Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts

Article abstract of DOI:10.1002/anie.201409928

Using rational design, an engineered myoglobinbased catalyst capable of catalyzing the cyclopropanation of aryl-substituted olefins with catalytic proficiency (up to 46800 turnovers) and excellent diastereo- and enantioselectivity (98-99.9%) was developed. This transformation could be carried out in the presence of up to 20 gL^-1 olefin substrate with no loss in diastereo- and/or enantioselectivity. Mutagenesis and mechanistic studies support a cyclopropanation mechanism mediated by an electrophilic, heme-bound carbene species and a model is provided to rationalize the stereopreference of the protein catalyst. This work shows that myoglobin constitutes a promising and robust scaffold for the development of biocatalysts with carbene-transfer reactivity.

Full text of DOI:10.1002/anie.201409928

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Angewandte
Communications
DOI: 10.1002/anie.201409928
Biocatalysis
Highly Diastereoselective and Enantioselective Olefin
Cyclopropanation Using Engineered Myoglobin-Based Catalysts**
Melanie Bordeaux, Vikas Tyagi, and Rudi Fasan*
Abstract: Using rational design, an engineered myoglobin-
based catalyst capable of catalyzing the cyclopropanation of
aryl-substituted olefins with catalytic proficiency (up to 46800
turnovers) and excellent diastereo- and enantioselectivity (98–
99.9%) was developed. This transformation could be carried
out in the presence of up to 20 gLꢀ1 olefin substrate with no
loss in diastereo- and/or enantioselectivity. Mutagenesis and
mechanistic studies support a cyclopropanation mechanism
mediated by an electrophilic, heme-bound carbene species and
a model is provided to rationalize the stereopreference of the
protein catalyst. This work shows that myoglobin constitutes
a promising and robust scaffold for the development of
biocatalysts with carbene-transfer reactivity.
this P450 enzyme catalyzing the cyclopropanation of styrene
in the presence of ethyl diazoacetate (EDA) with good
Z diastereoselectivity (up to 84% de) and good to high
enantioselectivity (90–99% eeZ).[1f,6] Our group recently
discovered that, along with other heme-containing proteins,
myoglobin is able to activate arylsulfonyl azides in intra-
[1g,7]
ꢀ
molecular nitrene C H insertion reactions,
suggesting
that this hemoprotein could also be useful for promoting
mechanistically related carbene-transfer processes. Herein we
report the rational design of engineered myoglobin-based
catalysts which can support the cyclopropanation of a variety
of aryl-substituted olefins with catalytic proficiency as well as
excellent E diasteroselectivity and enantioselectivity.
The oxygen-binding metalloprotein myoglobin contains
a heme (iron-protoporphyrin IX) cofactor coordinated at the
proximal side through a histidine residue. Because of its small
size (17 kDa) and robustness toward mutagenesis and other
structural modifications,[8] we selected this protein as a poten-
tially promising scaffold for developing biocatalysts to
promote non-native transformations such as nitrene-[1g,7] and
carbene-transfer reactions. In initial studies, we tested the
ability of sperm whale myoglobin (Mb) to catalyze the
cyclopropanation of styrene (1a) in the presence of EDA (2)
as the carbene source. Under reducing and anaerobic
conditions, Mb was found to effectively promote this reaction,
supporting about 180 turnovers and leading to (E)-ethyl 2-
phenylcyclopropanecarboxylate (3a and 3b) as the major
products (86% de; Table 1). Notably, this cyclopropanation
E
xpanding the scope of engineered and artificial biocatalysts
beyond the realm of chemical transformations catalyzed by
natural enzymes lies at the forefront of the biocatalysis field.[1]
Olefin cyclopropanation is a particularly valuable transfor-
mation owing to the occurrence of cyclopropyl moieties in
many bioactive natural and synthetic compounds. Further-
more, cyclopropanes constitute versatile intermediates for
a variety of synthetically useful ring-opening transforma-
tions.[2] A well-established chemical approach to olefin cyclo-
propanation involves transition-metal-catalyzed decomposi-
tion of diazo reagents followed by metallocarbenoid insertion
into C C bonds. A wide range of transition-metal com-
[3]
=
plexes have demonstrated utility in this respect, with the use
of chiral ligands enabling these reactions to proceed in an
asymmetric manner.[3] Despite this progress, achieving high
levels of both diastero- and enantioselectivity, also in
combination with high catalytic activity, has remained a sig-
nificant challenge in these processes, particularly in the
context of intermolecular cyclopropanation reactions in the
presence of acceptor-only carbene donors.[4]
Pioneering studies by Callot, Kodadek, and Woo demon-
strated the ability of metalloporphyrins to promote olefin
cyclopropanation in the presence of diazoacetates.[5] More
recently, Arnold and co-workers reported that a similar
reactivity is exhibited by P450BM3, with engineered variants of
activity compares well with that reported for the P450BM3
-
based variants in vitro (200–360 total turnovers)[1f] under
similar reactions conditions (0.02 mol% protein, 3:1 styrene/
EDA), while exhibiting complementary diastereoselectivity.
