Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical Reactivity

Article abstract of DOI:10.1021/acscatal.9b02272

Engineered myoglobins and other hemoproteins have recently emerged as promising catalysts for asymmetric olefin cyclopropanation reactions via carbene-transfer chemistry. Despite this progress, the transformation of electron-poor alkenes has proven to be very challenging using these systems. Here, we describe the design of a myoglobin-based carbene transferase incorporating a non-native iron-porphyrin cofactor and axial ligand, as an efficient catalyst for the asymmetric cyclopropanation of electron-deficient alkenes. Using this metalloenzyme, a broad range of both electron-rich and electron-deficient alkenes are cyclopropanated with high efficiency and high diastereo- A nd enantioselectivity (up to >99% de and ee). Mechanistic studies revealed that the expanded reaction scope of this carbene transferase is dependent upon the acquisition of metallocarbene radical reactivity as a result of the reconfigured coordination environment around the metal center. The radical-based reactivity of this system diverges from the electrophilic reactivity of myoglobin and most of the known organometallic carbene-transfer catalysts. This work showcases the value of cofactor redesign toward tuning and expanding the reactivity of metalloproteins in abiological reactions, and it provides a biocatalytic solution to the asymmetric cyclopropanation of electron-deficient alkenes. The metallocarbene radical reactivity exhibited by this biocatalyst is anticipated to prove useful in the context of a variety of other synthetic transformations.

Full text of DOI:10.1021/acscatal.9b02272

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Article
Stereoselective cyclopropanation of electrondeficient olefins with a
cofactor redesigned carbene transferase featuring radical reactivity
Daniela M Carminati, and Rudi Fasan
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b02272 • Publication Date (Web): 05 Sep 2019
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Stereoselective cyclopropanation of electrondeficient olefins
with a cofactor redesigned carbene transferase featuring radical
reactivity
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Daniela M. Carminatia and Rudi Fasan*,a
a Department of Chemistry, University of Rochester, Rochester, NY 14627, United States.
ABSTRACT: Engineered myoglobins and other hemoproteins have recently emerged as
promising catalysts for asymmetric olefin cyclopropanation reactions via carbene transfer
chemistry. Despite this progress, the transformation of electron-poor alkenes has proven very
challenging using these systems. Here, we describe the design of a myoglobin-based carbene
transferase incorporating a non-native iron-porphyrin cofactor and axial ligand, as an efficient
catalyst for the asymmetric cyclopropanation of electron-deficient alkenes. Using this
metalloenzyme, a broad range of both electron-rich and electron-deficient alkenes are
cyclopropanated with high efficiency and high diastereo- and enantioselectivity (up to >99% de
and ee). Mechanistic studies revealed that the expanded reaction scope of this carbene transferase
is dependent upon the acquisition of metallocarbene radical reactivity as a result of the
reconfigured coordination environment around the metal center. The radical-based reactivity of
this system diverges from the electrophilic reactivity of myoglobin and most of known
organometallic carbene transfer catalysts. This work showcases the value of cofactor redesign
toward tuning and expanding the reactivity of metalloproteins in abiological reactions and it
provides a biocatalytic solution to the asymmetric cyclopropanation of electrodeficient alkenes.
The metallocarbene radical reactivity exhibited by this biocatalyst is anticipated to prove useful in
the context of a variety of other synthetic transformations.
KEYWORDS: Cyclopropanation, electrondeficient olefins, myoglobin, carbene transfer catalysis,
radical mechanism, Hammett
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Introduction
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The metal-catalyzed cyclopropanation of alkenes with diazo compounds constitutes a
convenient and direct strategy for the construction of cyclopropane rings,1-4 which are key
structural motifs found in many drug molecules and biologically active natural products.5-8 While
several transition metal complexes have proven effective for executing olefin cyclopropanations
via carbene transfer chemistry,1-4 the development of biocatalytic strategies for realizing these
transformations is highly desirable toward the implementation of sustainable methods for chemical
synthesis. Over the past few years, iron-based heme-containing proteins and enzymes, such as
myoglobins9-13 and cytochrome P450s14-18, respectively, have been identified as promising
biocatalysts for promoting olefin cyclopropanation reactions with diazo compounds. In particular,
our group previously reported the ability of engineered variants of sperm whale myoglobin (Mb)
to catalyze the asymmetric cyclopropanation of vinylstyrenes in the presence of ethyl -
diazoacetate (EDA)9, 10 or other acceptor-only diazo compounds,11, 12 with high levels of diastereo-
and enantioselectivity. Artificial metalloenzymes have also been developed and investigated for
promoting abiological cyclopropanation reactions.19-25
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Recent experimental and computational studies have provided insights into the mechanism of
hemoprotein-catalyzed cyclopropanation reactions9, 26-28 and the role of the protein scaffold in
controlling the stereoselectivity of these transformations.29 These studies support a mechanistic
scenario in which an heme-carbenoid intermediate is initially formed upon reaction of the ferrous
hemoprotein with the diazo reagent. This intermediate was shown to possess electrophilic
reactivity9, 26 and be capable of engaging olefins and other nucleophiles30-32 in a carbene transfer
reaction manifold. In the context of iron-porphyrin and hemoprotein-catalyzed intermolecular
cyclopropanations with -diazo esters, computational and experimental analyses support a
catalytic mechanism involving a concerted, asynchronous insertion of the heme-bound carbene
into the C=C bond of the olefin substrate.27, 29
Despite the progress made in developing biocatalytic strategies for olefin cyclopropanation,
the scope of these transformations has been largely restricted so far to aryl-substituted and aliphatic
olefins, limiting the types of cyclopropane structures accessible using biocatalysis. In particular,
the cyclopropanation of electron-deficient olefins has remained a largely unmet goal in this area.
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In the presence of diazoesters, these transformations can give access to electrophilic cyclopropanes
carrying two electron-withdrawing groups, which represent valuable building blocks for medicinal
chemistry as well as key intermediates for various synthetic applications.33-35 While currently
lacking, biocatalytic approaches for realizing these transformations would be highly desirable.
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Modification of the metal coordination environment can have a profound effect on the
20, 21, 24, 36-48
functional properties in metalloenzymes/proteins.18,
In previous work, we
demonstrated how alteration of the heme cofactor environment in myoglobin through substitution
of the metal center,20, 49, 50 ‘proximal’ residue,20, 42 and/or porphyrin ligand,21 can alter the reactivity
of this metalloprotein in the context of non-native nitrene and carbene transfer reactions. Here, we
report the design of a cofactor-reconfigured myoglobin-based catalyst useful for the stereoselective
cyclopropanation of electron-deficient olefins with diazoesters. Mechanistic studies show how the
expanded reaction scope of this metalloprotein toward these challenging substrates is dependent
upon the acquisition of radical carbene transfer reactivity and a dramatic change in the
cyclopropanation mechanism, as a result of the combined effect of the non-native iron-porphyrin
cofactor and axial ligand integrated into this metalloprotein.
Results and Discussion
Design and Generation of Cofactor Reconfigured Carbene Transferase
The reactivity of myoglobin (Mb) in carbene transfer reactions stems from its hemin cofactor
(iron-protoporphyrin IX), which is coordinated at the iron center by the side-chain imidazolyl
group of a conserved histidine residue (His93 in sperm whale myoglobin) as the ‘proximal’ axial
ligand.51 The latter contributes to anchor the cofactor to the protein and participate in modulating
both the redox and the oxygen binding properties of this metalloprotein.51 While engineered Mb
have proven to be effective catalysts for the cyclopropanation of aryl-substituted olefins and
aliphatic olefins,9, 10, 12, 29 the cyclopropanation of electron-deficient olefins has proven challenging
using these systems. Notably, the cyclopropanation of electron-deficient alkenes has represented
a notorious challenge also for synthetic carbene transfer catalysts,52-54 a phenomenon attributed to
the electrophilic character of the metal-bound carbenes typically involved in these reactions.
