Regioselective Epoxidations by Cytochrome P450 3A4 Using a Theobromine Chemical Auxiliary to Predictably Produce N-Protected β- or γ-Amino Epoxides

Article abstract of DOI:10.1002/adsc.201700637

N-Protected β- and γ-amino epoxides are useful chiral synthons. We report here that the enzyme cytochrome P450 3A4 can catalyze the formation of such compounds in a regio- and stereoselective manner, even in the presence of multiple double bonds or aromatic substituents. To this end, the theobromine chemical auxiliary is used not only to control the selectivity of the enzyme, but also as a masked amine, and to facilitate product recovery. Theobromine predictably directed epoxidation at the double bond of the fourth carbon from the theobromine group. Unlike with most catalysts, the selectivity did not depend on electronic or steric factors but rather on the position of the olefin relative to the theobromine group. (Figure presented.).

Full text of DOI:10.1002/adsc.201700637

Title: Regioselective Epoxidations by Cytochrome P450 3A4 Using
a Theobromine Chemical Auxiliary to Predictably Produce N-
Protected β- or γ- Amino Epoxides
Authors: Vanja Polic, Kin Jack Cheong, Fabien Hammerer, and Karine
Auclair
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201700637
Link to VoR: http://dx.doi.org/10.1002/adsc.201700637

10.1002/adsc.201700637
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Regioselective Epoxidations by Cytochrome P450 3A4 Using a
Theobromine Chemical Auxiliary to Predictably Produce N-
Protected β- or γ- Amino Epoxides
Vanja Polic, Kin Jack Cheong, Fabien Hammerer and Karine Auclair*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8.
Fax: (+1)-514-398-3797; E-mail: k.auclair@mcgill.ca
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. N-Protected β- and γ-amino epoxides are useful
chiral synthons. We report here that the enzyme cytochrome
P450 3A4 can catalyze the formation of such compounds in
a regio- and stereo-selective manner, even in the presence of
multiple double bonds or aromatic substituents. To this end,
the theobromine chemical auxiliary is used not only to
control the selectivity of the enzyme, but also as a masked
amine and to facilitate product recovery. Theobromine
predictably directed epoxidation at the double bond of the
fourth carbon from the theobromine group. Unlike with most
catalysts, the selectivity did not depend on electronic or
steric factors but rather on the position of the olefin relative
to the theobromine group.
Keywords: Biocatalysis; Chemical Auxiliaries;
Cytochrome P450 3A4; Epoxidation; Theobromine.
shown that it directs hydroxylation at the fourth atom
away from the auxiliary with pro-R facial
selectivity.[17] In addition, the theobromine auxiliary
can also serve as a bait for the selective and quick
recovery of the product from complex biocatalytic
mixtures.[19] This is substantial since product recovery
typically accounts for ~80% of the production cost in
fermentation.
Epoxides are valuable building blocks in chemical
synthesis, with the potential to introduce two adjacent
chiral centers. These three-membered heterocycles
are often employed as versatile intermediates in
organic synthesis.[20] Available methods for
producing chiral epoxides are largely based on
transition-metal catalysis and/or the requirement of
pre-existing directing group in the substrate.[21–23]
Vinyl epoxides in particular are useful building
blocks that combine the conjugated reactivity of two
functional groups. One strategy to access this class of
intermediates involves direct monoepoxidation of the
corresponding diene; however, these reactions often
suffer from lack of regioselectivity or low selectivity
between monoepoxidation and bisepoxidation, or
may result in the formation of the undesired
regioisomer. Typically, the regioselectivity of diene
epoxidation is controlled by electronic factors, as
with peracid oxidants,[24] or by a directing group, as
with Sharpless epoxidation.[21,25] However, it is not
Introduction
Cytochrome P450 enzymes (P450s or CYPs) are a
superfamily of heme monooxygenases that are known
to
catalyze
diverse
reactions,
including
hydroxylations, epoxidations, sulfoxidations, aryl-
aryl couplings, Baeyer-Villiger oxidations, and
more.[1–7] Recently, even some non-natural enzymatic
reactions such as cyclopropanations[8,9] and C-H
aminations[10–15] were made available using
engineered P450. Many of these reactions are
otherwise difficult to achieve by traditional chemical
synthesis.
