Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

Article abstract of DOI:10.1021/acscatal.7b04423

The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450_BM3 that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatom-substituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.

Full text of DOI:10.1021/acscatal.7b04423

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.
Letter
pubs.acs.org/acscatalysis
Cite This: ACS Catal. 2018, 8, 2629−2634
Stereoselective Enzymatic Synthesis of Heteroatom-Substituted
Cyclopropanes
Oliver F. Brandenberg,† Christopher K. Prier,†,§ Kai Chen,† Anders M. Knight,‡ Zachary Wu,†
and Frances H. Arnold*,†
†Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena,
California 91125, United States
‡Division of Biology and Bioengineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California
91125, United States
S
* Supporting Information
ABSTRACT: The repurposing of hemoproteins for non-natural
carbene transfer activities has generated enzymes for functions
previously accessible only to chemical catalysts. With activities
constrained to specific substrate classes, however, the synthetic
utility of these new biocatalysts has been limited. To expand the
capabilities of non-natural carbene transfer biocatalysis, we
engineered variants of Cytochrome P450BM3 that catalyze the
cyclopropanation of heteroatom-bearing alkenes, providing valuable
nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or
five active-site mutations converted a single parent enzyme into
selective catalysts for the synthesis of both cis and trans heteroatom-
substituted cyclopropanes, with high diastereoselectivities and
enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed
evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.
KEYWORDS: biocatalysis, Cytochrome P450, cyclopropanes, carbene transfer
he cyclopropane ring is an important building block in
organic synthesis and a structural motif in diverse
reactions have been performed via carbene transfer with chiral
ruthenium,20−24 rhodium,25−27 and copper11,27 catalysts.
Aminocyclopropanes have also been accessed via alkene
cyclopropanation with nitro-substituted carbenoids, followed
by reduction of the nitro group.28,29 However, many of the
existing methods exhibit low catalyst turnover, proceed with
only moderate levels of stereocontrol, are limited to specific
substrate types, or require precious metals (e.g., Ru, Rh).
Furthermore, these methods generally provide only the trans-
cyclopropane products with high diastereoselectivity and
enantioselectivity.
We and others have demonstrated that hemoproteins can be
engineered to perform cyclopropanation reactions of olefins via
carbene transfer.30−35 The synthetic utility of these “carbene
transferases”, engineered by directed evolution of cytochromes
P450, myoglobins, and other proteins, has been demonstrated
through preparative-scale syntheses of cyclopropane-containing
pharmaceutical precursors.36−38 To date, however, almost all
cyclopropanation reactions catalyzed by hemoproteins have
required arene-substituted (styrenyl) olefins. In a first step
toward broadening the substrate scope of enzyme-catalyzed
T
bioactive compounds.1,2 Especially notable are heteroatom
(nitrogen, oxygen, or sulfur)-substituted cyclopropanes, which
appear in many natural products3 and biosynthetic inter-
mediates (Scheme 1A).4 In pharmaceuticals, introduction of
cyclopropylamines can substantially improve physicochemical
and pharmacokinetic properties,5 and the cyclopropylamine
functionality appears in the class of hepatitis C virus protease
inhibitors represented by simeprevir6 and grazoprevir.7 Novel
biomaterials have also been constructed using β-amino-
cyclopropane carboxylic acids (β-ACCs) and related com-
pounds as rigid building blocks in synthetic peptides.8,9
Heteroatom-bearing cyclopropanes are additionally useful
synthetic intermediates that engage in a variety of ring-opening
transformations.10−13
Enantiomerically enriched cyclopropylamines are most
commonly synthesized via Curtius or Hofmann rearrangements
from the corresponding enantioenriched cyclopropylcarboxylic
acids or amides.14,15 Chiral cyclopropylamines and cyclo-
propanols have also been synthesized via asymmetric phase-
transfer catalysis16,17 and enzymatic resolution.18,19 Alterna-
tively, forming the cyclopropane ring with control over
stereochemistry by catalytic, enantioselective cyclopropanation
of heteroatom-substituted alkenes represents a powerful
approach to this class of compounds (Scheme 1B). Such
Received: December 23, 2017
Revised: February 7, 2018
© XXXX American Chemical Society
2629
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634

ACS Catalysis
Letter
Scheme 1. (A) Examples of Cyclopropylamines and
Cyclopropanols in Natural Products, Pharmaceuticals, and
Biomaterials; (B) Proposed Enzymatic Synthesis of Chiral
a
Heteroatom-Substituted Cyclopropanes
a
Enzymatic carbene transfer to heteroatom-substituted alkenes
generates heteroatom-substituted cyclopropanes in a single step with
control over stereoselectivity.
