Enzymatic Construction of Highly Strained Carbocycles
Kai Chen
,
Xiongyi Huang
,
S. B. Jennifer Kan
,
Ruijie K. Zhang
, and
Frances H. Arnold
*
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology,
Pasadena, CA 91125, USA.
Abstract
Small carbocycles are structurally rigid and possess high intrinsic energy due to their significant
ring strain. These unique features lead to broad applications, but also create challenges for their
construction. We report the discovery and engineering of hemeproteins that catalyze the formation
of chiral bicyclobutanes, one of the most strained four-membered systems,
via
successive carbene
addition to unsaturated carbon–carbon bonds. Enzymes that produce cyclopropenes, putative
intermediates to the bicyclobutanes, were also identified. These genetically-encoded proteins are
readily optimized by directed evolution, function in
Escherichia coli
, and act on structurally
diverse substrates with high efficiency and selectivity, providing an effective route to many chiral
strained structures. This biotransformation is easily performed on preparative scale and the
resulting strained carbocycles can be derivatized, opening myriad potential applications.
One Sentence Summary
Heme enzymes engineered by directed evolution catalyze the asymmetric formation of highly
strained bicyclobutanes and cyclopropenes.
In cyclic organic molecules, ring strain arises from distortions of bond angle and bond
length, steric clashes of non-bonded substituents, and other effects (
1
). The simplest
carbocycles, cyclopropanes and cyclobutanes, possess ring strains of 26–28 kcal/mol (
2
).
Introducing carbon–carbon multiple bonds or bridges to these small ring systems induces
additional strain as well as structural rigidity. For example, cyclopropenes with an
endo
-
cyclic double bond bear a strain of 54 kcal/mol, whereas bicyclo[1.1.0]butanes, folded into
puckered structures, distinguish themselves as one of the most strained four-membered
systems with around 66 kcal/mol strain (Figure S1) (
2
). These carbocycles are particularly
attractive intermediates in chemical and materials synthesis, since they can undergo strain-
release transformations to furnish a myriad of useful scaffolds (
3
–
6
). The structural rigidity
imparted by strained rings in supramolecular materials can lead to interesting physical
properties, such as mechanical stability (
7
) and high glass-transition temperature (
8
). The
intrinsic energy of these strained structures can also be relieved in response to exogenous
force, which leads to radical changes in physical properties (
e.g
. conductivity), a feature
highly desirable for stimulus-responsive materials (
9
,
10
).
*
To whom correspondence should be addressed. frances@cheme.caltech.edu.
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High ring strain, however, greatly increases the difficulty of synthesis. A commonly used
method for preparing bicyclobutanes starts from dibromo-2-(bromomethyl)cyclopropane
substructures and utilizes organolithium reagents for lithium-halogen exchange followed by
nucleophilic substitution under rigorously anhydrous and cryogenic conditions (
3
). An
alternative route relies on the double transfer of a carbene to alkynes, but the few examples
in the literature are mostly limited to methylene carbene (
11
–
13
). Asymmetric bicyclobutane
construction is particularly challenging, with multiple chiral centers generated at the same
time (
14
,
15
) (Figure S2). Cyclopropene synthesis through enantioselective single carbene
addition to alkynes also requires chiral transition metal catalysts based on rhodium (
16
,
17
),
iridium (
18
) and cobalt (
19
). Development of a sustainable catalytic system that performs
with high efficiency and selectivity under ambient conditions would be a significant advance
for construction of these useful, highly strained carbocycles.
Enzymes, the catalytic workhorses of biology, are capable of accelerating chemical
transformations by orders of magnitude while exhibiting exquisite control over selectivity
(
20
). Although nature synthesizes various cyclopropane-containing products (
21
),
cyclopropene or bicyclobutane fragments are extremely rare (Figure S3) (
22
,
23
). This may
be attributed to the lack of biological machinery for synthesizing these motifs and/or the
instability of these structures under biological or natural product isolation/purification
conditions. Nevertheless, we envisioned that existing enzymes could be repurposed to forge
strained carbocycles by taking advantage of their catalytic promiscuity (
24
,
25
) in the
presence of non-natural substrates and by using directed evolution to increase the newly-
discovered activity and selectivity (
26
).
