Predictable stereoselective and chemoselective hydroxylations and epoxidations with P450 3A4

Article abstract of DOI:10.1021/ja200551y

Enantioselective hydroxylation of one specific methylene in the presence of many similar groups is debatably the most challenging chemical transformation. Although chemists have recently made progress toward the hydroxylation of inactivated C-H bonds, enzymes such as P450s (CYPs) remain unsurpassed in specificity and scope. The substrate promiscuity of many P450s is desirable for synthetic applications; however, the inability to predict the products of these enzymatic reactions is impeding advancement. We demonstrate here the utility of a chemical auxiliary to control the selectivity of CYP3A4 reactions. When linked to substrates, inexpensive, achiral theobromine directs the reaction to produce hydroxylation or epoxidation at the fourth carbon from the auxiliary with pro-R facial selectivity. This strategy provides a versatile yet controllable system for regio-, chemo-, and stereoselective oxidations at inactivated C-H bonds and demonstrates the utility of chemical auxiliaries to mediate the activity of highly promiscuous enzymes.

Full text of DOI:10.1021/ja200551y

ARTICLE
pubs.acs.org/JACS
Predictable Stereoselective and Chemoselective Hydroxylations
and Epoxidations with P450 3A4
Aaron T. Larsen, Erin M. May, and Karine Auclair*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montrꢀeal, Quꢀebec, Canada H3A 2K6
S Supporting Information
b
ABSTRACT: Enantioselective hydroxylation of one specific methylene in the presence of
many similar groups is debatably the most challenging chemical transformation. Although
chemists have recently made progress toward the hydroxylation of inactivated CꢀH bonds,
enzymes such as P450s (CYPs) remain unsurpassed in specificity and scope. The substrate
promiscuity of many P450s is desirable for synthetic applications; however, the inability to
predict the products of these enzymatic reactions is impeding advancement. We demon-
strate here the utility of a chemical auxiliary to control the selectivity of CYP3A4 reactions.
When linked to substrates, inexpensive, achiral theobromine directs the reaction to produce
hydroxylation or epoxidation at the fourth carbon from the auxiliary with pro-R facial
selectivity. This strategy provides a versatile yet controllable system for regio-, chemo-, and
stereoselective oxidations at inactivated CꢀH bonds and demonstrates the utility of
chemical auxiliaries to mediate the activity of highly promiscuous enzymes.
’ INTRODUCTION
Monooxygenases such as cytochrome P450 enzymes (P450s
or CYPs) arguably hold the greatest unexploited synthetic
potential of any family of enzymes.8 P450s are ubiquitous
enzymes with major roles in mammalian drug metabolism,
steroid biosynthesis, and bacterial biosynthesis of secondary
metabolites, which are an exceptional source of pharmaceutical
agents. Chemists have a special appreciation for P450s because of
their impressive ability to catalyze selective hydroxylations at
inactivated CꢀH bonds. To date, however, this potential has
remained highly untapped. Commercial applications using P450
enzymes are currently rare and limited to using whole cells.9ꢀ13
Synthetic applications of P450s have been hindered mainly by
the need for expensive cofactors, low turnover, and poor stability.
These limitations have recently been partly overcome by rational
and random mutagenesis,14ꢀ16 yet the use of free enzymes
remains nonideal for industrial processes. Research applications
of P450s are more accessible considering the smaller scale.