Drug metabolizing P450s are particularly attractive
for synthetic applications because of their high
substrate promiscuity. This is, however, contrasted
with our poor ability to predict their products.[16] To
circumvent this challenge, we have previously
reported that chemical auxiliaries can be used to
afford predictable regio- and stereo-selective
hydroxylations by human P450 3A4[17] and P450
2E1.[18] Thus, following attachment of the chemical
auxiliary to the substrate, the auxiliary is expected to
bind the enzyme in a way that orients the substrate
near the reactive iron species of the P450 such that
oxidation occurs consistently at the same distance
from the auxiliary. For example, when theobromine is
used as the chemical auxiliary with P450 3A4, it was
1
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
Table 1. Results of Cys-depleted mutant P450 3A4-
catalyzed reactions.
uncommon to obtain mixtures of epoxides along with
polymeric products.
Also of interest are N-protected β- and γ-amino
epoxides. Since β- and γ-amino 1,2-dioxygen motifs
(and related groups) are common in natural products
and in biologically active molecules,[26–31] new
methods providing easy access to diverse N-protected
β- and γ-amino epoxides are desirable.
Enzymes offer a green alternative to transition-
metal catalysts. Based on the success of theobromine
as a chemical auxiliary to allow prediction of the
regioselectivity of hydroxylation by P450 3A4,[17]
this system was further studied for its synthetic utility
in epoxidation of isolated and conjugated dienes. We
report herein that the use of P450 3A4 in combination
with the theobromine auxiliary provides a new route
to some difficult to access N-protected β- and γ-
amino epoxides and vinyl epoxides.
Results and Discussion
Epoxidation of isolated olefins
While compound 1 is oxidized to the racemic epoxide
16, compound 2 yields the enantiopure (>99%)
product 17 with the expected R-configuration at C4
(Table 1; structure and orientation of the theobromine
group, Tb, is shown in Scheme 1A).[17] Intrigued by
these results, we wondered if olefin substitution could
affect the regio- and stereoselectivity of P450 3A4.
Thus, a variety of theobromine derivatives with
isolated olefins or conjugated dienes in the side chain
were surveyed for selective epoxidation (see 3-15,
Table 1). Theobromine derivatives were prepared
with side chain double bonds located on either side of
the 4th carbon from theobromine, with either E or Z
configuration, with or without branching, additional
double bonds or aromatic substituents. The
compounds were conveniently prepared by
nucleophilic attack of the theobromine N-anion on
the desired tosylate or bromide precursor (Scheme
1A). Encouragingly, spectral binding studies of
selected compounds with P450 3A4 indicate that the
theobromine derivatives display typical Type 1 ligand
binding characteristics with spectral binding constant
(Ks) values ranging from 0.2 to 1.5 mM (Figure 1 and
S7). Type 1 spectra are characterized by a peak
around 390 nm and trough around 418 nm, resulting
from a heme iron spin shift from low to high spin
upon ligand binding. Type 1 binding spectra confirm
that the theobromine-containing compounds do
indeed bind the P450 3A4 active site, without directly
coordinating to the heme iron. Theobromine alone,
however, did not show evidence of active site binding
nor of coordination with the heme iron (Figure S8).
aTb is used to abbreviate the theobromine auxiliary. The
site of attachment is shown in Scheme 1A. bDetermined by
comparison with authentic standards. Refer to Supporting
Information for reaction conditions. All reactions were
c
performed at least in triplicate. Regioselectivity is shown
as percentage of epoxidation at the C3-C4 or C4-C5 double
bond with the major product shown. *denotes
chemoselectivity of the enzymatic reaction where
applicable. dEnantiomeric ratio of the major product.
ePercent conversion of substrate to oxidized product.
2
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
Scheme 1. Synthetic scheme for the preparation of 3-15
(A) and their respective racemic epoxides using mCPBA
(B). The alcohols were prepared as described elsewhere
(see main text).
Figure 1. Spectral binding characteristics of 5 with P450
3A4. A) Difference binding spectra of P450 3A4, with 5
displaying a peak at ~ 390 nm and a trough at ~420 nm,
which are characteristic of a type 1 ligand. B) Spectral
binding curve of P450 3A4 titrated with 5 to yield a
spectral binding constant (KS) of 0.22 ± 0.02 mM (fitting
in GraphPad).
Considering that human P450 3A4 is a
membrane-bound enzyme and is not very soluble in
vitro, it is expected to be more efficient and more
stable in a cellular environment, as in fermentation.
