Highly Diastereo- and Enantioselective Synthesis of Trifluoromethyl-Substituted Cyclopropanes via Myoglobin-Catalyzed Transfer of Trifluoromethylcarbene

Article abstract of DOI:10.1021/jacs.7b00768

We report an efficient strategy for the asymmetric synthesis of trifluoromethyl-substituted cyclopropanes by means of myoglobin-catalyzed olefin cyclopropanation reactions in the presence of 2-diazo-1,1,1-trifluoroethane (CF₃CHN₂) as

Full text of DOI:10.1021/jacs.7b00768

Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI
Communication
Highly diastereo- and enantioselective synthesis of trifluoromethyl-substituted
cyclopropanes via myoglobin-catalyzed transfer of trifluoromethylcarbene
Antonio Tinoco, Viktoria Steck, Vikas Tyagi, and Rudi Fasan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00768 • Publication Date (Web): 02 Apr 2017
Downloaded from http://pubs.acs.org on April 2, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Highly diastereo- and enantioselective synthesis of trifluoromethyl-
substituted cyclopropanes via myoglobin-catalyzed transfer of tri-
fluoromethylcarbene.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antonio Tinoco, Viktoria Steck, Vikas Tyagi and Rudi Fasan*
Department of Chemistry, University of Rochester, Rochester NY 14620, United States
ers.5 Despite these advances, the development of highly stereoseꢀ
ABSTRACT: We report on an efficient strategy for the
lective strategies for olefin cyclopropanation with 2ꢀdiazoꢀ1,1,1ꢀ
trifluoroethane as the carbene donor remains an outstanding chalꢀ
lenge. Here, we report a simple and efficient biocatalytic approach
for promoting these transformations that hinges upon the use of
engineered myoglobin variants in combination with a compartꢀ
mentalized reaction setup. This strategy could be applied to the
conversion of a broad range of vinylarene substrates, providing
access to trifluoromethylꢀsubstituted cyclopropanes with high
diastereoꢀ and enantioselectivity as well as stereocomplementary
selectivity.
asymmetric synthesis of trifluoromethylꢀsubstituted cycloproꢀ
panes by means of myoglobinꢀcatalyzed olefin cyclopropanation
reactions in the presence of 2ꢀdiazoꢀ1,1,1ꢀtrifluoroethane
(CF3CHN2) as the carbene donor. These transformations were
realized using a twoꢀcompartment setup in which ex situ generatꢀ
ed gaseous CF3CHN2 is processed by engineered myoglobin
catalysts expressed in bacterial cells. This approach was successꢀ
fully applied to afford a variety of transꢀ1ꢀtrifluoromethylꢀ2ꢀ
arylcyclopropanes in high yields (61ꢀ99%) and excellent diaꢀ
stereoꢀ and enantioselectivity (97ꢀ99.9% de and ee). Furthermore,
mirrorꢀimage forms of these products could be obtained using
myoglobin variants featuring stereodivergent selectivity. These
reactions provide a convenient and effective biocatalytic route to
the stereoselective synthesis of key fluorinated building blocks of
high value for medicinal chemistry and drug discovery. The preꢀ
sent studies expand the range of carbeneꢀmediated transforꢀ
mations accessible via metalloprotein catalysts and introduces a
potentially very general strategy for exploiting gaseous and/or
hardꢀtoꢀhandle carbene donor reagents in biocatalytic carbene
transfer reactions.