Unlike industrial settings, research environments require versa-
tile and promiscuous, yet controllable, catalysts. Mutagenesis has
mostly been used to modify the substrate specificity of
P450s,14ꢀ16 but rarely to increase their substrate promiscuity.17
Although numerous P450s are highly promiscuous, product
predictions are speculative at best. This problem is of special
concern for drug-metabolizing P450s and has beleaguered the
pharmaceutical industry for half a century.18
Natural systems are well recognized for streamlining the
synthesis of large and complex molecules by iterative CꢀC bond
formation reactions followed by tailoring oxidations at selective
positions. Although dramatic progress has recently been made,
such a strategy is not yet generally available to chemists, the main
difficulty being the poor selectivity achievable for oxidations at
inactivated CꢀH bonds. In particular, the stereoselective hydro-
xylation of one methylene group in the presence of similar
methylenes is probably the most challenging synthetic transfor-
mation at the present time. Recent successes by chemists in this
area include the regio- and stereoselective hydroxylation of
pleuromutilin1 and the total synthesis of deoxyerythronolide B
by late-stage CꢀH oxidations.2 The successful methodology uses
a bulky, electrophilic iron catalyst and hydrogen peroxide as the
oxidant but typically suffers from overoxidation and limited
functional group tolerance. The main difficulty arises in the fact
that selective hydroxylation of tertiary carbons is favored over
reactions at methylene groups. Recent advances in the hydro-
xylation of inactivated CꢀH bonds have thus focused on
reactions at tertiary sp3 carbons.3ꢀ7 The electron-rich nature of
tertiary CꢀH bonds and their relative rarity in comparison to
methylene and methyl carbons in natural products have made
them excellent targets for metal-catalyzed hydroxylation. To our
knowledge, however, no strategy using chemical catalysts has
been successful at achieving predictable hydroxylation at one
inactivated methylene CꢀH bond among others of similar
electronic properties. Enzymes remain unsurpassed at this task.
Not only are enzymes typically regio-, chemo-, and stereoselec-
tive, but they often perform well in mild aqueous conditions with
innocuous oxidants, providing an environment-friendly alterna-
tive to organometallic reagents.
The availability of a versatile catalyst for regio-, chemo-, and
stereoselective hydroxylations of inactivated methylene groups
would create a paradigm shift in synthetic chemistry. Such an
advance would however require that the catalyst show reasonable
Received: January 19, 2011
Published: April 29, 2011
r
2011 American Chemical Society
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Figure 1. Auxiliary design, attachment to the substrate, and cleavage from the product. (A) The auxiliary was designed to fit a binding pocket (the
specificity site) of CYP3A4 near the heme group and display the substrate to orient its pro-R face on the proximal face of the reactive iron species. Like a
ruler, the distance between the auxiliary and the iron is expected to control the position of hydroxylation relative to the auxiliary. (B) A desired auxiliary
should be easy to link to the substrate, yet easily cleaved from the product. The successful auxiliary described here is linked to the substrate via
substitution under basic conditions. (C) The hydrophobicity and the chromophoric properties of this auxiliary facilitate recovery of the product from the
enzymatic aqueous solution. Next the oxidized product is easily cleaved from the auxiliary.
substrate promiscuity, yet provide highly predictable selectivity.
Herein, we report a successful strategy to achieve CꢀH bond
hydroxylations and double bond epoxidations with predictable
regio-, chemo-, and stereoselectivity. To this end, we combined
the oxidative power of CYP3A4 with the positioning and
orienting effects of an inexpensive, easily functionalized, achiral,
cell-permeable auxiliary.
chemo-, regio-, and stereoselectivity. The optimal chemical
auxiliary should fit a binding pocket of CYP3A4 near the heme
group and display one selected CꢀH bond of the substrate
toward the reactive iron species (Figure 1A). Like a ruler, the
distance between the auxiliary and the iron should control the
position of hydroxylation. Facial selectivity is also expected to
arise from the orientation of the auxiliary relative to the heme
prosthetic group.
As inspiration for potential auxiliaries, we considered the
structures of several natural substrates of CYP3A4. A number
of unsuccessful auxiliaries were tested (e.g., coumarins and
fluorescein) until lisofylline attracted our attention. Lisofylline,
a molecule with multiple therapeutic activities,24ꢀ30 is metabo-
lized in part by human CYP3A4 via hydroxylation at the fourth
carbon from the theobromine group.31 We therefore envisioned
that theobromine (1; Figure 1B) may be a useful auxiliary if
oxidation is desired at the fourth atom of the substrate from the
point of attachment to the auxiliary.