cyclopropanation, we demonstrated recently that natural
hemoprotein diversity can be leveraged to identify and engineer
Cytochrome P450 and globin variants for the stereoselective
cyclopropanation of unactivated, aliphatic alkenes,39 an activity
previously achieved only by hemoproteins containing an
artificial iridium co-factor.40−42 Gober et al. also recently
reported engineered Cytochrome P450 variants for the
cyclopropanation of dehydroalanine residues in thiopeptides,43
thereby enabling a non-natural modification of a complex
natural product. We reasoned that diverse heteroatom-
substituted alkenes should be suitable coupling partners for
electrophilic metal carbenoids derived from the reaction of
diazo esters with genetically encoded heme proteins. This
reaction would extend the scope of biocatalytic cyclo-
propanation to encompass a new class of synthetically
important cyclopropanes (Scheme 1B). Here, we describe the
directed evolution of Cytochrome P450 variants that cyclo-
propanate heteroatom-bearing alkenes to yield chiral N-, O-,
and S-cyclopropanes. The resulting enzymes outperform
existing small-molecule catalysts, especially for challenging cis-
selective cyclopropanation reactions.
To gauge the potential of enzymatic heteroatom-substituted
cyclopropane synthesis, we first tested a set of hemoproteins for
cyclopropanation of N-vinylphthalimide (1) with ethyl
diazoacetate (EDA, 2) as the carbene precursor (Figure 1), a
reaction performed by free heme with a total turnover number
(TTN) of 3 and 28:72 diastereomeric ratio (dr) (cis:trans) (see
Table S2 in the Supporting Information). Enzymatic reactions
were carried out with whole Escherichia coli (E. coli) cells
expressing the hemoprotein variants and were analyzed for
reaction yield and stereoselectivity (see Table S2). Variants of
Cytochrome P450BM3 from Bacillus megaterium were found to
be active catalysts for synthesis of cyclopropane adduct 3,
including several “P411” variants that feature a cysteine-to-
serine mutation at the residue ligating the iron center.31 E. coli
expressing variant P411BM3-CIS31 demonstrated promising
diastereoselectivity (88:12 cis:trans), and this protein was
selected for further engineering by directed evolution to
enhance its catalytic activity.
Figure 1. Directed evolution of P411BM3-CIS for cis- and trans-
selective cyclopropanation of N-vinylphthalimide (1). Yields were
determined by liquid chromatography (LC) analysis. Enantioselectiv-
ities are given for the respective diastereomers. (A) Evolutionary
lineage for cis-selective N-vinylphthalimide cyclopropanation. Reac-
tions were performed with whole E. coli cells at OD600 = 30 expressing
the indicated variants, 30 mM N-vinylphthalimide (1), and 60 mM
EDA (2) under anaerobic conditions. (B) Evolutionary lineage for
trans-selective N-vinylphthalimide cyclopropanation. Reactions were
performed with whole E. coli cells at OD600 = 30 expressing the
indicated variants, 5 mM N-vinylphthalimide (1) and 10 mM EDA
(2).
L181, I263, T268, L437, and T438, positions previously shown
to influence P450-catalyzed non-natural carbene and nitrene
transfer activities (see Figure S3 in the Supporting
Information)30,36 and screened the resulting libraries for
cyclopropanation on N-vinylphthalimide with EDA. Several
mutations at these positions yielded improved catalysts (see
Table S3 in the Supporting Information); in addition, we found
that mutation I263G, located above the heme co-factor,
reversed diastereoselectivity from favoring cis-3 (88:12) to
favoring trans-3 (28:72), as the mutation I263A does in P450-
catalyzed styrene cyclopropanation.30 To identify the optimal
To increase the activity of P411BM3-CIS we performed
parallel site-saturation mutagenesis at active site residues V87,
2630
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634

ACS Catalysis
Letter
combination of activating mutations, we screened a recombi-
nation library encompassing a set of eight beneficial single
active-site mutations (Table S3). From this library, we
identified two variants with improved activity relative to
P411BM3-CIS as well as complementary trans and cis
diastereoselectivity: variant P411BM3-CIS L437F T438Q favors
the cis cyclopropane product (94:6 dr, 94% enantiomeric excess
(ee)), while P411BM3-CIS I263G L437F favors the trans (27:73
dr, −64% ee) (Figure 1).
