Dioxygenation of Long-Chain Alkadien(trien)ylphenols by Soybean Lipoxygenase

Article abstract of DOI:10.1021/jf980196a

Long-chain alkadien(trien)ylphenols, i.e., cardanols, cardols, and anacardic acids, as well as their acetylated and methylated derivatives were used in enzymatic dioxygenation catalyzed by soybean lipoxygenase-1 (LOX-1). Kinetic studies revealed the influence of functional groups, chain length, number of double bonds, and the distance of the double bonds from the terminal end. Free functional groups were found to be a structural prerequisite for high affinity and turnover number; exceptional kinetic parameters were obtained for ω-6 compounds possessing the same chain length as linoleic acid. The regioselectivity and enantioselectivity of LOX-1 catalysis were investigated with 3-[(Z,Z)-8′,11′-pentadecadienyl]phenol as the representative substrate, yielding 3-[12′-(S)-hydroperoxy-(Z,E)-8′,10′-pentadecadienyl]phenol as the main product. Our extensive chromatographic and spectroscopic studies comprised HPLC-MS/MS for differentiation of the regioisomers, ¹H NMR spectroscopy for determination of the double-bond configuration, GC-MS to evaluate the enantiomeric excess, and exciton-coupled circular dichroism for determination of the absolute configuration.

Full text of DOI:10.1021/jf980196a

J. Agric. Food Chem. 1998, 46, 2951−2956
2951
Dioxygen a tion of Lon g-Ch a in Alk a d ien (tr ien )ylp h en ols by Soybea n
Lip oxygen a se
Monika Roth, Birgit Gutsche, Markus Herderich, Hans-Ulrich Humpf, and Peter Schreier*
Lehrstuhl fu¨r Lebensmittelchemie, Universita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany
Long-chain alkadien(trien)ylphenols, i.e., cardanols, cardols, and anacardic acids, as well as their
acetylated and methylated derivatives were used in enzymatic dioxygenation catalyzed by soybean
lipoxygenase-1 (LOX-1). Kinetic studies revealed the influence of functional groups, chain length,
number of double bonds, and the distance of the double bonds from the terminal end. Free functional
groups were found to be a structural prerequisite for high affinity and turnover number; exceptional
kinetic parameters were obtained for ω-6 compounds possessing the same chain length as linoleic
acid. The regioselectivity and enantioselectivity of LOX-1 catalysis were investigated with 3-[(Z,Z)-
8′,11′-pentadecadienyl]phenol as the representative substrate, yielding 3-[12′-(S)-hydroperoxy-(Z,E)-
8′,10′-pentadecadienyl]phenol as the main product. Our extensive chromatographic and spectroscopic
1
studies comprised HPLC-MS/MS for differentiation of the regioisomers, H NMR spectroscopy for
determination of the double-bond configuration, GC-MS to evaluate the enantiomeric excess, and
exciton-coupled circular dichroism for determination of the absolute configuration.
Keyw or d s: Lipoxygenase; dioxygenation; cardanols; cardols; anacardic acids; exciton-coupled
circular dichroism (ECCD)
INTRODUCTION
kindly provided by Reinhard Zehnter, Laboratory of Organic
Chemistry, University of Bayreuth, Bayreuth, Germany. Li-
poxygenase (L-8383, Type I-S) was purchased from Sigma,
and linoleic acid was obtained from Fluka. All solvents
employed were of high purity at purchase and were redistilled
before use.
Nu clea r Ma gn etic Reson a n ce (NMR). NMR spectra (cf.
Table 2) were recorded on a Bruker WM 400 spectrometer (400
MHz) in CDCl3 and referenced to the solvent signal.
LC-MS a n d MS-MS An a lyses. Analysis of the acety-
lated compounds 1a -4a , methyl esters 5-6b/c, and regioiso-
meric hydroperoxides 7 and 8 was performed on a triple
quadrupole TSQ 7000 LC-MS/MS system (Finnigan MAT,
Bremen, Germany). Data acquisition and data evaluation
were carried out on a personal 5000/33 DEC station (Digital
Equipment, Unterfo¨hring, Germany) with ICIS 8.1 software
(Finnigan MAT). For HPLC, two Knauer 64 HPLC pumps
equipped with micropump heads were used.
