An Enzymatic Platform for the Highly Enantioselective and Stereodivergent Construction of Cyclopropyl-δ-lactones

Article abstract of DOI:10.1002/anie.202007953

Abiological enzymes offers new opportunities for sustainable chemistry. Herein, we report the development of biological catalysts derived from sperm whale myoglobin that exploit a carbene transfer mechanism for the asymmetric synthesis of cyclopropane-fused-δ-lactones, which are key structural motifs found in many biologically active natural products. While hemin, wild-type myoglobin, and other hemoproteins are unable to catalyze this reaction, the myoglobin scaffold could be remodeled by protein engineering to permit the intramolecular cyclopropanation of a broad spectrum of homoallylic diazoacetate substrates in high yields and with up to 99 % enantiomeric excess. Via an alternate evolutionary trajectory, a stereodivergent biocatalyst was also obtained for affording mirror-image forms of the desired bicyclic products. In combination with whole-cell transformations, the myoglobin-based biocatalyst was used for the asymmetric construction of a cyclopropyl-δ-lactone scaffold at a gram scale, which could be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes.

Full text of DOI:10.1002/anie.202007953

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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International Edition
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Accepted Article
Title: An enzymatic platform for the highy enantioselective and
stereodivergent construction of cyclopropyl-δ-lactones
Authors: Rudi Fasan, Xinkun Ren, Ningyu Liu, and Ajay L Chandgude
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202007953
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10.1002/anie.202007953
Angewandte Chemie International Edition
COMMUNICATION
An enzymatic platform for the highy enantioselective and
stereodivergent construction of cyclopropyl-δ-lactones
Xinkun Ren+, Ningyu Liu+, Ajay L. Chandgude, and Rudi Fasan*
Abstract: The development of enzymes capable of mediating
abiological reactions offers new opportunities for sustainable
chemistry. Here we report the development of biological catalysts
derived from sperm whale myoglobin that exploit a carbene transfer
mechanism for the asymmetric synthesis of cyclopropane-fused-δ-
lactones, which are key structural motifs found in many biologically
active natural products. While hemin, wild-type myoglobin, and other
hemoproteins are unable to catalyze this reaction, the myoglobin
scaffold could be remodeled by protein engineering to permit the
intramolecular cyclopropanation of a broad spectrum of homoallylic
diazoacetate substrates in high yields and with up to 99%
enantiomeric excess. Via an alternate evolutionary trajectory, a
stereodivergent biocatalyst was also obtained for affording mirror-
image forms of the desired bicyclic products. In combination with
whole-cell transformations, the myoglobin-based biocatalyst was
tetrahydropyran derivatives are key structural pharmacophores
found in numerous biologically active natural products including
microphyllandiolide, dinardokanshone B, 2-caren-8,4-olide and
chubularisin J (Figure 1).[2] Cyclopropyl-δ-lactones can be
accessed through the intramolecular cyclopropanation of
homoallylic diazoacetates but synthetic approaches to perform
these transformations have been notoriously scarce and
essentially limited to rhodium-based catalysts as reported by
Doyle and coworkers.[3] In addition, achieving high levels of
enantioselectivity using these systems has proven challenging
and variable levels of stereocontrol were observed depending on
the nature of substituents appended to the olefinic group
(Scheme 1a).[3] We therefore envisioned that the development of
potentially more general and enantioselective iron-based
biocatalysts for this transformation would offer an attractive and
sustainable alternative for the asymmetric construction of fused
readily applied to enable the asymmetric construction of
a
cyclopropyl-δ-lactone scaffold at a gram scale. The enzymatic product
could be chemically elaborated to furnish a variety of enantiopure
trisubstituted cyclopropanes, further highlighting the value of this
method to gain access to optically active cyclopropane-containing
scaffolds useful for the synthesis of bioactive molecules and natural
products.
cyclopropane-containing
intramolecular cyclopropanation.
