Stereodivergent Intramolecular Cyclopropanation Enabled by Engineered Carbene Transferases

Article abstract of DOI:10.1021/jacs.9b02700

We report the development of engineered myoglobin biocatalysts for executing asymmetric intramolecular cyclopropanations resulting in cyclopropane-fused γ-lactones, which are key motifs found in many bioactive molecules. Using this strategy, a broad range of allyl diazoacetate substrates were efficiently cyclized in high yields with up to 99% enantiomeric excess. Upon remodeling of the active site via protein engineering, myoglobin variants with stereodivergent selectivity were also obtained. In combination with whole-cell transformations, these biocatalysts enabled the gram-scale assembly of a key intermediate useful for the synthesis of the insecticide permethrin and other natural products. The enzymatically produced cyclopropyl-γ-lactones can be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes. This work introduces a first example of biocatalytic intramolecular cyclopropanation and provides an attractive strategy for the stereodivergent preparation of fused cyclopropyl-γ-lactones of high value for medicinal chemistry and the synthesis of natural products.

Full text of DOI:10.1021/jacs.9b02700

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Communication
Stereodivergent Intramolecular Cyclopropanation
Enabled by Engineered Carbene Transferases
Ajay L. L Chandgude, Xinkun Ren, and Rudi Fasan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b02700 • Publication Date (Web): 17 May 2019
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Journal of the American Chemical Society
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Stereodivergent Intramolecular Cyclopropanation Enabled by
Engineered Carbene Transferases
Ajay L. Chandgude†, Xinkun Ren†, and Rudi Fasan*
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Department of Chemistry, University of Rochester, 120 Trustee Road, Rochester, NY 14627, United States
the development of stereodivergent biocatalysts for a desired
transformation remains an important yet challenging endeavor.47,
ABSTRACT: We report the development of engineered
myoglobin biocatalysts for executing asymmetric intramolecular
cyclopropanations resulting in cyclopropane-fused -lactones,
which are key motifs found in many bioactive molecules. Using
this strategy, a broad range of allyl diazoacetate substrates were
efficiently cyclized in high yields with up to 99% enantiomeric
excess. Upon remodeling of the active site via protein
engineering, myoglobin variants with stereodivergent selectivity
were also obtained. In combination with whole-cell
transformations, these biocatalysts enabled the gram-scale
assembly of a key intermediate useful for the synthesis of the
insecticide permethrin and other natural products. The
enzymatically produced cyclopropyl--lactones can be further
elaborated to furnish a variety of enantiopure trisubstituted
48
Here, we report the successful development of engineered
myoglobin-based catalysts capable of promoting the
intramolecular cyclopropanation of allyl -diazoacetate
derivatives with high stereocontrol and complementary
enantioselectivity. This method provides efficient access to a
variety of bicyclic cyclopropane-fused -lactones, in both
enantiomeric forms and at a synthetically useful scale, for use as
pharmacophores or as key intermediates for the synthesis of
enantioenriched cyclopropane-containing molecules.
Scheme 1. Biocatalytic methods for olefin cyclopropanation
Previous Work:
cyclopropanes. This work introduces
a first example of
R2
EWG
Hemo-
biocatalytic intramolecular cyclopropanation and provides an
attractive strategy for the stereodivergent preparation of fused
cyclopropyl--lactones of high value for medicinal chemistry and
the synthesis of natural products.
