Origin of High Stereocontrol in Olefin Cyclopropanation Catalyzed by an Engineered Carbene Transferase

Article abstract of DOI:10.1021/acscatal.8b04073

Recent advances in metalloprotein engineering have led to the development of a myoglobin-based catalyst, Mb(H64V,V68A), capable of promoting the cyclopropanation of vinylarenes with high efficiency and high diastereo- and enantioselectivity. Whereas many enzymes evolved in nature often exhibit catalytic proficiency and exquisite stereoselectivity, how these features are achieved for a non-natural reaction has remained unclear. In this work, the structural determinants responsible for chiral induction and high stereocontrol in Mb(H64V,V68A)-catalyzed cyclopropanation were investigated via a combination of crystallographic, computational (DFT), and structure-activity analyses. Our results show the importance of steric complementarity and noncovalent interactions involving first-sphere active site residues, heme-carbene, and the olefin substrate in dictating the stereochemical outcome of the cyclopropanation reaction. High stereocontrol is achieved through two major mechanisms: first, by enforcing a specific conformation of the heme-bound carbene within the active site, and second, by controlling the geometry of attack of the olefin on the carbene via steric occlusion, attractive van der Waals forces, and protein-mediated π-π interactions with the olefin substrate. These insights could be leveraged to expand the substrate scope of the myoglobin-based cyclopropanation catalyst toward nonactivated olefins and to increase its cyclopropanation activity in the presence of a bulky α-diazo-ester. This work sheds light on the origin of enzyme-catalyzed enantioselective cyclopropanation, furnishing a mechanistic framework for both understanding the reactivity of current systems and guiding the future development of biological catalysts for this class of synthetically important, abiotic transformations.

Full text of DOI:10.1021/acscatal.8b04073

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Article
Origin of high stereocontrol in olefin cyclopropanation
catalyzed by an engineered carbene transferase
Antonio Tinoco, Yang Wei, Daniela M Carminati, John Bacik,
Eric J. Moore, Nozomi Ando, Yong Zhang, and Rudi Fasan
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04073 • Publication Date (Web): 28 Dec 2018
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Origin of high stereocontrol in olefin cyclopropanation
catalyzed by an engineered carbene transferase
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Antonio Tinocoa,†, Yang Weib,†, John-Paul Bacikc, Daniela M. Carminatia, Eric J. Moorea,
Nozomi Andoc, Yong Zhang*,b, and Rudi Fasan*,a
a Department of Chemistry, University of Rochester, Rochester, NY 14627, United States.
b Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ
07030, United States.
c Department of Chemistry, Princeton University, Princeton, NJ 08544, United States.
† These authors contributed equally to this work.
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ABSTRACT: Recent advances in metalloprotein engineering have led to the development of a
myoglobin-based catalyst, Mb(H64V,V68A), capable of promoting the cyclopropanation of
vinylarenes with high efficiency and high diastereo- and enantioselectivity. Whereas many
enzymes evolved in nature often exhibit catalytic proficiency and exquisite stereoselectivity, how
these features are achieved for a non-natural reaction has remained unclear. In this work, the
structural determinants responsible for chiral induction and high stereocontrol in
Mb(H64V,V68A)-catalyzed cyclopropanation were investigated via a combination of
crystallographic, computational (DFT), and structure-activity analyses. Our results show the
importance of steric complementarity and non-covalent interactions involving first-sphere active
site residues, heme-carbene, and the olefin substrate, in dictating the stereochemical outcome of
the cyclopropanation reaction. High stereocontrol is achieved through two major mechanisms.
First, by enforcing a specific conformation of the heme-bound carbene within the active site.
Second, by controlling the geometry of attack of the olefin on the carbene via steric occlusion,
attractive van der Waals forces and protein-mediated  interactions with the olefin substrate.
These insights could be leveraged to expand the substrate scope of the myoglobin-based
cyclopropanation catalyst toward non-activated olefins and to increase its cyclopropanation
activity in the presence of a bulky -diazo-ester. This work sheds first light into the origin of
enzyme-catalyzed enantioselective cyclopropanation, furnishing a mechanistic framework for
both understanding the reactivity of current systems and guiding the future development of
biological catalysts for this class of synthetically important, abiotic transformations.
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KEYWORDS: Olefin cyclopropanation, myoglobin, biocatalytic carbene transfer, heme carbenes, enantioselective
cyclopropanation, Density Functional Theory
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Introduction
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The development of engineered and artificial enzymes for ‘abiotic’ chemistry (i.e., reactions not
occurring in nature) has recently created new opportunities toward expanding the range of bond-
forming reactions accessible via biocatalysis.1-5 In particular, engineered heme-containing
proteins have emerged as a promising class of biological catalysts for mediating selective
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carbene transfer reactions, including olefin cyclopropanation,6-15 Y—H insertion (where Y = N,
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S, or Si),16-20 C—H functionalization,21-23 aldehyde olefination,24,
and other relevant
transformations.26, 27 Inspired by the exquisite chemo-, regio- and stereoselectivity of many
naturally occurring enzyme-catalyzed reactions,28-30 efforts in this area have often involved re-
engineering of the target protein and its active site to modulate the diastereoselectivity and/or
stereoselectivity of the carbene transfer process. The asymmetric cyclopropanation of olefins is
of particular interest as it provides access to conformationally constrained and stereochemically
rich cyclopropane rings, which represent key building blocks for the discovery and development
of drugs and other biologically active molecules.31 Using myoglobin (Mb) as hemoprotein
scaffold, our group has recently reported the development of an engineered biocatalyst,
Mb(H64V,V68A), which offers high catalytic activity (>10,000 turnovers) as well as excellent
diastereo- and enantioselectivity (>95-99% detrans and ee1S,2S) for the cyclopropanation of
vinylarenes in the presence of ethyl -diazoacetate (EDA) as the carbene donor.8 This Mb
variant, along with a stereocomplementary variant thereof, could be applied to the
enantioselective synthesis of a panel of cyclopropane-containing drugs at the gram scale.9 More
recently, the scope of the Mb(H64V,V68A) biocatalyst was extended to enable highly
enantioselective and efficient olefin cyclopropanations in the presence of other acceptor-only
diazo reagents, such as 2-diazotrifluoroethane32 and diazoacetonitrile.33 Because of its synthetic
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utility and ability to process a broad range of aryl-substituted olefins with a predictable trans-
(1S,2S)-selectivity,8, 9, 32-34 Mb(H64V,V68A) represents an ideal system for investigating general
stereoselectivity-determining factors in biocatalytic cyclopropanation beyond the confines of a
single substrate-enzyme pair.
