Cyclopropanations via Heme Carbenes: Basic Mechanism and Effects of Carbene Substituent, Protein Axial Ligand, and Porphyrin Substitution

Article abstract of DOI:10.1021/jacs.7b09171

Catalytic carbene transfer to olefins is a useful approach to synthesize cyclopropanes, which are key structural motifs in many drugs and biologically active natural products. While catalytic methods for olefin cyclopropanation have largely relied on rare transition-metal-based catalysts, recent studies have demonstrated the promise and synthetic value of iron-based heme-containing proteins for promoting these reactions with excellent catalytic activity and selectivity. Despite this progress, the mechanism of iron-porphyrin and hemoprotein-catalyzed olefin cyclopropanation has remained largely unknown. Using a combination of quantum chemical calculations and experimental mechanistic analyses, the present study shows for the first time that the increasingly useful C-C functionalizations mediated by heme carbenes feature an Fe^II-based, nonradical, concerted nonsynchronous mechanism, with early transition state character. This mechanism differs from the Fe^IV-based, radical, stepwise mechanism of heme-dependent monooxygenases. Furthermore, the effects of the carbene substituent, metal coordinating axial ligand, and porphyrin substituent on the reactivity of the heme carbenes was systematically investigated, providing a basis for explaining experimental reactivity results and defining strategies for future catalyst development. Our results especially suggest the potential value of electron-deficient porphyrin ligands for increasing the electrophilicity and thus the reactivity of the heme carbene. Metal-free reactions were also studied to reveal temperature and carbene substituent effects on catalytic vs noncatalytic reactions. This study sheds new light into the mechanism of iron-porphyrin and hemoprotein-catalyzed cyclopropanation reactions and it is expected to facilitate future efforts toward sustainable carbene transfer catalysis using these systems.

Full text of DOI:10.1021/jacs.7b09171

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Article
Cyclopropanations via Heme Carbenes: Basic Mechanism and Effects of
Carbene Substituent, Protein Axial Ligand, and Porphyrin Substitution
Yang Wei, Antonio Tinoco, Viktoria Steck, Rudi Fasan, and Yong Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09171 • Publication Date (Web): 22 Dec 2017
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Journal of the American Chemical Society
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Cyclopropanations via Heme Carbenes: Basic Mechanism and Ef-
fects of Carbene Substituent, Protein Axial Ligand, and Porphyrin
Substitution
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†
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ǂ
†
Yang Wei, Antonio Tinoco, Viktoria Steck, Rudi Fasan, * and Yong Zhang *
†
Department of Chemistry and Chemical Biology, Stevens Institute of Technology, 1 Castle Point on Hudson, Hoboken, NJ
0
7030, United States of America
ǂ
Department of Chemistry, University of Rochester, 120 Trustee Road, Rochester, NY 14627, United States of America
Supporting Information Placeholder
3
8-45
porphyrins
in cyclopropanation reactions have highlighted the
ABSTRACT: Catalytic carbene transfer to olefins is a useful
approach to synthesize cyclopropanes, which are key structural
motifs in many drugs and biologically active natural products.
While catalytic methods for olefin cyclopropanation have largely
relied on rare transition metal-based catalysts, recent studies have
demonstrated the promise and synthetic value of iron-based heme-
containing proteins for promoting these reactions with excellent
catalytic activity and selectivity. Despite this progress, the mech-
anism of iron-porphyrin and hemoprotein-catalyzed olefin cyclo-
propanation has remained largely unknown. Using a combination
of quantum chemical calculations and experimental mechanistic
analyses, the present study shows for the first time that the in-
creasingly useful C=C functionalizations mediated by heme car-
potential of exploiting earth-abundant and inexpensive first-row
transition metals for this important class of carbon-carbon bond
forming reactions. Furthermore, very recent studies have revealed
how iron-based, heme-dependent enzymes and proteins, such as
46-49
14, 50-52
cytochrome P450
and myoglobins,
constitute promising
biocatalysts for carbene-mediated cyclopropanation reactions. In
particular, excellent enantioselectivity (>90-99% ee) along with
high catalytic activity (>10,000 TON) and broad substrate scope,
have been recently achieved in olefin cyclopropanations using
engineered myoglobin variants.
applied to the stereoselective synthesis of cyclopropanes-
containing drugs at the multigram scale outperforming current
strategies to access these molecules. In addition to cyclopropa-
nation reactions, myoglobins and other hemoproteins have proven
useful for promoting a growing number of carbene-mediated
transformations, including carbene insertion into Y-H bonds (Y =
50, 51
These biocatalysts could be
5
1
II
benes feature an Fe -based, non-radical, concerted nonsynchro-
nous mechanism, with early transition state character. This mech-
IV
anism differs from the Fe -based, radical, stepwise mechanism of
heme-dependent monooxygenases. Furthermore, the effects of the
carbene substituent, metal coordinating axial ligand, and porphy-
rin substituent on the reactivity of the heme carbenes was system-
atically investigated, providing a basis for explaining experi-
mental reactivity results and defining strategies for future catalyst
development. Our results especially suggest the potential value of
electron-deficient porphyrin ligands for increasing the electro-
philicity and thus the reactivity of the heme carbene. Metal-free
reactions were also studied to reveal temperature and carbene
substituent effect on catalytic vs. non-catalytic reactions. This
study sheds new light into the mechanism of iron-porphyrin and
hemoprotein-catalyzed cyclopropanation reactions and it is ex-
pected to facilitate future efforts toward sustainable carbene trans-
fer catalysis using these systems.
53-57
58
N, S, Si, C),
rangements.
aldehyde olefinations, and sigmatropic rear-
5
9
Although iron porphyrin carbenes (IPCs) were implied in the
aforementioned carbene-mediated transformations and in some
cases directly shown to undergo cyclopropanation, the mecha-
nism of cyclopropanation reactions catalyzed by hemoproteins
and, more generally, Fe -porphyrins remains unknown. In biocat-
alytic cyclopropanations catalyzed by heme dependent proteins,
the ferrous species was indeed determined to be the catalytically
competent form of the metalloprotein.
investigations of cyclopropanations with formally ferric iron-
porphyrins suggest in situ reduction of the metalloporphyrin and
thus the active role of Fe -porphyrin carbene in these reactions.
3
1
II
4
7, 51
On the other hand,
II
29,
3
2, 36
Computationally, several different mechanisms have been
proposed for metal carbene cyclopropanations, including elimina-
60-62
tion from metallocyclobutane
and addition via both
6
3-65
66, 67
concerted
and stepwise
pathways. Since both the metal
Introduction
center and ligand environment greatly influence the operating
mechanism, it is critical to study the mechanism in newly devel-
oped systems such as the hemoprotein and Fe -porphyrin based
catalysts investigated here. Building on our recent progress in
developing DFT methods for accurate predictions of Fe -
Cyclopropanes are key structural motifs in many pharmaceu-
ticals and biologically active molecules, which underscores the
high synthetic values of catalytic methods for olefin cyclopropa-
nation via carbene transfer. Metalloporphyrin systems have
represented attractive catalysts for this type of reactions and major
efforts in this area have focused on metalloporphyrins incorporat-
ing noble metals, such as Rh,
owing to the well-known reactivity of second- and third-row tran-
1-5
II
6
-9
II
porphyrin carbenes’ experimental X-ray crystal structures, Möss-
bauer and NMR properties and IPC formations and C-H inser-
1
0-14
15-21
22-25
14, 26-28
Ru,
Os,
and Ir,
68-70
tions,
here we report the first quantum chemical investigation
of heme carbene-mediated cyclopropanation mechanism, which
was found to be different from Co porphyrin. The mechanistic
6-9
sition metals in carbene transfer reactions. More recently, the
successful development and application of Fe-
29-37
and Co-
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model derived from our computational analyses is supported by
Scheme 1. Target cyclopropanation reactions. (A) Concerted and stepwise
II
pathway for Fe -porphyrin catalyzed cyclopropanation of styrene. Oval
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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6
experiments with isotopically labeled probe substrates and radical
spin trapping reagents. In addition, this study performs the first
systematic analysis of the effects of carbene substituent, protein
axial ligand, porphyrin substituent on cyclopropanation reactions.
This study provides a mechanistic basis for explaining experimen-
tally observed reactivity trends as well as key insights for guiding
future development of iron-porphyrin based (bio)catalysts for
carbene-mediated cyclopropanations.
represents the porphyrin ligand. (B) Reactions 1-9 involving different
reactants (R ).
1
To this end, we initially investigated the cyclopropanation re-
action involving styrene and the iron-porphyrin complex
[
Fe(TPFPP)(C(Ph)CO
2
Et)] (TPFPP
=
meso-tetrakis(penta-
fluorophenyl)porphyrinato dianion), which was previously report-
31
ed by Che and coworkers. In this reaction (referred to as reac-
Results and Discussion
tion 1), two different pathways were considered and evaluated,
II
III
namely an Fe -based concerted pathway (in blue) and an Fe -
based radical stepwise pathway (in red) as shown in Scheme 1.
The TPFPP was modelled as a non-substituted porphyrin (Por) as
Cyclopropanation mechanism.
The IPC’s electronic nature is critical to understand its reac-
tivity. Previously, a decade-long debate has concerned whether
0
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4
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9
0
6
8, 69
II
0
IV
2- 31, 32,
done previously.
Experimental spin states of iron-containing
IPC is best described as Fe ß{:C(X)Y} or Fe ={C(X)Y} .
