Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

Article abstract of DOI:10.1016/j.chemphyslip.2013.01.002

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of ¹H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent δ-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and ¹H NMR.

Full text of DOI:10.1016/j.chemphyslip.2013.01.002

Chemistry and Physics of Lipids 167–168 (2013) 21–32
Contents lists available at SciVerse ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Steric analysis of epoxyalcohol and trihydroxy derivatives of
9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis
Christopher P. Thomasa,c, William E. Boeglina, Yoel Garcia-Diazb, Valerie B. O’Donnellc, Alan R. Brasha,∗
a Department of Pharmacology, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA
c Institute of Infection & Immunity, Tenovus Building, UHW, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R-
and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin
and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a
hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and
stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC–MS
analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid
hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S
and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a promi-
nent ␦-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer.
Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-
10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis
product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a prac-
tical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products
using a combination of HPLC, GC–MS and 1H NMR.
Received 31 October 2012
Received in revised form 6 December 2012
Accepted 6 January 2013
Available online xxx
Keywords:
9-HPODE
Trihydroxyoctadecenoic acid
Linoleic acid
Epoxyalcohol synthase
Epoxide hydrolase
Hematin
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
(fatty acid epoxyalcohols), with subsequent hydrolysis producing
a collection of trihydroxy-octadecenoates.
The oxygenation at C9 of linoleic acid is the gateway to a
diverse collection of oxylipin products (Andreou et al., 2009). C9
oxygenation in plants is catalyzed by 9-lipoxygenases (9-LOX),
well-represented among the LOX genes (Feussner and Wasternack,
2002). Linoleate 9-lipoxygenases are also known in prokaryotes
(Andreou et al., 2008; Gao et al., 2010; Zheng et al., 2008), and
are a catalytic activity of arachidonate 12R-LOX in higher animals
(Meruvu et al., 2005; Siebert et al., 2001; Zheng et al., 2011). Among
the many possible transformations of the resulting 9-hydroperoxy-
octadecenoic acid (9-HPODE), in keeping with other fatty acid
hydroperoxides are rearrangements to epoxy-hydroxy derivatives
The non-enzymatic transformation of fatty acid hydroperoxides
to epoxyalcohols is catalyzed by heme or transition metals, result-
ing in a mixture of isomers, as analyzed earlier utilizing 13-HPODE
(Dix and Marnett, 1985; Gardner et al., 1974; Hamberg, 1975). The
present study aims to address the heme transformations with 9-
HPODE as starting material. Enzymatic conversion results in regio-
and stereo-specific formation of epoxyalcohols. Lipoxygenases can
also catalyze epoxyalcohol synthesis (Garssen et al., 1976; Nigam
et al., 2004; Pace-Asciak et al., 1995; Yu et al., 2003), the trans-
formation being favored under anaerobic conditions (Zheng and
Brash, 2010). Heme and lipoxygenase catalysis involves formation
of alkoxyl and epoxyallylic radical intermediates, whereas oxygen
transfer from the hydroperoxide with epoxidation of a remote dou-
ble bond is effected by other enzymes. These include peroxygenase
(Blée et al., 1993; Hamberg and Hamberg, 1996), catalase-related
hemoproteins (Gao et al., 2009; Niisuke et al., 2009), as well as
putative P450-like enzymes that remain to be characterized, for
example in the fish fungus Saprolegnia parasitica (Hamberg et al.,
1986), potato leaves (Hamberg, 1999), and beetroot (Hamberg and
Olsson, 2011).
Abbreviations: CD, circular dichroism; COSY, correlation spectroscopy; DCM,
dichloromethane; DMP, dimethoxypropane; GC–MS, gas chromatography–mass
spectrometry; H(P)ODE, hydro(pero)xyoctadecadienoic acid; IPA, isopropyl alcohol;
LOX, lipoxygenase; RP-HPLC, reversed-phase high pressure liquid chromatography;
SP-HPLC, straight-phase high pressure liquid chromatography; TMS, trimethylsilyl;
UV, ultraviolet.
∗
Corresponding author at: Department of Pharmacology, Vanderbilt University
Medical Center, Nashville, TN 37232-6602, USA. Tel.: +1 615 343 4495;
fax: +1 615 322 4707.
Trihydroxy hydrolysis products are readily formed from fatty
acid epoxyalcohols, and in the case of potential 9-HPODE-derived
E-mail address: alan.brash@vanderbilt.edu (A.R. Brash).
0009-3084/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemphyslip.2013.01.002

22
C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
products, these triols are reported in such diverse settings as; an
in-vivo pathogen response in potato leaves (Göbel et al., 2002;
Hamberg, 1999), in brewing, where their formation contributes
to the bitter flavor of beer (Garbe et al., 2005; Hamberg, 1991b),
as products in mammalian blood vessels and leukocytes (Claeys
et al., 1985; Funk and Powell, 1985), and in preserved meat as a
marker of lipid peroxidation (Püssa et al., 2009). The adjuvant activ-
ity of 9S,12S,13S-trihydroxyoctadecenoic acid (commonly known
as pinellic acid) and its isomers in the administration of flu vac-
cine has also been reported (Shirahata et al., 2003). In addition, our
interests in the transformations of linoleate and its esters through
LOX activity in mammalian epidermis (Zheng et al., 2011) have
focused our attention on the preparation and characterization of
9-HPODE-derived epoxyalcohols and the resulting triols. There are
effective methods for their analysis in the literature, although the
separations cannot be related to typical HPLC methodology, which
was one impetus for our conducting the present study. Hamberg
(1991a) made assignments based on GLC and TLC mobilities cou-
pled with mass spectrometry and chemical degradation (oxidative
ozonolysis, followed by GLC analysis of the fragments). Shirahata
et al. (2006) prepared all possible stereo isomers of pinellic acid by
total synthesis and compared their spectral properties by NMR. The
use of an acetonide derivative to capture the vicinal 12,13-diol in
the 9,12,13-triol is part of this procedure and has been applied in
other studies to identify single naturally occurring fatty acid triols
by NMR (Bruno et al., 1992; Cardellina and Moore, 1980). We make
use of this procedure in our NMR assignments herein.
Accordingly, the objectives of the current study were (i) to
develop a practical method for production and purification of allylic
epoxyalcohols via hematin treatment of 9-hydroperoxy-linoleic
acid, (ii) to characterize the HPLC separation of the trihydroxy-
octadecenoates resulting from hydrolysis of the allylic alcohols, (iii)
to utilize a GC–MS and NMR approach for the assignment of the
trihydroxy-octadecenoate regio- and stereo-chemistry, (iv) to com-
pare the enzymatic synthesis, isomeric distribution and chirality of
epoxyalcohols and triols from 9R- and 9S-hydroperoxy-linoleic acid
in Beta vulgaris, a source of epoxyalcohol synthase (Hamberg and
Olsson, 2011), and (v) to characterize specific delta-ketols as prod-
ucts from the mild acid treatment of allylic trans-epoxyalcohols.