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Based on previous studies, myoglobin-catalyzed cyclopropanation is predicated to proceed via
the formation of a reactive heme-bound carbene intermediate, which arises from reaction of the
35-37
catalytically active ferrous hemoprotein with the diazo reagent (Figure 1).8,
Recent
experimental and computational analyses have further revealed that the reaction between this
species and the olefin substrate involves a concerted, asynchronous olefin insertion process
(Figure 1),36 which is distinct from the step-wise, radical cyclopropanation mechanism exhibited
by other metalloporphyrin systems.38, 39 Furthermore, these studies elucidated the beneficial role
of the axial imidazole ligand from the heme-ligating proximal histidine residue toward enhancing
the cyclopropanation reactivity of this metalloprotein.36 In other studies, replacement of the axial
histidine ligand with natural amino acids22 or the unnatural amino acid N-methyl-histidine40 was
found to be beneficial toward increasing the carbene transfer activity of this myoglobin variant
under non-reducing conditions.
FeIII
O
NHis
e-
OR
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COOR
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NHis
Ph
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NHis
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Figure 1. Mechanism of myoglobin-catalyzed olefin cyclopropanation with -diazo esters.
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Despite this progress, the structural determinants underlying protein-mediated chiral induction
and stereocontrol in these reactions have remained largely unknown. Recently, a computational
study was performed on two P450 variants with divergent cis:trans selectivity in styrene
cyclopropanation with EDA, but these analyses provided only a qualitative assessment of a
change in diastereoselectivity resulting from substitution of the proximal ligand (Cys vs. Ser).41
In this work, crystallographic, computational, and structure-activity analyses were carried out to
clarify the basis of high diastereo- and enantiocontrol in Mb(H64V,V68A)-catalyzed olefin
cyclopropanation. These studies shed first-time mechanistic insights into asymmetric
cyclopropanation catalyzed by a metalloprotein, thus providing a basis for rationalizing the
reactivity of current systems and guiding further reaction and catalyst development. Illustrating
this point, the insights gained from these analyses could be leveraged to further improve the
substrate scope of the myoglobin-based cyclopropanation catalyst across unactivated olefin
substrates and a bulky diazo reagent.
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Results and Discussion
Crystallographic analysis of Mb(H64V,V68A)
Compared to wild-type sperm whale myoglobin (Mb), Mb(H64V,V68A) features two amino
acid substitutions at the level of the ‘distal’ histidine residue (His64) and a second residue
(Val68) located on the distal side of the heme cofactor. Previous studies showed that the H64V
mutation mainly enhances the catalytic activity of the metalloprotein in the cyclopropanation of
styrene with ethyl -diazo-acetate (EDA) with minimal effect on the diastereo- and
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enantioselectivity of the reaction (93% de, 10% ee with Mb(H64V) vs. 86% de, 6% ee with wild-
type Mb for trans-(1S,2S)-configured product).9 The Val68Ala mutation, on the other hand, was
found to play a key role in enhancing both the diastereo- and enantioselectivity of the
metalloprotein in this reaction, as derived from direct comparison of the selectivity properties of
Mb(H64V,V68A) (99.9% de and 99.9% ee) with those of Mb(H64V).9
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To gain insights into the effects of these mutations on the protein structure, ferric
Mb(H64V,V68A) (acquo-complex) was crystallized and its X-ray crystal structure was
determined at 1.1 Å resolution. Superposition of the Mb(H64V,V68A) structure with that of wild
type Mb42 (CO-complex) crystallized in the same space group (P6), showed an RMSD of 0.3 Å
as calculated with Dali43, indicating that the protein three-dimensional fold is not greatly affected
by the mutations. Furthermore, hydrogen bond distances between the protein and heme as well as
a network of hydrophobic interactions appear to be maintained when comparing the Mb variant
to the wild-type structure (PDB: 1JW8) (Figure 2A). At the same time, the two mutations in
Mb(H64V,V68A) expand the volume of the heme distal pocket (Figure 2B), resulting in a
solvent accessible volume of 243 Å3 for Mb(H64V,V68A) compared with 125 Å3 for wild-type
Mb as calculated using DoGSiteScorer44 (Figure 3). In particular, the mutations create a larger
void above the solvent-exposed half (= rings C/D) of the porphyrin (H64V) and above ring A
and the meso position between rings A and D (V68A) (Figures 2C and 3A). This configuration
allows greater substrate accessibility to the distal pocket in the hemoprotein (Figure 3A).
Furthermore, the His64→Val mutation contributes to enhance the hydrophobicity near the heme-
bound iron. Altogether, these structural features are likely beneficial for promoting binding to the
diazo compound and olefin substrate, providing conditions conducive to the cyclopropanation
reaction. Further inspection of the crystal structure showed that the side chains of four residues,
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i.e., Val64, Ala68, Phe43, and Ile107, define the space above the plane of the iron porphyrin
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(Figures 2C).
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Figure 2. High-resolution crystal structure of sperm whale Mb(H64V,V68A) variant (top)
and comparison to wild-type Mb (bottom; PDB:1JW8). A) Arrangement of amino acids
surrounding the heme cofactor. The axial water ligand in Mb(H64V,V68A) and axial CO ligand
in Mb are displayed as sphere (red) and stick model, respectively. B) View of the molecular
surface representation around the heme binding site. C) Top view of the bound heme and nearby
amino acid residues, along with the labels for pyrrole N atoms and porphyrin rings. Carbon
atoms are shown as yellow (heme), gray (double-mutant structure), cyan (wild-type structure) or
salmon (mutated residues), nitrogens are blue and oxygens red. Inter-residue distances are
measured in angstroms and shown as dashed lines. In the top panel of (C), riding model
hydrogen atoms are shown as thin white sticks. The very high resolution of the data allowed for a
limited number of hydrogens to be discerned in the electron density maps, and thus their
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positions were added throughout the model, except in the case of solvent molecules. The 2mFo-
DFc electron density map surrounding the heme is shown at 1. See Table S1 for
crystallographic parameters.
