Engineering Cytochrome P450s for Enantioselective Cyclopropenation of Internal Alkynes

Article abstract of DOI:10.1021/jacs.0c01313

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out efficient cyclopropene synthesis via carbene transfer to internal alkynes. Directed evolution of a serine-ligated P450 variant, P411-C10, yielded a lineage of engineered

Full text of DOI:10.1021/jacs.0c01313

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Communication
Engineering cytochrome P450s for enantioselective
cyclopropenation of internal alkynes
Kai Chen, and Frances H. Arnold
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01313 • Publication Date (Web): 29 Mar 2020
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Engineering cytochrome P450s for enantioselective cyclopropenation
of internal alkynes
Kai Chen, Frances H. Arnold*
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a Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Supporting Information Placeholder
π-system and the planar heme cofactor, especially if the reaction
ABSTRACT: We report a biocatalytic platform of engineered
involves a concerted carbene-transfer mechanism.12 Recent
cytochrome P450 enzymes to carry out efficient cyclopropene
mechanistic studies have shown step-wise carbene-transfer
processes or even multiple pathways for the same type of reactions
with different engineered hemeproteins.13 We reasoned that proper
engineering of the enzyme active site may direct the desired
carbene transfer to proceed through a step-wise pathway, thereby
circumventing the steric issue.
synthesis via carbene transfer to internal alkynes. Directed
evolution of a serine-ligated P450 variant, P411-C10, yielded a
lineage of engineered P411 enzymes that together accommodate a
variety of internal aromatic alkynes as substrates for
cyclopropenation
with
unprecedented
efficiencies
and
stereoselectivities (up to 5760 TTN, and all with >99.9% ee). Using
an internal aliphatic alkyne bearing a propargylic ether group,
different P411 variants can selectively catalyze cyclopropene
formation, carbene insertion into a propargylic C‒H bond or [3+2]-
cycloaddition. This tunable reaction selectivity further highlights
the benefit of using genetically-encoded catalysts to address
chemoselectivity challenges.
Cyclopropenes, with endo-cyclic double bonds inside a three-
membered carbocycle, possess high strain energy, which enables
activity in different strain-release transformations for constructing
a myriad of useful molecular scaffolds.1 Carbene transfer to
alkynes represents one of the most straightforward approaches to
constructing cyclopropenes.1a,1b Small-molecule transition metal
complexes based on rhodium, iridium, cobalt and others have been
shown to catalyze carbene transfer to terminal alkynes to yield
enantio-enriched cyclopropenes.2‒4 However, enantioselective
carbene transfer to internal alkynes still remains largely
unexplored. Only two systems with chiral gold/silver5 or rhodium6
(co-)catalysts have been reported to take internal aromatic alkynes
for
asymmetric
cyclopropene
synthesis
with
good
stereoselectivities. These systems require precious metal catalysts
in relatively high loading together with complicated ligands and
have not been shown to work with internal aliphatic alkynes. We
wanted to develop an efficient biocatalytic platform that uses earth-
abundant iron to access internal cyclopropenes.
Figure 1. Enzymatic carbene transfer to alkynes for strained
carbocycle formation.
We initiated investigation of internal aromatic alkyne
cyclopropenation using ethyl diazoacetate (EDA) as the carbene
precursor and 1-phenylbutyne (1a) as the model alkyne substrate.
Screening various hemeprotein variants based on P450s, P411s
(P450 with axial ligating residue mutated to serine),14 cytochromes
c and globins in the form of whole Escherichia coli (E. coli) cell
catalysts identified a P411 variant, P411-C10, that formed the
desired internal cyclopropene (Figure 1). P411-C10 belongs to the
family of P411CHF (five amino acid substitutions away), which was
evolved for a carbene C‒H insertion reaction.15 Surprisingly, the
cyclopropene product synthesized by P411-C10 was determined to
be a single enantiomer, which suggests the enzyme scaffold binds
the alkyne and directs carbene transfer in a well-defined orientation.
