Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis

Article abstract of DOI:10.1016/j.bmc.2014.05.046

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard. Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon en route to various natural products further expanding the light-activated P450 BM3 biocatalysis and highlighting the advantages over traditional methods.

Full text of DOI:10.1016/j.bmc.2014.05.046

Accepted Manuscript
Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-
driven P450 BM3 biocatalyst yielding a valuable synthon for natural product
synthesis
Mallory Kato, Daniel Nguyen, Melissa Gonzalez, Alejandro Cortez, Sarah E.
Mullen, Lionel E. Cheruzel
PII:
S0968-0896(14)00404-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.046
BMC 11607
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
23 April 2014
21 May 2014
Please cite this article as: Kato, M., Nguyen, D., Gonzalez, M., Cortez, A., Mullen, S.E., Cheruzel, L.E., Regio- and
stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable
synthon for natural product synthesis, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/
j.bmc.2014.05.046
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Regio- and stereoselective hydroxylation of
10-undecenoic acid with a light-driven P450 BM3
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biocatalyst yielding a valuable synthon for natural product synthesis
Mallory Kato, Daniel Nguyen, Melissa Gonzalez, Alejandro Cortez, Sarah E. Mullen, and Lionel E. Cheruzel
One Washington Square, San Jose, CA 95192-0101, United States

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven
P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis
Mallory Katoa , Daniel Nguyena, Melissa Gonzaleza, Alejandro Corteza, Sarah E. Mullena, and Lionel E.
Cheruzela,∗
a San Jose State University, One Washington Square, San José, CA 95192-0101, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid
P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a
monohydroxylated product is detected by gas chromatography. Hydroxylation occurs
exclusively at the allylic position as confirmed from a synthesized authentic standard.
Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained
in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon
en route to various natural products further expanding the light-activated P450 BM3 biocatalysis
and highlighting the advantages over traditional methods.
Received in revised form
Accepted
Available online
Keywords:
Light-driven P450 BM3 biocatalyst
Stereoselective hydroxylation
Allylic oxidation
2009 Elsevier Ltd. All rights reserved.
(R)-9-hydroxy-10-undecenoic acid
Natural product synthesis
1. Introduction
have recently been extended beyond monooxygenation toward
olefin cyclopropanation6 and amination.7
Biocatalytic processes are gaining increasing importance in
organic synthesis from their unique selectivity advantages over
traditional methods.1 Over the last two decades, biocatalysis has
emerged as an important technology for meeting the demand for
green and sustainable synthesis of pharmaceuticals and other fine
chemicals. Among the various biocatalytic reactions comprising
hydrolytic, reductive and oxidative reactions, the selective
oxidations of unactivated C-H bonds catalyzed by cytochrome
P450 enzymes are particularly attractive.
The synthetic potential of P450 enzymes has long been
recognized8 and recently exploited in the synthesis of several
drugs such as the antimalarial artemisinin,9 important
metabolites10 and other fine chemicals.11 However their industrial
and biotechnological applications is still limited mainly due to
the dependence on the electron providing reductase and the need
of stoichiometric amounts of the expensive NAD(P)H cofactor.
To overcome these limitations, various approaches have
emerged. Great advances have been made in the use of whole cell
biocatalysis, for example in the large-scale production of
nootkatone from valencene by P450 enzymes12 or in the
oxidation of alpha-pinene.13 Other approaches have employed
NAD(P)H regeneration systems,14 artificial fusion P450
proteins,15 direct chemical16 or electrochemical reduction17, and
light-activated systems18 to drive P450 reactions.
The cytochrome P450 superfamily of heme-thiolate enzymes
is known to perform a myriad of C-H bond functionalizations,
often with high regio- and stereoselectivity, using molecular
dioxygen and two reducing equivalents.2 The consensus
mechanism involves two successive one-electron transfer steps
from the electron providing reductase to the heme center and
activation of molecular dioxygen to form several well-
characterized intermediates.3 The high-valent Fe(IV)-oxo
porphyrin radical species, Compound I, achieves the desired C-H
bond functionalization via a rebound mechanism.4
We have recently reported
a light-driven P450 BM3
biocatalyst (WT/L407C-Ru1) displaying high photocatalytic
activity and initial reaction rate in the hydroxylation of lauric
acid.19 This hybrid P450 BM3 enzyme exploits the photophysical
properties of a strategically positioned photosensitizer, i.e.
[Ru(OMebpy)2PhenA]2+ (Ru1) with OMebpy = 4,4’-methoxy-
2,2’-bipyridine and phenA = 5-acetamido-1,10-phenanthroline. A
highly reductive Ru(I) species is generated, under flash quench
reductive conditions, which provides the necessary two electrons
in successive one-electron transfer steps to the heme domain in
Among the large and diverse family of P450 enzymes, the
fatty acid hydroxylase CYP102A1 or P450 BM3 from Bacillus
megaterium with its fused reductase has been shown to be an
important design platform for biocatalysis due to the plasticity of
its active site and its ability to accommodate a wide range of
substrates upon protein engineering.5 In addition, P450 reactions
———
∗ Corresponding author. Tel.: +1-408-924-5283; fax: +1-408-924-4945; e-mail: lionel.cheruzel@sjsu.edu