7
1-74
IV
reactant R (1) and product P (1) (here the number in parenthesis
The Fe -based resonance structure, which is isoelectronic
1
1
IV
2-
after the reaction species symbol indicates the reaction number
and analogous to the Fe =O group involved in cytochrome
P450-catalyzed monooxygenation reactions, was discussed in IPC
studies and it was proposed to mediate olefin cyclopropanations
31, 85
used in this work) are singlet and triplet respectively,
which
31
were well reproduced in our recent studies of other related chemi-
69, 70
46, 50
cal reactions.
To determine the favored spin state for the tran-
by engineered P450s and hemoproteins.
However, our recent
6
8, 69
II
IV
sition state, both singlet and triplet pathways were considered. We
first investigated the concerted mechanism. Transition states lead-
ing to both the trans and cis product were obtained. Only the
pathways leading to trans-1R,2S and cis-1R,2R cyclopropanes
were studied, as the other two possible products are mirror images
of these isomers.
studies
show that it is the Fe -based and not the Fe -based
resonance structure that yields more accurate predictions of exper-
imentally determined Mössbauer, X-ray, and NMR properties of
IPCs, with calculated charges being consistent with the experi-
mentally found electrophilic reactivity of this species.
Challenging this view, a more recent computational study on iron-
porphyrin catalyzed N‒H insertion proposes that a diradical, anti-
ferromagnetically coupled Fe {•C(X)Y} complex with an open-
shell singlet (OSS) state is the most favored and thus the relevant
IPC intermediate implicated in the carbene transfer process.
Indirect support to the proposed carbon-centered radical catalytic
intermediate was drawn from previous studies describing a radical
mechanism for Co-porphyrin-catalyzed cyclopropanation
other (non-heme) systems,
experimentally. This mechanistic proposal is reminiscent of the
2
9, 31, 32, 50, 75
B
A
III
-
7
6
6
6, 77
and
78-80
albeit such claim was not verified
radical nature of P450-catalyzed C‒H hydroxylation and epoxida-
C
D
8
1
IV
2-
tion of certain olefins. The Fe =O species itself exhibits radi-
cal feature as indicated by ~0.9 e spin density of the oxygen atom
III 82, 83
III
bound to formal Fe .
ground state does not appear to be fully consistent with the Fe
feature of isolated IPCs characterized by UV/Vis and NMR spec-
At the same time, the Fe -based OSS
7
0
II
72, 73
31, 72, 84
troscopies,
X-ray crystallography,
and XANES, which
8
4
is directly sensitive to Fe oxidation state. In light of these diver-
gent views and proposal about the electronic structure of IPCs
involved in carbene transfer reactions and the unknown mecha-
nism of hemoprotein-catalyzed cyclopropanation, we set to inves-
tigate this reaction through a combination of computations and
experiments.
Figure 1. (A) TStrans; (B) TScis; (C) TS1trans; (D) TS1cis in reac-
tion 1. Atom color scheme: Fe – black, C – cyan, N – blue, O –
red, H – grey.
Our results show that the singlet transition state is less favora-
ble than the triplet transition state (see Table S4), which features a
II
Fe (S=1) center with non-radical character for carbene, styrene,
Ph
A
and porphyrin based on the calculated negligible spin densities
reported in Table S5. Using broken-symmetry initial setups of
C
C
3
2
R
2
CO Et
C
R
CO Et
2
III
C
1
Fe ferromagnetically and anti-ferromagnetically coupled with
Ph
Concerted
carbene radical for triplet and singlet transition states respectively
CO
R
2
Et
FeII
+
FeII
FeII
+
II
Ph
led to basically the same Fe -based results after geometry optimi-
II
R
1
R
2
TS
P
1
P
2
zations, suggesting the dominant Fe feature for the concerted
‡
Stepwise
mechanism. The smaller ΔG value for the pathway leading to the
trans product (18.58 kcal/mol) compared to that yielding the cis
Ph
Ph
R
Ph
isomer (20.75 kcal/mol) is consistent with the experimentally
3
1
CO
2
Et
R
CO
2
Et
R
2
CO Et
found trans selectivity of the reaction. This selectivity may be a
result of favorable π-π interactions between the phenyl rings of
the carbene moiety and styrene and one more short attractive
FeIII
FeIII
FeII
TS1
Int
TS2
…
B
O H interaction as illustrated in Figure 1A and 1B.
reaction: 1
: [Fe(Por)(C(Ph)CO
reaction: 4
: [Fe(TPFPP)(C(Ph)CO
reaction: 7
: [Fe(Por)(CHCO
reaction: 2
: [Fe(Por)(CHCO
reaction: 5
: [Fe(TPFPP)(CPh
reaction: 8
: [Fe(Por-meso-CN)(CHCO
reaction: 3
: [Fe(Por)(CPh
reaction: 6
: [Fe(Por)(CHCO
reaction: 9
Et)] R
2
: [Fe(Por-HBG)(CHCO Et)]
For the stepwise mechanism (red, Scheme 1), the first transi-
R
1
2
Et)]
R
1
2
Et)]
R
1
2
)]
tion state, TS1(1), which is the bottleneck step compared to
6
6
R
1
2
Et)]
R
1
2
)]
R
1
2
Et)(5-MeIm)]
TS2(1) in Co porphyrin cyclopropanation, was obtained for both
trans and cis isomers. Interestingly, in this case, with initial setups
of ferric center and radical carbene/styrene, the optimized struc-
R
1
2
Et)(SH-)]
R
1
2
1
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tures maintain some radical feature. For instance, the C3 carbon
diazoacetate (EDA, 2) as the carbene donor, and iron-tetraphenyl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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5
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5
5
6
atom (see Scheme 1 for atomic labels) has spin densities of 0.280
e and 0.384 e for triplet TS1trans(1) and TS1cis(1), and it has even
larger spin densities of -0.531/-0.567 e in the corresponding sin-
glet TS1trans(1) and TS1cis(1) (Table S5). The iron spin densities
of 1.373/1.375 e in these singlet transition states coupled with -
porphyrin (Fe(TPP)), hemin (iron-protoporphyrin IX or Fe(ppIX)),
or myoglobin (Mb) as the catalyst. As for the biocatalysts, we
evaluated both wild-type sperm Mb and its engineered variant
Mb(H64V,V68A), which displays significantly enhanced catalytic
activity (>10,000 vs. 180 turnover (TON) for Mb) as well as ex-
cellent trans-(1S,2S)-diastereo- and enantioselectivity (>99% de
and ee vs. 86% de and 0% ee for Mb) in the cyclopropanation of
0
.813/-0.809 e spin densities of the carbene carbon atom suggest
III
an Fe -based OSS feature, while the iron spin densities of ~1.9 e
and carbene carbon spin densities of ~0.1 e in the triplet transition
states indicate a major Fe feature. For the radical pathway, the
5
0
vinylarenes with EDA. To investigate the occurrence of step-
wise vs. concerted mechanism, we initially carried out the cyclo-
propanation reactions in presence of cis-β-deutero-styrene (1b,
Table 1), which was meant to probe the formation of a carbon-
centered radical intermediate (Int(1), Scheme 1). Indeed, a stereo-
specific reaction resulting in a cis configuration relative to the ‒D
and ‒Ph group in the corresponding cyclopropanation product
would be expected in the case of a concerted mechanism, whereas
(partial) cis/trans isomerization would be revealing of the inter-
mediacy of a radical species. Importantly, the isolated trans-2-
phenyl-1-ethyl carboxylate cyclopropane products from the reac-
tions catalyzed by Fe(TPP), wild-type Mb, and Mb(H64V,V68A)
II
III
II
Fe -based OSS transition states are more favorable than the Fe
triplet states by 3-8 kcal/mol (Table S4) and more favorable than
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
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5
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9
0
1
2
3
4
5
6
7
8
9
0
II
the Fe -based CSS transition states leading to the trans and cis
‡
products by 9.10 and 12.67 kcal/mol (ΔΔG ), respectively, which
make them indeed the most favorable states for this stepwise
III
mechanism. Regarding the geometries of these Fe -based OSS,
there is only one short distance between the carbene carbon and
the α and β carbons of styrene (RC1C2~2.2 Å, RC1C3~3.1 Å), as
shown in Figure 1C and 1D and Table S5, and as expected for the
‡
stepwise attack. Interestingly, the ΔG difference between trans
1
and cis transition states in this stepwise pathway is only 0.8
kcal/mol, which is smaller than the ~ 2 kcal/mol difference in the
concerted pathway and is similar to 0.3 kcal/mol difference for
styrene radical cyclopropanation catalyzed by Co porphyrin. As
shown in Figure 1C and 1D, the aromatic ring of styrene adopts a
conformation parallel to the plane of the porphyrin ring, which
differs from the tilted conformation observed in the concerted
pathway (Figure 1A and 1B). In this radical stepwise mechanism,
initial conformations analogous to those observed in the concerted
pathway and initial conformations in which the phenyl ring is
perpendicular to the porphyrin ring were also investigated. In all
cases, all conformations converged to that observed in the con-
certed pathway after geometry optimization. This suggests a pref-
all consisted of the syn addition product 3a, as determined by H
2
and H NMR spectroscopy (Table 1, Entries 1-2, 4). A detectable
albeit negligible amount of diastereomer 3b (<2%) was observed
with hemin (Entry 3). In stark contrast, a significant degree of
cis/trans isomerization (39.7%) was obtained in the same reaction
using Co(TPP) as the catalyst (Entry 5), a result that is consistent
with the previously reported radical mechanism of Co(TPP)-
6
6, 77
catalyzed cyclopropanation.
Importantly, the stereospecificity
of the cyclopropanation reaction catalyzed by Fe(TPP) and the
myoglobin variants strongly support a concerted, non-radical
pathway for the cyclopropanation reaction catalyzed by iron-
porphyrins.
Table 1. Biocatalytic and chemocatalytic cyclopropanation
reactions with cis-β-deutero-styrene.