We next chose to optimize the cis- and trans-selective P411
variants separately, with the aim of evolving highly active
catalysts for the selective synthesis of each cyclopropane
diastereomer. For the cis-selective variant, iterative site-
saturation mutagenesis and screening delivered variant
P411BM3-CIS L437F T438Q L75Y L181I; we refer to this
evolved cis-selective vinyl amide cyclopropanation catalyst as
P411-VACcis. This enzyme is a highly active and selective
catalyst: when used in whole E. coli cells (with typical enzyme
concentrations of 0.8 to 0.9 μM), P411-VACcis provides almost
full conversion at up to 30 mM substrate loading with 35 000
TTN, 97:3 dr and 97% ee (Figure 1A). Up to 40 000 TTN
were obtained at higher substrate or lower catalyst loadings (see
Figure S4 in the Supporting Information), which places P411-
VACcis among the most active carbene transfer hemoproteins
described to date.31,33,44
Figure 2. Kinetics of Cytochrome P411-catalyzed N-vinylphthalimde
cyclopropanation. (A) Apparent turnover frequency observed with
purified protein and whole-cell catalysts (“n.d.” = not determined). (B)
Reaction kinetics of whole-cell catalysts expressing P411-VACcis or
P411-VACtrans, recorded over 6 h. Results are the average of duplicate
reactions at each time point. Reaction conditions for P411-VACcis and
P411-VACtrans are as shown in Figure 1.
For the trans-selective variant P411BM3-CIS I263G L437F,
further site-saturation mutagenesis and screening yielded
variant P411BM3-CIS I263G L437F V87L L181R, which we
term P411-VACtrans. In whole E. coli cells, this variant catalyzes
cyclopropanation of N-vinylphthalimide with EDA in 86% yield
with 980 TTN, 3:97 dr, and 92% ee (Figure 1B).
thus strongly influenced by the reaction (cellular) environment,
as well as the protein sequence.
Kinetic analyses revealed that, in the whole-cell context,
P411-VACcis displays an apparent turnover frequency (TOF) of
390 min−1 over the first 10 min and maintains activity over
several hours (see Figure 2, as well as Figure S5 in the
Supporting Information). This long catalyst lifetime is notable
among hemoprotein carbene transferases, as many of those
described to date suffer from rapid inactivation through transfer
of the reactive carbene intermediate to protein side chains or
the porphyrin co-factor.44,45 Indeed, such “burnout” behavior
was observed for the evolved trans-selective variant P411-
VACtrans, which shows much lower TTNs than P411-VACcis and
loses its catalytic activity after 30−60 min (Figure 2B). Thus,
P411-VACcis may possess an active site structure that effectively
directs iron carbenoid reactivity toward the intended alkene
substrate while suppressing self-alkylation. We anticipate that
future work to identify biochemical and structural determinants
responsible for the long catalyst lifetime of P411-VACcis (and
the shorter lifetime of P411-VACtrans) will aid the design of
highly active carbene transfer catalysts for other substrates and
reaction classes.
To demonstrate the synthetic utility of the evolved cis- and
trans-selective variants, we performed preparative-scale syn-
theses with N-vinylphthalimide and EDA. From a 4.0 mmol
reaction with P411-VACcis, cyclopropane 3a was isolated in
96% yield (996 mg) with 97:3 dr and 97% ee, demonstrating
facile gram-scale cyclopropane synthesis enabled by this variant.
Using P411-VACtrans, a 0.125 mmol scale reaction delivered
88% yield (28.5 mg) of the trans-cyclopropane 3b with 3:97 dr
and 92% ee (Scheme 2). The reactions did not require a slow
Scheme 2. Syntheses of Phthalimide-Protected
Aminocyclopropanes with cis- and trans-Selective P411
Variants
Cyclopropanation of heteroatom-substituted alkenes also
proceeds with the purified P411 enzymes, although with
significantly lower efficiencies. In vitro, P411-VACcis catalyzes
N-vinylphthalimide cyclopropanation with a TOF of 22 min−1
and 31% yield, 620 TTN, 91:9 dr and 93% ee, demonstrating
significant decreases in all parameters, compared to whole-cell
conditions (see Figure 2A, as well as Table S4 in the
Supporting Information). This agrees with previous findings
of enhanced hemoprotein carbene transfer activity in whole
cells, compared to purified protein,31,44,46 which we speculate is
due to reduced EDA-induced protein alkylation and
inactivation in the intact cells. Catalyst activity and lifetime is
addition of substrates, and products were readily obtained in
high purity after extraction from the aqueous phase and silica
flash column chromatography. Absolute configurations were
assigned by crystallization of the trans-cyclopropane 3b, which
revealed the compound to be the (1S, 2S) isomer (see Figure
S1 in the Supporting Information). The cyclopropanation of
styrene by P411-VACcis was used to assign the configuration of
2631
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634

ACS Catalysis
Letter
the cis-cyclopropane 3a as (1S, 2R) (see Figure S2 in the
Supporting Information).
