Biocatalytic Strategy for Highly Diastereo- and Enantioselective Synthesis of 2,3-Dihydrobenzofuran-Based Tricyclic Scaffolds

Article abstract of DOI:10.1002/anie.201903455

2,3-Dihydrobenzofurans are key pharmacophores in many natural and synthetic bioactive molecules. A biocatalytic strategy is reported here for the highly diastereo- and enantioselective construction of stereochemically rich 2,3-dihydrobenzofurans in high enantiopurity (>99.9% de and ee), high yields, and on a preparative scale via benzofuran cyclopropanation with engineered myoglobins. Computational and structure-reactivity studies provide insights into the mechanism of this reaction, enabling the elaboration of a stereochemical model that can rationalize the high stereoselectivity of the biocatalyst. This information was leveraged to implement a highly stereoselective route to a drug molecule and a tricyclic scaffold featuring five stereogenic centers via a single-enzyme transformation. This work expands the biocatalytic toolbox for asymmetric C–C bond transformations and should prove useful for further development of metalloprotein catalysts for abiotic carbene transfer reactions.

Full text of DOI:10.1002/anie.201903455

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Accepted Article
Title: Biocatalytic strategy for highly diastereo- and enantioselective
synthesis of 2,3-dihydrobenzofuran based tricyclic scaffolds
Authors: Rudi Fasan, David Vargas, Rahul Khade, and Yong Zhang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903455
Angew. Chem. 10.1002/ange.201903455
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10.1002/anie.201903455
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Biocatalytic strategy for highly diastereo- and enantioselective syn-
thesis of 2,3-dihydrobenzofuran based tricyclic scaffolds
David A. Vargas,[a] Rahul L. Khade,[b] Yong Zhang,[b],* and Rudi Fasan[a],*
presence of acceptor-only diazo reagents, including -
Abstract: 2,3-Dihydrobenzofurans are key pharmacophores in
many synthetic and naturally occurring bioactive molecules. A
biocatalytic strategy is reported here for the highly diastereo- and
enantioselective construction of stereochemically dense 2,3-
dihydrobenzofurans in excellent enantiopurity (>99.9% de and ee),
5d]
diazoacetates,[5c,
2-diazotrifluoroethane[10]
and
diazoacetonitrile.[11] Building upon these findings and motivated
by shortcomings of current methodologies in the context of
carbene-mediated transformations as outlined above, we report
here the development of a Mb-based biocatalytic strategy for the
highly diastereo- and enantioselective cyclopropanation of
benzofurans with an acceptor-only carbene donor. This strategy
provides a new, efficient, and highly stereoselective route to afford
2,3-dihydrobenzofuran scaffolds of high value for medicinal
chemistry and drug synthesis.
high yields, and on
a preparative scale via benzofuran
cyclopropanation with engineered myoglobins. Computational
and structure-reactivity studies provide insights into the
mechanism of this unprecedented iron-catalyzed reaction,
enabling the elaboration of a stereochemical model that can
rationalize the high stereoselectivity of the metalloprotein catalyst.
This information could be further leveraged to implement a highly
stereoselective route to afford
dihydrobenzofuran-containing drug as well as a tricyclic scaffold
featuring five stereogenic centers via single-enzyme
a
precursor for
a
2,3-
a
transformation. This work expands the biocatalytic toolbox for
asymmetric C-C bond transformations and the mechanistic
insights accrued here should prove useful for further development
of metalloprotein catalysts for abiotic carbene transfer reactions.
Benzofuran and 2,3-dihydrobenzofuran scaffolds are key
structural motifs and pharmacophores in many bioactive natural
products and synthetic compounds (Figure 1).[1] Methods for the
synthesis and elaboration of these scaffolds are therefore of
significant interest in organic and medicinal chemistry.[1] The
metal-catalyzed cyclopropanation of benzofurans with diazo
reagents provides an attractive strategy for the preparation of 2,3-
dihydrobenzofurans.[2] While important advances in asymmetric
benzofuran cyclopropanations have been made using Rh-based
catalysts in the presence of donor-acceptor diazo reagents,[2-3] the
stereoselective cyclopropanation of benzofurans using readily
Figure 1. Biologically active molecules containing 2,3-dihydrobenzofuran
and benzofuran moieties.