‡
erence of the concerted pathway. In fact, the ΔG values for both
TS1trans(1) and TS1cis(1) in this stepwise pathway are higher than
the corresponding TStrans(1) and TScis(1) in the concerted path-
way by 4-7 kcal/mol (Table S6). The electronic energies, zero-
point energy corrected electronic energies, and enthalpies exhibit
the same trend, which clearly indicate that the radical, stepwise
pathway is unfavorable compared to the concerted pathway.
Based on these results, subsequent steps in the unfavorable, radi-
cal pathway were not further considered and we focused our at-
tention on the concerted pathway leading to the trans cyclopropa-
nation product, which is the favored product in the presence of
Reactionꢀa
b
Ratioꢀcꢀ
%ꢀꢀ
isomeriz.ꢀ
0%ꢀ
0%ꢀ
1.7%ꢀ
0%ꢀ
Entryꢀ
1ꢀ
Catalystꢀ
Yield ꢀ
Conditions ꢀ
3aꢀ:ꢀ3b ꢀ
30
50, 51
Mb(H64V,V68A)ꢀ
Mbꢀ
Aꢀ
Aꢀ
Aꢀ
Bꢀ
Bꢀ
48%ꢀ
3%ꢀ
24%ꢀ
14%ꢀ
5%ꢀ
1ꢀ:ꢀ0ꢀ
1ꢀ:ꢀ0ꢀ
1ꢀ:ꢀ0.017ꢀ
1ꢀ:ꢀ0ꢀ
synthetic iron porphyrin catalysts and myoglobins
2
ꢀ
As illustrated by the data in Figure 2-1, charge analysis shows
a significant charge transfer (CT) from the olefin substrate to the
carbene moiety, a result consistent with the experimentally deter-
3ꢀ
Fe(ppIX)ꢀ
Fe(TPP)ꢀ
Co(TPP)ꢀ
4
5
ꢀ
ꢀ
1ꢀ:ꢀ0.66ꢀ
39.7%ꢀ
aꢀ
3
1, 50
ReactionꢀconditionsꢀA:ꢀ200ꢀmMꢀ1,ꢀ400ꢀmMꢀEDA,ꢀ60ꢀµMꢀMbꢀvariantꢀ(orꢀhe-
min),ꢀ10ꢀmMꢀsodiumꢀdithioniteꢀinꢀ50ꢀmMꢀpotassiumꢀphosphateꢀbufferꢀ(pHꢀ7)ꢀ
containingꢀ 10%ꢀ DMF,ꢀ roomꢀ temp.,ꢀ 16ꢀ hours.ꢀ Reactionꢀ conditionsꢀ B:ꢀ 0.475ꢀ
mined electrophilicity of the latter.
As shown in Figure 3-1,
the most significant geometric change at the transition state
TStrans(1) relative to the reactants is the elongation of the Fe-
carbene distance (RFeC1) by about ~0.5 Å to accommodate attack
of the carbene group to the C=C bond of styrene. At this step,
both the elongation of the C=C bond (RC2C3) and the change in the
mmolꢀ1,ꢀ1.5ꢀequivꢀEDAꢀ(slowꢀaddition),ꢀ5ꢀmol%ꢀcatalystꢀinꢀCH
hours.ꢀ ꢀIsolatedꢀyieldꢀforꢀtrans-configuredꢀproductꢀrelativeꢀtoꢀPhꢀandꢀCO
2
Cl
2
,ꢀ40°C,ꢀ16ꢀ
Etꢀ
b
2
c
groupsꢀ (racemicꢀ forꢀ allꢀ reactionsꢀ exceptꢀ withꢀ Mb(H64V,V68A).ꢀ ꢀ Basedꢀ onꢀ
2
peakꢀintegrationꢀinꢀ HꢀNMRꢀspectrum.ꢀꢀꢀ
bond angle between the carbene substituents (Ph‒C‒CO
modest (0.013 Å and 4.2°, respectively). These minor structural
changes along with the 117.6° value for the Ph‒C‒CO Et bond
angle indicate that the carbene carbon in the transition state is still
2
Et) are
To further corroborate these conclusions, cyclopropanation
reactions with styrene (1a) and EDA (2) were then carried out in
the presence of the radical spin trapping agent 5,5-dimethyl-1-
pyrroline N-oxide (DMPO). As shown in Table 2, no effect in
product yield was observed for the reactions catalyzed by the
myoglobin variants or hemin, in the presence of DMPO when
compared to parallel reactions performed in its absence (Entries 1,
3 and 5 vs. 2, 4, and 6, respectively). In stark contrast, a dramatic
reduction in product yield (~90% reduction) was observed for the
Co(TPP)-catalyzed reaction upon addition of the radical trapping
reagent (Entry 7 vs. 8). These results thus agree with those ob-
tained with cis-β-deutero-styrene in evidencing a non-radical
2
2
3
largely sp -hydridized as in the reactant, rather than exhibiting sp
geometry as in the product. Altogether, these structural features
suggest that this is an early transition state. Furthermore, a differ-
ence of about 0.3 Å between the C1···C2 and C1···C3 distances
indicate the nonsynchronous character of the carbene addition
process to the olefin, as illustrated in Figure 3-1.
To verify these predictions experimentally, mechanistic stud-
ies were performed using styrene (1a) as the olefin substrate, ethyl
ACS Paragon Plus Environment

Journal of the American Chemical Society
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1
2
3
4
5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
8
9
Figure 2. (1-9) Atomic charge changes from reactants to transition state (in black) and charge transfers (in blue) as indicated by arrows and
numbers in parentheses in reactions 1-9.
mechanism for iron-porphyrin and hemoprotein-catalyzed cyclo-
propanation. Thus, these studies provide strong experimental sup-
port to the proposed concerted mechanism for IPC-mediated cy-
clopropanation, as derived from our computational analyses. Fur-
thermore, the small secondary kinetic isotope effect (KIE) deter-
mined for the Mb(H64V,V68A)-catalyzed cyclopropanation of
With the basic mechanism of IPC-mediated cyclopropanation
revealed above, we then extended our computational analyses to
the study of the carbene substituent effect. We first examined the
cyclopropanation of styrene with [Fe(Por)(CHCO
from EDA (reaction 2), a carbene donor reagent extensively in-
2
Et)] derived
46, 47, 49-51
vestigated in hemoprotein-
cyclopropanations.
and iron-porphyrin-catalyzed
Interestingly, the singlet transition
5
0
29-33, 36, 37
styrene versus perdeuterated d
8
-styrene (k
H
/k
D
= 0.96 ± 0.02)
suggests a minimal extent of rehybridization of the olefin carbon
atoms in the transition state, which is consistent with reagent-like
early transition-state predicted by our computations (Figure 3-1).
For this reaction a KIE of 0.99 was obtained by computation,
which is in excellent accord with the experimentally determined
state in this reaction is more favorable than the triplet state by ΔG
of 1.02 kcal/mol. This effect may be attributed to the reduced
steric hindrance of the CHCO Et vs. C(Ph)CO Et moiety, which
allows for a closer distance between the carbene carbon atom and
Fe (~0.5 Å) and thus a stronger field strength effect on the iron
center, which favors the low spin state. Furthermore, the comput-
ed ΔG for this step (12.47 kcal/mol) is significantly smaller (~6
kcal/mol) than that for the corresponding transition state in the
2
reaction with [Fe(Por)(C(Ph)CO Et)] (reaction 1), indicating a
more facile cyclopropanation reaction with the IPC intermediate
derived from ethyl diazoacetate (EDA). The elevated reactivity of
the latter may arise from the increased charge transfer from the
substrate to the carbene by 0.069 e due to shorter substrate-
carbene distance (ΔRC1C2 of 0.229 Å and ΔRC1C3 of 0.240 Å).
2
2
50
value (0.96).
‡
Table 2. Cyclopropanation reactions in the presence or absence of
free-radical spin trapping agent.