Despite its promising activity, wild-type Mb showed no
asymmetric induction in the cyclopropanation reaction, thus
leading to a racemic mixture for both the Z and E product as
observed for free hemin (Table 1).
Control experiments showed that the absence of reduc-
tant (dithionite) or the presence of air resulted in no
cyclopropanation product, thus indicating that ferrous myo-
globin is the catalytically active species and that O2 is
deleterious to this reactivity, likely because of the competition
with the diazo reagent for binding to the heme. Based on
these results and previous studies with metalloporphyrin
catalysts,[4d,5b,c,9] we hypothesized the Mb-catalyzed cyclo-
propanation reaction to involve a heme-bound carbene
intermediate formed upon reaction of EDA with the protein
in its reduced, ferrous state (Figure 1b). End-on[4d,5c,9b,10]
attack of the styrene molecule to this heme-carbenoid species
would then lead to the cyclopropanation product. While the
E selectivity of the Mb-catalyzed reaction clearly indicated
[*] Dr. M. Bordeaux,[+] Dr. V. Tyagi,[+] Prof. Dr. R. Fasan
Department of Chemistry, University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: fasan@chem.rochester.edu
[+] These authors contributed equally to this work.
[**] This work was supported by the U.S. National Institute of Health
grant GM098628. MS instrumentation was supported by the U.S.
NSF grant CHE-0946653.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409928.
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Table 1: Activity and selectivity of wild-type myglobin and its variants
the other hand, positions 43 or 68 were substituted with amino
toward styrene cyclopropanation with ethyl diazoacetate.[a]
acids carrying a larger [i.e., Mb(F43W), Mb(V68F)] or
smaller apolar side chain [i.e., Mb(F43V), Mb(V68A)], in
an attempt to affect the catalyst selectivity in the cyclo-
propanation reaction by varying the steric bulk on either side
of the heme (Figure 1a). Finally, a Mb(L29A) variant, which
contains a mutation at a remote position not expected to
directly interact with the heme-bound carbene during catal-
ysis, was used as a negative control.
Catalyst
Conv. [%][b]
TON
deE [%] eeE [%][c] eeZ [%][c]
Hemin
Mb
Mb(L29A)
Mb(H64V)
Mb(V68A)
29
36
38
73
56
52
41
45
91
>99
>99
>99[d]
47[e]
145
180
190
365
280
260
205
225
455
500
500
74
86
82
92
96
98
88
88
36
78
99.9
0
6
0
0
1
Analysis of these Mb variants revealed an important
effect of the active-site mutations on the activity and/or
selectivity of the hemoprotein toward styrene cyclopropana-
tion with EDA (Table 1). In particular, the H64V mutation
resulted in a twofold increase in the turnover number (TON),
the highest among this set of single mutants, while having
marginal effect on diastereo- and enantioselectivity. Con-
versely, all the mutations at the level of Phe43 and Val68
dramatically improved the enantioselectivity of the Mb
variant as compared to wild-type Mb, thus resulting in
formation of the 1S,2S-stereoisomer 3a with eeE values
ranging from 44 to 99.9%. The V68 substitutions also resulted
in an appreciable increase in both catalytic activity (TON)
and E diastereoselectivity. In contrast, the L29A mutation
had essentially no effect on either the cyclopropanation
activity or diastero- and enantioselectivity of the protein.
Thus, in accord with our design strategy, the H64V mutation
was particularly effective in enhancing Mb-dependent cyclo-
propanation activity, whereas the mutations at the level of
V68 and F43 were beneficial toward tuning its diastereo- and
enantioselectivity. To combine the beneficial effects of these
mutations, a series of Mb double mutants were prepared
(Table 1). Gratifyingly, variant Mb(H64V,V68A) was found
to exhibit high activity as well as excellent E diastereoselec-
tivity (> 99.9%) and 1S,2S enantioselectivity (> 99.9%), and
it was thus selected for further investigations.
