Controlled formation of mono- and dihydroxy-resolvins from EPA and DHA using soybean 15-lipoxygenase

Article abstract of DOI:10.1194/jlr.M036186

Resolvins and protectins are important anti-infl ammatory and pro-resolution compounds derived from the enzymatic oxidation of omega-3 fatty acids all -cis -5,8,11,14,17- eicosapentaenoic acid (EPA) and all -cis -4,7,10,13,16,19-docosahexaenoic acid (DHA). We have developed a simple, controlled method to synthesize an array of resolvin and protectin analogs from fatty acid starting materials using soybean 15-lipoxygenase. The conditions were optimized for the production of both mono- and dihydroxy derivatives, with enzyme concentration and pH found to have a signifi cant effect on the reaction products. The methods were applied to fi ve biologically important omega-3 and omega-6 fatty acid substrates. Mono- and dihydroxy compounds were successfully synthesized from all substrates and the products were characterized by normal phase (NP) HPLC , GC-MS, TOF-MS, UV-visible (UV-vis) spectroscopy, and NMR spectroscopy. The methods could be further applied to any polyunsaturated fatty acids containing the cis -1,4,7,10-undecatetraene moiety to produce a range of novel compounds with potential biological activity. Copyright

Full text of DOI:10.1194/jlr.M036186

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Controlled formation of mono- and dihydroxy-resolvins
from EPA and DHA using soybean 15-lipoxygenase
Eleanor P. Dobson,* Colin J. Barrow,* Jaroslav A. Kralovec,† and Jacqui L. Adcock1,*
Centre for Chemistry and Biotechnology,* Deakin University, Geelong, Victoria 3220, Australia;
and Ocean Nutrition Canada Ltd.,† Dartmouth, Nova Scotia B2Y 4T6, Canada
Abstract Resolvins and protectins are important anti-inflam-
matory and pro-resolution compounds derived from the
enzymatic oxidation of omega-3 fatty acids all-cis-5,8,11,14,17-
eicosapentaenoic acid (EPA) and all-cis-4,7,10,13,16,19-doco-
sahexaenoic acid (DHA). We have developed a simple,
controlled method to synthesize an array of resolvin and pro-
tectin analogs from fatty acid starting materials using soybean
15-lipoxygenase. The conditions were optimized for the pro-
duction of both mono- and dihydroxy derivatives, with en-
zyme concentration and pH found to have a significant effect
on the reaction products. The methods were applied to five
biologically important omega-3 and omega-6 fatty acid sub-
strates. Mono- and dihydroxy compounds were successfully
synthesized from all substrates and the products were char-
acterized by normal phase (NP) HPLC, GC-MS, TOF-MS,
UV-visible (UV-vis) spectroscopy, and NMR spectroscopy.
The methods could be further applied to any polyunsaturated
fatty acids containing the cis-1,4,7,10-undecatetraene moiety
to produce a range of novel compounds with potential bio-
logical activity.—Dobson, E. P., C. J. Barrow, J. A. Kralovec, and
J. L. Adcock. Controlled formation of mono- and dihydroxy-
resolvins from EPA and DHA using soybean 15-lipoxygenase.
J. Lipid Res. 2013. 54: 1439–1447.
fatty acids are pro-inflammatory (the lipoxins are an im-
portant exception) and those from omega-3 fatty acids
tend to be anti-inflammatory (the prostaglandins pro-
duced from EPA are pro-inflammatory, but less so than
those produced from ARA) (2, 4).
Inflammation is the body’s protective response to mi-
crobial invasion, injury, and other foreign particles (5).
Without inflammation, wounds and infections would
never heal and progressive tissue destruction would com-
promise the survival of the organism (5, 6). However, un-
controlled inflammation is a major contributing factor to
a number of conditions, including arthritis, Alzheimer’s
disease, multiple sclerosis, cardiovascular disease, cancer,
periodontal disease, and allergies (7). The noninflamma-
tory state does not arise passively from an absence of in-
flammatory stimuli; rather the resolution of inflammation
requires numerous biochemical signals to end the inflam-
matory response, including lipid mediators such as lipox-
ins, resolvins, protectins, and maresins (4, 6–8). Serhan
and coworkers in particular have undertaken ground-
breaking research in this field over the last thirty years,
uncovering different families of mediators, as well as char-
acterizing the compounds, their biological actions and
Supplementary key words polyunsaturated fatty acids • eicosanoid •
docosanoid • eicosapentaenoic acid • docosahexaenoic acid
Abbreviations: ARA, arachidonic acid, all-cis-5,8,11,14-eicosatetra-
enoic acid; DHA, all-cis-4,7,10,13,16,19-docosahexaenoic acid; 7,17-
diHDPAn-3, 7,17-dihydroxydocosapenta-8E,10Z,13Z,15E,19Z-enoic acid;
10,17-diHDPAn-6, 10,17-dihydroxydocosapenta-4Z,7Z,11E,13Z,15E-enoic
acid; 5,15-diHEPA, 5,15-dihydroxyeicosapenta-6E,8Z,11Z,13E,17Z-enoic
acid; 8,15-diHEPA, 8,15-dihydroxyeicosapenta-5Z,9E,11Z,13E,17Z-
enoic acid; 5,15-diHETE, 5,15-dihydroxyeicosatetra-6E,8Z,11Z,13E-
enoic acid; 8,15-diHETE, 8,15-dihydroxyeicosatetra-5Z,9E,11Z,13E-enoic
acid; DPAn-3, all-cis-7,10,13,16,19-docosapentaenoic acid; DPAn-6,
all-cis-4,7,10,13,16-docosapentaenoic acid; EPA, all-cis-5,8,11,14,
17-eicosapentaenoic acid; 17-HDPAn-3, 17-hydroxydocosapenta-
7Z,10Z,13Z,15E,19Z-enoic acid; 17-HDPAn-6, 17-hydroxydocosapenta-4Z,
7Z,10Z,13Z,15E-enoicacid;15-HEPA,15-hydroxyeicosapenta-5Z,8Z,11Z,
13E,17Z-enoic acid; 15-HETE, 15-hydroxyeicosatetra-5Z,8Z,11Z,13E-enoic
acid; LOX, lipoxygenase; NP-HPLC, normal phase HPLC; 15-sLOX,
soybean 15-lipoxygenase; 17S-H(P)DHA, 17S-hydro(pero)xydocosahexa-4Z,
7Z,10Z,13Z,15E,19Z-enoic acid; 10S,17S-diH(P)DHA, 10S,17S-dihydro
(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid; 7S,17S-diH(P)
DHA, 7S,17S-dihydro(pero)xydocosahexa-4Z,8E,10Z,13Z,15E,19Z-enoic
acid; SPE, solid phase extraction; UV-vis, UV-visible.
