Alternate Heme Ligation Steers Activity and Selectivity in Engineered Cytochrome P450-Catalyzed Carbene-Transfer Reactions

Article abstract of DOI:10.1021/jacs.8b09613

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out carbene-transfer reactions using a lactone-based carbene precursor. By simply altering the heme-ligating residue, we obtained two enzymes that catalyze olefin cyclopropan

Full text of DOI:10.1021/jacs.8b09613

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Communication
Alternate heme ligation steers activity and selectivity in
engineered cytochrome P450-catalyzed carbene transfer reactions
Kai Chen, Shuo-Qing Zhang, Oliver F. Brandenberg, Xin Hong, and Frances H. Arnold
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09613 • Publication Date (Web): 29 Oct 2018
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Journal of the American Chemical Society
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Alternate heme ligation steers activity and selectivity in engineered
cytochrome P450-catalyzed carbene transfer reactions
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Kai Chen,†a Shuo-Qing Zhang,†b Oliver F. Brandenberg,a Xin Hong,*b Frances H. Arnold*a
a Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
b Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China.
Supporting Information Placeholder
and stabilizing lactone-carbene intermediates, circumventing
unwanted β-hydride migration, and facilitating subsequent
carbene-transfer reactions with exquisite stereocontrol. This
proposed enzymatic approach would provide a concise route to a
broad array of lactone-containing products.
ABSTRACT: We report a biocatalytic platform of engineered
cytochrome P450 enzymes to carry out carbene-transfer reactions
using a lactone-based carbene precursor. By simply altering the
heme-ligating residue, we obtained two enzymes that catalyze
olefin cyclopropanation (Ser) or S–H bond insertion (Cys). Both
enzymes exhibit high catalytic efficiency and stereoselectivity, thus
enabling facile access to structurally diverse spiro[2.4]lactones and
α-thio-γ-lactones. Computational studies revealed the mechanism
of carbene S–H insertion and explain how the axial ligand controls
reactivity and selectivity. This work expands the catalytic
repertoire of hemeproteins and offers insights into how these
enzymes can be tuned for new chemistry.
We initially focused on constructing cyclopropane-containing
spiro-lactones using α-diazo-γ-lactone (LAD) and styrene (1a) as
substrates. Transfer of a cyclic carbene to an olefin double bond is
expected to form a spiro-carbon center. Quaternary stereogenic
centers frequently occur in biologically relevant molecules and
medicinal compounds,14 but are challenging to make due to the
highly congested nature of these centers.14,
Moreover, the
15
anticipated reaction produces a strained, three-membered ring and
furnishes two chiral centers in a stereoselective manner, which
necessitates precise control of the three-dimensional orientation of
two coupling partners. We believe this is a good challenge for an
enzyme catalyst.
Cytochromes P450, a class of heme-dependent proteins, natively
function as oxygenases and transfer oxygen to hydrocarbons or
heteroatoms.1, 2 Our group and others have repurposed P450s to
perform non-natural carbene- and nitrene-transfer reactions.3‒5 In
the presence of diazo reagents as carbene precursors,
iron‒carbenoid intermediates can form in the enzyme active site
and enable carbene addition to unsaturated carbon‒carbon bonds6
or insertion into heteroatom‒hydrogen bonds.7 Until now, these
catalysts have accepted a narrow set of diazo reagents (e.g., ethyl
2-diazoacetate),6 which limits the structural diversity of the
products. In addition, mechanistic profiles of carbene insertion into
X‒H bonds (e.g., X = S) remain underexplored,8 which has
impeded further development. Exploration of biocatalytic
scenarios using structurally diverse carbenes together with
mechanistic studies would significantly expand the synthetic value
of these methods and aid our understanding of how the enzyme
scaffold promotes high catalytic efficiency and selectivity.
O
TTN
4000
3000
2000
1000
+
O
99% de
N2
, 10 mM
1a
LAD
, 10 mM
E. coli harboring
P411
variants
97% de
M9-N buffer (pH 7.4)
room temp., 12 h
O
91% de
94% de
90% de
O
Ph
CIS
CIS
P411-
T268A
CIS
P411-
T438S
G8
G8S
P411-
P411-
P411-
2a
Figure 1. Comparison of P411 variants for forming spiro[2.4]-
lactone 2a. Reactions were performed in quadruplicate:
suspensions of E. coli expressing P411 variants (OD600 = 30), 10
mM 1a, 10 mM LAD, 25 mM D-glucose and 5 vol% EtOH in M9-N
buffer at room temperature under anaerobic conditions for 12
hours.
