Biocatalytic Strategy for the Highly Stereoselective Synthesis of CHF2-Containing Trisubstituted Cyclopropanes

Article abstract of DOI:10.1002/anie.202015895

The difluoromethyl (CHF₂) group has attracted significant attention in drug discovery and development efforts, owing to its ability to serve as fluorinated bioisostere of methyl, hydroxyl, and thiol groups. Herein, we report an efficient biocat

Full text of DOI:10.1002/anie.202015895

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Accepted Article
Title: Biocatalytic strategy for the highly stereoselective synthesis of
CHF2-containing trisubstituted cyclopropanes
Authors: Rudi Fasan, Daniela M. Carminati, Jonathan Decaens,
Samuel Couve-Bonnaire, and Philippe Jubault
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202015895
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10.1002/anie.202015895
Angewandte Chemie International Edition
COMMUNICATION
Biocatalytic strategy for the highly stereoselective synthesis of CHF2-
containing trisubstituted cyclopropanes
Daniela M. Carminati[a], Jonathan Decaens[b], Samuel Couve-Bonnaire[b], Philippe Jubault*[b] and Rudi
Fasan*[a]
Abstract: The difluoromethyl (CHF2) group has attracted significant
attention in drug discovery and development efforts, owing to its ability
to serve as fluorinated bioisostere of methyl, hydroxyl, and thiol
groups. Herein, we report an efficient biocatalytic method for the
highly diastereo- and enantioselective synthesis of CHF2-containing
trisubstituted cyclopropanes. Using engineered myoglobin catalysts,
a broad range of α-difluoromethyl alkenes are cyclopropanated in the
presence of ethyl diazoacetate to give CHF2-containing
cyclopropanes in high yield (up to >99%, up to 3,000 TON) and with
excellent stereoselectivity (up to >99% de and ee). By means of an
enantiodivergent myoglobin variant, the opposite enantiomer of the
cyclopropane product was also obtained. This methodology
represents a powerful strategy for the stereoselective synthesis of
high-value fluorinated building blocks for medicinal chemistry, as
exemplified through the formal total synthesis of a CHF2 isostere of a
TRPV1 inhibitor drug.
Despite the well recognized utility of cyclopropanes and
CHF2 groups in medicinal chemistry, the asymmetric synthesis of
difluoromethyl-containing cyclopropanes has remained largely
underdeveloped.[8] To the best of our knowledge, the only
example of an asymmetric synthesis of CHF2-containing
cyclopropanes with broad substrate scope involves rhodium-
catalyzed cyclopropanations in the presence of donor-acceptor or
acceptor-acceptor diazo reagents (Scheme 1a).[9a] Notably, no
chemo- or biocatalytic methods have been reported for the
asymmetric cyclopropanation of CHF2-containing olefins with
readily available acceptor-only diazo reagents.
Over the past few years, heme-containing proteins[10] and
artificial metalloenzymes[11] have emerged as promising systems
for catalyzing ‘abiological’ cyclopropanation reactions.[12] In
particular, our group has previously reported the high activity and
stereoselectivity of engineered myoglobins (Mb) toward
promoting the cyclopropanation of aryl-substituted olefins with
10c]
ethyl diazoacetate.[10b,
More recently, the scope of these
Due to their peculiar conformational properties,
cyclopropane rings contribute key structural motifs and
pharmacophores in many natural and synthetic bioactive
molecules.[1] Accordingly, there has been a significant interest in
developing methodologies for the synthesis of functionalized
cyclopropanes.[2] Along with cyclopropanes, fluorinated
substituents have been extensively exploited in medicinal
chemistry toward the discovery and development of new drugs.[3]
It is indeed well recognized that the introduction of fluorinated
substituents can significantly affect the pKa, lipophilicity, cell
permeability, and/or metabolic stability of a bioactive molecule,
often leading to significant improvements in its pharmacological
and/or pharmacokinetic properties.[3]
biocatalysts and myoglobin-catalyzed reactions was expanded to
include other diazo reagents carrying α-electron withdrawing
groups (Scheme 1b) as well as other olefins, producing
enantioenriched
cyclopropanes
amenable
to
further
diversification.[13] Despite this progress, fluorinated olefins have
remained elusive substrates for biocatalytic cyclopropanations.