order to activate molecular dioxygen at the heme center and
sustain catalysis upon light irradiation.
The proposed compounds were prepared by either of two
routes: 1) allylic oxidation of 10-UA using stoichiometric
amounts of SeO2 and tert-butyl peroxide21 followed by
methylation of the acid moiety using trimethylsilyldiazo-
methane22 or 2) methylation of 10-UA followed by allylic
oxidation of the ester.23 Both routes led to valuable intermediates,
i.e. racemic mixture of 9-hydroxy-10-undecenoic acid (( )-1) as
well as its methyl ester (( )-2), for the characterization of the
enzymatic product. The 1H and 13C NMR spectra for all the
synthesized compounds match those previously reported.
Our current efforts to expand the scope of reactivity and
substrate recognition of the light-driven enzymes led to the
investigation of 10-undecenoic acid (10-UA) as a substrate. With
its terminal double bond, this substrate presents the opportunity
to probe epoxidation and hydroxylation reactions. Under the
current photochemical conditions, the hydroxylation of 10-UA
resulted in only one monohydroxylated product (Figure 1). In
addition, the determination of its absolute configuration renders
the oxidized product a valuable synthon for the synthesis of
various natural products.
2.3. Light-driven enzymatic substrate oxidation
An aerated solution (10 mL) containing 2 µM of the hybrid
enzymes with 500 µM of 10-undecenoic acid (10-UA) and 100
mM sodium diethyldithiocarbamate (DTC) was exposed to
visible light irradiation from an Orion 1000 W Xenon arc lamp
with IR- and UV-cutoff filters. The solution was maintained at 20
°C using a water bath. After 90 min of light exposure, the
reaction mixture was quenched by addition of HCl and the
product was extracted with dichloromethane. A small aliquot
(500 µL) was evaporated and the residue was silylated using
N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA)/Pyridine for
GC/MS analysis.20 The remainder of the solution was subjected
to methylation using trimethylsilyldiazomethane. The methylated
product was separated from the starting material by flash
chromatography (Elution solvent: hexane/ethyl acetate 10:1,
Rf=0.4). Authentic samples prepared in Section 2.2 were used as
reference.
2.4. Determination of the stereoselectivity of the enzymatic
reaction
Figure 1. Schematic representation of the light-driven hybrid WT/L407C-
Ru1 P450 BM3 heme domain biocatalyst. Upon light activation, the Ru(II)
photosensitizer is quenched by sodium diethyldithiocarbamate (DTC) to
provide the necessary electrons to the heme domain, which performs the
selective hydroxylation of 10-undecenoic acid (10-UA).
Both the racemic methanoate ester (( )-2) from Section 2.2.
and the enzymatic hydroxylated methanoate ester from Section
2.3. were transformed to their Mosher ester following reported
procedure24 using (S)-(+)-α-Methoxy-α-trifluoromethylphenyl-
acetyl chloride (MTPA-Cl). Briefly, dry pyridine (2 µL) and
MTPA-Cl (2 µL) were added to a CDCl3 solution of the