Initial tests with wild-type myoglobin (Mb), its distal histidine
variant Mb(H64V), and a panel of other hemoproteins (e.g.,
P450s, cyt c, horseradish peroxidase) showed no activity in the
desired cyclopropanation of benzofuran (1) with ethyl α-
diazoacetate (2a) (Table S9). Using Mb(H64V) as background,
we then tested a mini-library of Mb variants in which the shape of
the heme pocket is systematically varied by substituting each of
the amino acid residues within this cavity (i.e., Leu29, Phe43,
Val68, Ile107) with an apolar amino acid of significantly different
size (e.g., Leu29 → Ala or Phe; Phe43 → Ile or Ser). Promisingly,
this strategy led to the successful identification of
Mb(H64V,V68A)[5c] as a viable catalyst for converting 1 to the
desired product 3a. Notably, neither Fe(TPP) nor hemin can
catalyze this reaction, highlighting the critical role of the protein
matrix in conferring benzofuran cyclopropanation reactivity to the
protein-embedded iron-porphyrin. Despite its modest catalytic
activity (19% conversion; Table 1, Entry 3), Mb(H64V,V68A) was
determined to exhibit excellent diastereo- and enantioselectivity
(>99.9% de and ee) in this reaction producing the (1R,1aR,6bS)-
configured isomer (vide infra) as the only detectable product.
Based on the previously established importance of the ‘gating’
Val64 residue in controlling access of the olefin substrate to the
heme pocket in Mb(H64V,V68A)-catalyzed cyclopropanation of
styrene,[12] the crucial V68A mutation (Table S9) was then
combined with smaller residues at position 64 (i.e., Ala, Gly), in
an effort to increase catalytic activity by favoring access of the
larger benzofuran substrate to the heme-carbene intermediate.
Gratifyingly, the corresponding variants Mb(H64A,V68A) and
Mb(H64G,V68A) showed a progressive improvement in catalytic
available and inexpensive acceptor-only diazoester reagents has
1e, 4]
proven very challenging.[1d,
In addition, iron-based
promoting metallo-carbenoid
(bio)catalysts
useful
for
cyclopropanations of benzofurans has thus far remained elusive.
Engineered heme-containing proteins have recently
emerged as promising biocatalysts for mediating selective
carbene transfer reactions, including olefin cyclopropanation,[5]
Y—H insertion (where Y = N, S, or Si),[6] C—H functionalization,[7]
aldehyde olefination,[8] and other relevant transformations.[9] In
particular, our group has previously reported the high activity and
selectivity of engineered variants of sperm whale myoglobin (Mb)
for promoting asymmetric olefin cyclopropanations in the
[a]
[b]
D. A. Vargas, Prof. Dr. R. Fasan
Department of Chemistry, University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: rfasan@ur.rochester.edu
Dr. R. L. Khade, Prof. Dr. Y. Zhang
Department of Chemistry and Chemical Biology, Stevens Institute of
Technology, Hoboken, NJ 07030 (USA)
E-mail: yong.zhang@stevens.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201903455
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COMMUNICATION
activity, while maintaining excellent stereoselectivity (>99.9% de
and ee; Table 1, Entries 4-5).
the benzofuran molecule with both electron-donating and
electronwithdrawing groups were well tolerated by the biocatalyst,
enabling the highly diastereo- and enantioselective (>99.9% de
and ee) synthesis of the corresponding tricyclic products 5a-c, 5e-
i with good to quantitative conversion (61-99%; Scheme 1).
Bulkier substrates such as the disubstituted derivative 4i and
naphtho[2,3-b]furan (4j) could be also efficiently processed by the
Mb-based carbene transferase to give 5i and 5j in enantiopure
form. Comparable results were observed for whole-cell
biotransformations using Mb(H64G,V68A)-expressing cells (e.g.,
90% yield and >99% de and ee for 5i). Furthermore, the
Table 1. Activity and selectivity of myoglobin variant in the
cyclopropanation of benzofuran with EDA and other diazoesters.[a]
Mb(H64G,V68A)-catalyzed
reaction
with
representative
substrates such as 5-fluoro- (4b), naphtho[2,3-b]furan (4j), and
6-methoxy-benzofuran (4e) could be readily performed on a
preparative scale to afford 55-90 mg of the corresponding
cyclopropanation products in 55% (5b), 51% (5j) and 67% (5e)
isolated yield, respectively. Crystallographic analysis of 5b, 5d,
and 5f (Figure S2-4), and chiral chromatography analysis of the
other products (Table S7), indicated that the Mb(H64G,V68A)
Equiv
Yield[b]
(TON)
%
de
%
ee
Entry
Catalyst
diazo
2a-d/
Temp.