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Figure 3. Pocket cavity space (mesh) above the heme cofactor in (A) Mb(H64V,V68A) and (B)
wild-type sperm whale myoglobin. The pocket volumes were calculated to correspond to 243 Å3
and 125 Å3, respectively, based on the crystallographic structure atom coordinates and a
difference of Gaussian filter method (DoGSiteScorer).44
Model of Mb(H64V,V68A) active site and DFT method
To gain insights into the effect of the protein matrix in controlling the stereoselectivity of Mb-
catalyzed cyclopropanation, a model of Mb(H64V,V68A) active site was built for performing
computational analysis of the carbene intermediate and transition states via Density Functional
Theory (DFT). Guided by crystallographic information, this model was chosen to include the full
heme cofactor (with the propionic groups substituted for propyl groups to avoid artificial H-
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bonding interactions), the axial His93 ligand, and the ‘first-sphere active site residues’45 Leu29,
Phe43, Phe46, Val64, Ala68, and Ile107 (C(amino acid)···Fe(heme) distances from 5.1 Å
(Ala68) to 13.2 Å (Phe46)); Figure 2A). Each residue was truncated at the Cα position and each
Cα atom fixed at the position found in the reference X-ray crystal structure to mimic the protein
environment, as done previously in the context of other metalloproteins.46, 47 The atoms in the
models were allowed to be optimized using a range-separated hybrid DFT method with
dispersion correction, B97XD,48 based on its excellent performance on heme carbenes and
other catalytic systems from previous methodological studies.35, 36, 49-51 Considering the large size
of this model, it was divided into high and low levels, with the former containing the core part of
the reaction system (iron porphyrin, His93 ligand, [:CHCO2Et] carbene, and styrene) and the low
level containing all other atoms. The basis set for the high level part of the system includes the
effective core potential (ECP) basis LanL2DZ52 for iron and the triple-zeta basis 6-311G(d) for
all other elements in this level, which was found to provide accurate predictions of various
experimental reaction properties of heme carbenes.35, 36, 51 For all atoms in the low level, 6-
31G(d) was used. More details about the DFT method can be found in the Supporting
Information.
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Analysis of carbene conformations
Using this system, an extensive conformational analysis was first conducted to identify the
most favorable orientation(s) of the carbene moiety in the active site of Mb(H64V,V68A). Given
the asymmetry of the protein active site and two possible alignments of carbene groups found in
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structurally characterized carbene-iron porphyrin complexes,53,
eight conformations were
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considered, in which the initial carbene plane is aligned either along each of the porphyrin N—
Fe axes or in-between each of the two neighboring N—Fe axes. These conformations are defined
by N1FeC1C dihedral angle values range from -135° to +180° with an interval of 45°, where N1
is the nitrogen atom of pyrrole ring A of the heme, C1 is the iron-bound carbon atom, and C is
the carbonyl carbon atom (see Table 1 for data entries and Figure 4A for atom labels). Since
rotation of the carbene-borne carboxylate group yields two additional conformers, herein referred
to as (+) and (-) based on HC1CO1 dihedral angle (Figure 4), a total of sixteen conformations
were calculated. After geometry optimization, the sixteen initial conformations could be divided
into four groups (Table 1), with the carbene-borne ester group occupying four distinct inter-
residue regions proximal to the porphyrin ring (Figure 2C), namely the regions between
Ala68…Ile107, between Ile107…Phe43, between Phe43…Val64, or between Val64…Ala68
(Table 1). Analyses of the relative energies associated with these conformational isomers
revealed that conformation 2(+) (Figure 5), in which the carbene ester group is located between
Ala68 and Ile107, is most favorable, whereas other conformations have ~3-10 kcal/mol higher
Gibbs free energies (G) (Table 1). The only exception was conformation 1(+), which after
optimization converges to a conformation geometrically and energetically identical to that
derived from 2(+). These results indicate that steric effects exerted by the protein matrix play a
major role in controlling the conformation of the heme-bound carbene within the protein active
site, with the lowest energy conformer, 2(+), occupying the region with the second largest inter-
residue distance (Figure 2C). Meanwhile, it became apparent that other factors also influence
the orientation of the carbene moiety. Indeed, conformations 3(+) and 6(+) are energetically
similar (G = +3.4 vs. 3.2 kcal mol-1 relative to 2(+)), despite the fact that the carbene moiety in
3(+) occupies a sterically less hindered region (~2.6 Å wider inter-residue distance; Figure 2C).
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Conversely, the carbene groups in conformations 3(-) and 6(+) occupy regions of similar steric
accessibility, yet the former has about 3 kcal/mol higher energy than the latter.
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Figure 4. Conformations of the heme-bound carbene upon rotation of the ester groups. The two
conformations are referred to as (+) (A) and (-) (B) in accordance to the sign of the HC1CO1
dihedral angle.
A more detailed analysis of the lowest-energy conformers within each group indicated that van
der Waals (vdW) interactions mediated by the carbene-borne ester group contribute to
stabilization of the carbene conformations. Indeed, the most favorable conformation 2(+)
encompasses a total of four short-distance contacts (2.4-2.6 Å) between the oxygen atoms of the
ester group (O1 and O2) and C—H bonds of neighboring residues (Ala68, Ile107, Leu29, Phe43)
(Figure 5A and Table S2). These distances are smaller than the sum of Bondi’s vdW radii for O
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and H (2.72 Å),55,
indicating the occurrence of favorable interatomic interactions. In
comparison, 3(+) and 6(+), i.e., the lowest-energy conformations within the I107…F43 and
V64…A68 regions, respectively, exhibit only two short-distance contacts of this type (Table
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S2). Conformer 3(-), i.e., the preferred conformer within the F43…V64 region, also displays two
short O(carbene)····H(residue) contacts, but the corresponding distances (2.6-2.7 Å) are longer
than those in 3(+) and 6(+), providing a plausible explanation for its lower stability compared to
the latter. For all optimized conformations, no short contacts were observed between the carbene
moiety and the side chains of Val64 and Phe46, indicating that Phe43, Leu29, Ala68, and Ile107
(Figure 2C) are primarily involved in affecting the orientation and stability of the heme-bound
carbene.