hydroxylated methanoate ester. The progress of the reaction was
followed by TLC (hexane/ethyl acetate 10:1). At the end of the
reaction, the products were purified by flash chromatography
(hexane/ethyl acetate 10:1). The resulting Mosher esters were
submitted to chiral analysis using the Chiralpak AD column
connected to a Dionex Summit HPLC system (Elution solvent:
92% hexane, 8% isopropylalcohol).
2. Materials and Methods
All reagents and solvents used in this work were of analytical
grade and purchased from Thermo Fisher Scientific and Sigma-
1
Aldrich. H and 13C NMR were recorded on a Varian 400 MHz
NMR spectrometer. The fatty acid analysis was performed, after
silylation of the acid and alcohol groups, on an Agilent 6890N
gas chromatography (GC) system equipped with a flame
ionization detector and an HP-5MS capillary column. The
stereochemistry of the fatty acid Mosher esters was determined
on a Chiralpak AD column (0.46 x 25 cm) connected to a Dionex
Summit HPLC system.
3. Results
3.1. Regioselective oxidation of 10-undecenoic acid by light-
driven WT/L407C-Ru1 enzyme
2.1. Hybrid enzyme generation
Following the flash quench reductive conditions previously
reported,20 we investigated the functionalization of 10-
undecenoic acid (10-UA) by the hybrid P450 BM3 WT/L407C-
Ru1 enzyme upon light activation. Determination of the binding
constant (Kd = 780 µM) reflects poorer substrate binding
compared to its saturated counterpart.20 While the oxidation of
dodecanoic acid led previously to three mono hydroxylated
The purification of the P450 BM3 heme domain mutant
(WT/L407C) expressed in E.coli and the generation of the hybrid
enzyme have been reported previously.19,20 Upon gel purification
of the WT/L407C heme domain, fractions with a Rz ratio
(A420/A280) greater than 1.5 were pooled. The hybrid enzyme
WT/L407C-Ru1 is assembled by reacting the iodoacetamide
derivative of the [Ru(OMebpy)2PhenA]2+ (Ru1) complex (with
products at subterminal positions,19 only
a sole mono-
OMebpy = 4,4’-methoxy-2,2’-bipyridine and phenA
= 5-
hydroxylated product is obtained with 10-UA as substrate (40%
yield) in the light-driven reaction as shown in Figure 2. The
nature of the hydroxylated product can be assigned to the 9-
hydroxy-10-undecenoic acid (1) based on the mass fragmentation
pattern after derivatization as established previously for the lauric
acid.20 An authentic sample of 1 was also prepared showing the
same retention time and fragmentation pattern, unambiguously
confirming the nature of the proposed hydroxylated product. The
initial reaction rate of 6 mol of product/mol of enzyme/min,
acetamido-1,10-phenanthroline) with an engineered non-native
cysteine residue (L407C) of P450 BM3 heme domain mutant.
The covalent attachment was confirmed by mass spectrometry,
UV-vis and fluorescence spectroscopic techniques, as well as by
chymotrypsin digestion of the hybrid enzymes.19,20
2.2. Synthesis of the racemic 9-hydroxy-10-undecenoate (( )-1)
and its methyl ester (( )-2)