Herein, we report the first example of a (bio)catalytic method for
the stereoselective synthesis of difluoromethyl-functionalized
cyclopropanes, which was made possible through the
cyclopropanation of electron poor α-difluoromethyl alkenes by
means of engineered myoglobin catalysts (Scheme 1).
Previous work:
Among fluorinated substituents, the CHF2 group has
recently attracted considerable attention due to its value in serving
as bioisostere for the methyl group,[4] which is widely exploited for
tuning the pharmacological properties of bioactive molecules.[5] In
addition, owing to its electronic and hydrogen bond donor
properties, the CHF2 group has also been exploited as a
functional mimic of hydroxyl or thiol groups.[6] For example, a
CHF2 moiety was employed to mimic a cysteine thiol group in
inhibitors of the hepatitis C virus NS3 protease, resulting in
analogs with enhanced potency.[7]
a)
O
CHF2
CF2H
Ar
[Rh(II)]
R2OC
R1
R1
+
R2
DCM, -50°C
Ar
N2
1 = Ar, NO2
R2 = OEt, 4-(OMe)-Ph
R
b)
[Mb]
+
R1
R2
N2
R1
R2
KPi (pH 7), RT
R1 = Ar, heterocycle,
R2 = CO2R,
CF3, CN
aliphatic, EWG
This work:
[a]
[b]
Dr. D. M. Carminati, Prof. Dr. R. Fasan
CHF2
[Mb], Na2S2O4
KPi (pH 7), RT
CO2Et
F2HC
Department of Chemistry, University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: rfasan@ur.rochester.edu
J. Decaens, Dr. S. Couve-Bonnaire, Prof. Dr. P. Jubault
Normandie Univ, INSA Rouen, UNIROUEN, CNRS, COBRA (UMR
6014), 76000 Rouen (France)
+
CO2Et
N2
R
R
Scheme 1. Biocatalytic cyclopropanation of α-difluoromethylated alkenes.
E-mail: philippe.jubault@insa-rouen.fr
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.202015895
Angewandte Chemie International Edition
COMMUNICATION
On the basis of these results, Mb(H64V,V68A) was selected
as the most promising biocatalyst for the target cyclopropanation
reaction. Further investigations showed that Mb(H64V,V68A)
catalyzes the formation of 3a with an initial rate of 520 TON/min
and the reaction reaches >75% completion in less than 5 min (SI
Figure S2). While fast, this rate is 2-fold lower than the
Mb(H64V,V68A)-catalyzed cyclopropanation of styrene with EDA
(initial rate of 1000 TON/min),[10b] possibly reflecting the lower
reactivity of the CHF2-containing substrate due to both electronic
Table 1. Myoglobin-catalyzed cyclopropanation of α-difluoromethyl-styrene
(2a) in the presence of ethyl 2-diazoacetate (EDA, 1).[a]
Yield
Entry
Protein
TON
d.e.
e.e.
(GC)
37%
20%
33%
38%
26%
97%
44%
21%
30%
87%d
67%
58%
1
2
Mb
185
100
165
190
130
485
220
105
3,000
435
335
290
79%
75%
97%
99%
99%
97%
71%
76%
96%
>99%
96%
90%
5%
4%
and
steric
effects.
Under
catalyst-limited
condition,
Mb(H64V)
Mb(H64V,V68A) was determined to support up to 3,000 turnovers
(TON) (Table 1, entry 9). Furthermore, the reaction could be
readily scaled up to afford 95 mg of enantiopure 3a (>99% de and
ee) in high isolated yield (87%) (Table 1, entry 10).
3
Mb(V68A)
91%
90%
98%
>99%
92%
-4%
4
Mb(H64G,V68A)
Mb(H64A,V68A)
Mb(H64V,V68A)
Mb(H64V,V68G)
Mb(H64V,V68F)
Mb(H64V,V68A)[b]
Mb(H64V,V68A)[c]
Mb(H64V,V68A)[e]
Mb(L29T,H64V,V68F)
5
6
We also tested the possibility to carry out this transformation
in whole cells, which is of practical relevance for industrial
7
8
processes.[14]
Under these conditions, the Mb(H64V,V68A)-
9
98%
>99%
>99%
-87%
catalyzed reaction in cellulo successfully produced the desired
cyclopropane 3a while maintaining high diastereoselectivity and
excellent enantioselectivity (96% de and >99% ee; Table 1, entry
11), albeit with reduced yield compared to the reaction with
purified protein (67% vs. 97%). Interestingly, these experiments
showed a bell-shape dependence of yield on cell density, with an
optimum at relatively low cell density (OD600 = 10) (SI Table S1).