R2
R1
proteins
+
R1
N2
EWG
EWG = CO2R, CF3, CN
This work:
R2
O
Engineered
myoglobins
O
O
O
H
H
H
Fused cyclopropyl-lactones are structural motifs found in
R2
R1
R2
R1
R1
O
many biologically active natural products (e.g., blepharolides,
or
O
N2
2
cedkathryn, laevinoids, sterelactones)1,
and synthetic
H
R1 = aromatic, aliphatic,
naphalene, heterocycle
R2 = H or Me
compounds.3 In addition, they constitute versatile intermediates
for the total synthesis of a diverse range of medicinally important
compounds, including basiliolide B, ambruticin S4-6 and others.7, 8
Because of their high synthetic value, significant efforts have
been devoted to developing methods for the preparation of these
molecular scaffolds, in particular through transition metal-
catalyzed intramolecular cyclopropanations.9-18 Despite this
progress, the development of catalytic protocols for asymmetric
intramolecular cyclopropanations involving an earth-abundant,
inexpensive, and non-toxic metal like iron has been difficult, with
success being reported thus far only with donor-acceptor diazo
compounds.19
up to 99 % yield
up to 96 % ee
up to 99 % yield
up to 99 % ee
In initial studies, we discovered that wild-type sperm whale
myoglobin (Mb) is able to catalyze the cyclization of trans-
cinnamyl-2-diazoacetate (1a) to give 2a (Table 1). Despite its
modest activity, which is comparable to that of free hemin (Table
S1), Mb exhibits good enantioselectivity toward formation of the
(1R,5S,6S)-configured intramolecular cyclopropanation product as
determined by single crystal X-ray diffractometry (80% ee; Table
1, Entry 1). Compared to Mb, other hemoproteins including
P450BM3, cytochrome c, and catalase show negligible activity (0-
2%) as well as lower enantioselectivity in this reaction (6-22% ee)
(Table S1). To develop a more efficient and selective biocatalyst
for this transformation, we screened a panel of Mb variants (~40)
featuring one to four mutations within the distal pocket of this
protein (i.e., at positions Leu29, Phe43, His64, Val68, Ile107;
Figure S1 and Table S2). These tests revealed the beneficial
effect of large-to-small substitutions at the level of Leu29 and
Val68 toward improving both enantioselectivity (80→90-93% ee)
and catalytic activity (32→163 TON; Entries 2-3) (Table 1;
Entries 2-3). Mb(H64V,V68A), a previously optimized Mb
variant for intermolecular cyclopropanation,21 showed higher
activity (32→82 TON) but similar enantioselectivity compared to
Mb (82% ee).
Engineered hemoproteins have recently emerged as
promising biocatalytic platforms for carbene transfer reactions.20-
37
Recently, our group have demonstrated that engineered
myoglobins (Mb) are capable of catalyzing the stereoselective
intermolecular cyclopropanation of vinylarenes in the presence of
diazo compounds with varied -electron withdrawing groups (-
COOR, -CF3, -CN), thus providing access to enantioenriched
disubstituted cyclopropanes (Scheme 1).21, 22, 24, 25, 38 Engineered
P450s20, 23, 26, 27 as well as artificial metalloenzymes32, 39-46 have
also proven useful for promoting intermolecular cyclopropanation
reactions. Despite this progress, biocatalytic strategies for
intramolecular cyclopropanations have so far been elusive. In
contrast to synthetic catalysts featuring an ‘open active site’,
achieving this task using an enzyme is challenged by the need of
orchestrating the intramolecular cyclopropanation reaction within
the confined environment of a protein’s active site. Furthermore,
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Table 1. Intramolecular cyclopropanation of cinnamyl 2-diazoacetate
was also achieved for the intramolecular cyclopropanation of the
sterically
(1a) with Mb and variants thereof.a
1
2
3
4
5
6
7
Table
2.
Substrate
scope
of
Mb(H64V,I107S)
and
O
O
Mb(L29A,H64V,V68A).a
H
H
Mb-catalyst
Ph
O
a
Mb(H64V,I107S) ( )
or
O
Ph
KPi (pH 7)
RT, 5 h
N2
O
H
O
1a
b
Mb(L29A,H64V,V68A) (
)
2a
O
R
R
O
KPi (pH 7), RT, 5 h
N2
H
Entry
catalyst
OD600 Yieldb TON
e.e.
1b - 1l
2b - 2l
8
9
1
2
Mb
-
-
13%
65%
65%
33%
99%
74%
99%
78%
32
163
162
82
80%
93%
90%
81%
96%
96%
96%
97%
Entr
y
Catal
yst
Yield
Mb(L29A)
Mb(V68A)
Product
TON
e.e.