Isola tion of Ca r d a n ols (1/2) a n d Ca r d ols (3/4). Ten
grams of CNSL was extracted three times with n-hexane, and
the organic phase was washed with water and dried over
anhydrous sodium sulfate. After evaporation of the organic
solvent, the residue was subjected to LC on a silica gel (Merck
silica gel 60, particle size 0.2-0.5 mm) column (80 × 4 cm).
Elution (3 mL/min) was performed in three steps using
mixtures (each 1 L) of n-pentane + ethyl acetate + acetic acid,
i.e., (i) 90 + 10 + 1, (ii) 80 + 20 + 1, and (iii) 50 + 50 + 1.
Fractions (20 mL each) 36-56 were combined, yielding 5 g of
cardanols, while fractions 95-125 contained 2 g of cardols.
Further separation of the cardanol and cardol isomers was
carried out by LC on silica gel (Merck silica gel 60, particle
size 0.2-0.5 mm) impregnated with 20% silver nitrate (Ever-
shed et al., 1982). Two grams of cardanols was applied to a
silver nitrate-loaded silica gel column (100 g) and eluted with
a n-hexane + ethyl acetate + acetic acid gradient employing
(i) 50 + 2 + 1 and (ii) 90 + 10 + 0.1 mixtures (500 mL each).
Fractions (20 mL each) 30-45 and 80-100 were combined,
yielding 250 and 1000 mg of 1 and 2, respectively. One gram
of cardol isomers was applied to an equally silver nitrate-
loaded silica gel column (100 g) and separated with a chloro-
form + ethyl acetate gradient using 60 + 40 and 50 + 50
Lipoxygenase (LOX) is a non-heme, iron-containing
dioxygenase that catalyzes the regioselective and enan-
tioselective oxidation of unsaturated fatty acids contain-
ing at least one (Z,Z)-1,4-pentadienoic moiety. Common
substrates such as linoleic acid and linolenic acid are
converted to the corresponding (E,Z)-2,4-conjugated
hydroperoxides (Veldink and Vliegenthart, 1984). The
actual knowledge in the field of LOX catalysis has
recently been summarized (Piazza, 1996).
Many phenolic compounds have been described to act
as inhibitors of soybean lipoxygenase-1 (LOX-1), among
them the constituents of cashew nut shell liquid (CNSL),
i.e., cardanols (1/2) and cardols (3/4) (Shobha et al.,
1994). However, anacardic acid (5) has been reported
to be a substrate for LOX-1 (Shobha et al., 1992). In
addition, as vinylphenols have also been found to be
accepted as substrates by LOX-1 (Markus et al., 1992),
it was interesting to study in detail long-chain alkadien-
(trien)ylphenols, exhibiting the (Z,Z)-1,4-pentadienoic
moiety but lacking the fatty acid structure, in order to
better understand the structural requirements for LOX-1
catalysis. In this paper, the results of our kinetic and
structural studies of the enzymatic dioxygenation of 1-4
as well as 5 and 6, including their acetylated and
methylated derivatives 1a -4a , 5a -c, and 6a -c, re-
spectively, are described.
EXPERIMENTAL PROCEDURES
Ch em ica ls. Cardanols (1/2) and cardols (3/4) were isolated
from technical cashew nut shell liquid (CNSL) (Imperial-Oel-
Import Handelsgesellschaft mbH, Hamburg, Germany). Ana-
cardic acids (5/6) and their methoxy derivatives (5a /6a ) were
* Corresponding author (telephone +49-931-8885481; fax
+49-931-8885484; e-mail schreier@pzlc.uni-wuerzburg.de).