bicyclo[4.1.0]
scaffolds
via
Previous work (Doyle):
O
H
R2
N2
Rh
-based catalysts
2(L*)4
O
R1
R2
O
R1
reflux, CH2Cl2
O
H
= Ph; R = H
2
4% to 73% ee
R
R1
1 = H, Me or Et; R
Cyclopropane-containing scaffolds have attracted significant
interest due to their importance for the design of pharmaceuticals,
occurrence in biologically active natural products, and value as
synthetic intermediates.[1] Fused cyclopropyl-δ-lactones and their
2 = H or Me 42% to 90% ee
This work:
O
O
R2
N2
H
H
H
O
O
R1
R2
O
R1
R2
R1
O
H
O
= aromatic, aliphatic,
O
R
90 to 99% ee
up to 99% yield
up to 99% ee
up to 99% yield
1 = H or Me
R2
O
O
O
O
H
HO
O
Scheme 1. Catalytic strategies for the synthesis of fused bicyclo[4.1.0]
scaffolds via intramolecular cyclopropanation
Microphyllandiolide
(terpenoid from Salvia microphylla)
2-Caren-8,4-olide
(anticancer activity)
OAc
HO
O
O
H
AcO
O
O
AcO
Over the past few years, we and others have demonstrated
the potential of engineered hemoproteins (myoglobin, cytochrome
P450s, cytochrome c)[4] and artificial metalloenzymes[5] for
promoting abiological carbene transfer reactions. Efforts in this
area have also expanded the range of diazo compounds
amenable to intermolecular cyclopropanation reactions.[6] More
O
OH
O
O
O HO
O
O
O
H
OAc
O
O
OAc
H
OAc
O
Dinardokanshone B
Chubularisin J
(potassium channel inhibitor)
(antidepressant)
recently,
the
possibility
of
mediating
intramolecular
Figure 1. Biologically active compounds containing fused bicyclo[4.1.0]
cyclopropanation reactions using engineered hemoprotein-based
biocatalysts was demonstrated.[7] Despite this progress, the
construction of cyclopropyl-δ-lactones through the cyclization of
homoallylic α-diazoacetates has proven elusive,[7a] reflecting the
challenge of orchestrating these energetically and sterically
demanding ring-closing reactions both with synthetic catalysts
and within the active site an enzyme (vide infra). In addition, no
iron-based catalysts have so far been reported for promoting
these transformations. Here, we report the successful
development of myoglobin-based biocatalysts capable of
scaffolds
[a]
[+]
Dr. X. Ren,[+],N. Liu[+] Dr. A.L.Chandgude, Prof. Dr. R. Fasan
Department of Chemistry, University of Rochester
120 Trustee Road, Rochester, NY 16427(USA)
E-mail: fasan@chem.rochester.edu
These authors contributed equally to this work
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.202007953
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COMMUNICATION
Table 1. Intramolecular cyclopropanation of (E)-4-phenylbut-3-en-1-yl 2-
diazoacetate (1a) with Mb and variants thereof. [a]
promoting the intramolecular cyclopropanation of homoallylic α-
diazoacetates with high enantioselectivity as well as
stereodivergent selectivity. These systems are shown to provide
an efficient and scalable approach to access enantiomeric pairs
of optically active cyclopropyl-δ-lactones as valuable synthons
for medicinal chemistry and natural product synthesis (Scheme
1).
H
N2
Mb(F43L,H64A,V68G,I107V)
O
O
KPi buffer (pH 7)
RT, 16 hrs, anaerobic
O
H
O
1a
2a
In initial experiments, we evaluated the ability of wild type
myoglobin (Mb) and a diverse panel of other hemoproteins,
including cytochromes P450 (P450BM3, XplA, BezE), catalase and
cytochromes c, to catalyze the cyclization of (E)-4-phenylbutenyl
2-diazoacetate (1a) to give the corresponding intramolecular
cyclopropanation products 2a/3a (Tables 1 and S1). However,
none of these metalloproteins show any detectable catalytic
activity toward this reaction (Table S1). Similar results were
obtained with two engineered Mb variants previously developed
for the intramolecular cyclopropanation of allyl α-diazoacetates
Entry
Catalyst
Hemin
OD600
-
Yield[b]
0%
TON
e.e.
n.d.
n.d.
n.d.
n.d.