The biotransformation of 1-15, however, was
performed with purified enzyme (as opposed to
whole-cells) in order to fully demonstrate the
involvement of cytochrome P450 3A4. For
expression and purification from Escherichia coli, the
enzyme is typically truncated to remove the
membrane-anchoring tail and a 4×His-tag is added to
facilitate purification. This variant is used here and
referred to as the wild type. We have also used a Cys-
depleted P450 3A4 mutant, previously generated in
our lab[32], from which we have replaced 5 native
cumene hydroperoxide (CHP). The use of CHP was
reported to replace the natural cofactor and redox
partner, NADPH and cytochrome P450 reductase
(CPR), and to afford higher yields.[34] No oxidation
was observed in the absence of the enzyme,
demonstrating that CHP is not oxidizing the double
bonds. As expected, control experiments with the
more expensive NADPH and CPR generated the
same products, only in lower yields compared to the
CHP-supported reaction (Figure 2B). In order to
further demonstrate the importance of the auxiliary,
substrate precursors without the theobromine moiety
were exposed to the enzyme under the same
conditions as above. None of them were turned over
by the enzyme in the absence of the theobromine
group. No oxidized product were detected (Figures
S4-S6), confirming the necessity of the auxiliary for
substrate recognition by P450 3A4, in agreement with
our earlier study.[17] After optimization, substrate
conversions of ca. 60% for the wild type enzyme and
ca. 90% for the Cys-depleted P450 3A4 mutant were
cysteine
residues
to
the
following:
mutant
C58T/C64A/C98S/C239S/C468G.
This
shows 2 fold improved activity with the substrates 7-
benzyloxy-4-trifluomethyl-coumarin and testosterone,
compared to the wild type enzyme[32], which may be
attributed to conformational changes as observed for
other P450 3A4 cysteine mutants.[33] Compounds 1-
15 were subjected to enzymatic transformation by
both the wild type and Cys-depleted P450 3A4
enzymes. For simplicity, the reactions were
performed in the presence of the surrogate cofactor
3
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
consistently obtained (Table 1 and Figure S3).
Enzyme regio- and stereoselectivities were unaffected
by the mutation. Most compounds were oxidized to
give one major product with a mass increase of +16
corresponding to epoxidation, as monitored by
HPLC-MS (Figure 2), with allylic hydroxylation
likely being the minor product. Racemic synthetic
epoxide standards were also synthesized by reaction
of 3-15 with mCPBA (Scheme 1B) for HPLC
comparison.
Table 1 lists all products obtained from the
enzymatic transformations. With isolated olefins, the
results indicate that the enzyme stereoselectivity is
not influenced by the location of the double bond.
Olefin substitution and geometry (E vs Z), however,
seem to affect the stereoselectivity of the reaction.
Interestingly, the Z-olefins (compounds 3, 4 and 5)
yielded products with very good enantiomeric ratio
(e.r.). In general, olefin derivatives with smaller
substituents and E geometry, such as methyl and
ethyl groups (see 6, 7, and 8), were transformed to the
corresponding epoxide (21, 22, and 23 respectively)
with poor enantioselectivity. 5,5-Substituted double
bonds (as in 9 and 10) or olefins conjugated to a
phenyl substituent (see 11 and 12) were also
transformed with low stereoselectivity. Remarkably
however, no aromatic hydroxylation products were
observed in the case of 11 and 12 (see Figure S1). In
most cases the chemoselectivity was good (>80%),
however it was sometimes diminished with the
presence of a methylene at the fifth carbon from the
theobromine moiety. In combination, these results
suggest that the optimal site of oxidation may be
between C4 and C5.
Figure 2. HPLC traces for representative P450 3A4 and
chemical transformations, monitored at 273 nm. Panel A
(non-chiral HPLC traces): a) P450 3A4 transformation of
13 using CHP and stopped before completion; b) product
of the chemical epoxidation of 13 with mCPBA; c) co-
injection of the products from the chemical and enzymatic
reactions of 13; d) unmodified compound 13. Panel B
(non-chiral HPLC traces): e) unmodified compound 5; f)
enzymatic transformation of 5 by P450 3A4 using
CPR/NADPH; g) enzymatic transformation of 5 by
cytochrome P450 3A4 using CHP. Panel C (chiral HPLC
traces): h) product of the chemical epoxidation of 5 with
mCPBA; i) P450 3A4 transformation of 5 using CHP and
stopped before completion.