S0021-8561(98)00196-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/30/1998

2952 J. Agric. Food Chem., Vol. 46, No. 8, 1998
Roth et al.
mixtures. Pooling of fractions 22-35 and 88-110 yielded 50
and 300 mg of 3 and 4, respectively. The chromatographic
and spectroscopic data of 1-4 corresponded to that of the
literature data (Roth and Rupp, 1995).
was extracted three times with diethyl ether. The combined
organic extract was dried over anhydrous sodium sulfate and
the solvent was evaporated under vacuum. Separation of the
regioisomeric hydroperoxides 7 and 8 was carried out by
normal phase HPLC on an Eurospher Si 100 column (Knauer;
250 × 4 mm; 5 µm) using pentane + ethanol (100 + 1) at a
flow rate of 1 mL/min (UV detection, 234 nm). Separation of
the isomeric hydroxides (cf. bottom) was performed analo-
gously.
Syn th esis of Acetyla ted Com p ou n d s 1a-4a . Fifty milli-
grams of 1a (2a , 3a , 4a ) was dissolved in 5 mL of dry pyridine,
5 mL of acetic acid anhydride was added, and the mixture was
stirred under nitrogen for 24 h at room temperature. The
remaining acetic acid anhydride was hydrolyzed by adding 20
mL of cold water, and the solution was extracted three times
with diethyl ether. The combined organic extracts were
washed three times with 0.1 N hydrochloric acid and twice
with a saturated solution of sodium carbonate and water each
and dried over anhydrous sodium sulfate. After evaporation
of the solvent, purification was carried out by preparative TLC
on precoated SIL G-100 UV254 TLC plates from Macherey-
Nagel (hexane + ethyl acetate, 80 + 20), and the structures
of 1a -4a were confirmed by HPLC-MS analysis. HPLC-
MS was performed with atmospheric pressure chemical ioniza-
tion (APCI) interface in the positive mode. The temperatures
of the heated vaporizer and inlet capillary in the nebulizer
interface were 400 and 200 °C, respectively. The current of
the APCI corona needle was set to 5.0 µA, resulting in 4.0 kV
of needle voltage. Nitrogen served both as the sheath (50 psi)
and auxiliary (10 units) gases. HPLC separations were carried
out on an Eurospher 100 C-18 (100 × 2 mm, 5 µm, Knauer)
employing different linear gradient programs at a flow rate
of 200 µL/min. Solvent A was 0.05% TFA in water; solvent B
was methanol. The scan range was 150-450 u.
On -Lin e HP LC-Electr ospr ay Tan dem Mass Spectr om -
etr y (HP LC-ESI-MS-MS) of Regioisom er ic Hyd r op er -
oxid es. The position of the hydroperoxy group in 7 and 8 was
determined by HPLC-ESI-MS-MS in positive mode without
derivatization as recently described by Schneider et al. (1997a).
The spray capillary voltage was set to 4 kV, and the temper-
ature of the heated inlet capillary was 180 °C. Nitrogen served
both as the sheath (482 MPa) and auxiliary gases. MS-MS
experiments were performed at a collision gas pressure of 0.24
Pa of argon. HPLC was carried out on an Eurospher 100 C-18
column (Knauer; 100 × 2 mm; 5 µm) with methanol + H2O
(5 mM NH4Ac) ) 85 + 15 as eluent at a flow rate of 0.2 mL/
min.
Red u ction of Hyd r op er oxid es 7 a n d 8. To a solution of
5 mg (0.015 mmol) of 7/8 in 50 mL of methanol was added 4.0
mg (0.105 mmol) of NaBH4. The mixture was stirred over 1 h
at 0 °C and ambient temperature each, then 0.1 N hydrochloric
acid was added, and the solution was extracted three times
with diethyl ether. The combined organic phase was washed
acid free with distilled water, and the solvent was evaporated
under vacuum.