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
32
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36
37
38
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43
44
45
46
47
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50
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52
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54
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56
57
58
59
60
3
-
O
4
Mb(H64V,V68A)
-
H
a
b
90
85
99%
95%
99%
96%
5
Mb(L29A,H64V,V68A)
Mb(L29A,H64V,V68A)
Mb(L29A,H64V,V68A)
Mb(H64V,I107S)
-
250
185
90
1
2
3
4
5
6
7
8
O
F
6c
7d
8
40
40
-
H
2b
O
H
195
a
b
75
60
83%
66%
97%
89%
O
Cl
99%
9d
Mb(H64V,I107S)
40
90
>99%
(83%)e
H
H
2c
2d
2e
2f
a
O
Reaction conditions: 5 mM cinnamyl 2-diazoacetate (2a), 20 M Mb
variant (or C41(DE3) E. coli cells at indicated OD600) in KPi buffer (50
mM, pH 7), 10 mM Na2S2O4 (protein only), r.t., 5 hours in anaerobic
chamber. b GC yield. c 15 min reaction time. d Using 2.5 mM 2a. e Isolated
yield.
a
b
90
77
99%
85%
94%
80%
O
Br
H
H
O
a
b
55
41
62%
45%
95%
68%
Based on this information, the beneficial L29A mutation was
introduced into Mb(H64V,V68A) to give the triple variant
Mb(L29A,H64V,V68A). Gratifyingly, this variant enabled the
quantitative conversion of 1a into the (1R,5S,6S)-configured
cyclopropane-fused -lactone 2a with high enantioselectivity (96%
ee) (Table 1, Entry 5). Moreover, about 74% product conversion
was reached in merely 15 minutes (Table 1, Entry 6 and Figure
S2). Following an alternative strategy for catalyst optimization,
we also screened an ‘active-site mutational landscape’ library
which samples all possible 19 amino acid substitutions at the
active-site positions Leu29, Phe43, Val68, and Ile107 (Figure S1)
in the Mb(H64V) background.22 From this library,
Mb(H64V,I107S) was identified as another efficient and highly
enantioselective catalyst (97% ee) for the synthesis of 2a from 1a
(Entry 7). Using either catalyst, the intramolecular
cyclopropanation reaction can be conveniently carried out in
whole cells using E. coli cells expressing these Mb variants
(Entries 6 and 8). Notably, in addition to quantitative conversion,
the whole-cell reactions with Mb(H64V,I107S)-containing E. coli
cells yielded 2a in high enantiopurity (>99% ee; Entry 9). A cell
density (OD600) of 40-60 was determined to be optimal for
granting high conversion and high enantioselectivity in these
biotransformations (Table S3). The whole-cell reaction with
Mb(H64V,I107S) could be readily scaled up to enable the
isolation of 180 mg of enantiopure 2a (>99% ee) in 83% isolated
yield (Entry 9), thus demonstrating the scalability of the
biocatalytic transformation.
O
H
O
H
a
b
82
50
90%
55%
96%
70%
O
CF3
H
H
O
a
b
70
75
77%
82%
90%
39%
O
CN
H
H
2g
O
a
b
85
64
95%
70%
99%
74%
O
H
2h
OMe
O
MeO
H
a
b
90
56
99%
62%
99%
68%
O
H
H
2i
2j
O
OMe
a
b
60
21
65%
23%
97%
81%
O
9
H
O
H
O
a
b
90
72
71%
95%
79%
98%
10
11
12
O
H
H
2k
O
To assess the substrate scope of these biocatalysts, a diverse
panel of allylic diazoacetate derivatives were then subjected to
Mb(H64V,I107S)-
a
b
90
90
99%
99%
10%
52%
O
or
Mb(L29A,H64V,V68A)-catalyzed
H
H
2l
cyclization in whole-cell reactions on a semi-preparative scale
(0.1 mmol) (Table 2). Substrates carrying para, meta, and ortho
substituents on the phenyl group (1b–1i) were all efficiently
processed by Mb(H64V,I107S), leading to the corresponding
bicyclic products 2b-2i in good to quantitative yields (62-99%).