To further investigate this aspect, we analyzed reaction 3, in
Reactionꢀa
Conditions ꢀ
b
c
d
d
Entryꢀ
Catalystꢀ
DMPO ꢀ Yield ꢀ
%ꢀde ꢀ
%ꢀee ꢀ
which [Fe(Por)(CPh
2
)] is used as the carbene source. As shown in
Table 3, the ΔG for this reaction is higher than that in the reac-
tion with [Fe(Por)(C(Ph)CO Et)] by 1.16 kcal/mol. Therefore, the
electron-withdrawing effect (EWG) of the CO Et group in reac-
tion 1 enhances the reaction rate as compared to the electron-
donating phenyl group in reaction 3. This result is consistent with
the substrate-to-carbene CT effect highlighted above, whereby the
EWG group in the carbene moiety facilitates the reaction, as re-
flected by an increased CT of 0.029 e for the transition state in-
2 2
volving [Fe(Por)(C(Ph)CO Et)] vs. [Fe(Por)(CPh )] (Figure 2-1
vs. 2-3). This result agrees with the experimentally observed
higher reactivity of [Fe(TPFPP)(C(Ph)CO Et)] compared to
2
[Fe(TPFPP)(CPh) ] in stoichiometric cyclopropanations. How-
ever, the reactivity difference in these experiments is more pro-
nounced than anticipated by our calculations, as indicated by the
2% yield (= 82 TON) obtained with [Fe(TPFPP)(C(Ph)CO Et)]
2
‡
1
2
3
4
5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Mb(H64V,V68A)ꢀ
Mb(H64V,V68A)ꢀ
Mbꢀ
Aꢀ
Aꢀ
Aꢀ
Aꢀ
Aꢀ
Aꢀ
Bꢀ
Bꢀ
Noꢀ
Yesꢀ
Noꢀ
Yesꢀ
Noꢀ
Yesꢀ
Noꢀ
Yesꢀ
86%ꢀ
82%ꢀ
27%ꢀ
35%ꢀ
8%ꢀ
13%ꢀ
76%ꢀ
8%ꢀ
99ꢀ
99ꢀ
89ꢀ
90ꢀ
87ꢀ
87ꢀ
49ꢀ
29ꢀ
99ꢀ
98ꢀ
3ꢀ
1ꢀ
0ꢀ
0ꢀ
0ꢀ
0ꢀ
2
2
Mbꢀ
Fe(ppIX)ꢀ
Fe(ppIX)ꢀ
Co(TPP)ꢀ
6ꢀ
7
8
ꢀ
ꢀ
Co(TPP)ꢀ
aꢀ
ReactionꢀconditionsꢀA:ꢀ10ꢀmMꢀstyrene,ꢀ20ꢀmMꢀEDA,ꢀ20ꢀµMꢀMbꢀvariantꢀ(orꢀ
hemin),ꢀ10ꢀmMꢀsodiumꢀdithioniteꢀinꢀ50ꢀmMꢀpotassiumꢀphosphateꢀbufferꢀ(pHꢀ
)ꢀcontainingꢀ10%ꢀDMF,ꢀroomꢀtemp.,ꢀ16ꢀhours.ꢀReactionꢀconditionsꢀB:ꢀ0.24ꢀ
7
mmolꢀstyrene,ꢀ1.5ꢀequivꢀEDAꢀ(slowꢀaddition),ꢀ5ꢀmol%ꢀCo(TPP)ꢀinꢀCH
2
Cl
2
,ꢀ40°Cd,ꢀꢀ
b
c
16ꢀhours.ꢀ ꢀWithꢀorꢀwithoutꢀ10ꢀequivꢀDMPOꢀrelativeꢀtoꢀstyrene.ꢀ ꢀGCꢀyield.ꢀ
Diastereomericꢀ(de)ꢀandꢀenantiomericꢀexcessꢀ(ee)ꢀforꢀtrans-(1S,2S)ꢀcyclopro-
paneꢀproduct.ꢀ
2
3
1
ꢀ
ꢀꢀ
Carbene substituent effect.
8
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1
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
6
7
8
9
Figure 3. (1-9) Key geometric parameters at transition state (in black) and changes from reactants to transition state (in blue) in reactions
1
-9. Atom color scheme: Fe - black, O - red, C - cyan, H – grey, F- purple, S- yellow.
a
Table 3. Key Energy, Charge, and Geometry Parameters.
the reactivity of the IPC intermediate in cyclopropanation reac-
tions.
‡ b)
TS
ΔG
kcal/ (kcal/
mol)
mol)
ΔG°
Q
(e)
CT
ΔQC1
(e)
ΔRC2C3
(Å)
R
C2C3
ΔRFeCl
(Å)
(
(Å)
To examine experimentally the carbene substituent effect on
the (bio)catalytic cyclopropanation reactions, side-by-side exper-
iments were performed using EDA vs. ethyl 2-diazo-2-phenyl-
acetate (EDPA, 6) as the carbene donor in the presence of
Mb(H64V,V68A), hemin or Fe(TPP) as the catalyst (Table 4).
For these reactions, 4-methoxy-styrene (5) was used as the olefin
substrate instead of the more volatile styrene because of the appli-
cation of elevated temperatures (50°C) for the reactions involving
EDPA. As summarized by the data in Table 4, these experiments
revealed that the Mb(H64V,V68A)-catalyzed cyclopropanation
reaction with the acceptor-only carbene donor (EDA) proceeds
with significantly higher rate (528-fold) and efficiency (140-fold
higher TON) at room temperature than the corresponding reac-
tions with EDPA at 50°C (Entry 1 vs 2). A similar reactivity trend
was observed for hemin and Fe(TPP), albeit both rates and cata-
lytic turnovers (TON) were drastically reduced in both cases
compared to the hemoprotein catalyst. Furthermore, unlike the
latter, hemin yielded no cyclopropanation product in the reaction
with EDPA at 50°C. These results are therefore consistent with
1
2
3
4
5
6
7
8
9
18.58 -35.50
12.47 -39.18
19.74 -30.43
16.25 -17.46
21.89 -11.69
7.49 -52.40
11.90 -41.27
9.24 -56.65
11.28 -66.71
0.158 -0.266
0.227 -0.148
0.129 -0.324
0.118 -0.245
0.161 -0.373
0.208 -0.122
0.223 -0.137
0.169 -0.142
0.180 -0.155
0.013
0.019
0.018
0.008
0.020
0.015
0.019
0.014
0.018
1.344 0.518
1.350 0.110
1.349 0.808
1.339 0.622
1.351 0.728
1.346 0.088
1.350 0.101
1.345 0.108
1.349 0.127
a
Results are for the most favorable trans products. Changes are those at
transition state compared with reactants.
and styrene, compared to the lack of reactivity for
Fe(TPFPP)(CPh) ] even at elevated temperatures and extended
2
reaction times. We attribute this difference to the porphyrin sub-
stituent effect, which was absent in our initial calculations. We
then calculated the cyclopropanation pathways using authentic
[
[Fe(TPFPP)(C(Ph)CO
reaction 5). As shown in Table 3, a similar trend was observed in
2 2
Et)] (reaction 4) and [Fe(TPFPP)(CPh) ]
2
the higher reactivity of the [Fe(Por)(CHCO Et)] vs.
(
‡
[Fe(Por)(C(Ph)CO Et)] intermediate as predicted by our computa-
these reactions but a significantly larger ΔΔG (5.64 kcal/mol)
was found for reaction 5 vs. reaction 4. This result is thus more in
line with the dramatic reactivity difference observed experimen-
tally. Altogether, the computational analyses described above
highlight the important role of the carbene substituent in affecting
2
tional analyses and previously observed using isolated IPC com-
31
31
plexes. As noted above, available experimental data demon-
strate the reduced reactivity of [Fe(Por)(CPh )] vs.
[Fe(Por)(C(Ph)CO Et)] in the cyclopropanation reaction.
2
2
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Table 4. Reactivity analysis of bio- and chemocatalytic cyclopropanation
reactions involving different diazo reagents.
3.23 kcal/mol compared to the heme mimic system lacking the
a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
axial ligand (reaction 2). This effect may arise from a more elec-
trophilic carbene (+0.01 e QC1, Table S10) and a weaker Fe-C
bond (elongated by 0.05 Å, Table S9). Similarly to the imidazole
−
ligand, a thiolate (HS ) ligand was also found to reduce the rela-
‡
tive energy of the transition state (ΔG ), as determined by analysis
−
of the same reaction using [Fe(Por)(CHCO
2
Et)(SH )] (reaction 7).
‡
However, the ΔΔG value in this case is smaller (1.19 kcal/mol,
Table 3) compared to the imidazole axial ligand in reaction 6. The
more favorable reaction rate associated with the neutral axial lig-
and may be a result of smaller energy cost of smaller geometry
change (see e.g. ΔRFeCl and ΔRClC2 in Table 3) and smaller charge
change (see e.g. QCT and ΔQCl in Table 3) in the early transition
states compared with the reactants. The relatively larger geometry
change and charge change due to a negatively charged ligand
probably comes from its relatively stronger trans effect and
charge donation effect. Compared to the charged thiolate ligand
RS ) or no axial ligand, the imidazole ligand appears to exert a
more favorable effect toward reducing the energy barrier not only
for the cyclopropanation step, but also for the carbene formation
step as reported recently. Such a synergistic effect may therefore
lie at the basis of the excellent role of the histidine proximal lig-
and in modulating biocatalytic cyclopropanation reactivity, as
evidenced by our results with the set of closely related, proximal
ligand Mb variants. The beneficial effect of this histidine-ligated
heme iron configuration is also apparent from the significantly
higher catalytic activity and rate of Mb(H64V,V68A) compared to
free hemin in the reactions described in Table 4, including those
with the less reactive acceptor/donor diazo reagent (EDPA).
Effect of porphyrin substitution.
Based on the results above, we envision that modification of
the porphyrin ligand could provide an important, future opportuni-
ty for tuning the carbene transfer reactivity of hemoprotein-based
catalysts, as evidenced above by calculations on reactions 4 and 5
using exact porphyrin substituents compared with non-substituted
porphyrins in reactions 1 and 2. Supporting this notion, we recent-
ly reported how replacement of heme with an iron-chlorin cofac-
tor in myoglobin can confer enhanced cyclopropanation reactivity
b
Rate ꢀ
c
Entryꢀ
Catalystꢀ
Tempꢀ
Productꢀ
TON ꢀ
(
TON/min)ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
ꢀ
ꢀ
Mb(H64V,V68A)ꢀ
Mb(H64V,V68A)ꢀ
-ꢀ
r.t.ꢀ
50°Cꢀ
50°Cꢀ
r.t.ꢀ
50°Cꢀ
r.t.ꢀ
7ꢀ
8ꢀ
8ꢀ
7ꢀ
8ꢀ
7ꢀ
8ꢀ
528ꢀ
1ꢀ
7,840ꢀ
55ꢀ
(0.2)ꢀ
16ꢀ
n.a.ꢀ
2ꢀ
0.02ꢀ
3ꢀ
1ꢀ
2ꢀ
4
5
6
7
ꢀ
ꢀ
ꢀ
ꢀ
Fe(ppIX)ꢀ
Fe(ppIX)ꢀ
Fe(TPP)ꢀ
Fe(TPP)ꢀ
n.a.ꢀ
0.2ꢀ-
86
4
r.t.ꢀ
5x10 ꢀ
-
(
aꢀ
Reactionꢀconditions:ꢀ10ꢀmMꢀ5,ꢀ10ꢀmMꢀEDAꢀ(2)ꢀorꢀEPDAꢀ(6),ꢀcatalystꢀ(0.01ꢀ
mol%ꢀ Mb(H64V,V68A);ꢀ 2ꢀ mol%ꢀ hemin;ꢀ 10ꢀ mol%ꢀ Fe(TPP)),ꢀ 10ꢀ mMꢀ sodiumꢀ
dithioniteꢀinꢀ50ꢀmMꢀpotassiumꢀphosphateꢀbufferꢀ(pHꢀ7)ꢀforꢀMbꢀvariantꢀandꢀ
b
ꢀforꢀFe(TPP),ꢀatꢀtheꢀindicatedꢀtemperature.ꢀ ꢀInitialꢀrateꢀoverꢀ1ꢀ
69
hemin,ꢀorꢀCH
minꢀ(EDA)ꢀandꢀoverꢀ30ꢀminꢀ(EDPA)ꢀ ꢀAfterꢀ16ꢀhours.ꢀN.a.ꢀ=ꢀnotꢀactive.ꢀꢀꢀ
2
Cl
2
c
Axial ligand effect.