1a : MS APCI (+) m/z 343 [M + H]+, 301 [343-H2CdCd
O]+, 189. 2a : MS APCI (+) m/z 341 [M + H]+, 299 [341-H2Cd
CdO]+, 217, 203, 189. 3a : MS APCI (+) m/z 401 [M + H]+,
359 [401 - H2CdCdO]+. 4a : MS APCI (+) m/z 399 [M + H]+,
357 [399 - H2CdCdO]+, 315 [399 - 2H2CdCdO]+.
Deter m in a tion of th e En a n tiom er ic Excess (ee) of 7.
NaBH4-reduced 7 was converted into its (-)-menthoxycarbonyl
(MC) derivatives, subjected to oxidative ozonolysis and meth-
ylated with diazomethane. The obtained diastereomers were
separated by gas chromatography (cf. below). Esterification
of the chiral hydroxy group was carried out in a mixture of
200 µL of dry toluene, 50 µL of pyridine, and 300 µL of (-)-
menthylchloroformate (1 µmol/µL in dry toluene). After 3 h
at 60 °C, the surplus (-)-menthylchloroformate was degraded
with methanol and the solvent was evaporated. For oxidative
ozonolysis, the MC derivatives were taken up in 0.5 mL of
dichloromethane and treated with an excess of ozone for 30
min at -20 °C. The solvent was removed under a stream of
nitrogen, the residue taken up in 500 µL acetic acid and 100
µL of hydrogen peroxide (30%), and the mixture incubated
overnight at ambient temperature. The resulting MC deriva-
tives of 2-hydroxypentanoic acid were extracted from the
aqueous solution with diethyl ether and methylated with
diazomethane at room temperature. As authentic standard,
racemic 2-hydroxypentanoic acid was esterified and methyl-
ated analogously. The resulting diastereomers (9) were
separated by gas chromatography on an achiral column (DB
wax; 30 m × 0.25 mm, 0.24 µm). The temperature program
was 3 min isothermal at 50 °C and then programmed at 4 °C/
min to 240 °C. Mass spectra (70 eV) were recorded in order
to identify the MC derivatives of methyl 2-hydroxypentanoate.
9: EI (+) MS m/z (%) 138 (20), 123 (16), 95 (46), 83 (53), 81
(58), 59 (40), 55 (100), 43 (69).
Syn th esis of Meth yl Ester s 5-6b/c. Methylation of the
carboxylic group in 5 and 6 as well as 5a and 6a was performed
in diethyl ether by use of diazomethane; the phenolic OH group
of 5/6 remained underivatized. After evaporation of the
solvent, purification was carried out by preparative TLC on
precoated SIL G-100 UV254 TLC plates from Macherey-Nagel
(pentane + diethyl ether + acetic acid, 80 + 20 + 1) and the
structures of the isolated methyl esters were confirmed by
HPLC-MS analysis with APCI interface in the positive mode
as described for the synthesis of the acetylated compounds.
5b: MS APCI (+) m/z 359 [M + H]+, 327 [359 - CH3OH]+,
309 [359 - CH3OH - H2O]+. 6b: MS APCI (+) m/z 387 [M +
H]+, 355 [387 - CH3OH]+, 337 [387 - CH3OH - H2O]+. 5c:
MS APCI (+) m/z 373 [M + H]+, 341 [373 - CH3OH]+, 323
[373 - CH3OH - H2O]+. 6c: MS APCI (+) m/z 401 [M + H]+,
369 [401 - CH3OH]+, 351 [401 - CH3OH - H2O]+.
Deter m in a tion of Kin etic P a r a m eter s. LOX-1 activity
was measured both polarographically (oxygen consumption)
using a Clark-type oxygen electrode and spectrophotometri-
cally at 234 nm (formation of the conjugated double bond; ꢀ )
25 000 M-1 cm-1). All essays were performed in 0.1 M borate
buffer (pH 9.0) using ethanolic stock solutions of the substrates
(1.0 mM for substrates 1-6/5a and 6a and 10.0 mM for
substrates 1a -4a /5b/5c/6b and 6c). In
a typical essay,
varying amounts (10-200 µL) of the substrate stock solution
were added to 2000 µL of buffer in a quartz cuvette. After
efficient mixing, 10 µL of enzyme stock solution (1 or 10 mg of
LOX/mL of borate buffer, pH 9.0) was added to start the
reaction. The substrates under study exhibited an initial lag
period which is common for lipoxygenase catalysis. The initial
rates were determined over a period of 1-5 min during the
linear increase of the reaction course, and the data were
processed with a software program (DNRPEASY, version 3.55)
on a personal computer (Duggleby, 1981) to give the Km and
kcat values.