Theobromine is achiral and inexpensive, contains a chromo-
phore, and can penetrate cells. Moreover, it has only one
nucleophilic group (a nitrogen atom) and is therefore readily
linked to various substrates (Figure 1B). Importantly, this auxiliary
is also easily cleaved off after enzymatic reactions, with the
generation of an amine or other functional groups depending on
’ RESULTS AND DISCUSSION
Despite decades of sustained investigations (mainly from the
pharmaceutical industry), the rules that govern binding selec-
tivity by CYP3A4 and other substrate-promiscuous P450s re-
main nebulous. Auxiliaries have been very successful in chemical
synthesis, and we envisaged that they may provide a new strategy
to control the selectivity of P450 enzymes such as CYP3A4
(Figure 1A). A few research groups have reported the use of
substrate engineering in biocatalysis, but mostly to increase
recognition of non-natural substrates, and not for rational control
of selectivity.19ꢀ22 To our knowledge, the only example with
P450 enzymes used a carbolide to “anchor” macrolides into P450
PikC.23 The method did not however control the site of
oxidation and generated multiple products. The auxiliary pre-
sented here is not only expected to anchor the substrate into the
enzyme but to also allow predictable control of the reaction
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Table 1. Results of CYP3A4 Enzymatic Transformations of
SubstrateꢀAuxiliary Molecules
Figure 2. Analysis of the CYP3A4 enzymatic transformations.
(A) HPLC trace of an enzymatic reaction mixture showing the oxidized
products and the substrateꢀauxiliary complex. (B) Chiral HPLC trace
showing the clean separation of the two enantiomers of the major
enzymatic product. See the Supporting Information for the complete list
of traces.
or epoxidation. No product resulting from overoxidation (carbonyl
product) could be detected (<0.1%). Furthermore, the presence of
characteristic theobromine fragments verified that all oxidations
occurred on the substrate portion of the molecule, and not on the
theobromine auxiliary. All products were isolated and purified by
achiral chromatography. HPLC analysis on a chiral column
(Figure 2B) was used to quantify the enantiopurity of each
enzymatic product. The chemical structure of the products and
absolute configuration at the oxidation site were determined by
comparison with synthetic authentic standards.
Careful analysis of the MS fragmentation patterns provided
information about the site of oxidation and was used to plan the
syntheses of authentic enantioenriched standards. Authentic
alcohol standards were synthesized using a divergent synthetic
scheme (Scheme 1A). The racemic synthetic alcohols thus
obtained were separated on a chiral HPLC column, and their
absolute configuration was determined by a well-established
technique32 involving esterification with enantiopure acids
of opposite configuration ((R)-MPA and (S)-MPA; MPA =
R-methoxyphenylacetic acid) to yield a pair of diastereoiso-
mers for each alcohol, followed by comparison of the related
diastereoisomers by NMR. Authentic epoxide standards were
synthesized by introducing chirality to terminal epoxides
using hydrolytic kinetic resolution33 followed by coupling of
the enantioenriched epoxides 34 and 35 to compound 1
(Scheme 1B). The fully characterized authentic enantioenriched
standards were assigned to CYP3A4-hydroxylated products on
the basis of their retention times and after coinjection on a chiral
HPLC column.
a Tb is used to abbreviate the theobromine auxiliary. b Determined by
comparison to authentic standards. See the Supporting Information for
reaction conditions. All reactions were performed at least in duplicate.
c Percentage of oxidation at the fourth carbon from Tb compared to all
other oxidation products. d Refers to the stereochemistry at the fourth
carbon from Tb (or at the second carbon from Tb for 19). e To a limit of
detection of 0.1%. f Not applicable.
the conditions used (Figure 1C). To explore the potential of
theobromine as a chemical auxiliary for the control of CYP3A4
oxidations, theobromine was functionalized with various small
substrates before reaction with CYP3A4. Alkanes and alkenes
were selected because of their notorious inertness and of their
multiple undifferentiated CꢀH bonds. Moreover, the expected
alcohol products represent versatile chiral synthons with a
potentially high commercial value. Under basic conditions
(Figure 1B) a series of alkyl halides were reacted with theobro-
mine to generate a range of substrateꢀauxiliary complexes
(compounds 2ꢀ13, Table 1) in very good yields.