Cytochromes P450 use an iron-heme complex as their catalytic
cofactor in their native oxygenase functions.7 Recently, directed
evolution has significantly expanded the catalytic repertoire of
P450 enzymes and other hemeproteins to include non-natural
carbene- and nitrene-transfer reactions, as described by our group
and others.8‒10 We recently reported an enzymatic platform of
engineered cytochrome P450 enzymes for stereoselective carbene
addition to terminal alkynes to forge cyclopropenes and
bicyclo[1.1.0]butanes (Figure 1).11 We hypothesized that P450
enzymes may achieve even more challenging transformations, such
as carbene transfer to internal alkynes for cyclopropene
construction. The major difficulty for internal alkyne
cyclopropenation lies in the severe steric clash between the linear
For the model reaction with 1a as the alkyne donor, C10 in the
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form of the whole-cell catalyst exhibited modest activity, with 55
substrates tested (1b to 1l), only cyclopropenes 2c, 2d, 2i and 2j
were synthesized efficiently, and most of the other alkynes with
substitutions on the aromatic ring showed poor to moderate
reactivities. Thinking that the evolved WIRF variant may have
acquired some specificity for the non-substituted aromatic ring or
for electron-rich alkynes, we decided to use a less reactive alkyne
substrate (compared to 1a), 1b, with an electron-deficient para-
chloro substitution, to further evolve the enzyme (Figure 2). A site-
saturation library targeting residue 332 afforded mutation S332G,
which boosted the total turnover by almost 5 fold. We reasoned that
the glycine substitution might help make space in the active site to
accommodate substrates with substitutions on the aromatic ring.
Mutagenesis of residues close to 332 was investigated, and two
additional beneficial mutations, G74A and E70K, yielded the final
WIRF_GAK variant with 2320 TTN for substrate 1b.
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total turnovers (TTN). Directed evolution targeting active-site
residues for site-saturation mutagenesis was performed to enhance
the overall catalytic efficiency (Figure 2). Residue 263, located
right above the heme cofactor (in the heme domain), was
previously found to play an important role in controlling carbene
transfer to phenylacetylene using other P411 variants.11 To our
delight, screening the enzyme library made by site-saturation
mutagenesis at residue 263 yielded a tryptophan mutation at this
site that improved TTN over 11 fold. Sequential mutagenesis
targeting sites in the loop regions led to beneficial mutations Q437I,
S72F and L436R and afforded the highly efficient variant WIRF,
with 2680 TTN towards the desired cyclopropene formation.
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P411 C10-WIRF’s scope of internal alkynes bearing different
aromatic rings or carbon chains was then evaluated. For the alkyne
Figure 2. Directed evolution of P411-C10 for internal cyclopropene synthesis. Reaction conditions: 10 mM alkyne, 10 mM EDA, E. coli
harboring P411-C10 variants (OD600 = 15 to 60), D-glucose (25 mM), M9-N buffer/EtOH (19:1), anaerobic, 6 h. Product formation was
quantified by gas chromatography (GC) and TTNs were determined based on P411 protein concentration. The heme-domain structure of
P411-E10 variant (pdb: 5UCW) was used to guide site-saturation mutagenesis; mutation sites are highlighted. See SI for details.
We revisited the substrate scope of this biocatalytic platform
using the whole lineage of cyclopropene-forming enzyme variants
(from C10 to WIRF and then to WIRF_GAK) (Figure 3). The
WIRF variant turned out to be efficient for non-substituted or
ortho-substituted aromatic alkynes (1a, 1c and 1d), catalyzing the
desired cyclopropene synthesis with 1200 to 2670 total turnovers,
while variants from later in the evolution showed impaired activity
with these substrates. Although we did not specifically evolve the
enzyme for activity on meta-substituted aromatic alkynes, variant
WIRF_G exhibited improved efficiency for a meta-methoxyl
alkyne substrate (1f), compared to WIRF. For aromatic alkynes
bearing para-substitutions or di-substitutions (1b and 1g to 1l), the
final variant WIRF_GAK catalyzes the desired transformations
with unprecedentedly high efficiency compared to all previously
reported systems for cyclopropene formation. For instance, an
electronically-withdrawing trifluoromethyl-substituted alkyne (1g)
was well-accepted by the enzymatic system. It is worth noting that
all of the internal cyclopropenes produced enzymatically were
determined to be single enantiomers (>99.9% ee for all), which
further supports our hypothesis that the engineered enzymes
impose a specific binding orientation of the alkyne substrate in the
protein active site, allowing for efficient carbene addition to triple
bonds with perfect stereocontrol.