Counterintuitively, a reduction in yield was observed at higher cell
densities (67%→45-48%), which may arise from partial
sequestration of the fluorinated substrate by the cell membrane
or other cellular components.
10
11
12
[a]Reaction conditions: 20 μM purified Mb variant, 10 mM styrene (2a), 20 mM
EDA (1), 10 mM Na2S2O4, in phosphate buffer (KPi 50 mM, pH 7), r.t., 16 hours.
Product yield, diastereomeric and enantiomeric excess were determined by
chiral GC-FID analysis. [b] Using 1 μM Mb(H64V,V68A). [c] 70 mg of 2a in 45-mL
scale. [d] Isolated yield. [e] Using whole cells (OD600 = 10).
We started this work by testing the catalytic activity of wild-
type sperm whale myoglobin (Mb) toward catalyzing the formation
of cyclopropane 3a starting from α-difluoromethyl styrene (2a) in
the presence of ethyl diazoacetate (EDA, 1) as carbene source
(Table 1, entry 1). While showing promising activity (37% yield),
wild-type Mb produced 3a with only moderate diastereoselectivity
(79% de) and very poor enantioselectivity (5% ee). Given the
previously established influence of these residues on the activity
To explore the generality of this biocatalytic method, several
α-difluoromethylated styrenes bearing different electron-donating
and withdrawing substituents at the ortho-, meta- and para-
position of the phenyl ring were tested in the Mb(H64V,V68A)-
catalyzed cyclopropanation with EDA (Scheme 2). Despite a
general reduction in yield (35-82% vs. 98%), substituents in meta-
and ortho- positions were well tolerated by the Mb(H64V,V68A)
catalyst, as evinced from the synthesis of 3b-3d with high to
excellent diastereo- and enantioselectivity (95-96% de and 89-
99% ee). A similar trend applies to para-substituted styrenes with
small to medium-sized substituents at the para position such as
fluoro (3i), methyl (3e), chloro (3j), and bromo (3k) groups, all of
which underwent Mb(H64V,V68A)-catalyzed transformation to
give the corresponding CHF2-substituted cyclopropanes with high
stereoselectivity (93-96% de, 82-94% ee) along with high yields
(93-96%; Scheme 2). In contrast, the presence of bulkier
substituents at the para position such as methoxy (3g), isopropyl
(3f) and dimethylamino (3h) groups were accompanied by a
noticeable reduction in yield and, for the latter two, also in
enantioselectivity (21-25% ee). Interestingly, this structure-
reactivity trend diverges significantly from that observed for the
Mb(H64V,V68A)-catalyzed cyclopropanation of styrenes with
EDA and other acceptor-only diazo reagents,[10c] thus indicating
an important effect of the CHF2 group on the substrate interaction
with the biocatalyst. Nevertheless, structural characterization of
3k by X-ray crystallography (Scheme 2; SI Figure S4) revealed
a (1R,2S) absolute configuration of the cyclopropane product,
which mirrors the (1S,2S)-stereoselectivity of Mb(H64V,V68A)
with styrene and EDA.[10b, 10c] Based on this information and the
previously reported stereochemical model for this reaction, we
posited that enlargement of the active site cavity at the level of
and stereoselectivity of myoglobin-catalyzed cyclopropanation
10c, 10h, 10i]
reactions,[10b,
we decided to screen a panel of Mb
variants with varying steric demands at the level of the distal
His64 residue (→Gly/Ala/Val) and Val68 (→Gly/Ala/Phe) (Table
1), which is located in close proximity to the heme center (SI
Figure S1). Among these variants, Mb(H64V,V68A) and
Mb(H64A,V68A) were found to exhibit significantly improved
diastereo- and enantioselectivity (97 to >99% de and ee)
compared to the wild-type protein (Table 1, entries 5-6). In
addition, Mb(H64V,V68A) offered also improved activity, resulting
in the nearly quantitative conversion (97%) of the fluorinated olefin
to the desired cyclopropanation product 3a. Comparison of the
results for the single variants Mb(H64V) and Mb(V68A) (Table 1,
entries 2-3) and other double mutant variants, the two mutations
in Mb(H64V,V68A) appear to have a clear synergistic effect in
improving the performance of the biocatalyst, with the Val68Ala
mutation mainly driving the improvement in stereoinduction and
the His64Val mutation being crucial for improving activity and
further refining its stereoselectivity (e.g., 91 → >99% ee). At
position 68, a further reduction in steric bulk (e.g., Ala→Gly)
reduces both diastereo- and enantioselectivity (entry 7 vs. 6),
while the introduction of a bulkier residue (Phe) produces a variant
with parent-like properties and a slight preference for the opposite
enantiomer (-4% ee; entry 8).
