of 6
Diverse Engineered Heme Proteins Enable Stereodivergent
Cyclopropanation of Unactivated Alkenes
Anders M. Knight,
S. B. Jennifer Kan,
Russell D. Lewis,
Oliver F. Brandenberg,
Kai Chen,
and Frances H. Arnold
*
,
,
Division of Biology and Bioengineering and
Division of Chemistry and Chemical Engineering, California Institute of Technology,
1200 East California Boulevard, MC 210-41, Pasadena, California 91125, United States
*
S
Supporting Information
ABSTRACT:
Developing catalysts that produce each stereo-
isomer of a desired product selectively is a longstanding
synthetic challenge. Biochemists have addressed this challenge
by screening nature
s diversity to discover enzymes that
catalyze the formation of complementary stereoisomers. We
show here that the same approach can be applied to a new-to-
nature enzymatic reaction, al
kene cyclopropanation via
carbene transfer. By screening diverse native and engineered
heme proteins, we identi
fi
ed globins and serine-ligated
P411
variants of cytochromes P450 with promiscuous activity for
cyclopropanation of unactivated alkene substrates. We then
enhanced their activities and stereoselectivities by directed
evolution: just 1
3 rounds of site-saturation mutagenesis and screening generated enzymes that transform unactivated alkenes
and electron-de
fi
cient alkenes into each of the four stereoisomeric cyclopropanes with up to 5,400 total turnovers and 98%
enantiomeric excess. These fully genetically encoded biocatalysts function in whole
Escherichia coli
cells in mild, aqueous
conditions and provide the
fi
rst example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated
alkenes.
INTRODUCTION
The biological world is a marvelous ensemble of chiral
molecules. From the amino acid and nucleoside building
blocks that form proteins and DNA to intricate natural
products produced by living organisms, chirality dictates how
molecules interact with living systems.
1
Modern medicines
often draw inspiration from chiral natural products.
2
Because
alternate stereoisomers can have very di
ff
erent biological
e
ff
ects,
3
characterization of novel bioactive compounds during
drug candidate screening should include testing each stereo-
isomer.
4
Developing stereodivergent syntheses, where a set of
complementary catalysts can generate every possible stereo-
isomer of the product, is therefore useful and is actively sought
after in catalysis.
5
Enzymes are green, sustainable options for
stereoselective catalysis, and stereocomplementary enzymes can
often be found in nature
s diversity: lipases,
6
ketoreductases,
7
and transaminases
8
chosen using genome mining,
9
for example,
have all a
ff
orded products with di
ff
erent stereoselectivities. We
show here that natural protein diversity can be leveraged in a
similar fashion to achieve stereodivergence for a new, non-
natural enzyme-catalyzed reaction, cyclopropanation of unac-
tivated alkenes via carbene transfer. Cyclopropanes are found in
a variety of natural products. They are commonly biosynthe-
sized through intramolecular cyclization of cationic or radical
intermediates catalyzed by terpene cyclases, desaturases, or
SAM-dependent enzymes. The formation of cyclopropane
moieties via carbene transfer to alkenes, widely used in
synthetic chemistry, is not known in nature.
10
Previous work from this group and others has shown that
iron
porphyrin (heme) proteins can be engineered to catalyze
the cyclopropanation of styrenyl alkenes with ethyl diazoacetate
(EDA,
1
).
11
14
This new-to-nature carbene transfer reaction
has been applied in the synthesis of key pharmaceutical
intermediates such as levomilnacipran,
15
ticagrelor,
16
,
17
and
tasimelteon.
17
Work from several groups has also shown that
cytochromes P450 and myoglobins can be engineered to switch
their diastereo- and/or enantioselectivity preference toward
styrenyl ole
fi
n cyclopropanation,
11
,
13
,
17
but none of these
precedents yielded all possible stereoisomers in high turnover
and selectivity (
Supplemental Table 1a
). In addition, alkene
cyclopropanation by heme proteins with the native iron
cofactor has been limited to styrenyl and other activated
alkenes. Unactivated, aliphatic alkenes are attractive feedstocks
for chemical synthesis, but their transformation to higher value
chiral products is challenging due to their inert nature, high
degree of conformational
fl
exibility, and limited steric and
electronic bias to guide stereocontrol.
