Efficient and specific conversion of 9-lipoxygenase hydroperoxides in the beetroot. formation of pinellic acid

Article abstract of DOI:10.1007/s11745-011-3592-7

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)- octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E) ,12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.

Full text of DOI:10.1007/s11745-011-3592-7

Lipids (2011) 46:873–878
DOI 10.1007/s11745-011-3592-7
COMMUNICATION
Efficient and Specific Conversion of 9-Lipoxygenase
Hydroperoxides in the Beetroot. Formation of Pinellic Acid
•
Mats Hamberg Ulrika Olsson
Received: 31 May 2011 / Accepted: 24 June 2011 / Published online: 10 July 2011
Ó AOCS 2011
Abstract The linoleate 9-lipoxygenase product 9(S)-hy-
droperoxy-10(E),12(Z)-octadecadienoic acid was stirred
with a crude enzyme preparation from the beetroot (Beta
vulgaris ssp. vulgaris var. vulgaris) to afford a product
consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-oc-
tadecenoic acid (pinellic acid). The linolenic acid-derived
hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octa-
decatrienoic acid was converted in an analogous way into
9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid
(fulgidic acid). On the other hand, the 13-lipoxygenase-
generated hydroperoxides of linoleic or linolenic acids
failed to produce significant amounts of trihydroxy acids.
Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-
octadecadienoic acid afforded the epoxy alcohol 12(R),
13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the
main product indicating the sequence 9-hydroperoxide ?
epoxy alcohol ? trihydroxy acid catalyzed by epoxy alcohol
synthase and epoxide hydrolase activities, respectively. The
high capacity of the enzyme system detected in beetroot
combined with a simple isolation protocol made possible by
the low amounts of endogenous lipids in the enzyme prep-
aration offered an easy access to pinellic and fulgidic acids
for use in biological and medical studies.
Abbreviations
EAS
Epoxy alcohol synthase
GC-MS
Gas-liquid chromatography-mass
spectrometry
GLC-FID Gas-liquid chromatography with flame-
ionization detection
HODE
HPODE
HPOTrE
MC
Hydroxyoctadecadienoic acid
Hydroperoxyoctadecadienoic acid
Hydroperoxyoctadecatrienoic acid
(–)-Menthoxycarbonyl
Me3Si
NMR
Trimethylsilyl
Nuclear magnetic resonance
SP-HPLC Straight-phase high-performance liquid
chromatography
UV
Ultraviolet
Introduction
The first characterization of unsaturated trihydroxy oxylipins
was published by Graveland in 1970 [1]. In this study,
lipoxygenase-catalyzed oxygenation of linoleic acid in
doughs and flour–water suspensions afforded hydroperox-
ides, hydroxides and epoxy alcohols as well as a fraction
consisting of a mixture of 9,12,13-trihydroxy-10-octadece-
noic and 9,10,13-trihydroxy-11-octadecenoic acids. Although
separation of the regio- and stereoisomeric trihydroxyocta-
decenoates was not achieved, it could be concluded that the
9,12,13-trihydroxy isomer was the dominating one and further
be deduced that this compound originated in linoleic acid
9-hydroperoxide (9-HPODE). Eight stereoisomers (four pairs
of enantiomers) of 9,12,13-trihydroxy-10-octadecenoic acid
are possible because of the three chiral carbons at C-9, C-12
and C-13. In 1991, two studies demonstrated that naturally
occurring 9,12,13-trihydroxy-10-octadecenoic acid has the
Keywords Fatty acid hydroperoxide Á Epoxy alcohol Á
Trihydroxy oxylipin Á Pinellic acid
M. Hamberg (&) Á U. Olsson
Division of Physiological Chemistry II,
Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden
e-mail: Mats.Hamberg@ki.se
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Lipids (2011) 46:873–878
Reference Oxylipins
9(S),12(S),13(S) configuration [2, 3]. More recently, 9(S),
12(S),13(S)-trihydroxy-10(E)-octadecenoic acid was isolated
from tubers of Pinellia ternata (crow-dipper), and the trivial
Regio- and stereoisomeric epoxy alcohols including methyl
12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoate were
prepared by incubating fatty acid hydroperoxides with per-
oxygenase from broad bean or oat seeds followed by isolation
by SP-HPLC [7]. 9(S),12(S),13(S)-Trihydroxy-10(E)-octa-
decenoic acid (pinellic acid), 9(S),12(S),13(S)-trihydroxy-
10(E),15(Z)-octadecadienoic acid (fulgidic acid) and other
isomeric trihydroxy oxylipins were prepared from linoleic
and linolenic acids as described in detail [7].
name pinellic acid was given to the compound [4]. The D15(Z)
-
unsaturated analog of pinellic acid, i.e. fulgidic acid, has also
been described [5].