Table 1. Substrate Scope of cis- and trans-Selective
Heteroatom-Substituted Alkene Cyclopropanation
a
We next tested the substrate scopes of P411-VACcis and
P411-VACtrans across a range of structurally diverse N-, O-, and
S-substituted olefins. We were delighted to find that P411-
VACtrans performs highly stereoselective cyclopropanation of N-
vinylcaprolactam, N-vinylpyrrolidone, and N-methyl-N-vinyl-
acetamide, delivering the corresponding cyclopropanes in
57%−65% isolated yield with up to 4:96 dr and 95% ee (see
Table 1). Variant P411-VACcis also catalyzes cyclopropanation
of these substrates, but with lower levels of enantiocontrol
between 4% ee and 59% ee (see Figure S6 in the Supporting
Information). The results varied across other substrates tested:
for phenyl vinyl sulfide, both catalysts failed to achieve good
diastereoselectivity or enantioselectivity. For phenyl vinyl ether,
we observed a switch in diastereoselectivity, with P411-VACcis
selectively providing the trans product (22:78 dr, 95% ee),
while P411-VACtrans yielded the cis product with low selectivity
(61:39 dr, 26% ee) (see Figure S6). Lastly, both variants accept
the bulky N-vinylcarbazole as a substrate, with P411-VACtrans
(6:94 dr, 94% ee) again outperforming P411-VACcis (44:56 dr,
22% ee) in both diastereoselectivity and enantioselectivity.
To improve the performance of P411-VACcis across these
different substrates, we performed one additional round of
mutagenesis and screening. A small library of 14 P411-VACcis
variants with single active-site mutations at residues V87, I263,
E267, and A328 (see Table S5 in the Supporting Information)
was screened for cyclopropanation activity on N-vinyl-
pyrrolidone, N-methyl-N-vinylacetamide, phenyl vinyl sulfide,
and phenyl vinyl ether. This approach yielded P411-VACcis
variants showing improvements in both dr and ee across these
substrates (Figure S6 and Table 1). For N-vinylpyrrolidone and
N-vinylcaprolactam, introduction of the V87T mutation
increased enantioselectivity from 4% ee to 74% ee and from
59% ee to 88% ee, respectively. Interestingly, the V87T
mutation also turned P411-VACcis into a competent catalyst for
cis-selective cyclopropanation of phenyl vinyl ether (72:28 dr,
87% ee). For N-methyl-N-vinylacetamide, ee was increased
from 28% to 97% through an E267N mutation. For cis-selective
cyclopropanation of phenyl vinyl sulfide, an A328N mutation
improved diastereoselectivity from 59:41 dr to 84:16 dr while
increasing the ee from 39% to 90%, thereby delivering a
cyclopropane core allowing extensive further functionalization
with high selectivity.47 Moreover, this approach yielded P411-
VACcis variants with good to excellent diastereoselectivities and
enantioselectivities for the trans-selective cyclopropanation of
phenyl vinyl ether and phenyl vinyl sulfide, through a V87I and
V87F mutation, respectively (Table 1, Figure S6). We thereby
generated a panel of P411 variants delivering diverse
heteroatom-substituted cyclopropanes with good to excellent
dr and ee (Table 1). Because high-resolution protein structures
of these variants are currently lacking, it is difficult to speculate
on the factors steering their enzymatic activity and cis- versus
trans-selectivity. We note, however, the important role of
position 87 in controlling diastereo- and enantioselectivity, as
previously reported.39,48,49 Given the malleability of the
P411BM3 active site observed in our studies, we expect that
the variants described here can be optimized further for highly
selective cyclopropanation of other, related substrates.
a
Reactions were performed on 0.125 mmol scale with whole E. coli
cells at OD600 = 50, 5 mM alkene, and 10 mM EDA. Isolated yields are
reported. Enantioselectivities are given for the major diastereomers.
applications and was previously inaccessible by enzymatic
cyclopropanation. The evolved P411 variants provide N,O,S-
cyclopropanes with high diastereoselectivities and enantiose-
lectivities, function under mild reaction conditions (at room
temperature in an aqueous buffer), and provide higher total
turnovers than reported for chemocatalysts for these trans-
formations (no more than 400 TTN).26 Together with recent
reports from this laboratory and others demonstrating
enzymatic cyclopropanation of unactivated, aliphatic alkenes39
In summary, this work demonstrates that Cytochrome P411
can synthesize heteroatom-bearing cyclopropanes, which is a
valuable product class that has been widely studied for their
unique physical properties as well as pharmaceutical
2632
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634

ACS Catalysis
Letter
and complex natural products,43 this work demonstrates that
hemoproteins are not limited to styrenyl substrates. The P411
catalysts, which consist of an iron co-factor and a protein
scaffold produced in bacterial cells without need for
purification, make these reactions a sustainable alternative to
approaches requiring precious metals. Furthermore, the enzyme
catalysts can go beyond existing chemical catalysts to generate
the cis-cyclopropanes with high stereoselectivity. This ex-
pansion of enzymatic activity from model substrates (styrenes)
to more diverse and functionalized starting materials such as
vinylamides will broaden the scope and potential applications of
non-natural biocatalysis in synthetic biology.