Monoepoxidation of dienes
To elucidate the scope of theobromine-mediated
epoxidation of dienes by P450 3A4, compounds 13,
14 and 15 were separately subjected to enzymatic
transformation. Both dienes 13 and 15 contain a
double bond between C2 and C3 (from theobromine),
whereas the double bonds in 14 are shifted by one
carbon further from the chemical auxiliary. While the
double bonds of compound 13 are all internal,
compounds 14 and 15 both have an internal and a
terminal olefin, the latter being more electron poor
than the former. When treated with mCPBA, a
commonly used reagent for epoxidation, compound
13 produced a mixture of regioisomers in a 3:1 molar
ratio that could not be separated by column
chromatography (Scheme 1B). On the other hand,
diene 13 was efficiently transformed by both P450
3A4 variants, in the presence of either CHP or
CPR/NADPH, into one single regioisomer, product
28 (Table 1, Figure 2A). Oxidation occurred at the
double bond between C4 and C5, as expected due to
the directing effect of theobromine that is based on
distance from the auxiliary rather than electronics.[17]
Exposure of diene 15 to the enzymatic reaction also
atom was introduced between C4 and C5. This is in
sharp contrast to the reaction with mCPBA where
again a mixture of products was observed. Enzymatic
reaction with diene 14 however, yielded a mixture of
regioisomers. Compound 29 was the main product
with oxidation at the C3-C4 bond, and the reaction
proceeded with very good enantioselectivity. The
minor product is likely due to oxidation at the C5-C6
bond. This result is again consistent with the optimal
site of oxidation being between C4 and C5.
yielded
a
single regioisomer with excellent
enantioselectivity, compound 30, where the oxygen
4
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
Product isolation using molecularly imprinted
polymers
on the distance from the chemical auxiliary, with the
double bond that includes the 4th carbon from
theobromine being oxidized. Interestingly this
method can differentiate electronically similar double
bonds and even favor epoxidation at the electron-
poorest double bond.
Since cytochrome P450 3A4 is a membrane-bound
enzyme and is more efficient and more stable in a
cellular environment, biocatalytic reactions should be
done with whole-cells as in fermentation processes.
We have previously demonstrated that molecularly
imprinted polymers (MIPs) can be used for the
selective recovery of theobromine-containing
compounds from complex biocatalytic mixtures with
great efficacy.[19] We thus verified if the theobromine
epoxide derivatives are compatible with MIPs (see
below for detailed conditions), and found that they
are indeed stable under the solid phase extraction
procedure used with MIPs. We achieved ca. 95%
recovery of epoxide 20 with <2% epoxide opening
observed under the acidic conditions.
Besides the fact that theobromine can serve to
control the regioselectivity of P450 3A4 and as a
protected amine, another advantage is that it can
facilitate product recovery with the use of MIPs, as
shown here with epoxides and previously for
hydroxylated products[19]. This is especially useful in
whole-cell biotransformations where the majority of
the cost goes to product recovery from the complex
mixture. Although the work presented here was done
with purified enzyme, large scale uses of P450
enzymes are likely to be done via fermentation to
maximize enzyme stability. MIPs also replace the
need for organic solvents during product recovery
and/or purification. Thus, combining the use of MIP
with whole-cell transformations should improve the
sustainability of the overall process. Finally, this
work promises to open up a new generation of
chemical auxiliaries for diene epoxidations with
complementary regioselectivities depending on the
enzyme and the auxiliary used.
Conclusion
Chemical auxiliaries have proven popular in
asymmetric synthesis; in particular, Evans
oxazolidone chiral auxiliaries have shown significant
utility in controlling the diastereoselectivity of
various reactions such as alkylation,[35] acylation[36]
and aldol condensations.[37] The auxiliaries are
normally removed after the reaction; however, they Experimental Section
are often expensive. In contrast, directing groups such
Expression and Purification of Wild-Type and Mutant
as the alcohol group required in Sharpless
epoxidation are typically part of the substrate and
cannot be removed easily. The results presented here
show that the use of P450 3A4 with the chemical
auxiliary theobromine is complementary to existing
chemical methods. Not only does P450 3A4 show
outstanding substrate promiscuity, theobromine is
also achiral and inexpensive. Furthermore, the
theobromine chemical auxiliary may also serve as a
masked amine, which can be revealed under reducing
conditions in high yield (80% as previously
shown).[17] This is non-negligible since existing
amino groups are most often protected in the
presence of epoxides.
P450 3A4
The expression plasmid for truncated P450 3A4 (E.C.
1.14.13.-) (pSE3A4His) was generously provided by Prof.
