P450-catalyzed asymmetric cyclopropanation of electron-deficient olefins under aerobic conditions

Article abstract of DOI:10.1039/c4cy00633j

A variant of P450 from Bacillus megaterium five mutations away from wild type is a highly active catalyst for cyclopropanation of a variety of acrylamide and acrylate olefins with ethyl diazoacetate (EDA). The very high rate of reaction enabled by histidine ligation allowed the reaction to be conducted under aerobic conditions. The promiscuity of this catalyst for a variety of substrates containing amides has enabled synthesis of a small library of precursors to levomilnacipran derivatives. This journal is

Full text of DOI:10.1039/c4cy00633j

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P450-catalyzed asymmetric cyclopropanation
of electron-deficient olefins under
aerobic conditions†
Cite this: Catal. Sci. Technol., 2014,
4, 3640
Received 15th May 2014,
Accepted 22nd June 2014
*
Hans Renata, Z. Jane Wang, Rebekah Z. Kitto and Frances H. Arnold
DOI: 10.1039/c4cy00633j
www.rsc.org/catalysis
A variant of P450 from Bacillus megaterium five mutations away
from wild type is a highly active catalyst for cyclopropanation of a
variety of acrylamide and acrylate olefins with ethyl diazoacetate
(EDA). The very high rate of reaction enabled by histidine ligation
allowed the reaction to be conducted under aerobic conditions.
The promiscuity of this catalyst for a variety of substrates
containing amides has enabled synthesis of a small library of
precursors to levomilnacipran derivatives.
FeII species under physiological conditions (Fig. 1).4 This key
finding afforded us the ability to conduct these reactions
using whole cells expressing the enzyme, an important
advantage of our system because it does not need exogenous
reductant or purified protein.
More recently, we discovered that mutation of the cysteine
at position 400 of P450-BM3 to histidine led to a further
dramatic increase in the rate of cyclopropanation of olefins.5
Through iterative site-saturation mutagenesis, we engineered
a P450-BM3 variant, BM3-Hstar (T268A-C400H-L437W-V78M-
L181V) five mutations away from wild type P450-BM3, which
catalyzed cyclopropanation of acrylamide 1 in greater than
92% yield (86% yield on preparative scale) with 92% enantio-
selectivity and 98 : 2 diastereoselectivity (Scheme 1). Conver-
sion of cyclopropane 2 to alcohol 3 constituted a formal
synthesis of levomilnacipran,6 the psychoactive enantiomer
of milnacipran and a selective serotonin and norepinephrine
reuptake inhibitor recently approved by the US Food and
Drug Administration.7
While there have only been a few reported examples of
cyclopropanation of electron-deficient olefins with transition
metal catalysts, these published reports showed broad gener-
ality on a variety of olefins and tolerance to electron-neutral
substituents of varying sizes.8 In contrast, enzymes often
require strong substrate binding for catalysis and thus
are highly specific for a particular molecule. For instance,
P450-BM3 only undergoes the requisite spin shift for molecu-
lar oxygen activation in the presence of a strongly bound sub-
strate like palmitic acid.9 While this exquisite selectivity can
be advantageous in some cases, it is also a significant syn-
thetic limitation because each evolved enzyme can only be
used for sterically similar substrates. However, as P450-BM3
contains a large hydrophobic binding pocket and strong sub-
strate binding is not required for unnatural cyclopropanation
activity, we hypothesized that our BM3-Hstar variant may
be a more general catalyst for a variety of acrylate and
acrylamide substrates. Furthermore, previous SAR studies of
milnacipran analogs have shown that many molecules within
Introduction
Expanding the range of synthetic transformations catalyzed
by biocatalysts will increase their usage by the synthetic com-
munity and aid in the discovery of new biologically active
molecules.1 Enantioselective cyclopropanation is a highly
sought after transformation as it can be used to construct
multiple stereocenters in one step and synthesize key compo-
nents of biologically relevant targets. While many catalysts
for cyclopropanation using transition metals have been devel-
oped,2 the cost and difficulty of these processes have limited
their use on scale in industry. As an alternative to these
methods, our laboratory has developed a biocatalytic method
for cyclopropanation of styrenes in the presence of diazo
compounds using engineered variants of cytochrome P450
from Bacillus megaterium (P450-BM3).3 The reaction takes
place in water at ambient temperature and the catalyst can
carry out tens of thousands of catalytic turnovers. Addition-
ally, we found that mutation of the proximal cysteine
ligand in P450-BM3 to serine (C400S) led to an increase in
the FeIII–FeII redox potential by 140 mV, thereby allowing
reduction of the FeIII resting state to the catalytically-active
Division of Chemistry and Chemical Engineering, California Institute of
Technology, 1200 E. California Blvd., MC-210-41, Pasadena, CA, USA.