1
2
3
4
0
0
0
0
Fe(TPP)Cl in DCM
Mb (WT)
0%
0%
Mb(H64V,I107S)
-
-
-
0%
Mb(F43Y,H64V,
V68A,I107V)
5
0%
0
n.d.
(Mb(H64V,I107S))[7a]
and
allyl
α-diazoacetamides
6
7
Mb(V68G)
1.1%
1.9%
1
2
16%
33%
(Mb(F43Y,H64V,V68A,I107V)),[7b] which were also found to be
catalytically incompetent toward this transformation (Table 1,
Entries 4-5). Considering the high efficiency of Mb(H64V,I107S)
toward mediating the cyclization of trans-cinnamyl-2-
diazoacetate,[7a] we attributed this result to the inherently higher
steric and entropic demands associated with mediating the
intramolecular cyclopropanation of 1a to give the cyclopropane-
fused-bicyclo[4.1.0] product 2a within the active site of the
hemoprotein when compared to the allylic counterpart.
Furthermore, neither iron-tetraphenylporphyrin (Fe(TPP)) nor free
hemin showed any activity toward conversion of 1a into 2a/3a
(Table 1, Entry 1-2), which is in contrast to their previously
observed ability to catalyze the cyclopropanation of allyl α-
diazoacetates.[7a] This result indicated that the former reaction is
energetically unfavorable in the presence of iron-porphyrins as
catalysts, even in the absence of steric constraints around the
metal active site.
Mb(H64A,V68G)
Mb(H64A,V68G,
I107V)
8
9
-
-
45%
63%
>99%
92%
66%
56
79
61%
99%
99%
99%
99%
Mb(F43L,H64A,
V68G, I107V)
Mb(F43L,H64A,
V68G,I107V)
10
11
12[c]
20
5
316
1164
208
Mb(F43L,H64A,
V68G,I107V)
Mb(F43L,H64A,
V68G,I107V)
20
[a] Reaction conditions: 2.5 mM (E)-4-phenylbut-3-en-1-yl 2-diazoacetate (1a),
20 μM Mb variant (or C41(DE3) E.coli cells at indicated OD600) in KPi buffer
(50 mM, pH 7), 10 mM Na2S2O4 (protein only), RT, 16 hrs in anaerobic chamber.
[b] GC yield based on the calibration curves by authentic standard. [c] reaction
time: 2 min
Next, we screened a diverse ‘in-house’ library of ~80 Mb
variants containing up to four amino acid substitutions in their
heme pocket but the large majority (98%) of these proteins
showed either no activity (0% yield, 59/82 variants) or negligible
activity (<1% yield; 21/82 variants) in the reaction with 1a (Table
S2). Within this panel of Mb variants, however, Mb(V68G) and
Mb(H64V,V68G) were identified as having some appreciable
activity toward formation of 2a although both the yields (1-2%) and
enantioselectivity of these reactions were low (16% and 4% ee,
respectively) (Table 1, Entries 6-7). Interestingly, these Mb
variants share a space-creating mutation (Val→Gly) at position
68, whose side-chain is projected toward the meso position
between rings A and D of the hemin cofactor (Figure S1).[8] These
results suggested that enlargement of the active site cavity in Mb
is beneficial for promoting the cyclization reaction, supporting the
notion about the high sensitivity of this reaction to steric factors.
This aspect is further highlighted by the lack of activity of
Mb(V68A) and Mb(H64A,V68A) (Table S2), which feature only a
subtle increase in steric bulk (H→Me) at position 68 compared to
Mb(V68G) and Mb(H64A,V68G), respectively.