18
State-of-the-art methods for enantioselective unactivated
alkene cyclopropanation often rely on noble metals,
19
21
with
Received:
November 10, 2017
Published:
February 21, 2018
Research Article
Cite This:
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2018, 4, 372
377
© 2018 American Chemical Society
372
DOI:
10.1021/acscentsci.7b00548
ACS Cent. Sci.
2018, 4, 372
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This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
only a few examples of Co-
22
and Cu-based
23
catalysts
(
Supplemental Table 1b
); no iron-based catalyst for the
enantioselective intermolecular cyclopropanation of unactivated
alkenes has been reported. Recent work by Hartwig and others
has shown that heme-binding proteins metalated with an
arti
fi
cial iridium-porphyrin cofactor can be leveraged for
these
20
,
21
,
24
and other challenging carbene transfer reactions,
25
where the protein active site enhances the stereoselectivity and
rate of the iridium-catalyzed reaction. Our group has previously
used directed evolution of heme proteins to access carbene
transfer reactions performed with noble-metal catalysts, such as
carbon
silicon bond formation
26
and intermolecular C
H
amination.
27
We therefore set out to create a collection of
genetically encoded biocatalysts equipped with the native heme
cofactor for stereodivergent unactivated alkene cyclopropana-
tion, taking advantage of the natural diversity of heme proteins
to identify suitable starting activity and stereoselectivity.
RESULTS AND DISCUSSION
In order to
fi
nd initial enzymatic activity for unactivated alkene
cyclopropanation, we collected a panel of 11 heme proteins
from thermophilic and hyperthermophilic bacteria and archaea
(
Supplemental Table 2
), as thermostable proteins can better
withstand the potentially destabilizing e
ff
ects of mutations and
are therefore more
evolvable
.
28
They are also often easier to
work with and better tolerate polar organic solvents used to
solubilize substrates. These heme proteins were initially tested
with mutations to the putative distal ligand and active-site
entrance loop (similar mutations have been found by Fasan and
co-workers to enhance myoglobin-catalyzed cyclopropanation
of vinylarenes
12
) for the cyclopropanation of 1,7-octadiene
(
Supplemental Table 2
). Protein variants that showed catalytic
activity were subsequently tested with their mutations removed
for 1-octene cyclopropanation. Through these experiments,
wild-type
Aeropyrum pernix
protoglobin (
Ape
Pgb WT, UniProt
ID: Q9YFF4) and wild-type
Rhodothermus marinus
nitric oxide
dioxygenase (
Rma
NOD WT, UniProt ID: D0MGT2) were
found to have low but measurable cyclopropanation activity on
1-octene (
2a
), catalyzing the reaction with 18 and 27 total
turnovers per enzyme active site (TTN). Notably,
Ape
Pgb WT
and
Rma
NOD WT displayed complementary diastereoselec-
tivity, preferentially producing
cis
(1
R
,2
S
)-
3a
and
trans
(1
S
,2
S
)-
3a
, respectively.
In addition to searching globin protein diversity for this novel
reactivity, we also investigated heme proteins obtained in
previous directed evolution studies. A panel of 36 variants of a
Bacillus megaterium
cytochrome (P450
BM3
) engineered for
other non-natural carbene and nitrene transfer reactions
14
was tested for the ability to cyclopropanate
2a
and 4-phenyl-1-
butene (
2b
).
2b
was chosen as a substrate for library screening,
because the UV
visible phenyl group enables screening by
HPLC-UV. BM3 variant P411-CIS L437F T438Q L75Y L181I
(P411-UA, DNA sequence in
Supplemental Table 12
) showed
signi
fi
cant activity and selectivity for production of
cis
(1
S
,2
R
)-
3a
, the third of the four possible isomers. This variant of a
serine-ligated
P411
(P411-CIS
29
) had been engineered for
cyclopropanation reactivity on e
lectron-rich, non-styrenyl
alkenes such as
N
-vinyl amides.