REFERENCES
■
(1) Wong, H. N. C.; Hon, M.-Y.; Tse, C.-W.; Yip, Y.-C.; Tanko, J.;
Hudlicky, T. Use of cyclopropanes and their derivatives in organic
synthesis. Chem. Rev. 1989, 89, 165−198.
(2) Chen, D. Y.-K.; Pouwer, R. H.; Richard, J.-A. Recent advances in
the total synthesis of cyclopropane-containing natural products. Chem.
Soc. Rev. 2012, 41, 4631−4642.
(3) Geng, X.; Jin, L.; Shimada, M.; Kim, M. G.; Mackey, D. The
phytotoxin coronatine is a multifunctional component of the virulence
armament of Pseudomonas syringae. Planta 2014, 240, 1149−1165.
(4) Van de Poel, B.; Van Der Straeten, D. 1-aminocyclopropane-1-
carboxylic acid (ACC) in plants: More than just the precursor of
ethylene! Front. Plant Sci. 2014, 5, 640.
(5) Talele, T. T. The “Cyclopropyl Fragment” is a Versatile Player
that Frequently Appears in Preclinical/Clinical Drug Molecules. J.
Med. Chem. 2016, 59, 8712−8756.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscatal.7b04423.
■
S
(6) Rosenquist, A.; Samuelsson, B.; Johansson, P.-O.; Cummings, M.
D.; Lenz, O.; Raboisson, P.; Simmen, K.; Vendeville, S.; de Kock, H.;
Nilsson, M.; Horvath, A.; Kalmeijer, R.; de la Rosa, G.; Beumont-
Mauviel, M. Discovery and Development of Simeprevir (TMC435), a
HCV NS3/4A Protease Inhibitor. J. Med. Chem. 2014, 57, 1673−1693.
(7) Harper, S.; McCauley, J. A.; Rudd, M. T.; Ferrara, M.; DiFilippo,
M.; Crescenzi, B.; Koch, U.; Petrocchi, A.; Holloway, M. K.; Butcher, J.
W.; Romano, J. J.; Bush, K. J.; Gilbert, K. F.; McIntyre, C. J.; Nguyen,
K. T.; Nizi, E.; Carroll, S. S.; Ludmerer, S. W.; Burlein, C.; DiMuzio, J.
M.; Graham, D. J.; McHale, C. M.; Stahlhut, M. W.; Olsen, D. B.;
Monteagudo, E.; Cianetti, S.; Giuliano, C.; Pucci, V.; Trainor, N.;
Fandozzi, C. M.; Rowley, M.; Coleman, P. J.; Vacca, J. P.; Summa, V.;
Liverton, N. J. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus
NS3/4a Protease Inhibitor. ACS Med. Chem. Lett. 2012, 3, 332−336.
(8) Gnad, F.; Reiser, O. Synthesis and Applications of β-
Aminocarboxylic Acids Containing a Cyclopropane Ring. Chem. Rev.
2003, 103, 1603−1623.
Crystallographic data (CIF)
Experimental procedures, supplementary data, calibration
curves, and characterization of cyclopropanation prod-
ucts (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: frances@cheme.caltech.edu.
ORCID
Oliver F. Brandenberg: 0000-0001-5662-1234
Christopher K. Prier: 0000-0003-0902-1636
Kai Chen: 0000-0002-3325-3536
Anders M. Knight: 0000-0001-9665-8197
Frances H. Arnold: 0000-0002-4027-364X
■
(9) Koglin, N.; Zorn, C.; Beumer, R.; Cabrele, C.; Bubert, C.; Sewald,
N.; Reiser, O.; Beck-Sickinger, A. G. Analogues of Neuropeptide Y
Containing β-Aminocyclopropane Carboxylic Acids are the Shortest
Linear Peptides That Are Selective for the Y1 Receptor. Angew. Chem.,
Int. Ed. 2003, 42, 202−205.
(10) Schneider, T. F.; Kaschel, J.; Werz, D. B. A new golden age for
donor-acceptor cyclopropanes. Angew. Chem., Int. Ed. 2014, 53, 5504−
5523.