Compounds 2ꢀ13 were next allowed to react with CYP3A4 and
cofactors in buffer. Product formation and substrate conversion
were monitored by HPLC and LCꢀMS (Figure 2A). For all
productsdetected, themassincreasecorrespondedtohydroxylation
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Scheme 1. Synthetic Schemes for the Preparation of Selected
Chiral Alcohol (A) and Epoxide (B) Authentic Standardsa
influence the structure and ratio of products (see the Supporting
Information for comparative HPLC traces). After optimization
of the conditions, substrate conversion rates were typically
around 70% on the basis of HPLC (Figure 2A and Supporting
Information), and isolated yields ranged from 40% to quantita-
tive (Table 1).
As predicted, oxidation is detected at the fourth carbon
position relative to the theobromine auxiliary for all compounds
listed in Table 1, except for compounds 2 and 3, which were
designed not to have a fourth methylene carbon. Moreover,
oxidation proceeds with a distinct preference for the pro-R face of
the substrate and stops at the alcohol or the epoxide. Over-
oxidation products are not detected (on the basis of LCꢀMS
analysis of crude mixtures). We expected compound 2 to be too
short (no fourth carbon) to achieve the necessary proximity to
the heme iron active species, and as expected it is not transformed
by CYP3A4 (<0.1%). Compound 3 was designed to have a
methyl group at the fourth carbon from the auxiliary. There are
very few reports of methyl oxidations by this enzyme;35 however,
the absence of detectable products for the CYP3A4 oxidation of 3
(<0.1%), together with epoxide formation of compound 12 (also
four-carbon chain but terminal sp2 CH2 instead of CH3),
suggests that electronic factors contribute to the lack of reactivity
at the methyl group of 3. The reduced reactivity of methyl
compared to methylene groups is expected from the bond
dissociation energy and also well understood for radical pro-
cesses such as those catalyzed by iron-based small molecules.36,37
Overall, the fourth-carbon rule consistently allows prediction of
which methylene group is hydroxylated by CYP3A4.
The scope of this system was further investigated with
compounds containing branching, unsaturations, or a hetero-
atom at or near the position of oxidation. The tertiary carbon of
compound 6 is oxidized by CYP3A4 to a single product (>95%),
compound 16, indicating the potential utility of this system in the
production of enantiopure tertiary alcohols. Racemic com-
pounds 7 and 8 are also hydroxylated at the fourth carbon from
the auxiliary (products 17 and 18), in spite of the branching point
at either the third or second carbon, respectively. It was inter-
esting to see that the enzyme accepted both enantiomers of
compounds 7 and 8 as substrates, although one is transformed
with a slight preference over the other (5:2 and 4:3 for 7 and 8,
respectively, absolute configuration not determined). The
branching point of compound 9 forms a heterocyclic ring. As
anticipated, CYP3A4 proceeds to hydroxylate 9 at the fourth
carbon from the auxiliary, with very high selectivity for one fourth
carbon (counting clockwise, R to the ether) over the other
(counting counterclockwise, β to the ether). This preference was
expected on the basis of electronics. Hydroxylation at that
position yields a hemiacetal which spontaneously opens to the
corresponding γ-hydroxy aldehyde (19). The chirality of this
product arises from the hydroxyl group, the configuration of
which is already determined at the branching point before
enzymatic transformation. Here again CYP3A4 accepts both
enantiomers of racemic 9, and as expected for a quantitative
transformation, a racemic mixture is obtained. At low conversion
rate, however, analysis of the mixture shows a slight preference
for one enantiomer (configuration not determined; see the
Supporting Information). Although CYP3A4 may not be suitable
for resolutions, its tolerance for different enantiomers of the
same substrate further demonstrates its substrate promiscuity,
which has the advantage here of allowing the generation of
different diastereoisomers for comparison. Also in line with the
a Racemic alcohols were separated by chiral HPLC. Their absolute
stereochemistries were determined by comparing 1H NMR spectra after
functionalization with (R)- or (S)-R-methoxyphenylacetic acid. Com-
pound names with “a” were prepared from (R)-(ꢀ)-R-methoxyphe-
nylacetic acid, and those labeled with “b” were prepared from (S)-(þ)-
R-methoxyphenylacetic acid. For the definition of Co(OAc)Salen,
see ref 33.