30
Site-saturation mutagenesis libraries were generated and
screened to increase the activities and selectivities of the
di
ff
erent enzymes. Because crystal structures of
Ape
Pgb and
Rma
NOD have not been reported, homology models were
built to help us identify residues within the putative distal heme
pocket, where carbenoid formation and substrate binding are
predicted to take place (
Supplemental Figure 2
). P411-UA
residues were selected based on the crystal structure of its
P411-CIS predecessor (PDB ID: 4H23). Individual site-
saturation libraries were screened for increased activity and
diastereoselectivity using
2b
and
1
as substrates. Variants with
enhanced diastereoselectivity in the production of
3b
were
regrown in larger scale, and their activities were tested in the
cyclopropanation of
2b
and
2a
with
1
. Enzyme variants with
the greatest overall selectivity enhancements against
3a
and
3b
were used as parents in the next rounds of site-saturation
mutagenesis and screening. A single mutation (Q52V) gave
Rma
NOD near-perfect stereoselectivity for producing
trans
(1
S
,2
S
)-
3a
. Three mutations (W59A Y60G F145W, or
AGW
) gave
Ape
Pgb the ability to make
cis
(1
R
,2
S
)-
3a
with
89:11 diastereomeric ratio (dr) and 99% enantiomeric excess
(ee). During screening to increase P411-UA
s
cis
diastereose-
lectivity, a single mutation, V87F, was found to completely
invert the diastereoselectivity from 89:11
cis
(1
S
,2
R
)-
3a
to 4:96
trans
(1
R
,2
R
)-
3a
,a
ff
ording the fourth and
fi
nal stereoisomer we
needed. Residue 87 is known to modulate the stereoselectivity
of P450
BM3
for oxygenation of various substrates.
31
With initial screening of 11 new and 36 previously
engineered heme proteins, followed by just one to three
rounds of site-saturation mutagenesis, we discovered four
protein variants capable of cy
clopropanating unactivated
alkenes (
Rma
NOD Q52V,
Ape
Pgb W59A Y60G F145W (=
Ape
Pgb AGW), P411-UA-V87C, and P411-UA-V87F), each of
which produced a distinct stereoisomer of the desired product
3a
with 89:11 to <1:99 dr and 96% to >99% ee (
Figure 1
). The
enzyme activities against unactivated alkenes are comparable to
those of the state-of-the-art catalysts, with 100
490 TTN for
3a
and as high as 2,400 TTN for
3b
, the substrate against which
the enzymes were screened. The system is straightforward and
easy to use: the protein-expressing bacterial cells need only be
resuspended to the desired concentration and the alkene and
diazo ester added directly under an anaerobic atmosphere.
When the reaction is complete, the product is extracted into
organic solvents for analysis or puri
fi
cation. While these
enzymes were optimized for use in whole cells, they also
function to some degree in lysates and as puri
fi
ed proteins
(
Supporting Information
).
The four engineered biocatalysts were tested on a range of
alkenes. Their activities and selectivities were high on
unbranched aliphatic alkenes similar to those for which they
were engineered, but their substrate scope extends to sterically
hindered and electron-de
fi
cient alkenes as well (
Figure 2
).
Though the activity and stereoselectivity di
ff
ered on di
ff
erent
substrates, each catalyst accepted most of the substrates tested.
It is likely that activity on speci
fi
c substrates can be optimized
further, if desired, as has been shown in many other directed
evolution studies.
32
,
33
We have shown that these protein variants function with the
commonly used diazo carbene precursor EDA, which is
especially useful because the ester moiety can be further
derivatized, e.g., to form amides and other esters. It can also be
reduced to alcohols and aldehydes, which are versatile synthetic
handles for accessing a broad range of functional groups.