1/RT
1
2
3
hemin
Mb
2a
2a
2a
0
0
-
-
-
-
1/RT
1/RT
Mb(H64V,V68A)
19%
(24)
29%
(36)
39%
(49)
45%
(56)
76%
(95)
>99%
(125)
13%
(16)
>99
>99
4
5
Mb(H64A,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
Mb(H64G,V68A)
2a
2a
2a
2a
2a
2b
2c
2d
2a
2a
1/RT
1/RT
2/RT
2/RT
2/3oC
2/3oC
2/3oC
2/3oC
2/3oC
2/3oC
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
exhibits
a
remarkably conserved stereoselectivity toward
formation of the exo (1R,1aR,6bS)-configured tricyclic product.
This combination of exquisite stereocontrol and broad substrate
scope has little precedent in the context of new-to-nature enzyme-
catalyzed reactions.[13]
6
7[c]
8[c]
9[c]
Scheme
1.
Substrate
scope
for
Mb(H64G,V68A)-catalyzed
cyclopropanation of benzofurans with EDA.[a]
10[c]
11[c]
12[c-d]
13[c]
36%
(45)
>99%
(125)
89%
(445)
78%
(59)
Mb(H64G,V68A)
(E. coli cells)[e]
[a] Conditions: 20 μM catalyst, 2.5 mM benzofuran, 2.5 or 5 mM diazo compound,
10 mM Na2S2O4, 16 hr, anaerobic conditions. [b] GC yield. [c] Slow diazo addition.
[d] 5 μM protein. [e] OD600 = 50.
Using Mb(H64G,V68A), further improvements in both the
catalytic turnovers (TON) and product conversions were then
obtained via optimization of pH, EDA:benzofuran ratio, and
temperature (Table S10). Under optimal conditions, full
conversion of benzofuran to enantiopure 3a was achieved using
0.8 mol% of Mb(H64G,V68A) (Entry 8). Various other diazo esters
(2b-c) are compatible with this reaction, furnishing 3b-d with
excellent levels of stereoselectivity (>99.9% de and ee; Entry 9-
11), albeit in reduced yields in the case of the bulkier diazo
reagents 3b and 3c. With EDA, high product conversion (89%)
was obtained also with a 4-fold lower catalyst loading (0.2 mol%),
corresponding to a TTN of 445 (Entry 12). Moreover, comparable
yields of 3a were achieved using E. coli expressing
Mb(H64G,V68A) (Entry 13), thus demonstrating the possibility to
performing this biocatalytic transformation using whole cells.
While offering excellent enantioselectivity and involving an iron-
based biocatalyst, the trans-selectivity of Mb(H64G,V68A) nicely
complements the cis-selectivity of a Ir-salen catalyst previously
reported by Katsuki and coworkers for a related transformation.[4a]
Encouraged by these results, we further investigated the
substrate scope of the Mb(H64G,V68A) biocatalyst using
variously substituted benzofurans (Scheme 1). Substitution on
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[a] As in Table 1 Entry 8. [b] 15 mM EDA. [c] Using whole-cells (OD600 = 50).
Figure 2. Gibbs free energy diagram for the heme-carbene reactions with benzofuran and indole. ΔG values calculated at the B97XD/6-
311G(d)-LANL2DZ level (see SI for details). NMI = N-methyl-imidazole. Dotted bars and values in brackets correspond to transition states in
the protein active site model (see SI for details).
