Regiocontrol of soybean lipoxygenase oxygenation. Application to the chemoenzymatic synthesis of methyl 15(S)-HETE and Methyl 5(S),15(S)-diHETE

Full text of DOI:10.1021/jo960239r

9062
J . Org. Chem. 1996, 61, 9062-9064
Sch em e 1. Su bstr a te Recogn ition by SBLOX,
Regiocon tr ol of Soybea n Lip oxygen a se
Ad a p ted fr om th e Mod els Develop p ed by Leh m a n
a n d Ha ta n a k a (A, Nor m a l Or ien ta tion , 15
Oxygen a tion ; B, Rever se Or ien ta tion , 5
Oxygen a tion )
Oxygen a tion . Ap p lica tion to th e
Ch em oen zym a tic Syn th esis of Meth yl
15(S)-HETE a n d Meth yl 5(S),15(S)-d iHETE
Dominique Martini, Ge´rard Buono, and Gilles Iacazio*,†
ENSSPICAM, URA 1410 du CNRS, Av. Escadrille
Normandie-Niemen, 13397 Marseille Cedex 20, France, and
Laboratoire de Microbiologie, Faculte´ des Sciences de St
J e´roˆme, Av. Escadrille Normandie Niemen, 13397 Marseille
Cedex 20, France
Received February 6, 1996
Lipoxygenases (LOX’s) are dioxygenases that catalyze
the stereospecific incorporation of molecular oxygen into
the 1(Z),4(Z)-pentadienyl moiety of polyunsaturated fatty
acids.1 We have shown recently2,3 that soybean LOX
(SBLOX) can vigorously function at high linoleic acid
concentration (0.1 M) under oxygen pressure. After
reduction of the formed hydroperoxides (HPOD), coriolic
acid [(9Z,11E,13S)-13-hydroxy-9,11-octadecadienoic acid]
is obtained as a main product, in good yield (83%) and
with high ee (98%). As pointed out by Zhang and Kyler,4
SBLOX has attracted little attention from a synthetic
point of view,4,5 despite its extremely high selectivity. In
an effort to extend its use, we have investigated the
ability of this enzyme to functionalize arachidonic acid
at the normal n-6 position but also on carbon number 5,
a carbon normally not attacked by this LOX.6 This
approach is illustrated by the chemoenzymatic synthesis
of methyl (5S,6E,8Z,11Z,13E,15S)-5,15-dihydroxy-6,8,-
11,13-eicosatetraenoic acid [methyl 5(S),15(S)-diHETE]
(4),7 a naturally occuring eicosanoid with physiological
properties.8,9
Syn th esis of Meth yl 15(S)-HETE. Arachidonic acid
(0.1 M, 913.5 mg) was treated with SBLOX (120 mg,
Fluka AG) under the optimized conditions determined
previously for linoleic acid.3 After 15 min, the reaction
was stopped, and the formed hydroperoxides (∼99%, UV
determination) were reduced with triphenylphosphine
(TPP) and then esterified with diazomethane to afford
† Faculte´ des Sciences de St J e´roˆme.
(1) For general reviews on LOX's, see: (a) Galliard, T.; Chan, H.
W.-S. Biochemistry of Plants; Academic Press: New York, 1980; Vol.
4, p 131. (b) Veldink, G. A.; Vliegenthart, J . F. G. Advances in organic
biochemistry; Elsevier Scientific Publishing Co.: Amsterdam, 1984; Vol.
6, p 139. (c) Corey, E. J . Stereochemistry of organic and bioorganic
transformations; Bartmann, W., Sharpless, K. B., Eds.; VCH Publish-
ers: Weiheim, 1986; p 1. (d) Vick, B. A.; Zimmerman, D. C. Biochem-
istry of plants; Academic Press: New York, 1987; Vol. 9, p 53. (e)
Yamamoto, S. Free Radical Biol. Med. 1991, 10, 149.