To overcome these limitations, we envisioned the possibility of enhancing the electrophilic
reactivity of the heme-carbene intermediate through modification of the heme cofactor and
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proximal ligand, as these are bound to directly influence the electronic properties of this reactive
species in Mb-mediated cyclopropanation.27 As the parent scaffold for harboring these
substitutions, we selected our previously reported variant Mb(H64V,V68A) (1, Figure 1a) due to
its excellent stereoselectivity (>90-99% de and ee) and broad substrate scope in the
cyclopropanation of vinylarenes with EDA.9, 10 Based on the considerations outlined above, we
decided to substitute the native heme in Mb(H64V,V68A) (1) with iron-2,4-diacetyl
deuteroporphyrin IX (Fe(DADP)) (Figure 1a), an electron-deficient heme analog amenable to
incorporation into this hemoprotein.37 The incorporation of Fe(DADP) was previously shown to
increase the Fe3+/Fe2+ reduction potential (Eo; couple) of Mb,37 a result consistent with the effect
of the electronwithdrawing acetyl groups conjugated to the tetrapyrrole ring toward destabilizing
the ferric form of the metalloprotein. This non-native cofactor was thus expected to potentially
enhance the electrophilic character of the heme-carbene intermediate in the context of
cyclopropanation reactions. Given the critical role of the proximal axial ligand in affecting the
functional properties of myoglobin, we designed a second variant in which the Fe(DADP) cofactor
is coordinated at the axial position by the non-canonical amino acid N-methyl-histidine (NMH,
Figure 1a) in place of the native histidine ligand (His 93). This substitution was also reported to
increase the Eo of Mb,46 an effect attributed to the loss of a hydrogen bond interaction between the
proximal His ligand and a serine residue within the proximal pocket of the protein.55, 56 The
aforementioned cofactor and axial ligand modifications were thus envisioned to potentially
cooperate toward affecting the reactivity of the Mb-based as a carbene transferase, with the change
in redox properties providing a proxy for such additive or synergistic effect.
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The
corresponding
Mb
variants,
Mb(H64V,V68A)[Fe(DADP)]
(2)
and
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3), could be successfully produced and isolated from E.
coli in good yields (23 and 5 mg protein/L culture, respectively). Incorporation of the non-native
Fe(DADP) cofactor was effectively achieved by adapting a protocol previously used for the
recombinant production of cofactor-substituted variants of Mb20, 21 and other hemoproteins,57
whereas NHM was introduced at the axial position via amber stop codon suppression58 in E. coli
cells co-expressing an orthogonal aminoacyl-tRNA/tRNA pair specifically evolved for genetic
incorporation of this non-canonical amino acid.59
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Characterization of Fe(DADP)-based Mb variants
Characterization
of
Mb(H64V,V68A)[Fe(DADP)]
(2)
and
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Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) by UV-vis spectroscopy showed a small blue-shift
of the Soret band of the Fe(DADP) complex upon incorporation into these proteins (412-415 nm
vs. 417 nm for Fe(DADP) alone; Figure 1b and 1c). Incubation of the Mb variants with sodium
dithionite induces a shift of the Soret band from 415 nm to 434 and from 412 nm to 430 nm,
respectively, corresponding to reduction of the protein from the ferric to the ferrous form. Further
incubation with carbon monoxide resulted in a further shift of the Soret band to 434 and 430 nm,
respectively, corresponding to CO-bound complex. Overall, the spectral features of the cofactor-
substituted variants in their ferric, ferrous, and CO-bound form are similar, albeit not identical, to
each other and compared to the heme-containing counterpart (Figure S1).
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Figure 1. a) High-resolution crystal structure29 of sperm whale Mb(H64V,V68A) (1) and structure
of histidine- and N-methyl-histidine-ligated Fe(DADP) cofactor. b) Absorption spectra for ferric,
ferrous and CO-bound form of Mb(H64V,V68A)[Fe(DADP)] (2). c) Absorption spectra for ferric,
ferrous and CO-bound form of Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3). See Figure S1 for
spectra corresponding to Mb(H64V,V68A) (1).
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To assess the impact of the cofactor and axial ligand substitution on the stability of the protein,
their apparent melting temperature (Tm) was determined via thermal denaturation experiments
using circular dichroism (CD) (Figure 2 and S2). These studies revealed that incorporation of the
Fe(DADP) cofactor has no deleterious effect on the stability of the protein compared to heme-
containing counterpart (Tm = 66.3°C for Mb(H64V,V68A)[Fe(DADP)] (2) vs. 66.0°C for
Mb(H64V,V68A) (1)60). Although substitution of the proximal His with N-methyl-histidine causes
a slight destabilization of the protein (Tm = -2.9°C), the Mb(H64V,V68A,H93NMH)[Fe(DADP)]
(3) variant retains significant thermostability (Tm = 63.4°C; Figure 2), indicating that both
structural modifications are well tolerated by the Mb scaffold.
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The effect of these cofactor modifications on the Fe3+/Fe2+ reduction potential (E°) of the
metalloprotein was then investigation using a UV-vis spectrochemical method.61 For comparison
purposes, wild-type sperm whale Mb as well as a variant incorporating only the proximal ligand
substitution, Mb(H64V,V68A,H93NMH), were included in these analyses. As expected, these
experiments showed an shift of the redox potential toward more positive values for both the
Fe(DADP)- and NMH-containing variant (E°Fe3+/Fe2+ = 59 ± 1 mV and 77 ± 6 mV, respectively;
Figure S3b-c), compared to the parent protein Mb(H64V,V68A) (1) (E°Fe3+/Fe2+ = 54 ± 1 mV;
Figure S3a). Using the same spectrochemical assay, wild-type Mb was determined to have an E°
value of 47 ± 1 mV, which is very similar to those previously reported in the literature using other
methods.37, 62 Notably, the combination of the Fe(DADP) cofactor with the NMH axial ligand leads
to a dramatic increase of the redox potential, resulting in a E° value of 146 ± 3 mV for the
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) variant (Figure S3d). The nearly 100 mV increase
in E° for the latter variant compared to the +6 to +23 mV increases for the Fe(DADP)- and NMH-
containing variant, respectively, indicated a clear synergistic role of the non-native cofactor and
axial ligand toward affecting the redox potential of the metalloprotein.
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Figure 2. Thermal denaturation curves for Mb(H64V,V68A) (1), Mb(H64V,V68A)[Fe(DADP)]
(2) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) as determined by circular dichroism (222).