Biosynthesis of pinellic acid in plants can take place by
lipoxygenase-catalyzed oxygenation of linoleic acid into
9(S)-HPODE, which can be further converted into the
epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-oc-
tadecenoic acid in the presence of epoxy alcohol synthase
(EAS) [6, 7] or peroxygenase [8]. Pinellic acid is formed
from the epoxy alcohol by action of epoxide hydrolase
activity, which catalyzes regio- and stereospecific opening
at C-12 of the C-12/C-13 epoxide [7, 8]. Also linoleic acid
13-hydroperoxide (13-HPODE) can be converted to
9,12,13-trihydroxyoctadecenoates as shown in chemical
model systems [9, 10].
Enzyme Preparation
Beetroots (red beets) obtained from a local market were
peeled and the flesh portion finely diced in 6 volumes/
weight of ice-cold 0.1 M phosphate buffer pH 6.7. The
mixture was homogenized at 0 °C for 3 9 30 s using a
Polytron and then filtered through gauze. The filtrate was
used for all preparative incubations with fatty acid hydro-
peroxides. For ultraviolet (UV) spectrophotometric assay,
the filtrate was diluted 1:7.7 (v/v) with 0.1 M phosphate
buffer pH 6.7.
Pinellic acid and other trihydroxyoctadecenoates are
produced in plants during wounding and infection by
fungal pathogens [11–13]. Interestingly, such trihydroxy
oxylipins inhibit growth of fungi and germination of spores
[12, 13] and may play a role in plants’ defense against
pathogenic fungi. Further interest in trihydroxyoctadece-
noates stems from their presence in beer [3, 14, 15], in
which beverage they may contribute a bitter taste [16].
More recent studies on pinellic acid are focussed on the use
of this trihydroxy oxylipin as an oral adjuvant for nasal
influenza vaccines [4, 17–19].
Incubations and Treatments
For generation of oxidation products for structural work,
fatty acid hydroperoxides (300 lM) were stirred with
enzyme preparation (5 mL) at 23 °C for 5 or 30 min. The
mixtures were acidified to pH 5 and rapidly extracted with
75 mL of diethyl ether. Material obtained after evaporation
of the solvent was methyl-esterified by treatment with
diazomethane and either analyzed by SP-HPLC or
trimethylsilylated and analyzed by gas–liquid chromatog-
raphy-mass spectrometry (GC–MS) or gas–liquid chro-
matography with flame-ionization detection (GLC-FID).
The present paper describes a new EAS-epoxide
hydrolase pathway in the beetroot by which 9-lipoxyge-
nase-generated fatty acid hydroperoxides are converted
into specific trihydroxy acids. Based on the results, a pro-
cedure for easy preparation of pinellic acid of [99.5%
stereochemical purity was elaborated. This enzymatic
approach offers an alternative to earlier described prepa-
rations of pinellic acid by organic chemical synthesis [17,
20–23].
Preparation of Pinellic Acid (free acid form
of compound II)
A typical preparation of pinellic acid (batches of 15–20 mg)
was carried out as follows. Beetroots were peeled and the
flesh portion finely diced (40 g). Potassium phosphate buffer
(240 mL) was added, and homogenization was performed at
0 °C using a Polytron. Following filtration through gauze,
179 mL of the filtrate was stirred with 500 lM 9(S)-HPODE
(27.9 mg) at 23 °C for 1 h. The incubation mixture was
acidified to pH 3 and extracted with ethyl acetate. The
extracted product (53 mg) was dissolved in 3 mL of 2-pro-
panol/chloroform 1:2 (v/v); this solution was divided and
loaded on two 0.5-g aminopropyl cartridges (Supelco,
Bellefonte, PA). Neutral lipids were eluted with 6 mL of the
Experimental Procedures
Fatty Acid Hydroperoxides
9(S)-HPODE, 13(S)-HPODE, 9(S)-HPOTrE and 13(S)-
HPOTrE were prepared by lipoxygenase-catalyzed oxy-
genation of linoleic or linolenic acids [7] followed by
purification by straight-phase high-performance liquid
chromatography (SP-HPLC). [1-14C]9(S)-HPODE (specific
radioactivity 15.1 kBq/lmol) was prepared in the same
way starting with [1-14C]linoleic acid (Perkin-Elmer,
Boston, MA).
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Lipids (2011) 46:873–878
875
same solvent mixture, whereas pinellic acid was obtained by
elution with 15 mL of ethyl acetate/acetic acid 98:2 (v/v).