Our group has recently reported the development of engineered
myoglobin catalysts for promoting a variety of carbene transfer
reactions,6 including olefin cyclopropanation.7 These transforꢀ
mations are believed to be mediated by an electrophilic hemeꢀ
bound carbenoid species generated upon reaction of a diazo comꢀ
pound with the heme cofactor embedded in the protein.7a This
species is amenable to reaction with a number of nucleophiles
(olefins, amines, thiols, phosphines) in order to form new CꢀC and
Cꢀheteroatom bonds, with the protein active site providing an
asymmetric environment to affect the stereoselectivity of the
carbene transfer process.6ꢀ7 In addition to myoglobin (Mb), other
biocatalysts8 have been investigated for promoting carbene transꢀ
fer reactions, but the scope of biocatalytic carbene transfer reacꢀ
tions has so far been restricted to αꢀdiazoacetates as carbene
donor reagents. Given the high reactivity of myoglobin variants
toward the acceptorꢀonly carbene donor reagent ethyl diazoacetate
(EDA, 1), we envisioned that 2ꢀdiazoꢀ1,1,1ꢀtrifluoroethane (DTE,
2) could provide a potentially viable carbene precursor for myoꢀ
globinꢀcatalyzed cyclopropanations. A wellꢀknown challenge
associated with the use of DTE in carbene transfer reactions,
however, is the difficulty in handling this reagent due to its high
toxicity and volatility. In situ generation of DTE via diazotization
of 2,2,2ꢀtrifluoroethylamine has provided an attractive approach
for utilizing this reagent in various reaction manifolds,4c,9 but
these conditions are too harsh for proteinꢀbased catalysts. To
overcome this problem, we envisioned the possibility to utilize a
twoꢀcompartment reaction setup, in which ex situ produced DTE
from a ‘reagent generation chamber’ is carried through a solution
containing the myoglobin catalyst by inert gas (Figure 1). This
system would effectively segregate the two incompatible reacꢀ
tions, thereby preserving the stability of the biocatalyst while
eliminating the need for isolating and handling DTE.
Trifluoromethylꢀsubstituted cyclopropanes constitute attractive
synthons in medicinal chemistry as they combine the conformaꢀ
tional rigidity of threeꢀmembered rings with the unique and often
highly beneficial features of fluorinated substituents.1 Reflecting
this notion, these structural motifs have represented key building
blocks for the design and development of several bioactive moleꢀ
cules and investigational drugs, including cannabinoid receptor
antagonists, HCV NS5B polymerase inhibitors, and potassium
channel activators, among others.1a Various methods have been
investigated to afford these structures, most of which involve
starting materials that already incorporate the trifluoromethyl
group.2 A convenient and most direct strategy to access this class
of compounds is through the addition of trifluoromethylcarbene to
an olefin. Early studies in this area have entailed the use of trifluoꢀ
romethylcarbene generated by photolytic decomposition of 2ꢀ
diazoꢀ1,1,1ꢀtrifluorethane (CF3CHN2), resulting in a mixture of
products.3 More synthetically useful strategies for olefin trifluoꢀ
romethylcyclopropanation with this reagent were made available
only recently using transition metal catalysis.4 To date, however,
only a few studies have addressed the problem of developing
enantioselective variants of these transformations.4a,5 In a first
report, Simmoneaux and coworkers described the use of chiral
metalloporphyrins for the asymmetric cyclopropanation of styꢀ
renes in the presence of 2ꢀdiazoꢀ1,1,1ꢀtrifluoroethane, but only
moderate enantioselectivity (30ꢀ79% ee) was observed using this
system.4a Higher selectivity was more recently achieved in similar
reactions using Co(III)ꢀsalen complexes by Carreira and coworkꢀ
To implement this approach, we initially tested the possibility
to carry out the myoglobinꢀcatalyzed cyclopropanation of pꢀ
chloroꢀstyrene (3) in the presence of ex situ generated EDA. The
latter was produced by diazotization of glycine ethyl ester (4) in
the presence of sodium nitrite and acid (Table 1).10 For these
experiments, the Mb(H64V,V64A) variant was chosen as the
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 5
found to occur with excellent diastereoꢀ and enantioselectivity
(99.9% de and ee).
1
2
3
4
Table 1. Mb(H64V,V68A)ꢀcatalyzed cyclopropanation of pꢀ
chlorostyrene in the presence of ex situ generated EDA and DTE.a
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Compartmentalized reaction setup for coupling myoꢀ
globinꢀcatalyzed cyclopropanation with ex situ generation of 2ꢀ
diazoꢀ1,1,1ꢀtrifluoroethane (DTE).
Equiv
Entry
Catal.
Prod.