Cyclopropanation Activity
In order to evaluate the catalytic activity of Mb(H64V,V68A)[Fe(DADP)] (2) and
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) as cyclopropanation catalysts, these proteins were
tested in a model reaction with styrene (4) and ethyl diazoacetate (EDA) under “standard reaction
conditions” (10 mM styrene, 20 mM EDA, 10 mM sodium dithionite (Na2S2O4), 0.1 mol% protein,
anaerobic conditions). As summarized in Table 1, both proteins produce the desired product 5a in
quantitative yield and with excellent diastereo- and enantiomeric excess (>99% de and >98-99%
ee; Table 1, Entry 2-3). The high cyclopropanation activity exhibited by
Mb(H64V,V68A)[Fe(DADP)] (2) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) and the fact
that both reactions proceed cleanly without the formation of carbene dimerization byproducts
(diethyl maleate and fumarate) indicate that the [Fe(DADP)] cofactor can efficiently activate the
diazo compound (EDA) promoting the carbene transfer reaction. In addition, both catalysts
preserve the high stereoselectivity of the parent Mb(H64V,V68A) (1) variant, indicating that in
either case the cofactor and cofactor/axial ligand substitution do not disrupt the active site structure
of the protein.29
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Since modification of the porphyrin cofactor and/or proximal ligand can affect the sensitivity
of Mb-based carbene transferases to oxygen and/or non-reducing conditions,20, 21, 42, 47 the
cyclopropanation activity of the [Fe(DADP)]-based variants was also tested in the presence and in
the absence of oxygen and/or reductant (Na2S2O4) (Table 1 and Table S1). While the catalytic
activity of Mb(H64V,V68A) (1) decreases in the presence of oxygen (62% vs. >99% yield),
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) maintains high cyclopropanation activity (85%
yield) under aerobic and reducing conditions (Table 1, Entry 6 vs. 4). This activity is higher than
in the presence of the Fe(DADP) cofactor or the His93NMH substitution alone (Table S1, Entry
3), indicating a combined effect of these substitutions toward increasing the oxygen tolerance of
the biocatalyst. Unlike Mb(H64V,V68A) (1), both [Fe(DADP)]-based variants and in particular
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) exhibit high cyclopropanation activity (86-100%
yield) also under non-reducing (and anaerobic) conditions (Table 1, Entry 7-9).
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As the [Fe(DADP)]-containing variants are prepared as ferric proteins, the catalytic activity
under non-reducing conditions can be explained based on the ability of these variants to undergo
in situ reduction by the EDA reagent to produce the catalytically active ferrous form42 or,
alternatively, by their ability to catalyze this reaction also in their ferric form, as previously
observed for an iron-chlorin e6 containing Mb variant.21 To gain insights into this aspect, the same
reactions were carried out in the presence of CO, which binds to the ferrous but not the ferric form
these proteins as confirmed by UV-vis spectroscopy (Figure 1b-c and Figure S4). CO binding to
the iron center is expected to inhibit cyclopropanase activity. Under these conditions, the
cyclopropanation product 5a was obtained only in trace amounts (Table S1, Entry 8). Control
experiments ruled out that CO-mediated reduction63 of this protein occurs within a timescale
relevant for observation of cyclopropanation activity (i.e., <30-45 min) (Figure S4). These results
suggest that in situ generated ferrous form may contribute to the observed catalytic activity of the
Fe(DADP)-containing variants under non-reducing conditions. Given the lower activity of
Mb(H64V,V68A) (1) under these conditions, it is possible that the Fe(DADP) cofactor make these
variants susceptible to reduction by EDA, which can act as a mild reducing agent,64-67 a behavior
that may be at least in part attributed to their increased redox potential (Eo) compared to the heme-
containing parent protein.
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Table 1. Catalytic activity of Mb(H64V,V68A) (1) and cofactor substituted variants thereof in the
cyclopropanation of styrene with EDA.a
7
8
9
OEt
0.1 mol% catalyst
N2
CO2Et
+
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reaction conditions (X),
KPi (pH 7)
O
4
5a
Entry
1
Catalyst
Xb
-/red
-/red
-/red
O2/red
O2/red
O2/red
-/-
Yield
>99%
>99%
>99%
62%
TON
% de
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
% ee
>99
>99
98
1
2
3
1
2
3
1
2
3
3
3
3
>1000
>1000
>1000
620
2
3
4
>99
>99
98
5
39%
390
6
85%
850
7
10%
150
>99
>99
>99
>99
>99
>99
8
-/-
86%
865
9
-/-
>99%
12%
>1000
125
10
11c
12d
O2/-
-/-
>99%
40%
2155
4010
-/-
a Reaction conditions: 10 mM styrene (4), 20 mM EDA, 10 M purified Mb variant in KPi 50
mM (pH 7), r.t., 16 hours. Yield, diastereomeric and enantiomeric excess determined by chiral
b
GC-FID analysis using 1,3-benzodioxole as internal standard. Variable parameter (X): “-
/red”=anaerobic, 10 mM Na2S2O4; “-/-“=anaerobic, no reductant; “O2/red”=aerobic, 10 mM
c
d
Na2S2O4; “O2/-“=aerobic, no reductant. Reaction performed using 5 M protein. Reaction
performed using 1 M protein.
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Reactivity toward Aryl- and Heteroatom-Substituted Olefins
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Based on the positive results from the studies above, we then assessed the catalytic performance
of the Fe(DADP)-based variants toward the cyclopropanation of aryl-substituted olefins under
non-reducing and anaerobic conditions. As shown in Scheme 1, a diverse panel of para-, ortho-,
meta-, and - substituted styrene derivatives were efficiently converted to the desired
cyclopropanation products 5b-h in good to excellent yield, and excellent trans-diastereoselectivity
(98 to >99% de) and (1S,2S)-enantioselectivity (98% to >99% ee). Both electron-donating (5b-e)
and electron-withdrawing groups (5f-h) in the phenyl ring were well tolerated. In addition, the
sterically more demanding -methylstyrene and 2-vinyl-naphtalene were also efficiently
converted to the corresponding cyclopropane 5i and 5j with high diastereo- and enantioselectivity.
Moreover, unlike Mb(H64V,V68A)[Fe(DADP)] (2), Mb(H64V,V68A,H93NMH)[Fe(DADP)]
(3) showed also good activity toward non-styrenyl substrates such as phenylvinylether and
phenylvinylsulfane, producing 5k, and 5l, respectively, in good to high yield and with high
stereoselectivity. The number of catalytic turnovers (TON) observed with these substrates are 2-
to 3-fold higher than those obtained with Mb(H64V,V68A) (1) under reducing conditions. The
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unactivated
olefin
substrate
(allyloxy)benzene
was
also
converted
by
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) into the cyclopropane product 5m In this case, the
Fe(DADP)-based catalyst also offers improved stereoselectivity (>99% de and ee) compared to
the parent enzyme (95% de, 86% ee).
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Scheme
1.
Substrate
scope
of
Mb(H64V,V68A)[Fe(DADP)]
(2)
and
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3).a
7
8
9
R1
R1
R
2
3
cat. or
+
CO2Et
CO2Et
R
N2
KPi (pH 7)
anaerobic
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yield%, TON, de%, ee%
CO2Et
CO2Et
CO2Et
5d
5b
5c
98%, >1000, 98%, >99%
>99%, >1000, >99%, 99%
>99%, >1000, 97%, >99%
>99%, >1000, 98%, 99%
2:
3:
99%, >1000, 99%, 99%
>99%c, >1000, 99%, 98%
CO2Et
CO2Et
CO2Et
MeO
F
Cl
5e
5f
5g
99%, >1000, 98%, >99%
>99%, >1000, 99%, 97%
98% >1000, 98%,> 99%
>99%, >1000, >99%, 99%
99%, >1000, 99%, >99%
99%, >1000, 99%, 98%
2:
3:
CO2Et
CO2Et
CO2Et
Br
5h
5i
5j
>99%, >1000, 99%, 99%
99%, >1000, >99%, 98%
80%, 820, 97%,> 99%
>99%, >1000, 97%, 99%
99%, >1000, 99%, 99%
>99%, >1000, >99%, 99%
2:
3:
O
CO2Et
S
CO2Et
O
CO2Et
5kb
5lb
5mb
5%, 55, >99%, >99%
54%, 545, 99%, 85%
9%, 90, >99%, >99%
85%, 850, 98%, 97%
1%, 15, >99%, >99%
26%, 260, 99%, >99%
2:
3:
a Reaction conditions: 10 mM alkene, 20 mM EDA, 10 M Mb variant 2 or 3 in KPi buffer (50
mM, pH 7), r.t., 16 hrs, anaerobic conditions. Yield and diastereomeric excess were determined
by chiral GC-FID analysis. Enantiomeric excess was determined by chiral GC-FID or SFC
analysis. b 50 mM alkene, 20 mM EDA, 20 M catalyst 3.
