of 6
Stereoselective Enzymatic Synthesis of Heteroatom-Substituted
Cyclopropanes
Oliver F. Brandenberg,
Christopher K. Prier,
,
§
Kai Chen,
Anders M. Knight,
Zachary Wu,
and Frances H. Arnold
*
,
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena,
California 91125, United States
Division of Biology and Bioengineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California
91125, United States
*
S
Supporting Information
ABSTRACT:
The repurposing of hemoproteins for non-natural
carbene transfer activities has generated enzymes for functions
previously accessible only to chemical catalysts. With activities
constrained to speci
fi
c substrate classes, however, the synthetic
utility of these new biocatalysts has been limited. To expand the
capabilities of non-natural carbe
ne transfer biocatalysis, we
engineered variants of Cytochrome P450
BM3
that catalyze the
cyclopropanation of heteroatom-bearing alkenes, providing valuable
nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or
fi
ve active-site mutations converted a single parent enzyme into
selective catalysts for the synthesis of both
cis
and
trans
heteroatom-
substituted cyclopropanes, with high diastereoselectivities and
enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed
evolution for e
ffi
cient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.
KEYWORDS:
biocatalysis, Cytochrome P450, cyclopropanes, carbene transfer
T
he cyclopropane ring is an important building block in
organic synthesis and a structural motif in diverse
bioactive compounds.
1
,
2
Especially notable are heteroatom
(nitrogen, oxygen, or sulfur)-substituted cyclopropanes, which
appear in many natural products
3
and biosynthetic inter-
mediates (
Scheme 1
A).
4
In pharmaceuticals, introduction of
cyclopropylamines can substantially improve physicochemical
and pharmacokinetic properties,
5
and the cyclopropylamine
functionality appears in the class of hepatitis C virus protease
inhibitors represented by simeprevir
6
and grazoprevir.
7
Novel
biomaterials have also been constructed using
β
-amino-
cyclopropane carboxylic acids (
β
-ACCs) and related com-
pounds as rigid building blocks in synthetic peptides.
8
,
9
Heteroatom-bearing cyclopropanes are additionally useful
synthetic intermediates that engage in a variety of ring-opening
transformations.
10
13
Enantiomerically enriched cyclopropylamines are most
commonly synthesized via Curtius or Hofmann rearrangements
from the corresponding enantioenriched cyclopropylcarboxylic
acids or amides.
14
,
15
Chiral cyclopropylamines and cyclo-
propanols have also been synthesized via asymmetric phase-
transfer catalysis
16
,
17
and enzymatic resolution.
18
,
19
Alterna-
tively, forming the cyclopropane ring with control over
stereochemistry by catalytic, enantioselective cyclopropanation
of heteroatom-substituted alkenes represents a powerful
approach to this class of compounds (
Scheme 1
B). Such
reactions have been performed via carbene transfer with chiral
ruthenium,
20
24
rhodium,
25
27
and copper
11
,
27
catalysts.
Aminocyclopropanes have also been accessed via alkene
cyclopropanation with nitro-substituted carbenoids, followed
by reduction of the nitro group.
28
,
29
However, many of the
existing methods exhibit low catalyst turnover, proceed with
only moderate levels of stereocontrol, are limited to speci
fi
c
substrate types, or require precious metals (e.g., Ru, Rh).
Furthermore, these methods generally provide only the
trans
-
cyclopropane products with high diastereoselectivity and
enantioselectivity.
We and others have demonstrated that hemoproteins can be
engineered to perform cyclopropanation reactions of ole
fi
ns via
carbene transfer.
30
35
The synthetic utility of these
carbene
transferases
, engineered by directed evolution of cytochromes
P450, myoglobins, and other proteins, has been demonstrated
through preparative-scale syntheses of cyclopropane-containing
pharmaceutical precursors.
36
38
To date, however, almost all
cyclopropanation reactions catalyzed by hemoproteins have
required arene-substituted (styrenyl) ole
fi
ns. In a
fi
rst step
toward broadening the substrate scope of enzyme-catalyzed
Received:
December 23, 2017
Revised:
February 7, 2018
Published:
February 24, 2018
Letter
pubs.acs.org/acscatalysis
Cite This:
ACSCatal.
2018, 8, 2629
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© 2018 American Chemical Society
2629
DOI:
10.1021/acscatal.7b04423
ACSCatal.