Previous studies on hemoprotein-catalyzed cyclopropanation
suggested an important role of the iron binding axial ligand to-
ward affecting the reactivity of these biocatalysts. This effect can
be evinced, for example, from the higher cyclopropanation activi-
ty exhibited by engineered myoglobins, which bear a histidine
or by serine- and histidine-ligated P450s,
compared to P450 featuring a native, cysteinate-ligated heme.
Clear structure-reactivity trends cannot be extracted from these
reports, however, due to the use of different protein scaffolds (i.e.,
Mb vs. P450s; different engineered variants), reaction conditions,
5
0, 51
47, 48
ligated heme,
4
6
4
7, 48
and/or their investigation in whole-cell biotransformations,
whose performance is affected by multiple factors such as the
intracellular environment and protein expression levels. To better
examine the impact of the axial ligand on hemoprotein-catalyzed
cyclopropanation reactivity, two variants of Mb(H64V,V68A)
were prepared in which the proximal histidine residue (His93) is
substituted for cysteine (as in P450s) or phenylalanine, which is
unable to coordinate the heme iron. After expression and isolation
from E. coli, the proximal ligand variants Mb(H64V,V68A,H93C)
and Mb(H64V,V68A,H93F) were found to exhibit distinct spec-
troscopic features, compared to each other and to
Mb(H64V,V68A), with respect to their Soret and Q bands in the
ferric, ferrous and/or CO-bound form (Figure S1). These spectral
differences reflect the different heme iron coordination environ-
ment present in these metalloproteins. Next, the catalytic rates of
these biocatalysts for styrene cyclopropanation with EDA were
determined via time course experiments. These experiments
showed the histidine-ligated Mb(H64V,V68A) catalyzes this reac-
tion at a significantly higher faster rate (995 TON/min) than the
87
under aerobic conditions. The effect of porphyrin substituents on
the enzymatic activity of hemoproteins is apparent also in the
88, 89
context of other reactions.
Thus, to help guide future catalyst
and reaction development, we set to examine the styrene cyclo-
propanation pathway using an electrondeficient iron-porphyrin
carrying four electronwithdrawing cyano (‒CN) groups at the four
meso positions (reaction 8), which based on the newly revealed
cyclopropanation mechanism discussed above were expected to
enhance carbene’s electrophilicity. As anticipated, the carbene
carbon was found to bear a more positive charge by 0.029 e than
the corresponding carbene atom in the unsubstituted porphyrin
system of reaction 2 (Table S10). This change results in improved
‡
reactivity, as suggested by the reduced ΔG of 7.49 kcal/mol
compared to 12.47 kcal/mol in reaction 2. This ligand modifica-
tion is thus expected to dramatically increase the rate of the cy-
clopropanation step. Figure 3-8 vs. 3-2 indicates slightly smaller
geometric changes in reactions 8 vs. 2 As this reaction has early
.
transition state feature, smaller structural changes are associated
cysteine-ligated
counterpart
(235
TON/min)
or
the
Mb(H64V,V68A,H93F) variant, which lacks metal coordination
at the axial position (210 TON/min). Based on these results, the
following order of reactivity was derived for the proximal ligand
Mb variants: Mb(H64V,V68A) >> Mb(H64V,V68A,H93C) >
Mb(H64V,V68A,H93F).
‡
with lower energy barriers, supporting the reduced ΔG of reac-
tion 8 vs. 2.
To investigate how this differences in enzymatic activity may
correlate with the reactivity of the IPC intermediate, we compared
the calculated energy profile for styrene cyclopropanation in the
2
Et)] (reaction 2) with that involving
Fe(Por)(CHCO Et)(5-MeIm)] (reaction 6), in which 5-
methylimidazole (5-MeIm) mimics the axial His ligand (proximal
His) present in myoglobin and Mb(H64V,V68A). These analyses
show that the energy barrier (ΔG ) for reaction 6 is lowered by
Whereas these computational analyses provide insights into
the steric (reactions 4 and 5) and electronic (reaction 8) effects of
porphyrin substituents on cyclopropanation, previous work by
Zhang and coworkers showed how the introduction of an hydro-
gen-bonding group (HBG) at the meso position (Figure 2-9) im-
proves the performance of a Co-porphyrin catalyst in cyclopropa-
presence of [Fe(Por)(CHCO
[
2
6
6
nation reactions. This non-covalent interaction was found to
decrease the carbene formation barrier in both Co- and Fe-
‡
66
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6
9
porphyrins due to carbene stabilization via the hydrogen bond
between carbene’s carboxylate moiety and the amide group of the
HBG. Since the effect of the latter on the cyclopropanation path-
way remains undefined, we studied the styrene cyclopropanation
compound to liberate the free carbene followed by its reaction
with styrene, and pathway VI, which involves a direct concerted
cyclopropanation with concomitant release of N . As shown in
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2, the former pathway (V) is relatively more favorable
than pathway VI by 10.28 kcal/mol, with carbene formation by
thermal decomposition representing the RDS. Based on these
analyses, pathways I and V are the preferred ones in the presence
and in the absence of the metal catalyst, respectively, with the
metal-assisted pathway I being more favorable by 20.91 kcal/mol.
Since preformed IPCs have been also investigated experimental-
2
reaction involving the CHCO Et complex of the HBG-substituted
porphyrin, reaction 9. As shown in Table 3, the HBG group de-
creases the ΔG for the cyclopropanation by 0.57 kcal/mol, which
together with the ΔG reduction of 2.25 kcal/mol for the carbene
‡
‡
6
9
formation step may contribute to promoting catalytic perfor-
6
6
mance as found experimentally for Co porphyrins. As the side
chain of many natural amino acid residues (e.g., Ser, Thr, Asn,
Tyr, etc.) can establish H-bond interactions, these findings are
relevant toward the future design and engineering of biocatalysts
with improved cyclopropanation reactivity.
3
1
ly, pathways III and IV, which start from preformed IPCs and
involves metal-assisted cyclopropanation and carbene dissocia-
tion/free carbene cyclopropanation, respectively, were also com-
pared. As observed for pathway I vs. II, the metal-assisted cyclo-
propanation pathway III is largely preferred over pathway IV by
28.43 kcal/mol (Scheme 2). As the temperature is raised from
room temperature to 80°C, a condition applied to some reactions
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
R =0.93
3
1
with preformed IPC and metal-free reactions with diazo com-
9
1
pounds, the viable metal-assisted pathways (I and III) feature
increased energy barriers by 2-3 kcal/mol, while the non-metal-
assisted (thermal decomposition) pathways (V and IV) have re-
duced barriers by ~2 kcal/mol. These changes notwithstanding,
the metal-assisted pathways remain more favorable. In the pres-
2
ence of the donor-acceptor carbene, C(Ph)CO Et, the energy bar-
rier difference for the RDS in metal-free pathway V vs. the metal-
assisted pathway I is reduced by 4.30 kcal/mol at room tempera-
ture and further lowered by 3.04 kcal/mol at elevated temperature
used for experimental studies of preformed IPC involved cyclo-
propanations. The contribution from the metal-free pathway is
even further enhanced in the case of the donor-donor carbene,
‡
Figure 4. Plot of ΔQC1 vs. ΔG .
Upon investigation of the effect of various structural features
(carbene substituent, proximal ligand, porphyrin substituents) on
IPC mediated cyclopropanation pathways, we noted the existence
of a good correlation between the Gibbs free energy of activation
C(Ph) (Table 5). This trend is consistent with the improved per-
2
formance of more electron-rich donor-acceptor diazo compounds
2
90
and the carbene carbon charge change (R =0.93, Figure 4). This
in metal-free reactions at high temperatures. Overall, these stud-
result further highlights the importance of the carbene electro-
philicity in cyclopropanation catalyzed by heme-based carbene.
As seen from Figure 4, smaller charge changes are associated with
lower energy barriers, further supporting an early transition state
character for the reactive intermediates in these reactions.
ies show that all reactions starting from either the diazo reagent or
II
IPC feature the Fe -assisted process as the most favorable path-
way, with the catalytic roles being two folds to help both carbene
formation and cyclopropanation. At the same time, the contribu-
tion from the metal-free pathway can increase as the temperature
is raised and particularly so for carbenes bearing electron-
donating substituents.
Overall reactions and comparison with metal-free reactions.