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4
Stereoselective Cyclopropanation of Electron-Deficient Olefins
5
6
7
8
The high cyclopropanation activity of Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) toward
the monohalogenated styrenes (products 5f, 5g, and 5h in Scheme 1) was in line with our goal of
enhancing the reactivity of Mb-based systems toward electron-poor olefins and encouraged us to
assess its scope toward more challenging substrates such as trifluoromethyl-, nitro-, and
pentafluoro-styrenes (Scheme 2). Gratifyingly, the corresponding cyclopropanation products 5n-
q could be obtained in high (69-85%) to quantitative yield (5o), along with high diastereo- and
enantioselectivity (98 to >99% de; 86 to >99% ee). Notably, 4-vinylbenzensulfonamide, whose
cyclopropanation failed using Mb(H64V,V68A) (1) or commonly adopted organometallic carbene
transfer catalysts such as Rh2(OAc)4 and Fe(TPP)Cl (Table S2), was successfully cyclopropanated
in the presence of Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) to afford 5r in 48% yield and
>99% de and 84% ee (Scheme 2).
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Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) could be successfully applied to the
stereoselective cyclopropanation of a variety of other electron-deficient non-styrenyl olefins.
While acrylate esters are notoriously challenging substrates for transition metal-catalyzed
cyclopropanation,52, 68 phenyl and benzyl acrylate could be converted to the desired products 5s
and 5t, respectively, in good to high yields (49-89%) and high enantiopurity (98-99% de; 99% ee).
Vinyl benzoate, which is unreactive in the presence of Fe(TPP)Cl and Mb(H64V,V68A) (1) and
only poorly reactive toward Rh2(OAc)4 (Table S3), was also efficiently cyclopropanated by
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) to give enantiopure 5u in quantitative yield with 99%
de and 97% ee. Time-course experiments showed this reaction proceeds rapidly, with >90% of the
final product being formed within the first 10 minutes and that it is faster than the cyclopropanation
of styrene (Figure S5). It is also worth noting that this transformation can provide an expedite
route (i.e., via hydrolysis of 5u) to cyclopropanols, which are valuable building blocks for drug
synthesis. Finally, the Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation of N-
vinyl-amides and succinamides was also successful, affording the corresponding cyclopropanes
5v and 5w in good to moderate yields (75-33%) and high stereoselectivity. Comparable selectivity
but
somewhat
reduced
yield
(by
20-30%)
were
obtained
for
the
Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed reactions with the electrondeficient olefins in
the presence of sodium dithionite, indicating an optimal performance of this catalyst under non-
reducing conditions.
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Scheme 2. Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation of
electrondeficient olefins.a
3
cat.
(0.01-0.2 mol%)
+
CO2Et
N2
EWG
EWG
CO2Et
KPi (pH 7)
anaerobic
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yield, TON, de%, ee%
O2N
CO2Et
CO2Et
CO2Et
F3C
O2N
5n
5o
5p
>99%, >1000, >99%,>99%
85%, 865, 98%, >99%
69%, 685, >99%, >99%
F
F
F
CO2Et
CO2Et
F
H2NO2S
F
5q
5r
48%b, 240 , >99%, 85%
84%, 840, 99%, 86%
O
O
O
CO2Et
O
CO2Et
CO2Et
O
O
5s
82%c, 920, 99%, 99%
5t
5u
49%c, 485, 98%, 99%
80%c, 795, 99%, 97%
99%d, 490, >99%, 98%
O
O
N
CO2Et
N
CO2Et
O
5w
33%c, 335, 99%, 95%
5v
75%b, 370, >99%, 99%
a Reaction conditions: 10 mM alkene, 20 mM EDA, 10 M catalyst 3 in KPi buffer (50 mM, pH
7), r.t., 16 hrs, anaerobic conditions. Yield and diastereomeric excess were determined by chiral
b
GC-FID analysis. Enantiomeric excess was determined by chiral GC-FID or SFC analysis. 20
mM alkene, 10 mM EDA, 20 M 3. c 50 mM alkene, 20 mM EDA, 20 M 3. d 2.5 mM alkene, 2.5
mM EDA, 5 M 3.
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8
9
In general, Mb(H64V,V68A)[Fe(DADP)] (2) and Mb(H64V,V68A,H93NMH) proved to be
inferior catalysts for the cyclopropanation of the electrodeficient alkenes compared to
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3). For example, cyclopropane 5r and 5u were obtained
in <20% yields using either of these catalysts compared to 50-80% yields obtained with
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) under the same reaction conditions (Tables S2 and
S3). Mirroring the observed effect on other functional properties (i.e. E0, sensitivity to
reductant/oxygen), these results point at a synergistic effect of the Fe(DADP) cofactor and axial
N-methyl-histidine ligand toward conferring high reactivity toward these challenging substrates.
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Mechanistic Studies
To gain insights into the mechanistic basis for the divergent reactivity of
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) compared to His-ligated heme counterpart, we
measured the relative rates of these catalysts for the cyclopropanation of a series of para-
substituted styrenes vs. styrene via competition experiments. Consistent with our previous
observations,9 styrene derivatives carrying electrondonating groups (i.e., -Me, -OMe) showed
higher reactivity than styrene in the Mb(H64V,V68A)-catalyzed reactions, whereas electron-
deficient styrenes (e.g., p-CF3, p-NO2) exhibited reduced cyclopropanation rates compared to
styrene (Table 2, Figure S6). Plots of the corresponding log(kX/kH) values against the Hammett
constants69 p and + gave relatively good linear correlations, yielding a p value of -1.2 (R2 =
0.84) and a + value of -0.77 (R2 = 0.77), respectively (Figure 3a). Similar plots generated after
excluding p-bromo-styrene, which showed the largest deviation from the trend, yielded even
stronger linear correlations (R2 = 0.88-0.93) without affecting the  values p = -1.2; + = -0.79).
The large negative  values is consistent with the electrophilic nature of the metal carbene
intermediate involved in these reactions.9, 27
In stark contrast to the reactivity trend exhibited by Mb(H64V,V68A) (1), the rate of
Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation is accelerated by the
presence of electron-withdrawing groups in the styrene substrates, whereas reduced
cyclopropanation rates are observed with the electron-rich styrenes relative to unsubstituted
styrene (Table 2, Figure S7). The corresponding log(kX/kH) values showed no correlation with the
polar substituent constant p or + (R2 = 0.07-0.1) (Figure 3b). The non-linearity of these plots,
along with our previous observations concerning the radical mechanism of hemoprotein-catalyzed
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nitrene transfer reactions,70 prompted to us to consider the potential involvement of intermediates
with significant radical character in Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed
cyclopropanation.
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Table 2. Relative rates scales for the cyclopropanation of para-substituted styrene catalyzed by
Mb(H64V,V68A) (1) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) and sigma and sigma-dot
scales for para substituents.a
Mb variant
+
CO2Et
+
CO2Et
EDA, Na2S2O4
KPi/MeOH (8:2)
Y
(Y)
kX / kH
kX / kH
Substituent
p

mb
•JJ
(catalyst 1)
(catalyst 3)
-OMe
-Me
-Cl
1.04
0.61
0.39
0.66
0.16
0.04
0.65
0.89
1.12
1.39
0.28
3.43
-0.27
-0.17
0.23
0.23
0.54
0.78
-0.778
-0.77
-0.29
0.11
0.13
0.49
0.86
0.23
0.15
0.22
0.23
-0.01
0.36
-0.31
0.11
0.15
0.61
0.79
-Br
-CF3
-NO2
a p and p are the Hammett constants69 and mb and •JJ are Jiang’s polar71 and spin-delocalization
constants72, respectively, for para-substituted aryl derivatives.