Based on the results above, Mb(V68G) was chosen as the
starting point for the development of a biocatalyst with improved
activity and enantioselectivity for this intramolecular
cyclopropanation reaction via protein engineering. To this end,
starting from Mb(V68G), iterative rounds of single-site site-
saturation mutagenesis (NNK codon) were performed by
sequentially targeting each of the active site residues Leu29,
Phe43, His64, and Ile107, which surround the heme active center
(Figure 2a). The resulting libraries were expressed in multi-well
plates and screened as whole cells using 1a as the substrate. The
most promising ‘hits’ were validated by characterizing the
corresponding Mb variants in purified form prior to the next round
of mutagenesis and screening. Using this approach, three
beneficial mutations corresponding to H64A, I107V, and F43L
(Figure 2b) were accumulated in three consecutive rounds of
directed evolution, resulting in a ~60-fold improvement in yield (1
→63%) and a dramatic enhancement in enantioselectivity from
16% ee to 99% ee toward formation of 2a (Table 1, Entry 9). The
(1S,6S,7S)-configuration of 2a was assigned on the basis of
crystallographic analysis of related products 2e and 2i (Figures
2c, Tables S6-S7) described further below.
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10.1002/anie.202007953
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COMMUNICATION
Figure 2. Engineering of myoglobin scaffold for cyclopropyl-δ-lactone synthesis. (a) Crystal structure of sperm whale myoglobin (pdb 1VXA) and close-up
view of the heme distal pocket (box). The heme cofactor (green) and the active site residues targeted for mutagenesis (orange) are shown as stick models.
(b) Evolutionary path leading to an enantioselective biocatalyst for the intramolecular cyclopropanation of 1a to give 2a. The bar graph reports the GC yields
of the reactions with the different variants. See Table S3 for further details. (c) Crystal structures of compound 2e and 2i. (d) Time course experiments for
Mb(F43L,H64A,V68G,I107V)-catalyzed synthesis of 2a using whole cells (C41(DE3) cells, OD600 = 20) and 2.5 mM 1a in oxygen-free potassium phosphate
buffer (50 mM, pH 7.0).
Table 2. Substrate scope for Mb(F43L,H64A,V68G,I107V)-catalyzed
intramolecular cyclopropanation of homoallylic α-diazoacetats [a]
A whole-cell biotransformation with E. coli cells expressing
Mb(F43L,H64A,V68G,I107V)
N2
H
the Mb(F43L,H64A,V68G,I107V) biocatalyst enabled the
quantitative conversion of 1a into 2a with excellent
enantioselectivity (99% ee; Table 1, entry 10). A cell density
(OD600) of 20 was determined to be sufficient for achieving
quantitative yield of the desired product 2a, corresponding to 316
catalytic turnovers (TON) supported by the intracellular enzyme
(Table 1, Entry 10; Table S4). By lowering the cell density (OD600
20 → 5), a nearly four-fold higher TON value of 1,160 was
obtained while maintaining excellent enantioselectivity (99% ee)
and high yields (92%) (Table 1, entry 11). Thus, in addition to
offering higher enantioselectivity and involving an iron-based
catalyst, the catalytic activity (TON) of this Mb-based system is
10- to 20-fold higher than that previously achieved using Rh-
based organometallic catalysts (TON ~ 50-80),[3] highlighting its
superior efficiency over synthetic methods previously available for
attaining this transformation. Furthermore, time-course
experiments showed that the Mb-catalyzed whole-cell reaction
reaches ~70% product conversion in 2 min (Table 1, entry 12)
and quantitative conversion of 1a to 2a in less than 10 min (Figure
2d), thus exhibiting fast reaction kinetics. Additional experiments
(vide infra) demonstrated that the whole-cell biotransformation
can be volumetrically scaled up to the gram scale without
noticeable loss in yield and enantioselectivity, thus demonstrating
the utility of this biocatalytic method for preparative scale
synthesis.