In the past several years, we and others have engineered natural hemeproteins to catalyze
reactions not known in nature (
27
–
32
). We hypothesized that carbene transfer to triple bonds
with a heme-dependent enzyme might afford highly strained cyclopropene and
bicyclobutane structures and do so enantioselectively. We anticipated several challenges at
the outset, especially in bicyclobutane formation, as it involves two sequential carbene
additions to the alkyne substrate: 1) the enzyme would need to bind the alkyne in a specific
conformation in order to transfer the carbene enantioselectively; 2) the high-energy
cyclopropene intermediate generated by the first carbene addition would need to be accepted
and stabilized by the protein; 3) compared to methylene carbene used previously, a
substituted carbene (
e.g
. with an ester group) might hinder access of the cyclopropene to the
iron-carbenoid; and 4) the protein would also be expected to possess precise stereocontrol
over the second carbene transfer step regardless of structural differences between the initial
alkyne and the cyclopropene intermediate. Despite these challenges, we decided to
investigate whether a starting enzyme with this unusual and non-natural activity could be
identified, and whether its active site could be engineered to create a suitable environment
for substrate binding, intermediate stabilization, and selective product formation.
We first tested whether free heme (± bovine serum albumin (BSA)), which is known to
catalyze styrene cyclopropanation (
27
), could transfer carbene to an alkyne. Reactions using
ethyl diazoacetate (EDA) and phenylacetylene (
1a
) as substrates in neutral buffer (M9-N
minimal medium, pH 7.4) at room temperature, however, gave no cyclopropene or
bicyclobutane product. Next, a panel of hemeproteins including cytochromes P450,
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cytochromes P411 (P450 with an axial serine ligand), cytochromes
c
and globins in the form
of
E. coli
whole-cell catalysts were tested for the desired transformation under anaerobic
conditions (
32
), but none were fruitful (Figure 1C and Table S1). Interestingly, a P411
variant obtained in a previous cyclopropanation study, P411-
S1
I263W (see Supplemental
Materials for sources, sequences and mutations), afforded a furan product (
3b
) with a total
turnover number (TTN) of 210. Since other furan analogs have been identified as adducts of
carbenes and alkynes (
33
), we were curious as to how furan
3b
was generated. Preliminary
kinetic study of the enzymatic reaction suggested that the enzyme first synthesized an
unstable cyclopropene (
3a
), which subsequently rearranged to the furan either spontaneously
or with assistance from the enzyme (Figure 1B and S5). This result provided strong evidence
that the P411 hemeprotein is capable of transferring a carbene to an alkyne, which is, to our
knowledge, an activity not previously reported for any protein or even any iron complex.
To divert the enzymatic reaction to bicyclobutane formation, the enzyme is required to
transfer a second carbene to cyclopropene intermediate
3a
before the cyclopropene
rearranges to the undesired furan product (Figure 1B). We thus tested P411 variants closely
related to P411-
S1
I263W. We reasoned that amino acid residue 263, which resides in the
distal pocket, above the heme cofactor, might modulate the rate of this step and that the
bulky tryptophan (Trp) side chain at this site may be blocking the second carbene transfer. A
P411-
S1
variant with phenylalanine (Phe) instead of Trp at this position (I263F) in fact
catalyzed bicyclobutane formation at a very low level (< 5 TTN) (Table S1). Variant ‘
P4
’
with 3 additional mutations relative to P411-
S1
I263F (V87A, A268G and A328V) (
28
)
synthesized the desired bicyclobutane
2a
with 80 TTN and with the formation of furan
adduct substantially suppressed (
2a
:
3b
> 50: 1, Figure 1C). Another related P411 variant,
E10
(=
P4
A78V A82L F263L), which was engineered from
P4
for nitrene transfer
reactions (
29
), catalyzed the desired transformation with > 6-fold higher activity (530 TTN,
Figure 1E). NMR analysis revealed an
exo
,
endo
-configuration of the enzymatically-
produced bicyclobutane
2a
, which is distinct from the only reported achiral
endo
,
endo
-
isomer, made using an osmium-porphyrin complex (
34
,
35
). We chose this P411-
E10
variant
as the starting template for directed evolution of a more efficient bicyclobutane-constructing
enzyme.