Lactones are an important class of organic moieties with
applications in synthetic chemistry,9 materials science10 and
medicinal chemistry.11 A few studies have demonstrated that α-
diazo lactones can be utilized by rhodium complexes for carbene-
transfer reactions.12 However, these rhodium‒carbenes are prone to
β-hydride migration,13 which gives rise to undesired unsaturated
lactones; optimization of the rhodium catalysts and cryogenic
reaction conditions are required to attenuate these side reactions.12a
Additionally, no enantioselective versions of these transformations
have been reported. Our previous work on engineering
hemeproteins for transfer of acyclic carbenes highlighted the power
of directed evolution to shape protein active sites and greatly
improve both reactivity and selectivity.6a‒e We therefore speculated
that aptly engineered hemeproteins would be capable of generating
We began by testing a panel of cytochrome P411 (Ser-ligated
P450) variants, expressed in Escherichia coli (E. coli) and used as
whole-cell catalysts, with LAD and 1a as substrates (Figure 1).
Variant P411-CIS,16 previously engineered to react with ethyl 2-
diazoacetate and transfer the corresponding α-mono-substituted
carbene to styrene to forge a cis-cyclopropane, showed modest
reactivity towards spiro-lactone formation, with 120 ± 10 total
turnovers (TTN) and 90% de. Several P411 variants with various
active site mutations (e.g., T268A or T438S) were tested but did
not exhibit substantial improvements in activity (Figure S1).
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However, to our delight, P411-CIS double mutant L437F T438Q
enzymatic reactions are also readily scalable and maintain
stereoselectivity and high reactivity. Products 2b and 2c were
prepared in hundred-milligram quantities with good isolated yields
(71% yield, 6500 TTN and 95% yield, 6970 TTN). The absolute
configurations of products 2b and 2f were unambiguously assigned
as (1S, 2S) by X-ray crystallography.19
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2
3
4
5
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7
8
(referred to as P411-G8), previously evolved for carbene transfer
to heteroatom-substituted olefins,17 was found to have 10-fold
higher reactivity, providing 1320 ± 190 TTN while also showing
improved stereoselectivity (97% de). A variant with two additional
mutations, L75Y and L181I (referred to as P411-G8S18), was an
even better biocatalyst, furnishing the desired spiro-lactone product
2a in 3090 ± 120 TTN, 99% de and >98% ee. Control experiments
showed that free heme does not catalyze this reaction.
Having established this biocatalytic platform for the efficient
synthesis of spiro-lactones, we speculated that the active-site
environment of P411-G8S may be especially suited to stabilize the
lactone-carbenoid intermediate. We were eager to examine whether
this P411 variant can catalyze the transfer of this lactone-carbene
to other functionalities, such as thiols. Carbene S‒H insertion is
poorly developed among carbene-transfer reactions; one challenge
is that thiols can poison transition-metal catalysts through
coordination. To date, only a few catalytic systems have been
developed for asymmetric carbene S–H insertion, and these exhibit
limited catalytic efficiency20 and/or low stereoselectivity.21 Fasan
and coworkers reported carbene S‒H insertion with up to 49% ee
by an engineered myoglobin.7b Whereas carbene Si–H and N–H
insertions with iron-porphyrin (bio)catalysts have been revealed to
undergo concerted Si‒H insertion22 and nucleophilic attack by
amine at the carbene center,8c the mechanism of carbene S–H
insertion remains obscure.23 We thus decided to take a closer look
at lactone-carbene S–H insertion with the current enzymatic
system.
To examine whether the serine axial ligand of this P411 variant
played a significant role in promoting in vivo cyclopropanation
activity, we mutated serine back to the canonical P450 axial ligand
cysteine and found that the resulting P450-G8S variant synthesized
2a with much lower activity (380 ± 20 TTN) but the same
stereoselectivity (Figure S2).