(11) Reissig, H.-U.; Zimmer, R. Donor−Acceptor-Substituted
Cyclopropane Derivatives and Their Application in Organic Synthesis.
Chem. Rev. 2003, 103, 1151−1196.
(12) Shaw, M. H.; Bower, J. F. Synthesis and applications of
rhodacyclopentanones derived from C−C bond activation. Chem.
Commun. 2016, 52, 10817−10829.
(13) Fumagalli, G.; Stanton, S.; Bower, J. F. Recent Methodologies
That Exploit C−C Single-Bond Cleavage of Strained Ring Systems by
Transition Metal Complexes. Chem. Rev. 2017, 117, 9404−9432.
̂ ́
(14) Charette, A. B.; Cote, B. Stereoselective Synthesis of All Four
Isomers of Coronamic Acid: A General Approach to 3-Methanoamino
Acids. J. Am. Chem. Soc. 1995, 117, 12721−12732.
(15) Miyamura, S.; Itami, K.; Yamaguchi, J. Syntheses of Biologically
Active 2-Arylcyclopropylamines. Synthesis 2017, 49, 1131−1149.
(16) Belyk, K. M.; Xiang, B.; Bulger, P. G.; Leonard, W. R., Jr.;
Balsells, J.; Yin, J.; Chen, C. Enantioselective Synthesis of (1R,2S)-1-
Amino-2-vinylcyclopropanecarboxylic Acid Ethyl Ester (Vinyl-ACCA-
OEt) by Asymmetric Phase-Transfer Catalyzed Cyclopropanation of
(E)-N-Phenylmethyleneglycine Ethyl Ester. Org. Process Res. Dev.
2010, 14, 692−700.
(17) Lou, S.; Cuniere, N.; Su, B.-N.; Hobson, L. A. Concise
asymmetric synthesis of a (1R,2S)-1-amino-2-vinylcyclopropanecar-
boxylic acid-derived sulfonamide and ethyl ester. Org. Biomol. Chem.
2013, 11, 6796−6805.
Present Address
§Present address: Merck Research Laboratories, Merck & Co.,
P.O. Box 2000, Rahway, NJ 07065, USA.
Author Contributions
The manuscript was written with contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding
This work was supported in part by the National Science
Foundation, Division of Molecular and Cellular Biosciences
(Grant No. MCB-1513007) and the Defense Advanced
Research Projects Agency Biological Robustness in Complex
Settings Contract HR0011-15-C-0093. O.F.B. acknowledges
support from the Deutsche Forschungsgemeinschaft (DFG
Grant No. BR 5238/1-1) and the Swiss National Science
Foundation (SNF Grant No. P300PA-171225). C.K.P. thanks
the Resnick Sustainability Institute for a postdoctoral fellow-
ship. A.M.K. and Z.W. acknowledge support from the NSF
Graduate Research Fellowship (Grant No. 1745301), and
A.M.K. acknowledges support from Caltech’s Center for
Environmental Microbial Interactions.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Stephan Hammer for help with chiral separations
of cyclopropylamines and all members of the Arnold
Laboratory for stimulating comments and discussions. We
thank Dr. David Rozzell for suggestions on cyclopropanation
substrate scope.
(18) Beaulieu, P. L.; Gillard, J.; Bailey, M. D.; Boucher, C.; Duceppe,
J.-S.; Simoneau, B.; Wang, X.-J.; Zhang, L.; Grozinger, K.; Houpis, I.;
Farina, V.; Heimroth, H.; Krueger, T.; Schnaubelt, J. Synthesis of
(1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic Acid Vinyl-ACCA)
2633
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634

ACS Catalysis
Letter
Derivatives: Key Intermediates for the Preparation of Inhibitors of the
Hepatitis C Virus NS3 Protease. J. Org. Chem. 2005, 70, 5869−5879.
(19) Bassan, E. M.; Baxter, C. A.; Beutner, G. L.; Emerson, K. M.;
Fleitz, F. J.; Johnson, S.; Keen, S.; Kim, M. M.; Kuethe, J. T.; Leonard,
W. R.; Mullens, P. R.; Muzzio, D. J.; Roberge, C.; Yasuda, N.
Multikilogram-Scale Synthesis of a Chiral Cyclopropanol and an
Investigation of the Safe Use of Lithium Acetylide−Ethylene Diamine
Complex. Org. Process Res. Dev. 2012, 16, 87−95.
(20) Abu-Elfotoh, A.-M.; Phomkeona, K.; Shibatomi, K.; Iwasa, S.
Asymmetric Inter- and Intramolecular Cyclopropanation Reactions
Catalyzed by a Reusable Macroporous-Polymer-Supported Chiral
Ruthenium(II)/Phenyloxazoline Complex. Angew. Chem., Int. Ed.
