Gram-Scale Synthesis of Chiral Cyclopropane-Containing Drugs and Drug Precursors with Engineered Myoglobin Catalysts Featuring Complementary Stereoselectivity

Article abstract of DOI:10.1002/anie.201608680

Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations, but variants featuring predictable, complementary stereoselectivity in these reactions have remained elusive. In this study, a rationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity. The stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations, these stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine, Tasimelteon, Ticagrelor, and a TRPV1 inhibitor) in high yield and with excellent diastereo- and enantioselectivity (98–99.9% de; 96–99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs.

Full text of DOI:10.1002/anie.201608680

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International Edition: DOI: 10.1002/anie.201608680
German Edition: DOI: 10.1002/ange.201608680
Biocatalysis
Hot Paper
Gram-Scale Synthesis of Chiral Cyclopropane-Containing Drugs and
Drug Precursors with Engineered Myoglobin Catalysts Featuring
Complementary Stereoselectivity
Priyanka Bajaj+, Gopeekrishnan Sreenilayam+, Vikas Tyagi, and Rudi Fasan*
Abstract: Engineered hemoproteins have recently emerged as
promising systems for promoting asymmetric cyclopropana-
tions, but variants featuring predictable, complementary ste-
reoselectivity in these reactions have remained elusive. In this
study, a rationally driven strategy was implemented and
applied to engineer myoglobin variants capable of providing
access to 1-carboxy-2-aryl-cyclopropanes with high trans-
(1R,2R) selectivity and catalytic activity. The stereoselectivity
of these cyclopropanation biocatalysts complements that of
trans-(1S,2S)-selective variants developed here and previously.
In combination with whole-cell biotransformations, these
stereocomplementary biocatalysts enabled the multigram syn-
thesis of the chiral cyclopropane core of four drugs (Tranylcy-
promine, Tasimelteon, Ticagrelor, and a TRPV1 inhibitor) in
high yield and with excellent diastereo- and enantioselectivity
(98–99.9% de; 96–99.9% ee). These biocatalytic strategies
outperform currently available methods to produce these
drugs.
(cis), 95% ee (1R,2S)).[5] Unfortunately, varying cis/trans
ratios and degrees of stereoselectivity were observed with
these enzymes in the presence of other styrene deriva-
tives.[3a,5] We previously reported the development of an
engineered myoglobin variant, Mb(H64V,V68A), that is
capable of catalyzing the cyclopropanation of styrene with
EDA with excellent trans diastereoselectivity and (1S,2S)
enantioselectivity (99.9% de, 99.9% ee).[4] Promisingly, the
high trans-(1S,2S) selectivity of this Mb variant extended to
the cyclopropanation of a variety of styrene derivatives (97–
99.9% de, 96–99.9% ee).[4]
The ability to access both enantiomeric forms of a target
cyclopropane pharmacophore is critical in the context of the
synthesis of bioactive molecules, as such stereoisomers often
exhibit remarkably divergent pharmacological and/or toxicity
profiles.[1] However, developing stereo- or enantiocomple-
mentary variants of an enzyme is far from being a trivial
task,[6] as mirror-image forms of these biomolecules are not
readily available.[7] Reflecting this notion, cyclopropanation
biocatalysts that can reliably offer complementary stereose-
lectivity have thus far remained unavailable.[3,5,8] Herein, we
report the development and characterization of a panel of
engineered Mb catalysts that provide access to trans-(1R,2R)-
configured 1-carboxy-2-aryl-cyclopropanes with high selec-
tivity and catalytic activity across a broad range of olefin
substrates. We further demonstrate that these Mb-catalyzed
reactions can be carried out and scaled up using whole-cell
systems. Using these stereocomplementary biocatalysts in
combination with whole-cell transformations, the asymmetric
synthesis of the cyclopropane core of four different drugs,
featuring both trans-(1S,2S) and trans-(1R,2R) configurations,
was accomplished at the multigram scale.
Previously, we found that mutations at the five amino acid
positions defining the distal pocket in Mb (i.e., Leu29, Phe43,
His64, Val68, and Ile107; see the Supporting Information,
Figure S1) significantly affected the activity and selectivity of
this hemoprotein in carbene[4,9] and nitrene[10] transfer
reactions. In particular, mutation of the distal histidine
residue (H64V) was shown to have a general activity-
enhancing effect in these reactions, possibly owing to an
increased accessibility of the heme pocket to the reactants.