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Table 1. Relative energies and initial and optimized dihedral angles for the 16 carbene
conformations within Mb(H64V,V68A) active site. The conformations are numbered 1 through 8
and (+)/(-) according to the initial N1FeC1C and HC1CO1 dihedral angles, respectively, and
grouped according to the inter-residue region occupied by the carbene-borne ester group. The
reported energy values are relative to the lowest-energy conformation, 2(+).
Initial
Optimized
Confor
mation
Region
∆E ∆EZPE ∆H ∆G
HC1CO1
N1FeC1C N1FeC1C
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(kcal/ (kcal/ (kcal/ (kcal/
mol) mol) mol) mol)
(°)
(°)
(°)
I107…F43
1(-)
2(-)
3(+)
4(+)
3(-)
4(-)
5(+)
5(-)
6(-)
6(+)
7(+)
8(+)
7(-)
8(-)
1(+)
2(+)
0.0
15.4
-113.2
3.5
3.2 1.4 10.4
7
8
9
45.0
90.0
44.8
140.3
133.1
125.0
124.3
-148.0
168.4
-122.5
-96.2
-94.7
-101.9
-70.0
-63.3
40.4
-97.7
102.0
97.3
-1.1 -1.8 -3.4 4.3
0.4
2.5
0.4 -0.3 3.4
2.6 0.9 8.8
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60
135.0
90.0
F43…V64
V64…A68
-98.0
-100.6
99.1
-2.2 -2.2 -4.2 6.1
135.0
180.0
180.0
-135.0
-135.0
-90.0
-45.0
-90.0
-45.0
0.0
0.6
3.1
1.0
3.5
0.8 -0.2 6.2
2.3 0.9 6.4
0.6 -0.7 6.3
3.0 1.4 7.5
-84.4
-91.0
92.2
-0.9 -1.4 -2.5 3.2
0.3 -0.5 -1.6 3.9
90.2
84.8
0.6
0.6 -1.3 6.3
A68…I107
-104.6
-102.0
95.5
-2.3 -2.6 -3.8 2.7
-2.3 -2.8 -5.2 7.0
-0.2 -0.9 -0.7 0.3
0.00 0.00 0.00 0.00
45.0
43.1
92.9
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Figure 5. Side view (A) and top view (B) of the most favorable conformation 2(+) for the heme-
bound carbene in Mb(H64V,V68A) active site.
These results are consistent with previous observations that the improved stereoselectivity of
Mb(H64V,V68A), compared to wild-type Mb, is largely dictated by the V68A mutation rather
than the H64V mutation.9 Altogether, the analyses above showed that the overall best
conformation 2(+) arises from a good steric complementary between the carbene group and the
inter-residue space above the plane of the heme cofactor (Figure 5) combined with a larger
number of favorable non-covalent interactions between the carbene and surrounding residues.
Stereodivergent pathways to cyclopropanation product
Having identified the most favorable orientation of the carbene group within the
Mb(H64V,V68A) active site, we investigated the transition states leading to the four possible
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stereoisomers of the cyclopropanation product (i.e., TS1S,2S, TS1R,2R, TS1S,2R, TS1R,2S). For these
analyses we used the singlet concerted reaction pathway, as the relevance of this mechanism for
Mb-catalyzed cyclopropanation was established previously.36 The optimized structures for the
four TS and corresponding Gǂ are shown in Figure 6 and Table 2, respectively. Based on
these data, the Mb(H64V,V68A)-catalyzed reaction involving styrene and EDA is predicted to
occur with a diastereomeric excess (de) of 99.9% and an enantiomeric excess (ee) of 99.8% for
the trans-(1S,2S) configured cyclopropanation product. These values are in excellent agreement
with the experimentally determined values of 99.9% de and 99.9% ee (Table 3, Entry 1).8 The
trans isomers have significantly lower barriers than the cis isomers (Gǂ from -5.2 to -5.9 kcal
mol-1; Table 2). This energetic difference was also observed in a protein-free model of heme-
catalyzed cyclopropanation36 and originates from favorable  interactions and vdW contacts
occurring in the TStrans vs. TScis. Whereas this result is consistent with the trans preference of
iron-porphyrins in this reaction,57-59 this effect is greatly amplified within the Mb(H64V,V68A)
active site due to steric clashes between the styrene ring and the side chain of Leu29, thus
providing a basis for the significantly higher trans-diastereoselectivity exhibited by
Mb(H64V,V68A) compared to hemin alone (99.9% vs. 87% de)8. To further probe this
conclusion, a Mb(H64V,V68A)-derived variant was prepared in which the steric bulk at the level
of Leu29 is reduced via substitution of this amino acid residue with alanine. The resulting Mb
variant, Mb(L29A,H64V,V68A), was found to catalyze the cyclopropanation of styrene with
EDA with 10-fold lower trans-diastereoselectivity compared to Mb(H64V,V68A) (50:1 vs.
>500:1 trans:cis ratio; Table 3, Entry 10 vs. 1), thereby providing experimental support to the
functional role of Leu29 side chain in controlling the diastereoselectivity (and enantioselectivity,
vide infra) of the cyclopropanation reaction.
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Side-by-side comparison of TS1S,2S vs. TS1R,2R structures provided further insights into
the determinants responsible for the high (1S,2S)-enantioselectivity of Mb(H64V,V68A). Most
notably, the TS1S,2S structure revealed a  stacking interaction between the phenyl ring of
styrene and that of Phe43 (Figure 6, panel A, red line), which is absent in all of the other
transition states (Figure 6, panels B and D). This interaction is made possible through a ~90°
rotation of Phe43 phenyl ring around the C-C bond (2 bond), as evinced from comparison of
the structure of TS1S,2S with the crystal structure of Mb(H64V,V68A) (Figure 6A vs. 2A).