Sigma) for 1 h at room temperature. After a low speed centrifuga-
tion (500 rpm, 10 min) the supernatant was poured through 2 layers
of cheesecloth into a graduated cylinder. Sodium acetate (1 M) pH
5.5 was then added to 10% volume. The resulting enzyme solu-
tion was then suspended in a water bath at 15 ◦C. Linoleic acid in
ethanol (1 g) was added drop wise with a strong stream of oxygen
blowing on the surface of the solution and the reaction stirred for
90 min. The resulting solution was acidified to pH 4.0 and extracted
with dichloromethane (DCM). The DCM was then washed twice
with half volume of H2O and taken to dryness. The 9S-HPODE was
purified using SP-HPLC with a Beckman Ultrasphere silica 10 ␮m
column with an isocratic mobile phase of hexane/IPA/acetic acid
(100:2:0.1).
2.2. Reaction of 9S-HPODE with hematin
A hematin solution (5 mg/ml) was prepared by grinding the
crystals to a fine powder and stirring with 0.1 M K2HPO4 adjusted to
pH 11.0 with NaOH. 9S-HPODE (20 mg in 0.5 ml ethanol) was mixed
with 0.1 M K2HPO4 (20 ml, pH 8.5), 1 ml of the hematin solution
was added and the reaction left for 15 min at room temperature.
The sample was cooled on ice, two volumes of ice-cold DCM were
added, then a predetermined mix of 1 M KH2PO4 (5 ml) and 1 M HCl
solution, sufficient to bring the pH to ∼4.5, followed immediately by
vigorous mixing of the phases. The aqueous layer was removed and
the DCM washed twice with ice-cold water. The DCM was trans-
ferred to a fresh vial, dried under N2, and the sample stored at
−20 ◦C in DCM:MeOH (2:1, v/v) prior to initial purification on an
open-bed silica Bond-Elut cartridge.
2.2.1. Silica cartridge purification of extract
The extract from the hematin reaction was pre-dissolved
in ethyl acetate (2 ml) then hexane (8 ml) was added and the
sample immediately applied to a 1 g Bond-Elut silica cartridge pre-
conditioned with 25% ethyl acetate in hexane. A further 10 ml rinse
of the sample vial with ethyl acetate (25%) in hexane was added and
followed by a further 10 ml 25% ethyl acetate. The products were
then eluted with 100% ethyl acetate (10 ml), and the sample was
dried under N2 and stored for analysis in DCM:MeOH, 2:1.
2.2.2. HPLC purification of hematin products
2. Experimental
The range of products generated from the hematin reaction
was analyzed on an Agilent 1100 series HPLC using a Thomson
2.1. Generation of 9R-HPODE and 9S-HPODE substrates
˚
Advantage Silica 150 A 5 ␮ column (250 mm × 4.6 mm) an iso-
cratic solvent of hexane:IPA:acetic acid (100:2:0.02), a flow rate
of 1 ml/min, with diode array detection at wavelengths of 205, 220,
235 and 270 nm.
2.1.1. 9R-HPODE
Linoleic acid (10 mg) in 0.5 ml ethanol was stirred into 200 ml
50 mM Tris pH 7.5, 150 mM NaCl, 20 mM CHAPS with a strong oxy-
gen stream on the surface of the solution. Anabaena LOX enzyme
(a linoleate 9R-LOX, (Zheng et al., 2008)) was added as 150 ␮l of a
20 mg/ml solution; reaction was complete in 2 min as evidenced by
UV analysis of the conjugated diene chromophore of the product.
The solution was then acidified to pH 4.0 and extracted with ethyl
acetate, washed twice with half volume of water, taken to dryness
and redissolved in a small volume of methanol for storage prior
to HPLC. The 9R-HPODE was purified by semi-preparative SP-HPLC
using a Beckman Ultrasphere silica 10 ␮m column, with isocratic
hexane/IPA/acetic acid (100:2:0.1), a flow rate of 4 ml/min, with UV
monitoring at 235 nm.
2.3. HPLC separation of trihydroxy-octadecenoate products from
allylic epoxyalcohol hydrolysis
2.3.1. Acid hydrolysis of
9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate
To the allylic epoxide dissolved in acetonitrile (5 ml), 1% aqueous
acetic acid (20 ml) was added and mixed thoroughly. After stand-
ing at room temperature for 30 min, the hydrolysis products were
extracted in a C18 Bond Elute cartridge, washed with two volumes
of H2O and eluted with MeOH.
2.3.2. Purification of trihydroxy-octadecenoate products
2.1.2. 9S-HPODE
Initial RP-HPLC analysis employed
a
Waters Symme-
9S-HPODE was prepared using the linoleate 9S-LOX in potato
(Galliard and Phillips, 1971). Yukon Gold potatoes were diced and
blended with 255 ml water. To eliminate starch, the solution was
then stirred with 250 ␮l Tween 80, 320 ␮l Viscozyme L (Sigma)
and 160 ␮l Amyloglucosidase (from Aspergillus niger 300 units/ml,
try C18 column (250 mm × 4.6 mm) eluted isocratically with
methanol/water/acetic acid (80/20/0.01) at 1 ml/min. The 205 nm
absorbing peaks in the triol region of the chromatogram at ∼4 min
retention time were pooled and subsequently resolved using a
Chiralpak AD column (250 mm × 4.6 mm) with an isocratic mobile

C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
23
phase of hexane/methanol/ethanol/acetic acid (86.5/4.5/9/0.1) run
at 1 ml/min. The peak designated as II was further separated using
a Chiralpak OD-H column (250 mm × 4.6 mm) with a mobile phase
of hexane/ethanol/acetic acid, (97.5/2.5/0.1) run at 1 ml/min.
unstable epoxyalcohol products were extracted with diethyl ether
eliminating the requirement for the Sep-Pak chromatography.
2.5.1. Assignment of regio- and stereo-chemistry
1H NMR and GC–MS analysis was carried out as Section 2.4
for triol analysis. Deuterated benzene was used as solvent (locked
on the 7.15 ppm signal of residual C6H6), except for underivatized
triols and their methyl esters, recorded in deuterated methanol
(locked on 3.34 ppm). Epoxyalcohol methyl esters were converted
to the trimethylsilyl derivative by treatment with BSTFA for 1 h (or
overnight) at room temperature.
For CD analysis, approximately 50 ␮g of epoxyalcohol methyl
ester was converted to the 9-O-benzoate ester (1 ␮l benzoyl chlo-
ride, ∼1 mg dimethylaminopyridine in 30 ␮l pyridine, 15 min at
room temperature) and repurified by RP-HPLC (MeOH/H2O, 95:5
solvent, flow rate 1 ml/min, retention time approximately 7 min) as
described (Schneider et al., 1997). The samples were re-dissolved
in acetonitrile and the concentration adjusted to give 1 AU at the
226 nm UV chromophore. CD spectra (200–300 nm) were recorded
using a Jasco J-700 spectropolarimeter (3 scans of each sample
averaged, scan rate 30 nm/min).