2010, 49, 8439−8443.
(21) Chanthamath, S.; Phomkeona, K.; Shibatomi, K.; Iwasa, S.
Highly stereoselective Ru(II)−Pheox catalyzed asymmetric cyclo-
propanation of terminal olefins with succinimidyl diazoacetate. Chem.
Commun. 2012, 48, 7750−7752.
(22) Chanthamath, S.; Nguyen, D. T.; Shibatomi, K.; Iwasa, S. Highly
Enantioselective Synthesis of Cyclopropylamine Derivatives via
Ru(II)-Pheox-Catalyzed Direct Asymmetric Cyclopropanation of
Vinylcarbamates. Org. Lett. 2013, 15, 772−775.
(23) Chanthamath, S.; Mandour, H. S. A.; Tong, T. M. T.;
Shibatomi, K.; Iwasa, S. Highly stereoselective cyclopropanation of
diazo Weinreb amides catalyzed by chiral Ru(II)−Amm−Pheox
complexes. Chem. Commun. 2016, 52, 7814−7817.
(24) Xie, M.-S.; Zhou, P.; Niu, H.-Y.; Qu, G.-R.; Guo, H.-M.
Enantioselective Intermolecular Cyclopropanations for the Synthesis
of Chiral Pyrimidine Carbocyclic Nucleosides. Org. Lett. 2016, 18,
4344−4347.
(25) Denton, J. R.; Davies, H. M. L. Enantioselective Reactions of
Donor/Acceptor Carbenoids Derived from α-Aryl-α-Diazoketones.
Org. Lett. 2009, 11, 787−790.
Ligated Cytochrome P450 Enables the Enantioselective Formal
Synthesis of Levomilnacipran. Angew. Chem., Int. Ed. 2014, 53,
6810−6813.
(37) Bajaj, P.; Sreenilayam, G.; Tyagi, V.; Fasan, R. Gram-Scale
Synthesis of Chiral Cyclopropane-Containing Drugs and Drug
Precursors with Engineered Myoglobin Catalysts Featuring Comple-
mentary Stereoselectivity. Angew. Chem., Int. Ed. 2016, 55, 16110−
16114.
(38) Hernandez, K. E.; Renata, H.; Lewis, R. D.; Kan, S. B. J.; Zhang,
C.; Forte, J.; Rozzell, D.; McIntosh, J. A.; Arnold, F. H. Highly
Stereoselective Biocatalytic Synthesis of Key Cyclopropane Inter-
mediate to Ticagrelor. ACS Catal. 2016, 6, 7810−7813.
(39) Knight, A. M.; Kan, S. B. J.; Lewis, R. D.; Brandenberg, O. F.;
Chen, K.; Arnold, F. H.: Diverse engineered heme proteins enable
stereodivergent cyclopropanation of unactivated alkenes. ACS Cent.
Sci., in press. DOI: 10.1021/acscentsci.7b00548.
(40) Key, H. M.; Dydio, P.; Clark, D. S.; Hartwig, J. F. Abiological
catalysis by artificial haem proteins containing noble metals in place of
iron. Nature 2016, 534, 534−537.
(41) Key, H. M.; Dydio, P.; Liu, Z.; Rha, J. Y.-E.; Nazarenko, A.;
Seyedkazemi, V.; Clark, D. S.; Hartwig, J. F. Beyond Iron: Iridium-
Containing P450 Enzymes for Selective Cyclopropanations of
Structurally Diverse Alkenes. ACS Cent. Sci. 2017, 3, 302−308.
(42) Reynolds, E. W.; Schwochert, T. D.; McHenry, M. W.; Watters,
J. W.; Brustad, E. M. Orthogonal Expression of an Artificial
Metalloenzyme for Abiotic Catalysis. ChemBioChem 2017, 18,
2380−2384.
(43) Gober, J. G.; Ghodge, S. V.; Bogart, J. W.; Wever, W. J.;
Watkins, R. R.; Brustad, E. M.; Bowers, A. A. P450-Mediated Non-
natural Cyclopropanation of Dehydroalanine-Containing Thiopep-
tides. ACS Chem. Biol. 2017, 12, 1726−1731.
(44) Renata, H.; Lewis, R. D.; Sweredoski, M. J.; Moradian, A.; Hess,
S.; Wang, Z. J.; Arnold, F. H. Identification of Mechanism-Based
Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineer-
ing of Improved Enzymes. J. Am. Chem. Soc. 2016, 138, 12527−12533.
(45) Wolf, M. W.; Vargas, D. A.; Lehnert, N. Engineering of RuMb:
Toward a Green Catalyst for Carbene Insertion Reactions. Inorg.