This mutation also slightly increases the trans-(1S,2S) selec-
tivity of Mb for styrene cyclopropanation with EDA (93% de
(trans), 10% ee (1S,2S) compared to 86% de (trans), 6% ee
(1S,2S) for wild-type Mb). This effect could be combined with
that of a stronger trans-(1S,2S) selectivity inducing mutation,
i.e., V68A (96% de (trans), 68% ee (1S,2S)), to yield the
Catalytic methods for the cyclopropanation of olefins cover
a prominent role in organic and medicinal chemistry owing to
the recurrence of cyclopropane motifs among biologically
active natural products and pharmaceuticals.[1] Significant
progress has been made in the development of synthetic
methods for asymmetric cyclopropanation, in particular
through the transition-metal-catalyzed insertion of carbenoid
species into carbon–carbon double bonds.[2] More recently,
the Arnold group and our own laboratory have shown that
engineered cytochrome P450s[3] and myoglobins (Mb),[4]
respectively, constitute promising catalysts for mediating the
cyclopropanation of styrenes in the presence of a-diazoace-
tate reagents, thus providing a biocatalytic alternative for this
valuable transformation. Variants of the bacterial cytochrome
P450BM3 were found to favor cis selectivity in the cyclo-
propanation of styrene in the presence of ethyl a-diazoacetate
(EDA; P450BM3-CIS-T438S: 86% de (cis), 97% ee (1S,2R)).[3a]
By utilizing a different P450 enzyme, opposite enantioselec-
tivity was achieved by Brustad and co-workers for the
cis cyclopropanation of this substrate, albeit with more
moderate diastereoselectivity (P450Biol-T238A: 42% de
[*] Dr. P. Bajaj,[+] Dr. G. Sreenilayam,[+] Dr. V. Tyagi, Prof. Dr. R. Fasan
Department of Chemistry, University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: rfasan@ur.rochester.edu
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201608680.
aforementioned
highly
trans-(1S,2S)-selective
Mb(H64V,V68A) catalyst (Figure 1, gray path).[4] These
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Table 1: Activity and selectivity of representative myoglobin variants for
styrene cyclopropanation with EDA.[a]
Catalyst
Conv. TON detrans
eetrans eecis
[%][b]
[%]
[%][c]
[%][c]
Mb
Mb(H64V)
36
47
60
93
57
53
82
61
58
180
235
300
460
285
265
410
305
290
265
86
93
93
95
>99.9
>99.9
ꢁ6
ꢁ10
70
59
72
0
8
18
39
–
–
–
–
–
Mb(L29S,H64V)
Mb(L29T,H64V)
Mb(H64V,V68F)
Mb(H64V,V68L)
Mb(H64V,V68S)
Mb(L29T,H64V,V68L)
Mb(L29T,H64V,V68F)
70
>99.9 ꢁ99
>99.9
>99.9
99
92
92
95
Figure 1. Structure–reactivity-guided design of Mb cyclopropanation
biocatalysts with complementary stereoselectivity. The path in gray is
described in Ref. [4].
Mb(L29T,F43W,H64V,V68F) 53
–
[a] Reaction conditions: 20 mm Mb variant, 10 mm styrene, 20 mm EDA,
10 mm dithionite, 16 h. See also Tables S2 and S3. [b] Based on GC
analysis, relative to the olefin. [c] trans=(1R,2R), cis=(1S,2R). Values
determined by gas chromatography or supercritical fluid chromatogra-
phy on a chiral stationary phase.
results supported our hypothesis that additive effects can be
leveraged to fine-tune the stereoselectivity of Mb in cyclo-
propanation reactions. At the same time, none of the active-
site Mb variants (> 10) examined in these earlier studies
showed any preference for formation of the trans-(1R,2R)
product, indicating that achieving this type of stereoselectivity
would require exploration of a wider active-site sequence
space. On the basis of these considerations, we sought to
implement a systematic active-site mutagenesis approach
combined with structure–reactivity-guided design to achieve
our goal of developing trans-(1R,2R)-selective Mb catalysts.
Accordingly, starting from Mb(H64V) as the parent
protein, each of the remaining four active-site positions was
systematically mutated to any of the other 19 amino acids by
site-directed mutagenesis. This process resulted in a library of
76 Mb(H64V)-derived variants, which were tested individu-
ally for their activity (turnover number, TON) and selectivity
in the model cyclopropanation reaction with styrene and
EDA (Table S2). Nearly half of these Mb variants (33/76;
43%) could be expressed in E. coli in correctly folded form as
determined by the signature Soret band for their CO-bound
complex (lmax ꢀ 420 nm). Moreover, the large majority of
these proteins (75%) was catalytically competent (TON > 50)
towards styrene cyclopropanation (Table S2), highlighting the
robustness of the Mb active site to mutagenesis. More
importantly, screening of the library led to the identification
of a number of Mb variants with significantly improved
trans-(1R,2R) selectivity (Table 1) compared to the parent
protein Mb(H64V) or wild-type Mb. Most effective in
favoring such selectivity was the introduction of a leucine or
phenylalanine residue in place of Val68 (i.e., 86!99% de,
(ꢁ6)!70–72% ee (1R,2R)). Residue 68 lies along the side of
the heme group and is expected to be in close proximity to the
putative heme-bound carbene intermediate implicated in the
cyclopropanation reaction.[4] Interestingly, substitution of the
more remote Leu29 with either serine or threonine was also
beneficial towards enhancing trans-(1R,2R) selectivity, as
indicated by the 93–95% de and 59–70% ee values obtained
with Mb(L29S,H64V) and Mb(L29T,H64V) (Table 1). Muta-
tions of positions Phe43 or Ile107 had noticeable but
comparatively weaker effects on favoring trans-(1R,2R)
selectivity (16–55% ee; Table S2). Moreover, a number of
highly trans-(1S,2S)-selective variants were identified among
the
Val68
site-saturation
sub-library,
including
Mb(H64V,V68S) and Mb(H64V,V68C), which produce 3b
in > 99% de and ee (Tables 1 and S2).
To obtain Mb catalysts with further improved trans-
(1R,2R) selectivity, the beneficial mutations at position 68
(V68L, V68F) were combined with those at position 29
(L29T, L29S) and 107 (I107H) to yield a set of triple-site
variants (Table S3). Gratifyingly, this process resulted in the
identification
of
two
closely
related
variants,
Mb(L29T,H64V,V68L) and Mb(L29T,H64V,V68F), with
excellent trans diastereoselectivity (99% de) along with
high 1R,2R stereoselectivity (92% ee; Table 1). Next, these
Mb variants were further mutated at position Ile107 or Phe43,
with the choice of the target substitutions being guided by the
structure–reactivity relationship (SRR) data collected during
screening of the initial library. Although none of the resulting
quadruple-site variants with substitutions at position 107
showed improved trans-(1R,2R) selectivity, the introduction
of the F43W mutation in the Mb(L29T,H64V,V68F) back-
ground led to the desired additivity effect. Indeed, the
corresponding Mb(L29T,F43W,H64V,V68F) variant catalyzes
the transformation of styrene and EDA into 3a with excellent
diastereo- and enantioselectivity (> 99% de, 95% ee;
Table 1). It should be noted that refinement of the trans-
(1R,2R) selectivity came with no loss in catalytic efficiency as
indicated by the comparable TONs achieved with the trans-
(1R,2R)-selective Mb variants and the parent protein (265–
460 vs. 235 TON). Figure 1 provides a graphical representa-
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tion of the SRR-driven strategy implemented here for the
(product 7b). Importantly, for all of these substrates, the
development of stereocomplementary Mb cyclopropanation
catalysts. As anticipated, achieving high trans-(1R,2R) selec-
tivity involved a more extensive remodeling of the Mb active
site (4 mutations) than required for obtaining high trans-
(1S,2S) selectivity (2 mutations).
The substrate scope of the trans-(1R,2R)-selective Mb
catalysts (called RR1–RR5, Table S3) was then probed
against substituted styrene derivatives. As shown in Table 2,
quantitative product conversion (95–99%) was achieved with
different para-, meta-, ortho-, and alpha-functionalized styr-
enes (4b–6b, 8b–10b), with more moderate conversion
(45%) being observed only with 4-trifluoromethylstyrene
desired trans-(1R,2R)-configured cyclopropane products
were obtained with good to excellent diastereoselectivity
(92–99% de) and enantioselectivity (49–99% ee) with one or
more of the Mb variants. Next, we extended these analyses to
other aryl-substituted olefins, including pyridine, thiophene,
N-methylimidazole, and benzothiazole derivatives. These
molecules comprise N- and S-containing heterocycles, which
are notoriously problematic in the context of cyclopropana-
tion reactions with rhodium- or other metal-based complexes
owing to catalyst poisoning effects. For example, the reaction
of 1-methyl-2-vinylimidazole with EDA and Rh2(OAc)4 as
the catalyst failed to give any product (see the Supporting
Information for details). Notably, all of these olefins could be
efficiently processed by the engineered Mb catalysts, resulting
in product conversions of up to 84% (11b–15b, Table 2).
Furthermore, the corresponding trans-(1R,2R) cyclopropane
products could be obtained with good to very good selectiv-
ities (65–99% de, 65–98% ee). For all of these transforma-
tions, complementary stereoselectivity is provided by the
trans-(1S,2S)-selective Mb(H64V,V68A) variant (Table S4).
Whole-cell biotransformations constitute a convenient
approach to enhance the scalability of biocatalytic processes
supported by heme-dependent enzymes.[3b,11] Importantly, we
established that the Mb(H64V,V68A)-catalyzed cyclopropa-
nation of styrene with EDA to give 3b can be readily achieved
using E. coli cells (BL21DE3) expressing this Mb variant,
resulting in high product conversion (68–100%) even under
aerobic conditions (Table S5). Furthermore, the substrate
loading in these whole-cell reactions could be increased up to
0.5m styrene and 0.5m EDA in a 1:1 ratio while maintaining
high conversion (62%) and excellent selectivity (99.9% de,
99.9% ee; Table S5).
Aryl-substituted trans cyclopropanes are found in several
marketed and investigational drugs (Scheme 1),[1] including
the MAO inhibitor Tranylcypromine ((ꢂ)-16, formerly Par-
nate),[12] the melatonin receptor agonist Tasimelteon (20),[13]
the TRPV1 antagonist 24,[14] and the platelet aggregation
inhibitor Ticagrelor (28, Brilinta).[15] Although Tranylcypro-
mine has been used in clinical settings in racemic form, the
two enantiomers display different biological potencies.[16] The
other drugs are used in enantiomerically pure form. Each of
these molecules features a cyclopropane unit that can be
retrosynthetically derived from either a trans-(1R,2R)- ((ꢁ)-
16, 19, 28) or trans-(1S,2S)-configured 1-carboxy-2-aryl-cyclo-
propane ((+)-16, 24). As such, these drugs were chosen as
relevant targets to assess the synthetic utility of the stereo-
complementary cyclopropanation biocatalysts developed
herein.
Table 2: Substrate scope for trans-(1R,2R)-selective Mb variants.[a]
Product
Cat.[b]
Conv.
[%]
TON
de
[%]
ee
[%]
R1 =4-CH3 (4b)
R1 =4-MeO (5b)
R1 =4-Cl (6b)
RR5
RR5
RR2
RR4
RR4
RR3
>99[c]
95[c]
165
96
97
97
97
92
63
74
49
64
84
160
>500
75
>500
>500
>99
R1 =4-CF3 (7b)
R1 =3-Me (8b)
R1 =2-Me (9b)
45[c]
>99
>99
>99
>99
RR3
RR4
RR2
RR2
RR1
>99[c]
45[c]
84
165
75
78
80
96
88
99
78
88
65
67
98
420
390
95
78
56[c]
Accordingly, to obtain the dextrorotatory form of Tranyl-
cypromine, a large-scale reaction with E. coli cells expressing
Mb(H64,V68A) was carried out in the presence of 2.9 g
styrene and 3.1 g EDA (1 equiv), resulting in the isolation of
3b as the only product (99.9% de, 99.9% ee) in 91% yield
(4.7 g; Scheme 1a). Under similar reaction conditions, but
using cells expressing Mb(L29T,H64V,V68L), 1.2 g of the
opposite enantiomer 3a were obtained in 80% yield and
excellent selectivity (99.9% de, 95% ee). Both intermediates
can be converted into the desired (+)- and (ꢁ)-Tranylcypro-
RR4
54[c]
90
65
71
[a] Reaction conditions: 20 mm Mb variant (0.2 mol%), 10 mm styrene,
20 mm EDA, 10 mm dithionite, 16 h. [b] For the nomenclature of the
RR1–RR5 variants, see Figure 1 and Table S3. [c] With 0.6 mol% Mb
catalyst.
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desired 1R,2R-configured cyclopropanation product 19 was
obtained in 99.9% de and 96% ee and in 91% isolated yield
(0.96 g), thus furnishing a key intermediate en route to
Tasimelteon (20).[17]
The stereoselective cyclopropanation of 22 into 23 was
denoted by Pfizer chemists as “the most challenging step” in
the preparation of the advanced drug candidate 24,[14] owing
to the presence of a pyridine functional group and formation
of a trisubstituted cyclopropane.[2k] Upon screening over 120
different catalytic conditions, the best results were obtained
using Ru(iPr-PyBox) as the catalyst to give 23 in 40% de and
65% ee.[14] Using whole cells expressing Mb(H64V,V68A),
0.83 g of 23 could be obtained in high yield (75%) with much
greater diastereo- and enantioselectivity (99.9% de, 99.9%
ee; Scheme 1c). The cyclopropanation product was further
processed to afford 24 (see the Supporting Information for
details).
Lastly, we targeted the synthesis of (1R,2R)-ethyl 2-(3,4-
difluorophenyl)cyclopropanecarboxylate 26 (Scheme 1d),
which can be readily hydrolyzed to 27, a key synthetic
intermediate for the preparation of Ticagrelor.[15] Of note, this
intermediate is currently accessible through synthetic routes
involving no less than four to five steps.[15,18] Conveniently, the
key cyclopropane building block 26 could be obtained in
a single step in high yield (94%) and selectivity (98% de,
58% ee) by whole-cell cyclopropanation of commercially
available 3,4-difluorostyrene with cells expressing the
(1R,2R)-selective Mb(L29T,H64V,V68L) variant. Taken
together, these results demonstrate the promise and scalabil-
ity of stereoselective myoglobin-catalyzed cyclopropanations
in the context of relevant and challenging building blocks for
drug synthesis.
In summary, we have reported the successful design and
application of myoglobin-based cyclopropanation biocata-
lysts that are capable of offering high trans selectivity along
with complementary stereoselectivity across a broad panel of
aryl-substituted olefins. Furthermore, we have demonstrated
that these myoglobin-mediated transformations can be per-
formed in the context of whole-cell systems, which further
simplifies their use for synthetic applications. The biocatalytic
systems developed here have enabled the stereoselective
synthesis of multiple cyclopropane-containing drugs at the
preparative scale, offering superior performance over cur-
rently available methods for asymmetric cyclopropanation
(i.e., (+)- and (ꢁ)-16, 20, and 24) or granting a more concise
route to their preparation (28). Along with the growing
number of abiotic reactions accessible using engineered
myoglobins,[4,9,10,19] these results support the promise of
these metalloproteins for the asymmetric synthesis of chiral
drugs and synthons at a practical scale.
Scheme 1. Total and formal syntheses of a) (+)- and (ꢁ)-Tranylcypro-
mine, b) Tasimelteon, c) TRPV1 inhibitor 24, and d) Ticagrelor by
myoglobin-catalyzed cyclopropanation reactions in whole cells. See the
Supporting Information for details on the synthetic steps. DPPA=di-
phenylphosphoryl azide, PTSA=para-toluenesulfonic acid, T3P=pro-
pane phosphonic acid anhydride, TFA=trifluoroacetic acid.
mine, respectively, by conversion into the corresponding acyl
azides followed by a stereoretentive Curtius rearrangement[16]
and hydrolysis (93% yield over three steps for (+)-16; see the
Supporting Information for details). By comparison, the same
cyclopropanation reaction catalyzed by two prominent
Cu(Box) catalysts was reported to proceed with high stereo-
selectivity (90–99% ee) but only moderate diastereoselectiv-
ity (46–50% de (trans)).[2d,e]
The asymmetric cyclopropanation of olefin 18 to give 19
represents a key step in the synthesis of Tasimelteon as
reported by chemists at Bristol–Myers Squibb.[17] Under best
performing conditions, this transformation was realized in
91% de and 85% ee using Nishiyamaꢀs Ru(iPr-PyBox)
catalyst in the presence of excess EDA. After affording
olefin intermediate 18 according to published procedures,[17]
this compound was subjected to Mb-catalyzed cyclopropana-
tion using whole cells expressing the trans-(1R,2R)-selective
variant Mb(L29T,H64V,V68F,I107L) in the presence of
stoichiometric amounts of EDA (Scheme 1b). Notably, the
Acknowledgements
This work was supported by the U.S. National Institute of
Health grant GM098628. LC-MS instrumentation was sup-
ported by the U.S. NSF grant CHE-0946653.
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Received: September 5, 2016
Revised: November 2, 2016
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biocatalysis
P. Bajaj, G. Sreenilayam, V. Tyagi,
R. Fasan*
&&&& — &&&&
Gram-Scale Synthesis of Chiral
Cyclopropane-Containing Drugs and
Drug Precursors with Engineered
Myoglobin Catalysts Featuring
Complementary Stereoselectivity
Chiral cyclopropanes ꢀ la carte: Myoglo-
bin-based cyclopropanation catalysts
featuring complementary stereoselectiv-
ity were developed for the synthesis of 1-
carboxy-2-aryl-cyclopropanes. The engi-
neered hemoproteins were applied in
whole-cell reactions to afford cyclopro-
pane-containing drugs and precursors
thereof at the gram scale, in high yield,
and with excellent diastereo- and enan-
tioselectivity.
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!