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Numerous transformations have been developed to furnish
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diverse molecular structures from versatile cyclopropane building
blocks.1,2b,2c,6,7 Here, we also derivatized the enzymatically-
synthesized cyclopropenes by hydrogenation and ester reduction to
afford an all-cis cyclopropane product (Figure 4), which is
otherwise difficult to prepare due to the cis-stereochemistry of the
three substituents on the cyclopropane ring.
Compared to internal aromatic alkynes described above, internal
aliphatic alkynes are typically more challenging targets for
enantioselective cyclopropene formation in terms of reactivity and
selectivity. As the aryl groups on aromatic alkynes can provide a
stabilizing effect through the conjugated system in the carbene
transfer process, purely aliphatic alkynes without additional
intramolecular effects may suffer from a higher energy barrier for
carbene transfer. Additionally, alkyl groups at the two ends of the
triple bond are less easy to distinguish than the alkyl and aryl
groups on aromatic alkynes. Until now, no systems have been
reported for enantioselective cyclopropene synthesis with internal
aliphatic alkynes. However, we believed that enzymes can
accomplish this, as the enzyme active site is a chiral environment
that can recognize minor steric differences for chiral induction.16
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Testing the evolved enzymes for a cyclopropenation reaction
with internal aliphatic alkyne 1m was not fruitful, as only trace
activity was observed. However, with the parent enzyme P411-C10
we observed the desired cyclopropene product 2m (Figure 5) with
modest activity (43 TTN). This might be because the whole enzyme
lineage was evolved for a set of structurally different aromatic
alkynes. Further screening of variants in the C10 family identified
a triple mutant of C10, C10_VLC, which catalyzed the formation
of internal cyclopropene 2m with improved activity (64 TTN) and
perfect stereocontrol (>99% ee). We anticipate that further
evolution will lead to more efficient enzymes for internal aliphatic
cyclopropene construction, as we have demonstrated for aromatic
alkynes.
Figure 3. Substrate scope of internal aromatic alkynes for
cyclopropene formation. Reactions were performed in
quadruplicate under the following conditions: 10 mM alkyne, 10
mM EDA, E. coli harboring P411-C10 variants (OD600 = 10 to 20),
D-glucose (25 mM), M9-N buffer/EtOH (19:1), anaerobic, 16 h.
Product formation was quantified by gas chromatography (GC) and
TTNs were determined based on protein concentration. See SI for
details.
To further demonstrate the utility of this highly stereoselective
enzymatic platform, we carried out large-scale preparation of
internal cyclopropenes (Figure 4). With a simple modification of
the reaction conditions using the diazo reagent in excess (2.4
equivalents added in three portions), we obtained high isolated
yields of the desired cyclopropene products at mmol scale (90% for
2d with variant WIRF, and 87% for 2g with variant WIRF_GAK).
Interestingly, the enzyme turnovers of the large-scale reactions are
typically higher than those obtained with analytical-scale ones,
indicating that the evolved enzymes in whole cells might still retain
(partial) activity after the reactions and the turnovers were limited
by consumption of the diazo substrate.
Figure 5. Cyclopropenation of internal aliphatic alkynes and
chemoselectivity study with substrate 1n. Reactions were
performed in quadruplicate under the following conditions: 10 mM
alkyne, 10 mM EDA, E. coli harboring P411 variants (OD600 = 15
to 20), D-glucose (25 mM), M9-N buffer/EtOH (19:1), anaerobic,
16 h. C10_VLC: C10 T327V Q437L S332C; C11: C10 G74T
S118M L162F L401I Q437L; L8: C10 A87P A264S E267D T327P
S332A Q437L; C10_PVV: C10 Q437P T327V A87V. See SI for
details.