At high temperatures, diazo reagents compounds can undergo
9
0
Table 5. Energies for rate determining steps in pathways I-IV.
thermal decomposition leading to cyclopropanation, C‒H inser-
9
1
92
tion, and N‒H insertion, in the absence of a metal catalyst. To
compare and contrast the energy profiles of iron porphyrin-
catalyzed vs. metal-free cyclopropanation, we performed a sys-
tematic examination of four metal-involved pathways and two
c)
C(X)Y
X=H, Y=CO
T
r.t.
From
Diazo
Pathway
∆G
a)
2
Et
I
13.59
34.50
V
IPC
Diazo
IPC
III 10.46
IV 38.89
metal-free pathways using [CHCO
Scheme 2). Pathway I entails iron-porphyrin catalysed decompo-
sition of EDA to form the IPC [Fe(Por)(CHCO Et)], followed by
2
Et] as the carbene moiety
(
8
0°C
I
V
15.78
33.90
2
the cyclopropanation step according to the most favorable mecha-
nism as described earlier. Comparison of the computed ΔG for
III 13.41
IV 36.22
‡
b)
the cyclopropanation and carbene formation step as determined
X=Ph, Y=CO
2
Et
r.t.
Diazo
IPC
I
V
15.74
31.45
6
9
here and previously, respectively, suggests that the latter is the
rate determining step (RDS) in this reaction pathway. Compared
to pathway I, pathway II involves thermal decomposition of the
IPC intermediate, followed by styrene cyclopropanation by the
free carbene. As illustrated in Scheme 2, the carbene dissociation
step becomes rate determining in this pathway, with a 15 kcal/mol
higher energy barrier compared to the RDS in pathway I. In this
case, although the triplet state of the free carbene is more favora-
ble than the singlet state (Table S13), in agreement with previous
III 16.87
IV 32.02
60°C
r.t.
Diazo
IPC
I
V
17.73
31.30
III 18.58
IV 30.15
I
b)
X=Ph, Y=Ph
Diazo
IPC
18.28
30.67
V
91
III 18.20
IV 33.57
calculations, the singlet carbene was considered for the pathway
energy calculations since it is the most relevant species to react
with singlet styrene to form the singlet cyclopropanation product,
6
0°C
Diazo
IPC
I
V
20.20
30.51
9
1
as done previously. In the case of metal-free reactions with di-
azo compound, two pathways were considered, namely pathway
V, which involves initial thermal decomposition of the diazo
III 19.74
IV 31.80
a)
b)
c)
Protein environment. Benzene. unit: kcal/mol
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Page 8 of 14
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Cyclopropanation pathways with and without metal. The reported data are calculated at room temperature in protein environment.
Conclusions
novel insights into of effects of the carbene substituent, porphyrin
substituent, and the axial ligand on the activation barriers of IPC-
mediated cyclopropanation and on the reactivity of hemoprotein-
based cyclopropanation biocatalysts. In particular, our results
demonstrate the importance of an electron-deficient carbene (ac-
ceptor-only > acceptor/donor > donor/donor) as well as that of an
imidazole axial ligand bound to the heme iron toward favoring
cyclopropanation reactivity. Furthermore, our analyses predict the
potentially beneficial effect of exploiting protein-mediated H-
This work shows for the first time that the increasingly useful
C=C functionalization mediated by heme carbenes features a Fe -
II
based, non-radical, concerted nonsynchronous mechanism, as
supported by our DFT calculations and experimental mechanistic
IV
studies. This mechanism is thus distinct from the typical Fe -
based, radical, stepwise mechanism of heme-dependent enzymes
i.e., cytochrome P450s). The present studies also provide key and
(
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Journal of the American Chemical Society
bond interactions and/or electron-deficient porphyrin ligands to-
tion metals in future studies, for which ECP basis is more readily
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ward further enhancing such reactivity, thereby providing valua-
ble guidelines for future catalyst development efforts in the ex-
panding area of hemoprotein-mediated carbene transfer chemistry.
Incidentally, these studies demonstrate the feasibility of perform-
ing biocatalytic cyclopropanation reactions in the presence of
poorly reactive donor-acceptor diazo reagents such as EDPA.
This finding is relevant since biocatalytic carbene transfer reac-
tions have so far been limited to acceptor-only diazo reagents and
paves the way to future studies to explore the scope of this trans-
formation. Finally, we report a first comparative analysis of met-
al-catalyzed vs. thermally induced cyclopropanation, which fur-
ther highlights the importance of heme-based catalysts and the
critical role of forming electrophilic heme carbenes in these reac-
tions. Overall, these studies are expected to facilitate the devel-
opment of sustainable iron-porphyrin based (bio)catalysts for
cyclopropanations and, possibly, other important carbene-
mediated transformations.
available and commonly used. The details of these methodologi-
cal studies are in Supporting Information.
Among the nine reactions studied here, for reactions 2 and 6-9,
singlet spin state are favored for both reactants and transition
states, while in reactions 1 and 3-5, because singlet is favored for
reactants and triplet is favorable for transition states, they exhibit
9
9-102
the so-called two state reactivity
and there are minimum en-
ergy crossing points (MECPs) between these two spin states in
9
9-102
these reactions. However, previous work
also shows that
these MECPs are of lower energy than the reaction transition
states, i.e. they are not rate-limiting. So, the singlet reactants here
after passing MECPs to become triplet reactants still need more
energy to overcome the barriers of triplet transition states (the
most favorable pathways here) to form products. Accordingly, the
focus of this first computational mechanistic work in this area is
on the rate-limiting reaction transition states, which is the most
important part to understand the reactions and help future experi-
mental work and catalyst design. Indeed, such transition states
studied here well reproduced experimental reactivity results as
described below.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL PROCEDURES
Computational Analyses. Nine styrene cyclopropanation re-
actions (see Scheme 1) and additional comparative reactions (see
Scheme 2) were computationally investigated. All calculations
W
The KIE calculations with tunneling effect correction (KIE )
1
03
were done using the following formulae reported recently:
(
!∆!! ! ∆!! /!")
KIE
E
= e
(1)
9
3
!!"
were performed using Gaussian 09. All models investigated in
this work were subject to full geometry optimizations without any
KIE
W
= ꢀꢁꢂ ×
(2)
!
!
!"
!!
!!
)!
3
1
(
symmetry constraints in the experimentally used solvents with
Qt (Tunneling correction) = 1 +
(3)
9
4
!"
the PCM method, i.e. benzene for reactions 1-5 and 8-9 and for
reactions 6-7, a dielectric constant of 4.0 was used to simulate the
where h is Planck’s constant, υ is the imaginary frequency of the
transition state, k is Boltzmann’s constant, T is temperature, and
subscripts of H and D indicate hydrogen and deuterium respec-
tively.
95
protein environment effect as done previously. The frequency
analysis was used to verify the nature of the stationary points on
respective potential energy surfaces and to provide zero-point
energy corrected electronic energies (EZPE’s), enthalpies (H’s),
and Gibbs free energies (G’s) at 1 atm and experimental reaction
temperatures, i.e. 333.15 K for reactions 1-5 and 8-9, and room
temperature (r.t.) for reactions 6-7. The atomic charges and spin
densities reported here are from the Natural Population Analysis
(NPA) and Mulliken schemes respectively, as implemented in
Gaussian 09. Relative Gibbs free energies and selected geometry,
charge, and spin density results were discussed here, while all
absolute values of electronic energies (E’s), zero-point energy
corrected electronic energies (EZPE’s), enthalpies (H’s), Gibbs free
energies (G’s), key geometric parameters, charges, spin densities,
General Procedures and Analytical Methods. All chemicals
and reagents were purchased from commercial suppliers (Sigma-
Aldrich, AlfaAeser, Cambridge Isotope Laboratories, Inc.) and
used without any further purification, unless otherwise stated. 2-
Diazo-2-phenyl-acetate (6) was prepared following reported pro-
1
04
cedures. Authentic standards for 4 and 7 were prepared as de-
5
1 1
13
2
scribed previously. H, C, and H NMR spectra were measured
1
on a Bruker DPX-400 instrument (operating at 400 MHz for H,
00 MHz for C, and 60 MHz for H) or a Bruker DPX-500 in-
strument (operating at 500 MHz for H and 125 MHz for C).
13
2
1
1
13
Tetramethylsilane (TMS) served as the internal standard (0 ppm)
1
for H NMR, CDCl
3
was used as the internal standard (77.0 ppm)
3
D structures and coordinates of optimized structures of the most
1
3
2
for C NMR and for H NMR (7.26 ppm). Gas chromatography
GC) analyses were carried out using a Shimadzu GC-2010 gas
favorable conformations and spin states as well as other details
are in Supporting Information (SI).
(
chromatograph equipped with an FID detector and a Chiral Cy-
closil-B column (30 m X 0.25 mm X 0.25 mm film). Separation
method for cyclopropanation reactions: 1 mL injection, injector
temp.: 200 °C, detector temp.: 300 °C. Gradient: column tempera-
All calculations were done using a range-separated hybrid
9
6
DFT method with dispersion correction, ωB97XD, based on its
excellent performance on heme carbenes and other catalytic sys-
68-70, 97
tems from previous methodological studies.
This ωB97XD
-1
ture set at 120 °C for 3 min, then to 150 °C at 0.8 °C min , then
method was found to yield accurate predictions of various exper-
imental spectroscopic properties, structural features, and reactivity
-1
to 245 °C at 25 °C min . Total run time: 46.30 min. HPLC anal-
6
8-70
yses were performed on a Shimadzu LC-2010A-HT equipped
with a VisionHT C18 column and a UV–vis detector. Injection
volume: 20 µL. Flow rate: 1 mL/min. Gradient: 30% acetonitrile
results of iron porphyrin carbenes.