Table 1 lists all products obtained from CYP3A4 transforma-
tions for each of compounds 2ꢀ13. Our research group has
reported that the reaction rate of human CYP3A4 is improved by
up to 30% when the natural cofactors, NADPH and cytochrome
P450 reductase (CPR), are replaced with the inexpensive cumene
hydroperoxide (CHP) or sodium percarbonate (SPC).34 To take
advantage of this, enzymatic transformations were carried out
using CHP as a cofactor surrogate. A series of separate experi-
ments using NADPH/CPR revealed that the choice of cofactor
used did not affect the stereoselectivity of the reaction, nor did it
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Figure 4. Results of docking studies. Proposed substrateꢀauxiliary
orientation relative to the heme group in the enzyme pocket, as
suggested by docking studies (see the text for details).
Figure 3. Results of CYP3A4 enzymatic transformations of substrates
lacking the auxiliary. None of these compounds were transformed under
the same conditions described for Figure 2. From Chefson et al.39 for the
last seven compounds.
study.39 None of the molecules were transformed by the enzyme to
any detectable level (<0.1%) without the theobromine auxiliary.
Finally, to determine if the above-described enzymatic selec-
tivity was specific to CYP3A4, enzymatic reactions were per-
formed with human CYP2D6. Although compound 12 was
transformed to the corresponding epoxide, 5 and 13 were
hydroxylated to mixtures of compounds, and other substrateꢀ
auxiliary systems were not oxidized to any detectable level by
CYP2D6. Reported crystal structures40ꢀ43 and drug metabolism
data for these two enzymes also support a different selectivity.
In an attempt to better understand the reasons for the
observed regio- and stereoselectivites, we performed in silico
docking studies using the entire scope of substrateꢀauxiliary
complexes, 2ꢀ13, and a crystal structure of CYP3A4 available in
the protein database.41ꢀ43 Although P450 enzymes are known to
be difficult to handle with most in silico applications,44 this
docking survey led to a structure that is consistent with our
results. Figure 4 shows the auxiliary bound in proximity to the
CYP3A4 heme group with the oxidized CꢀH bond nearest to
the heme iron distal side.
fourth-carbon rule and the pro-R facial selectivity is the formation
of compounds 20 and 21 from 10 and 11, respectively. Con-
sidering that epoxidation of double bonds is thermodynamically
favored over sp3 CꢀH bond hydroxylation, these examples
demonstrate the functional group tolerance of the system.