determined after 1 min reaction, is much lower than the one
observed for the light-driven hydroxylation of lauric acid19 but
similar to the one reported for the full-length enzyme using
NADPH.25
6.1 min as shown in Figure 3. Following similar procedures, we
investigated the stereochemistry of the hydroxylated product
from the light-activated enzymatic reaction. Mainly the R
enantiomer was obtained (85% ee) consistent with the previous
report on the saturated fatty acid counterpart.26
Figure 3. Chromatographic resolution of the enantiomers from the synthetic
standard (blue) and the monohydroxylated product generated during the light
Figure 2. A) Gas chromatograms of the trimethylsilylated mixture from the
light-driven reaction (blue) and authentic sample (red). 12-
hydroxydodecanoic acid was used as internal standard (I.S., 10 nmol) for
product quantification. B) Mass fragmentation of the peak at 14.3 min with
the corresponding structure indicating the major fragments observed.
reaction (red) after conversion to their respective Mosher esters.
4. Discussion
P450 BM3 enzyme is known to catalyze the regio- and
stereoselective oxidation of various saturated and unsaturated
long chain fatty acids.5 Interestingly, terminally unsaturated long
chain fatty acids have not received much attention. Early report
indicated that terminal unsaturated substrates of various chain
lengths resulted in rapid enzyme inactivation.27 Chen et al.
recently reported that allylic hydroxylation of 10-undecenoic acid
(10-UA) is preferred under turnover conditions with small
amount of epoxidation at the terminal unsaturation.25 Depending
on the method of Compound I generation in this study, a change
in the product ratio between hydroxylation and oxidation is
observed. We have been interested in studying the oxidation of
the 10-undecenoic acid using our light-activated P450 BM3 heme
domain biocatalyst, WT-L407C-Ru1. We recently demonstrated
that this light-activated approach is valuable in the hydroxylation
of lauric acid, a C12 fatty acid yielding high photocatalytic
activity and initial reaction rate.19 In addition, the ratio of
hydroxylated products in the photocatalytic reaction is identical
to that observed with the wild-type heme domain.
3.2. Synthesis of the racemic standard
A racemic mixture of the proposed hydroxylated product (1)
can also be prepared synthetically from the allylic oxidation of
10-undecenoic acid. The compound is easily obtained using
stoichiometric amount of Selenium dioxide and tert-
butylhydroperoxide in reasonable yield (40%) after stirring for 48
hours at room temperature.21 Once purified, Compound 1 can be
prepared for stereochemical analysis by converting to its methyl
ester (2) using trimethylsilyldiazomethane and further to its
Mosher ester (3) using (S)-(+)-α-Methoxy-α-trifluoromethyl-
phenylacetyl chloride (MTPA-Cl) in CDCl3 according to Scheme
1.
3.3. Investigation of the stereoselectivity of the hydroxylated
product
The synthetic racemic Mosher esters (3) from Section 3.2
Under the photocatalytic conditions previously developed, the
oxidation of 10-UA by the light activated WT/L407C-Ru1 hybrid
enzyme leads to a single monohydroxylated product as shown in
the GC chromatogram (Figure 2). From the mass fragmentation
pattern, the hydroxylation occurs exclusively at the 9 position as
also confirmed with the synthetic analog. It is worth noting that
under the photocatalytic conditions, no reduction of the starting
material is observed. In addition, no epoxidation product is
detected in the GC chromatograms. As shown previously,
Scheme 1. Synthetic routes to convert 9-hydroxy-10-undecenoic acid (1)
obtained from either light driven enzymatic reaction (a) or synthetically (b) to
its methyl (2) and Mosher (3) esters.
were then analyzed on a chiral HPLC column leading to the
elution of the R enantiomer at 5.1 min and the S enantiomer at

6. Acknowledgments
absence of light or reductive quencher do not show any product
formation.
The authors would like to thank the National Institute of
Health (GM095415) for financial support. This research was also
supported by an award from Research Corporation for Science
Advancement. L.C. thanks the National Science Foundation
(MRI grant 0923573) and San José State University for the use of
mass spectrometry facilities in the PROTEIN LAB.
While obtaining a single hydroxylated product is attractive,
the possibility of having one of the two enantiomers from the
enzymatic reaction would represent a high added value. Besides
being a natural product, cochadylonic acid,28 the (R)-9-hydroxy-
10-undecenoic acid ((+)-1) is also a useful synthon for the
synthesis of various natural products as illustrated in Scheme 2.
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a
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