Interestingly, both Mb(H64V,V68A) and Mb(H64G,V68A) exhibit
a divergent chemoselectivity in the reaction with benzofuran vs.
the structurally related indole, the latter leading exclusively to the
C(3)—H functionalization product.[7c] Both reactions can occur (or
compete) with these heterocycles in the presence of other
catalytic systems[14] (for Rh: see Figure S6). To gain insights into
the mechanism of the present reaction and the basis for this
Despite these similarities, the relative energy of TSins vs. TSzw
differ significantly between the two heterocycles. For indole,
TSN/zw is indeed 1.5 kcal/mol lower in energy than TSN/ins (Gǂ =
6.8 vs. 8.3 kcal/mol; Figure 2), indicating that C(3)—H
functionalization by the heme-carbene is favored over
cyclopropanation with this substrate.[15] In contrast, the energy
barriers for the two pathways in the benzofuran reaction are
comparable (Gǂ = 11.4-11.7 kcal/mol TSO/ins); Figure 2).
However, when the active site residues surrounding the heme-
carbene are included in the calculations (see SI for details), the
C2=C3 insertion step becomes significantly more favorable (Gǂ =
10.6 (TSO/ins) vs. 17.6 kcal/mol (TSO/zw); Figure 2), suggesting a
key role of the protein matrix in favoring this reaction. To further
discriminate which process is operative in the metalloprotein, we
reasoned that the two pathways should be differentially impacted
by substitution at the level of the C2 and C3 position of
benzofuran, as given by the differential steric constraints around
these sites in the corresponding TS structures (Figure 2).
Experimentally, Mb(H64G,V68A) was found to show significant
cyclopropanation activity on 3-methyl-benzofuran, but no activity
on 2-methyl-benzofuran (Scheme 2). These results are
consistent with the C2=C3 insertion mechanism, as this is
expected to be significantly more tolerant to steric encumbrance
at the C3 vs. the C2 site compared to the stepwise mechanism.
These conclusions are further corroborated by the results with 2-
methyl-indole, which can be converted by the enzyme with
modest but measurable activity (Scheme 2). Since TSO/zw and
TSN/zw have similar geometries (Figure 2), the distinct reactivity
trend observed with the 2-substitued heterocycles further
divergent
chemoselectivity, both transformations were
investigated via Density Functional Theory (DFT) using a model
of histidine-ligated Fe-porphyrin (see SI for details). In each case,
both a mechanism involving a direct C2=C3 carbene insertion
(‘ins’) and one proceeding via a zwitterionic (‘zw’) intermediate
were considered (Figure 2). For the indole reaction, a direct
carbene C—H insertion was ruled out based on isotope labeling
experiments conducted with both Mb(H64V, V68A)[7c] and
Mb(H64G,V68A) (Figure S5). For both reaction pathways, the
calculated TS geometries with benzofuran vs. indole are similar.
Consistent with a nucleophilic attack through the C3 position of
the heterocycle, the TS structures for the stepwise pathway
shows a shorter distance (~0.3 Å) between the C3 atom and the
carbene C1 carbon atom compared to the C2···C1 distance (2.22
vs. 2.48 Å for benzofuran and 2.30 vs. 2.67 Å for indole; Figure
2). In both TSO/zw and TSN/zw, the plane of the heterocycle is
perpendicular to the carbene ester group, projecting the C2—H
and C3—H bonds toward the porphyrin ring. In comparison, the
TS structures for the C2=C3 insertion pathway show a significantly
different orientation of the heterocycle ring with respect to the
carbene group (Figure 2). Moreover, the C2 atom is now closer
to the carbene C1 compared to the C3 atom (C2···C1(2.22/2.30
Å) < C3···C1(2.52/2.38 Å) for benzofuran/indole; Figure 2).
supports
a
mechanistic scenario whereby benzofuran
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cyclopropanation proceeds via a concerted, asymmetric carbene
insertion with nucleophilic attack to the carbene initiating from the
C2 end of the C2=C3 bond (i.e., via TSO/ins).
and enantiopure form as a potential precursor for a highly
stereoselective synthesis of Merck’s antidiabetic drug 6 (Scheme
3b). Of note, the latter was previously prepared as a mixture of
stereoisomers, requiring the isolation of the desired enantiomer
by chiral chromatography.[1d]
Scheme 2. Reactivity studies with 2- and 3-methyl benzofurans.
In conclusion, we reported here the first example of a
biocatalytic (and iron-based) strategy for asymmetric benzofuran
cyclopropanation to afford stereochemically rich 2,3-
dihydrobenzofuran scaffolds of high value for medicinal chemistry.