2.4. Assignment of regio- and stereo-chemistry of
trihydroxy-octadecenoates via 1H NMR and GC–MS analysis
Methyl esters were prepared by dissolving the products in
MeOH (typically 10 ␮l), followed by addition of several drops of
ethereal diazomethane (∼100 ␮l); after mixing and standing for
30 s on ice the sample is taken to dryness under a stream of nitro-
gen. Methyl esters of the triols derived from hydrolysis of methyl
9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate were purified
as a group by SP-HPLC using a Thomson Advantage Silica col-
umn (25 cm × 0.46 cm), a solvent of hexane/IPA (90:10, by vol.) run
at 1 ml/min, with UV detection at 205 nm and a retention time
of 9–10 min. Acetonide derivatives were prepared by addition of
DMP:acetone (1:1) containing 1 mM p-toluene sulfonic acid to the
dry methyl ester derivatives (reaction is complete within 5 min at
room temperature); the acid was then neutralized using an equal
volume of triethylamine (1.5 mM) in DCM and the sample taken
to dryness. Individual methyl ester DMP derivatives were purified
by HPLC using a Thomson Scientific silica column (25 × 0.46 cm),
a solvent of hexane/IPA (97:3, v/v), and a flow rate of 1 ml/min
with UV detection at 205 nm; the methyl ester DMP derivatives
eluted with retention times close to 5 min. Resolution of mixtures
of triol methyl ester DMP derivatives was achieved using a solvent
of hexane/IPA (100:1, by vol.).
GC–MS analyses were run using an Agilent/J&W DB-5MS col-
umn (25 m × 0.2 mm, 0.33 ␮m film) in a ThermoFinnigan DSQ
instrument operated in EI mode (ion source temperature 250 ◦C,
electron energy 70 eV) set to full scans from m/z 50 to 650.
1H NMR spectra of the DMP derivatized trihydroxy methyl esters
were recorded in C6D6 (150 ␮l solvent in a 3 mm tube) at 25 ◦C using
a Bruker 600 MHz instrument equipped with a 5 mm Z-gradient
TCI Cryo-probe and using pulse program zg with parameter sett-
ings including a pulse length 8.50 ␮s, time domain 32768, and a
relaxation time of 2 s.
3. Results
3.1. Generation and isolation of epoxyalcohols from hematin
treatment of 9-HPODE
Fig. 1A illustrates the RP-HPLC separation of the hematin prod-
ucts from 9S-HPODE. These include four 9,10-epoxy-13-hydroxy
isomers appearing late in the chromatogram, of which the more
prominent are the two trans epoxide isomers, identified earlier
as 9S,10S-trans-epoxy-13S-hydroxy followed by the 9S,10S,13R
diastereomer (Zheng et al., 2011). On either side of the first of
these are the two less abundant 9,10-cis-epoxide isomers labeled
as peaks 6 and 8, the stereochemistry of which can be deduced from
earlier studies as the 9S,10R-cis-epoxy-13R-hydroxy diastereomer
followed by the 9S,10R,13S (Gao et al., 2009). We also identified
two 9S,10S-trans-epoxy-11-hydroxy diastereomers (Fig. 1A, peaks
4 and 5): the 11S-erythro isomer elutes earlier at ∼9.3 min, and the
11R-threo-hydroxy isomer co-elutes with the first cis-epoxide at
11.0 min (see Supplementary Tables S9 and S10 for 1H NMR assign-
ments); this is consistent with previous findings that the erythro
diastereomers are the less polar on SP-HPLC or TLC (Corey and
Mehrotra, 1983; Corey et al., 1983; Dix and Marnett, 1985; Gardner
and Kleiman, 1981; Vasiljeva et al., 1993).
2.5. Enzymatic synthesis of epoxyalcohols and triols from
9S/R-HPODE with B. vulgaris extract
A single beet was peeled and diced, 100 g was added to ice cold
0.1 M potassium phosphate pH 6.7 (600 ml) and blended using a
Polytron homogenizer for 3 × 30 s or until a smooth consistency
was obtained. The solution was filtered through 2 layers of gauze
and used directly for incubation with HPODE. Metabolizing activity
was monitored by disappearance of the UV absorbance at 235 nm
using 130 ␮l of homogenate in 870 ␮l buffer and 50 ␮M HPODE sub-
strate as described (Hamberg and Olsson, 2011). For larger scale
incubations, 4 mg of either 9R- or 9S-HPODE in 200 ␮l methanol
was added to the beetroot homogenate (26 ml) with stirring at
room temperature for 1 h. The incubations were then acidified to
pH 3.0 with 1N HCl and extracted with 2 volumes of ethyl acetate.
The ethyl acetate was washed twice with half a volume of water,
taken to dryness, and the recovered products resuspended in iso-
propanol/chloroform (1:2). The sample was then loaded onto a
Waters Sep-Pak Plus NH2 cartridge. The reaction products were
eluted in 15 mL ethyl acetate/acetic acid (98:2). HPLC purification
was then performed using a Waters Symmetry C18 column with an
isocratic 1 ml/min flow of methanol/water/acetic acid (80:20:0.01).
Generation of the epoxyalcohol product was carried out in a similar
way with the following deviations: (1) reaction time was reduced
to 5 min to prevent conversion by epoxide hydrolase and (2) the
3.2. Separation and characterization of trihydroxyoctadecenoate
isomers from the acid hydrolysis of 9S,10S-trans-epoxy-11E,
13S-hydroxyoctadecenoate
To prepare a series of trihydroxy standards for analytical work,
we resolved the products of mild acid hydrolysis of 9S,10S-
trans-epoxy-11E-13S-hydroxyoctadecenoate. After epoxide ring
opening, attack of water on either face of the molecule at C10
or C12 is expected, giving rise to two 9S,10(R/S),13S- and two
9S,12(R/S),13S-triols. Using an RP-HPLC system in which the
epoxyalcohol starting material elutes at 7 min, a ␦-ketol by-
product (Section 3.4) elutes at ∼5.5 min, the four trihydroxy
isomers co-elute around 4 min (Fig. 1B). Although the four triols
are diastereomers, not enantiomers, use of a Chiralpak AD chi-
ral column proved most helpful for their separation, and using a
combination of successive chromatographic steps the four were
resolved: Chiralpak AD separation of the collected trihydroxy iso-
mers allows separation of 3 of the 4 trihydroxy products (labeled I,
II and III) (Fig. 1C). Peak II is further separated to peaks IIa and IIb

24
C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
using the Chiralpak OD-H column (Fig. 1D). Each peak was collected
and analyzed individually.
9(S)-HODE
3
A
205nm
220nm
235nm
270nm
900
9(S),10(S)-trans-
epoxy,13(S)-
hydroxy
700
500
300
100
9(S),10(S)-trans-
epoxy,13(R)-
hydroxy
3.2.1. GC–MS analysis of triol isomers
(peak 7)
The four trihydroxyoctadecanoate methyl esters from acid
hydrolysis of 9S,10S-trans-epoxy-13S-hydroxyoctadecenoate were
treated with dimethoxypropane/toluene sulphonic acid, thus cou-
pling the vicinal diol as the dimethoxypropane (DMP) acetonide
derivative, then silylated on the free hydroxyl with BSTFA and ana-
lyzed by GC–MS. This clearly identified triols I and IIb as having
a 13-OTMS group (prominent m/z 173), and IIa and III as 9-OTMS
(m/z 259):
1
(peak 9)
6
2
8
4
5
4
6
8
10
12
14
16
min
B
Trihydroxy isomers
400
300
200
100
0
TriOH Peak I and IIb
The mass spectrum of the methyl ester, Me3Si, DMP derivative
of peaks I and IIb showed ions at m/z 441 (M+ – 15; loss of
.CH3), 295 (C1–C13 – 90; loss of Me3SiOH), 199 (C11–C18), 173
(C13–C18), 103 (Me3SiO+ CH2), 99 (C13–C18 Me3Si+) and 73
(Me3Si+). These fragment ions indicate a 9,10,13 trihydroxyoc-
tadecenoate (Fig. 2A and C).