Although generally known for their roles in energy
storage and membrane structure (1), polyunsaturated
fatty acids (PUFAs) such as arachidonic acid, all-cis-
5,8,11,14-eicosatetraenoic acid (ARA, C20:4n-6), all-cis-5,
8,11,14,17-eicosapentaenoic acid (EPA, C20:5n-3), and
all-cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, C22:
6n-3) are also metabolized into a range of potent lipid
signaling molecules (2, 3). These compounds are in-
volved in regulating cellular processes including inflam-
mation, allergies, and blood clotting. Important families
of mediators metabolized from each fatty acid are sum-
marized in Fig. 1. Most of the metabolites of omega-6
This research was funded by the Australian Research Council and Ocean Nutri-
tion Canada.
1 To whom correspondence should be addressed.
Manuscript received 23 January 2013 and in revised form 26 February 2013.
e-mail: jadcock@deakin.edu.au
Published, JLR Papers in Press, March 7, 2013
DOI 10.1194/jlr.M036186
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of six tables.
Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 54, 2013
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Fig. 1. Important families of lipid mediators of inflammation and resolution, and their precursor fatty acids.
biosynthetic pathways (9–14). These compounds are all
di- and trihydroxy fatty acids with conjugated double bond
systems. The omega-3 and omega-6 fatty acid precursors
cannot be produced in the body and must be obtained
from the diet (15).
This enzyme is probably the most widely studied and well-
characterized LOX enzyme (26). It is one of at least eight
lipoxygenase isoforms found in soybeans, the crystal struc-
ture is known and the enzyme is available commercially
(31, 36, 37).
PUFAs and the mediators synthesized from them are
potentially important nutraceuticals and pharmaceutical
targets for the prevention and treatment of several chronic
immune diseases (16). For example, lipoxin analogs are
already being developed as drugs for asthma and other in-
flammatory airway diseases (17), and resolvin analogs are
being tested for the treatment of dry eye and other retinal
diseases (Resolvyx) (18). These oxygenated fatty acids are
products of a number of enzyme systems; primarily lipoxy-
genases (LOXs), cyclooxygenases, and cytochrome P450
epoxygenases (2, 3, 19). Synthesis often involves multiple
enzymes and cell types (19, 20). The products are mainly
mono-, di-, and trihydroxylation products of fatty acids
and the bioactivity and potency is dependent upon multi-
ple factors including the double bond configuration, and
the location and chirality of hydroxy groups (19, 21).
Therefore, there is a need to synthesize these compounds
with a high degree of regio- and stereospecificity. A small
number of organic synthesis methods have been published
[for examples see (22–25)], but these involve challenging
and time-consuming multistep reactions with various ste-
reocenters and cis/trans double bonds. The use of enzymes
as biocatalysts is an attractive option to overcome the dif-
ficulties of organic synthesis for the production of a range
of known and novel lipid mediators (26, 27).
Lipoxygenases are involved in the biosynthetic pathways
of many lipid mediators including leukotrienes, lipoxins,
resolvins, protectins, and maresins, and are therefore a
valuable enzyme class to investigate as biocatalysts (26). Li-
poxygenases are nonheme iron-containing dioxygenases
that catalyze the regioselective and enantioselective oxida-
tion of certain polyunsaturated fatty acids containing one
or more cis,cis-1,4-pentadienoic moieties to give the corre-
sponding hydroperoxy derivatives (26, 28–31). Lipoxyge-
nases with different selectivities have been identified in a
range of species, and many are available commercially.
If we can gain a better understanding of the reactions,
we can potentially create products with higher purity and
in higher yield and manipulate the reaction conditions to
produce a larger range of compounds, using much sim-
pler techniques than those required for organic synthesis.
Lipoxygenase enzymes have previously been investigated
for the production of hydroxy fatty acids, with most work
utilizing soybean 15-lipoxygenase-1 (15-sLOX)-1 (32–35).
Single dioxygenation reactions catalyzed by lipoxyge-
nase enzymes have mainly used linoleic acid (C18:2n-6) as
substrate, with a significant amount of work also done on
␣-linolenic acid (C18:3n-3) and arachidonic acid, but less
work has been done using other substrates (34, 35, 38–42).
The first report of using a lipoxygenase enzyme to catalyze
a double dioxygenation was by Bild et al. in 1977 (43, 44).