Yieldc
TON % de % ee
4 or 5 b
catalyst based on its high activity and selectivity toward cycloproꢀ
panation of arylꢀsubstituted olefins with EDA.7a Initial tests inꢀ
volving purified Mb(H64V,V64A) in the reaction chamber proꢀ
duced only small amounts of the cyclopropanation product 6
(Table 1, Entry 1), a result we attributed to protein inactivation by
the gas stream flowing through the reaction mixture. Given our
recent finding that Mbꢀcatalyzed cyclopropanation reactions can
be realized using whole cell systems,7b a suspension of E. coli
cells expressing the Mb(H64V,V64A) variant was used as the
biocatalytic system in the reaction chamber. Gratifyingly, this
modified setup resulted in the accumulation of significantly larger
amounts of 6 (Table 1, Entry 2). Further improvement of the
reaction was possible by varying the cell density of the whole cell
transformation and the molar ratio between the olefin substrate
and the EDA precursor (Table 1, Entries 3ꢀ4). Under optimized
conditions (i.e., OD600 = 40, corresponding to a cell density of
~3.5 g cdw Lꢀ1, 10 equivalents of glycine ethyl ester relative to the
olefin), nearly quantitative conversion of the pꢀchloroꢀstyrene
substrate to 6 was observed within 12 hours (Table 1, Entry 5).
Importantly, neither the reaction setup nor the whole cell setting
was found to have a noticeable impact on the stereoselectivity of
the Mb(H64V,V64A)ꢀcatalyzed reaction, leading to the formation
of the transꢀ(1S,2S)ꢀconfigured cyclopropanation product in
excellent diastereoꢀ and enantiomeric excess (98.5ꢀ99.9% de,
99.8ꢀ99.9% ee).
1
2
3
4
5
6
7
protein
cellsd
cells
6
6
2
4%
47%
80%
75%
>99%e
22%
180
560
365
340
500
99.9
97.2
99.9
98.5
99.9
99.8
99.9
99.8
99.9
99.9
99.9
99.9
2
5
6
cells
6
10
10
5
cells
6
protein
cells
7b
7b
1,110 98.5
520 99.9
5
92%
(67%)
a
Reactions were carried out at a 20 mLꢀscale with 30 mM 4ꢀchloroꢀstyrene (3),
purified Mb variant (20 ꢀM) or Mb(H64V,V68A)ꢀexpressing E. coli (BL21(DE3))
cells (OD600 = 40) in KPi 50 mM (pH 7), RT, 5 hours. Diazo reagent was supplied
b
using indicated equivalents of 4 or 5 (slow addition over 4 hours). Relative to
c
olefin. As determined by GC using calibration curves generated with isolated
product as reference. Isolated yields are reported in brackets. d OD600 = 20. e Reaction
time: 12 hours.
To explore the scope of this reaction, a series of styrene derivaꢀ
tives and arylꢀsubstituted olefins was investigated. As shown by
the data in Table 2, all of these substrates could be efficiently
converted to the corresponding trifluoromethylꢀcontaining cycloꢀ
propanes (54ꢀ99%) using Mb(H64,V68A)ꢀexpressing cells in the
twoꢀcompartment setup described in Figure 1. These included
metaꢀ and paraꢀsubstituted styrene derivatives containing both
electronꢀdonating and electronꢀwithdrawing groups. 4ꢀ
Nitrostyrene displayed lower reactivity toward Mbꢀcatalyzed
cyclopropanation than electronrich styrene derivatives (e.g., prodꢀ
ucts 9b, 11b, 12b), which is line with the electrophilic character
of the putative hemeꢀcarbenoid intermediate expected to mediate
these reactions.7a As demonstrated by the results with 14b and
15b, the Mbꢀcatalyzed transformation could be readily extended
to other arylꢀsubstituted olefins such as 2ꢀvinylꢀnaphthalene and
3ꢀ(propenꢀ2ꢀyl)thiophene, supporting the broad substrate scope of
the Mb(H64,V64A) variant. In contrast to protocols involving
chiral metalloporphyrins,4a the biocatalytic reactions proceed
efficiently using the olefin as the limiting reagent, which increases
their attractiveness for the transformation of valuable starting
materials. Importantly, using Mb(H64V,V68A)ꢀexpressing cells,
the cyclopropanation product was afforded in most cases with
high diastereosteroselectivity (99.9% de) and enantioselectivity
(92ꢀ99.9% ee). As an exception, moderate enantioselectivity (28ꢀ
57% ee) was observed with the toluene derivatives 11a and 12a
(Table 2, Entries 4ꢀ5). For these compounds, however, excellent
diastereoꢀ and enantioselectivity (97ꢀ99.9% de and ee) could be
achieved using cells expressing Mb(H64V,V68G), a Mb variant
previously identified as having similar selectivity properties to
Mb(H64V,V68A) in cyclopropanation reactions with EDA.7b
Altogether, the stereoselectivity offered by these Mbꢀbased cataꢀ
lysts exceeds those provided by the most selective synthetic cataꢀ
lysts reported to date for the asymmetric synthesis of related CF3ꢀ
containing cyclopropanes (84ꢀ91% ee).5 Compared to the latter,
the Mbꢀcatalyzed transformations also involve shorter reaction
times (5 vs. 14 hours) and higher catalytic efficiency (e.g., 520 vs.