Deter m in a tion of th e Absolu te Con figu r a tion . The
chromophoric derivatives of 7 were prepared as follows:
2-naphthoyltriazole (1 mg, 4.6 µmol) and redistilled 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (0.7 mL, 4.6 µmol) were
added to a solution of sodium borohydride-reduced 7 (1 mg,
3.0 µmol) dissolved in 0.2 mL of dry acetonitrile in a 2-mL
conical glass vial under argon atmosphere. The reaction
mixture was stirred at ambient temperature overnight, con-
centrated, and purified by TLC (pentane + diethyl ether ) 80
+ 20) to yield chromophoric derivative 10. The UV and CD
spectra of 10 were recorded in acetonitrile on a Perkin-Elmer
Lambda 4B UV/vis spectrometer and a J asco J -600 spectro-
polarimeter, respectively.
P r od u ct Isola tion fr om LOX-1 Ca ta lysis of 1. Dioxy-
genation of 20 mg (0.067 mmol) of 1 by LOX-1 (5 mg; 5 × 10-4
mmol) was carried out in 200 mL of 0.1 M borate buffer (pH
9.0) under oxygen flux (1 mL/min) over 4 h. The reaction was
stopped by acidification to pH 3 (hydrochloric acid), then
sodium chloride was added up to saturation, and the mixture

Dioxygenation of Long-Chain Phenols
J. Agric. Food Chem., Vol. 46, No. 8, 1998 2953
Ta ble 1. Str u ctu r es of Com p ou n d s Stu d ied a s Su bstr a tes for LOX-1 Ca ta lysis a n d Th eir Kin etic P a r a m eter sa
R2
R3
R1
R4
compd
R1
R2
R3
R4
Km, µM
kcat, min-1
linoleic acid
1
1a
2
2a
3
3a
4
4a
5
5a
5b
5c
6
6a
6b
6c
19
20
757
21
261
23
550
21
245
18
27
136
186
22
802
76
68
67
64
115
84
129
53
267
170
22
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′,14′-pentadecatrienyl
(Z,Z)-8′,11′,14′-pentadecatrienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′,14′-pentadecatrienyl
(Z,Z)-8′,11′,14′-pentadecatrienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-pentadecadienyl
(Z,Z)-8′,11′-heptadecadienyl
(Z,Z)-8′,11′-heptadecadienyl
(Z,Z)-8′,11′-heptadecadienyl
(Z,Z)-8′,11′-heptadecadienyl
H
H
H
H
H
H
H
H
OH
OAc
OH
OAc
OH
OAc
OH
OAc
OH
OMe
OH
OMe
OH
OMe
OH
H
H
H
H
OH
OAc
OH
OAc
H
H
H
H
H
H
H
H
COOH
COOH
COOMe
COOMe
COOH
COOH
COOMe
COOMe
21
679
849
58
21
209
431
OMe
40
a
Data taken from the polarographic assays; photometric results are consistent.