The latter eluates from the two columns were combined
and taken to dryness affording a white solid (23.6 mg;
yield, 80%). Analysis of this material by GC–MS and
GLC-FID revealed 97% trihydroxyoctadecenoate and 3%
other materials (mainly 9-HODE). Regio- and stereochem-
ical analysis of the trihydroxyoctadecenoate [24] showed
98% 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid
(pinellic acid) and 2% of other isomeric trihydroxyocta-
decenoates. An aliquot of this material was crystallized
from methanol/diethyl ether to provide pinellic acid,
[99.5% pure according to GC–MS and GLC-FID, m.p.
105.5–106.5 °C (earlier reported, 104–106 °C [17, 21]).
Results and Discussion
Isolation of oxidation products
[1-14C]9(S)-HPODE (300 lM) was stirred at 23 °C for
30 min with the enzyme preparation from beetroot, and the
methyl-esterified product was subjected to SP-radio-HPLC.
A major peak of radioactivity due to compound II appeared
(95% of the chromatographed radioactivity) (Fig. 1b).
Reducing the time of incubation to 5 min resulted in lower
formation of compound II, and instead the less polar
compound I was the main product (Fig. 1a).
Incubation of 9(S)-HPOTrE (300 lM) with enzyme
preparation for 30 min followed by extraction, methyl-
esterification and isolation by SP-HPLC afforded the
trihydroxyester compound III ([90%). As described for
9(S)-HPODE, when the incubation time was reduced to
5 min, the yield of compound III was lowered, and instead
an epoxy alcohol methyl ester was the main product; this
however, was not further characterized.
Instrumental Methods
For SP-HPLC, a column of Nucleosil 50-5 (200 9 4.6 mm)
purchased from Macherey–Nagel (Du¨ren, Germany) was
used. The column effluent was passed through serially con-
nected detectors for measurement of UV absorbance (210 or
234 nm) and radioactivity. For isolation of methyl esters of
epoxy alcohols and trihydroxy acids, solvent systems of
2-propanol/hexane (1.5:98.5, vol/vol) and 2-propanol/hex-
ane (7:93, vol/vol), respectively, were used. The flow rate
was 2 mL/min.
Structure of Compound I
No absorption band in the range 210–300 nm was observed
in the UV spectrum of compound I demonstrating the dis-
appearance of the conjugated diene of the incubated 9(S)-
HPODE. On SP-HPLC, compound I comigrated with methyl
12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoate, sug-
gesting the identity of compound I with this epoxy alcohol or
an isomer thereof. The mass spectrum of the Me3Si deriva-
tive of compound I showed a molecular ion at m/z 398 (rel-
ative intensity, 2%) and further ions at m/z 383 (3; M?—15;
loss of ÁCH3), 259 [18; Me3SiO?=CH-(CH2)7-COOCH3],
241 [87; M? - 157; loss of Á(CH2)7-COOCH3], 225 (21), 99
For GC–MS, a Hewlett-Packard model 5970B mass
selective detector connected to a Hewlett-Packard model
5890 gas chromatograph equipped with a 12-m phenyl-
methylsilicone capillary column was used.
GLC-FID was carried out with a Hewlett-Packard model
5890 gas chromatograph equipped with a flame-ionization
detector and a 25-m methyl silicone capillary column.
Helium at a flow rate of 25 cm/s was used as the carrier
gas. Retention times were converted into C-values as
described [25].
A
UV spectra were recorded on a Hitachi (Tokyo, Japan)
model U-2000 UV/VIS instrument. Assay of EAS activity
was performed by continuous measurement of the absor-
bency at 235 nm.
Compound I
Compound II
1H-NMR spectra were recorded on deuteriochloroform
solutions using a Bruker 400 MHz instrument. Tetra-
methylsilane was used as internal chemical shift reference.
B
Compound II
Chemical Methods for Structure Determination
0
10
20
30
40
50
Retention time (min)
Catalytic hydrogenation of epoxy alcohols and trihydr-
oxyesters [26], oxidative ozonolysis [27], and acid-pro-
moted hydrolysis of allylic epoxy alcohols [24] were
performed as indicated. Steric analysis of epoxy alcohols
and trihydroxyesters was carried out as described in detail
[24].