Figure 4. Preparative-scale synthesis of internal cyclopropenes
and further derivatization. See SI for details.
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As the parent P411-C10 enzyme was initially engineered for a
previous demonstration of enzyme-controlled reaction selectivity
between C‒H insertion and cyclopropanation,18 highlight how
enzyme catalysis can solve chemoselectivity problems in synthetic
methodology.
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carbene C‒H insertion reaction, we took a deeper look at the
chemoselectivity between cyclopropenation and C‒H insertion.15
Internal alkyne substrate 1n, bearing a propargylic ether group, was
found to mainly undergo a carbene insertion reaction into the
propargylic C‒H bond with high enantioselectivity with catalyst
P411CHF; a cyclopropene product was also detected as a minor
product (see SI for details). However, P411-C10 reversed the
chemoselectivity to favor the cyclopropene 2n as the major
product; and a third product observed in low proportion in this latter
reaction was confirmed to be a furan derivative, 2n-2, which may
be generated through a [3+2]-cycloaddition.11,17 After intensive
screening of variants in the families of P411CHF and P411-C10, we
discovered two related variants, P411-C11 and P411-L8, which
could catalyze the C‒H insertion reaction and the cyclopropenation
reaction with even higher activity and selectivity (compared to
P411CHF and P411-C10, respectively), as shown in Figure 5. And
In conclusion, we have developed a versatile biocatalytic
platform based on engineered cytochrome P411 enzymes that
offers access to an array of structurally diverse internal
cyclopropenes through carbene transfer to internal alkynes. This
biocatalytic system was evolved rapidly to take internal aromatic
alkynes as substrates and furnish the desired cyclopropenes with
unprecedentedly high stereoselectivities (>99.9% ee for all). This
enzymatic platform is also readily scalable for the production of
cyclopropenes in preparative quantities, with even higher
efficiencies compared to the analytical-scale reactions.
Enantioselective cyclopropenation of internal aliphatic alkynes was
also shown to be possible. The versatility and tunability of these
biocatalysts has been demonstrated, with chemoselectivity that can
be switched among cyclopropenation, carbene C‒H insertion and
[3+2] cycloaddition. Ongoing studies with this family of P411-C10
variants will help to define the catalytic potential of C10 as a highly
promiscuous carbene transferase for non-native transformations.
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a
C10 triple mutant, C10_PVV, was found to flip the
chemoselectivity to favor formation of the furan product. These
variants are closely related, differing by only a few amino acid
substitutions, but gave very different chemoselectivities without
any specific enzyme evolution. These results, together with our
(2) a) Protopopova, M. N.; Doyle, M. P.; Müller, P.; Ene, D. High
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ASSOCIATED CONTENT
Supporting Information
Experimental details, and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
frances@cheme.caltech.edu (F.H.A.)
ORCID
Kai Chen: 0000-0002-3325-3536
Frances H. Arnold: 0000-0002-4027-364X
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by NSF Division of Molecular and
Cellular Biosciences grant MCB-1513007, US Army Research
Office Institute for Collaborative Biotechnologies cooperative
agreement W911NF-19-2-0026, and US Army Research Office
Institute for Collaborative Biotechnologies contract W911NF-19-
D-0001. K.C. thanks the Resnick Sustainability Institute at Caltech
for fellowship support. We thank R. K. Zhang, N. P. Dunham, D.
J. Wackelin, Y. Yang and M. Garcia-Borràs for helpful discussions
and comments.
(5) Briones, J. F.; Davies, H. M. L. Gold(I)-catalyzed asymmetric
cyclopropenation of internal alkynes. J. Am. Chem. Soc. 2012, 134, 11916–
11919.
(6) a)
A
recent example of Rh-catalyzed internal alkyne
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Zheng, M.-M.; Xue, X.-S.; Marek, I.; Zhang, F.-G.; Ma, J.-A. Catalytic
enantioselective cyclopropenation of internal alkynes: Access to
difluoromethylated three-membered carbocycles. Angew. Chem., Int. Ed.
2019, 58, 18191–18196. b) See ref 2a for preliminary results on the
rhodium-catalyzed asymmetric cyclopropenation of internal alkynes in poor
enantioselectivity.