Based on these promising results, we next investigated the posꢀ
sibility to supply the Mb catalyst in the whole cell system with
gaseous DTE as the carbene donor. In this case, 2,2,2ꢀ
trifluoroethylamine (5) was subjected to the diazotization reaction
in the reagent generation chamber (Table 1). To our delight,
excellent conversion of pꢀchloroꢀstyrene to the desired trifluoroꢀ
methylꢀsubstituted cyclopropane 7b (92%) was observed in less
than five hours (Entry 7, Table 1) and using only 5 equivalents of
the diazo reagent precursor 5. Because of the higher volatility of
7b compared to 6, product recovery in this case was improved by
addition of a water trap downstream of the reaction vessel. No
product was detected using cells not expressing the Mb variant,
thus demonstrating the direct role of the hemoprotein in mediating
the carbene transfer reaction. Under the applied conditions, the
Mb variant was estimated to support about 520 catalytic turnovers
(TON), as determined based the hemoprotein concentration in the
reaction mixture measured via a COꢀbinding assay. This TON
value is higher than that observed in the presence of ex situ generꢀ
ated EDA under identical operational conditions (Entry 7 vs. 3,
Table 1). Higher yields (GC) and TON were also observed in the
presence of DTE vs. EDA when purified Mb(H64V,V64A) was
applied as the catalyst (Entry 6 vs. 1). These differences are likely
to reflect the inherent reactivity of the diazo reagents as well as
differences in the rate and efficiency with which the ex situ generꢀ
ated reagents are transferred from the generation chamber to the
reaction vessel under the applied experimental setup (Figure 1).
Importantly, Mb(H64V,V68A)ꢀcatalyzed synthesis of 7b was
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
~10 TON for 7b). In addition to protecting it from inactivation
configured product. Consistent with this scenario, the
1
2
3
4
5
6
7
8
(Entry 7 vs. 6, Table 1), the use of whole cells bypasses the need
for purification and isolation of the myoglobin catalyst, which
further simplifies the overall procedure from a technical standꢀ
point. From these reactions, the CF3ꢀsubstituted cyclopropane
products could be isolated in good yields (43ꢀ82%, Table 2). The
synthetic utility of these transformations was further evidenced by
a larger scale reaction with pꢀmethoxyꢀstyrene, from which 0.1 g
of 9b was obtained in 76% isolated yield and excellent diastereoꢀ
and enantiomeric excess (99.9% de, 99.9% ee),
Mb(H64V,V68G) variant, which features a similar ‘largeꢀtoꢀ
small’ mutation at position 68 (Figure S1), is also transꢀ(1S,2S)
selective (Table 2, Entries 4ꢀ5). An implication of the proposed
model is that the aryl ring of the olefin projects toward the solꢀ
ventꢀexposed side of the active site, a molecular arrangement that
is consistent with Mb(H64V,V68A) ability to process structurally
diverse vinylarene substrates with consistently high transꢀ(1S,2S)
selectivity (Table 2).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Substrate scope of myoglobinꢀcatalyzed olefin cycloproꢀ
panation with ex situ generated DTE.a
Figure
2.