Both electron withdrawing groups and electron donating groups
were well tolerated, with high enantioselectivity being maintained
across all of these substrates (90-99% ee; Entries 1-8). This
include the ortho-substituted product 2i, indicating a good
tolerance of this catalyst to steric hindrance in proximity to the
olefinic bond. Good conversion and enantioselectivity (97% ee)
O
a
b
79
80
87%
89%
5%
38%
O
H
H
2m
2n
O
F
O
a
b
39
62
43%
68%
74%
51%
13
H
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a
Reaction conditions: 2.5 mM diazoacetate, Mb-expressing E. coli
His64→Val) and the ring A/D side of the heme (Val68→Ala). In
b
(OD600 = 40) in KPi buffer (50 mM, pH 7), 40 mL-scale, r.t., 3-5 hours.
GC or SFC yield. See Table S5 for additional data.
1
2
3
4
5
6
7
8
contrast, Mb(F43A/H64W/V68F) features significantly increased
steric occlusion at these positions (Leu29; His64→Ala;
Val68→Phe), but an enlarged cavity at the level of the opposite
side of the cofactor (i.e., ring B/D via Phe43→Ala).
demanding naphtyl-based substrate 1j (Entry 9). Compared
to Mb(H64V,I107S), Mb(L29A,H64V,V68A) displays a similarly
broad substrate scope, albeit with overall reduced activity and/or
enantioselectivity toward this set of substrates. Notably, both Mb
variants exhibit a consistent (1R,5S,6S) stereoselectivity across
this diverse panel of substrates, as evinced from crystallographic
analysis of 2a, 2c and 2d (Figure S3-5) and the similar
chromatographic behavior of the other products in chiral SFC or
GC.
These results prompted us to investigate challenging
substrates such as diazoacetates equipped with unactivated olefins
(1l, 1m) or multiple olefinic groups (1k, 1m, 1n). Notably, all
these substrates were efficiently cyclized by either Mb variant to
generate 2k-n in good to quantitative yields (68-99%; Entries 10-
13). Interestingly, Mb(L29A,H64V,V68A) offered significantly
higher enantioselectivity in the transformation of diazoacetates
with aliphatic substituents compared to Mb(H64V,I107S), thus
complementing its scope across this group of substrates. The
results with 2n and the nerol derivative 2m also demonstrated the
high regioselectivity of the biocatalysts toward formation of the
cyclopropane--lactones in the presence of competing olefinic
groups. In addition to 2m, other (Z)-allylic diazoacetates (1o-p)
could be efficiently cyclized with good enantioselectivity (88-89%
ee), but modest diastereoselectivity (Scheme S1). Methyl-
zation of position 43 and 64 via two additional rounds of
mutagenesis
and
screening.
The
resulting
variant,
Mb(F43A,H64W,V68F),
catalyzes
the intramolecular
cyclopropanation of 1a to give 3a in 89% ee and quantitative
yield (Table S4). To assess its substrate scope, this biocatalyst
was then challenged with the panel of diazoacetate substrates
described in Table 2. To our delight, all these substrates were
converted by Mb(F43A/H64W/V68F) to give enantioenriched 3a-
n in up to 96% ee and 41-99% yields (Scheme 2). In each case,
Mb(F43A,H64W,V68F) exhibits opposite enantioselectivity
compared to the (1R,5S,6S)-selective variants (Table 2), thus
furnishing a stereodivergent catalyst for this reaction.
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
Scheme
2.
Substrate
scope
of
(1S,5R,6R)-selective
Mb(F43A,H64W,V68F).
O
O
H
Mb(F43A,H64W,V68F)
5 mol%
R
O
O
R
N2
KPi (pH 7), Na2S2O4
RT, 3-5 h, anaerobic
H
1a - 1l
3a - 3l
O
O
H
O
H
H
H
O
substituted
cinnamyl
2-diazoacetates
and
homoallylic
O
F
O
Cl
diazoacetates could not be processed by the current biocatalysts
(Scheme S2), defining targets for future catalyst development.