Fig. 1 SP-radio-HPLC analysis of methyl-esterified incubation prod-
ucts obtained after incubation of [1-14C]9(S)-HPODE (300 lM) with
enzyme preparation (5 mL) from beetroot. a Incubation at 23 °C for
5 min. b Incubation at 23 °C for 30 min. The column was eluted with
2-propanol/hexane (1.5:98.5, vol/vol) (0–20 min) followed by 2-pro-
panol/hexane (7:93, vol/vol) (20–50 min) at a flow rate of 2 mL/min
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Lipids (2011) 46:873–878
R
R
HO
OH
OH
HO
OH
OH
Isomer 6a
(C-22.36)
Isomer 7a
(C-22.58)
6%
15%
11
R
9
12
13
10
HO
O
Compound I
61%
18%
R
R
HO
HO
OH
HO
HO
OH
Isomer 11a
(C-22.63)
Isomer 10a (Compound II)
(C-22.46)
Scheme 1 Structures, percentages and C-values of isomeric trihydroxyesters produced upon acid-catalyzed hydrolysis of compound
I. Numbering of trihydroxyesters corresponds to numbers used in Ref. [24]. R = (CH2)7-COOCH3
[100; ?O:C-(CH2)4-CH3], and 73 (80; Me3Si?), thus
indicating the presence of an epoxide group at C-12/C-13
and a hydroxyl group at C-9. The mass spectrum and C-value
were identical to those of the Me3Si derivative of authentic
methyl 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadece-
noate [7]. Oxidative ozonolysis performed on the (-)-
menthoxycarbonyl (MC) derivative of compound I followed
by methyl-esterification afforded the MC derivative of
dimethyl 2(S)-hydroxy-1,10-decanedioate, in this way
demonstrating the presence of a double bond in the D10
position and showing that the C-9 alcohol had the ‘‘S’’ con-
figuration. Oxylipins possessing an allylic epoxy alcohol
structure undergo facile hydrolysis into predictable patterns
of trihydroxyesters under mildly acidic conditions [10, 24],
and accordingly a sample of 1 mg of compound I was treated
with methanol/1 mM hydrochloric acid (1:50, v/v) at 23 °C
for 10 min. Regio- and stereochemical analysis [24] of the
resulting trihydroxyesters (Me3Si derivatives) by GC–MS
and GLC-FID revealed the presence of the following com-
pounds (abundance, C-value, numbering according to Ref.
[24]): methyl 9(S),12(S),13(S)-trihydroxy-10(E)-octadece-
noate (61%, C-22.46, isomer 10a), methyl 9(S),12(R),13(S)-
trihydroxy-10(E)-octadecenoate (18%, C-22.63, isomer
11a), methyl 9(S),10(R),13(S)-trihydroxy-11(E)-octadece-
noate (15%, C-22.58, isomer 7a), and methyl 9(S),10(S),
13(S)-trihydroxy-11(E)-octadecenoate (6%, C-22.36, iso-
mer 6a) (Scheme 1). Acid treatment of authentic methyl
12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoate pro-
duced an identical result, thus conclusively demonstrating
the identity of compound I with this epoxy alcohol.
Structure of compound II
The mass spectrum of the Me3Si derivative of compound II
showed ions at m/z 545 (0.4%; M?-15; loss of ÁCH3), 460
[15; M?-100; rearrangement with loss of OHC-(CH2)4-
CH3], 439 [2; M?-(31 ? 90); loss of ÁOCH3 plus
Me3SiOH], 387 (4; M? - 173; loss of ÁCH(OSiMe3)-(CH2)4-
CH3), 259 [16; Me3SiO?=CH-(CH2)7-COOCH3], 230 [5;
rearrangement with loss of OHC-CH = CH–CH(OSiMe3)-
CH(OSiMe3)-(CH2)4-CH3], 173 [100; Me3SiO?=CH-
(CH2)4-CH3], 147 (11; Me3Si-O?=SiMe2), 103 (11;
Me3SiO?=CH2), and 73 (69; Me3Si?), thus indicating a
9,12,13-trihydroxyoctadecenoate. The C-value (22.46) was
identical to that of the corresponding derivative of authentic
methyl 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoate
but differed from those of other possible trihydroxyocta-
decenoate isomers (cf. Ref. [24]). Oxidative ozonolysis
performed on the tris-MC derivative of compound II fol-
lowed by methyl-esterification affordedthe MC derivativeof
dimethyl 2(S)-hydroxy-1,10-decanedioate, thus locating the
double bond to the D10 position and assigning the ‘‘S’’ con-
figuration to C-9. 1H-NMR spectral data of compound II are
given in Table 1. The coupling constant observed for the
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Lipids (2011) 46:873–878
877
Table 1 1H-NMR data for compound II
1,6
1,4
1,2
1
Proton
d (ppm)
Multiplicity
J (Hz)
2
2.31
t
7.4
3
1.62
m
m
m
q
0,8
0,6
0,4
0,2
0
4–7,14–17
1.25–1.45
1.53
8
9
4.15
6.1, 12.2
5.8, 15.5