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E. coli harboring
O
O
P411-G8S
variant
+
O
R
O
R
M9-N buffer (pH 7.4)
room temp., 12 h
N2
, 10 mM
1
LAD
2
, 10 mM
O
O
O
O
MeO
O
O
O
O
MeO
Me
2a
2b
2c
2d
, 4000 TTN
, 3090 TTN
, 10470 TTN
, 11150 TTN
99.0% de 98.3% ee
>99.9% de, 99.6% ee
>99.9% de, 99.8% ee
>99.9% de, 99.6% ee
,
O
O
O
O
F
E. coli harboring
G8S variants
O
O
O
O
O
O
S
S
H
+
Cl
Br
F3C
O
O
M9-N buffer (pH 7.4)
room temp., 40 min
2ha
, 1650 TTN
2e
2f
2g
, 1590 TTN
, 3660 TTN
>99.9% de, 99.7% ee
, 3170 TTN
N2
, 10 mM
>99.8% de, 99.4% ee
99.3% de, 99.8% ee
98.6% de, 98.5% ee
3a
LAD
4a
, 10 mM
O
O
84% ee
1800
1200
90
60
1610 TTN
O
O
2jab
, 1740 TTN
96.1% de, >99.9% ee
2i
, 2610 TTN
>99.9% de, 99.1% ee
Preparative-scale synthesis:
O
O
O
MeO
O
600
30
0
15% ee
300 TTN
MeO
2b
2c
, 6970 TTN, 95% isolated yield
416 mg @ 2.0 mmol scale
, 6500 TTN, 71% isolated yield
124 mg @ 0.8 mmol scale
0
G8S
G8S
P411-
P450-
O
O
O
Figure 3. Lactone-carbene S‒H insertion: comparison of
reactivity and selectivity of P411-G8S and P450-G8S, and
overlay of P450-CIS (PDB: 4h24, in green) and P411-CIS
(PDB: 4h23, in purple) active-site structures. Reactions were
performed in quadruplicate: suspensions of E. coli expressing G8S
variants (OD600 = 30), 10 mM thiol, 10 mM LAD, 25 mM D-
glucose and 5 vol% EtOH in M9-N buffer at room temperature
under anaerobic conditions for 40 minutes.
O
Br
2f
2j
, 1230 TTN, 66% isolated yield
35.8 mg @ 0.25 mmol scale
, 1810 TTN, 64% isolated yield
42.4 mg @ 0.25 mmol scale
Figure 2. Scope of spiro[2.4]lactones with variant P411-G8S. a5
mM olefin, 5 mM LAD. Absolute configuration not determined.
See SI for details.
b
With this highly active and selective P411-G8S variant, we next
explored the scope of olefin substrates for spiro-lactone
construction (Figure 2). Electron-rich styrenyl olefins (1b and 1c)
are particularly good substrates, with over 10,000 TTNs. Halides
(1e, 1f and 1h) are also well-tolerated, offering opportunities for
further derivatization through coupling methods. Fluorine-
containing substrates, despite the strong electron deficiency, are
also accepted (e.g., 1g). Additionally, steric bulk (e.g., 2-vinyl
naphthalene 1i) did not have a significant influence on reactivity.
All tested styrenyl substrates gave (E)-spiro[2.4]lactone products
with high-to-perfect stereoselectivity (98.6 to >99.9% de, and 98.3
to 99.8% ee). Unactivated aliphatic olefins such as 4-phenyl butene
are also accepted by this biocatalytic system with 1740 TTN.
Interestingly, NMR analysis revealed a (Z)-configuration of
product 2j, which indicates potentially different binding modes for
aromatic vs. aliphatic olefins in the enzyme’s active site. The
Using P411-G8S as a whole-cell catalyst, thiophenol 3a reacted
with LAD to give the desired α-thio-γ-lactone product 4a in 300 ±
30 TTN, but with poor enantioselectivity (15 ± 2% ee). In an effort
to improve reactivity and selectivity, we wanted to explore how the
identity of the heme axial ligand affected the S‒H insertion
process.24 We thus tested this reaction with a library of mutant
enzymes made by site-saturation mutagenesis of the axial-ligand
residue S400 in P411-G8S. To our delight, simply replacing the
axial serine with cysteine (the native ligating residue in P450
enzymes) substantially improved reactivity and selectivity, to 1610
± 60 TTN and 84 ± 1% ee (Figure 3). We next evaluated the
substrate range of this cysteine-ligated P450-G8S variant for
lactone-carbene S‒H insertion (Figure 4). Thiophenols with meta-
OMe and para-halide substituents were successfully converted into
the corresponding α-thio-γ-lactone products (4b to 4e) with good
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TTNs and enantioselectivities. However, para-(fluorinated)alkyl
calculations. The generation of iron-carbenoid species is
1
2
3
4
5
6
7
8
groups (3f to 3h) or a bulkier aromatic ring (3j) gave lower
reactivity and selectivity, suggesting that steric hindrance may
affect substrate orientation. Products 4d and 4e were also
synthesized in hundred-milligram scale under modified conditions;
good isolated yields were obtained, with slightly improved
enantioselectivity.
reasonably facile, according to previous experimental and
8e, 22
computational studies.8b,
Our computational investigations
thus focused on the insertion steps. Simplified iron-porphyrin
models (cat1 and cat2) were used, where SMe and OMe were
chosen to mimic deprotonated Cys and Ser ligands.25, 8f
The free energy diagram of the most favorable pathway for S–H
insertion is shown in Figure 5. The insertion occurs in a stepwise
fashion, involving one hydrogen-atom-transfer (HAT) step and
subsequent C–S bond formation with a thiyl radical. Both cat1 and
cat2 showed similar reactivities for S–H bond insertion, with free
energy barriers of 19.4 kcal/mol and 18.5 kcal/mol, respectively.