They used 15-sLOX and arachidonic acid to produce 8,15-
dihydroperoxy-5,9,11,13-eicosatetraenoic acid. Compared
with single dioxygenation reactions, only a small number
of double dioxygenation reactions have been studied, and
almost all of these have used arachidonic acid or DHA as
substrate (13, 32, 45–54); only one paper also utilized both
all-cis-4,7,10,13,16-docosapentaenoic acid (DPAn-6) and
all-cis-7,10,13,16,19-docosapentaenoic acid (DPAn-3) (33),
and one paper reported the formation of a conjugated
triene from EPA (55), although the structure was not
determined.
In this paper we are studying double dioxygenation re-
actions catalyzed by 15-sLOX, initially using DHA as a
model substrate for the optimization of reaction condi-
tions. We then apply the reaction to a range of biologically
important omega-3 and omega-6 PUFA substrates and
characterize the products. We also compare the affinity of
the enzyme for each of the substrates. This paper includes
the first characterization of products produced by the dou-
ble dioxygenation of EPA catalyzed by a LOX enzyme, and
demonstrates the usefulness of LOXs for the production
of a range of compounds structurally related to resolvins.
MATERIALS AND METHODS
Materials
Soybean 15-lipoxygenase (EC 1.13.11.33, P1, 13.7 megaunits/ml,
50 mg of protein/ml, 270 kilounits/mg of protein) and prosta-
glandin B2 were purchased from Cayman Chemical (Ann Arbor,
MI). The fatty acids DHA, EPA, ARA, DPAn-3, and DPAn-6 were
purchased from Nu-Chek Prep (Elysian, MN). Sodium tetrabo-
rate and hydrochloric acid were purchased from APS Chemicals
Ltd. (Seven Hills, NSW, Australia). Sodium borohydride, Su-
pelcleanTM LC-18 solid phase extraction (SPE) tubes (3 ml, 0.5 g,
Supelco Analytical), SPE vacuum apparatus (Supelco Analytical),
2,2-dimethoxypropane (98%, Fluka), platinum(IV) oxide, N,O-bis
(trimethylsilyl)trifluoroacetamide (with 1% trimethylchlorosilane),
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pyridine, and (trimethylsilyl)diazomethane (2 M in diethyl ether)
Multiple injections per sample were performed and the purified
were all purchased from Sigma-Aldrich (Castle Hill, NSW, Aus-
tralia). Acetic acid was purchased from BDH Lab Supplies (UK).
Chloroform, ethanol, and isopropanol were purchased from
ChemSupply (Gillman, SA, Australia). Methanol was purchased
from Merck (Kilsyth, Vic, Australia). Heptane (95%) was pur-
chased from RCI LabScan Limited (Bangkok, Thailand). Deuterated
methanol was purchased from Cambridge Isotope Laboratories,
Inc. (Andover, MA).
fractions pooled.
Under these conditions, fatty acid starting materials elute first,
monohydroxy products elute second, and dihydroxy products
elute last. These methods enabled the separation of 7,17-dihy-
droxy DHA and 10,17-dihydroxy DHA (both produced from the
lipoxygenase catalyzed dioxygenation of DHA), which could not
be achieved by reversed phase HPLC (47).
GC-MS
Lipoxygenase catalyzed reactions
GC-MS analysis was performed on an Agilent 6890N gas chro-
matograph with a 5975 mass selective detector (electron impact
ionization). Separations were performed on a DB-5MS column
(Agilent, 30 m × 0.25 mm, 0.25 ␮m film thickness) with helium
carrier gas (1.5 ml/min). Samples were injected (2 ␮l) into the
inlet which was kept at 250°C with a split ratio of 20:1. The oven
temperature program was as follows: 100°C for 2 min, increased
to 300°C at 10°C/min, held at 300°C for 8 min. Monohydroxy-
and dihydroxy-fatty acids were hydrogenated and silylated prior
to injection. To hydrogenate, the sample was prepared in 2 ml
ethanol with 10 mg PtO2 catalyst under a stream of hydrogen
with stirring for 25 min, followed by filtration (0.45 ␮m filter)
and evaporation of the solvent under nitrogen. Compounds were
then silylated in a mixture of N,O-bis(trimethylsilyl)trifluoroacet-
amide with 1% trimethylchlorosilane (200 ␮l) and pyridine (200 ␮l)
at 100°C for 1 h.
Lipoxygenase catalyzed reactions were performed following
the method of Butovich and colleagues (32, 47), with minor
modifications. For the synthesis of monohydroxy fatty acids, the
PUFA substrate (0.1 mM) was prepared in borate buffer (50 mM,
pH 12) from a stock solution (10 mM in ethanol), and the reac-
tion initiated by the addition of 15-sLOX (2 mg added per 10 ml
substrate solution). The reaction mixture was stirred for 15 min
at room temperature, and then the hydroperoxides were reduced
by the addition of sodium borohydride (1 M, 0.2 ml added per 10 ml
substrate solution). The solution was acidified by the dropwise
addition of glacial acetic acid (50 ␮l added per 10 ml substrate
solution) after 15 min and left with gentle stirring until foaming
stopped. Products were extracted by chloroform solvent extrac-
tion or SPE. For SPE, C18 cartridges were conditioned according
to the manufacturer’s instructions. The reaction mixture was
loaded onto the cartridge and the cartridge was washed with de-
ionized water and dried under vacuum. The products were then
eluted with a small quantity of ethanol. No further purification
was required.
This method did not give full resolution of the different dihy-
droxy isomers (other columns were tested and did not provide an
improvement in separation), however mass spectra were used to
determine the location of the hydroxyl groups.
For the synthesis of dihydroxy fatty acids, the PUFA substrate
(0.1 mM) was prepared in borate buffer (50 mM, pH 9) from a
stock solution (10 mM in ethanol), and the reaction initiated by
the addition of 15-sLOX (5 mg added per 10 ml substrate solu-
tion). The reaction then proceeded as above. Products were
extracted by SPE or solvent extraction. Products were then meth-
ylated with (trimethylsilyl)diazomethane (200 ␮l, 2 M in diethyl
ether) and methanol (200 ␮l) for 1 h at room temperature.
Methylated products were purified by preparative normal phase
(NP) HPLC (see below).
UV-visible spectrophotometry
UV-visible absorption spectra were collected for purified prod-
ucts. Spectra were collected in acetonitrile using a Varian Cary 300
Bio UV-visible spectrophotometer with a 2.0 mm path length quartz
cuvette. Spectra were collected from 200 nm to 300 nm in double
beam mode with a bandwidth of 1 nm and scan rate of 0.2 nm/sec.
High-resolution mass spectrometry
The molecular masses of the purified products were deter-
mined using high-resolution mass spectrometry with an Agilent
LC/MSD time-of-flight spectrometer. Direct injection of prod-
ucts in acetonitrile was performed with an Agilent 1200 Series
HPLC binary pump and autosampler modules with a mobile
phase of acetonitrile (flow rate of 1 ml/min). Mass spectrometer
conditions were as follows: drying gas, nitrogen (7 l/min, 350°C);
nebulizer gas, nitrogen (16 psi); capillary voltage, 4.0 kV; vapor-
izer temperature, 350°C; and cone voltage, 60 V. Spectra were
collected using an electrospray ionization source operating in ei-
ther negative or positive mode and data were processed with Agi-
lent MassHunter Qualitative Analysis software.
NP-HPLC
An Agilent 1200 series HPLC (Agilent Technologies Australia,
Mulgrave, Vic, Australia) in normal phase mode, equipped with
a solvent degasser, quaternary pump, autosampler, thermostated
column compartment, diode array detector, and fraction collec-
tor was used for all HPLC analyses.
Analytical separations were carried out on a Supelcosil LC-
Diol column (Supelco, 25 cm × 3 mm, 5 ␮m particle size; Sigma-
Aldrich) with an isocratic mobile phase of 95% solvent A (heptane
with 0.1% (v/v) acetic acid and 0.1% (v/v) 2,2-dimethoxypro-
pane) and 5% isopropanol. The flow rate was 0.5 ml/min and
the column temperature was maintained at 10°C. The sample
was prepared in mobile phase and 10 ␮l injected. The signal was
monitored at multiple wavelengths: 210 nm to detect fatty acids,
234 nm to detect conjugated dienes, and 270 nm to detect conju-
gated trienes.
Preparative separations were carried out on methylated prod-
ucts using a LiChrospher silica column (Merck, 12.5 cm × 4 mm,
5 ␮m particle size) with a mobile phase gradient of 1% isopropa-
nol to 5% isopropanol in heptane over 20 min, and held constant
at 5% isopropanol for an additional 10 min. The flow rate was
1 ml/min and the column temperature was maintained at 30°C.
The samples were prepared in mobile phase and 100 ␮l in-
jected. Fraction collection was triggered by the signal at 250 nm.
NMR experiments
NMR experiments were performed on a Bruker Ascend 500
MHz Fourier transform (FT) NMR spectrometer or a JEOL
Eclipse 400 MHz FT-NMR spectrometer. 10–50 mg of purified
product was dissolved in CD3OD and spectra were collected at
room temperature. The following experiments were performed:
1
1
1
1D H-NMR; 2D H, H-COSY; 1D 13C-NMR; 1D 13C-DEPT-135;
2D 1H, 13C-HMQC and 2D 1H, 13C-HMBC. Bruker NMR data was
processed using MestreNova version 7.1.0 and JEOL NMR data
was processed with Delta JEOL USA processing software.
Enzyme kinetics measurements
The rate of formation of conjugated trienes from the differ-
ent fatty acid substrates catalyzed by 15-sLOX was measured by
Formation of resolvin analogs using soybean 15-lipoxygenase
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TABLE 1. Reaction variables studied for the 15-sLOX catalyzed
monitoring the absorbance of the reaction mixture at 270 nm.
To initiate the reaction, 1.5 mg of 15-sLOX (in 1 ml water) was
added to a quartz cuvette containing 2 ml substrate (1 ␮M–0.3 mM)
in oxygenated borate buffer (75 mM, pH 9). The solution was
quickly mixed and absorbance measured every 0.2 min for 2 min.
Reactions were performed at room temperature. Spectra were
collected using a Varian Cary 300 Bio UV-visible spectrophotom-
eter with a 10 mm path length quartz cuvette. All measurements
were repeated three times. Data was fitted to the Michaelis-
Menten kinetic model, r = Vmax[S]/(Km + [S]) by least squares fit
to give kinetic parameters (33).
dioxygenation of DHA
Variable
Initial Conditions
Range Studied
Enzyme amount
Substrate concentration
Temperature
5 mg
0.10 mM
20°C
1–6 mg
0.05–0.50 mM
0–40°C
pH
9
7–13
Buffer concentration
Oxygenation
50 mM
No sparging
30–70 mM
Nitrogen sparging
No sparging
Oxygen sparging
0–60 min
Time
15 min
Reaction volume, 10 ml.
RESULTS
two maxima at approximately 225 and 244 nm (in heptane:
isopropanol, 95:5) (47).
Optimization of reaction conditions using DHA
The dioxygenation of DHA catalyzed by 15-sLOX can
produce the following compounds as major products: 17S-
hydro(pero)xydocosahexa-4Z,7Z,10Z,13Z,15E,19Z-enoic
acid (17S-H(P)DHA), 10S,17S-dihydro(pero)xydocosahexa-
4Z,7Z,11E,13Z,15E,19Z-enoic acid (10S,17S-diH(P)DHA),
and 7S,17S-dihydro(pero)xydocosahexa-4Z,8E,10Z,13Z,
15E,19Z-enoic acid (7S,17S-diH(P)DHA) (Fig. 2) (13, 32,
33, 47). 7S,17S-diHDHA is known as resolvin D5 (RvD5)
and has demonstrated pro-resolving activity (56), while
10S,17S-diH(P)DHA (called PDX) is an isomer of (neuro)
protectin D1 and also has demonstrated bioactivity (45).
The reaction proceeds as follows:
Enzyme and substrate concentrations. Under our condi-
tions, at low enzyme concentrations (<5 mg/10 ml) the
DHA is not fully converted into dihydro(pero)xy products,
and some monohydro(pero)xy-DHA still remains (no
DHA remained at the end of the reaction under any of
the enzyme concentrations tested). The conversion of
monohydro(pero)xy-DHA to dihydro(pero)xy products
increases with increasing enzyme concentration until full
conversion is achieved (5 mg/10 ml).
At low substrate concentrations (р0.10 mM) DHA fully
converts into the two dihydro(pero)xy products and all
DHA is consumed. The amount of product generated is
proportional to the starting amount of DHA. At DHA con-
centrations above 0.10 mM, the formation of dihydro(pero)
xy products is completely inhibited. The DHA is still fully
consumed; some is converted into 17-H(P)DHA, and two
new products are also formed. The first compound (only
detected for reactions using 0.50 mM DHA) eluted early
15-sLOX
O2
15-sLOX
O2
DHA o17S-H(P)DHA o
10S,17S-diH(P)DHA+7S,17S-diH(P)DHA
The amount of product formed and the ratio of different
products depend upon the reaction conditions. To exam-
ine this further, we monitored the product profiles under
different reaction conditions (Table 1) using NP-HPLC
(Fig. 3). The two dihydro(pero)xy compounds can easily
be distinguished by their absorption spectra, as 10S,17S-
in NP-HPLC (at 3.5 min), ␭
= 240 nm, and was un-
max
changed by the sodium borohydride reduction. The sec-
ond major product formed at both 0.2 mM and 0.5 mM
DHA, and eluted after 17-H(P)DHA, but before the
diH(P)DHA contains a conjugated triene and has a ␭
of approximately 270 nm, while 7S,17S-diH(P)DHA con-
max
dihydro(pero)xy compounds, at 6.6 min, ␭
= 275 nm;
max
after reduction with sodium borohydride the peak shifts to
9.5 min, ␭max = 235 nm. Adding more enzyme to compen-
sate for the increase in DHA and maintain a constant
substrate:enzyme ratio did not change the product profile.
It was suspected that these products were formed due to
insufficient oxygen being present in the solution to dioxy-
genate the higher quantities of DHA. This was confirmed
by bubbling oxygen through the solution during the reac-
tion; the additional products were no longer detected,
only mono- and dihydro(pero)xy-DHA products were
formed. The first eluting compound is expected to be a
fatty acid dimer with a conjugated diene formed during
dimerization (57), while the second compound was iso-
latedandidentifiedas17-oxoheptadeca-4Z,7Z,10Z,13Z,15E-
pentaenoic acid.
tains two conjugated dienes and has a split pattern with
Reaction temperature. At low temperatures (<20°C),
the monohydro(pero)xy-DHA was not fully converted
into dihydro(pero)xy products. Between 10°C and 30°C,
there was not a significant difference in the amount of
Fig. 2. Chemical structures of DHA, 17S-H(P)DHA, 10S,17S-
diH(P)DHA, and 7S,17S-diH(P)DHA.
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Fig. 4. Effect of pH on products formed in the dioxygenation of
DHA catalyzed by 15-sLOX.
concentration did not have a significant effect on the reac-
tion over the range studied.
Oxygen concentration. Saturating the buffer with oxygen
before the addition of enzyme did not have any effect on
the product profile at this substrate concentration. How-
ever, it does have an effect under conditions where oxygen
is the limiting reagent, including high substrate concen-
trations. Nitrogenating the buffer before the reaction and
performing the reaction in a sealed container to limit oxy-
gen from the reaction, completely stopped the formation
of dihydro(pero)xy compounds and significantly reduced
the formation of 17-H(P)DHA. Instead, the main product
formed was the same compound seen in reactions with
high substrate concentrations, 17-oxoheptadeca-4Z,7Z,
10Z,13Z,15E-pentaenoic acid. Small amounts of other un-
identified products were also detected.
Fig. 3. A: Example NP-HPLC chromatogram, showing the reac-
tion products formed in the dioxygenation of 0.10 mM DHA cata-
lyzed by 2 mg 15-sLOX (10 ml reaction volume, pH 9). B: UV-vis
absorption spectra of products (from HPLC-DAD). PGB2, prosta-
glandin B2; I.S., internal standard.
dihydro(pero)xy product produced, but at 40°C, there
was a significant drop in the amount of product formed
(approximate 30% decrease). There was no indication of
increased side reactions or nonenzymatic oxidation of
DHA at higher temperatures, as has been previously re-
ported (32). The ratio of 10,17-diH(P)DHA to 7,17-diH(P)
DHA increased up to 30°C then decreased.
Reaction time. Under the initial conditions shown in
Table 1, the reaction was performed and aliquots taken at
regular intervals to monitor the time course of the reac-
tion. It was found that all DHA was converted into 17-H(P)
DHA immediately (within 10 s) after the addition of the
enzyme. The 17-H(P)DHA was quickly converted into
dihydro(pero)xy products and the reaction was complete
within 5 min.