ꢀ1
2
ꢀ1
ꢀ1
26
15
3
71
13
ꢀ6
ꢀ6
ꢀ6
68
>99.9
44
34
67
>99.9
>99.9
99.9
99.9
Mb(V68F)
Mb(F43V)
Mb(F43W)
Mb(F43V,V68A)
Mb(F43V,V68F)
Mb(H64V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
10000[d] 99.9
46800[e] 99.9
[a] Reactions conditions: 20 mm Mb (or hemin), 30 mm styrene, 10 mm
EDA, 10 mm dithionite, 16 h. [b] The conversion is based on GC analysis
and relative to the limiting reagent. [c] trans=1S,2S and cis=1R,2S as
determined by GC analysis using a chiral stationary phase. [d] Reaction
conditions: 20 mm protein, 0.2m styrene, 0.4m EDA, 10 mm dithionite,
1 h. [e] Reaction conditions: 2 mm protein, 0.2m styrene, 0.4m EDA,
10 mm dithionite, 16 h.
Mb(H64V,V68A)-catalyzed cyclopropanation was deter-
mined to follow Michaelis–Menten kinetics, with estimated
Km values of about 2 mm and 5 mm for styrene and EDA,
respectively (see Figure S1 in the Supporting Information).
To further optimize this transformation, the impact of the
olefin/diazoester ratio on the efficiency of the reaction was
first examined. These experiments revealed an increase in
TON as the EDA to styrene ratio was raised from 1:5 to 6:1,
with significant amounts (37% of total products) of dimeri-
zation byproducts (diethyl maleate and fumarate) accumu-
lating only in the presence of a large (sixfold) excess of the
diazo compound (see Figure S2). Overall, a twofold excess of
EDA over styrene was found to be optimal for maximizing
cyclopropanation turnovers while minimizing dimerization
(< 1% of total products). Notably, by using this reagent ratio
and a catalyst loading of 0.01 mol%, quantitative conversion
of the olefin could be achieved in the presence of up to 0.2m
styrene (20 glꢀ1) within one hour (see Table S2). Further-
more, despite the reaction being biphasic at this reagent
concentration, excellent levels of diastereo- and enantiose-
lectivity (99.9% ee, 99.9% de) were maintained, thus
indicating that the Mb variant is stable under these reaction
conditions (release of hemin from the protein would indeed
lead to racemization). Quantitative conversion of the olefin in
Figure 1. a) Active site of sperm whale myoglobin (pdb 1A6K). The
residues targeted for mutagenesis are Phe43, His64, and Val68.
b) Proposed mechanism for myoglobin-catalyzed styrene cyclopropana-
tion with diazo esters.
that a trans heme-carbene/styrene arrangement is preferred,
the lack of enantioselectivity also suggested that the native
Mb scaffold is unable to dictate facial selectivity for the
styrene approach to the heme-carbene intermediate. Accord-
ingly, we reasoned that mutation of the amino-acid residues
lying at the periphery of the porphyrin cofactor could provide
a means to improve the diastereo- and enantioselectivity of
this catalyst, possibly by imposing only one modality of attack
of the styrene molecule on the heme-carbene group.
Upon inspection of the sperm whale Mb crystal struc-
ture,[11] residues Phe43, His64, and Val68, were selected as
promising targets for mutagenesis because of their close
proximity to the distal face of the heme (Figure 1a).
Specifically, a Mb variant where the distal histidine (His64)
is mutated to Val, Mb(H64V), was considered as this
ꢀ
mutation was previously found to increase the C H amina-
tion activity of this protein on bulky arylsulfonyl azides.[7] On
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Table 2: Substrate scope for Mb(H64V,V68A)-catalyzed cyclopropana-
tion.
these reactions also suggested that the TONs supported by
the Mb catalyst are in excess of 10000. To examine this aspect,
reactions at high substrate loading (0.2m styrene, 0.4m EDA)
were repeated using decreasing amounts of the hemoprotein
(20 to 1 mm; see Table S2). At a catalyst loading of
0.001 mol%, Mb(H64V,V68A) was found to support about
30000 turnovers after 1 hour and over 46000 total turnovers
after overnight incubation with styrene and EDA (Table 1).
Notably, high total turnover numbers (TTNs) were main-
tained using stoichiometric amounts of reductant relative to
the Mb catalyst (TTN = 10600). Time-course experiments
also revealed that Mb(H64V,V68A)-catalyzed cyclopropana-
tion proceeds very rapidly, with an initial rate of 1000
turnovers minꢀ1 over the first 10 minutes and an average
rate of 500 turnovers minꢀ1 over the first hour of the reaction.