(7) a) Ortiz de Montellano, P. R. ed., Cytochrome P450: structure,
mechanism, and biochemistry (Springer International Publishing: Cham,
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by P450: a) Brandenberg, O. F.; Fasan, R.; Arnold, F. H. Exploiting and
engineering hemoproteins for abiological carbene and nitrene transfer
reactions. Curr. Opin. Biotechnol. 2017, 47, 102‒111. b) Prier, C. K.;
Arnold, F. H. Chemomimetic biocatalysis: Exploiting the synthetic
potential of cofactor-dependent enzymes to create new catalysts. J. Am.
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Brown, T. R.; Arnold, F. H. Enantioselective intramolecular C–H amination
catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo.
Angew. Chem., Int. Ed. 2013, 52, 9309–9312. b) Hyster, T. K.; Farwell, C.
C.; Buller, A. R.; McIntosh, J. A.; Arnold, F. H. Enzyme-controlled
nitrogen-atom transfer enables regiodivergent C–H amination. J. Am. Chem.
Soc. 2014, 136, 15505–15508. c) Singh, R.; Kolev, J. N.; Sutera, P. A.;
Fasan, R. Enzymatic C(sp3)–H amination: P450-catalyzed conversion of
carbonazidates into oxazolidinones. ACS Catal. 2015, 5, 1685–1691. d)
(12) A similar rationalization in iron-porphyrin-catalyzed internal alkene
cyclopropanation: Wolf, J. R.; Hamaker, C. G.; Djukic, J.-P.; Kodadek, T.;
Woo, L. K. Shape and stereoselective cyclopropanation of alkenes
catalyzed by iron porphyrins. J. Am. Chem. Soc. 1995, 117, 36, 9194–9199.
(13) Mechanistic studies on hemeprotein catalyzed carbene-transfer
reactions: a) Zhang, Y. Computational investigations of heme carbenes and
heme carbene transfer reactions. Chem. Eur. J. 2019, 25, 13231‒13247. b)
Sharon, D. A.; Mallick, D.; Wang, B.; Shaik, S. Computation sheds insight
into iron porphyrin carbenes’ electronic structure, formation, and N–H
insertion reactivity. J. Am. Chem. Soc. 2016, 138, 9597‒9610. c) Wei, Y.;
Tinoco, A.; Steck, V.; Fasan, R.; Zhang, Y. Cyclopropanations via heme
carbenes: Basic mechanism and effects of carbene substituent, protein axial
ligand, and porphyrin substitution. J. Am. Chem. Soc. 2018, 140,
1649‒1662. d) Carminati, D; Fasan, R. Stereoselective cyclopropanation of
electron-deficient olefins with a cofactor redesigned carbene transferase
featuring radical reactivity. ACS Catal. 2019, 9, 9683‒9697. e) ref 10d.
(14) Coelho, P. S.; Wang, Z. J.; Ener, M. E.; Baril, S. A.; Kannan, A.;
Arnold, F. H.; Brustad, E. M. A serine-substituted P450 catalyzes highly
efficient carbene transfer to olefins in vivo. Nat. Chem. Bio. 2013, 9, 485–
487.
(15) Zhang, R. K.; Chen, K.; Huang, X.; Wohlschlager, L.; Renata, H.;
Arnold, F. H. Enzymatic assembly of carbon–carbon bonds via iron-
catalysed sp3 C–H functionalization. Nature 2019, 565, 67–72.
(16) See ref 11e for an example of an engineered P411 enzyme
constructing a methyl-ethyl stereocenter.
(17) Cui, X.; Xu, X.; Wojtas, L.; Kim, M. M.; Zhang, X. P.
Regioselective synthesis of multisubstituted furans via metalloradical
cyclization of alkynes with α-diazocarbonyls: Construction of
functionalized α-oligofurans. J. Am. Chem. Soc. 2012, 134, 19981–19984.
(18) See refs 10d, 10g and 13 for examples of tunable chemo-
selectivities with engineered hemeproteins.
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graphic abstract
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