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10.1002/anie.202015895
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COMMUNICATION
benzodioxole as internal standard. [b] Using Mb(H64G,V68A) as catalyst. CCDC
entry for 3k: 1893087
His64, such as in Mb(H64G,V68A), should better accommodate
the bulkier para-substituted styrenes. Gratifyingly, the
Mb(H64G,V68A) variant proved indeed to be a superior catalyst
for the synthesis of 3f-3h, offering higher diastereo- and
enantioselectivity for these transformations (96-99% de, 41-85%
ee).
The high activity of Mb(H64V,V68A) in the cyclopropanation
of p-(fluoro)-α-CHF2-fluorostyrene (2i) encouraged us to test
additional electron-deficient alkenes, which are challenging
substrates for carbene transfer catalysts due to the typically
electrophilic character of their metallo-carbene intermediate.[11h,
Scheme 2. Substrate scope of Mb(H64V,V68A)-catalyzed cyclopropanation of
α-difluoromethyl-olefins. [a]
15]
Notably, electrondeficient α-difluoromethyl-styrenes carrying
CF3-, formyl-, or a cyano substituent in the ring could efficiently
converted into the corresponding cyclopropanation products 3l,
3m, and 3n, respectively, in good yields (up to 80%) and excellent
stereoselectivity (up to 99% de and 96% ee). Moreover, the
absence of side reactions with 4-(formyl)−α-CHF2-styrene
demonstrated the chemoselectivity and compatibility of the
biocatalytic method with a substrate containing a reactive
aldehyde group.
Substrate scope was then extended to aromatic O- and S-
containing heterocycles which are widely used in medicinal
chemistry.[3e] Specifically, benzofuran- and thiophene-containing
substrates were converted into product 3o and 3p, respectively,
with high diastereo- and enantioselectivity (72-92% de, 95 to
>99% ee). Of note, Mb(H64V,V68A) also retained high activity
toward 2o (54% yield) despite the relatively large size of this
substrate. An unactivated alkene such as 4-phenyl-2-
(difluoromethyl) butene (2q) was also tested. While
Mb(H64V,V68A) was able to afford the desired cyclopropane
product 3q in low yield (Scheme 2), the Mb(H64G,V68A) variant
proved to be a superior biocatalyst for this transformation, offering
higher yield (15% vs. 5%) along with higher enantioselectivity
(51% vs. 25% ee) and excellent diastereoselectivity (>99% de),
thus demonstrating the utility of these biocatalysts also in the
context of unactivated olefins.
Having established the generality of Mb(H64V,V68A) for the
synthesis of cis-(1R,2S)-difluoromethylated cyclopropanes, we
investigated the possibility to obtain an enantiodivergent
biocatalyst for this reaction. As shown for Mb(H64V,V68F) in
Table 1, the substitution of Val68 with Phe resulted in a modest
but detectable inversion of enantioselectivity toward formation of
the (1S,2R)-configured cyclopropane (Table 1, entry 8). Based on
this result, a panel of V68F-containing engineered myoglobins
were tested and found to exhibit enhanced (1S,2R)-
enantioselectivity
(SI
Table
S2).
Among
them,
Mb(L29T,H64V,V68F) was able to produce the desired (1S,2R)-
enantiomer of 3a in good yield (58%) and high enantiomeric
excess (90% de and -87% ee) (Table 1, entry 12), thereby
demonstrating the feasibility of achieving enantiodivergent
selectivity in this myoglobin-catalyzed reaction.
To further explore the scope of this methodology in the
context of fluoromethylated olefins, Mb(H64V,V68A) was
challenged
with
α-fluoromethyl-styrene
(4)
and
α-
(trifluoromethyl)-styrene (6). Notably, both substrates were
efficiently converted into the desired CH2F- and CF3-substituted
cyclopropanes 5 and 7, respectively, in high yield and excellent
diastereo- and enantioselectivity (96-99% de and 98 to >99% ee;
Scheme 3a-b), which further highlighted the broad substrate
profile of this enzyme.