Reports of cyclopropanation via tri
fl
uoromethylcarbene transfer
to alkenes,
34
as well as carbene transfer to silanes and boranes
have shown that enzymes can also be engineered to use
di
ff
erent carbene precursors.
26
,
35
ACS Central Science
Research Article
DOI:
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ACS Cent. Sci.
2018, 4, 372
377
373
The small-molecule-catalyzed enantioselective preparation of
cyclopropyl esters from electron-de
fi
cient alkenes has pre-
viously been limited to making the
trans
-cyclopropanes,
36
whereas strategies to directly access 1-keto-2-ester or 1,2-diester
cis
-cyclopropanes (or their corresponding carboxylates) via
enantioselective cyclopropanation are unknown. The biocata-
lysts, in contrast, enable access to the
cis
-1-keto-2-ester and
cis-
1,2-diester products in a single, intermolecular step using an
Escherichia coli
based platform (
cis
-
3c
,
cis
-
3g
,
Figure 2
). Some of
these products are precursors to valuable compounds: cyclo-
propyl esters of unbranched, aliphatic alkenes are used in
fragrances, for example, including the essential odorants in
frankincense.
37
Notably, the enzymes catalyze the reaction on
2-vinylpyridine (
2h
), which is a di
ffi
cult substrate for many
catalysts due to pyridine
s propensity to coordinate to and
inhibit metal centers. This cyclopropanation product is a
precursor for an orphan GPR88 agonist.
38
A similar vinyl-
pyridine substrate was used in myoglobin-catalyzed cyclo-
propanation.
17
Enzymes are chemoselective and can generate desired
products without additional steps to protect and deprotect
other reactive functional groups on the same molecule. As
shown in
Figure 3
, the enzymes described here, for example,
can selectively cyclopropanate terminal alkenes in the presence
of alcohol and carboxylic acid functional groups which often
undergo competitive O
H insertion reactions with small-
molecule carbene transfer catalysts like rhodium acetate
dimer.
39
Ape
Pgb AGW performed par
ticularly well with
unprotected 7-octen-1-ol (
2i
) and 7-octen-1-oic acid (
2j
),
yielding products
cis
-
3i
and
cis
-
3j
at 77% and 64% isolated yield,
respectively, in preparative-scale reactions. When functional
groups cannot be protected easily, chemo- and regioselectivity
is even more important. In the cases of (
E
)-penta-1,3-diene
(
2k
) and (
Z
)-penta-1,3-diene (
2l
), all four engineered proteins
cyclopropanate the terminal alkene with perfect regioselectivity,
likely due to higher accessibility of this double bond in each
enzyme
s active site. The diastereoselectivity varied for
3k
and
3l
, though the enantioselectivity for the major isomer remained
high. As the electronic properties of
2k
and
2l
are similar, the
di
ff
erence in stereoselectivity likely re
fl
ects steric constraints of
the enzyme active sites.
Citing the need for a greater reactivity of the metal center to
cyclopropanate unactivated alkenes, Hartwig, Clark, and co-
Figure 1.
Stereoselective enzymatic cyclopropanation of the aliphatic
alkene
2a
and
1
to obtain each of four stereoisomers of cyclopropane
product
3a
with diastereoselectivies from 89:11 to <99:1 dr and
enantioselectivies from 96% to >99% ee. Reaction conditions: whole
E.
coli
cells in M9-N bu
ff
er, 25 mM glucose, 10 mM
2a
, direct addition of
20 mM
1
under anaerobic conditions, 5% ethanol cosolvent. The
diastereoselectivity ratio (dr) is given as
cis
:
trans
, and the enantiomeric
excess (ee) is given for the major diastereomer. Catalysts used:
rhodium acetate dimer (Rh
2
(OAc)
4
) to form the racemic authentic
standard, two variants of the engineered, serine-ligated cytochrome
P450
BM3
(P411-UA-V87C and P411-UA-V87F),
Aeropyrum pernix
protoglobin W59A Y60G F145W (
Ape
Pgb AGW), and
Rhodothermus
marinus
nitric oxide dioxygenase Q52V (
Rma
NOD Q52V). Protein
sequences are available in the
Supporting Information
. Abbreviations
used: RT, room temperature;
n
Hex,
n
-hexyl.