Intriguingly, this protein-olefin substrate interaction can explain the broad substrate scope of
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8
Mb(H64V,V68A) across aryl-substituted olefins, whereas no reactivity was observed with
alkenes lacking an aromatic substituent (e.g., 1-octene),8, 9 despite the latter are viable substrates
for hemoprotein-catalyzed cyclopropanation.13 In addition to the aforementioned  interaction,
two vdW contacts between the carbene moiety and the styrene substrate are present in TS1S,2S
(O1(carbene)…H(styrene): 2.5/2.5 Å), in contrast with only one in TS1R,2R
(O2(carbene)…H(styrene): 2.6 Å). In TS1S,2S, other favorable vdW force involve the O2 atom and
residue Phe43 (2.6 Å) and Leu29 (2.7 Å), compared to only one in TS1R,2R (O2…H(Ala68): 2.6
Å). Regarding the two transition states leading to the cis-configured cyclopropanation products,
only TS1S,2R displays a favorable O1(carbene)…H(styrene) contact (2.6 Å) that stabilizes the
substrate. While this interaction is similar to that present in TS1R,2R, TS1S,2R involves more
important steric clashes between the carbene group and Ile107, as suggested by the shorter
H(carbene)…H(Ile107) distance of 2.3 Å vs. 4.0 Å in TS1R,2R. Altogether, these analyses indicate
that (i) stabilization of the carbene conformation as a result of residue-carbene interactions and
(ii) close-range weak interactions between the active site residues and the styrene substrate,
jointly contribute to direct and favor the attack of styrene to the si face of the heme-bound
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carbene, thereby dictating the high trans-diastereoselectivity and (1S,2S)-enantioselectivity of
the Mb(H64V,V68A)-catalyzed cyclopropanation reaction. Furthermore, in TS1S,2S the phenyl
ring of styrene is projected toward the opening connecting the heme pocket to the solvent, which
is expanded by the H64V mutation (Figure 3A vs. 3B). This arrangement is expected to make
the chirality induced by Mb(H64V,V68A) in the cyclopropanation reaction rather independent of
structural variations at the level of the aromatic ring of the olefin, which can explain the
preserved trans-(1S,2S) selectivity of Mb(H64V,V68A) across a broad range of aryl-substituted
olefins as observed in previous studies.8
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Table 2. Energy barriers (kcal/mol) for the four stereodivergent pathways in Mb(H64V,V68A)-
catalyzed styrene cyclopropanation with EDA. ΔΔGǂ values are relative to TS1S,2S
.

ΔE
-17.7
-12.9
-14.6
-8.1
ΔEZPE
-14.4
-11.0
-11.7
-6.6
ΔH
-15.8
-13.5
-14.5
-8.0
ΔG
8.8
ΔΔG
0.0
TS1S,2S
TS1R,2R
TS1S,2R
TS1R,2S
12.9
14.0
14.7
4.2
5.2
5.9
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Figure 6. Free energy diagram for the cyclopropanation steo and optimized structures for the
transition states leading to the four possible stereoisomeric products in Mb(H64V,V68A)-
catalyzed styrene cyclopropanation with EDA: (A) TS1S,2S, (B) TS1R,2R, (C) TS1S,2R, and (D)
TS1R,2S.
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Structure-reactivity studies with -diazoester derivatives
Whereas the mechanistic scenario presented above is able to explain the enantioselectivity of
Mb(H64V,V68A) both qualitatively and quantitively, additional experiments were performed in
order to probe its validity. To this end, the Mb(H64V,V68A)-catalyzed styrene cyclopropanation
reaction was carried out with a series of probe diazo reagents (2b-2f, Table 3), which feature
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varying steric demands at the proximal (C1’) (2b, 2c) and distal carbon atom (C2’) of the alkyl
group (2d), as well as isosteric substitutions within the ester group (2e, 2f). Compared to EDA
(2a), the reactions with isopropyl (2b) and tert-butyl -diazoacetate (2c) show a progressive
decrease in yield, catalytic turnovers (>500 (3a) → 420 (3b) → 330 TON (3c); Table 3, Entries
2-3 vs. 1), turnover rate (kcat: 585 (3a) → 103 (3b) → 45 min-1 (3c)) and (1S,2S)-
enantioselectivity (99% (3a) → 83% (3b) → 59% ee (3c) as the steric bulk at the C1’ atom is
increased. In contrast, a more moderate reduction in all of these parameters was observed with
2’,2’,2’-trichloro-ethyl -diazoacetate (2d), which bears increased steric bulk at the C2’ atom
(Entry 4). These results are consistent with the preferred conformation of the heme-bound
carbene, 2(+) (Figure 4), in which close carbene-residue (Ile107) contacts occur at the level of
the C1’ atom, whereas a larger space is available around the C2’ atom. These structural
differences are expected to impose a lower tolerance of the C1’ over the C2’ position toward
substitution with larger group(s) (i.e., H→CH3/Cl) in the context of the cyclopropanation
reaction, an effect clearly reflected by the experimentally observed trend in catalytic activity and
selectivity across the diazoesters 2a-2d.
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Based on the results above, it was surmised that increasing the active site space around residue
68 should better accommodate diazo reagents with a bulkier alkyl chain such as 2c. To probe
these predictions, a variant carrying a Ala→Gly substitution at this position, Mb(H64V,V68G),
was tested in the styrene cyclopropanation reaction in the presence of 2a, 2b, or 2c. Gratifyingly,
the reaction with tert-butyl -diazoacetate (2c) shows a significantly higher trans-
diastereoselectivity compared to those with EDA (2a) or isopropyl -diazoacetate (2b) (95% vs.