E-mail: frances@cheme.caltech.edu
† Electronic supplementary information (ESI) available: Experimental
procedures for protein-catalyzed reactions and characterization of new com-
pounds. See DOI: 10.1039/c4cy00633j
3640 | Catal. Sci. Technol., 2014, 4, 3640–3643
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probe the steric and electronic restriction the enzyme scaf-
fold places on the cyclopropanation reaction.
When we combined a variety of small- to medium-sized
acrylamides with EDA in the presence of Escherichia coli cells
expressing BM3-Hstar, we observed formation of the desired
cyclopropanes in more than 90% yield with excellent
diastereoselectivity and enantioselectivity (Table 1). Notably,
Weinreb amide 6b, a valuable intermediate for further trans-
formations,11 could be synthesized in excellent yield and
selectivity. Unsymmetrical amides such as 5f could also be
cyclopropanated in good yields. Previous biological studies
with a racemic sample of tetrahydroquinolinyl analog of
milnacipran have shown that it is very active and highly
selective for inhibition of epinephrine and serotonin mono-
amine transporters.12 A precursor to this potent molecule, 6g,
can be prepared in 50% yield and good selectivity with BM3-
Hstar. While the yield of this reaction is lower than what was
observed for smaller amide substrates, our method provides
a facile method for rapidly accessing enantioenriched 6g.
Moving to the aryl group of the acrylamide, we found that
both sterically- and electronically-demanding aryl substitu-
ents are well-tolerated (Table 2). Acrylamides containing
electron-rich aryl groups (7a–c) provided the corresponding
cyclopropane products in good to high yield and great stereo-
selectivity and even substrates containing p-Cl or p-CF3
Fig. 1 Evolution of P450-BM3 for cyclopropanation activity by mutation
of the axial ligand at position 400.
Scheme 1 Biocatalytic cyclopropanation of acrylamide 1 en route to
formal synthesis of levomilnacipran.
this family are active against monoamine transporters.10
Thus, we were interested in employing the BM3-Hstar
biocatalyst for enantioselective synthesis of levomilnacipran
analogs.
Table 1 Scope of acrylamides with variation on the amide moietya b c
,
,
Results and discussion
To examine the generality of cyclopropanation by BM3-Hstar,
a library of acrylamides was synthesized. The substituent
on the amide moiety was varied by conducting Schotten–
Baumann reactions on atropic acid with the appropriate
amines (Scheme 2A). In parallel, a range of phenylacetic acid
derivatives was converted to the corresponding diethyl carb-
oxamide, followed by condensation with paraformaldehyde to
arrive at the appropriate acrylamides (Scheme 2B). Variation
on both the amide and the aryl moieties would allow us to
a
Reactions were carried out with whole cells expressing BM3-Hstar
(2.0 μM), glucose (250 mM, 50 μM), acrylamide (10 μM), EDA (10 μM)
b
in M9-N buffer (500 μL) at room temperature. Yields and dr were
determined by gas chromatography of the crude reaction mixture
calibrated for the appropriate products using 0.4 μmol of
2-phenylethanol as internal standard. Enantioselectivity was deter-
mined by chiral super-critical fluid chromatography (SFC). Relative
and absolute configurations were assigned based on analogy to 2.
Scheme 2 Preparation of acrylamide library from the appropriate
carboxylic acid containing variation at A) the amide moiety or B) the
aryl group.
c
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Table 2 Scope of acrylamides with variation on the aryl ringa b c
,
,
Table 3 Comparison of anaerobic and aerobic reaction with BM3-Hstar
Anaerobic yield Aerobic yield
Aerobic yield/anaerobic
yield
Product (%)
(%)
6a
6b
6c
6d
6e
6f
6g
8a
8b
8c
8d
8e
8f
98
99
98
97
93
75
50
80
76
82
83
77
80
98
98
98
98
95
92
75
45
71
68
72
61
62
54
95
1.0
0.99
1.0
0.98
0.99
1.0
0.90
0.89
0.89
0.88
0.73
0.81
0.68
0.97
10
a
our evolved protein may be tuned for this particular func-
tional group.