Supporting this hypothesis, a very good linear correlation (R2 = 0.86) was obtained between
the log(kX/kH) values and Jiang’s spin-delocalization substituent constants •JJ72 (Figure 3c; Table
3). An even stronger correlation (R2 = 0.93) was achieved using the dual-parameter equation
log(kX/kH) = XX + ••, which takes into consideration of both the polar (X) and spin-
delocalization effects (•) of the substituents.73-75 Fitting of this equation to the data via multiple
linear regression using Jiang’s mb71 polar scale and •JJ72 radical scale yielded a mb and •JJ value
of 0.18 and 2.6, respectively (Figure 3d, Table 3). While the improved correlation achieved with
the extended equation compared to the radical scale alone suggests the participation of both polar
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and spin effects in affecting the rate of the reaction, the small mb/•JJ ratio of 0.06 indicates a
dominance of the spin-delocalization effect over the polar effect.75 In stark contrast, dual-
parameter fitting of the Mb(H64V,V68A) (1) data yielded a large negative mb (-0.79 vs. +0.18
for 3; Table 3) and a mb/•JJ above the unity (= 1.5), which have been attributed to reactions
dominated by polar effects.75
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Table 3. Rho and R2 values for single- and dual-parameters correlations of log(kX/kH) values for
Mb(H64V,V68A) (1) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) with sigma and sigma-dot
parameters. N.a. = not applicable due to non-linear correlation.
R2
X
mb
•JJ
mb/•JJ
Catalyst
Parameters
1
1
1
1
3
3
3
3
-1.2
-
-
-
-
0.84
0.77
0.05
0.84
0.10
0.07
0.86
0.93
p
+
-0.77
-
-
•JJ
-
-
-
n.a.
-0.52
-
-
mb/•JJ
p
-0.79
1.5
n.a.
n.a
-
-
-
+
-
-
-
-
-
•JJ
2.7
2.6
mb/•JJ
-
0.18
0.06
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Figure 3. Hammett plots. (a) Plot of log(kX/kH) for Mb(H64V,V68A) (1) vs.  constant. (b-d)
Plots of log(kX/kH) for Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) vs.  constant (b), •JJ
constant (c) and 0.18mb+2.6•JJ (d).
To further probe the occurrence of
a
radical mechanism in the
Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation process, reactions were
carried out using EDA and the isotopically labelled substrate cis--deuterostyrene (d-4a) (see
Experimental details), where a (partial) loss of stereospecificity would reveal the occurrence of a
radical intermediate. Insightfully, this reaction led to the formation of d-5a in low yield as a 1.2:1
mixture of cis and trans isomers with respect to the -D and -Ph groups, as determined based on 1H
2
and H NMR spectra (Scheme 3). In contrast, the same reaction in the presence of
Mb(H64V,V68A) (1) proceeds stereospecifically with no scrambling of the deuterium label. In
comparison, ~40% isomerization was observed for the same reaction using Co(TPP) as the
catalyst,27 a result in line with the radical cyclopropanation mechanism previously established for
this system.76, 77 Altogether, these experiments provided strong evidence for the intermediacy of a
radical intermediate in the Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) reaction.
To further corroborate this conclusion, the Mb(H64V,V68A,H93NMH)[Fe(DADP)]-
catalyzed reaction with styrene and EDA was carried out in the presence and in absence of the
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5
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7
8
9
radical spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (Table 4). In the presence
of DMPO, a significant reduction (by 58%) in the yield of the cyclopropane product 5a was
observed under reducing conditions compared to a parallel reaction lacking the spin trapping agent
(Entry 7 vs. 6). A similar effect but with a higher degree of inhibition (77% reduction in yield) was
observed under non-reducing conditions. Very similar results were obtained from parallel
experiments involving 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) in place of
DMPO as the spin trapping agent (Table 4, Entries 3 and 8). In contrast, the Mb(H64V,V68A)-
catalyzed cyclopropanation reaction showed only a small reduction in yield upon addition of
DMPO or TEMPOL (Table 4, Entries 2-3 vs. 1). In line with these results, kinetic experiments
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showed
a
drastic
reduction
of
the
cyclopropanation
rate
of
Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) in the presence of DMPO compared to a reaction
without it (38 vs. 670 turnovers min-1), whereas the cyclopropanation rate of Mb(H64V,V68A) (1)
was only minimally affected by the spin trapping agent (510 vs. 550 turnovers min-1; Figure S10).
To probe the step at which DMPO-mediated inhibition occurs, inhibition experiments were
performed in the presence of EDA but in the absence of styrene, under which conditions the
carbene dimerization products diethyl malonate/maleate are produced. Insightfully, nearly full
suppression (>95%) of the carbene dimerization product was observed in the presence of DMPO
with Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) (Table 4, Entry 10 vs. 9), whereas only little
inhibition (<20%) was observed with Mb(H64V,V68A) (1) (Table 4, Entry 5 vs. 4). Taken
together, the Hammett analyses and radical rearrangement/trapping experiments converged in
supporting (a) the occurrence of
a
step-wise radical-based mechanism for the
Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed olefin cyclopropanation reaction and (b) the
intermediacy of a metallocarbene species with significant radical character, as evidenced by its
susceptibility to inhibition by spin trapping agents.
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Scheme 3. Enzymatic cyclopropanation reactions with cis--deuterostyrene (d-4a).
5
6
7
8
D
H
H
D
H
O
1
3
Catalyst or
H
+
+
OEt
CO2Et
CO2Et
D
9
N2
10
11
12
13
14
15
16
17
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d-4a
d-5acis
d-5atrans
 (2H-NMR): 1.72 ppm
 (2H-NMR): 1.32 ppm
d-5acis d-5a
ratio
:
trans
1 : 0
1
3
catalyst
catalyst
1.2 : 1
Table 4. Inhibition experiments with spin trapping agents in styrene cyclopropanation reaction
catalyzed by Mb(H64V,V68A) (1) or Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3).a
Entry
Catalyst
Styrene
Inhibitor
Yield
% de
% ee
1
2
1
1
1
1
1
3
3
3
3
3
Yes
Yes
Yes
No
None
DMPO
TEMPOL
None
>99%
89%
99
99
>99
-
>99
>99
99
-
3
90%
4
95%b
80%b
>99%
42%
5
No
DMPO
None
-
-
6
Yes
Yes
Yes
No
>99
>99
>99
-
>99
99
99
-
7
DMPO
TEMPOL
None
8
40%
9
88%b
8%b
10
No
DMPO
-
-
a Reaction conditions: 10 mM styrene, 20 mM EDA, 100 mM DMPO (or TEMPOL), 20 μM Mb
variant, 10 mM sodium dithionite in 50 mM potassium phosphate buffer (pH 7) containing 10%
DMF, r.t., 16 hours. Yield, diastereomeric and enantiomeric excess were determined by chiral GC-
FID analysis. b Yield of diethyl malonate and maleate determined by chiral GC-FID analysis.
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On the basis of these results, a plausible mechanism for this reaction is proposed in Scheme
4. Under reducing conditions, the ferrous form of the protein (I) reacts with the diazo reagent to
form an iron-bound carbene intermediate (II). While the structural and electronic features of this
species awaits further elucidation, this intermediate clearly possesses distinct reactivity compared
to the heme-bound carbene intermediate27 and possesses significant radical character, as evidenced
by its reactivity toward the radical trapping agent. Upon generation of this species, metallocarbene-
radical activation of the olefin substrates is envisioned to ensue, leading to a carbon-centered
radical intermediate (III). Ring closure leads to the cyclopropane product and regenerates the
catalyst (Scheme 4).