Because the side chain of residue 263 influenced formation of the bicyclobutane product, we
performed site-saturation mutagenesis (SSM) of variant
E10
at position 263 and screened
whole
E. coli
cells expressing the mutated proteins for improved production of
bicyclobutane
2a
. The enzyme having leucine at this position (263L) was the most active;
other amino acid residues either lowered the reactivity towards bicyclobutane formation
and/or delivered more furan product. In parallel, two additional residues in
E10
, V78 and
S438, were also targeted by SSM. Aromatic residues were found to be activating at 78, with
a phenylalanine or tyrosine mutation giving 1.5 – 2-fold improvement over
E10
. This
beneficial mutational effect may stem from a
π
-
π
stacking interaction between the side
chain and the alkyne substrate or the cyclopropene intermediate. A single S438A mutation
on a loop residing above the heme also significantly increased the activity, giving >2.5-fold
increase in turnover. Finally, recombination of V78F/Y and S438A mutations led to the
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discovery of even more powerful biocatalysts for bicyclobutane formation (
e.g
. 1880 TTN
with
E10
V78F S438A, Figure 1E and S9).
With the evolved
E10
V78F S438A variant in hand, we next assayed the bacterial catalyst
against a panel of aromatic alkyne coupling partners. Biotransformations with 10 different
substrates were performed on 0.1 – 0.2 mmol scale. These preparative-scale reactions
proceeded smoothly to furnish the corresponding bicyclobutanes with up to 1760 TTN and
80% yield (Figure 2A). Additionally, three alkynes,
1k
,
1l
and
1m
, were transformed in
mmol scale, and bicyclobutanes were isolated in hundred-milligram quantities,
demonstrating that the biocatalytic transformation is readily scalable. Among the 13
different substrates, the engineered P411 hemeprotein did not exhibit strong preference
toward specific electronic or steric features. Electron-deficient halides (
2b
–
2d
), which can
be used as pre-functionalities for further transformations as well as electron-rich alkyl or
alkoxyl groups (
2e
–
2h
and
2k
) at
meta
- or
para-
position of the phenyl group were accepted
by the enzyme. Even heterocyclic substrates such as thiophene (
2j
) served as suitable alkyne
partners, albeit with lower reactivity.
Free functionalities, including alcohols (
2i
and
2m
) and a second alkyne (
2l
), are well-
preserved, providing an additional opportunity for derivatization of these products. A
terminal alkyne allows copper-catalyzed click chemistry, through which bicyclobutane
2l
can be modified with a simple sulfonyl azide (
4a
) or even decorated with biologically
relevant fragments, such as a phenylalanine derivative (
4b
). An unprotected hydroxyl group
could also offer the possibility of linkage to useful structures. Additionally, in order to probe
the enantiopurity of bicyclobutane products, we derivatized
2l
and
2m
with
L
-azido-
phenylalanine and (
R
)-Mosher’s acid, respectively. The diastereomeric excess of these
derivatized products would inform us the enantiomeric ratio of the bicyclobutanes. In fact,
we observed only one diastereomer of derivatized bicyclobutanes
4b
and
4c
by NMR.
Furthermore, the dicarboxylic esters on the bicyclobutane structure can be reduced easily
with a mild reducing reagent, LiBH4, to give diol product
4d
with the strained ring structure
preserved. The diol product
4d
allowed for the unequivocal confirmation of the
bicyclobutane structure and determination of the absolute configuration through X-ray
crystallography.
We next asked whether the enzyme could stop at the cyclopropene product if less reactive
aliphatic alkynes are used. To this end, we examined enzyme variants from the P411-
S1
lineage for cyclopropene formation, using phenylbutyne (
5a
) and EDA as starting reagents.
Encouragingly,
P4
catalyzed the desired transformation with 260 TTN and 95.5: 4.5
er
.