[a] Reaction conditions: 20 μM Mb(H64V,V68A), 10 mM alkene, 20 mM EDA (1),
10 mM Na2S2O4 in KPi 50 mM (pH 7), r.t., 16 hours. Yield, diastereomeric and
enantiomeric excess determined by chiral GC-FID analysis using 1,3-
Finally, we tested the utility of the present biocatalytic
approach toward the generation of a difluoromethyl bioisostere of
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10.1002/anie.202015895
Angewandte Chemie International Edition
COMMUNICATION
a drug molecule. To this end, we targeted the synthesis of 10
(Scheme 3c), which corresponds to an analog of a TRPV1
inhibitor drug candidate developed by Pfizer[16] in which the
Acknowledgements
This work was supported by the U.S. National Institute of Health
grant GM098628. The authors are grateful to Dr. William
Brennessel for assistance with crystallographic analyses. MS and
X-ray instrumentation are supported by U.S. National Science
Foundation grants CHE-0946653 and CHE-1725028. This work
was partially supported by Normandie Université (NU), the
Région Normandie, the Centre National de la Recherche
Scientifique (CNRS), Université de Rouen Normandie (URN),
INSA Rouen Normandie, Labex SynOrg (ANR-11-LABX-0029)
Innovation Chimie Carnot (I2C) and CNRS through the
International Emerging Action program. J.D. thanks the Labex
SynOrg (ANR-11-LABX-0029).
methyl group is replaced by
a
CHF2 group. Using
Mb(H64G,V68A) as the catalyst, α-difluoromethyl-substituted
olefin 8 could be successfully cyclopropanated in a semi-
preparative scale reaction to afford 9 (50 mg, 36%) with high
stereoselectivity (99% de, 98% ee). This key intermediate can be
then converted into the final product 10 in only two steps using
established routes.[10c, 16]
a
CH2F
FH2C
EDA, Mb(H64V,V68A)
Na2S2O4, KPi (pH 7)
CO2Et
5
4
>99% yield, 500 TON,
99% de, 98% ee
b
References
CF3
F3C
EDA, Mb(H64V,V68A)
Na2S2O4, KPi (pH 7)
CO2Et
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H
N
SO2Me
F2HC
H
(S)
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(R)
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N
O
F3C
difluoro analog of TRPV1 inhibitor
10
(
)
Scheme 3. Biocatalytic cyclopropanation of α-CH2F- and α-CF3-styrene and
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In summary, we reported the first example of a biocatalytic
system for the asymmetric cyclopropanation of difluoromethylated
alkenes. Using two engineered myoglobins, Mb(H64V,V68A) and
Mb(H64G,V68A),
a
broad
panel
of
trisubstituted
difluoromethylcyclopropanes were synthesized with good
efficiency (up to 99% yield) and high to excellent stereoselectivity
(up to >99% de and ee) using ethyl 2-diazoacetate as carbene
donor. The scope of these biocatalysts extends to include the
stereoselective cyclopropanation of unactivated olefins as well as
mono- and trifluoro-methylated olefins, as exemplified through the
successful synthesis of enantioenriched 3q, 5 and 7, respectively.
The possibility of achieving enantiodivergent selectivity in this
transformation
was
also
demonstrated,
with
a
stereocomplementary myoglobin variant showing up to -87% ee
for this transformation. Finally, this strategy could be readily
applied to enable the highly stereoselective synthesis of a key
synthon for the chemoenzymatic synthesis of a difluoromethyl
isostere of a drug candidate. This methodology is expected to
create new opportunities for the biocatalytic asymmetric synthesis
of high-value fluorinated building blocks for organic and medicinal
chemistry.
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Entry for the Table of Contents
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Daniela M. Carminati, Jonathan
Decaens, Samuel Couve-Bonnaire,
Philippe Jubault and Rudi Fasan
Page No. – Page No.
Biocatalytic strategy for the highly
stereoselective synthesis of CHF2-
containing trisubstituted
cyclopropanes
CHieF2 cyclopropanes: A biocatalytic method was developed for the highly diastereo- and
enantioselective synthesis of CHF2-substituted cyclopropanes via myoglobin-catalyzed carbene transfer.
These biocatalysts offer broad substrate scope, enantiodivergent selectivity and could be applied to
produce a difluoromethyl bioisostere of a drug candidate.
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