205nm
-ketol
Epoxyhydroxy
substrate
2
4
6
8
10
12
min
TriOH IIa and III.
II
I
Analyzed as the methyl ester, DMP, Me3Si ether derivatives
the following ions were detected; m/z 441 (M+ – 15; loss
of CH3), 356 (M+ – 100, rearrangement followed by loss of
OHC-(CH2)4-CH3), 298 (C1–C13 Me3Si+ CH3), 259 (C1–C9), 209
(C9–C18 Me3SiOH), 103 (Me3SiO+ CH2), 73 (Me3Si+). The m/z
259 ion representing ␣-cleavage next to the 9-OTMS group
is diagnostic for the 9,12,13 trihydroxyoctadecenoate (Fig. 2B
and D).
C
80
60
40
20
0
205nm
III
3.2.2. 1H NMR of DMP derivative of products from acid hydrolysis
of 9S,10S-trans-epoxy-11E, 13S-hydroxyoctadecenoate
10
15
20
25
IIa
50
30
35
40
45
min
Table 1 details the diagnostic chemical shift, multiplicity and
coupling constants for all 4 trihydroxy isomers formed (See also
Supplementary Tables S1–S4). From the GC–MS analysis, I and IIb
are 9,10,13-trihydroxy, and IIa and III are the 9,12,13-trihydroxy
epimers. As these products are generated from 9S,10S-epoxy-13S-
hydroxy, the difference in the NMR data is due to the chirality of
either the 10-hydroxy or 12-hydroxy. The results are consistent
with the unknown hydroxy groups in I and III having the same rel-
ative configuration (I, J9,10 = 7.5 Hz and III, J12,13 = 7.62 Hz with both
the H10 and H12 chemical shifts at 4.06 ppm). The chemical shifts
of the acetonide methyl groups (I and III – 1.47 and 1.45 ppm) indi-
cate the syn-diol configuration (Bruno et al., 1992; Cardellina and
Moore, 1980). Trihydroxy IIa and IIb show the opposite chirality of
the unconfirmed hydroxyl (IIa, J12,13 = 6.84 Hz and IIb, J9,10 = 6.72 Hz
with both the H10 and H12 chemical shifts at 4.41 ppm). The ace-
tonide methyl groups in this case indicate an anti-diol (IIa and
IIb – 1.56 and 1.375 ppm). All products have a trans double bond
(J10,11/11,12 = 15.5 Hz).
Peak III co-eluted with pinellic acid, (the beetroot product
from 9S-HPODE (Hamberg and Olsson, 2011), see the follow-
ing Section 3.3) in the Chiralpak AD separation and also shares
the same 1H NMR and GC–MS data, confirming its structure as
pinellic acid, 9S,12S,13S-trihydroxyoctadec-10E-enoic acid. Triol I
shares the same 1H NMR profile as III (Table 1) and can therefore
be assigned as 9S,10S,13S-trihydroxyoctadec-11E-enoic acid. The
stereochemistry of IIa, the second 9,12,13-triol, has an 1H NMR
spectrum consistent with an anti-diol and is therefore 9S,12R,13S-
trihydroxyoctadec-10E-enoic acid. The second 9,10,13-triol, IIb,
also shares this anti-diol and can be assigned as 9S,10R,13S-
trihydroxyoctadec-11E-enoic acid.
II
IIb
D
60
40
20
0
205nm
30
40
60
70
80 min
Fig. 1. Isolation of epoxyalcohols and trihydroxyoctadecenoic acids. (A) SP-HPLC
analysis of products from reaction of 9S-HPODE with hematin. The four allylic
epoxy alcohols are the smaller 9,10-cis-epoxy-11E-13-hydroxy products (peak
6, 9S,10R,13R, and peak 8, 9S,10R,13S), and the more prominent trans epox-
ides (peak 7, 9S,10S,13S, and peak 9, 9S,10S,13R). Other products appearing
on the chromatogram include the following: (1) 9-keto-octadec-10E,12Z-dienoic
acid, (2) 9S,10S-epoxy-13-oxo-octadec-11E-enoic acid, (3) 9S-HODE, (4) 9S,10S-
trans-epoxy-11S-erythro-hydroxy-octadec-12Z-enoic acid, and (5) 9S,10S-trans-
epoxy-11R-threo-hydroxy-octadec-12Z-enoic acid. Column: Thomson Advantage
˚
Silica 150 A 5 ␮ column, 25 cm × 0.46 cm; solvent: hexane/IPA/HAc 100/2/0.02 (by
vol.), flow rate 1 ml/min. (B) RP-HPLC analysis after mild acid hydrolysis of the
9S,10S-trans-epoxy-13S-hydroxy isomer with unresolved triols (4 min), ␦-ketol
(5.5 min), and unreacted epoxyalcohol (6.7 min). Column: Waters Symmetry C18
column 250 mm × 4.6 mm; solvent: methanol/water/acetic acid (80/20/0.01) with
UV detection at 205 nm. (C) Further separation of the collected triols using a
Chiralpak AD column produced three peaks, designated I, II and III. Solvent:
hexane/methanol/ethanol/acetic acid (86.5/4.5/9/0.1), 1 ml/min, UV detection at
205 nm. (D) Resolution of II was achieved using a Chiralpak OD-H column yielding IIa
and IIb. Solvent: hexane/ethanol/acetic acid, (97.5/2.5/01), 1 ml/min, UV detection
at 205 nm.

C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
25
Fig. 2. Regio isomer assignment of 9S-HPODE-derived triols. EI mass spectra of the triols as the methyl ester, dimethoxypropane, trimethylsilyl ether derivative (M+• 456). A
prominent m/z 173 ion indicates a 13-OTMS structure and is diagnostic of a 9,10,13-triol (I and IIb, panels A and C) with other characteristic ions at m/z 327, 295, 270, 228,
227 and 212. A prominent ion at m/z 259 indicates a 9-OTMS and is diagnostic of a 9,12,13-triol (IIa and III, panels B and D) with other characteristic ions at m/z 356, 298,
241 and 209.
3.2.3. SP-HPLC resolution of the triols as the methyl ester DMP
derivative
To further test the capabilities of this method, we prepared the
four diastereomeric triols from acid hydrolysis of the 9S,10S,13R-
epoxyalcohol (the second of the two major epoxyalcohols from
the hematin reaction with 9S-HPODE). These are separated into
an additional three new peaks in this system (Fig. 3B). As indicated
in the figure legend, the first of these new peaks (marked with an
asterisk) contains two triols which can be resolved by SP-HPLC of
the methyl ester derivative.
As suggested by the results above, chromatographic separa-
tion of the four triols from the 9S,10S,13S epoxyalcohol is quite
challenging as they are poorly resolved in typical RP-HPLC and SP-
HPLC systems. In a late development in this work, we found that
the triols are widely resolved by SP-HPLC of the methyl ester DMP
derivative (Fig. 3A).