A)
Crystal
structure
of
(1S,2S)ꢀ2ꢀ
(trifluoromethyl)cyclopropyl)ꢀ4ꢀnitroꢀbenzene product (10b). B)
Plausible geometry for vinylarene attack to hemeꢀbound trifluoꢀ
romethylcarbene intermediate leading to the (1S,2S)ꢀconfigured
cyclopropane product.
Entry
1
Product
OD600
80
Yield b
% de
% ee
Given the parallelism between the stereoselectivity exꢀ
hibited by Mb(H64V,V68A) with EDA and DTE, we surmised
that the stereoselectivity of Mb variants in cyclopropanation
reactions with EDA could serve as a general predictor for their
stereoselectivity in the cyclopropanation of related substrates with
DTE. Supporting this notion, a very good correlation (R2 = 0.911)
was found between the enantiomeric excess exhibited by a series
of active site Mb variants in the cyclopropanation of pꢀmethoxyꢀ
styrene with EDA vs. that of parallel reactions with DTE as the
carbene donor (Figure 3A). In addition to Mb(H64V,V68A), this
panel comprised other Mb variants with high transꢀ(1S,2S)ꢀ
selectivity (Mb(H64V,V68G), Mb(H64V,V68S)) as well as a
series of recently developed Mb catalysts with high transꢀ
(1R,2R)ꢀselectivity in olefin cyclopropanation with EDA (variants
RR1 through RR5).7b Mb variants that span a range of enantioseꢀ
lectivity values from 13% ee (wildꢀtype Mb) to 86% ee
(Mb(V68A)) in the latter reaction were included as additional
references (Table S1). As illustrated by the data of Figure 3A and
Table S1, the diastereoꢀ and enantioselectivity of the trifluoroꢀ
methylcarbene transfer reaction was found to closely mirror the
diastereoꢀ and enantioselectivity of these Mb variants in the αꢀ
diazoester carbene transfer reaction (average Iꢁ(de)I = 3.3%;
average Iꢁ(ee)I = 14.4%; Table S1). Based on these analyses,
RR2 (= Mb(H64V,V68L,L29T)) was selected as a promising
candidate for gaining access to the mirrorꢀimage forms of prodꢀ
ucts 7b-14b. Gratifyingly, reactions with RR2ꢀexpressing E. coli
cells produced the desired transꢀ(1R,2R) trifluoromethylꢀ
substituted cyclopropanes with excellent diastereoselectivity (98%
de for 9c, >99.5% de for the others) and high enantioselectivity
(80ꢀ92% ee; Figure 3B; Figure S2). As an exception, 14c was
obtained only in 21% ee. However, this compound could be synꢀ
thesized in higher enantiomeric excess (58% ee) using RR4, as
anticipated by the high transꢀ(1R,2R)ꢀselectivity of this Mb variꢀ
ants in olefin cyclopropanation with EDA (Figure 3A). Altogethꢀ
er, these experiments demonstrate the versatility of the Mb scafꢀ
fold toward providing access to both enantiomeric forms of the
target trifluoromethylꢀcontaining cyclopropanes. They also supꢀ
port the predictable reactivity of these biocatalysts in the presence
of structurally and electronically related carbene donors.
69%
(68%)
99.9
99.9
92%
(76%)
2
3
4
5
80
80
99.9
99.9
99.9
99.9
54%
(43%)
40
80
85%
88%
(78%)
76%
>99%
(82%)
96
31
96 c
97 c
40
80
99.8
28
99.9 c
99.9 c
70%
(58%)
7
8
80
99.9
99.9
92
>99%
(71%)
40
99.9
a
Reactions were carried out at 20 mLꢀscale with Mb(H64V,V68A)ꢀexpressing E.
coli at indicated cell density, 8ꢀ30 mM olefin in KPi 50 mM (pH 7), room temperaꢀ
ture, 5 hours. DTE was supplied using 5 equiv. 5 (slow addition over 4 hours). b As
determined by GC using calibration curves generated with isolated product as
c
reference. Isolated yields are reported in brackets.
Mb(H64V,V68G).