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Under non-reducing conditions, a similar mechanism may be operative after generation of the
catalytically competent ferrous form via in situ reduction of the ferric protein by the diazo
compound. This reductive process could be facilitated by the increase in the redox potential
associated with the incorporation of NMH-ligated Fe(DADP) cofactor compared to the histidine-
ligated heme counterpart. While the CO inhibition experiments would support this hypothesis, a
cyclopropanation pathway mediated by the ferric form of this protein is also considered viable.
Fe(III)-based catalysts capable of promoting cyclopropanation reactions have been described67, 78-
81 and we previously reported a Mb variant functioning as cyclopropanase in its ferric state.21 This
alternative scenario is suggested by various pieces of experimental data, including the higher
cyclopropanation activity of Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) with the
electrondeficient olefins and the higher degree of DMPO-induced inhibition observed under non-
reducing vs. reducing conditions. Future studies are warranted to elucidate these aspects in more
detail.
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Scheme 4. Proposed mechanism for Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed
cyclopropanation reaction.
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O
O
N
N
N
N
EtO2C
Ph
FeII
CO2Et
N2
-
-O2C
N
N
CO2
I
Ph
N2
H
O
CO2Et
H
CO2Et
O
O
O
N
N
N
N
N
N
N
N
Fe
Fe
-
-O2C
N
CO2
-
-O2C
N
CO2
II
Ph
N
N
H
O
CO2Et
IV
O
Ph
N
N
N
N
Fe
-
-O2C
N
CO2
N
III
Further Discussion
The radical-based stepwise mechanism operating in the present system is reminiscent of that
reported for olefin cyclopropanation catalyzed by Zhang and de Bruin’s Co-porphyrins76, 77 and an
open-shell iron alkylidene complex recently reported by Deng and coworkers,82 both of which
systems were found to be reactive toward electron-deficient alkenes.52, 82 The reactivity of the
Fe(DADP)-based biocatalyst 3 toward electron-poor olefins (Scheme 2) sets it apart from the
electrophilic reactivity of Fisher-type metallocarbenes involved in cyclopropanation reactions
catalyzed by Mb ( = - 1.2; Figure 3a) and various organometallic catalysts, including iron- ( =
-0.6865;  = -0.4183), ruthenium- ( = -0.4484), and iridium-porphyrins (+ = -0.2285),
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mononuclear Rh (+ = -0.4086) and copper complexes ( = - 0.8587;  = -0.7686;  = -0.7488).
Furthermore, the positive mb value (0.18), along with the large •JJ (2.6), obtained for the
Fe(DADP)-based catalyst suggest the involvement of a metallocarbene radical intermediate with
some nucleophilic character. Such characteristics, which arise from a synergistic role of the
Fe(DADP) cofactor and NMH axial ligand, can explain the expanded substrate scope of the
cofactor engineered biocatalyst toward electron-poor (5n-5w; Scheme 2) and unactivated olefins
(5m; Scheme 1) in addition to electron-rich alkenes (5a-5l; Scheme 1).
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As supported by our results, reconfiguration of the first coordination sphere around the iron
center via the porphyrin and axial ligand substitutions induces a dramatic change in the mechanism
of the intermolecular olefin cyclopropanation investigated here, going from a (predominantly)
concerted carbene transfer process dominated by polar effects and mediated by an electrophilic
heme-carbene intermediate,27 to a stepwise radical mechanism dominated by spin delocalization
effects and mediated by
a
metallocarbene intermediate with radical-type, dual
nucleophilic/electrophilic character. While ligand-induced changes in reaction mechanisms have
been in some cases observed for transition metal-catalyzed transformations,89, 90 this is the first
instance, to the best of our knowledge, where this effect is achieved in the context of a
metalloprotein.
It should be further noted that we previously observed a latent radical reactivity of free hemin
in intermolecular cyclopropanation reactions with EDA (2% isomerization with -
deuterostyrene)27, whereas more recent studies from our laboratory showed evidence for the
occurrence of radical-mediated rearrangements in Mb-catalyzed intramolecular cyclopropanation
reactions.91 Based on this knowledge and the present work, we set forth the hypothesis that
hemoproteins may possess a dual (or multistate) reactivity in carbene transfer reactions, possibly
involving distinct intermediates and reaction pathways, akin to that proposed for metal- and
metalloenzyme-catalyzed oxo transfer processes.92, 93 Importantly, the present studies demonstrate
how modification of the first coordination sphere around the iron can provide an effective strategy
for enhancing the radical-type carbene transfer reactivity of this system, enabling cyclopropanation
reactions not readily accessible using their native cofactor-containing counterparts. Further studies
are warranted to elucidate the nature of the catalytic intermediates involved in these reactions and
better understand the role played by the cofactor/axial ligands in favouring the observed radical-
type reactivity, which is only rarely found among organometallic carbene transfer catalysts.76, 82
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Conclusion
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In summary, we have described the design and development of a metalloprotein catalyst
useful for the stereoselective cyclopropanation of both electron-rich and electron-poor alkenes. A
variety of electrondeficient styrenes and alkenes, which are either poorly reactive or unreactive
toward heme-based protein catalysts, were effectively cyclopropanated by this system with high
catalytic activity as well as high diastereo- and enantioselectivity. Our mechanistic studies show
that the expanded reaction scope of this biocatalyst compared to the heme-based counterpart is
dependent upon the acquisition of radical-type carbene transfer reactivity as a result of the
reconfigured primary coordination sphere around the iron center through a N-methyl-histidine-
ligated Fe(DADP) cofactor. Hammett analyses and radical rearrangement/trapping experiments
provide evidence for the involvement of a radical-based, stepwise mechanism in these reactions,
which differs from the concerted nonsynchronous carbene transfer mechanism previously found
to dominate Mb- and iron-porphyrin-catalyzed intermolecular cyclopropanations with
diazoesters.94, 95 This study highlights the value of cofactor engineering for tuning the reactivity
and expanding the reaction scope of metalloproteins in the context of abiological reactions.
Furthermore, in view of the unique and attractive features of radical-mediated reactions,96-99 the
metallocarbene radical reactivity exhibited by the present system is expected to prove useful in the
context of other synthetically valuable transformations.
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EXPERIMENTAL DETAILS
9
General Information. All chemicals and reagents were purchased from commercial
suppliers (Sigma-Aldrich, Alfa Aesar, Frontier Scientific Inc, Chem-Impex Int.) and use without
any further purification, unless otherwise stated. All dry reactions were carried out under argon
pressure in oven-dried glassware with magnetic stirring using standard gas-tight syringes, cannula,
and septa. 1H and 13C NMR spectra were measured on a Bruker DPX-400 instrument (operating
at 400MHz for 1H, 100 MHz for 13C) or a Bruker DPX- instrument (operating at 500 MHz for 1H
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13
and 125 MHz for C). Column chromatography purification was carried out using AMD Silica
Gel 60 230-400 mesh. Thin Layer Chromatography (TLC) was carried out using Merck Millipore
TLC silica gel 60 F254 glass paltes. Gas chromatography (GC) analyses were carried out using a
Shimadzu GC-2010 gas chromatograph equipped with a FID detector, and a Cyclosil-B column
(30 m x 0.25 mm x 0.25μm film). Supercritical Fluid Chromatography (SFC) analysis were
performed using a JASCO Analytical and Semi-Preparative SFC instrument equipped with a
column oven (35 °C), photodiode array detector, a backpressure regulator (12.0 MPa), a carbon
dioxide pump and a sample injection volume of 3 μL.