26
C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
Table 1
Diagnostic MS and 1H NMR characteristics of triol hydrolysis products of 9S,10S-epoxy-13S-hydroxy-octadec-11E-enoic acid.
Hydrolysis
Products
Epoxyalcohol precursor: 9S,10S,12S
Product
stereochemistry
GC–MS (DMP)-MeTMS
diagnostic ions
1H NMR DMP
geminal (OH)
proton
1H NMR DMP-methyl
(ppm)
I
9S,10S,13S
9,10-syn (threo)
H10 – 4.06 ppm
J9,10 = 7.7 Hz
1.45
1.47
m/z 173
J10,11 = 7.5 Hz
IIa
IIb
III
9S,12R,13S
12,13-anti (erythro)
H12 – 4.42 ppm
J12,13 = 6.8 Hz
J11,12 = 6.8 Hz
1.38
1.56
m/z 209 and 259
m/z 173
9S,10R,13S
9,10-anti (erythro)
H10 – 4.42 ppm
J9,10 = 7.0 Hz
J10,11 = 6.7 Hz
1.38
1.56
9S,12S,13S
12,13-syn (threo)
H12 – 4.06 ppm
J12,13 = 7.6 Hz
J11,12 = 7.6 Hz
1.45
1.47
m/z 209 and 259
m/z 241 and 99
9-hydroxy, 13-keto
Diagnostic signal
H12 – doublet at
2.73 ppm
␦-ketol
R
HO
O
J11,12= 7.0 Hz
R = HO2C(CH2)7–
.
3.3. Enzymatic conversion of 9S- and 9R-HPODE by beetroot
extract to epoxyalcohols and triols
(Me3SiO+ CH2), 73 (Me3Si+). The presence of ions at both m/z 173
ion (C13–C18, typical of 13-OTMS) and m/z 259 (C1–C9, typical of
9-OTMS) also fails to resolve the issue. Indeed, Gardner and Kleiman
(1981) have shown that both m/z 173 and 259 are prominent ions
Hamberg and Olsson reported the efficient enzymatic trans-
formation of 9S-HPODE by a filtered homogenate of B. vulgaris
(beetroot) to 9S-hydroxy-12R,13S-cis-epoxy-octadec-10E-enoic
acid and upon longer incubation its enzymatic hydrolysis to
9S,12S,13S-trihydroxy-octadec-10E-enoate (pinellic acid); 13S-
HPODE was not metabolized (Hamberg and Olsson, 2011). In
extending these experiments we found that 13R-HPODE is also not
metabolized, while 9R-HPODE was converted with only a slightly
lower rate of reaction compared to 9S-HPODE. The 9S-HPODE enan-
tiomer gave products as reported (Hamberg and Olsson, 2011),
a single epoxyalcohol product after 5 min incubation (data not
shown), and one prominent triol peak following its hydroly-
sis during 60 min incubation (RP-HPLC, peak at 4 min retention
time, Fig. 4A with GC–MS, Fig. 4B and 1H NMR, Table 2 and
Supplementary Table S5, confirming the structure). The transfor-
mations of 9R-HPODE produced one main epoxyalcohol with two
minor isomers upon short-term incubation (5 min), Fig. 4C. Incu-
bation for 60 min yielded their enzymatic hydrolysis products with
the polarity of triols (Fig. 4E), resolved on a Chiralpak AD column
into one predominant trihydroxy isomer with two minor products
(Fig. 4E, inset).
A
200
Methyl ester, DMP derivatives,
triols from 9S,10S,13S EpOH
150
triol I
9S,10S,13S
22%
triol IIb
9S,10R,13S
40%
100
50
triol IIa
triol III
9S,12R,13S
9S,12S,13S
26%
12%
205 nm
0
B
50
from 9S,10S,13R EpOH
triol I
triol
IIb
triol
25
0
*
triol
IIa
III
205 nm
10
0
5
15
20
25
30
3.3.1. Identification of the main 9R-HPODE-derived epoxyalcohol
formed in beetroot
Retention time (min)
The epoxyalcohol produced by 9R-HPODE incubation with beet-
root was isolated as per methods as a single main product eluting
at 6 min on RP-HPLC (Fig. 4C). The 1H NMR spectrum clearly estab-
lished the presence of a trans double bond with allylic cis-epoxide
(J = 4 Hz) and carbon-bearing hydroxyl on either side (Table 2,
Supplementary Table S6). However, due to the symmetry of struc-
ture in this type of linoleate derivative, the NMR does not establish
whether the epoxyalcohol is 9,10-epoxy-11E-13-hydroxy or the
reversed 9-hydroxy-10E-12,13-epoxy.
Fig. 3. SP-HPLC resolution of triols as the methyl ester DMP derivative. (A) The four
triol hydrolysis products of methyl 9S,10S-epoxy-13S-hydroxyoctadec-11E-enoate
were purified as a group by SP-HPLC, derivatized to the acetonide, and analyzed
using a Thomson Advantage Silica 150 A 5 ␮ column (25 cm × 0.46 cm) with a sol-
˚
vent of hexane/IPA 100/1 (by vol.) at a flow rate 1 ml/min, with UV detection at
205 nm. The relative proportions of the four triols are indicated as percentages. (B)
The same four products were chromatographed with the triols (methyl ester DMP
derivative) from the second major epoxyalcohol of the hematin reaction, the 13R
diastereomer, 9S,10S-epoxy-13R-hydroxyoctadec-11E-enoate (the shaded peaks).
The four 13R triols resolve as two pairs by SP-HPLC of the methyl ester deriva-
tive using the same Thomson silica column with a solvent of hexane/IPA 90/10
(by vol.), retention times 8.8 min (9S,10R,13R + 9S,12S,13R) and 11.5 min (9S,10S,
13R + 9S,12R,13R). As the Me DMP derivative, 9S,10R,13R + 9S,10S,13R coelute in the
peak marked with an asterisk, followed by the 9S,12S,13R and 9S,12R,13R isomers.