(2) Iacazio, G.; Langrand, G.; Baratti, J .; Buono, G.; Triantaphylides,
C. J . Org. Chem. 1990, 55, 1690.
methyl (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eico-
satetraenoic acid [methyl 15(S)-HETE] (1) (see Scheme
1A) in 83% yield (798 mg), with high purity (>97%,
normal-phase HPLC, 1H NMR) and very high ee (>99%,
chiral phase HPLC).10 This reaction is rapid and ex-
tremely stereoselective and proves the general ability of
SBLOX to generate in high yields n-6(S)-HPOD of
natural polyunsaturated fatty acids, when used at high
substrate concentration under oxygen pressure.2,3,11
Syn th esis of Meth yl 5(S),15(S)-d iHETE. We were
then interested in the further functionalization of methyl
15(S)-HETE since this compound still bears two 1(Z),4-
(3) Martini, D.; Iacazio, G.; Ferrand, D.; Buono, G.; Triantaphylides,
C. Biocatalysis 1994, 11, 47.
(4) Zhang, P.; Kyler, K. S. J . Am. Chem. Soc. 1989, 111, 9241.
(5) For other examples of the use of SBLOX in organic synthesis,
see: (a) Corey, E. J .; Su, W. G.; Cleaver, M. B. Tetrahedron Lett. 1989,
30, 4181. (b) Maguire, N. M.; Mahon, M. F.; Molloy, K. C.; Read, G.;
Roberts, S. M.; Sik, V. J . Chem. Soc., Perkin Trans. 1 1991, 2054. (c)
Dussault, P.; Lee, I. Q. J . Org. Chem. 1995, 60, 218.
(6) Three groups have reported the bis oxygenation of arachidonic
by SBLOX.5a,15,18 In these three papers, after reduction of hydroper-
oxides, 8,15-diHETE is the main (65%,5a 60%15) or the exclusive18
product of the reaction, the other product being, in the former two
cases, 5,15-diHETE.
(7) For the chemical synthesis of 5(S),15(S)-diHETE see: Nicolaou,
K. C; Weber, S. E. J . Am. Chem. Soc. 1984, 106, 5734.
(8) Maas, R. L.; Turk, J .; Oates, J . A.; Brash, A. R. J . Biol. Chem.
1982, 257, 7056.
(9) Morita, E.; Schro¨der, J .-M.; Christophers, E. J . Immunol. 1990,
144, 1893.
(10) Ku¨hn, H.; Wieser, R.; Lankin, V. Z.; Nekrasov, A.; Alder, L.;
Schewe, T. Anal. Biochem. 1987, 160, 24.
(11) The chemoenzymatic synthesis of 1 has already been reported
but at a much lower substrate concentration (1.6 × 10-3 M) and in
lower yield (50%); see: Baldwin, J . E.; Davies, D. I.; Hughes, L.;
Gutteridge, N. J . A. J . Chem. Soc., Perkin Trans. 1, 1979, 115.
S0022-3263(96)00239-3 CCC: $12.00 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 25, 1996 9063
(Z)-pentadienyl systems, which could be enzymatically
oxygenated. The challenging problem is to force SBLOX
to recognize 1 as a substrate, but with a reverse orienta-
tion, in order to conduct a 5 lipoxygenation. Indeed, as
shown in Scheme 1A, SBLOX is thought to position the
reactive pentadienyl system of the substrate with the aid
of two pockets, a hydrophobic and a hydrophylic one,
receiving, respectively, the methyl ending and the car-
boxylic ending chains of the natural substrate. According
to the models developed by Lehman12 and Hatanaka,13
methyl 15(S)-HETE should be orientated with the methyl
ester chain in the hydrophobic pocket to be oxygenated
at the 5 position. In order to secure this orientation, the
second pocket should also be filled, something that could
be done as proposed previously by Zhang and Kyler4 by
adding a carboxylic ending chain, such as a succinyl
group, taking advantage in our case of the 15 hydroxy
functionality as shown in Scheme 1B. As such, methyl
15-succinyl-15(S)-HETE (2) bears the two important
features to be recognized and oxygenated at the 5 position
by the enzyme. To test this hypothesis, 2 has been
synthesized in 90% yield from 1 and succinic anhydride
for 5 days in refluxing CH2Cl2 with DMAP as catalyst.
Preliminary experiments have proved that 2 is ef-
fectively a substrate of SBLOX. In order to isolate the
product(s) of the reaction, oxygenation of 2 has been
conducted in a 2 L fermentor vessel at a concentration
of 10-3 M.14 After 10 min, the substrate is totally
consumed as judged by HPLC, and the formed hydrop-
eroxides extracted and reduced with TPP, affording
methyl 15-succinyl-5(S),15(S)-diHETE (3) as a main
product in 78% yield. 3 is then hydrolyzed (LiOH, THF/
H2O, 1/1, 48 h) and methylated (CH2N2), affording methyl
5(S),15(S)-diHETE (4) in 59% yield from 2 and 44% from
arachidonic acid. The structure of 4 has been established
HETE, but also to conduct a 5 lipoxygenation on the
carbon skeleton of arachidonic acid. This second oxy-
genation involves a suitable structural modification of 1
in order to change the natural orientation of the substrate
in the active site of the enzyme. Methyl 5(S),15(S)-
diHETE is then easily accessible by very simple chem-
istry with a de of more than 98%.
Exp er im en ta l Section
Gen er a l P r oced u r es. Unless otherwise noted, materials
were obtained from commercial suppliers and were used without
further purification. SBLOX was purchased from Fluka. Dichlo-
romethane (CH2Cl2) was distilled from calcium hydride. 1H
NMR spectra were recorded at 200 MHz, and 13C spectra were
proton decoupled at 50 MHz. Chemical shifts are reported as
ppm downfield of tetramethylsilane. J values are given in Hz.
The synthesis of 1 was carried out in a high-pressure reactor,3
and the synthesis of 4 was conducted in a fermentor.3
Meth yl 15(S)-HETE (1). Exactly 913.5 mg (3 × 10-3 mol)
of arachidonic acid was weighted in the reactor PTFE beaker,
and then 30 mL of 0.1 M of borate buffer (referred to as dissolved
Na2B4O7‚10H2O) pH 11 was added and allowed to reach a
temperature of 5 °C. Then 0.12 g of soybean lipoxygenase was
added, the vessel closed and pressurized at 2.5 bar of pure
oxygen, and the stirring set at maximum speed (1600 rpm). After
15 min the reaction mixture was withdrawn from the reactor,
diluted with 300 mL of brine, acidified to pH 3 with citric acid,
and extracted with diethyl ether (3 × 500 mL). The combined
organic phases were dried (MgSO4, 0 °C) and the hydroperoxides
reduced overnight (0 °C) with 0.866 g of TPP (3.3 mmol). The
solvent volume was then reduced to approximatively 30 mL by
evaporation and the product esterified with diazomethane.
Purification of the crude product by silica gel chromatography
eluting with 4:6 hexane/diethyl ether afforded 0.798 g (83%) of
a colorless oil: [R]20 ) +24.2 (c ) 0.67, MeOH), UV (EtOH)
D
λmax ) 237 nm, ꢀ ) 29 900 mol-1‚cm-1‚L; IR (thin film) 3428,
3013, 2931, 2859, 1740, 1438, 986, 952, 913 cm-1; 1H NMR (200
MHz, CDCl3) δ 0.89 (t, 3H), 1.20-1.90 (bm, 11H), 2.12 (dt, 2H,
J ) 6, 6), 2.33 (t, 2H, J ) 7), 2.81 (dd, 2H, J ) 6, 6), 2.96 (dd,
2H, J ) 6, 6), 3.67 (s, 3H), 4.17 (dt, 1H, J ) 7, 7), 5.40 (m, 5H),
5.70 (dd, 1H, J ) 7, 15), 6.00 (dd, 1H, J ) 11, 11), 6.54 (dd, 1H,
J ) 11, 15); 13C NMR (50 MHz) δ 14.1, 22.7, 24.8, 25.2, 25.6,
26.1, 26.6, 31.8, 33.4, 37.4, 51.6, 72.8, 125.2, 127.6, 128.1, 128.6,
128.8, 129.0, 130.1, 136.8, 174.2. Anal. Calcd for C21H34O3: C,
75.40; H, 10.25. Found: C, 75.28; H, 10.18.
1
from its UV, IR, MS, and H and 13C NMR spectra, which
are in full accordance with the previously reported ones.15
The S absolute configuration at the newly formed asym-
metric carbon was determined by a previously reported
method,16 based on the chromatography of diastereoiso-
meric (-)-menthoxy carbonate derivatives of dimethyl
2-hydroxyadipate. GC analysis of the mixture of dias-
tereoisomers showed that the second oxygenation per-
formed by SBLOX is essentially diastereoselective.17
Con clu sion . We have shown that SBLOX could be
used not only as a normal n-6 specific LOX, to generate
in high yield and with high selectivity methyl 15(S)-
Meth yl 15-Su ccin yl-15(S)-HETE (2). To a solution of 0.5
g (1.49 mmol) of compound 1 and 0.05 g (0.4 mmol) of dimethy-
laminopyridine in 15 mL of dichloromethane was added a
solution of 0.449 g (4.49 mmol) of succinic anhydride in 15 mL
of the same solvent. The reaction mixture was then heated to
reflux for 5 days under nitrogen. The crude product was
concentrated and purified by silica gel chromatography, eluting
with 6:4:0.5 diethyl ether/hexane/methanol, to afford 0.585 g
(90%) of a colorless oil: IR (thin film) 3012, 2930, 2859, 1737,
1713, 1436, 1170, 987, 953, 726 cm-1; 1H NMR (200 MHz, CDCl3)
δ 0.80 (t, 3H), 1.20 (bs, 6H), 1.4-1.7 (m, 4H), 2.02 (dt, 2H, J )
6), 2.24 (t, 2H, J ) 7), 2.55 (t, 4H, J ) 5), 2.71 (dd, 2H, J ) 6,
6), 2.86 (dd, 2H, J ) 6, 6), 3.59 (s, 3H), 5.1-5.4 (m, 6H), 5.50
(dd, 1H, J ) 7, 15), 5.87 (dd, 1H, J ) 11, 11), 6.45 (dd, 1H, J )
11, 15); 13C NMR (50 MHz) δ 14.0, 22.5, 24.7, 24.7, 25.6, 26.1,
26.5, 29.0, 29.2, 31.5, 33.5, 34.5, 51.5, 75.3, 127.4, 127.7, 127.8,
128.7, 128.7, 129.1, 131.2, 131.4, 171.5, 174.2, 178.0. Anal. calcd
for C25H38O6: C, 69.09; H, 8.81. Found: C, 69.01; H, 8.75.
Meth yl 15-Su ccin yl-5(S),15(S)-d iHETE (3). A solution of
2 (0.276 g, 0.64 mmol) in 5 mL of dioxane and 635 mL of borate
buffer 0.1 M, pH 9 were placed in the fermentor and allowed to
reach a temperature of 5 °C. Then 0.162 g of soybean lipoxy-
(12) Hatanaka, A. Phytochemistry 1993, 34, 1201.
(13) Lehman, W. D. Free Radical Biol. Chem. 1994, 16, 241.
(14) At higher substrate concentrations (3 × 10-3 or 10-2 M), the
formed hydroperoxide is unstable in the reaction medium. This
phenomenon could be related to the known ability of lipoxygenase to
transform 5-HPETE in leucotriene LTA4 when activated at a lipidic
interface (see: Riendeau, D.; Falgueyret, J .-P.; Meisner, D.; Sherman,
M. M.; Laliberte´, F.; Street, I. P. J . Lipid Med. 1993, 6, 23). Indeed,
as shown by UV spectroscopy, this unstability (disparition of the UV
band at 235 nm) is accompanied by the formation of lipoxin-like
compounds (appearance of three bands at 280, 302, and 320 nm
characteristic of conjugated tetraenic structure). An explanation of this
phenomenon could be the general ability of LOX’s to transform
5-HPOD of arachidonic acid or derivatives via an epoxidation/hydroly-
sis process, similar to the one involved in the biosynthesis of leucotriene
LTA4 and lipoxins. In our case, an increase in the concentration of 2
could create
a water/oil interface, at which SBLOX activates to
generate the 15-succinyl equivalent of lipoxins. It should be noted that
the same phenomenon has been observed when ethyl (15-succinyl)-
(17) Authentic racemic samples were obtained through oxidative
ozonolysis of the (-)-menthoxy carbonate of 1-cyclohexenol followed
by methylation and preparation of the authentic S sample by the same
procedure, but starting from enzymatically generated optically pure
1(S)-cyclohexenol (see: Gupta, A. K.; Kazlauskas, R. J . Tetrahedron:
Asymmetry 1993, 4, 879).
15(S)-HETE has been used instead of 2, but at
a much lower
concentration (5 × 10-5 M).
(15) Van Os, C. P. A.; Rijke-Schilder, G. P. M.; Van Halbeek, H.;
Verhagen, J .; Vliegenthart, J . F. G. Biochim. Biophys. Acta 1981, 663,
177.
(18) Bild, G. S.; Ramadoss, C. S.; Lim, S.; Axelrod, B. Biochem.
Biophys. Res. Commun. 1977, 74, 949.
(16) Hamberg, M. Anal. Biochem. 1971, 43, 515.

9064 J . Org. Chem., Vol. 61, No. 25, 1996
Notes
genase was added, the vessel closed and pressurized at 1 bar of
pure oxygen, and the stirring set at maximum speed (1000 rpm).
After 10 min, the reaction mixture was withdrawn from the
reactor, saturated with NaCl, acidified to pH 3 with citric acid,
and extracted with diethyl ether (3 × 600 mL). The combined
organic phases were dried (MgSO4, 0 °C) and the hydroperoxides
reduced overnight (0 °C) with 0.183 g of TPP (0.7 mmol). The
crude product was concentrated and purified by silica gel
chromatography, eluting with 7:3:0.25 diethyl ether/hexane/
methanol, to afford 0.223 g (78%) of a colorless oil: IR (thin film)
3217, 3013, 2932, 2862, 1734, 1718, 1437, 1169, 986, 955, 911,
acidified to pH 3 with citric acid, and extracted with diethyl ether
(3 × 50 mL). The combined organic phases were dried (MgSO4,
0 °C), the solvent volume was then reduced to approximatively
30 mL by evaporation, and the product was methylated with
diazomethane. Purification of the crude product by silica gel
chromatography eluting with 3:7 hexane/diethyl ether afforded
0.132 g (76%) of a colorless oil: [R]20D ) +18.4 (c ) 0.66, MeOH);
UV (EtOH) λmax ) 243 nm, ꢀ ) 34 000 mol-1‚cm-1‚L; IR (thin
film) 3392, 3010, 2931, 2859, 1738, 1438, 986, 955, 913, 734 cm-1
;
1H NMR (200 MHz, CDCl3) δ 0.90 (t, 3H), 1.30 (bs, 6H), 1.5-1.8
(m, 6H), 2.36 (t, 2H, J ) 7), 3.08 (dd, 2H, J ) 8, 8), 3.67 (s, 3H),
4.19 (dt, 2H, J ) 6,6), 5.43 (dt, 2H, J ) 7, 11), 5.6-5.8 (m, 2H),
6.01 (dd, 2H, J ) 11, 11), 6.58 (dd, 2H, J ) 11, 15); 13C NMR
(50 MHz) δ 14.0, 20.7, 22.6, 25.1, 26.5, 31.7, 33.7, 36.5, 37.3,
51.5, 72.0, 72.5, 124.9, 125.3, 128.1, 128.3, 129.2, 129.6, 136.2,
136.8, 174.2. Anal. calcd for C21H34O4: C, 71.96; H, 9.78.
Found: C, 71.96; H, 9.75.
734 cm-1 1H NMR (200 MHz, CDCl3) δ 0.88 (t, 3H), 1.28 (bs,
;
6H), 1.5-1.8 (bm, 6H), 2.34 (t, 2H, J ) 7), 2.64 (bs, 4H), 3.08
(dd, 2H, J ) 7, 7), 3.67 (s, 3H), 4.23 (dt, 2H, J ) 7, 7), 5.2-5.8
(bm, 2H), 5.98 (dd, 2H, J ) 7, 15), 6.5-6.7 (bm, 2H), 7.4-7.7
(bm, 2H); 13C NMR δ 14.0, 20.9, 22.5, 24.8, 26.6, 28.8, 29.3, 31.5,
33.8, 34.5, 36.4, 51.6, 72.0, 75.2, 125.2, 127.6, 128.0, 128.4, 129.4,
130.5, 131.5, 136.2, 171.8 174.3, 176.4. Anal. Calcd for
C
25H38O7: C, 66.64; H, 8.50. Found: C, 66.58; H, 8.46.
Meth yl 5(S),15(S)-d iHETE (4). To a solution of 0.223 g
Ack n ow led gm en t. This work is supported by a
grant from the “Ministe`re de la Recherche et de
l’Enseignement” (MRE 90320) to D.M.
(0.49 mmol) of 3 in 20 mL of THF/H2O 1:1 was added 0.082 g of
LiOH‚H2O (1.96 mmol). The solution was then stirred at 20 °C
for 48 h. The reaction mixture was diluted with 30 mL of brine,
J O960239R