Using cells expressing
To elucidate the stereoselectivity of the Mb(H64V,V68A) cataꢀ
lyst in these reactions, product 10b was crystallized and deterꢀ
mined to have a transꢀ(1S,2S) configuration by Xꢀray diffraction
analysis (Figure 2A). The same configuration was indirectly
assigned to 7b and the other cyclopropanation products of Table
2 based on the similar order of elution for the corresponding
enantiomers upon resolution via chiral GC or SFC (see Figure
S2). The stereoselectivity of Mb(H64V,V68A) in the cyclopropaꢀ
nation reactions with DTE thus mirrors that observed in the reacꢀ
tions with EDA.7a Leveraging this insight and in consideration of
the structural and electronic similarities between EDA and DTE, a
stereochemical scenario analogous to that proposed for
Mb(H64V,V68A)ꢀcatalyzed cyclopropanation of arylꢀsubstituted
olefins with EDA7a is proposed. According to this model, the
‘cavity’ created by the Val68→Ala substitution (Figure S1) can
facilitate orientation of the bulkier ‒CF3 group (cp. to ‒H) in the
hemeꢀbound carbene intermediate in proximity to the N2 atom of
the heme cofactor (Figure 2B). This active site configuration
enforces high facial selectivity during endꢀon attack of the olefin
to this reactive species, giving rise to the observed transꢀ(1S,2S)ꢀ
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
Corresponding Author
1
2
* rfasan@ur.rochester.edu
3
4
5
6
7
8
9
ACKNOWLEDGMENT
This work was supported in part by the U.S. National Institute of
Health grant GM098628 and in part by the National Science
Foundation grant CHEꢀ1609550. The authors are grateful to Dr.
William Brennessel (U. Rochester) and Dr. Eric Reinheimer
(Rigaku Oxford Diffraction) for assistance with crystallographic
analyses.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
(1) (a) Grygorenko, O. O.; Artamonov, O. S.; Komarov, I. V.;
Mykhailiuk, P. K. Tetrahedron 2011, 67, 803; (b) Bos, M.; Poisson, T.;
Pannecoucke, X.; Charette, A. B.; Jubault, P. Chem. Eur. J. 2017, 23, in
press (doi: 10.1002/chem.201604564).
(2) (a) Katagiri, T.; Yamaji, S.; Handa, M.; Irie, M.; Uneyama, K.
Chem. Commun. 2001, 2054; (b) Wang, Y.; Zhao, X. M.; Li, Y. H.; Lu,
L. Tetrahedron Lett. 2004, 45, 7775; (c) Risse, J.; FernandezꢀZumel, M.
A.; Cudre, Y.; Severin, K. Org. Lett. 2012, 14, 3060; (d) Duncton, M. A.
J.; Singh, R. Org. Lett. 2013, 15, 4284; (e) Zhu, C. L.; Yang, L. J.; Li, S.;
Zheng, Y.; Ma, J. A. Org. Lett. 2015, 17, 3442.
Figure 3. Stereocomplementary selectivity. A) Correlation
between enantioselectivity of Mb variants (n = 12) in cyclopropaꢀ
nation of pꢀmethoxyꢀstyrene with EDA vs. DTE. See Table S1
for additional data. B) Diastereoꢀ and enantioselectivity of transꢀ
(1R,2R)ꢀselective Mb variant RR2 in cyclopropanation of arylꢀ
substituted olefins in the presence of DTE. See Table S2 for
details. * = with RR4ꢀexpressing cells.
(3) Atherton, J. H.; Fields, R. J. Chem. Soc. C 1967, 1450.
(4) (a) Le Maux, P.; Juillard, S.; Simonneaux, G. Synthesis 2006,
1701; (b) Mykhailiuk, P. K.; Afonin, S.; Ulrich, A. S.; Komarov, I. V.
Synthesis 2008, 1757; (c) Morandi, B.; Carreira, E. M. Angew. Chem. Int.
Ed. 2010, 49, 4294.
(5) Morandi, B.; Mariampillai, B.; Carreira, E. M. Angew. Chem. Int.
Ed. 2011, 50, 1101.