H
H
3a
3b
3c
99% yield, 89% ee
99% yield, 89% ee
57% yield, 76% ee
48
While challenging to obtain,47,
stereocomplementary
O
H
O
H
O
H
biocatalysts are key assets for the synthesis of drugs and complex
molecules.22, 49-55 To develop a stereodivergent biocatalyst for this
reaction, wild-type Mb was subjected to iterative rounds of site-
saturation mutagenesis (a.k.a. ISM)56 directed to the active-site
residue Leu29, Phe43, His64, Val68, Ile107 (Figure S1). The
resulting libraries were screened in whole cells using cinnamyl-2-
diazoacetate (1a) as the substrate. Partial inversion of
enantioselectivity was initially achieved via a Val→Phe mutation
O
Br
O
CF3
O
H
H
H
3f
98% yield, 79% ee
3e
3d
99% yield, 84% ee
99% yield, 76% ee
O
MeO
O
H
O
H
H
O
O
O
CN
at position 68 (80%
improvement of the desired (1S,5R,6R)-selectivity was then
obtained through optimi
→ -10%; Figure 1). Progressive
H
H
H
3i
3h
3g
OMe
41% yield, 83% ee
80% yield, 90% ee
99% yield, 81% ee
O
H
O
O
H
H
Figure 1. Evolutionary paths to stereodivergent intramolecular
cyclopropanation biocatalysts.
OMe
O
O
O
O
H
H
3k
H
3l
3j
98% yield, 51% ee
80% yield, 96% ee
95% yield, 79% ee
O
H
F
O
H
3n
96% yield, 27% ee
Based on these considerations, we propose a stereochemical
model for the Mb(L29A/H64V/V68A)-catalyzed reaction
whereby intramolecular attack to the re face of the carbene is
favored by accommodating the ester group and phenyl group into
the cavities created by V68A and L29A/H64V, respectively
(Figure 2). This mode of attack is likely disfavored in the case of
the (1S,5R,6R)-selective variant Mb(F43A/H64W/V68F) due to
steric hindrance provided by the bulky Trp/Phe residues at
positions 64/68, whereas attack to the si face of the carbene may
be further facilitated by accommodating the phenyl group into the
cavity created by the F43A mutation (Figure 2). While further
Upon mapping their mutations onto Mb structure (Figure
S1), the stereocomplementary Mb variants clearly feature a
distinct
active-site
configuration.
The
mutations
in
Mb(L29A/H64V/V68A) expand the distal cavity in
correspondence to the upper side of the pocket (Leu29→Ala;
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computational and structural studies are warranted to probe these
point, enantiopure 2a produced with Mb(H64V,I107S) was
reduced with LiAlH4 to give the cis-hydroxymethyl-substituted
cyclopropane 6 in 89% yield in a single step (Scheme 3). On the
other hand, alkaline hydrolysis of 2a or its treatment with benzyl
amine in the presence of LiCl afforded the trisubstituted
cyclopropanes 7 and 8 in 94% and 81% yield, respectively.
Finally, hydrazinolyis of 2a followed by treatment with nitrous
acid furnish the cyclopropane-fused urethane 9 in 78% yield. In
all cases, these transformations occur with minimal (8; 98% ee) to
no erosion (7, 9; 99% ee) of enantiopurity (Scheme 3b).
In summary, the first example of biocatalytic intramolecular
olefin cyclopropanation was accomplished through the
engineering of myoglobin-based catalysts capable of offering high
enantioselectivity as well as stereodivergent selectivity for the
asymmetric construction of bicyclic cyclopropane--lactones from
allyl diazoacetates. These biocatalytic transformations can be
performed in whole cells, at a gram scale, and they can be applied
to gain stereoselective access to key intermediates for the
synthesis of cyclopropane-containing natural products and a
variety of highly valuable trisubstituted cyclopropane synthons for
medicinal chemistry and drug discovery. This work paves the way
to the development of hemoprotein-based catalysts for other types
of intramolecular carbene transfer reactions.