Buffer pH and concentration. Reaction pH is known to
have a significant effect on lipoxygenase reactions (26, 28,
34, 42–44, 53–55, 58) and this was also found for the double
dioxygenation reactions studied in this paper (Fig. 4). To-
tal conversion of 17-H(P)DHA to dihydro(pero)xy com-
pounds was observed at pH 9 and below. The total amount
of dihydro(pero)xy compounds produced increased to a
maximum at pH 9, however, all the initial substrate is not
accounted for at low pH. That is, there is less product, but
no starting material remains. The fate of the missing sub-
strate at low pH is currently unknown, as no new products
were detected by NP-HPLC.
From pH 9 to 12, the amount of dihydro(pero)xy com-
pounds produced decreased, until there was none pro-
duced at pH 12 and above. Over this same range the
amount of monohydro(pero)xy-DHA increases from un-
detectable at pH 9 to a maximum at pH 12.5. At pH 13 the
amount of 17-H(P)DHA produced decreased, and signifi-
cant amounts of unreacted DHA remained. There were
also small amounts of two unidentified compounds pro-
duced at pH 11 and above, most likely containing a conju-
gated diene, and of similar polarity to the dihydro(pero)xy
products. The ratio of 10,17-diH(P)DHA to 7,17-diH(P)
DHA increases up to pH 10 then drops severely. The buffer
Reaction with a range of substrates
From the above set of experiments, two sets of optimized
reaction conditions were chosen: one for the production
of monohydro(pero)xy products and one for the produc-
tion of dihydro(pero)xy products (summarized in Table 2).
The optimized reaction conditions were applied to a num-
ber of polyunsaturated fatty acid substrates (ARA, EPA,
DPAn-6, DPAn-3, and DHA) and all compounds were
found to be suitable substrates for the lipoxygenase en-
zyme. The reaction products are summarized in Table 3. It
is interesting to note that the reaction with both docosap-
entaenoic acids (DPAn-6 and DPAn-3) resulted in the for-
mation of different product ratios compared with the
other substrates. While the reactions with ARA, EPA, and
DHA resulted in approximately equal amounts of both
dihydro(pero)xy products, the reaction of DPAn-3 produced
Formation of resolvin analogs using soybean 15-lipoxygenase
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TABLE 2. Optimized reaction conditions for the production of
monohydro(pero)xy and dihydro(pero)xy products
bond protons located at the second position of the triene.
Therefore the double bonds adjacent to the hydroxylated
carbons were both identified as trans,cis, and the absolute
stereochemistry was identified as E,Z,E.
Monohydro(pero)xy
Dihydro(pero)xy
Enzyme amount
Substrate concentration
Temperature
pH
Buffer concentration
Oxygenation
Time
2 mg
0.10 mM
20°C
pH 12
50 mM
5 mg
0.10 mM
20°C
pH 9
50 mM
The structure of the conjugated diene of all five monohy-
droxy products, 15-hydroxyeicosatetra-5Z,8Z,11Z,13E-enoic
acid (15-HETE), 15-hydroxyeicosapenta-5Z,8Z,11Z,13E,17Z-
enoic acid (15-HEPA), 17-DPAn-6, 17-DPAn-3, and 17-H-
DHA, were confirmed as cis,trans based on the coupling
constants of the double bond protons (32, 33). In 15-HETE
and 15-HEPA, the value of J11,12 was ف11 Hz, indicating
the presence of a cis configuration double bond. Whereas
the coupling constant of J13,14 was approximately 15.2 Hz,
characteristic of trans geometry. Likewise in 17-HDHA,
17-hydroxydocosapenta-7Z,10Z,13Z,15E,19Z-enoic acid (17-
HDPAn-3), and 17-hydroxydocosapenta-4Z,7Z,10Z,13Z,15E-
enoic acid (17-HDPAn-6), J13,14 was approximately 11 Hz
and J15,16 was 15.0–15.2 Hz.
No oxygenation
15 min
No oxygenation
15 min
only 7,17-dihydroxydocosapenta-8E,10Z,13Z,15E,19Z-enoic
acid (7,17-diHDPAn-3), and the reaction of DPAn-6 pro-
duced significantly more 10,17-dihydroxydocosapenta-4-
Z,7Z,11E,13Z,15E-enoic acid (10,17-diHDPAn-6) than 7,
17-diHDPAn-6 (approximately 3:1). All compounds were
characterized by NP-HPLC, GC-MS, TOF-MS, UV-visible
(UV-vis) spectrometry (Table 4), and NMR (supplemen-
tary Tables I–VI).
Reaction kinetics
NMR
The reaction kinetics for the formation of conjugated
trienes was monitored by UV-vis spectroscopy at 270 nm. The
results are shown in Table 5 (DPAn-3 was not included be-
cause it did not produce significant amounts of the conju-
gated triene product). Because of the strong overlap of the
UV absorption spectra of the monohydroxy products and the
conjugated diene dihydroxy products, direct monitoring of
the formation of these products was not possible (47).
From the kinetics results, DHA and DPAn-6 appear to
be the best substrates for the formation of a dihydro(pero)
xy conjugated triene product. The reaction proceeded
fastest with these substrates (Vmax and kcat), with better ef-
ficiency and specificity (kcat/Km), and with higher substrate
capture (Vmax/Km). However, the low Km value for AA sug-
gests the enzyme has a higher affinity for this substrate.