However, we found that the iron-alkyl intermediates (int3 and
int10) can undergo a reversible C–Fe bond homolytic cleavage (via
TS6 and TS12), which erodes the enantioenriched alkyl-substituted
stereocenter. The two pathways, radical rebound and C–Fe bond
cleavage, are differentiated by the axial coordination. With SMe
ligation, C–S bond formation is more favorable than C–Fe bond
homolytic cleavage (TS4, ΔG≠ = 6.7 kcal/mol vs. TS6, ΔG≠ = 10.4
kcal/mol), while the opposite is true for the OMe-ligated
intermediate int10 (TS11, ΔG≠ = 7.3 kcal/mol vs. TS12, ΔG≠ = 6.2
kcal/mol). These results indicate that Cys-ligated P450 enzymes
should achieve much higher enantioselectivities than Ser-ligated
P411 enzymes, which matches the experimental observations.
E. coli harboring
P450-G8S variant
O
O
S
S
H
+
Ar
O
O
Ar
M9-N buffer (pH 7.4)
room temp., 40 min
9
N2
, 10 mM
3
LAD
4
, 10 mM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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45
46
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49
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O
O
O
O
S
MeO
S
S
S
O
O
O
O
F
Cl
4d
4a
84% ee
4b
89% ee
4c
86.5% ee
86% ee
, 1610 TTN,
, 1820 TTN,
, 1770 TTN,
, 1960 TTN,
O
O
O
O
S
S
S
S
O
O
O
O
Br
4e
Me
4f
iPr
4g
F3
C
83.5% ee
75% ee
45% ee
4h
60% ee
, 300 TTN,
, 1820 TTN,
, 1090 TTN,
, 420 TTN,
O
O
MeO
MeO
S
S
O
O
4i
84.5% ee
4j
71.5% ee
, 330 TTN,
, 530 TTN,
Preparative-scale synthesis:
The axial coordination has a limited effect on C–S rebound, but
strongly controls the feasibility of C–Fe bond dissociation.26 Since
oxygen has a much higher electronegativity than sulfur, the anionic
cat2 is more stable than the sulfur-coordinated cat1. Therefore, C–
Fe homolytic cleavage with OMe ligation, which releases cat2, has
a lower activation barrier and is more exothermic than that with
sulfur coordination. The difference in exothermicity drives the
change in reaction barrier, which eventually leads to the difference
in enantioselectivities of the two catalysts.
O
O
S
S
O
O
Cl
4d
Br
4e
88% ee
86.5% ee
, 1980 TTN,
, 2190 TTN,
438 mg
@ 2.4 mmol
386 mg
@ 1.5 mmol
, 80% isolated yield
, 94% isolated yield
3e
for 2 h)
3d
(with 1.5 eq.
for 2 h)
(with 1.5 eq
Figure 4. Scope of α-thio-γ-lactones with variant P450-G8S. See
SI for details.
It struck us as interesting that the P411/P450-G8S variants
behaved so differently in the S–H insertion and cyclopropanation
reactions. According to a previous structural study of P411-CIS and
P450-CIS variants that also differed solely at the axial-ligand
residue, the cysteine-to-serine mutation maintains almost identical
active-site geometries (Figure 3). As also demonstrated in previous
work on in vivo reactions using P411 variants,16 the endogenous
reductant NADPH can act as an electron donor to reduce FeIII to
FeII, which is required for the iron cofactor to activate diazo
compounds to form iron-carbenoid intermediates. Cysteine-ligated
heme, on the other hand, has a lower reduction potential of the
ferric state and thus results in poor reduction efficiency of the iron
center by NADPH. Consequently, P411-G8S exhibits significantly
higher cyclopropanation activity compared to P450-G8S, though
both show similar selectivities. However, for carbene S–H
insertion, thiol substrates can also serve as a strong reductant and
enable P450 variants to form iron-carbenoids. But this did not
explain the significant disparity between P411-G8S and P450-G8S
in both reactivity and selectivity, causing us to speculate that the
axial ligand was exerting a heretofore unobserved effect on the
reaction mechanism.