6.2, 15.5
6.1
10
5.83
dd
dd
dd
m
t
11
5.72
0
50
100
150
200
250
300
350
400
450
12
3.95
Time (sec)
13
3.48
Fig. 2 UV-spectrophotometric assay of hydroperoxidase activity in
the beetroot. Enzyme preparation diluted 1:7.7 (v/v) with potassium
phosphate buffer pH 6.7 (2 mL) was treated at 23 °C with the
following fatty acid hydroperoxides: 9(S)-HPODE (51 lM; filled
circles), 9(S)-HPOTrE (52 lM; filled squares), 13(S)-HPODE
(46 lM; filled diamonds), and 13(S)-HPOTrE (45 lM; filled trian-
gles). The absorbance at 235 nm was monitored versus time
18
0.90
6.9
OCH3
OH
3.67
s
2.29
s
Recorded at 400 MHz in CDCl3 with tetramethylsilane as internal
chemical shift reference
olefinic protons at C-10 and C-11 (J = 15.5 Hz) confirmed
that the D10 double bond had the ‘‘E’’ configuration. Final
identification of compound II as methyl 9(S),12(S),13(S)-
trihydroxy-10(E)-octadecenoate (methyl ester of pinellic
acid) was provided by melting point data, i.e. 92.0–92.5 °C;
earlier reported 92.5–93.0 °C [21].
R
HOO
9(S)-HPODE
Epoxy alcohol synthase
Structure of Compound III
The trihydroxyester formed following incubation of
9(S)-HPOTrE gave analytical results identical to those
previously recorded for methyl 9(S),12(S),13(S)-trihy-
droxy-10(E),15(Z)-octadecadienoate (methyl ester of ful-
gidic acid) with regard to C-value and mass spectrum [7].
Proof for the identity of compound III with this oxylipin,
i.e., the D15(Z)-unsaturated analog of compound II, was
provided by results of catalytic hydrogenation, which
produced methyl 9(S),12(S),13(S)-trihydroxyoctadecano-
ate, and oxidative ozonolysis, which produced methyl
hydrogen 2(S)-hydroxy-1,10-decanedioate (cf. Ref. [7]).
R
HO
O
12(R),13(S)-epoxy-9(S)-hydroxy-
10(E)-octadecenoic acid
Epoxide hydrolase
+ H O
2
R
Substrate Specificity of the Beetroot EAS Activity
HO
HO
OH
As described above, 9(S)-HPODE and 9(S)-HPOTrE were
both rapidly converted when added to the enzyme prepa-
ration from beetroot. However, neither 13(S)-HPODE nor
13(S)-HPOTrE were similarly converted as judged by
analysis of the incubation products by SP-HPLC or GC–
MS. In order to further study this point, UV spectropho-
tometric assays of the rate of hydroperoxide consumption
by the beetroot enzyme preparation were performed. As
seen in Fig. 2, a rapid disappearance of the conjugated
diene chromophore absorbing at 235 nm was noted for the
two 9-hydroperoxides, whereas the two 13-hydroperoxides
were essentially stable during the incubation period.
Pinellic acid
Scheme 2 Sequence of reactions in the conversion of 9(S)-HPODE
into pinellic acid (free acid form of compound II). R = (CH2)7-
COOH
Conclusion
Beetroot contains a strong EAS activity, which is active on
linoleic and linolenic acid 9-hydroperoxides, as well as an
epoxide hydrolase activity, which stereospecifically con-
verts the epoxy alcohol produced into a trihydroxy acid
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(Scheme 2). The former activity is reminiscent of the EAS
enzymes earlier studied in the fungus Saprolegnia parasi-
tica [6] and in potato leaves [7], however, whereas these
enzymes produce two isomeric epoxy alcohols from each
hydroperoxide, the beetroot EAS forms a single one. A
related conversion has been studied using an enzyme
preparation from oat seeds, in which case the EAS activity
was due to a peroxygenase [8]. The epoxide hydrolase
activity found in beetroot appears to be related to similar
activities earlier found in potato leaves [7] and oat seeds
[8].
The nature of the beetroot EAS and epoxide hydrolase is
currently under investigation and the results will be
reported shortly. Meanwhile, the high catalytic activities of
the two enzymes, combined with an uncomplicated pro-
tocol for product isolation, offer a practical way to prepare
considerable amounts of pinellic and fulgidic acids.
Acknowledgments The expert technical assistance of Mrs.
G. Hamberg is gratefully acknowledged. This work was supported by
a grant from the Swedish Research Council (project 2009-5078).
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