Protons were assigned using integrated 1D 1H-NMR and
1
1
2D H, H-COSY spectra to detect correlation between
neighboring protons. The classification of primary (CH3),
secondary (CH2), and tertiary (CH) carbons was deter-
mined using the 13C-DEPT-135 experiments. Protons were
then assigned to carbons using 2D 1H, 13C-HMQC spectra
1
showing single through-bond relationships, and 2D H,
13C-HMBC spectra to identify relationships through either
two or three bonds. NMR data is provided in the supple-
mentary material (Tables I–VI).
The predicted E,Z,E double bond structure of the con-
jugated triene of 8,15-dihydroxyeicosatetra-5Z,9E,11Z,
13E-enoic acid (8,15-diHETE), 8,15-dihydroxyeicosapen-
ta-5Z,9E,11Z,13E,17Z-enoic acid (8,15-diHEPA), 10,17-di-
HDPAn-6, and 10,17-diHDHA was confirmed by several
1
characteristic proton and carbon resonances in 1D H-
NMR, 1D 13C-NMR, and 2D 1H, 1H-COSY spectra (32, 33).
First a proton resonance with a chemical shift (␦) of 6.12–
6.24 ppm was absent in the 1D H-NMR spectrum. This
DISCUSSION
1
The dioxygenation of polyunsaturated fatty acids con-
taining all-cis-1,4,7-octatriene and all-cis-1,4,7,10-undeca-
tetraene moieties catalyzed by 15-sLOX has been shown to
produce double dioxygenation products as well as the ex-
pected single dioxygenation products (13, 32, 33, 43–55).
It has been proposed that the reaction proceeds through
an initial, very fast, and specific dioxygenation reaction to
produce the omega-6 hydroperoxy product, where the
chemical shift is indicative of a conjugated E,E double
bond fragment, therefore absence confirms E,Z geometry.
1
1
Second, the 2D H, H COSY experiments showed a cor-
relation between protons of both CH–OH (1H ␦ ف4.1
ppm) carbons and protons of the adjacent –CH= with ␦
ف5.7 ppm, a characteristic chemical shift of trans protons.
No correlation was observed between the CH–OH protons
with protons at ␦ 5.9–6.0 ppm, indicative of the cis double
TABLE 3. Major products from the lipoxygenase catalyzed dioxygenation of a range
of polyunsaturated fatty acids
Monohydroxy
Product
Dihydroxy Product
(conjugated diene)
Dihydroxy Product
(conjugated triene)
Substrate
ARA, 20:4n-6
EPA, 20:5n-3
DPAn-6, 22:5n-6
DPAn-3, 22:5n-3
DHA, 22:6n-3
15-HETE
15-HEPA
17-HDPAn-6
17-HDPAn-3
17-HDHA
5,15-diHETE
5,15-diHEPA
8,15-diHETE
8,15-diHEPA
a
—
10,17-diHDPAn-6
b
7,17-diHDPAn-3
7,17-diHDHA
—
10,17-diHDHA
a 7,17-diHDPAn-6 was detected as a minor product only.
b 10,17-diHDPAn-3 not detected.
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TABLE 4. Product characterization
TOF-MS
Substrate
ARA
Product
GC-MS,a Major Ions
Expected (m/z) Actual (m/z)
UV-vis (nm)
15-HETE
5,15-diHETE
8,15-diHETE
15-HEPA
5,15-diHEPA
8,15-diHEPA
17-HDPAn-6
10,17-diHDPAn-6
17-HDPAn-3
7,17-diHDPAn-3
17-HDHA
73, 117, 173, 401, 457
73, 173, 261, 399, 489, 545
73, 173, 303, 359, 489, 545
73, 117, 173, 401, 457
73, 173, 261, 399, 489, 545
73, 173, 303, 359, 489, 545
73, 117, 173, 429, 485
73, 173, 331, 359, 427, 517, 573
73, 117, 173, 429, 485
73, 117, 173, 289, 402, 517, 573
73, 117, 173, 429, 485
357.24002c
373.23493c
373.23493c
317.21222b
371.21928c
371.21928c
345.24297b
399.25113c
345.24297b
399.25058c
343.22787b
397.23493c
397.23493c
357.22519
373.22115
373.23395
317.57493
371.20427
371.20417
345.37750
399.48680
345.37657
399.24808
343.44719
397.21042
397.23907
236.6
243.4 (shoulder at 226.9)
269.0 (suppressed maxima at 259.6 and 279.5)
235.4
244.3 (shoulder at 225.8)
269.5 (suppressed maxima at 260.4 and 280.3)
232.8
269.0 (suppressed maxima at 259.8 and 280.0)
236.5
243.6 (shoulder at 227.6)
236.8
243.8 (shoulder at 227.6)
269.8 (suppressed maxima at 260.4 and 280.5)
EPA
DPAn-6
DPAn-3
DHA
7,17-diHDHA
10,17-diHDHA
73, 117, 173, 289, 402, 517, 573
73, 117, 173, 331, 359, 427, 517, 573
a All products analyzed as hydrogenated, trimethylsilyl derivatives.
b Free acid [M-H]Ϫ ion in negative ion mode.
c Methyl ester [M+Na]+ ion in positive ion mode.
(anionic) substrate enters the enzyme methyl group first.
The second dioxygenation is slower and less specific, add-
ing a hydroperoxy group at either the omega-13 or omega-16
position to form a dihydro(pero)xy product. In this in-
stance, it is proposed that the (neutral) substrate enters
the enzyme carboxylic acid group first (42, 52). This is sup-
ported by the current results examining the optimum pH
of the reaction, where the second dioxygenation was
shown to be suppressed at high pH values (55), as the fatty
acid would be fully deprotonated (pKa of DHA under reac-
tion conditions is ف7.4), and the anionic form of the sub-
strate is unable to enter the enzyme acid group first.