Overall, the catalytic efficiency of this engineered Mb rivals
that of some of the most active transition-metal catalysts
reported to date for similar transformations (TTN of 11–
98000),[4d,12] while offering greater diastero- and stereocontrol
(compared to 75–94% de and 83–98% ee).[4d,12] Furthermore,
unlike the latter, slow addition of the diazo reagent was not
required in the Mb-catalyzed reactions to minimize the
undesired dimerization reaction.
To examine the substrate scope of the Mb variant,
a variety of styrene derivatives and other olefin substrates
were subjected to Mb(H64V,V68A)-catalyzed cyclopropana-
tion in the presence of EDA. Using a catalyst loading of
0.07 mol%, efficient cyclopropanation of para- (1b–e), meta-
(1 f), and ortho-substituted (1g) styrenes could be achieved
with yields ranging from 69 to 92% (Table 2). Importantly,
excellent levels of E diastereoselectivity and, when measur-
able, of 1S,2S enantioselectivity were observed in each case,
thus highlighting the broad scope of the Mb-based catalyst in
terms of activity and selectivity across the substituted styrene
derivatives. At lower catalyst loadings (0.001 mol%),
Mb(H64V,V68A) was found to support TTNs ranging from
7760 to 14500 on these substrates. Analysis of reactions with
a-methylstyrene (1h) and trans-b-methylstyrene showed
efficient and highly diastereo- and enantioselective cyclo-
propanation only in the case of the former, thus suggesting
that b substitutions on the alkene group are not tolerated by
the Mb catalyst. Among alternative alkene substrates,
N-methyl-3-vinyl-indole (1i) could be converted into the
corresponding cyclopropanation product with high selectivity,
although the efficiency of this reaction was compromised by
the instability of this substrate in water. In contrast, no
appreciable cyclopropanation activity was observed in the
presence of 1-hexene or trans-penta-1,3-diene, thus evidenc-
ing the chemoselective reactivity of the Mb-based catalyst
toward aryl-substituted olefins versus aliphatic ones.
Substrate Product
Conv. [%]
(TON)[a]
TTN[b] de
ee
[%] [%]
77
(1150)
1b
1c
7760
99.6 n.a.[c]
89
(1330)
11670
99.9 n.a.[c]
99.8 99.9
99.9 99.9
99.8 99.9
99.4 99.9
97.2 96
92
(1380)
1d
1e
1 f
1g
1h
1i
12280
8660
69
(1035)
73
(1095)
14500
11700
8510
85
(1275)
86
(1290)
10
(150)
1790
97.4 n.a.[c]
[a] Reactions conditions: 20 mm Mb, 30 mm styrene, 60 mm EDA. Yield is
based on GC conversion. [b] Reactions conditions: 2 mm Mb, 200 mm
styrene, 400 mm EDA. [c] Enantiomers could not be resolved. n.a.=not
available.
cyclopropanations operated by electrophilic metal carbene
intermediates.[4d,5c,9b,12a,13] Furthermore, a plot of the log(kX/
kH) values against the Hammett constants s+ for the
corresponding para substituents yielded a reasonably good
(R2 = 0.79) linear correlation with a small negative 1+ of
ꢀ0.34 ꢁ 0.07 (see Figure S3). This value is comparable to that
measured for similar reactions with iron porphyrin catalysts
(1+ = ꢀ0.41[9b]) and it is suggestive of a partial positive charge
build-up at the benzylic carbon atom in the transition state.
Unlike the latter, however, no significant secondary isotope
effect [kH/kD = 0.96 ꢁ 0.02 (see Figure S4) compared to
0.87][5c] was observed for the Mb(H64V,V68A)-catalyzed
cyclopropanation of styrene versus [D8]styrene. Thus, while
these results point at subtle mechanistic differences between
the two systems, the Hammett analyses support a general
mechanism for the Mb-catalyzed cyclopropanation involving
an electrophilic heme-carbene species analogous to that
proposed for iron porphyrin catalysts.[5c,9b]
To gain insights into the mechanism of the Mb-catalyzed
cyclopropanation, the relative rates (i.e., kX/kH ratios) for
=
cyclopropanation of para-substituted styrenes (p-XC6H4CH
CH2, 1b–e) versus styrene were estimated from competition
experiments in the presence of Mb(H64V,V68A) as the
catalyst and methanol (20%) as a cosolvent. Electron-
donating substituents were found to accelerate the cyclo-
propanation reaction, while electron-withdrawing substitu-
ents lead to reduced rates, a phenomenon consistent with
To rationalize the activity and selectivity enhancements
brought about by the mutations in Mb(H64V,V68A), a model
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of this protein was generated based on the available structure
of the closely related Mb(H64V) variant.[14] Inspection of the
model revealed a wider opening leading the distal cavity
because of the H64V mutation (see Figure S5a,b), which is
likely to increase the accessibility of the heme center to the
diazoester and olefin substrate. The V68A mutation, on the
other hand, expands the size of the distal cavity above the
nitrogen atom N2 of the heme group (Figure S5c,d). In the
crystal structure of Fe-(porphyrin)-carbene complexes,[9b] the
carbene moiety is roughly aligned (15–208 deviation) with the
to fully substantiate it, the proposed model can justify the
stereochemical outcome of the Mb(H64V,V68A)-catalyzed
cyclopropanation reactions and qualitatively predict the
effect of structural modifications at the level of the diazo
reagent.