Further evolution was performed on
P4
to improve its catalytic efficiency. We first targeted
position 87, known for its importance to substrate recognition in P450-catalyzed oxidations
(
36
). A87F (290 TTN, 3.0: 97.0
er
) and A87W (240 TTN, 97.1: 2.9
er
) were found to exert
the opposite enantio-preference, suggesting that residue 87 also controls substrate
orientation for non-native carbene chemistry. Using
P4
A87F and
P4
A87W as parents,
single- and double-site-saturation mutagenesis were conducted sequentially to improve both
reactivity and selectivity (Figure 3A, S11 and S13). The final
K10
and
C6
variants
performed with >10-fold higher reactivity compared to the initial
P4
variant and with
excellent stereocontrol (99.55: 0.45
er
and 99.95: 0.05
er
, respectively).
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To evaluate the substrate range of the evolved P411 variants for cyclopropene construction,
we focused on P411-
C6
and examined structurally diverse aliphatic alkynes. Enzymatic
reactions with 12 alkynes in preparative scale (up to 5.0 mmol scale) afforded the desired
cyclopropenes with TTNs ranging from hundreds to thousands and good to excellent
stereoselectivities (Figure 3B and 3C). Alkynes with a linear carbon chain (
5b
) or cyclic
fragments (
5g
,
5h
and
5j
) all served as good substrates. Different functional groups,
including ether (
5f
,
5i
and
5l
), ester (
5d
), acetal (
5e
), chloride (
5k
), and free hydroxyl (
5m
),
were well-tolerated. Further optimization of reaction conditions with slow addition of EDA,
for example, would likely improve the isolated yields, as we demonstrated for cyclopropene
6h
(66% yield, Figure 3B; and 94% yield, Figure 3C).
Cyclopropenes are used as synthetic building blocks (
4
,
37
), bio-orthogonal imaging
precursors (
38
), and monomers in polymer synthesis (
39
). Our ability to construct these
motifs using bacteria at scale allows us to further explore their potential utility in diverse
fields. Here we present two simple transformations of cyclopropenes to build a multi-
substituted cyclopropane
7a
and a fused ring system, bicyclo[4.1.0]heptene
7b
(Figure 3C),
both of which are substructures common in pharmaceutical candidates and bioactive natural
products (
21
).
In conclusion, we have developed a biocatalytic platform for the construction of highly
strained bicyclobutanes and cyclopropenes through directed evolution of a serine-ligated
cytochrome P450 (P411) enzyme. That the protein could be quickly adapted to produce
these highly strained structures (2–6 mutational steps) highlights the evolvability of the P411
scaffold and its potential to direct the construction of complex motifs. The protein enabled
the desired transformations through activation of iron-carbenoid for carbene addition to
alkynes, stabilization of the reactive cyclopropene intermediate (in bicyclobutane formation),
and precise stereocontrol of the carbene transfer processes. Biotransformations with the
evolved enzymes have a surprisingly broad substrate scope with high reactivity and
selectivity, providing a route to more than 25 products in preparative scale. This biocatalytic
system grants facile access to versatile molecular architectures rarely seen in nature,
expanding the set of chemical structures available to biological systems.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported in part by the Gordon and Betty Moore Foundation through Grant GBMF2809 to the
Caltech Programmable Molecular Technology Initiative, and the National Science Foundation, Office of Chemical,
Bioengineering, Environmental and Transport Systems SusChEM Initiative (Grant CBET-1403077). R.K.Z. was
supported by a National Science Foundation Graduate Research Fellowship (NSF GRFP; Grant DGE-1144469), is
a trainee in the Caltech Biotechnology Leadership Program, and has received financial support from the Donna and
Benjamin M. Rosen Bioengineering Center. Any opinions, findings, and conclusions or recommendations
expressed in this material are those of the author(s) and do not necessarily reflect the views of the funding
organizations. We thank D. K. Romney, S. C. Hammer and S.-Q. Zhang for helpful discussions and comments on
the manuscript. We are grateful to C. K. Pier, O. F. Brandenberg and A. M. Knight for sharing hemeprotein variants
and to K. Ding (Anderson Lab, Caltech) and J. Li (Grubbs Lab, Caltech) for the generous donation of materials and
reagents. We also thank S. Virgil and the Caltech Center for Catalysis and Chemical Synthesis, N. Torian and the
Caltech Mass Spectrometry Laboratory, and M. K. Takase and L. Henling and Caltech X-ray Crystallography
Chen et al.