Consistent with previous results, higher enzyme con-
centrations are required to catalyze the formation of
dihydro(pero)xy products than are required to perform a
single dioxygenation (32, 43, 48). This may be because at
lower enzyme concentrations, enzyme deactivation/inhi-
bition occurs before there is a chance to perform the sec-
ond dioxygenation, or because the much slower kinetics
of the reaction requires more enzyme to increase the prob-
ability of the reaction occurring.
At high substrate concentrations, the formation of
dihydro(pero)xy compounds is completely inhibited and the
formation of monohydro(pero)xy compounds is significantly
reduced. This could not be rectified by the addition of more
enzyme. Therefore, it is most likely that at high substrate
concentrations, oxygen becomes the limiting reagent and
prevents the reaction from occurring (42, 59). Instead,
other products are formed, including one compound iden-
tified as 17-oxoheptadeca-4Z,7Z,10Z,13Z,15E-pentaenoic
acid, and a second compound proposed to be a DHA dimer
(with conjugated diene). It is most likely that these products
are formed through the anaerobic lipoxygenase pathway,
where a substrate free radical can be released from the en-
zyme without oxygenation. This can occur, because hydro-
gen abstraction occurs before molecular oxygen enters the
reaction (60). This free radical can then react with other
fatty acid molecules, radicals, and oxygen. Products such as
aldehydes, ketones, and fatty acid dimers have been re-
ported to form under anaerobic conditions (26, 57). The
formation of these products could be prevented by bub-
bling oxygen through the solution during the reaction
when high substrate concentrations were used. It has been
reported previously, that oxygen pressure can allow the use
of higher substrate concentrations (34). Similar products,
especially 17-oxoheptadeca-4Z,7Z,10Z,13Z,15E-pentaenoic
acid, were also observed in reactions where oxygen had
been removed by sparging the buffer with nitrogen prior to
adding the enzyme. At low oxygen concentrations small
amounts of nonspecific mono- and dihydro(pero)xy prod-
ucts also formed (34, 60).
Although it has previously been reported that lipoxyge-
nase catalyzed reactions should be carried out at low tem-
peratures to prevent the formation of nonspecific oxidation
and isomerization products (due to nonenzymatic reac-
tions) (32, 34), we observed that at higher temperatures
the reaction proceeded faster without an increase in un-
wanted side reactions. However, at 40°C, there was a de-
crease in the amount of product formed, either due to
deactivation of the enzyme or degradation of the product.
pH was found to be a critical variable in the lipoxyge-
nase catalyzed reactions studied. The double dioxygenation
reaction can be completely suppressed by performing the
reaction at pH 12, which is much higher than the optimum
TABLE 5. Reaction kinetics for the 15-sLOX catalyzed double dioxygenation of a range of
polyunsaturated fatty acid substrates
Parameter
ARA
EPA
DPAn-6
DHA
4.2
18.8
0.22
0.8
7.0
53.3
0.13
1.3
34.9
62.4
0.56
6.5
26.6
49.5
0.54
4.9
Vmax (␮M/min)
Km (␮M)
Vmax/Km (minϪ1
)
kcat (minϪ1
)
kcat/Km (␮MϪ1/minϪ1
)
0.04
0.02
0.10
0.10
Formation of resolvin analogs using soybean 15-lipoxygenase
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for the enzyme [pH 9 (43, 44, 58)], but for the purposes of
regio- and stereospecific manner. We have demonstrated
the use of this enzyme in a simple and controlled method
for the synthesis of both mono- and dihydroxy resolvin
analogs from a range of biologically important polyunsatu-
rated fatty acids. Compounds of this class are important, as
many have been demonstrated to have significant bioactiv-
ity, especially in relation to modulating the inflammatory
response. The reaction can be controlled by modifying
reaction conditions; dihydro(pero)xy products can be
formed by using high enzyme concentrations and dihydro
(pero)xy product formation can be completely inhibited
by using high pH to form highly pure monohydro(pero)
xy products instead. It is very important to maintain ade-
quate oxygen levels throughout the reaction to prevent
the formation of anaerobic reaction products.
synthesizing standards, ensures a more pure product. The
optimum pH found for the double dioxygenation reaction
(pH 9) is higher than the pH optimum reported previ-
ously for the second dioxygenation when using the mono-
hydroperoxy compound as the starting material instead of
the fatty acid [pH 7.5 (44, 58)]. This was thought to be due
to the need to first form 17S-HPDHA when using DHA as a
starting material, which occurs at higher pHs due to the
requirement of a charged substrate, while the second dioxy-
genation requires a neutral substrate. However, under
our conditions, we found the optimum pH for the trans-
formation of 17S-HPDHA to dihydro(pero)xy products
was also pH 9, although the maximum was not as dis-
tinct and similar amounts of dihydro(pero)xy products
were formed at pH 7 and 8 (results not shown). Higher
pHs also improve the solubility of the substrate, which may
be required under these conditions where no surfactant is
used.
Under our conditions, with relatively high enzyme con-
centrations (compared with previous work), the initial di-
oxygenation reaction to form 17S-H(P)DHA happened
almost instantaneously. The second dioxygenation was
also fast, consuming all 17S-H(P)DHA within 5 min, after
which the product profile remained constant.
Under certain reaction conditions, such as at high
temperatures and at low pH, there was a decrease in the
amount of dihydro(pero)xy products produced, without a
corresponding increase in another compound as detected
by NP-HPLC. At this point the reason for this is unknown,
but we propose that it could be caused by the formation of
volatile breakdown products that are lost before analysis
or are not detected by NP-HPLC, or the formation of polar
compounds that are removed by SPE prior to analysis. We
are continuing to investigate this.
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Formation of resolvin analogs using soybean 15-lipoxygenase
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