The mass spectrum of the methyl ester TMS ether derivative
(Fig. 4D) showed ions at m/z 398 (M+), 327 (C1–C13), 297, 259
(C1–C9), 237 (327-Me3SiOH), 199 (C1–C10), 173 (C13–C18), 103

C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
27
A
1600
B
100
205nm
235nm
220nm
270nm
73
TMS
O
H
C
O
3
CH
O
3
O
80
60
40
20
0
O
259
1200
800
CH
200
H
C
3
3
131
100
0
241
103
100
209
259
298
5
10
15
20 min
356
381
400
155
185
314
441
0
10
12
14
16
18
min
2
4
6
8
200
300
400
500
m/z
C
D
73
100
80
60
40
20
0
205nm
235nm
TMS
O
220nm
270nm
1600
1200
800
400
0
H C
O
3
CH
3
O
O
103
173
155
259
129
237
327
199
297
300
398
400
2.5
5
7.5
10
12.5
15
17.5
20 min
100
200
m/z
E
1200
800
400
0
F
100
73
205nm
235nm
220nm
270nm
TMS
O
H
C
O
3
700
CH
80
60
40
20
O
3
O
O
259
298
CH
H
C
3
3
300
131
5
10
15
20
min
242159
209
103
100
356
381
155
314
300
441
0
200
400
500
m/z
min
2.5
5
7.5
10
12.5
15
17.5
Fig. 4. Isolation and structural characterization of products from the incubation of 9R- and 9S-HPODE with B. vulgaris extract. (A) RP-HPLC (as per Section 2.3.2) of products
from 60 min incubation of 9S-HPODE: trihydroxy product(s) elute at 4 min with unreacted 9-HPODE substrate and 9-HODE eluting at 15 and 17.5 min respectively. Inset:
separation using a Chiralpak AD column confirms the generation of one major trihydroxy isomer. (B) EI mass spectrum of the methyl ester DMP TMS ether derivative with a
diagnostic ion at m/z 259 indicating a 9-OTMS group and a 9,12,13-trihydroxyoctadecenoate structure. (C) RP-HPLC of the epoxyalcohol (retention time, 6 min) from a 5 min
incubation of 9R-HPODE with B. vulgaris. (D) EI mass spectrum (methyl ester TMS ether derivative) of the 9-hydroxy-12,13-epoxyoctadecenoic acid product. (E) RP-HPLC of
products from the 60 min incubation of 9R-HPODE with two triol peaks eluting at 4 min. Inset: the triols were resolved using a Chiralpak AD column as major (17 min) and
minor (24 min) isomers. (F) EI spectrum of the major isomer from 9R-HPODE, a 9,12,13-trihydroxyoctadecenoate, analyzed as in (B).
in the mass spectra of the methyl ester TMS ether derivative of
9-hydroxy-12,13-epoxyoctadecenoates.
beetroot 9R-HPODE product or be the enantiomer. Finally, we
assigned this 13S-HPODE-derived cis-epoxyalcohol with the 9R-
hydroxy configuration by CD analysis (Gonnella et al., 1982;
Schneider et al., 1997) (Fig. 5), establishing it as identical to the
beetroot product which therefore has the structure, 9R-hydroxy-
12R,13S-cis-epoxyoctadec-10E-enoic acid (Scheme 1).
Based on its biosynthesis in beetroot and by analogy to the
metabolism of 9S-HPODE (Hamberg and Olsson, 2011), an oxygen
transfer mechanism converting 9R-HPODE to 9R-hydroxy-12,13-
cis-epoxyoctadecenoate is anticipated. This was confirmed by
comparison of the beetroot 9R-HPODE-derived epoxyalcohol with
the two 9,10-cis-epoxy-13-hydroxy diastereomers obtained from
hematin treatment of 9-HPODE (Section 3.1), and the corre-
sponding two from 13S-HPODE (9-hydroxy-10E-12,13-cis-epoxy).
By RP-HPLC, three resolved from the beetroot product and one
of 13S-HPODE cis-epoxyalcohols co-chromatographed (data not
shown). This one must have the same regio- and stereochemistry as
3.3.2. Identification of the main 9R-HPODE-derived triols formed
in beetroot
The major triols from the 9S- and 9R-HPODE incubations with
beetroot show indistinguishable mass spectra (Fig. 4B, 4F), indicat-
ing they are the same regio isomers. Analyzed as the methyl ester,
DMP, TMS ether derivatives the following ions were detected; m/z

28
C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
Table 2
Diagnostic MS and 1H NMR characteristics of 9S/R-hydroperoxy-10E, 12Z-octadecadienoic acid and Beta vulgaris incubation products.
Incubation products
Hydroperoxide precursor: 9R
Product
stereochemistry
GC–MS (DMP)-MeTMS
diagnostic ions
1H NMR DMP geminal
(OH) proton
1H NMR DMP-methyl
(ppm)
9R-hydroxy,12R,13S-
m/z 259
cis-epoxide
5 min incubation
epoxy
H12 – 3.25 ppm
J12,13 = 4.4 Hz
9R,12S,13S
12,13-syn (threo)
m/z 209 and 259
H12 – 4.06 ppm
J12,13 = 7.3 Hz
J11,12 = 7.3 Hz
1.46
1.48
60 min incubation
9S-hydroxy,12R,13S-
m/z 259
cis-epoxide
5 min incubation
epoxy
H12 – 3.25 ppm
J12,13 = 4.4 Hz
9S,12S,13S
12,13-syn (threo)
m/z 209 and 259
H12 – 4.06 ppm
J12,13 = 7.1 Hz
J11,12 = 7.3 Hz
1.46
1.47
60 min incubation
R = HO2C(CH2)7–.
441 (M+ – 15; loss of CH3), 356 (M+ – 100, rearrangement fol-
lowed by loss of OHC-(CH2)4-CH3), 298 (C1–C13 Me3Si+ CH3),
259 (C1–C9), 209 (C9–C18 Me3SiOH), 103 (Me3SiO+ CH2), 73
(Me3Si+). The presence of the ion at m/z 259, diagnostic of the
9-OTMS group, identifies these products as 9,12,13 trihydroxy
(Table 2).
also support this conclusion (Bruno et al., 1992; Cardellina and
Moore, 1980). This reversal of configuration at C12 upon hydroly-
sis of the epoxyalcohols is expected through the actions of epoxide
hydrolase (Arand et al., 2005). Finally, the coupling constants for
both these products show the double bond is trans (J10,11 = 15.5 Hz).
Therefore, the 9S-HPODE-derived triol, pinellic acid, is 9S,12S,13S
as already reported (Hamberg and Olsson, 2011), and the main triol
from 9R-HPODE is 9R,12S,13S-trihydroxy-10E-octadecenoic acid
(Scheme 1).
Table 2 includes a summary of the diagnostic 1H NMR data for
the major 9S- and 9R-HPODE-derived triols as the methyl ester
DMP derivative (with full details given in Supplementary Tables
S5 and S7). The original 9R/13S or 9S/13S configurations in the par-
ent epoxyalcohols should be retained, and therefore the unresolved
issue is the chirality at C12. The chemical shifts of the C12 proton
and coupling constant for H12,13 are almost identical (4.06 ppm,
J12,13 = 7.1 or 7.3 Hz), indicating the same relative configuration in
the vicinal diol: the substituents are trans to each other on the ace-
tonide ring and so the hydroxyls must be in the syn configuration
and are 12S,13S. The chemical shifts of the acetonide methyl groups
3.4. Identification of a specific ı-ketol product from mild acid
hydrolysis of 9S,10S-trans-epoxy-11E,13S-hydroxyoctadecenoate
Upon RP-HPLC analysis of acid-treated 9S,10S-trans-epoxy-
11E-13S-hydroxy-octadecenoate, we consistently observed a peak,
more polar than the parent epoxyalcohol yet considerably less
polar than the triol hydrolysis products, and exhibiting only end
9R-HPODE
9S-HPODE
Hematin
9S
major products
9R
R
10S
13R
R
OOH
R
OOH
9S
9S
O
10S
OH
13S
(Hamberg &
Epoxyalcohol synthase (Beta vulgaris)
Olsson, 2010)
R
R
R
O
O
O
13S
OH
12R
13S
12R
9R
9S
13R
R
R
O 10R
9S
OH
OH
OH
Epoxide hydrolase (Beta vulgaris)
13S
O 10R
9S
OH
HO
OH
HO
OH
9S
12S
13S
9R
12S
13S
H+
each gives 4 triols
mild acid
R
R
hydrolysis
OH
OH
Scheme 1.