The basis set includes the
9
8
effective core potential (ECP) basis LanL2DZ for iron and the
triple-zeta basis 6-311G(d) for all other elements, which was
found to provide accurate predictions of various experimental
(
0.1% TFA) in water (0.1% TFA) for 3 min, then increased to
69, 70
90% over 58 min.
reaction properties of heme carbenes.
The use of a much larg-
Synthesis of cis-β-deutero-styrene (1b). Cis-β-deutero-
styrene was prepared in two steps from phenylacetylene according
to the synthetic scheme shown in Figure S2. To a flame-dried 125
mL round-bottom flask was added phenylacetylene (3.3 mL, 30.0
mmol, 1.0 eq.) and anhydrous THF (20.0 mL). The mixture was
stirred in an ice-water bath (0 °C) under argon pressure, and n-
butyllithium (2.5 M in hexanes, 15.6 mL, 39.0 mmol, 1.3 eq.) was
added drop-wise via a gas-tight syringe over 15 minutes. The
er 6-311++G(2d,2p) basis for all non-metal atoms was found to
yield similar results and thus further support the efficient use of
70
the current basis set in reaction studies. The alternative use of an
all-electron basis for the metal center was found here to result in
qualitatively same conclusions of the geometric, electronic, and
energetic features, and therefore supports the use of LanL2DZ
basis set here, which may help direct comparisons with late transi-
9
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Journal of the American Chemical Society
Page 10 of 14
reaction mixture was stirred at 0 °C for 1 hour, then D
2
O (3.0 mL)
(IPTG) and 0.3 mM δ-aminolevulinic acid (ALA). The cells were
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was added slowly. The resulting mixture was stirred overnight at
room temperature under argon pressure. The crude reaction mix-
ture was passed through a pad of anhydrous MgSO , using a me-
4
pelleted by centrifugation (4,000 rpm, 4 °C, 20 min) and then
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. Cell suspensions
were thawed at room temperature, lysed by sonication, and clari-
fied by centrifugation (14,000 rpm, 20 min, 4 °C). 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 col-
umn, the protein was buffer exchanged against 50 mM KPi buffer
(pH 7.0) using 10 kDa Centricon filters. Myoglobin concentration
was determined using an extinction coefficient of ε408 = 157
dium-porosity fritted-glass funnel and rinsing with pentane (10
mL, three times). The solvent was evaporated under reduced pres-
1
sure to afford phenylacetylene-d
500 MHz, CDCl
1
(2.93 g, 95% yield). H-NMR
(
3
): ꢀ 7.49-7.47 ppm (m, 2H), 7.34-7.28 ppm (m,
3H). C-NMR (125 MHz, CDCl ): 132.1 ppm, 128.8 ppm, 128.3
1
3
3
ppm, 122.2 ppm, 83.3 ppm (t, J = 7.5 Hz), 77.3 ppm (t, J = 38.3
Hz). GC-MS m/z (% relative intensity): 104 (57.4), 103 (100), 78
(
18.8), 77 (17.4). To a flame-dried 50 mL round-bottom flask was
added zirconocene hydrochloride (Schwartz’ reagent) (2.052 g,
.93 mmol, 1.1 eq.), and the flask was purged with argon for 1
minute. Anhydrous dichloromethane (15 mL) was added, and the
flask was placed in an ice-water bath (0 °C). Phenylacetylene-d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
1
−
1
−1
−1
−1
(0.744 g, 0.80 mL, 7.2 mmol, 1.0 eq.) was added via syringe, and
the reaction mixture was stirred at room temperature in the dark
for 2 hours. Water (1.0 mL) was added, and the reaction mixture
was stirred for additional 2 hours. The resulting mixture was
mM cm for ferric Mb(H64V,V68A), ε406 = 145 mM cm for
−
1
−1
ferric Mb(H64V,V68A,H93F), and ε399 = 118 mM cm for fer-
ric Mb(H64V,V68A,H93C), as determined using the hemochrome
1
05
assay.
passed through a pad of anhydrous MgSO
4
, using a medium-
Reactions with cis-β-deutero-styrene. The Mb- and hemin-
catalyzed reactions were carried out at 2 mL-scale using 60 µM
catalyst, 0.2 M cis-ꢀ-d -styrene (1b), 0.4 M ethyl diazoacetate
1
(EDA), 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, 10% dimethylforma-
mide (DMF) was purged with argon for 3 minutes in a septum-
capped vial. In a separate vial, a buffered solution containing
myoglobin (Mb) or hemin (80 mM in DMF) was carefully purged
in tandem, and the two solutions were then mixed via cannula.
porosity fritted-glass funnel and rinsing with pentane (5 mL, three
times). The resulting suspension was passed through a silica gel
pad using a medium-porosity fritted-glass funnel and eluting with
pentanes. The filtrate was concentrated under reduced pressure
and an ice-water bath to afford cis-β-deutero-styrene (1b) (0.404
1
g, 53% yield; >98:2 d.r. ( H-NMR)) as a light-yellow oil. Note: to
avoid substantial loss of the volatile product, a trace amount of
1
solvent was not removed (ca 5% pentanes). H-NMR (500 MHz,
CDCl
3
): ꢀ 7.43 ppm (d, J = 7.5 Hz, 2H), 7.35 ppm (t, J = 7.4 Hz,
2H), 7.27 ppm (m, 1H), 6.73 ppm (dt, J = 10.8 Hz, 2.1 Hz, 1H),
1
Reactions were initiated by the addition of cis-ꢀ-d -styrene (42.1
1
3
5.25 ppm (d, J = 10.9 Hz, 1H). C-NMR (125 MHz, CDCl
37.6 ppm, 136.8 ppm, 128.5 ppm, 127.8 ppm, 126.2 ppm, 113.7
ppm (t, J = 23.6 Hz). GC-MS m/z (% relative intensity): 105
100.0), 104 (40.5), 79 (21.7), 78 (20.1).
Synthesis of ethyl
3
): ꢀ
mg, 0.400 mmol, 1.0 eq.), followed by addition of EDA (84.0 µL,
0.800 mmol, 2.0 eq.) with a syringe. The reaction was stirred at
room temperature under argon pressure for 16 hours. The reaction
product was extracted with dichloromethane (2 mL, three times),
and the organic layers were collected and dried over anhydrous
sodium sulfate. The organic solvent was removed by rotary evap-
oration, and the crude trans-2-phenyl-1-ethyl carboxylate cyclo-
propane product (2a/b) was purified via flash column chromatog-
raphy using silica gel and 5% EtOAc/hexanes as the eluent to
afford the desired product as a clear, colorless oil. For the
1
(
2-(4-methoxyphenyl)-1-
phenylcyclopropanecarboxylate (8). An authentic standard for
compound 8 was prepared via Rh-catalyzed cyclopropanation as
follows. To a flame dried 10 mL round bottom flask, equipped
with
a
stir bar, were added 4-methoxy-styrene (100 mg,
(OAc) (3 mg, 6.4 µmol, 2 mol%), and
0
.75 mmol, 2.3 eqiv.), Rh
2
4
1
Fe(TPP)- and Co(TPP)-catalyzed reactions, cis-ꢀ-d -styrene (50.0
anhydrous DCM (3 mL) under argon. After that, a solution of
ethyl 2-diazo-2-phenylacetate (62 mg, 0.32 mmol, 1.0 eqiv.) in
anhydrous DCM (1 mL) was slowly added dropwise over a period
of 30 min. The resulting reaction mixture was stirred at room
temperature for 16 hours. The solvent was removed under reduced
pressure and the crude product purified via column chromatog-
raphy on silica gel using 0-5% diethyl ether in pentanes to yield
ethyl 2-(4-methoxyphenyl)-1-phenylcyclopropanecarboxylate as a
mg, 0.475 mmol, 1.0 eq.) and either Fe(TPP)Cl (16.7 mg, 0.024
mmol, 0.05 eq.) or Co(TPP) (16.1 mg, 0.024 mmol, 0.05 eq.)
were added to a flame-dried 50 mL two-neck round bottom flask.
The flask was purged with argon for 1 minute, and anhydrous
dichloromethane (15 mL) was added. Ethyl-2-diazoacetate (75.0
µL, 0.713 mmol, 1.5 eq.) was dissolved in anhydrous dichloro-
methane (3 mL), and was added slowly to the reaction mixture
over 4 hours. The reaction flask was heated to a reflux (40°C)
under an argon atmosphere. After 16 hours, the crude reaction
mixture was concentrated under reduced pressure, and the crude
trans-2-phenyl-1-ethyl carboxylate cyclopropane product (2a/b)
was purified as described above. Isolated yields were 2.4 mg
(3.1%) for WT Mb, 37.0 mg (48.3%) for Mb(H64V,V68A), 19.1
mg (24.9%) for hemin, 12.8 mg (14.1%) for Fe(TPP), and 4.5 mg
white crystalline solid (79 mg, 0.27 mmol, 85% yield). R
f
= 0.24
, 500 MHz): δ
.12 (s, 3H, Har), 7.03 (s, 2H, Har), 6.69 (d, 2H, Har), 6.60 (d, 2H,
ar), 4.18-4.08 (m, 2H, -CH CH ), 3.69 (s, 3H, -OCH ), 3.05 (m,
H, HBn), 2.15 (m, 1H, CCCH ), 1.79 (m, 1H, CCCH ), 1.18 (t,
). C NMR (CDCl , 125 MHz): δ 174.0, 158.2,
1
(
5% diethyl ether in pentanes). H NMR (CDCl
3
7
H
2
3
3
1
2
2
1
3
3H, -CH
2
CH
3
3
1
3
2
35.1, 132.1, 129.1, 128.6, 127.7, 126.9, 113.3, 61.3, 55.2, 37.4,
2.6, 20.4, 14.3. GC-MS m/z (% relative intensity): 297 (21.9),
96 (100.0), 267 (14.8), 251 (26.0), 250 (81.7), 249 (82.2), 224
(
(
5.0%) for Co(TPP). Characterization data for enantiopure 2a
1
from Mb(H64V,V6A) reaction): H-NMR (400 MHz, CDCl
3
): ꢀ
7
.30 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2
(
22.2), 223 (94.8), 222 (20.2), 221 (70.1).