In summary, this work demonstrates that myoglobin
constitutes a versatile and robust scaffold for the develop-
ment of highly active and selective olefin cyclopropanation
catalysts. By rational design, an engineered Mb variant
capable of catalyzing the cyclopropanation of a variety of
aryl-substituted olefins with an unprecedented combination
of catalytic proficiency (10–46800 TON) and excellent
E diastereo- and enantioselectivity (> 99%) was obtained.
The practical utility of this biocatalyst is further highlighted
by its ability to operate at high reagent concentration (i.e.,
0.2–0.4m) and in presence of organic cosolvents (e.g., 20%
MeOH). Mutagenesis and Hammett analyses support the
intermediacy of an heme-bound electrophilic carbene species
in these reactions, analogous, albeit not identical, to that
operating in cyclopropanation reactions catalyzed by iron
porphyrins in organic solvents. Importantly,
ꢀ
ꢀ
diagonal N Fe N bonds of the porphyrin ring. Assuming
a similar geometry is adopted by the heme-bound carbene,
four orientations of this group are possible, that is, two
projecting the ester moiety toward the protein core (i.e.,
above heme atoms N2 or N3; Figure 1) and two projecting it
toward the solvent-exposed face of the heme cofactor (i.e.,
above heme atoms N1 or N4). Among the possible arrange-
ments for an end-on attack of styrene to this intermediate (see
Figure S6), the one featuring the carbene ester group above
heme N2 and the styrene phenyl group extending toward the
the much greater reactivity and selectivity
offered by the Mb catalyst as compared to
free hemin highlights the key role of the
protein matrix in modulating the catalytic
efficiency and stereochemical outcome of
the reaction. Finally, a model was presented
for rationalizing the selectivity of the Mb-
based catalyst which could be useful for
Figure 2. Proposed geometries for styrene approach to the putative heme-carbene complex
leading to the (1S,2S)-ethyl 2-phenylcyclopropanecarboxylate stereoisomer. The orientation
of the heme rings is the same as in Figure 1.
further tuning this scaffold in order to access
other cyclopropane stereoisomers. Based on
the present results, we anticipate that myo-
globin-derived catalysts can prove useful for
solvent (geometry A, Figure 2; see Figure S6) is consistent
with the experimentally observed E and 1S,2S selectivity of
the catalyst and appears sterically feasible. While leading to
the same cyclopropane stereoisomer, geometry B (Figure 2
and S6) imposes steric clashes between the styrene ring and
Phe43. Thus, the V68A mutation could favor A by better
accommodating the carbene ester group in proximity of N2,
thereby explaining the dramatic effect of this mutation
toward improving 1S,2S enantioselectivity (6!99.9% ee).
This scenario would also explain the remarkable tolerance of
Mb(H64V,V68A)-induced selectivity to variations on the aryl
group of the olefin (which is solvent-exposed in A) but not at
the b-position, because of steric clashes between the b sub-
stituent and the carbene ester group and/or heme porphyrin
ring. Another implication of this model is that an increase in
steric bulk at the level of the alkyl ester group or of the a-
carbon atom in the diazo reagent is expected to cause
a decrease in diastereo- and enantioselectivity, as these
changes would disfavor A over other geometries (Figure S6).
In agreement with these predictions, Mb(H64V,V68A)-cata-
lyzed styrene cyclopropanation with tert-butyl diazoacetate
(12) or ethyl diazopropanoate (13) yielded the corresponding
1S,2S cyclopropane products (14a, 15a) with lower diaste-
reoselectivity (82% de and 74% de, respectively) and
drastically reduced enantioselectivity (58% ee and 1% ee,
respectively). Thus, while further studies are clearly necessary
a variety of other synthetically valuable carbene-transfer
reactions.
Received: October 9, 2014
Published online: December 23, 2014
Keywords: biocatalysis · carbenes · iron · protein engineering ·
.
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