R2
E. coli whole cells
R1
R2
O
R1
O
KPi buffer (pH 7)
RT, 2 hrs, anaerobic
O
H
O
-
-
1b 1j
2b 2j
Entry
1
Product
Yield[b]
TON
251
ee
H
F
79%
(65%)
O
97%
94%
97%
99%
90%
92%
94%
H
O
2b
2c
2d
2e
H
Cl
Br
88%
(71%)
O
2
3[c]
4
278
57
H
O
H
54%
(42%)
O
O
H
O
H
F3C
55%
(40%)
174
38
H
O
H
36%
(25%)
5[c]
O
H
O
2f
H
95%
(77%)
O
O
6
300
316
H
O
O
2g
H
99%
(82%)
7
H
2h
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10.1002/anie.202007953
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COMMUNICATION
H
variants favor the formation of the (1R,6R,7R) enantiomer 3a
(Table S2), we envisioned the possibility to develop an
enantiocomplementary Mb-based catalyst for the synthesis of the
cyclopropane-fused δ-lactones. To this end, Mb(V68G) and
Mb(H64A,V68G) were chosen as the starting points for these
directed evolution campaigns in view of their higher catalytic
activity compared to the aforementioned Mb variants, which
exhibit only marginal cyclopropanation activity on 1a (<1% yield,
Table S2). Gratifyingly, a progressive improvement of the desired
(1R,6R,7R)-selectivity was obtained by subjecting both parent
enzymes to iterative rounds of site-saturation mutagenesis
directed to the yet unmodified active site residues (Figure 3). In
both evolutionary pathways, a Ile→Phe mutation at position 107,
which is located in the ‘back side’ of the heme distal pocket with
respect to its solvent exposed side (Figure 2a), played a critical
role in favoring stereoinduction toward formation of enantiomer 3a
as evinced from the increase in (1R,6R,7R)-enantioselectivity in
the case of Mb(H64L,V68G) (-21 → -89% ee) and the inversion
of enantioselectivity in the Mb(H64A,V68G) background (33% ee
→ -74% ee; Figure 3). Further mutagenesis of these I107F-
containing variants led to the identification of biocatalysts with
99%
(84%)
O
8
316
52
99%
91%
H
OMe
O
2i
H
41%
(32%)
9[d]
O
H
O
2j
[a]
Reaction conditions: 2.5 mM substrate, Mb(F43L,H64A,V68G,I107V)-
expressing E.coli (OD600 = 20) in KPi buffer (50 mM, pH 7), 80 mL-scale, RT,
2 hrs. [b] Product conversion as determined by GC. Yields of isolated products
are reported in brackets. Errors are within 10%. [c] Using OD600 = 60. [d] Using
1 mM substrate, 200 mL-scale.
To
examine
the
substrate
tolerance
of
Mb(F43L,H64A,V68G,I107V), we then tested the activity of the
biocatalyst in the intramolecular cyclopropanation of a diverse
panel of homoallylic α-diazoacetate derivatives under the
optimized conditions described above and at a preparative scale
(0.2 mmol) (Table 2). To our delight, both electron-withdrawing
and electron-donating groups on the aromatic ring were accepted
by the Mb variant, which exhibited high to excellent
enantioselectivity (90-99% ee) across all of the tested substrates
(1b-1i). Substrates carrying a halogen functional group (1b to 1d)
useful for further derivatization were all efficiently processed to
afford the corresponding bicyclic products (2b to 2d) in good
yields (54% to 88%). Similar results were obtained with fluorinated
substrates such as 1e, which was transformed into 2e in 55%
yield and 99% ee. Encouraged by these results, we then surveyed
the effects of substitutions at the para, meta and ortho positions
on the aromatic ring using a methyl group as the probe substituent
(Table 2, Entries 5-7). Whereas all substrates were readily
cyclized by the Mb catalyst, the para-substituted methylated
substrate 1f was found to be the least favorable substrate,
resulting in the formation of 2f in 36% yield and 90% ee (Table 2,
Entry 5). In comparison, the Mb(F43L,H64A,V68G,I107V)-
catalyzed cyclization of the meta- and ortho-substituted
regioisomers 1g and 1h proceeded with significantly higher
efficiency, resulting in their quantitative or near-quantitative
conversion to the cyclopropyl-δ-lactones 2g and 2h (95-99%)
along with high(er) enantioselectivity (92-94% ee) (Table 2,
Entries 6-7). The efficient conversion of substrate 1i into 2i and
excellent enantioselectivity of this reaction (99% ee; Table 2,
Entry 8) further highlighted the tolerance of the Mb catalyst to
steric hindrance in close proximity to the olefinic group. Finally,
we extended these studies to an aliphatic diazoacetate substrate
containing an unactivated olefinic group such as 4-methylpent-3-
en-1-yl 2-diazoacetate (1j). Albeit in moderate yield (41%), this
substrate was successfully cyclized by the Mb catalyst to afford 2j
in 91% ee. Notably, the Mb(F43L,H64A,V68G,I107V)-catalyzed
intramolecular cyclopropanation reaction was determined to
exhibit a conserved (1S,6S,7S)-stereoselectivity across this
diverse panel of substrates, as evinced from crystallographic
analysis of 2e and 2i (Figure 2c) and the similar chromatographic
behavior of the other products in chiral GC (Figure S2).