(6) (a) Sreenilayam, G.; Fasan, R. Chem. Commun. 2015, 51, 1532;
(b) Tyagi, V.; Bonn, R. B.; Fasan, R. Chem. Sci. 2015, 6, 2488; (c) Tyagi,
V.; Fasan, R. Angew. Chem. Int. Ed. 2016, 55, 2512; (d) Tyagi, V.;
Sreenilayam, G.; Bajaj, P.; Tinoco, A.; Fasan, R. Angew. Chem. Int. Ed.
2016, 55, 13562.
(7) (a) Bordeaux, M.; Tyagi, V.; Fasan, R. Angew. Chem. Int. Ed.
2015, 54, 1744; (b) Bajaj, P.; Sreenilayam, G.; Tyagi, V.; Fasan, R.
Angew. Chem. Int. Ed. 2016, 55, 16110.
(8) (a) Coelho, P. S.; Brustad, E. M.; Kannan, A.; Arnold, F. H.
Science 2013, 339, 307; (b) Renata, H.; Wang, Z. J.; Kitto, R. Z.; Arnold,
F. H. Catal. Sci. Technol. 2014, 4, 3640; (c) Wang, Z. J.; Renata, H.;
Peck, N. E.; Farwell, C. C.; Coelho, P. S.; Arnold, F. H. Angew. Chem.
Int. Ed. 2014, 53, 6810; (d) Weissenborn, M. J.; Low, S. A.; Borlinghaus,
N.; Kuhn, M.; Kummer, S.; Rami, F.; Plietker, B.; Hauer, B.
Chemcatchem 2016, 8, 1636; (e) Srivastava, P.; Yang, H.; Ellisꢀ
Guardiola, K.; Lewis, J. C. Nat. Commun. 2015, 6, 7789; (f) Dydio, P.;
Key, H. M.; Nazarenko, A.; Rha, J. Y.; Seyedkazemi, V.; Clark, D. S.;
Hartwig, J. F. Science 2016, 354, 102; (g) Key, H. M.; Dydio, P.; Clark,
D. S.; Hartwig, J. F. Nature 2016, 534, 534; (h) RiozꢀMartinez, A.;
Oelerich, J.; Segaud, N.; Roelfes, G. Angew. Chem. Int. Ed. 2016, 55,
14136.
(9) (a) Morandi, B.; Carreira, E. M. Angew. Chem. Int. Ed. 2010, 49,
938; (b) Morandi, B.; Carreira, E. M. Angew. Chem. Int. Ed. 2011, 50,
9085; (c) Hyde, S.; Veliks, J.; Liegault, B.; Grassi, D.; Taillefer, M.;
Gouverneur, V. Angew. Chem. Int. Ed. 2016, 55, 3785.
(10) (a) Barrett, A. G. M.; Braddock, D. C.; Lenoir, I.; Tone, H. J.
Org. Chem. 2001, 66, 8260; (b) Wurz, R. P.; Charette, A. B. Org. Lett.
2002, 4, 4531.
In summary, we have described the development of a biocataꢀ
lytic strategy for the asymmetric synthesis of trifluoromethylꢀ
substituted cyclopropanes via myoglobinꢀcatalyzed addition of
trifluoromethylcarbene to olefins. These transformations could be
applied to a variety of vinylarene substrates, offering unpreceꢀ
dented levels of diastereoꢀ and enantioselectivity. Furthermore,
both enantiomers of the target CF3ꢀcontaining products could be
accessed using Mb catalysts with complementary stereoselectiviꢀ
ty, whose choice was guided by their reactivity with EDA. The
reactions presented here provide access to enantioenriched fluoriꢀ
nated building blocks of high value for medicinal chemistry. This
study provides a firstꢀtime demonstration that carbene donor
reagents other than αꢀdiazoesters can be engaged in biocatalytic
carbene transfer reactions. This finding, combined with the
demonstrated feasibility of coupling these reactions with ex situ
generated diazo compounds, is anticipated to enable extension of
the present approach to a variety of other carbene precursors in
order to expand the scope of carbeneꢀmediated transformations
accessible with myoglobins and other metalloprotein catalysts.
Further studies in this direction are currently underway in our
laboratory.
ASSOCIATED CONTENT
Supporting Information
Supporting information includes Supplementary Tables and Figꢀ
ures, experimental procedures, compound characterization data
andcrystallographic data.
AUTHOR INFORMATION
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment