A diastereoselective P450-catalyzed epoxidation reaction: Anti versus syn reactivity This Letter is dedicated to the memory of Harry Wasserman

Article abstract of DOI:10.1016/j.tetlet.2015.03.076

The achiral cyclohexene derivative dimethyl cis-1,2,3,6-tetrahydrophthalate has been subjected to oxidation catalyzed by cytochrome P450 monooxygenase P450-BM3, leading to diastereoselective epoxidation rather than oxidative hydroxylation. This reaction occurs with 94% diastereoselectivity in favor of the anti-epoxide, in contrast to m-CPBA which delivers unselectively a 70:30 mixture of anti/syn diastereomers. The experimental results are nicely explained on a molecular level by docking experiments and molecular dynamics computations.

Full text of DOI:10.1016/j.tetlet.2015.03.076

Tetrahedron Letters 56 (2015) 3435–3437
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A diastereoselective P450-catalyzed epoxidation reaction: anti versus
syn reactivity
⇑
Adriana Ilie, Richard Lonsdale, Rubén Agudo, Manfred T. Reetz
Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein Str., 35032 Marburg, Germany
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The achiral cyclohexene derivative dimethyl cis-1,2,3,6-tetrahydrophthalate has been subjected to oxida-
tion catalyzed by cytochrome P450 monooxygenase P450-BM3, leading to diastereoselective epoxidation
rather than oxidative hydroxylation. This reaction occurs with 94% diastereoselectivity in favor of the
anti-epoxide, in contrast to m-CPBA which delivers unselectively a 70:30 mixture of anti/syn diastere-
omers. The experimental results are nicely explained on a molecular level by docking experiments and
molecular dynamics computations.
Received 4 December 2014
Revised 10 March 2015
Accepted 18 March 2015
Available online 21 March 2015
This Letter is dedicated to the memory of
Harry Wasserman
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Biocatalysis
P450 monooxygenase
Olefin epoxidation
Diastereoselectivity
Oxidation
Cytochrome P450 monooxygenases (CYPs) have been used for a
long time in order to perform regio- and stereoselective oxidative
hydroxylation of organic compounds (RH ? ROH), molecular oxy-
gen O2 serving as the oxidant under mild conditions.1 The mecha-
nism involves abstraction of a hydrogen atom by a catalytically
active high-spin heme-Fe@O intermediate (so-called Compound
I; Scheme 1) with formation of the respective R-radical followed
by rapid CAO bond formation. Whenever wild-type (WT) CYPs
are not regio- or stereoselective in the hydroxylation of a given
substrate, protein engineering using rational design2 or directed
evolution3 can be applied in order to manage the synthetic prob-
lem. These protein engineering techniques provide (bio)catalysts
which are generally complementary to synthetic reagents or
catalysts.4
Compounds containing olefinic double bonds are often
hydroxylated at the allylic positions by CYP-catalysis, but they
may also undergo epoxidation.1,5 The reasons for the preference
of one reaction type versus the other are not fully understood,
but the specific pose of a given substrate in the CYP binding pocket
is crucial in determining its oxidative ‘fate’. The mechanism of
epoxidation of alkenes by CYPs has not been studied as intensively
as oxidative hydroxylation. A hydroperoxo–Fe-heme complex has
been postulated in some cases, but Compound I is generally
believed to be the catalytically active species.5,6 A concerted pro-
cess is generally favored based on stereochemical results, although
in rare cases a two-step radical mechanism may be involved.
Irrespective of which mechanism is preferred, the stereochemistry
of epoxide formation will be determined by the face of the double
bond that is placed closest to the heme species.
When prochiral olefins are epoxidized, enantioselectivity is
relevant, while in the case of chiral olefins, the control of
diastereoselectivity constitutes the challenge (in addition to the
regioselectivity problem). A special example is the P450cam-cat-
alyzed epoxidation of chiral 5,6-dehydrocamphor which results
in the selective formation of 5,6-exo-epoxycamphor.5b
In the present study we report a different type of diastereose-
lectivity, namely syn/anti-selectivity in the epoxidation of an achi-
ral substituted cyclohexene derivative. In this system it was
possible to explain the origin of diastereoselectivity on a molecular
level using docking and molecular dynamics (MD) computations.
We employed P450-BM3 as the catalyst, a well-known self-suffi-
cient CYP from Bacillus megaterium1,7 which has been used very
often in oxidative hydroxylation, but less so in epoxidation.1,5
Commercially available dimethyl cis-1,2,3,6-tetrahydrophtha-
late (1) was chosen as the substrate (Scheme 2), which can be pre-
pared by Diels–Alder reaction of butadiene and dimethyl maleate.8
WT P450-BM3 proved to be 78% regioselective in favor of
⇑
Corresponding author. Tel.: +49 (0)6421 28 25500; fax: +49 (0)6421 28 25620.
E-mail address: reetz@mpi-muelheim.mpg.de (M.T. Reetz).
http://dx.doi.org/10.1016/j.tetlet.2015.03.076
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

3436
A. Ilie et al. / Tetrahedron Letters 56 (2015) 3435–3437
Why does compound 1 undergo epoxidation to a greater extent
O
O2C(H2C)2
O2C(H2C)2
than oxidative hydroxylation, and why is anti-selectivity the pre-
ferred mode of attack? In order to address these questions, induced
fit docking and MD simulations were performed. Guided by the
crystal structure of P450-BM3 (PDB code 1JPZ),11 substrate 1 was
positioned in the active site using induced fit docking.12 The pose
which places the substrate closest to the heme has both the double
bond and one of the allylic hydrogen atoms of 1 close to the heme
(Fig. 2). In a previous QM/MM study of epoxidation and hydrox-
ylation of alkenes by P450cam, a preference for epoxidation over
allylic hydroxylation was observed when the two oxidation sites
were equally accessible to Compound I.13 If epoxidation is intrinsi-
cally preferred over hydroxylation, as is expected given that no
hydroxylated product is observed upon reaction of substrate 1
with m-CPBA, this would explain the observation that the epoxide
is the major product for the enzyme catalyzed reaction. A docking
pose was observed in which the methyl group of one of the ester
substituents is close to the heme. As no products are observed in
which either of the two methyl groups undergo hydroxylation, this
is not consistent with the observed reactivity. It is possible that
hydrogen abstraction is not energetically feasible from this posi-
tion, as it would lead to the formation of a primary radical
intermediate.
N
N
IV N
Fe
S
N
Enzyme
Scheme 1. Compound I [Ferryl (FeIV)-oxo-
p
porphyrin cation radical].
OH
CO2Me
WT
CO2Me
CO2Me
CO2Me
O
P450-BM3
+
CO2Me
CO2Me
1
2
3
O
CO2Me
CO2Me
O
MeO2C
MeO2C
2-anti
2-syn
Scheme 2. Oxidation products resulting from WT P450-BM3 catalyzed oxidation of
substrate 1.10
Now we turn our attention to the stereoselectivity of the epox-
ide formation. As already pointed out, the species responsible for
oxidation of alkenes by P450s is widely accepted to be
Compound I.1,5 When the Compd I oxygen atom is added to the
model, the docking pose is consistent with the formation of 2-anti.
No docking poses were observed in which oxidation would occur
on the opposite side of the double bond, hence providing an expla-
nation for the relatively low observed amount of 2-syn. To explore
the conformational flexibility of this enzyme/substrate complex,
two unrestrained MD simulations were performed starting from
the above mentioned docking pose.14 The substrate remains close
to the observed docking pose during both of the two 26 ns
epoxidation leading to 2, while allylic hydroxylation with forma-
tion of 3 in essentially racemic form occurred to a smaller extent
(22%) as revealed by GC analysis. Oxidative demethylation was
not observed. Importantly, 94% diastereoselectivity favoring the
anti-configuration 2-anti was observed (Fig. 1, top). This com-
pound constitutes an intermediate in the synthesis of effective
inhibitors of glycosidases.9 It was prepared as a mixture of anti/
syn-diastereomers by m-CPBA-mediated epoxidation, which had
to be separated by column chromatography.9 Upon repeating this
reaction, we indeed obtained a 70:30 mixture of 2-anti and 2-
syn, respectively (Fig. 1, bottom).
pA
O
OH
CO2Me
55
CO2Me
CO2Me
O
50
MeO2C
CO2Me
MeO2C
45
3
2-syn
2-anti
40
35
30
25
7 .6
7 .8
8
8 .2
8 .4
8 .6
8 .8
9
pA
120
100
80
60
40
20
7 .6
7 .8
8
8 .2
8 .4
8 .6
8 .8
9
Figure 1. GC chromatograms of oxidation products using 1 as starting material. Top: oxidation products in the reaction catalyzed by WT P450-BM3 using resting cells;
bottom: oxidation products in the transformation performed by chemical reaction using m-CPBA as oxidant. GC conditions: 30 m DB1, inner diameter 0.25 mm; pressure:
2 bar H2; injector: 230 °C; oven: temperature gradient: from 50 to 300 °C with 12 °C/min FID detector: 350 °C. The peak assigned to substrate 1 was detected at retention time
6.81.

A. Ilie et al. / Tetrahedron Letters 56 (2015) 3435–3437
3437
2. Recent examples of rational design of P450 monooxygenases: (a) Sowden, R. J.;
Yasmin, S.; Rees, N. H.; Bell, S. G.; Wong, L.-L. Org. Biomol. Chem. 2005, 3, 57–64;
(b) Seifert, A.; Antonovici, M.; Hauer, B.; Pleiss, J. ChemBioChem 2011, 12, 1346–
1351; (c) Nguyen, K. T.; Virus, C.; Günnewich, N.; Hannemann, F.; Bernhardt, R.
ChemBioChem 2012, 13, 1161–1166; (d) Venkataraman, H.; de Beer, S. B. A.; van
Bergen, L. A. H.; van Essen, N.; Geerke, D. P.; Vermeulen, N. P. E.; Commandeur,
J. N. M. ChemBioChem 2012, 13, 520–523.
3. Review of directed evolution of P450 monooxygenases: Roiban, G.-D.; Reetz, M.
T. Chem. Commun. 2015. http://dx.doi.org/10.1039/c4cc09218j.
4. (a) Reetz, M. T. J. Am. Chem. Soc. 2013, 135, 12480–12496; (b) Nestl, B. M.;
Hammer, S. C.; Nebel, B. A.; Hauer, B. Angew. Chem., Int. Ed. 2014, 53, 3070–
3095; (c) Clouthier, C. M.; Pelletier, J. N. Chem. Soc. Rev. 2012, 41, 1585–1605.
5. Examples of P450-catalyzed olefin epoxidation: (a) Vaz, A. D. N.; McGinnity, D.
F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555–3560; (b) Gelb, M. H.;
Malkonen, P.; Sligar, S. G. Biochem. Biophys. Res. Commun. 1982, 104, 853–858;
(c) Kolev, J. N.; Zaengle, J. M.; Ravikumar, R.; Fasan, R. ChemBioChem 2014, 15,
1001–1010; (d) Jin, S.; Makris, T. M.; Bryson, T. A.; Sligar, S. G.; Dawson, J. H. J.
Am. Chem. Soc. 2003, 125, 3406–3407; (e) Li, A.; Wu, S.; Adams, J. P.; Snajdrova,
R.; Li, Z. Chem. Commun. 2014, 8771–8774; (f) Farinas, E. T.; Alcalde, M.; Arnold,
F. Tetrahedron 2004, 60, 525–528; (g) Miller, V. P.; Freutel, J. A.; Ortiz de
Montellano, P. R. Arch. Biochem. Biophys. 1992, 298, 697–702; (h) Roiban, G.-D.;
Agudo, R.; Reetz, M. T. Tetrahedron 2013, 69, 5306–5311.
6. Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004, 104, 3947–3980.
7. (a) Narhi, L. O.; Fulco, A. J. J. Biol. Chem. 1986, 261, 7160–7169; (b) Munro, A. W.;
Leys, D. J.; McLean, K. J.; Marshall, K. R.; Ost, T. W. B.; Daff, S.; Miles, C. S.;
Chapman, S. K.; Lysek, D. A.; Moser, C. C.; Page, C. C.; Dutton, P. L. Trends
BioChem. Sci. 2002, 27, 250–257; (c) Clodfelter, K. H.; Waxman, D. J.; Vajda, S.
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Figure 2. Induced fit docking pose of substrate 1 in the active site of WT P450-BM3.
The docking pose places the substrate in an orientation in which oxidation will
result in formation of 2-anti.
10. Experimental procedure for the P450-BM3 catalyzed epoxidation of substrate 1: An
Erlenmeyer flask (100 mL) containing LB (20 mL) and kan (50 lg/mL) was
inoculated with a colony of WT P450 and incubated 6 h at 37 °C with gentle
shaking. The pre-culture was further inoculated into TB (400 mL) containing
kan (50 lg/mL) and allowed to grow at 37 °C until O.D. of 0.8–0.9 at 600 nm
was reached. IPTG was added to a final concentration of 0.2 mM and the
culture grown at 30 °C during 16 h with gentle agitation. Cells were pelleted by
centrifugation (15 min, 5000 r.p.m. at 4 °C). The supernatant was discarded
and the pellet was resuspended in 50 mL of reaction buffer [phosphate buffer
simulations. The active site of WT P450-BM3 is hydrophobic and,
while no hydrogen bonding interactions are observed between
the substrate and active site, the shape of the active site clearly
influences the orientation in which the double bond can approach
Compd I, to the extent that oxidation can only occur on one face of
the double bond.
In summary, we have discovered that P450-BM3 is an efficient
catalyst for the regio- and stereoselective epoxidation of the
achiral cyclohexene derivative dimethyl cis-1,2,3,6-tetrahydroph-
thalate (1) with preferential formation of the anti-configured epox-
ide 2-anti. The observed anti-diastereoselectivity was explained on
a molecular level by docking experiments and MD simulations,
resulting in a reasonable model. The product is a useful inter-
mediate in the synthesis of inhibitors of certain glycosidases.9
(pH 7.4, 100 mM), NADP+ (300
lM) and glucose (100 mM)]. Resuspended cells
were transferred to a 250 mL Erlenmeyer flask, and starting material (200
lL,
1.16 mmol) was added. Reaction was carried out at 25 °C for 20 h with mild
agitation. The reaction mixture was extracted with ethyl acetate (400 mL) and
the resulting colorless oil was loaded on chromatographic column (EA/PE 1:1).
Spectroscopic data of the anti stereoisomer are in agreement with published
data.9 yield 23% (58 mg) anti-2: 1H NMR (300 MHz, CDCl3) d 2.20–1.96 (m, 4H),
2.76 (t, 3J = 6.2 Hz, 2H), 3.07 (s, 2H), 3.53 (d, 3J = 2.6 Hz, 6H, OCH3). 13C NMR
(75 MHz, CDCl3) d 173.19, 51.69, 51.23, 37.39, 24.55. HRMS (EI) calcd for
C
10H14O5 [M]+: 214.0834; found: 214.0893.
11. Haines, D. C.; Tomchick, D. R.; Machius, M.; Peterson, J. A. Biochemistry 2001,
40, 13456–13465.
12. (a) Schrödinger Suite 2014-2 Induced Fit Docking protocol; Glide version 6.3;
Schrödinger, LLC: New York, NY, 2013. Prime version 3.6, Schrödinger, LLC,
New York, NY, 2014; (b) Farid, R.; Day, T.; Friesner, R. A.; Pearlstein, R. A. Bioorg.
Med. Chem. 2006, 14, 3160–3173; (c) Sherman, W.; Day, T.; Jacobson, M. P.;
Friesner, R. A.; Farid, R. J. Med. Chem. 2006, 49, 534–553; (d) Sherman, W.;
Beard, H. S.; Farid, R. Chem. Biol. Drug Des. 2006, 67, 83–84.
Acknowledgments
Financial support by the Max-Planck-Gesellschaft and the
Arthur C. Cope Foundation is gratefully acknowledged.
13. (a) Lonsdale, R.; Harvey, J. N.; Mulholland, A. J. J. Phys. Chem. Lett. 2010, 1,
3232–3237; (b) Lonsdale, R.; Harvey, J. N.; Mulholland, A. J. J. Phys. Chem. B
2010, 114, 1156–1162.
References and notes
14. MD simulations were performed using AMBER14,15 using the same settings
and minimization/equilibration protocol outlined in Ref. 16.
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E. M.; Guengerich, F. P. Biochim. Biophys. Acta Gen. Subj. 2007, 1770, 314–329;
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(d) Ortiz de Montellano, P. R. Chem. Rev. 2010, 110, 932–948; (e) Shaik, S.;
Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. Chem. Rev. 2010, 110, 949–
1017; (f) Whitehouse, C. J. C.; Bell, S. G.; Wong, L.-L. Chem. Soc. Rev. 2012, 41,
1218–1260; (g) Lewis, J. C.; Coelho, P. S.; Arnold, F. H. Chem. Soc. Rev. 2011, 40,
2003–2021; (h) Holtmann, D.; Fraaije, M. W.; Arends, I. W. C. E.; Opperman, D.
J.; Hollmann, F. Chem. Commun. 2014, 13180–13200; (i) Bernhardt, R.; Urlacher,
V. B. Appl. Microbiol. Biotechnol. 2014, 98, 6185–6203.
15. Case, D. A.; Babin, V.; Berryman, J. T.; Betz, R. M.; Cai, Q.; Cerutti, D. S.;
Cheatham, T. E., III; Darden, T. A.; Duke, R. E.; Gohlke, H.; Goetz, A. W.; Gusarov,
S.; Homeyer, N.; Janowski, P.; Kaus, J.; Kolossváry, I.; Kovalenko, A.; Lee, T. S.;
LeGrand, S.; Luchko, T.; Luo, R.; Madej, B.; Merz, K. M.; Paesani, F.; Roe, D. R.;
Roitberg, A.; Sagui, C.; Salomon-Ferrer, R.; Seabra, G.; Simmerling, C. L.; Smith,
W.; Swails, J.; Walker, R. C.; Wang, J.; Wolf, R. M.; Wu, X.; Kollman, P. A. AMBER
14; University of California: San Francisco, 2014.
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