Figure 2.
Cyclopropanation substrate scope. (a) Activity and
selectivity of each protein variant against
3a
,
3b
, and
3c
. (b) Activity
and selectivity against a variety of alkenes. The diastereoselectivity
ratio (dr) is given as cis:trans, and the enantiomeric excess (ee) is
given for the major diastereomer. Enzyme variant used is denoted by
superscripts: 1,
Ape
Pgb AGW; 2,
Rma
NOD Q52V. General reaction
conditions: whole
E. coli
cells (OD
600
=5(
Ape
Pgb AGW,
Rma
NOD
Q52V), OD
600
= 20 (P411-UA-V87C, P411-UA-V87F)) in M9-N
bu
ff
er, 25 mM glucose, 10 mM alkene, direct addition of 20 mM
1
under anaerobic conditions, 5% ethanol cosolvent. Modi
fi
ed reaction
conditions:
3g
OD
600
= 20;
3e
OD
600
= 10.
Rma
NOD Q52V
3a
,
OD
600
= 10. Analytical yields for these reactions are given in
Supplemental Table 8
. The absolute con
fi
gurations of products
3b
and
3c
are assigned by analogy to the
3a
products (see the
Supporting
Information
section
Determination of absolute con
fi
gurations of the
cyclopropane products
for details). Chiral separation conditions
reported in the
Supporting Information
.
*
The benzyl ester of
3c
has
IUPAC naming priority, and therefore the chiral carbon numbering is
reversed for these compounds.
ACS Central Science
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DOI:
10.1021/acscentsci.7b00548
ACS Cent. Sci.
2018, 4, 372
377
374
workers showed that heme proteins could bind an arti
fi
cial
iridium cofactor in place of iron heme for carbene transfer
chemistry.
20
They showed that a protein
s active site can confer
selectivity to noble-metal, small-molecule catalysts that can
already catalyze the reaction.
20
,
21
,
25
Use of an arti
fi
cial iridium
cofactor (Ir(Me)PIX) required the lysis, puri
fi
cation, and
in
vitro
metalation of the apoprotein with the Ir(Me)PIX, all of
which add time and cost to catalyst preparation. Though it may
be possible to incorporate the new metal
in vivo
,
40
the synthetic,
noble-metal cofactor is more expensive than the native heme,
which is manufactured by the cell and loaded into the catalyst
during protein expression
in vivo
. The use of iridium is also not
ideal due to the negative impact mining and re
fi
ning precious
metals has on the environment.
41
The Ir(Me)PIX system,
however, has the advantage that it can cyclopropanate internal
alkenes
21
and catalyze carbene insertion into C
H bonds,
25
neither of which have been reported yet with heme proteins.
There is precedent that iron
porphyrin catalysts are capable
of unactivated alkene cyclopropanation. Two decades ago, Woo
and co-workers showed that iron
meso
-tetrakis-
(penta
fl
uorophenyl)porphyrin chloride (Fe(PFP)Cl) can cata-
lyze the reaction of 2-ethyl-1-butene and EDA with 390 TTN;
they reported the formation of cyclopropane products using 1-
decene as well.
42
In fact, we observed that heme in aqueous
bu
ff
er, with no protein, can catalyze the formation of
3a
, albeit
with only 0.4 TTN. This basal activity is greatly enhanced and
stereoselectivity is enforced by the protein environment,
allowing the heme proteins described here to cyclopropanate
a range of alkenes from electron-rich conjugated dienes to
electron-de
fi
cient vinyl ketones and acrylates with high
diastereo- and enantioselect
ivity. The primary factor in
determining activity appears to be the binding of the alkene
in a productive con
fi
guration: the heme
s local protein
environment can be molded to enhance activity and selectivity
by optimizing the substrate binding modes. Di
ff
erent local
heme environments can be accessed by screening natural and
engineered protein diversity. Directed evolution then
fi
ne-tunes
these features.