J. R. Halpert (University of Connecticut). The Cys-
depleted mutant was prepared as previously described by
our lab and had the following additional mutations: C58T,
C64A, C98S, C239S, C468G.[32] Expression and
purification were performed as previously described.[34]
Expression and Purification of Cytochrome P450
Reductase (CPR)
The expression plasmid for cytochrome P450 reductase
(pOR263) was generously provided by Prof. Charles B.
Kasper (University of Wisconsin-Madison). CPR was
expressed and purified as described elsewhere.[34] Protein
concentration was measured by the Bradford method
(Pierce 23236). The concentration of holo-CPR was
estimated from its flavin content using an extinction
coefficient of 21.4 mM-1 cm-1 at 456 nm.[38]
For P450 3A4 transformations, it has been shown
that when the 4th carbon from theobromine is a
methylene or a methine, hydroxylation is favored at
the pro-R C-H bond, even when more reactive groups
(e.g. double bonds or other tertiary carbons) are
present elsewhere in the molecule.[17] We report here
that when the 4th carbon from theobromine is part of
a double bond, i.e. between C3 and C4, or C4 and C5,
an epoxide is consistently produced in high yield. In
the case of molecules with a single olefin, better
stereoselectivity is observed for Z-double bonds or
terminal double bonds between C4 and C5.
The approach reported here is especially useful
with dienes and proceeds with predictable
regioselectivity, in a way that is complementary to
the current chemical toolbox. In contrast to chemical
catalysts, the site of epoxidation by P450 3A4 is
independent of electronic factors, and depends solely
Optimized Enzymatic Transformation Conditions
P450 3A4 (1.2 nmol, 12 µM final concentration) and
substrate (20 nmol, 200 µM final concentration) were
mixed in potassium phosphate buffer (KPi, 0.1 M, pH 7.4)
and preincubated at 27oC and 250 rpm for 5 min (reaction
tubes were placed on their side in an orbital
shaker/incubator). The enzymatic reaction was initiated
with the addition of cumene hydroperoxide (CHP, 60 nmol,
600 µM final concentration). Additional CHP (60 nmol)
was added after 15 min and the reaction mixture was
incubated for an additional 1.5 h. Alternatively, a mixture
of cytochrome P450 reductase (16 µM) and NADPH (1
mM) can be used instead of CHP. Controls were run in the
absence of each of CHP, substrate or enzyme. The reaction
was terminated by extraction of the substrate and product
in dichloromethane (4 × 0.5 mL). The extracts were
5
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
combined and the solvent was evaporated. Alternatively,
substrate and product can be extracted directly using MIP
as described below. The residue was redissolved in
acetonitrile (200 µL), filtered and analyzed by HPLC-MS.
[3] F. P. Guengerich, A. W. Munro, J. Biol. Chem. 2013,
288, 17065–17073.
[4] F. P. Guengerich, Chem. Res. Toxicol. 2001, 14, 611–
650.
MIP Extraction of Epoxides
[5] E. M. Isin, F. P. Guengerich, Biochim. Biophys. Acta
2007, 1770, 314–329.
The MIP was prepared as previously described[19] using as
the template a theobromine derivative with a 6-carbon
chain hydroxylated at C4, as a stable mimic of the epoxide.
The template was removed as usual.[19] To test the method,
a mixture containing a known amount of 20 (1 mg, 2.5 mL,
1.5 mM in H2O) was loaded onto the MIP (1.5 mL) by
centrifugation (3,000 × g for 5 min at 4°C), leaving a dry
solid phase. The MIP was then washed with H2O (3 × 2.5
mL) by centrifugation (3,000 × g at 4°C for 5 min each).
The aqueous eluate was discarded, leaving a dry solid
phase. Finally, the epoxide was eluted from the solid phase
with ethanol:acetic acid (9:1, 3 × 2.5 mL). The eluate was
pooled and diluted to 25 mL with H2O, before analysis by
LC-UV-MS (injecting 100 µL).
[6] R. Fasan, ACS Catal. 2012, 2, 647–666.
[7] H. M. Girvan, A. W. Munro, Curr. Opin. Chem. Biol.
2016, 31, 136–145.
[8] M. Carta, R. Malpass-Evans, M. Croad, Y. Rogan, J. C.
Jansen, P. Bernardo, F. Bazzarelli, N. B. McKeown,
Science. 2013, 339, 303–307.
[9] P. S. Coelho, Z. J. Wang, M. E. Ener, S. A. Baril, A.