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,
Protein Expression. The Mb variants were expressed in E.
coli BL21(DE3) or E. coli C41(DE3) cells as follows. After trans-
1
3
5
.2 Hz, 1H), 1.30 (t, J = 7.2 Hz, 3H) ppm. C-NMR (100 MHz,
formation, cells were grown in TB medium (ampicillin, 100 mg
−
1
CDCl ): ꢀ 173.2, 140.0, 128.3, 126.3, 126.0, 60.5, 25.9, 23.9, 16.8
L ) at 37 °C (200 rpm) until OD600 reached 0.6. Cells were then
3
2
(
3
t, J = 25 Hz), 14.1 ppm. H-NMR (60 MHz, CDCl ): ꢀ 1.32 ppm
induced with 0.50 mM isopropyl-β-D-1-thiogalactopyranoside
1
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(
s, 1H). GC-MS m/z (% relative intensity): 191 (35.3), 146 (31.7),
extracted with 400 µL of DCM, the organic layer was removed
via evaporation and the residue was dissolved in 300 µL methanol
and 20 µL DMSO, followed by HPLC analysis. Control experi-
ments with no catalyst showed negligible background (thermally
induced) formation of 8 at 50°C, this amount accounting for
<0.5% of the product formed in the presence of
Mb(H64V,V68A). The Fe(TPP)-catalyzed reactions were carried
out on a 400 µL-scale using 1 mM Fe(TPP), 10 mM sodium di-
thionite, 10 mM styrene, and 10 mM diazo compound (ethyl 2-
diazoacetate or ethyl 2-diazo-2-phenylacetate) in anhydrous
DCM. In a typical procedure, 40 µL Fe(TPP) (from a 2.5 mM
stock solution in anhydrous DCM) were added to a flame dried,
sealed vial under argon and purged with argon for 3 min. Subse-
quently, 380 µL of anhydrous DCM were added, and 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 diazo
compound (from a 0.4 M stock solution in ethanol) with a syringe.
The reaction mixture was stirred at room temperature and product
formation was monitored at different time points over 180 min
and then after 16 hours. The EDA reactions were immediately
analyzed by GC-FID after adding 20 µL of internal standard (ben-
zodioxole, 50 mM in ethanol). The EDPA reactions were stopped
by rapidly removing the organic layer under high vacuum (<1-2
min), after which the residue was dissolved in 300 µL methanol
and 20 µL DMSO and analysed by HPLC. Rates and catalytic
turnovers were determined by means calibration curves prepared
using authentic standards of 7 and 8 prepared synthetically.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
145 (26.7), 135 (24.7), 118 (100.0), 117 (31.2), 116 (62.0), 92
(
Co(TPP)-catalyzed reaction product shows a 1.5:1 mixture of 3a
3
and 3b diastereomers ( H-NMR (60 MHz, CDCl ): ꢀ 1.72 ppm (s,
1
2
20.5). The H-NMR and H-NMR spectrum of the isolated
2
1
2
0
.66H), 1.32 ppm (s, 1H)). In contrast, the H-NMR and H-NMR
spectra of the isolated products from the reactions with Mb and
Fe(TPP) shows a single diastereomer (3a) (see spectra in Sup-
porting Information).
Radical spin trap experiments. The Mb- and hemin-
catalyzed reactions were carried out on a 400 µL scale using 20
µM catalyst, 10 mM styrene, 20 mM ethyl diazoacetate (EDA),
with or without 100 mM 5,5-dimethyl-1-pyrroline-N-oxide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
DMPO), 10 mM sodium dithionite. In a typical reaction, a solu-
tion containing sodium dithionite (100 mM stock solution) in
potassium phosphate buffer (50 mM, pH 7, 10% dimethylforma-
mide (DMF)) was purged with argon for 3 minutes in a septum-
capped vial. In a separate vial, a buffered solution containing
myoglobin (Mb) or hemin (80 mM in DMF) was carefully purged
in tandem, and the two solutions were then mixed together via
cannula. Reactions were initiated by the addition of styrene (40
µL, 100 mM stock in DMF, 1.0 eq.), followed by the addition of
DMPO (40 µL, 1 M stock in DMF, 10.0 eq.), and EDA (40 µL,
200 mM stock in DMF, 2.0 eq.) with a syringe. The reaction was
stirred at room temperature, under argon pressure for 16 hours.
For product analysis, internal standard (20 µL of benzodioxole at
1
00 mM in ethanol) was added to the reaction mixture, followed
by extraction with dichloromethane (400 µL), and analysis by
GC-FID. For the Co(TPP)-catalyzed reaction, Co(TPP) (8.1 mg,
0
Reactions with proximal ligand Mb variants. Reactions
were carried out on a 400 µL scale using 1 µM Mb(H64V,V68A),
.012 mmol, 0.05 eq.), styrene (25.0 mg, 0.240 mmol, 1.0 eq.),
or
5
µM
for
(Mb(H64V,V68A,H93F)
and
and DMPO (0.272g, 2.40 mmol, 10.0 eq.) were added to a flame-
dried 25 mL two-neck round bottom flask. The flask was purged
with argon for 1 minute, and anhydrous dichloromethane (3 mL)
was added. Ethyl-2-diazoacetate (38.0 µL, 0.360 mmol, 1.5 eq.)
was dissolved in anhydrous dichloromethane (2 mL), and was
added slowly to the reaction mixture over 4 hours. The flask was
heated to a reflux (40 °C) under argon atmosphere. After 16
hours, internal standard (20 µL of benzodioxole at 100 mM in
ethanol) was added to a 400 µL aliquot of the reaction mixture.
The mixture was washed with 400 µL of saturated NaCl solution,
followed by centrifugation (14,000 rpm) and analysis of the or-
ganic layer by GC-FID.
(
Mb(H64V,V68A,H93F), 20 mM styrene, 0.5-80 mM ethyl di-
azoacetate (EDA), 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, 20%
MeOH) was purged with argon for 3 minutes in a septum-capped
vial. In a separate vial, a buffered solution containing the Mb
variant was carefully purged in tandem, and the two solutions
were then mixed together via cannula. Reactions were initiated by
the addition of styrene (10 µL, 800 mM stock solution in EtOH),
followed by the addition EDA (80-800 mM stock solutions in
EtOH) with a syringe. The reactions were stirred at room tempera-
ture under argon pressure for 30 seconds, and then immediately
quenched with 100 µL HCl (2 N) via syringe. The reactions were
analyzed by GC-FID as described above. The reported rates refer
to the initial rates of product formation measured at near-
saturation conditions (80 mM EDA).
Reactivity studies with EDA vs. EDPA. The hemin- and
Mb-catalyzed reactions were carried out at a 400 µL-scale using
2
00 µM hemin or 1 µM Mb(H64V,V68A), 10 mM 4-methoxy-
styrene, 10 mM diazo compound (ethyl 2-diazoacetate (EDA) or
ethyl 2-diazo-2-phenylacetate (EDPA)), and 10 mM sodium di-
thionite in potassium phosphate buffer (50 mM, pH 7.0). In a
typical procedure, a buffered solution containing sodium dithio-
nite was degassed by bubbling argon into the mixture for 3 min in
a sealed vial. A solution containing the catalyst (hemin in DMSO;
Mb(H64V,V68A) in buffer) was carefully degassed 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
ASSOCIATED CONTENT
Supporting Information
Computational details of methods, absolute energies, key geomet-
ric parameters, charges, spin densities, 3D structures and coordi-
nates of optimized structures of the most favorable conformations
and spin states, as well as experimental UV-vis spectra, and NMR
spectra, are available free of charge via the Internet at
http://pubs.acs.org.
0
1
.4 M stock solution in ethanol), followed by the addition of
0 µL of diazo compound (from a 0.4 M stock solution in etha-
nol) with a syringe. The reaction mixture was stirred at room tem-
perature (ethyl 2-diazoacetate) or 50 °C (ethyl 2-diazo-2-
phenylacetate) before quenching with 100 µL of 2 N HCl. Product
formation (7 or 8) was monitored at different time points over 180
min and then after 16 hours. The EDA reactions were analysed by
adding 20 µL of internal standard (benzodioxole, 50 mM in etha-
nol) to the reaction mixture, followed by extraction with 400 µL
of DCM, and analysis by GC-FID. The EDPA reactions were
AUTHOR INFORMATION
Corresponding Author
yong.zhang@stevens.edu; rfasan@ur.rochester.edu
Notes
The authors declare no competing financial interests.
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Lai, T. S.; Chan, F. Y.; So, P. K.; Ma, D. L.; Wong, K. Y.;
ACKNOWLEDGMENT
Che, C. M. Dalton Trans. 2006, 4845-4851.
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This work was supported by the U.S. National Science Founda-
tion (NSF) grant CHE-1300912 awarded to Y.Z. and by the U.S.
National Institute of Health (NIH) grant GM098628 awarded to
R.F.. V.T. acknowledges support from the Ford Foundation Grad-
uate Fellowship Program. MS instrumentation at the University of
Rochester was supported by the NSF grant CHE-0946653.
(33)
Carminati, D. M.; Intrieri, D.; Caselli, A.; Le Gac, S.; Boitrel,
B.; Toma, L.; Legnani, L.; Gallo, E. Chem.-Eur. J. 2016, 22, 13599-
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