concentration of P411 protein in the biotransformation was calculated accordingly.
tres is a notoriously difficult problem in asymmetric catalysis40,41
.
We envisioned that engineered haem proteins could provide a pow-
erful platform to address this challenge (Fig. 4c). Indeed, directed
evolution of P411Diane1 led to P411Diane4 bearing five additional muta-
tions (L82C, L181V, I327T, A330M and Q437G), culminating in
the enantioconvergent formation of a methyl–ethyl stereocentre
(5g) in 82% yield, 2,120 TTN and 87% e.e. This result represents a
rare example of solving the methyl–ethyl problem using a C(sp3)–H
functionalization strategy.
Mechanistic and computational investigations provided further
insight into this enantioconvergent C(sp3)–H amination process.
First, we prepared sulfamoyl azide substrates that bear a stereochem-
ically well-defined olefin moiety ((Z)-1d and (E)-1d) and subjected
them to enzymatic reactions (Fig. 5a). Partial scrambling of the ole-
fin geometry was observed in both the (E)- and (Z)-substrates, with
the (Z)-substrate providing a substantial amount of the scrambled
product that bears a thermodynamically more stable (E)-olefin.
The erosion of C=C stereochemistry is consistent with the forma-
tion of a carbon-centred radical at the allylic position and does not
agree with a concerted C–H insertion mechanism. Consistent with
literature reports on related iron-based catalyst systems38,39,42, these
findings support a radical mechanism for this cytochrome P450-
catalysed C(sp3)–H amination process.
C–H amination reactions using whole E. coli cells that harbour P411. The
suspensions of E. coli cells that express the appropriate haem protein variant in
M9-N buffer (typically OD600 =30) were kept on ice. In another centrifuge tube,
a solution of d-glucose (250mM in M9-N) was prepared. All solutions were then
transferred into an anaerobic chamber for reaction setup. A GOx oxygen depletion
solution (20μl of stock solution) containing 14,000Uml–1 catalase and 1,000Uml–1
glucose oxidase in M9-N buffer), d-glucose (40μl of 250mM stock solution in
M9-N buffer), the suspension of E. coli expressing P411 (320μl) and the sulfamoyl
azide substrate (20μl, typically 400mM stock solution in EtOH) were added in
succession to a 2ml vial. The vials were sealed and shaken at room temperature
and 500r.p.m. for 12–20h and then analysed by gas chromatography–mass
spectrometry or liquid chromatography–mass spectrometry.
Data availability
All data necessary to support the paper’s conclusions are available in the main text
and the Supplementary Information. Solid-state structures of 2a, 4a, 5a and 5f are
available free of charge from the Cambridge Crystallographic Data Centre under
reference nos CCDC 1905551, 1905553, 1905552 and 1905554. Plasmids encoding
the enzymes reported in this study are available for research purposes from F.H.A.
We thus postulate that this enantioconvergent amination com-
prises a stereoablative hydrogen atom transfer and an enantioselec-
tive C–N bond formation. As described in Fig. 5b, reaction of the
ferrous haem cofactor with the racemic azide substrate leads to an
open-shell iron nitrenoid intermediate. Subsequent stereoablative under a material transfer agreement with the California Institute of Technology.
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∆G
Me
O
(∆H)
kcal mol–1
Ph
Me
H
S
N
167.0°
N
1.33
1.33
O
H
2.40
2.76
Me
[Fe]
N
O
[Fe]
N
2.03
TS1
(triplet)
S
Me
Ph
O
15.7
(9.1)
TS2
(quintet)
7
TS2
TS1
(triplet)
O
O
6.3
(4.5)
S
Me
0.0
(0.0)
HN
N
8
(triplet)
Ph
Me
Me
Me
5a
–12.1
Me
N
N
N
(–13.2)
Me
O
Ph
N
Ph
N
[Fe]
Fe
[FeII]
O
S
S
N
N
N 0.84
9
HN
O
O
(quintet)
[FeIII
]
Fe 1.16
[FeIII
]
OMe
–38.4
(–27.1)
7
8
Fig. 6 | Free energy profile of the iron porphyrin-catalysed C(sp3)–H amination. Density functional theory calculations were performed at the B3LYP-
D3(BJ)/6-311+G(d,p)–LANL2TZ(f)/SMD(chlorobenzene)//B3LYP-D3(BJ)/6-31+G(d)–LANL2DZ level of theory. The Mulliken spin densities of iron and
nitrogen for the key iron nitrenoid 7 are shown in blue.
16. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering
Received: 12 April 2019; Accepted: 15 August 2019;
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Author contributions
Y.Y. designed the overall research with F.H.A. providing guidance. Y.Y. and I.C.
designed and performed the initial screening of haem proteins and directed evolution
experiments. Y.Y. designed and performed the substrate scope study and mechanistic
study. X.Q. carried out the computational studies with P.L. providing guidance. Y.Y. and
F.H.A. wrote the manuscript with the input of all other authors.
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Competing interests
A provisional patent application (inventors Y.Y. and I.C.) has been filed through the
California Institute of Technology. The provisional patent covers the development
and application of engineered cytochromes P450 for the synthesis of chiral diamine
derivatives by C–H amination.
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Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-019-0343-5.
Correspondence and requests for materials should be addressed to P.L. or F.H.A.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
Acknowledgements
published maps and institutional affiliations.
This work was supported by the NSF (grant nos MCB-1513007 for F.H.A. and CHE-
1654122 for P.L.). Y.Y. thanks the National Institutes of Health for a postdoctoral
© The Author(s), under exclusive licence to Springer Nature Limited 2019
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