C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
29
satisfactorily in RP-HPLC solvent containing 0.01% glacial acetic
acid, prior formation of the methyl esters (diazomethane) and elim-
ination of the acetic acid improves the subsequent recovery of
intact allylic epoxyalcohol products from the solvent.
Identification of hydrolysis products. In linoleate-derived triols
arising from intermediate allylic epoxyalcohols, the structural sym-
metry of the 9,10,13- and 9,12,13-trihydroxy products confounds
their definitive assignment solely by NMR. On the other hand,
these regioisomers are readily distinguished by GC–MS of the DMP
derivative (Fig. 2, Table 1 and Supplementary Tables S1–S4). Epox-
ide ring opening under mild acid hydrolysis induces formation of
an intermediate allylic carbocation with the hydroxyl configura-
tions at 9 and 13 fixed with respect to the substrate. When the
structure of the epoxyalcohol is known, therefore, the remaining
issue of stereochemistry in the resulting triols is the configuration
of the hydroxy at C10 or C12. In 1H NMR of the DMP derivative, the
chemical shift of the allylic hydroxyl in the vicinal diol is diagnos-
tic of the syn or anti configuration. Thus, the chemical shift of the
allylic hydroxyl in the syn vicinal diols (products I and III, Table 1)
is 0.4 ppm upfield in comparison to anti (products IIa, IIb), which is
sufficient to define the complete stereochemistry.
Fig. 5. CD analysis of the hydroxyl configuration in 9-hydroxy-12,13-cis-
epoxyalcohols CD spectra were recorded using a Jasco-700 spectropolarimeter
on the 9-O-benzoate esters of the 9-hydroxy-10E-12,13-cis-epoxy-octadecenoate
methyl esters in acetonitrile solution with the concentration adjusted to give 1.0 AU
at 227 nm in the UV (methods in Section 2.5.1). Interaction of the chromophores
of the 9-O-benzoate ester and the 10E bond produces either a positive first Cotton
effect (top) associated with a positive interaction (+) in the Newman projection, and
allowing designation of the 9S configuration, or the negative Cotton effect, negative
Newman projection, designating 9R.
The chemical shifts of the geminal methyl groups on the
acetonide ring in the DMP derivatives also provide structural infor-
mation, confirming the chirality of the unknown hydroxyl (cf.
(Bruno et al., 1992; Cardellina and Moore, 1980; Shirahata et al.,
2006)). When the 9,10 or 12,13 hydroxyls are syn, the chemical
shifts of the acetonide methyl groups are almost superimposable,
only ∼0.02 ppm apart. When the vicinal diol hydroxyls are anti, the
methyl groups are in a cis configuration and the chemical shifts dif-
fer by ∼0.2 ppm. Thus, for products I and III (Table 1), the acetonide
methyls have chemical shifts of 1.45 and 1.47 ppm, whereas they
are separated in IIa and IIb with values of 1.38 and 1.56 ppm.
Four additional triols with the 9S,10(R or S),13R and 9S,12(R
or S),13R configurations are available by acid hydrolysis of the
second trans-epoxyalcohol from 9S-HPODE (9S,10S-epoxy-11E-
13R-hydroxy). We show that these can be resolved by SP-HPLC of
the methyl ester DMP derivative (Fig. 3B). Although not addressed
in this study, the two trans-epoxyalcohols derived from the enan-
tiomeric hydroperoxide, 9R-HPODE, will give rise to eight triol
enantiomers to the products resolved here. As an alternative to
the reported degradative methods of chiral analysis via ozonolysis
(Hamberg, 1991a, 1991b; Garbe et al., 2005), chiral HPLC column
methods could be explored as a means to their assignment.
Proportions of the hydrolysis products. Hamberg has reported the
relative proportions of the four products of mild acid hydrolysis
from a linoleate-derived 9,10-cis-epoxy-13-hydroxy epoxyalcohol:
61% and 15% attack on the allylic epoxy carbon (favoring rever-
sal of configuration), 15% and 8% on the carbon ␣ to the hydroxyl
(favoring formation of the erythro vicinal diol) (Hamberg, 1991a).
He found similar proportions, 61:18:15:6, in hydrolysis of the
12,13-cis-epoxy-9-hydroxy precursor of pinellic acid (Hamberg
and Olsson, 2011). We found different proportions for mild acid
hydrolysis of an analogous trans-epoxy product, methyl 9S,10S-
trans-epoxy-11E-13S-hydroxy-octadecenoate. Our best estimate
comes from resolution of the four diastereomers as the methyl ester
DMP derivative showing a ratio of 22:26:40:12 for products I, IIa,
IIb, and III respectively (Fig. 3A). A point of agreement with hydrol-
ysis of the cis-epoxyalcohols is that the highest abundance triol
is formed via attack on the allylic epoxy carbon with reversal of
configuration (product IIb in our case), and the lowest is the threo
vicinal diol ␦ to the original epoxide (product III in our case).
Stereospecificity of the beetroot epoxyalcohol synthase. Here we
showed that 9R-HPODE, enantiomeric to the 9S-HPODE studied
by Hamberg and Olsson is also a good substrate for the beet-
root enzymes (Hamberg and Olsson, 2011) (whereas neither of
the 13-HPODE enantiomers react). The initial major product from
absorbance in the UV (Fig. 1B). After further purification as the
methyl ester (Chiralpak AD column), the main component was
identified by NMR as a ␦-ketol. The significant chemical shifts,
multiplicity and coupling constants for the ␦-ketol product are
detailed in Table 1 (Supplementary Table S8). Diagnostic fea-
tures of the spectrum include the doublet signal for the CH2
at 2.7 ppm (J11,12 = 7.02 Hz), with coupling only to the one trans
double bond (J = 15.5 Hz), with no additional coupling due to the
adjacent ketone and a second CH2 triplet representing H14 near
H2 (Supplementary Table S8). Due to the symmetry typical of
many linoleate-derived products in the NMR, it was not possi-
ble to distinguish the 9-oxo-11E-13-hydroxyoctadecenoate from
the 9-hydroxy-10E-13-oxo isomer. In this case, however, GC–MS
analyses were definitive. The methyl ester, methoxime, TMS ether
derivative gave a pair of closely eluting peaks for the syn and anti
isomers on GC with slightly differing ion abundances on MS (Fig. 6A
and B); both peaks gave ions at m/z 429 (M+•) 396 (M+-CH3O), 356
(C1–C13), 285 (C1–C11), 270 (C9–C18), 168 (C10–C18), 73 (Me3Si+).
GC–MS of the methyl ester, TMS derivative yielded one main peak
showing two main ions at m/z 241 (C9–C18) and 99 (C13–C18)
(Table 1, Fig. 6C and D) (Gardner and Kleiman, 1979). These results,
together with the NMR data, identify the ␦-ketol as 9-hydroxy-13-
oxo-octadec-10E-enoic acid.