Furthermore, in all cases, the desired cyclopropyl-δ-lactone
product could be readily isolated from the whole-cell reactions,
resulting in up to 84% isolated yields (Table 2).
further
improved
(1R,6R,7R)-enantioselectivity,
namely
Mb(F43Y,H64A,V68G,I107F) and Mb(F43H,H64L,V68G,I107F),
which catalyze the intramolecular cyclopropanation of 1a to give
3a in 98% ee and 99% ee, respectively (Figure 3, Table S3).
Notably, the improvement in (1R,6R,7R)-enantioselectivity across
these variants is accompanied by an increase in catalytic activity,
resulting in significantly improved yields for the reactions
catalyzed
by
Mb(F43Y,H64A,V68G,I107F)
and
Mb(F43H,H64L,V68G,I107F) (43-69%) compared to the parent
enzymes (1-2% yield) (Figure 3). Also noteworthy is the fact that
the beneficial mutations accumulated in the (1R,6R,7R)-selective
variants show a consistent trend with respect to increasing the
steric bulk at position 107 (Ile107 →Phe) and introducing aromatic
groups with H-bond donor/acceptor capability at position 43
(Phe43 →Tyr/His). These features are in stark contrast with the
effects of the mutations beneficial toward inducing (1S,6S,7S)
enantioselectivity, which reduce the steric bulk at both the 107
and 43 positions (i.e., Ile107 →Val and Phe43 →Leu; Figure 2b).
These structure-selectivity trend highlight the differential
demands in terms of active site configuration that are required for
steering the enantioselectivity of the Mb-catalyzed intramolecular
cyclopropanation reaction in opposite directions.
Albeit rare,[9] enantiocomplementary biocatalysts are highly
valuable for the synthesis of drugs and complex molecules since
mirror-image forms of bioactive molecules often display distinct
pharmacological activities. Upon noting that some of the Mb
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10.1002/anie.202007953
Angewandte Chemie International Edition
COMMUNICATION
H
R2
N2
R2
Mb(F43Y,H64A,V68G,I107F)
Na2S2O
O
R1
O
R1
4, KPi (pH 7),
H
O
O
RT, 2 hrs, anaerobic
1a - 1l
3a - 3l
H
H
H
H
H
F
Cl
O
O
O
H
O
O
O
3c
3a
3b
99% yield, 98% ee
85% yield, 99% ee*
47% yield, 78% ee
80% yield, 73% ee
H
H
H
Br
F3C
O
O
O
H
H
H
O
O
O
3d
3e
99% yield, 90% ee*
3f
99% yield, 80% ee
52% yield, 87% ee
H
H
H
O
O
O
H
O
3g
3h
58% yield, 97% ee*
99% yield, >99% ee
H
H
O
O
H
OMe
H
O
O
3j
3i
99% yield, 97% ee
75% yield, 51% ee
Scheme 2. Substrate scope of the (1R,6R,7R)-selective biocatalyst
Mb(F43Y,H64A,V68G,I107F). Reaction conditions: 1 mM substrate, 20 µM
protein, 10 mM Na2S2O4, in KPi buffer (50 mM, pH 7), RT, anaerobic, 2 hours.
Reported yields correspond to GC yields. * Using Mb(F43H,H64L,V68G,I107F).
Figure 3. Development of enantiodivergent biocatalysts. The diagram shows
the evolutionary paths leading to the (1R,6R,7R)-selective myoglobin variants
for the synthesis of 3a from 1a. The bar graphs report the GC yields of the
reactions with the different variants. See Table S3 for further details.