Synthetic Procedures. Detailed procedures for the synthesis of Fe(DADP)Cl, cis--
deuterostyrene and cyclopropanes 5a-w are provided in the Supporting Information.
Growth Media. Cell cultures were grown in enriched M9 medium which was prepared
follows. For 1 L, 779 mL deionized H2O were mixed with 200 mL M9 salts (5X) solution, 10 mL
casamino acids (20% m/v), 10 mL glycol, 1 mL MgSO4 (2M), and 100 μL CaCl2 (1M). The M9
(5X) solution was prepared by dissolving 15 g Na2HPO4, 7.5 g K2HPO4, 2.5 g NaCl, 5 g NH4Cl
in 1 L deionized H2O and sterilized by autoclaving. The casamino acids, glycol and MgSO4
solutions were autoclaved separately. The CaCl2 solution was sterilized by filtration. Enriched M9
agar plates were prepared by adding 17 g agar to 1 L of enriched M9 media containing all of the
aforementioned components at the specified concentrations with the exception of CaCl2, which
was added immediately prior to plating. To media and plates, ampicillin was added to a final
concentration of 100 mg/L and chloramphenicol was added to a final concentration of 34 mg/L.
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Protein Expression. Cloning of MbH64V,V68A was described previously.9
Mb(H64V,V68A) was expressed in E.coli C41(DE3) cells as follows. After transformation, cells
were grown in TB medium (ampicillin, 100 mg/L) at 37°C (200 rpm) until OD600 reached 0.6.
Cells were then induced with 0.25 mM isopropyl--D-1-thiogalactopyranoside (IPTG) and 0.3
mM -aminolevulinic acid (ALA). After induction, cultures were shaken at 180 rpm and 27 °C
and harvested after 20 h by centrifugation at 4,000 rpm at 4°C. The cells were resuspended in 20
mL of Ni-NTA Lysis Buffer (50 mM KPi, 250 mM NaCl, 10 mM histidine, pH 8.0). Resuspended
cells were frozen and stored at -80°C until purification.
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Expression of Cofactor-Substituted Myoglobin variants. Mb(H64V,V68A)Fe(DADP)
was expressed in E.coli C41(DE3) cells using pET22_Mb(H64V,V68A) as follows. The cells were
plated on a lysogeny broth agar plate containing ampicillin and a single colony of freshly
transformed cells was cultured in 5 mL of lysogeny broth medium containing ampicillin. The
overnight cultures were transferred to 1 L enriched M9 medium containing ampicillin, followed
by incubation at 37°C with shaking (180 rpm). At OD600 of 1.4, cells were induced with 0.25 mM
IPTG and the induced culture was shaken at 27°C and rpm 180 for additional 16 to 20 hours. After
harvesting, the cells were pelleted by centrifugation, resuspended in 20 mL of Ni- NTA Lysis
Buffer (50 mM KPi, 250 mM NaCl, 10 mM histidine, pH 8.0) and 20 μL of a cofactor solution
(30 mg/mL in DMF) were added. Cells were lysed by sonication and the cell lysate was clarified
by
centrifugation
(14,00
rpm,
4°C,
30
min).
For
expression
of
Mb(H64V,V68A,H93NMH)Fe(DADP), pET29b_Mb(H64V,V68A,H93NMH) and pEVOL-
PyIRS(NMH) were used to transform E.coli BL21(DE3) and the cells were plated on a lysogeny
broth agar plate containing ampicillin and chloramphenicol. A single colony of freshly transformed
cells was cultured overnight in 5 mL of lysogeny broth medium containing ampicillin and
chloramphenicol. The overnight cultures were transferred to 1 L enriched M9 medium containing
ampicillin and chloramphenicol, followed by incubation at 37°C with shaking (180 rpm). At OD600
of 0.4, cell cultures were condensed by centrifugation (4,000 rpm, 4°C, 30 min) followed by
resuspension of the cell pellets in 200 mL of enriched M9 medium. To the condensed expression
cultures, N-3-methyl-L-histidine (NMH) and arabinose were added to a final concentration of X
mM and X mM, respectively, and then cultures were shaken at 24°C and 180 rpm for 20 minutes
before induction with 0.25 mM IPTG. The induced culture was shaken at 24°C and rpm 180 for
additional 16 to 20 hours. After harvesting, the cells were pelleted by centrifugation, resuspended
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in 20 mL of Ni- NTA Lysis Buffer (50 mM KPi, 250 mM NaCl, 10 mM histidine, pH 8.0) and 20
μL of a cofactor solution (30 mg/mL in DMF) were added. Cells were lysed by sonication and the
cell lysate was clarified by centrifugation (14,00 rpm, 4°C, 30 min).
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Protein Purification. The clarified lysate was transferred to a Ni-NTA column
equilibrated with Ni-NTA Lysis Buffer. The resin was washed with 50 mL of Ni-NTA Lysis Buffer
and then 50 mL of Ni-NTA Wash Buffer (50 mM KPi, 250 mM NaCl, 20 mM histidine, pH 8.0).
Proteins were eluted with Ni-NTA Elution Buffer (50 mM KPi, 250 mM NaCl, 250 mM histidine,
pH 7.0). After elution from the Ni-NTA column, the protein was buffer exchanged against 50 mM
KPi buffer (pH 7.0) using 10 kDa Centricon filters. The concentration of the Mb variants (ferric
form) was determined using the following extinction coefficients: 410=157 mM-1cm-1 for
Fe(ppIX)-containing variants and 470=60 mM-1cm-1 for Fe(DADP)-containing variants, which
was determined using the pyridine hemocrome assay.100
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Tm determination. Thermal denaturation experiments were carried out using a JASCO J-
1100 CD spectrophotometer equipped with variable temperature/wavelength denaturation analysis
software and samples of purified Mb variant at 3 μM in 50 mM potassium phosphate buffer (pH
7.0). Thermal denaturation curves were measured by monitoring the change in molar ellipticity at
222 or 228 nm over a temperature range from 13°C to 100°C. The temperature increase was set to
0.5°C per minute with an equilibration time of 10 seconds. Data integration time for the melt curve
was set to 4 seconds with a bandwidth of 1 nm. Linear baselines for the folded (θf) and unfolded
state (θu) were generated using the low temperature (θf = mf T + bf ) and high temperature (θu = mu
T + bu) equations fitted to the experimental data before and after global unfolding, respectively
Using these equations, the melt data were converted to fraction of folded protein (Ff) vs.
temperature plots and the resulting curve was fitted to a sigmoidal equation (θfit) via nonlinear
regression analysis in SigmaPlot from which apparent melting temperatures were derived. The
reported mean values and standard errors were derived from experiments performed at least in
duplicate.
Reduction potential determination. Reactions were carried out on a 1 mL scale in a
solution of KPi (50 mM, pH 7) containing xanthine (30 mM stock solution); protein; dye; catalase
(10 mg/mL stock solution); a mixture of glucose (1 M stock solution)/glucose oxidase (175 μM
stock solution), and xanthine oxidase (175 μM stock solution). In a sealed vial, a solution of buffer
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containing glucose (5 mM final concentration), glucose oxidase (50 μg/mL final concentration),
and xanthine (300 μM final concentration) was degassed by bubbling argon for 3 min. A buffered
solution containing the myoglobin variant and dye was carefully degassed in a similar manner in
a sealed cuvette (the concentration of the dye was adjusted by titration to give an absorbance which
is approximately equal to that of the highest absorbance band in the protein spectra). The two
solutions were then mixed together via cannula, and then catalase (5 μg/mL final concentration)
and xanthine oxidase (50 nM final concentration) were added to initiate the two-electron oxidation
of xanthine to uric acid and the corresponding reduction of protein and dye. The reactions were
monitored by UV–Vis spectrophotometry and the data were plotted. The reduction potential was
determined by adding the standard reduction potential of dye to the value of the y intercept
obtained by fitting the data to the Nernst equation (Eq. 1).