In addition to excellent stereoselectivity, this method offers good
substrate scope, scalability, and compatibility with whole-cell
biotransformations. Our studies yielded valuable insights into the
mechanism of this reaction which helped elucidate
metalloprotein-mediated stereoinduction and informed the
application of this methodology to enable the highly
Based on these results, TSO/ins was docked into the structure
of Mb(H64G,V68A), as modeled based on the crystal structure of
Mb(H64V,V68A).[12] In the resulting complex (Figure 3), which is
consistent with the stereochemical outcome of the
cyclopropanation reaction, the carbene ester group extends into
an inner cavity between Ala68 and Ile107, whereas the
benzofuran molecule approaches the heme-bound carbene via
an opening created by the Gly residue at position 64. This
arrangement enforces attack of the heterocycle in trans with
respect to the carbene-borne ester group and to the si face of the
carbene group (the re face being shielded by Phe43; Figure 3),
thereby explaining the absolute diastereo- and enantioselectivity
of the biocatalyst in this reaction. This model is also consistent
with (a) the beneficial effect of the large-to-small mutations in
position 64 (His << Val < Ala < Gly; Table 1), as these should
facilitate access of the benzofuran to heme-bound carbene
through the solvent-exposed side of the heme group (ring C/D),
and (b) the tolerance of the biocatalyst to substitutions on the
aromatic ring of benzofuran, as suggested by the available space
surrounding these positions, especially at positions 6 and 7, which
are solvent-exposed (Figure 3).
enantioselective synthesis of
a
drug precursor and
a
sterochemically rich tricyclic scaffold bearing five stereogenic
centers via a single-enzyme transformation. The present findings
expand the biocatalytic toolbox for abiotic reactions[13] and should
aid further development of metalloprotein catalysts for selective
carbene transfer reactions.
Scheme 3. Mb(H64G,V68A)-catalyzed stereoselective synthesis of 5k
and antidiabetic drug 6.
Acknowledgements
This work was supported by the U.S. National Institute of Health grant
GM098628 (R.F) and a National Science Foundation grant CHE-
1300912 (Y.Z). D.V. acknowledges support from the NSF Graduate
Fellowship Program. The authors are grateful to Dr. William
Brennessel for assistance with crystallographic analyses and Noah
Gubernick for technical assistance. MS and X-ray instrumentation are
supported by U.S. National Science Foundation grants CHE-0946653
and CHE-1725028.
Figure 3. Model of TSO/ins complex (cyan/purple) in Mb(H64G,V68A)
structure.
Leveraging these insights, a double, Mb(H64G,V68A)-
catalyzed asymmetric cyclopropanation was accomplished using
7-vinyl-benzofuran (4k) to give 5k in high enantiopurity (>99.9 de
and ee) (Scheme 3a). Time course experiments confirmed that
cyclopropanation of vinyl group occurs first, followed by C2=C3
cyclopropanation Notably, this transformation enables the
creation of five stereogenic centers with high stereoselectivity
using a single enzyme. Finally, Mb(H64G,V68A) could be applied
to access 0.3 g of the tricyclic compound 5d in 76% isolated yield
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[15] NMR monitoring of the enzymatic reactions with indole (or N-methyl-indole)
and EDA showed no evidence of a cyclopropane precursor to the C3-
alkylation product, disfavoring an alternative mechanism involving
cyclopropanation/ring opening pathway as observed in ref. 14b.
a
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10.1002/anie.201903455
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
David A. Vargas, Rahul L. Khade, Yong
Zhang, and Rudi Fasan
Page No. – Page No.
Biocatalytic strategy for highly
diastereo- and enantioselective syn-
thesis of 2,3-dihydrobenzofuran
based tricyclic scaffolds
Forging rings with iron: A novel biocatalytic strategy for the highly stereoselective
cyclopropanation of benzofurans is reported. This approach enables the efficient
construction of enantiopure 2,3-dihydrobenzofuran-based tricyclic scaffolds useful
for the synthesis of pharmaceuticals and bioactive natural products. Computational
and structure-activity studies provide insights into the mechanism of this reaction
and the protein-mediated control of its stereochemical outcome.
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