RESULTS AND DISCUSSION
The kinetic parameters determined for the LOX-1
catalyzed dioxygenation of the long-chain alkadien-
(trien)ylphenolic substrates under study are summa-
rized in Table 1. Despite their lack of a carboxylic
moiety, both long-chain phenols 1 and 2 and resorcinol
derivatives 3 and 4 were accepted as substrates by LOX-
1. These compounds exhibited Km values similar to
those of linoleic acid (Km ) 19 µM), reflecting the high
affinity of LOX-1 toward compounds containing free
phenolic groups. The corresponding acetylated com-
pounds 1a -4a showed drastically increased Km values
(Km > 200 µM). At the pH conditions (pH ) 9.0) applied
for the enzymatic reaction, phenolic hydroxy groups are
deprotonated like the carboxylic group of the unsatu-
rated fatty acids, the common natural substrates of
LOX-1. If these functional groups are blocked, e.g., by
acetylation, no dissociation is possible and, as a conse-
quence, LOX-1 affinity decreases significantly. Obvi-
ously, (a) free phenolic hydroxy group(s) mimic(s) the
carboxylic group of fatty acids, which is generally
considered as a structural prerequisite for high LOX-1
affinity.
Compounds 1, 3, and 5, possessing free functional
groups and bearing the same (Z,Z)-8′,11′-pentadecadi-
enyl side chain, exhibited comparable Km values, while
the turnover number (kcat) increased from the phenolic
(1) to the resorcinol (3) and the salicylic acid (5)
compounds. Evidently, the presence of a carboxylic
group enhances the catalytic efficiency of the enzyme,
and compounds with several free phenolic groups are
slightly better substrates of LOX-1 than those with only
one free functional group.
F igu r e 1. Comparison of the different carbon chains (R1) of
the substrates under study.
observed. As shown in Figure 1, the distance between
the aromatic ring (the carboxylic group in the case of
linoleic acid) and the pentadiene moiety is fixed for all
compounds under study, whereas the side chain of
linoleic acid and compounds 6/6a -c is two carbon atoms
elongated in comparison to all other substrates. Com-
pounds 6/6a -c and linoleic acid are ω-6 compounds,
while 1, 1a , 3, 3a , and 5/5a -c are ω-4 compounds. Our
results indicate that next to the presence of free
functional groups, two further factors can be considered
responsible for the remarkable kinetic parameters of 6
and 6a , i.e., the chain length and the ω-6 position of
the double bond. These observations are in accordance
with previous studies carried out by Ku¨hn et al. (1990)
and Hatanaka (1993). The first mentioned authors have
reported ω-6 compounds to be optimal substrates for
LOX-1, exhibiting excellent kcat values and regioselec-
tivity. Hatanaka (1993) has investigated the kinetic
parameters of a series of C14-C24 carboxylic acids
containing a likewise fixed pentadiene carboxyl moiety.
His results have revealed the highest LOX-1 activity
for linoleic acid and considerably less activity for all
longer or shorter carboxylic acids.
Comparison of the kinetic parameters of 1 with 2 and
3 with 4, respectively, indicates that neither Km nor kcat
was influenced by the presence of two or three double
bonds.
Exceptional kinetic parameters were obtained for the
salicylic acid compounds 6 and 6a , reaching not only
Km but also kcat values similar to those of linoleic acid.
With these substrates, distinctly higher turnover num-
bers than with any other substrate under study were
The regio- and enantioselectivities of LOX-1 catalysis
were investigated with 1 as a representative example
(Figure 2). The analytical procedure for the identifica-
tion of the reaction products is outlined in Figure 3.
Separation of the regioisomeric hydroperoxides 7 and
8 was carried out by normal phase HPLC on an
Eurospher Si 100 column as shown in Figure 4, and the
position of the hydroperoxy group was determined by
on-line LC-ESI-MS/MS without derivatization

2954 J. Agric. Food Chem., Vol. 46, No. 8, 1998
Roth et al.
F igu r e 5. Product ion spectrum of 3-[12′-hydroperoxy-(Z,E)-
8′,10′-pentadecadienyl]phenol (7), precursor ion m/z 350 [M +
NH4]+ (ESI positive mode, -7 eV, 0.24 Pa of argon as collision
gas). Inset: fragmentation pattern explaining the character-
istic ions observed.
F igu r e 2. LOX-1-catalyzed dioxygenation of 3-[(Z,Z)-8′,11′-
pentadecadienyl]phenol (1).