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[
]
[
]
퐷푦푒표푥
푃푟표푡푒푖푛표푥
(Eq. 1)
퐸푚.퐷푦푒
+
푅푇ln
= 퐸푚.푃푟표푡푒푖푛
+
푅푇ln
(
)
(
)
[
]
[
]
n퐹
n퐹
퐷푦푒푟푒푑
푃푟표푡푒푖푛푟푒푑
The absorbance values corresponding to the protein (based on the Soret band of the oxidised form)
and the dye (Figure S3) were used to determine the ratio of concentrations of oxidized (ox) to
reduced (red) form of both protein and dye at each stage of the experiment (Eq. 2).
A ― Amin
Amax ― A
[oxidised]
[reduced]
(Eq. 2)
=
Cyclopropanation reactions. General cyclopropanation reactions were carried out at a
400 μL-scale using 10 μM Mb variant, 10 mM styrene and 20 mM EDA. In a typical procedure,
potassium phosphate buffer (50 mM, pH 7.0) was degassed by bubbling argon into the mixture for
3 min in a sealed vial. A buffered solution containing the myoglobin variant was carefully degassed
in a similar manner in a separate vial. The two solutions were then mixed together via cannula.
Reactions were initiated by addition of 10 μL of styrene (from a 0.4 M stock solution in ethanol),
followed by the addition of 10 μL of EDA (from a 0.8 M stock solution in ethanol) with a syringe,
and the reaction mixture was stirred for 16 hours at room temperature, under positive argon
pressure. Aerobic reactions were carried out without degassing the solution with argon and in open
reaction vessels. Reactions with reductant were performed in a similar manner adding to the
potassium phosphate buffer 40 μL of sodium dithionite (100 mM stock solution in buffer).
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Product analysis. The reactions were analyzed by adding 20 μL of internal standard
(benzodioxole, 100 mM in methanol) to the reaction mixture, followed by extraction with 400 μL
of dichloromethane (DCM) and analyzed by GC − FID (see the Analytical Methods in the
Supporting Information). Calibration curves of the different cyclopropane products were
constructed using synthetically produced authentic standards (see the Synthetic Procedures in the
Supporting Information). All measurements were performed at least in duplicate. For each
experiment, negative control samples containing either no enzyme or no reductant were included.
For stereoselectivity determination, the samples were analyzed by chiral GC−FID or chiral SFC
as described in the Supporting Information
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Kinetic experiments. For the kinetic measurement, reactions were carried out on a 400
μL-scale in KPi buffer (pH 7.0) using 5 μM Mb variant, 2.5 mM vinyl benzoate (from a 0.2 M
stock solution in ethanol) and 2.5 mM EDA (from a 0.2 M stock solution in ethanol). At regular
intervals 20 μL of solution were collected and quenched with 50 μL of 0.2 M HCl. Aliquots were
analysed by adding 20 μL of internal standard (benzodioxole, 50 mM in ethanol) followed by
extraction with 400 μL of dichloromethane and analysis by gas chromatography (GC). For the
Hammett analyses, reactions were carried out on a 400 μL scale in KPi buffer (pH 7.0) and 10%
ethanol using 1 μM or 2 μM Mb variant (see Figure S6 and S7 for details), 1.25 mM substituted
styrene (from a 0.1 M stock solution in ethanol), 1.25 mM styrene (from a 0.1 M stock solution in
ethanol) and 2.5 mM EDA (from a 0.2 M stock solution in ethanol). The reactions were quenched
after 2, 4 and 5 min by adding 100 μL of 0.2 M HCl and 20 μL of internal standard (benzodioxole,
50 mM in ethanol) to the reaction mixture. The products were extracted with 400 μL of
dichloromethane and analysed by gas chromatography (GC). Representative kinetic plots
corresponding to these experiments are reported in Figure S6 and S7.
Radical Spin Trap Experiments. Reactions were carried out on a 400 μL-scale using 10
μM Mb variant, 10 mM styrene, 20 mM EDA with or without 100 mM 4-hydroxy-2,2,6,6-
tetramethylpiperidine 1-oxyl (TEMPOL) or 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), and 10
mM sodium dithionite. In a typical reaction, a solution containing sodium dithionite (100 mM
stock solution) in potassium phosphate buffer (50 mM, pH 7.0, 10% dimethylformamide (DMF))
was degassed by bubbling argon into the mixture for 3 min in a septum-capped vial. A buffered
solution containing the myoglobin variant was carefully degassed in a similar manner in a separate
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vial. The two solutions were then mixed together via cannula. Reactions were initiated by addition
of 10 μL of styrene (from a 0.4 M stock solution in DMF), followed by the addition of radical trap
reagent (40 μL, 1M stock solution in DMF) and 10 μL of EDA (from a 0.8 M stock solution in
DMF) with a syringe. The reaction was stirred for 16 hours at room temperature, under positive
argon pressure. For product analysis, 20 μL of internal standard (benzodioxole, 50 mM in ethanol)
were added to the reaction mixture followed by extraction with 400 μL of dichloromethane and
analysis by gas chromatography (GC).
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Reactions with cis--deutero-styrene. Reactions were carried out at 2 mL-scale using 60
μM Mb variant, 0.2 M cis--d1-styrene, 0.4 M ethyl diazoacetate (EDA). In a typical reaction,
potassium phosphate buffer (50 mM, pH 7.0, 10% DMF) was degassed by bubbling argon into the
mixture for 3 min in a sealed vial. A buffered solution containing the myoglobin variant was
carefully degassed in a similar manner in a separate vial. The two solutions were then mixed
together via cannula. Reactions were initiated by addition of cis--d1-styrene (42.1 mg, 0.40
mmol), followed by the addition of EDA (84.0 mL, 0.80 mmol) with a syringe. The reaction
mixture was stirred for 16 hours at room temperature, under positive argon pressure. The reaction
products were extracted with dichloromethane (2 mL, three rimes), and the organic layers were
collected and dried over MgSO4. The organic solvent was removed by rotary evaporation, and the
crude was purified via flash chromatography (silica gel, hexane:AcOEt 99:1). The analytical data
for 3a-d and 3b-d are in accord with those reported in literature. Enantiopure d-5acis: 1H NMR (400
MHz, CDCl3):  7.30 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2 Hz, 2H), 4.20
(q, J = 14.4 Hz, 7.2 Hz, 2H), 2.53 (dd, J = 9.2 Hz, 4.0 Hz, 1H), 1.91 (t (=dd), J = 4.0 Hz, 1H), 1.61
(dd, J = 8.9 Hz, 5.2 Hz, 1H), 1.30 (t, J = 7.2 Hz, 3H) ppm. 2H NMR (60 MHz, CDCl3):  1.32 ppm
(s, 1H). Enantiopure d-5atrans: 1H NMR (400 MHz, CDCl3):  7.28-7.21 (m, 2H), 7.16 (t, J = 7.1
Hz, 1H), 7.06 (d, J = 7.2 Hz, 2H), 3.83 (q, J = 7.1 Hz, 2H), 2.54 (t, J = 9.0 Hz, 1H), 2.03 (t, J =
8.5 Hz, 1H), 1.28 (d, J = 7.0 Hz, 1H), 0.93 ppm (t, J = 7.1 Hz, 3H). 2H NMR (60 MHz, CDCl3): 
1.72 ppm (s, 1H).
29
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