To further assess the synthetic value of the present strategy,
The substrate scope of Mb(F43Y,H64A,V68G,I107F) was
further investigated by challenging this biocatalyst with the panel
of diazoacetate substrates described in Table 2. These
experiments show that Mb(F43Y,H64A,V68G,I107F) is able to
efficiently process these substrates producing the desired
(1R,6R,7R)-configured cyclopropane-δ-lactones in good to
excellent enantiomeric excess (73 to >99% ee; Scheme 2), thus
exhibiting a broad substrate tolerance along with a consistent,
a
large-scale
biotransformation
with
Mb(F43L,H64A,
V68G,I107V)-expressing E. coli cells was carried out in the
presence of 650 mg of 1a (3 mmol). This reaction enabled the
isolation of 480 mg of enantiopure 2a (99% ee) in 85% isolated
yield (Scheme 3), thus demonstrating the scalability of this
biocatalytic method. The enzymatically produced cyclopropane-
δ-lactone was then chemically reduced with LiAlH4 to give the cis
hydroxymethyl/ethyl-substituted cyclopropane 4 in 89% yield and
99% ee in a single step (Scheme 3). Furthermore, alkaline
hydrolysis of 2a afforded the trisubstituted cyclopropane 5 in 94%
isolated yield as a single enantiomer. These results thus
demonstrate the versatility of these bicyclic enzymatic products
toward gaining access to optically active trisubstituted
cyclopropanes of value for medicinal chemistry and total
synthesis.[1].
stereodivergent
selectivity
compared
to
Mb(F43L,H64A,V68G,I107V). The aliphatic substrate 1j was also
efficiently converted by the (1R,6R,7R)-selective catalyst to give
3j albeit with reduced enantioselectivity (51% ee) compared to the
other substrates. The Mb variant Mb(F43H,H64A,V68G,I107F)
was also found to be a competent and enantioselective catalyst
for these reactions, offering superior enantioinduction for the
synthesis of 3e and 3g (90-97% ee vs. 50-71% ee) compared to
Mb(F43Y,H64A,V68G,I107F).
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10.1002/anie.202007953
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N2
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94% yield, 99% ee
Scheme 3. Gram-scale synthesis and chemoenzymatic diversification of
cyclopropyl-δ-lactones.
In summary, we have developed an efficient, versatile, and
sustainable biocatalytic platform for the enantioselective
synthesis of cyclopropyl-δ-lactones, which are key motifs in
bioactive molecules (Figure 1) as well as versatile intermediates
for the preparation of trisubstituted cyclopropanes (Scheme 3).
While neither wild-type myoglobin nor its cofactor (hemin) are able
to catalyze this intramolecular cyclopropanation reaction, two
biocatalysts capable of executing these reactions with high
enantioselectivity as well as stereodivergent selectivity across a
broad range of substrates were obtained through re-design of the
active site of this hemoprotein. These biocatalytic transformations
can be carried out using whole cell systems, which eliminates the
need for protein purification, and could be readily performed at the
gram scale, which further demonstrates their value for synthetic
applications. This study expands the range of synthetically
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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.
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Conflict of interest
The authors declare no conflict of interest.
Keywords: intramolecular cyclopropanation • δ-lactones •
carbene transfer • myoglobin • biocatalysis
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10.1002/anie.202007953
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Lords of the Rings: A
Xinkun Ren+, Ningyu Liu+, Ajay
L. Chandgude, and Rudi Fasan*
biocatalytic strategy for the
stereodivergent synthesis of
cyclopropyl-δ-lactones via the
intramolecular cyclopropanation
of homoallylic diazoacetates is
reported. Myoglobin was re-
engineered into two
Page No. – Page No.
An enzymatic platform for the
highly enantioselective and
stereodivergent construction
of cyclopropyl-δ- lactones
enantiocomplementary
biocatalysts capable of
producing a range of aryl- and
alkyl-substituted cyclopropane-
fused δ-lactone scaffolds useful
for medicinal chemistry and
natural product synthesis.
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