Metalloporphyrin catalysts have been used in synthetic
chemistry for decades, but nature has used them for millions of
years. Present in all forms of life on Earth, heme-binding
proteins have diverse functions as well as promiscuous activities
for which they were never selected, such as the ability to form
reactive carbene intermediates. We have taken advantage of this
natural diversity to
fi
nd catalysts for reactions not known to be
catalyzed in biology, but that are synthetically useful and are
driven by a synthetic carbene precursor (EDA).
While biocatalysts often possess very high selectivity, this
selectivity can be synthetically limiting. A single enzyme may
make only a single isomer, but access to other isomers may be
equally important. Natural diversity can be leveraged e
ff
ectively
for this challenge. A combination of natural diversity and
directed evolution let us realize the stereodivergent cyclo-
propanation of unactivated and electron-de
fi
cient alkenes in
mild, aqueous conditions with a fully genetically encoded heme
protein expressed in bacteria. This set of biocatalysts can serve
as starting points for green, sustainable synthesis of valuable
cyclopropanated products.
ASSOCIATED CONTENT
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website
at DOI:
10.1021/acscentsci.7b00548
.
Materials and experimental methods, detailed protein
engineering strategies for each variant, and compound
characterization (
PDF
)
AUTHOR INFORMATION
Corresponding Author
*
E-mail:
frances@cheme.caltech.edu
.
ORCID
Anders M. Knight:
0000-0001-9665-8197
S. B. Jennifer Kan:
0000-0001-6371-8042
Russell D. Lewis:
0000-0002-5776-7347
Oliver F. Brandenberg:
0000-0001-5662-1234
Kai Chen:
0000-0002-3325-3536
Frances H. Arnold:
0000-0002-4027-364X
Notes
The authors declare no competing
fi
nancial interest.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
Division of Molecular and Cellular Biosciences (Grant MCB-
1513007) and the O
ffi
ce of Chemical, Bioengineering,
Environmental and Transport Systems SusChEM Initiative
(Grant CBET-1403077). The authors thank Dr. Nathan
Dalleska, Aurapat Ngamnithiporn, and Dr. Scott C. Virgil for
analytical chiral GC support, and Dr. Stephan C. Hammer and
Figure 3.
Selective, preparative-scale cyclopropane syntheses from various aliphatic alkenes and dienes. Preparative-scale reactions against substrate
s
with free alcohol (7-octen-1-ol,
2i
), free carboxylic acid (7- octen-1-oic acid,
2j
), the two geometric isomers of 1,3-pentadiene (
2k
,
2l
), and ketone
(5-hexen-2-one,
2m
). The diastereoselectivity ratio (dr) is given as
cis
:
trans
, and the enantiomeric excess (ee) is given for the major diastereomer.
The yields are reported for isolated products. Enzyme variant used is denoted by superscripts: 1,
Ape
Pgb AGW; 2,
Rma
NOD Q52V. Reaction and
chiral separation conditions are available in the
Supporting Information
.
ACS Central Science
Research Article
DOI:
10.1021/acscentsci.7b00548
ACS Cent. Sci.
2018, 4, 372
377
375
Dr. Xiongyi Huang for helpful discussions and critical reading
of the manuscript. A.M.K. gratefully acknowledges support
from Caltech
s Center for Environmental Microbial Inter-
actions and the NSF Graduate Research Fellowship (Grant No.
DGE-1745301). R.D.L. is supported by an NIH
National
Research Service Award training grant (5 T32 GM07616).
O.F.B. acknowledges support from the Deutsche Forschungs-
gemeinschaft (Grant No. BR 5238/1-1) and the Swiss National
Science Foundation (Grant No. P300PA-171225). A provi-
sional patent application has been
fi
led through the California
Institute of Technology based on the results presented here.
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