1
2
3
4
5
6
7
8
stereochemical models,38 it is instructive to observe how complete
remodeling of Mb active site was not only required for achieving
stereodivergent selectivity in the intramolecular cyclopropanation
reaction, but also with respect to enantioselective Mb-based
biocatalysts previously developed for the intermolecular version22
of this transformation (Table S4).
O
O
Ph
Mb(L29A,H64V,
V68A)
Fe
Mb(F43A,H64W,V68F)
His
Val64
Ala29
Leu29
9
Trp64
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
H
H
Ph
Ala68
O
Phe43
Phe68
O
Ph
Ala43
H
H
H
O
O
H
Fe
Fe
Si face attack
Re face attack
H
H
Ph
Ph
O
O
H
H
O
O
2a
3a
Figure 2. Stereochemical model for intramolecular
cyclopropanation catalyzed by the stereodivergent Mb variants.
ASSOCIATED CONTENT
Supporting Information
Gem-dimethyl substituted cyclopropanes are found in several
bioactive natural products, including the insecticide permethrin.
To further demonstrate the synthetic utility of the present strategy,
a large scale biotransformation with Mb(L29A,H64V,V68A)-
expressing E. coli cells was carried out in the presence of 1.5 g 3-
methylbut-2-en-1-yl 2-diazoacetate (4). This reaction enabled the
stereoselective synthesis of dimethyl cyclopropane 3-
oxabicyclo[3.1.0]hexan-2-one 5 in 81% ee and 83% isolated yield
(Scheme 3A). Further elaboration of this key intermediate via
known methods57, 58 can furnish the pyrethroid natural products
chrysanthemic acid, permethrin and phenothrin.
Supplementary tables, figures, experimental procedures,
characterization data, and crystallographic data are available in
the Supporting Information.
AUTHOR INFORMATION
Corresponding Author
* rfasan@ur.rochester.edu
ORCID
Scheme 3. Formal total synthesis of pyrethroid natural products (A)
and chemoenzymatic synthesis of trisubstituted cyclopropanes (B).
Ajay L. Chandgude: 0000-0001-8236-9626
Xinkun Ren: 0000-0001-6645-9074
Rudi Fasan: 0000-0003-4636-9578
O
a)
O
Author Contributions
4
N2
Mb(L29A,H64V,V68A)
KPi (pH 7), RT, 3 hr
†A.L.C. and X.R. contributed equally to this work.
O
H
H
Notes
H
H
H
ref 55
ref 56
X
X
O
O
O
The authors declare no competing financial interest.
O
OH
H
R
5
Chrysanthemic
acid
1.02 g
Phenothrin
Permethrin
R = CH2-Ph-2-Ph, X = CH3
R = CH2-Ph-2-Ph, X = Cl
ACKNOWLEDGMENT
83% isolated yield
81% ee
This work was supported by the U.S. National Institute of Health
grant GM098628. X.R. is supported by Sunivo LLC (US). 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.
b)
O
H
O
H
LiOH,
94%
H
O
LiAlH, THF
H2O/THF
HO
HO
H
HO
H
89%
2a
>99% ee
H
OH
7
6
99% ee
LiCl, DCM
BnNH2
2) NaNO2
1) NH2NH2
MeOH
81%
78%
O
H
H
H
O
N
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N
H
HO
O
H
H
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Graphic Table of Content
Mb
Mb
O
O
R2
O
H
H
H
R2
R1
R2
Mb(H64V,I107S)
O
Mb(F43A,H64W,V68F)
KPi (pH 7), RT
O
R1
O
R1
KPi (pH 7), RT
N2
H
up to 99% yield
up to 99% ee
up to 99% yield
up to 96% ee
R1 = aryl, alky, heterocycle
R2 = H, Me
R
R
R
R
H
O
H
H
H
H
H
H
H
HN
O
NH
Ph
OH
O
OH
OH
OH
OH
O
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