F igu r e 6. Product ion spectrum of 3-[8′-hydroperoxy-(E,Z)-
9′,11′-pentadecadienyl]phenol (8), precursor ion m/z 350 [M +
NH4]+ (ESI positive mode, -7 eV, 0.24 Pa of argon as collision
gas). Inset: fragmentation pattern explaining the character-
istic ions observed.
12′-hydroperoxide 7, resulting from the cleavage of the
C11′-C12′ bond as shown in Figure 5. In the case of the
8′-hydroperoxide 8, distinguishing product ions m/z 219
and 203 arose from the fission of the corresponding C8′-
C9′ bond and subsequent loss of H2O and H2O2, respec-
tively (Figure 6). Thus, the regioisomers were identified
as the 12′-hydroperoxide 7 and 8′-hydroperoxide 8 at
an 82:18 ratio.
F igu r e 3. Analytical procedure: isolation and characteriza-
tion of products formed by LOX-1-catalyzed dioxygenation of
3-[(Z,Z)-8′,11′-pentadecadienyl]phenol (1).
Further characterization was carried out after reduc-
tion of the instable hydroperoxides with sodium boro-
hydride to the corresponding alcohols. In order to
determine the configuration of the double bonds, a 400-
1
MHz H NMR spectrum of the 12′-hydroxide in CDCl3
was recorded and decoupling experiments were carried
out. The 1H NMR data are summarized in Table 2. The
8′-H (5.45 ppm) showed a doublet of a triplet based on
its coupling with 9′-H (J ) 10.8 Hz) and the two protons
of 7′-C (J ) 7.9 Hz), 9′-H (5.99 ppm) coupled with 8′-H
(J ) 10.8 Hz) and 10′-H (J ) 11.0 Hz), resulting in a
doublet of a doublet. The 10′-H (6.50 ppm) and 11′-H
F igu r e 4. Normal phase HPLC separation of hydroperoxides
formed by LOX-1 -catalyzed dioxygenation of 3-[(Z,Z)-8′,11′-
pentadecadienyl]phenol (1). Product separation was performed
on an Eurospher Si 100 column (250 × 4 mm, 5 µm); flow, 1
mL/min; eluent, pentane + ethanol ) 100 + 1, monitored by
UV detection at 234 nm.
(5.68 ppm) also formed doublets of doublets (J 11′-10′
)
15.1 Hz). With the coupling constant of 15.1 Hz
between 10′-H and 11′-H being characteristic of a trans
double bond and J ) 11.0 Hz between 8′-H and 9′-H
being typical of a cis double bond, the double-bond
configuration of 7 was determined as 8′(Z), 10′(E).
To evaluate the ee, NaBH4-reduced 7 was converted
into its (-)-MC derivatives, subjected to oxidative
ozonolysis and esterified with diazomethane (Van Aarle,
1993). Separation of the resulting diastereomers, i.e.,
(-)-MC derivatives of 9, by GC on an achiral phase
(Schneider et al., 1997a). Low-energy collision-induced
dissociation of precursor ions [M + NH4]+ with m/z 350
led to characteristic product ions for both hydroperox-
ides, due to a cleavage of the carbon bearing the
hydroperoxy moiety and the adjoining double-bond
carbon. Product ion m/z 243 was characteristic of the

Dioxygenation of Long-Chain Phenols
J. Agric. Food Chem., Vol. 46, No. 8, 1998 2955
Ta ble 2. 1H NMR (400 MHz) Da ta of Na BH4-Red u ced
3-[12′-Hyd r op er oxy-(Z,E)-8′,10′-p en ta d eca d ien yl]p h en ol
(Solven t: CDCl3)
δ, ppm
7.14
proton
5-H
multiplicity
dd
J , Hz
J 5-6 ) 7.7
J 5-4 ) 7.7
J 4-5 ) 7.7
J 2-6 ) 1.6
J 6-5 ) 7.7
J 6-2 ) 1.7
J 10′-11′ ) 15.1
J 10′-9′ ) 11.0
J 9′-10′ ) 11.0
J 9′-8′ ) 10.8
J 11′-10′ ) 15.1
J 11′-12′ ) 7.0
J 8′-9′ ) 10.8
J 8′-7′ ) 7.9
n.d.