69-77% de; Table 3, Entries 7-9), indicating a better match between the bulkier tert-butyl group
on the carbene and the further enlarged cavity between residue 68 and 107 (Figure S1), and
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consequently, a less optimal match between the latter and the smaller alkyl groups in the 2a- and
2b-derived carbenes. This effect is also evident from the enhanced styrene cyclopropanation
activity of Mb(H64V,V68G) in the presence of tert-butyl -diazoacetate compared to
Mb(H64V,V68A) with respect to yield (99% vs. 66%) and TON (>500 vs. 330). At the same
time, the enantioselectivity of this reaction is drastically reduced compared to that with the other
-diazo acetates, leading to formation of the two enantiomeric trans-cyclopropanes in nearly
equal amounts (2% vs. 76-96% ee). These results can be rationalized in view of the calculated
transition states leading to the (1S,2S)- and (1R,2R)-configured cyclopropane products (Figure 6,
panels A and B). Indeed, the bulkier tert-butyl group significantly increases steric hindrance at
the level of the si face of the heme-bound carbene compared to an ethyl or isopropyl group, as
evidenced by a model of TS1S,2S involving heme-bound [:CHCO2(tBu)] carbene (Figure S1).
This effect is expected to disfavor styrene attack to the si vs. the re face of the carbene, resulting
in the drastically reduced enantioselectivity for this reaction as observed experimentally.
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Consistent with the effect of steric bulk at the level of the carbene ester group on the catalyst
stereoselectivity, the cyclopropanation reaction with 2’,2’,2’-trifluoro-ethyl -diazoacetate (2e)
was found to proceed with comparably high efficiency and trans-(1S,2S)-selectivity as with 2a
(Table 3, Entry 5 vs. 1), as expected given the comparable size of the trifluoroethyl group vs.
ethyl group. At the same time, kinetic analyses showed that the apparent Michealis-Menten
constant (KM) of Mb(H64V,V68A) for 2e is nearly 4-fold lower than that for EDA (6.4 vs. 21.1
mM; Table 3). This effect is consistent with the trifluoroethyl group projecting toward the
hydrophobic core of the protein as in 2(+). Indeed, while being isosteric to EDA, the increased
lipophilicity of 2e should lead to a tighter interaction with the hemoprotein than EDA, a
phenomenon that would be reflected by a decrease in the corresponding KM value. Corroborating
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this trend and thus further substantiating the mechanistic model presented above, a progressive
decrease in KM was observed for the Mb(H64V,V68A)-catalyzed cyclopropanation reaction with
ethyl (2a) vs. isopropyl (2b) vs. tert-butyl -diazoacetate (21.1 → 7.3 → 3.0 mM; Table 3).
Furthermore, a reasonably good correlation (R2: 0.63) could be found between the KM values of
Mb(H64V,V68A) for 2a-2f and the hydrophobicity of these molecules, as estimated based on the
octanol:water partition coefficient of the corresponding acetate (thio)esters (Figure S3).
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Finally, the functional role of the ester group in protein-induced asymmetric induction was
probed using 2f, i.e., an EDA analog in which the ester oxygen atom (O1) is substituted for
sulfur. Despite this subtle (isosteric) substitution, the Mb(H64V,V68A)-catalyzed styrene
cyclopropanation reaction with 2f shows a significant reduction in yield (41% vs. 99%) and kcat
(62 vs. 585 min-1), along with a 20-fold lower selectivity for formation of the (1S,2S) enantiomer
compared to that with EDA (e.r. 11:1 (84% ee) vs. 200:1 (99.9% ee); Table 3, Entry 6 vs. 1). To
illuminate the basis of this effect, a model of the Mb(H64V,V68A) active site with heme-bound
[:CHCOSEt] carbene was built with the COSEt group located within the A68…I107 region
(same as the ethyl ester group in 2(+)), followed by optimization (Figure S2A). Based on the
computed transition state energies for the four stereoisomers in Table S3, the predicted de and ee
values for this reaction are 99.9% and 85%, respectively, which are in excellent agreement with
the experimental data (99% de and 84% ee). While the optimized TS1S,2S structure (Figure S2B)
was found to display some basic features of TS1S,2S obtained for the reaction with EDA, such as
the π-π interaction between styrene and Phe43 and attractive O…H interactions between carbene
and active site residues, only two attractive O(carbene)…H or S(carbene)…H interactions are
established with Phe43 (2.7 Å) and styrene (2.9 Å) respectively, as opposed to four in the TS1S,2S
structure for the reaction with EDA (Table S2). Furthermore, the contact distances with the
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[:CHCOSEt] group are longer than those with the EDA-derived carbene. These relatively less favorable
interactions may contribute to the increased computed ΔGǂ for TS1S,2S in the cyclopropanation reaction
with [:CHCOSEt] compared to that with [:CHCO2Et]. This difference is consistent with the
9
experimentally observed lower catalytic activity and rate of the Mb(H64,V68A)-catalyzed
cyclopropanation reaction with 2f compared to those with EDA (Table S4). In contrast with this
reduced reactivity for TS1S,2S due to the O→S substitution, the formation of TS1R,2R (Figure
S2C) actually becomes more favorable (Tables 2 and S3) as a result of three attractive
O(carbene)…H interactions with styrene (2.6/2.6 Å) and Ile107 (2.6 Å) compared to only two of
such interactions found for TS1R,2R in the reaction with EDA. Altogether, these effects reduce the
difference between the TS1S,2S and TS1R,2R barrier leading to reduced enantioselectivity for the
(1S,2S) product, as observed experimentally. Overall, these results corroborate the involvement
of the ester group in influencing the stereochemical outcome of the reaction as revealed by the
computational analyses for EDA and highlight the importance of weak interactions between the
carbene and the surrounding protein environment in controlling reactivity and selectivity in the
cyclopropanation reaction.
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Table 3. Activity, selectivity, and kinetic parameters for the cyclopropanation of styrene with -
5
6
diazoester derivatives catalyzed by Mb(H64V,V68A) and variants thereof.a
7
8
9
Mb variant
O
(0.2 mol%)
Y
R
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13
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R
Y
+
Na2S2O4
KPi buffer (pH 7.0)
anaerobic, r.t.
O
N2
Y
R
3a-3e
1
2a
)
O
O
O
O
O
S
CH2CH3 (
2b
CH(CH3)2 (
)
2c
C(CH3)3 (
)
2d
CH2CCl3 (
)
2e
CH2CF3 (
)
2f
CH2CH3 ( )
c
c
kcat
(min-1)
KM
Entry
1
Catalyst
Diazo Yieldb TON
% de % ee
(mM)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68G)
Mb(H64V,V68G)
Mb(H64V,V68G)
Mb(L29A,H64V,V68A)
2a
2b
2c
2d
2e
2f
99%
84%
66%
99%
99%
41%
99%
77%
99%
99%
>500
420
99.9
93
93
83
96
99
77
69
95
96
99.9
83
59
92
96
84
96
76
2
585
21.1
2
3
103
45
7.3
3.0
3.2
6.4
8.1
n.d.
n.d.
n.d.
n.d.