Chem. 2017, 56, 5623−5635.
(46) Kan, S. B. J.; Huang, X.; Gumulya, Y.; Chen, K.; Arnold, F. H.
Genetically programmed chiral organoborane synthesis. Nature 2017,
552, 132−136.
(47) Chawner, S. J.; Cases-Thomas, M. J.; Bull, J. A. Divergent
Synthesis of Cyclopropane-Containing Lead-Like Compounds, Frag-
ments and Building Blocks through a Cobalt Catalyzed Cyclo-
propanation of Phenyl Vinyl Sulfide. Eur. J. Org. Chem. 2017, 2017,
5015−5024.
(48) Oliver, C. F.; Modi, S.; Sutcliffe, M. J.; Primrose, W. U.; Lian, L.
Y.; Roberts, G. C. A Single Mutation in Cytochrome P450 BM3
Changes Substrate Orientation in a Catalytic Intermediate and the
Regiospecificity of Hydroxylation. Biochemistry 1997, 36, 1567−1572.
(49) Vottero, E.; Rea, V.; Lastdrager, J.; Honing, M.; Vermeulen, N.
P.; Commandeur, J. N. Role of residue 87 in substrate selectivity and
regioselectivity of drug-metabolizing cytochrome P450
CYP102A1M11. J. Biol. Inorg. Chem. 2011, 16, 899−912.
(26) Sambasivan, R.; Ball, Z. T. Screening Rhodium Metallopeptide
Libraries “On Bead”: Asymmetric Cyclopropanation and a Solution to
the Enantiomer Problem. Angew. Chem., Int. Ed. 2012, 51, 8568−8572.
́
(27) Garcia, P.; Diez, D.; Anton, A. B.; Garrido, N. M.; Marcos, I. S.;
Basabe, P.; Urones, J. G. Stereoselective Synthesis of Cyclopropanols.
Mini-Rev. Org. Chem. 2006, 3, 291−314.
(28) Moreau, B.; Charette, A. B. Expedient Synthesis of Cyclo-
propane α-Amino Acids by the Catalytic Asymmetric Cyclopropana-
tion of Alkenes Using Iodonium Ylides Derived from Methyl
Nitroacetate. J. Am. Chem. Soc. 2005, 127, 18014−18015.
(29) Lindsay, V. N. G.; Lin, W.; Charette, A. B. Experimental
Evidence for the All-Up Reactive Conformation of Chiral Rhodium-
(II) Carboxylate Catalysts: Enantioselective Synthesis of cis-Cyclo-
propane α-Amino Acids. J. Am. Chem. Soc. 2009, 131, 16383−16385.
(30) Coelho, P. S.; Brustad, E. M.; Kannan, A.; Arnold, F. H. Olefin
Cyclopropanation via Carbene Transfer Catalyzed by Engineered
Cytochrome P450 Enzymes. Science 2013, 339, 307−310.
(31) Coelho, P. S.; Wang, Z. J.; Ener, M. E.; Baril, S. A.; Kannan, A.;
Arnold, F. H.; Brustad, E. M. A serine-substituted P450 catalyzes
highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol.
2013, 9, 485−487.
(32) Heel, T.; McIntosh, J. A.; Dodani, S. C.; Meyerowitz, J. T.;
Arnold, F. H. Non-natural Olefin Cyclopropanation Catalyzed by
Diverse Cytochrome P450s and Other Hemoproteins. ChemBioChem
2014, 15, 2556−2562.
(33) Bordeaux, M.; Tyagi, V.; Fasan, R. Highly Diastereoselective and
Enantioselective Olefin Cyclopropanation Using Engineered Myoglo-
bin-Based Catalysts. Angew. Chem., Int. Ed. 2015, 54, 1744−1748.
(34) Tinoco, A.; Steck, V.; Tyagi, V.; Fasan, R. Highly Diastereo- and
Enantioselective Synthesis of Trifluoromethyl-Substituted Cyclo-
propanes via Myoglobin-Catalyzed Transfer of Trifluoromethylcar-
bene. J. Am. Chem. Soc. 2017, 139, 5293−5296.
(35) Brandenberg, O. F.; Fasan, R.; Arnold, F. H. Exploiting and
engineering hemoproteins for abiological carbene and nitrene transfer
reactions. Curr. Opin. Biotechnol. 2017, 47, 102−111.
(36) Wang, Z. J.; Renata, H.; Peck, N. E.; Farwell, C. C.; Coelho, P.
S.; Arnold, F. H. Improved Cyclopropanation Activity of Histidine-
2634
DOI: 10.1021/acscatal.7b04423
ACS Catal. 2018, 8, 2629−2634