In conclusion, we have developed a powerful biocatalytic
platform based on engineered cytochrome P411/P450 enzymes that
enables facile access to a broad array of structurally diverse γ-
lactone derivatives including spiro[2.4]lactones and α-thio-γ-
lactones. This convergent biosynthetic scheme offers a succinct
method to construct complex lactone-based structures, as an
alternative to nature’s strategy of de novo assembly of lactone
moieties. Interestingly, by simply altering the heme ligation (serine
or cysteine) in the cytochrome P450 variants, we were able to
rapidly optimize the two carbene-transfer reactions,
cyclopropanation and S‒H insertion, with unprecedented levels of
efficiency and stereo-control. To understand how axial ligation
affects both selectivity and reactivity in carbene S‒H insertion, we
conducted a DFT study and described two mechanistic pathways
involving a radical coupling process between a thiyl radical and a
heme-bound alkyl species or a free alkyl radical. The different
electronic properties of serine and cysteine ligands lead to opposite
preferences in the reaction pathways, and distinct product profiles.
The development of novel reactions combined with thorough
mechanistic analyses expands the biocatalytic repertoire of
hemeproteins and also provides insights into how these enzymes
can be tuned for further exploration of new chemistries.
To explore the effects of axial coordination on the S–H insertion
reaction in more detail, we used density functional theory (DFT)
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G in kcal/mol
SMe
OMe
1
2
3
4
5
6
7
8
9
PhS
H
O
PhS
O
H
O
O
N
N
N
N
N
N
Fe
Fe
N
N
TS12
TS6
TS1
1
OMe
TS4
O
SMe
O
PhS
TS9
O
(triplet)
TS2
H
O
(triplet)
H
O
18.5
PhS
O
19.4
H
O
N
N
N
N
N
N
N
N
N
N
N
H
O
Fe
Fe
Fe
N
N
N
N
TS2
N
TS9
PhSH
PhSH
Fe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
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60
SMe
OMe
TS11
OMe
TS6
(sextet)
TS12
(triplet)
1.7
(sextet)
SMe
TS4
4.0
0.6
(OSS)
0.3
PhS
0.0
O
0.0
PhS
O
5.4
O
O
6.4
O
N
N
N
N
N
N
N
Fe
O
N
N
N
N
N
N
N
12.2
13.1
H
O
Fe
N
Fe
Fe
H
O
N
O
H
O
H
N
N
N
O
O
OMe
Fe
N
N
N
SMe
N
OMe
SMe
Fe
int8
(OSS)
int1
N
(OSS)
cat2
(quintet)
cat1
(quintet)
int7
int7
OMe
SMe
int10
(doublet)
int3
(doublet)
47.5
46.3
O
O
O
O
H
SPh
H
SPh
int5
int5
(favored)
HAT
Radical Rebound
HAT
Radical Rebound (disfavored)
Stereoretentive
(favored)
CFe Bond Homolytic Cleavage (disfavored)
CFe Bond Homolytic Cleavage
Loss of Stereogenic Center
O
O
O
O
G = 7.4 kcal/mol
G = 34.1 kcal/mol
+
PhS
H
H
SPh
int5
int7
Figure 5. Free energy diagram for the most favorable reaction pathways of S–H insertion. Gibbs free energy obtained at the
B3LYP/def2-TZVP//B3LYP/6-31G(d)-LANL2DZ level. The most stable spin states are shown in parentheses. OSS is open-shell singlet.
“Thousand Youth Talents Plan”, and the “Fundamental Research
Funds for the Central Universities” is gratefully acknowledged.
ASSOCIATED CONTENT
O.F.B. acknowledges support from the Swiss National Science
Foundation (P300PA_171225). Calculations were performed on
the high-performance computing system at the Department of
Chemistry, Zhejiang University. We thank X. Huang, D. K.
Romney, R. K. Zhang, S. B. J. Kan, R. D. Lewis in the Arnold lab
and M. Garcia-Borràs in the K. Houk Lab, UCLA for helpful
discussion and comments. We also thank N. Torian and the Caltech
Mass Spectrometry Laboratory, and L. M. Henling and the Caltech
X-ray Crystallography Facility for analytical support, and B. M.
Stoltz for use of a polarimeter.
Supporting Information
Experimental details, and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
hxchem@zju.edu.cn (X.H.) and frances@cheme.caltech.edu
(F.H.A.)
ORCID
REFERENCES AND NOTES
Kai Chen: 0000-0002-3325-3536
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Oliver F. Brandenberg: 0000-0001-5662-1234
Xin Hong: 0000-0003-4717-2814
Frances H. Arnold: 0000-0002-4027-364X
Author Contributions
†K.C. and S.-Q.Z. contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Financial support by NSF Division of Molecular and Cellular
Biosciences grant MCB-1513007, National Natural Science
Foundation of China (21702182), Zhejiang University, the Chinese
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