330
4
>500
>500
205
295
320
62
5
6
7
2a
2b
2c
2a
>500
385
n.d.
n.d.
n.d.
n.d.
8
9
>500
>500
10
84
a Reaction conditions: 10 mM styrene (1), 20 mM diazo reagent (2a-2f), 10 mM Na2S2O4, 20 M
purified Mb variant in phosphate buffer (50 mM, pH 7.0), under anaerobic conditions, room
b
c
temperature, 16 hours. GC yield. Apparent kinetic parameters for the carbene donor at sub-
saturating styrene concentration (20 mM) under aerobic conditions.
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Expanding the substrate scope of Mb(H64V,V68A)
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Previous studies showed Mb(H64V,V68A) catalyzes the cyclopropanation of a broad
range of vinyl arenes, but lacks activity on olefin substrates such as 1-octene and cyclohexene.8
These findings previously suggested that the substrate scope of this biocatalyst may be limited to
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electronrich aryl-substituted olefins, owing to their higher reactivity toward the electrophilic
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heme-carbene intermediate mediating these reactions.8,
However, in view of the now
revealed role of protein-mediated  interactions in favoring the cyclopropanation reaction with
styrene, we hypothesized that unactivated olefins bearing an aromatic substituent could also
constitute viable substrates for this biocatalyst. In agreement with our hypothesis, a
Mb(H64V,V68A)-catalyzed reaction in the presence of 4-phenyl-but-1-ene (5a) and EDA
showed that this substrate can be accepted by the Mb variant, yielding the corresponding
cyclopropanation product 5a with high trans-selectivity (95% de; 75% ee; Table 4, Entry 2). In
contrast, no activity was detected with 4-bromo-but-1-ene (4a). Since the C=C double bonds in
4a and 5a share similar electronic properties, the activity observed with 5a can be directly
ascribed to the beneficial effect of the aromatic substituent in favoring the Mb(H64V,V68A)-
mediated cyclopropanation of the unactivated olefin. Building upon these findings, the substrate
scope of the carbene transferase could be further extended to the transformation of other non-
styrenyl olefin substrates such as 6a-8a, enabling the synthesis of the corresponding
cyclopropanation products 6b-8b in modest to good yields (18-65%), and good to excellent
diastereo- and enantioselectivity (80-98% de; 74-99% ee) (Table 4, Entries 3-5). Altogether,
these results demonstrate how the mechanistic insight gained from the present studies have
enabled expansion of the substrate profile of Mb(H64V,V68A) as a cyclopropanation
biocatalyst.
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Table 4. Activity and selectivity for Mb(H64V,V68A)-catalyzed cyclopropanation of non-
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styrenyl olefins with EDA.a
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9
Mb(H64V,V68A)
O
(0.2-0.5 mol%)
O
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+
R
O
R
Na2S2O4
N2
2a
O
KPi buffer (pH 7.0)
anaerobic, r.t.
4a-8a
4b-8b
Entry
1
Olefin
Product
Yieldb TON
% de % ee
Br
Br
CO2Et
0%
0
-
-
4a
4b
CO2Et
2
3
13%
25
95
75
5a
5b
O
O
CO2Et
18%
65%
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95
98
86
99
6a
6b
O
4c
325
O
S
CO2Et
CO2Et
7a
7b
S
5c
40%
200
80
74
8a
8b
a Reaction conditions: 20 mM olefin, 10 mM EDA, 10 mM Na2S2O4, 50 M purified Mb variant
in phosphate buffer (50 mM, pH 7.0), under anaerobic conditions, room temperature, 16 hours. b
GC yield. c Using 20 M catalyst.
Conclusions
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To conclude, using crystallographic, computational, and reactivity/mutagenesis analyses, we
have gained first-time insights into the origin and structural determinants responsible for chiral
induction and high stereocontrol in olefin cyclopropanation catalyzed by an engineered
metalloprotein. These studies show that the excellent diastereo- and enantioselectivity of
Mb(H64V,V68A)-catalyzed cyclopropanation is granted through protein-mediated control on
both (i) the conformation of the heme-bound carbene; and (ii) the geometry and spatial
orientation of the transition state, via a combination of steric effects (i.e., shape
complementarity) as well as non-covalent vdW and  interactions between first-sphere active
site residues, the heme-bound carbene, and the olefin substrate. Importantly, the mechanistic
scenario developed here is able to reproduce experimentally observed enantioselectivities and
can explain structure-reactivity trends with different protein variants and diazo reagents. In
addition, the mechanistic insights gained through these studies could be leveraged to (i) expand
the substrate scope of the Mb(H64V,V68A) carbene transferase to include unactivated olefins
(5a, 6a) and (ii) design an alternative Mb-based biocatalyst, Mb(H64V,V68G), with higher
reactivity toward a bulkier diazo reagent than EDA (tert-butyl -diazoacetate). The present
findings enhance our understanding of enzyme-catalyzed asymmetric cyclopropanation and are
expected to have important implications toward guiding the future development of biological
catalysts for this important class of C—C bond forming reactions.
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EXPERIMENTAL DETAILS
General Information. All chemicals and reagents were purchased from commercial
suppliers (Sigma-Aldrich, ACS Scientific, Alfa Aesar, J.T. Baker) and used without any further
purification, unless otherwise stated. All reactions were carried out under argon pressure in oven-
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dried glassware with magnetic stirring using standard gas-tight syringes, cannulae and septa. 1H,
13C and 19F NMR spectra were measured on a Bruker DPX-400 instrument (operating at 400
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MHz for H, 100 MHz for 13C, and 375 MHz for 19F) or a Bruker DPX-500 instrument
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(operating at 500 MHz for H and 125 MHz for C). Tetramethylsilane (TMS) was used as the
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internal standard (0 ppm) for H NMR spectra, CDCl3 was used as the internal standard (77.0
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ppm) for C NMR spectra, and trifluorotoluene served as the internal standard (0 ppm) for F
NMR spectra. Flash column chromatography purification was carried out using AMD Silica Gel
60 Å 230-400 mesh. Thin Layer Chromatography (TLC) was carried out using Merck Millipore
TLC silica gel 60 F254 glass plates.