Indeed no epoxidation product was detected (<0.1%) for the
transformation of compound 11 by CYP3A4, and LCꢀMS
fragmentation suggests that the terminal epoxide is a minor
product (<35%) of oxidation of 10 by CYP3A4 (this explains the
low isolated yield, 45%, for product 20). The functional group
tolerance of this enzyme was long established from the selective
hydroxylation of approximately half of all clinical drugs, many of
which have numerous orthogonal functional groups.38
Enantioselective epoxidation of terminal olefins is especially
challenging.33 The use of our auxiliary to control the selectivity of
terminal epoxidations was investigated. Two compounds were
designed to include a double bond either between C-3 and C-4
(compound 12) or between C-4 and C-5 (compound 13). Both
underwent CYP3A4-catalyzed epoxidation at the terminal dou-
ble bond. Lower conversion rates account for the suboptimal
isolated yields (40ꢀ50%). Further optimization of the reaction
conditions may be needed for epoxidations. Unreacted starting
material could however be isolated and recycled. While com-
pound 13 generated the enantiopure (>99%) product 23 with
the expected R-configuration at the chiral carbon, compound 12
was oxidized to the racemic epoxide 22. This is consistent with
the stereoselectivity rules proposed above: stereocontrol is
believed to take place at the fourth carbon from the auxiliary,
which is not a prochiral carbon in compound 12.
’ CONCLUSION
In summary, we have established a proof-of-concept for the
utility of chemical auxiliaries to control the selectivity of enzy-
matic reactions. We have demonstrated that inexpensive achiral
theobromine (1) is a powerful chemical auxiliary for CYP3A4
transformations, allowing prediction of the site of oxidation and
the facial selectivity. In all cases, oxidation at the fourth carbon
from the auxiliary and Pro-R facial selectivity are favored. The
isolated yields were on the order of ∼70% with purified enzyme
at synthetically useful scales, yet we believe that this strategy
should also find use with whole-cell transformations. Indeed,
theobromine and derivatives similar to compounds 2ꢀ13 are
known to cross cell membranes.45 This strategy provides easy
access to small, enantioenriched or enantiopure, alcohols and
epoxides that are otherwise difficult to access, often requiring
either expensive enantioselective separation or low-yielding
hydrolytic kinetic resolution. Although limited to substrates
not considerably larger than the auxiliary, this method has the
added advantages of not yielding overoxidation products and of
being functional group tolerant (intrinsic to this enzyme).
To demonstrate the synthetic utility of our strategy, scaled up
experiments were performed. We were pleased to find that the
transformation of 100 mg of 4 affords 14 in 63% isolated yield
after purification by flash chromatography (see the Supporting
Information for details). Moreover, the regioselectivity and
enantioselectivity are similar at both scales.
The necessity of the auxiliary was verified by reacting CYP3A4
with substrates lacking the theobromine group. A number of
commercially available molecules resembling the substrate part
of compounds 2ꢀ13 were tested for transformation by CYP3A4
(Figure 3). Also included in Figure 3 are data from a previous
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Chemical methods for selective oxidations at methylene groups1,2
preferentially lead to ketone formation and show poor functional
group tolerance. Conversely, no carbonyl-containing products
were detected here (<0.1%), and this enzyme has a well-established
extended functional group tolerance.38 This was confirmed here
with the selective methylene hydroxylation in the presence of a
nearby double bond and of the different functionalities present on
the auxiliary itself. P450 enzymes, including CYP3A4, are known
to catalyze N- and O-demethylation, yet none of the N-methyl
groups of the theobromine auxiliary were affected during our
transformations. We believe that new auxiliaries can be designed to
afford different regio- and stereoselectivities for CYP3A4 transfor-
mations. This approach should also apply to other P450s, and to
enzymes from other families, to produce a series of biocatalyst/
auxiliary systems, each with complementary selectivities. This
strategy should therefore find broad application in synthesis.
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Corresponding Author
karine.auclair@mcgill.ca
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This work was funded by the National Science and Engineering
Research Council of Canada (NSERC), Merck Frosst Canada Ltꢀee,
Boehringer Ingelheim Canada, and AstraZeneca Canada. A.T.L. was
supported by scholarships from the NSERC, the Dr. Richard H.
Tomlinson Foundation, and the Winnipeg Police Association. We also
thank the Center in Green Chemistry and Catalysis for its support and
A. B. Charette (Universitꢀe de Montrꢀeal, Canada) for suggestions.
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