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Facility for analytical support, and the Brian Stoltz lab for use of their polarimeter and chiral gas chromatography.
A provisional patent application has been filed through the California Institute of Technology based on the results
presented here. Crystallographic coordinates and structure factors have been deposited with the Cambridge
Crystallographic Data Centre (
https://www.ccdc.cam.ac.uk/
) under reference number 1815089 for compound
4d
.
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Fig. 1.
(A) Overall reaction of carbene transfer to an alkyne catalyzed by an engineered
hemeprotein. (B) Proposed catalytic cycle of carbene transfer to phenylacetylene to
form cyclopropene and bicyclobutane structures. (C) Screening of hemin and
hemeprotein catalysts for bicyclobutane formation
(BSA = bovine serum albumin; for
sources, sequences, and mutations in
Bacillus megaterium
P411-
S1
and other proteins, see
Supplementary Materials).
(D) X-ray crystal structure of P411-E10 (PDB ID: 5UCW)
and view of its distal heme region
. The heme axial ligand is S400, and amino acid residues
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V78, L263 and S438 are shown as gray sticks.
(E) Directed evolution of P411-E10 for
bicyclobutane formation
(using phenylacetylene and EDA as substrates; numbers refer to
total turnovers to product (TTN) measured). Experiments were performed on analytical scale
using suspensions of
E. coli
expressing P411-
E10
variants (OD600 = 10–30), 10 mM
phenylacetylene, 10 mM EDA, 5 vol% EtOH, M9-N buffer (pH 7.4) at room temperature
under anaerobic conditions for 6 h. Reactions performed in quadruplicate. Here TTN refers
to the total desired product, as quantified by gas chromatography (GC), divided by total
hemeprotein. (Note: because bicyclobutane formation requires two carbene transfers, the
number of carbene transfers the hemeprotein catalyzes is 2 x TTN in these reactions.)
Further details on reaction conditions and data analysis are provided in the Supplementary
Materials.
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Fig. 2.
(A) Scope of P411-E10 V78F S438A-catalyzed bicyclobutane formation
. Standard
conditions of preparative-scale reactions (0.1 – 0.2 mmol scale, unless otherwise indicated):
suspension of
E. coli
(OD600=15 – 20) expressing P411
E10
-V78F S438A, 1.0 equiv
aromatic alkyne, 2.0 – 4.0 equiv EDA, 10 – 15 mM
D
-glucose, 1 – 5 vol% EtOH, M9-N
buffer (pH 7.4) at room temperature under anaerobic conditions for 12 hours. Isolated yields.
TTN determined based on isolated yields.
(B) Derivatization of bicyclobutane products
. a)
and b) copper-catalyzed click cyclization of
2l
with azide substrates; c) esterification of
2m
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with Mosher’s acid; d) reduction of
2k
to diol with LiBH4. Further details on reaction
conditions and data analysis are provided in the Supplementary Materials.
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Fig. 3.
(A) Evolutionary trajectory of P411-P4 variants for stereodivergent cyclopropenation
of aliphatic alkynes. (B) Scope of P411-C6-catalyzed cyclopropene formation
.
Conditions of preparative-scale reactions (0.08 – 0.4 mmol scale): suspension of
E. coli
expressing P411-
C6
or
K10
(OD600 = 10 – 32), 10 – 150 mM alkyne, 1.0 – 4.0 equiv EDA
(6.0 equiv for
5m
), 10 – 15 mM
D
-glucose, 1 – 5 vol% EtOH, M9-N buffer (pH 7.4) at room
temperature under anaerobic conditions for 12 hours. Isolated yields. TTN determined based
on isolated yields and enantiomeric ratio (
er
) determined by chiral HPLC.
(C) Enzymatic
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cyclopropenation at mmol scale and derivatization of corresponding products
. a)
copper-catalyzed addition to cyclopropene
6a
for synthesizing a multi-substituted
cyclopropane; b) Diels-Alder reaction of cyclopropene
6h
with 1,3-
di
Me-butadiene to form
a fused ring system. Further details on reaction conditions and data analysis are provided in
the Supplementary Materials.
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