Kannan, F. H. Arnold, E. M. Brustad, Nat. Chem. Biol.
2013, 9, 485–487.
Spectral Binding Studies
[10] J. A. McIntosh, P. S. Coelho, C. C. Farwell, Z. J.
Wang, J. C. Lewis, T. R. Brown, F. H. Arnold, Angew.
Chemie - Int. Ed. 2013, 52, 9309–9312.
All UV/Vis spectra of the enzyme-ligand complexes were
obtained on a Cary 5000 UV/Vis spectrophotometer. P450
3A4 (2 μM, 22 μL from a 17.8 μM stock), KPi buffer (178
μL, 0.1 M at pH = 7.4) were added to both the reference
and sample cuvettes, and a blank was taken. In separate
experiments, substrates 3, 5, 13, 14 and 15 were titrated
into the sample cuvette from stock solutions in acetonitrile
such that the final acetonitrile concentration never
exceeded 3% (v/v). This acetonitrile concentration had no
detectable effect on the P450 spectra. Equal volumes of
acetonitrile were added to the reference cuvette and the
difference spectra were acquired from 300−500 nm. The
difference in absorbance at the peak and the trough was
plotted against substrate concentration to obtain a binding
curve. Spectral dissociation constants (Ks) were extracted
from the binding curves by fitting to the following
equation:
[11] Z. J. Wang, N. E. Peck, H. Renata, F. H. Arnold,
Chem. Sci. 2014, 5, 598–601.
[12] T. K. Hyster, C. C. Farwell, A. R. Buller, J. A.
McIntosh, F. H. Arnold, J. Am. Chem. Soc. 2014, 136,
15505–15508.
[13] R. Singh, J. N. Kolev, P. A. Sutera, R. Fasan, ACS
Catal. 2015, 5, 1685–1691.
[14] J. N. Kolev, K. M. O’Dwyer, C. T. Jordan, R. Fasan,
ACS Chem. Biol. 2014, 9, 164–173.
[15] R. Singh, M. Bordeaux, R. Fasan, ACS Catal. 2014, 4,
546–552.
|∆A| = (|∆Amax|∙|S|) / (Ks + |S|)
[16] B. C. Jones, D. S. Middleton, K. Youdim, in Prog.
Med. Chem., Elsevier, 2009, pp. 239–263.
Here ΔA is the difference in absorbance between the peak
and the trough, ΔAmax is the maximum reachable value of
ΔA at saturating substrate concentrations, [S] is the
substrate concentration, and Ks is the apparent spectral
dissociation constant. Fitting was performed by using the
GraphPad Software.
[17] A. T. Larsen, E. M. May, K. Auclair, J. Am. Chem.
Soc. 2011, 133, 7853–7858.
[18] A. Menard, C. Fabra, Y. Huang, K. Auclair,
Chembiochem a Eur. J. Chem. Biol. 2012, 13, 2527–
2536.
Acknowledgements
[19] A. T. Larsen, T. Lai, V. Polic, K. Auclair, Green
Chem. 2012, 14, 2206–2211.
This work was funded by grants to K.A. from the National
Science and Engineering Research Council of Canada (NSERC)
and the Center in Green Chemistry and Catalysis (CGCC). K.J.C
thanks the CGCC for a scholarship. V.P. was supported by
scholarships from the Dr. Richard H. Tomlinson Foundation and
CGCC. We would also like to thank Dr. J. R. Halpert for
providing the cytochrome P450 3A4 plasmid, and Dr. C. B.
Kasper for the CPR plasmid.
[20] D. J. Ramon, M. Yus, Curr. Org. Chem. 2004, 8,
149–183.
[21] T. Katsuki, K. B. Sharpless, J. Am. Chem. Soc. 1980,
102, 5974–5976.
[22] W. Zhang, J. L. Loebach, S. R. Wilson, E. N.
Jacobsen, J. Am. Chem. Soc. 1990, 112, 2801–2803.
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6
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Advanced Synthesis & Catalysis
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10.1002/adsc.201700637
Advanced Synthesis & Catalysis
FULL PAPER
Regioselective Epoxidations by Cytochrome
P450 3A4 Using a Theobromine Chemical
Auxiliary to Predictably Produce N-Protected β-
or γ- Amino Epoxides
Adv. Synth. Catal. Year, Volume, Page – Page
Vanja Polic, Kin Jack Cheong, Fabien Hammerer
and Karine Auclair*
8
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