Reactions were carried out with whole cells expressing BM3-Hstar
(2.0 μM), glucose (250 mM, 50 μM), acrylamide (10 μM), EDA (10 μM)
in M9-N buffer (500 μL) at room temperature. Yields and dr were
b
In our previous report, we hypothesized that the much
improved rate of cyclopropanation of acrylamide 1 catalyzed
by BM3-Hstar would out-compete inhibition by molecular
oxygen, thereby allowing the cyclopropanation reaction to be
performed under aerobic conditions. To test the generality of
this behavior, cyclopropanation of 5a–g, 7a–f, and 9 was
performed under ambient atmosphere without degassed
buffer or glucose. A comparison of the reaction for each sub-
strate under aerobic versus anaerobic conditions is presented
in Table 3. We observed that reactions with substrates 5a–g
showed minimal loss in yield when conducted under aerobic
conditions. However, the effect of oxygen inhibition became
more appreciable when the substituents on the aryl ring were
varied. In particular, electron-withdrawing substituents and
the naphthyl group gave the most significant drop in yield
when the reaction was run under aerobic conditions. Presum-
ably, the more electron-deficient nature of the olefin and the
increase in steric bulk (in the case of naphthyl substrate 7f)
lead to a slower rate of cyclopropanation and as a result, inhi-
bition by atmospheric oxygen is competitive.
determined by gas chromatography of the crude reaction mixture
calibrated for the appropriate products using 0.4 μmol of
2-phenylethanol as internal standard. Enantioselectivity was deter-
mined by chiral SFC. Relative and absolute configurations were
assigned based on analogy to 2.
c
electron withdrawing substituents (7d and 7e, respectively)
were readily cyclopropanated with BM3-Hstar, albeit with
lower yields. Additionally, increasing the size of the aryl
group to naphthyl did not diminish the yield of the reaction.
BM3-Hstar is surprisingly insensitive to the size and shape
of the acrylamide, considering that substrates like 5a and 5g
differ by seven carbons and even those with rigid substitu-
ents like naphthyl (7f) react readily within the protein. Inter-
estingly, the diastereo- and enantioselectivity of the
cyclopropanation remained consistent for all of the sub-
strates examined, suggesting that the enzyme facilitates the
approach of the olefin to the putative iron carbenoid gener-
ated at the P450-heme prosthetic group in the same orienta-
tion for all of the substrates examined. However, changing
the acrylamide to the analogous acrylate (Scheme 3), despite
having little effect on the yield, led to diminished
diastereoselectivity and enantioselectivity of the reaction.
This result suggests that the amide group is important for
stereocontrol in cyclopropanation with BM3-Hstar and that
Conclusions
In summary, we demonstrated that P450-catalyzed cyclo-
propanation can be used for rapid synthesis of a variety of
enantioenriched precursors to levomilnacipran derivatives
and that the engineered enzyme BM3-Hstar is a relatively
general catalyst for this transformation. Our findings showed
that this catalyst tolerates substrates with substituents on
both the amide and the aryl components. In all the cases
examined, the diastereoselectivity of the reaction is excellent
and the enantioselectivity ranges from moderate to high,
implying that the protein still plays an important role in
orienting substrates that it was not specifically evolved for. In
Scheme 3 Reaction of acrylate 9 with EDA catalyzed by BM3-Hstar.
3642 | Catal. Sci. Technol., 2014, 4, 3640–3643
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addition, we have demonstrated that this biocatalytic cyclo-
propanation of electron-deficient alkenes could be conducted
under aerobic conditions, without loss of yield for many of
the cases examined. Our findings suggest that the activity of
P450-BM3 variants for unnatural modes of reactivity may be
more promiscuous than for natural monooxygenation.
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Acknowledgements
We thank Dr. S. Virgil and the Center for Catalysis and
Chemical Synthesis (3CS) at Caltech for assistance with chiral
SFC. This research was supported by the Gordon and Betty
Moore Foundation through grant GBMF2809 to the Caltech
Programmable Molecular Technology Initiative. Z. J. Wang
was supported by
a Ruth L. Kirschtein NRSA post-
doctoral fellowship from the National Institutes of
Health (5F32EB015846). We thank Mr. C. C. Farwell and
Dr. J. A. McIntosh for helpful discussions. We thank
Dr. K. M. Engle for assistance with variable temperature
NMR. The content of this paper is solely the responsibil-
ity of the authors and does not represent the official
views of any of the funding agencies.
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