2018, 8, 2629
2634
This is an open access article published under an ACS AuthorChoice License, which permits
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cyclopropanation, we demonst
rated recently that natural
hemoprotein diversity can be leveraged to identify and engineer
Cytochrome P450 and globin variants for the stereoselective
cyclopropanation of unactivated, aliphatic alkenes,
39
an activity
previously achieved only by hemoproteins containing an
arti
fi
cial iridium co-factor.
40
42
Gober et al. also recently
reported engineered Cytochrome P450 variants for the
cyclopropanation of dehydroalanine residues in thiopeptides,
43
thereby enabling a non-natural modi
fi
cation of a complex
natural product. We reasoned that diverse heteroatom-
substituted alkenes should be suitable coupling partners for
electrophilic metal carbenoids derived from the reaction of
diazo esters with genetically encoded heme proteins. This
reaction would extend the sco
pe of biocatalytic cyclo-
propanation to encompass a new class of synthetically
important cyclopropanes (
Scheme 1
B). Here, we describe the
directed evolution of Cytochrome P450 variants that cyclo-
propanate heteroatom-bearing alkenes to yield chiral N-, O-,
and S-cyclopropanes. The resulting enzymes outperform
existing small-molecule catalysts, especially for challenging
cis
-
selective cyclopropanation reactions.
To gauge the potential of enzymatic heteroatom-substituted
cyclopropane synthesis, we
fi
rst tested a set of hemoproteins for
cyclopropanation of
N
-vinylphthalimide (
1
)withethyl
diazoacetate (EDA,
2
) as the carbene precursor (
Figure 1
), a
reaction performed by free heme with a total turnover number
(TTN) of 3 and 28:72 diastereomeric ratio (dr) (
cis
:
trans
) (see
Table S2
in the Supporting Information). Enzymatic reactions
were carried out with whole
Escherichia coli
(
E. coli
) cells
expressing the hemoprotein variants and were analyzed for
reaction yield and stereoselectivity (see
Table S2
). Variants of
Cytochrome P450
BM3
from
Bacillus megaterium
were found to
be active catalysts for synthesis of cyclopropane adduct
3
,
including several
P411
variants that feature a cysteine-to-
serine mutation at the residue ligating the iron center.
31
E. coli
expressing variant P411
BM3
-CIS
31
demonstrated promising
diastereoselectivity (88:12
cis
:
trans
), and this protein was
selected for further engineering by directed evolution to
enhance its catalytic activity.
To increase the activity of P411
BM3
-CIS we performed
parallel site-saturation mutagenesis at active site residues V87,
L181, I263, T268, L437, and T438, positions previously shown
to in
fl
uence P450-catalyzed non-natural carbene and nitrene
transfer activities (see
Figure S3
in the Supporting
Information)
30
,
36
and screened the resulting libraries for
cyclopropanation on
N
-vinylphthalimide with EDA. Several
mutations at these positions yielded improved catalysts (see
Table S3
in the Supporting Information); in addition, we found
that mutation I263G, located above the heme co-factor,
reversed diastereoselectivity from favoring
cis-
3
(88:12) to
favoring
trans
-
3
(28:72), as the mutation I263A does in P450-
catalyzed styrene cyclopropanation.
30
To identify the optimal
Scheme 1. (A) Examples of Cyclopropylamines and
Cyclopropanols in Natural Products, Pharmaceuticals, and
Biomaterials; (B) Proposed Enzymatic Synthesis of Chiral
Heteroatom-Substituted Cyclopropanes
a
a
Enzymatic carbene transfer to he
teroatom-substituted alkenes
generates heteroatom-substituted cyclopropanes in a single step with
control over stereoselectivity.
Figure 1.
Directed evolution of P411
BM3
-CIS for
cis
- and
trans
-
selective cyclopropanation of
N
-vinylphthalimide (
1
). Yields were
determined by liquid chromatography (LC) analysis. Enantioselectiv-
ities are given for the respective diastereomers. (A) Evolutionary
lineage for
cis
-selective
N
-vinylphthalimide cyclopropanation. Reac-
tions were performed with whole
E. coli
cells at OD
600
= 30 expressing
the indicated variants, 30 mM
N
-vinylphthalimide (
1
), and 60 mM
EDA (
2
) under anaerobic conditions. (B) Evolutionary lineage for
trans
-selective
N
-vinylphthalimide cyclopropanation. Reactions were
performed with whole
E. coli
cells at OD
600
= 30 expressing the
indicated variants, 5 mM
N
-vinylphthalimide (
1
) and 10 mM EDA
(
2
).
ACS Catalysis
Letter
DOI:
10.1021/acscatal.7b04423
ACSCatal.
2018, 8, 2629
2634
2630