Molecular Cloning. pET22b(+) (Novagen) was used as the recipient plasmid vector for
expression of all of the myoglobin variants. In this construct, the gene encoding for sperm whale
myoglobin carrying a neutral D122N mutation60 is C-terminally fused to a polyhistidine tag and
it is under the control of an IPTG-inducible T7 promoter. The preparation of plasmids encoding
for Mb(H64V,V68A) and Mb(H64V,V68G) was described previously and that encoding for
Mb(L29A,H64V,V68A) was prepared using a similar procedure8, 9 and the mutagenizing primers
in Table S4. A pET22-based plasmid containing the gene encoding for Mb(H64V,V68A)
without the poly-histidine tag was prepared in a similar manner using Myo_XbaI_for and
XhoI_NoHisTag_rev primers.
Protein Expression and Purification. Engineered Mb variants were expressed in E. coli
C41(DE3) cells as described previously.9 Briefly, cells were grown in TB medium (ampicillin,
−1
100 mg L ) at 37 °C (200 rpm) until OD600 reached 1.0-1.2. Cells were then induced with 0.25
mM β-d-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-aminolevulinic acid (ALA). After
induction, cultures were shaken at 180 rpm and 27 °C and harvested after 18-20 hours by
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centrifugation at 4000 rpm at 4 °C. After cell lysis by sonication, the cell lysate was loaded on a
Ni-NTA column equilibrated with Ni-NTA Lysis Buffer (50 mM KPi, 250 mM NaCl, 10 mM
histidine, pH 8.0). After washing with 50 mL Ni-NTA Lysis Buffer and 50 mL of Ni-NTA
WashBuffer (50 mM KPi, 250 mM, NaCl, 20 mM imidazole, pH 8.0), the protein was eluted
with Ni-NTA Elution Buffer (50 mM KPi, 250 mM, NaCl, 250 mM histidine, pH 7.0). The
protein solution was buffer exchanged against potassium phosphate buffer (50 mM, pH 7.0) and
the protein concentration was determined using ε410 = 156 mM-1 cm-1 as the extinction
coefficient.
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Protein Expression and Purification for Crystallization. Freshly transformed
BL21(DE3) cells expressing HisTag-free Mb(H64V,V68A) were used to inoculate 5 mL LB
medium containing ampicillin (100 mg/L), followed by growth overnight at 37°C with shaking.
The overnight culture was used to inoculate 1L of Terrific Broth medium containing ampicillin
and supplemented with 0.3 mM delta-aminolevulinic acid, followed by incubation at 37°C with
shaking (180 rpm). At OD600 = 0.6-0.8, cells were induced with 0.5 mM IPTG and incubated at
27°C with shaking (180 rpm) for 20-24 hours. Cells were harvested by centrifugation (4,000
rpm, 4°C, 20 minutes) and then resuspended in 20 mL Tris HCl buffer (20 mM, pH 8.0)
containing 1 mM EDTA. After sonication and clarification of the lysate via centrifugation
(14000 rpm, 4°C, 20 minutes), the protein was purified using a three-step purification process
according to a modified version of a reported procedure.61 An initial salting out purification was
performed by slowly adding solid ammonium sulfate to the clarified lysate in an Erlenmeyer
flask with stirring on ice. At 65% m/v saturation, the solution was incubated for one hour in ice,
followed by centrifugation (14000 rpm, 4°C, 45 minutes). The supernatant was transferred to a
clean Erlenmeyer flask and solid ammonium sulfate was slowly added to the supernatant to reach
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95% m/v saturation with constant stirring on ice, followed by incubation for one hour in ice and
centrifugation (14000 rpm, 4°C, 45 minutes). The red-colored pellet containing precipitated
Mb(H64V,V68A) was resuspended in a minimal volume of Tris HCl buffer (pH 8.0) containing
1 mM EDTA. The resuspended pellet was then dialyzed against 20 mM Tris HCl buffer (pH 8.0)
and 1 mM EDTA using a 5 kDa cut-off membrane overnight. The protein was then purified by
gel filtration chromatography using a Superdex 75 10/300 GL column (GE Healthcare) and
isocratic elution with 20 mM Tris HCl buffer (pH 8.4) containing 1 mM EDTA, following
absorbance at 409 nm. The protein was then further purified via ion-exchange chromatography
using a DEAE Sepharose column (GE Healthcare) under isocratic elution with Tris HCl (pH 8.4)
containing 1 mM EDTA. Purified Mb(H64V,V68A) was concentrated using a 10 kDa cut-off
centrifugal filter to a final concentration of 4 mM and its purity was confirmed by SDS-PAGE.
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Crystallization, Data collection, Processing, and Refinement. A crystal of the ferric
Mb(H64V,V68A) complexed with water was grown at room temperature using the hanging-drop
vapor-diffusion method by mixing 1 L of reservoir buffer (2 M ammonium sulfate, 0.2 M Tris
pH 8.6) with 1 L of protein in buffer (20 mM Tris pH 8.0, 1 mM EDTA). The crystal was
cryoprotected by soaking it in a drop containing reservoir buffer supplemented with 25% sucrose
for 5 minutes prior to being flash-cooled in liquid nitrogen. Data were collected at Cornell High
Energy Synchrotron Light Source (CHESS) F1 station using a Pilatus3 6M detector. Data was
integrated using iMosflm62 and scaled and merged using Scala63. Rigid body fitting of the
crystallographic structure 1JW842 was used for initial molecular replacement phase calculations
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(PHENIX). This was followed by further rounds of refinement using PHENIX and COOT65.
Data processing and refinement statistics are presented in Table S1. Structure coordinates were
deposited with the Protein Data Bank under the accession number 6M8F. For comparative
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