6.75
6.66
6.65
4-H
2-H
6-H
d
d
dd
6.50
5.99
5.68
5.45
10′-H
9′-H
dd
dd
dd
dt
11′-H
8′-H
4.20
2.56
2.19
2.12
1.60
1.34-1.24
0.90
12′-H
1′-H
m
t
J 1′-2′ ) 7.7
7′-H
m
m
m
m
t
n.d.
n.d.
n.d.
n.d.
13′-H
2′-H
3′-6′, 14′-H
15′-H
J 15′-14′ ) 7.0
F igu r e 8. UV and CD spectra of the 2-naphthoyl derivative
of 3-[12′-(S)-hydroxy-(Z,E)-8′,10′-pentadecadienyl]phenol in
acetonitrile.
tive Cotton effect at 243 nm and the second negative
Cotton effect at 226 nm indicate that the two axes of
the 2-naphthoate and diene chromophore adopt a right-
handed screw, as is the case for (S)-configurated deriva-
tives. Thus, 3-[12′-(S)-hydroperoxy-(Z,E)-8′,10′-penta-
decadienyl]phenol was characterized as the main product
(67%) of the LOX-1-catalyzed dioxygenation of 1 by
extensive chromatographic and spectroscopic studies.
The enzyme exhibits distinct regio- and enantioselec-
tivities toward phenolic compound 1, which can be
considered to be a fine substrate for LOX with regard
to its kinetic parameters. The selective dioxygenation
of the phenolic compounds under study reveals that the
carboxylic function in LOX-1 substrates can be replaced
by phenolic structures. This finding extends previous
studies, in which N-linoleoylamides were selectively
converted by LOX-1 (Van der Stelt et al., 1997).
F igu r e 7. Gas chromatographic separation of the diastereo-
meric (-)-MC derivatives of methyl 2-hydroxypentanoate. (A)
(-)-MC derivatives of racemic methyl 2-hydroxypentanoic acid
as the authentic standard; (B) (-)-MC derivatives of enantio-
merically enriched methyl 2-hydroxypentanoate, derived from
enantiomerically enriched 3-[12′-hydroxy-(Z,E)-8′,10′-penta-
decadienyl]phenol. Column: DB wax (30 m × 0.25 mm, 0.24
µm); temperature program, 50 °C [3 min] f 4 °C/min f 240
°C [10 min].
(Figure 7) revealed an ee of 64%. The identity of the
diastereomers was confirmed by GC and GC/MS analy-
ses using derivatized racemic 2-hydroxypentanoic acid
as the authentic standard. In addition, the ECCD
method (Nakanishi and Berova, 1994) was applied to
determine the absolute configuration of 7, as recently
described for similar hydroxylated dienes (Schneider et
al., 1997b). In order to introduce an exciton coupling
chromophore, the chiral hydroxy group was acylated
with 2-naphthoyltriazole. The UV and CD spectra of
the 2-naphthoate derivative 10 are shown in Figure 8.
The CD spectrum, revealing a positive split CD curve
with extrema at 243 nm (∆ꢀ +40.0) and 226 nm (∆ꢀ
-41.0), arises from exciton coupling between the two
transition moments of the chromophores. The π f π*
transition of the diene, which is polarized along the long
axis of the chromophore, couples with the 1Bb transition
band of the 2-naphthoate chromophore. The first posi-
ACKNOWLEDGMENT
We are grateful to Reinhard Zehnter, Laboratory of
Organic Chemistry, University of Bayreuth, for gener-
ously providing anacardic acids and their methoxy
derivatives and to Imperial-Oel-Import Handelsgesell-
schaft mbH, Hamburg, Germany, for the donation of
technical cashew nut shell liquid. We thank Dagmar
Ilge for the isolation of cardols and cardanols and Elke
Richling for her skillful assistance with LC/MS meas-
urements.
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