4. Discussion
Utility of hematin method. In the hematin treatment of fatty
acid hydroperoxides under the conditions we describe, the allylic
epoxyalcohols are recovered as major products when suitable
precautions are taken to avoid their facile hydrolysis. To avoid
hydrolysis of these highly acid-labile allylic epoxyalcohols, the
samples are kept on ice during acid extraction, the organic solvent
is added prior to acidification (and thus is ready for mixing the
instant after acid is added), and the acidification itself uses a pre-
determined quantity of 1 M KH2PO4 and HCl to produce the desired
pH, immediately followed by organic extraction; subsequent wash-
ing of the organic phase with water prior to evaporation is another
essential precaution. Although the products chromatograph

30
C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
100
80
60
40
20
A
0
8
9
10
11
12
168
Time (min)
100
O
CH
3
B
TMS
O
H
C
O
N
168
3
80
60
40
20
0
CH
O
3
285
238
159
270
73
203.90
396
400
285
356
350
429
100
150
200
250
300
450
500
m/z
10
0
C
8
0
6
0
4
0
2
0
0
9.
0
9.5
10.0
10.5
Time (min)
241
100
D
99
80
60
40
20
0
TMS
H
C
O
O
O
3
CH
O
3
241
99
207
200
143
150
299
300
383
100
250
350
400
m/z
Fig. 6. Identification of a ␦-ketol. (A) Total ion current chromatogram of the methyl ester, methoxime, TMS ether derivative of the ␦-ketol formed during acid hydrolysis of
9S,10S-trans-epoxy,13S-hydroxy-octadec-11E-enoic acid showing the closely eluting syn and anti isomers at ∼9.8 min. (B) EI mass spectrum of the first-eluting isomer with
diagnostic ions at m/z 168 and 285. (C) Total ion current chromatogram of the methyl ester, TMS ether derivative. (D) The mass spectrum of the main peak has diagnostic
ions at m/z 99 and 241 (Gardner and Kleiman, 1979).
the 9R-hydroperoxide is the analogous 9R-hydroxy-12R,13S-cis-
epoxide, similarly formed by epoxidation at 12,13. Subsequent
hydrolysis gave a major product formed via epoxide hydrolase.
Recently it was shown that cis epoxides present in epoxye-
icosatrienoic acids (EETs) are converted to the syn-diol by soluble
epoxide hydrolase present in rat red blood cells; conversely the
trans epoxides became anti-diols (Jiang et al., 2008). These results
are in line with our findings. It is logical, therefore, that the main
product from the enzymatic hydrolysis of 9R-hydroxy-12R,13S-
trans-epoxide is indeed the 9R,12S,13S-triol.
To assign the stereoconfigurations in the primary beetroot
epoxyalcohol product from 9R-HPODE, NMR was used to estab-
lish the hydroxy-trans double bond-cis-epoxy partial structure.
Again, because of the symmetry in linoleate-derived struc-
tures, NMR did not reveal the orientation of this unit in the
fatty acid. Similarly, GC–MS is not definitive because the usual

C.P. Thomas et al. / Chemistry and Physics of Lipids 167–168 (2013) 21–32
9,10-trans-epoxy-11E-13-hydroxy
31
HO2C
HO2C
O
H OH
H+
H+
δ+
symmmetrical carbocation
O
δ+
H OH
H
HO2C
HO
H
H OH
hydride shift to
C10 or C12, forming
9-hydroxy-10E-13-oxo
and 9-oxo-11E-13-hydroxy
HO2C
HO2C
HO
H
H O
H
-H
HO
O
δ-ketol, 9-hydroxy-10E-13-oxo
Scheme 2.
“diagnostic” ions for 9-OTMS (m/z 259) or 13-OTMS (m/z 173)
were both present in the EI mass spectrum of the methyl ester
TMS derivative (cf. (Gardner and Kleiman, 1981)). We assigned the
configuration by (i) comparison of its HPLC mobility to each of the
potential cis-epoxy diastereomers prepared by hematin treatment
of 9-HPODE (two 9,10-cis-epoxy-11E-13-hydroxy diastereomers)
and 13S-HPODE (two 9-hydroxy-10E-12R,13S-cis-epoxides). The
9R-HPODE-derived beetroot product co-chromatographed on RP-
HPLC only with one of the two 12R,13S-epoxides; (ii) their
9-hydroxy configuration was then established by CD analysis
(Gonnella et al., 1982), proving that the beetroot product is 9R-
hydroxy-10E-12R,13S-cis-epoxy-octadec-10E-enoic acid. Although
we did not pursue the matter, another potential application of
the CD method is assignment of the stereochemistry of the free
hydroxyl in the DMP derivative of the triols.
the same molecular weight as the starting epoxyalcohol and poten-
tial ␦-ketol product.
Acknowledgements
Financial support was provided by European Union FP7, Marie
Curie Outgoing Fellowship (C.P.T.), by NIAMS grant AR-51968 (to
A.R.B.) and a voucher for use of the mass spectrometry resource
through the Vanderbilt-Ingram Cancer Center Support grant (P30
CA068485). We would also like to thank Drs. Wade Calcutt and Don-
ald Stec for their assistance with GC–MS and 1H NMR respectively,
and Nicholas M. Adams and Dr. David Wright for the CD analysis.
Appendix A. Supplementary data
Mechanism of transformation to delta-ketol. We observed that
mild acid hydrolysis of the trans-epoxyalcohol, 9S,10S-epoxy-11E-
13S-hydroxy-octadecenoate, formed not only the distinctly more
polar triols, but also a peak of intermediate polarity on RP-HPLC
(Fig. 1B) that we identified by NMR and GC–MS as mainly one
␦-ketol. In the 1970s, Gardner reported formation of two iso-
meric ␦-ketols from treatment of either 9-HPODE or 13-HPODE
with FeCl3/cysteine (Gardner and Kleiman, 1979), but there has
been little attention to such products since. Gardner described
that a single epoxyalcohol intermediate treated with a Lewis
acid (BF3-etherate) gave rise to similar quantities of two iso-
meric ketols, 9-hydroxy-10E-13-oxo and 9-oxo-11E-13-hydroxy.
We found a 10-fold relative abundance of the 9-hydroxy-13-
oxo isomer over the 9-oxo-13-hydroxy. Whereas Gardner and
Kleiman implicated a symmetrical carbocation intermediate to
account for the appearance of apparently similar amounts of two
diastereomers, our results suggest asymmetry in the reaction
intermediate, giving the bias toward predominantly one out-
come (Scheme 2). Although delta-ketols have not appeared in
the recent literature, given the similarity in the EI mass spectra
of the methyl ester TMS ether derivatives of certain epoxyalco-
hols and delta-ketols (Gardner and Kleiman, 1979), they are a
candidate that might provide an alternative explanation for the
appearance of unlikely epoxyalcohol isomers in the analysis of
human skin fatty acids (Zheng et al., 2011). Even more probable,
we suspect a delta-ketol(s) accounts for the peak of intermediate
polarity illustrated in the RP-HPLC profile in our acid hydrolysis
of the arachidonate-derived epoxyalcohol, 8R-hydroxy-11R,12R-
trans-epoxy-eicosatrienoic acid (Yu et al., 2003); in that case LC–MS
analysis confirmed that the intermediate polarity product(s) has
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.
2013.01.002.
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