Synthesis of the 16S,17S-Epoxyprotectin Intermediate in the Biosynthesis of Protectins by Human Macrophages

Article abstract of DOI:10.1021/acs.jnatprod.5b00574

The n-3 polyunsaturated fatty acids act as substrates during the resolution phase of acute inflammation for the biosynthesis of specialized pro-resolving lipid mediators. One premier example is the C22-dihydroxy-polyunsaturated fatty acid protectin D1 (1). The human 15-lipoxygenase type I, via stereoselective processes and with docosahexaenoic acid as the substrate, enables the formation of this specialized pro-resolving lipid mediator. Herein, based on results from LC/MS-MS metabololipidomics, support is presented for the apprehended biosynthesis of 1 in human macrophages occurring via the intermediate 16S,17S-epoxyprotectin (5). Stereochemically pure 5 was obtained using the Katsuki-Sharpless epoxidation protocol, establishing the chirality at the C16 and C17 atoms, one Z-selective reduction, and E- and Z-stereoselective Wittig reactions. In addition, information on the nonenzymatic aqueous hydrolysis products and the half-life of 16S,17S-epoxyprotectin (5) is presented.

Full text of DOI:10.1021/acs.jnatprod.5b00574

Article
pubs.acs.org/jnp
Synthesis of the 16S,17S‑Epoxyprotectin Intermediate in the
Biosynthesis of Protectins by Human Macrophages
Marius Aursnes,† Jørn E. Tungen,† Romain A. Colas,‡ Iliyan Vlasakov,‡ Jesmond Dalli,‡
Charles N. Serhan,*,‡ and Trond V. Hansen*,†
†School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO Box 1068 Blindern, N-0316 Oslo, Norway
‡Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine,
Harvard Institutes of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, United
States
S
* Supporting Information
ABSTRACT: The n-3 polyunsaturated fatty acids act as
substrates during the resolution phase of acute inflammation
for the biosynthesis of specialized pro-resolving lipid
mediators. One premier example is the C22-dihydroxy-
polyunsaturated fatty acid protectin D1 (1). The human 15-
lipoxygenase type I, via stereoselective processes and with
docosahexaenoic acid as the substrate, enables the formation of
this specialized pro-resolving lipid mediator. Herein, based on results from LC/MS-MS metabololipidomics, support is presented
for the apprehended biosynthesis of 1 in human macrophages occurring via the intermediate 16S,17S-epoxyprotectin (5).
Stereochemically pure 5 was obtained using the Katsuki−Sharpless epoxidation protocol, establishing the chirality at the C16 and
C17 atoms, one Z-selective reduction, and E- and Z-stereoselective Wittig reactions. In addition, information on the
nonenzymatic aqueous hydrolysis products and the half-life of 16S,17S-epoxyprotectin (5) is presented.
everal chemical mediators were recently identified that have
Sthe ability to initiate, modulate, and dampen acute
inflammation as well as stimulate resolution.1,2 Recent efforts
have established that the return to homeostasis, i.e., catabasis,3
is due to the effective catalytic action by lipoxygenases and
cyclooxygenases on the dietary n-3 polyunsaturated fatty acids
(PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA). During the resolution phase of acute inflammation
these enzymatic processes stereoselectively form several distinct
families of autacoids coined specialized pro-resolving mediators
(SPMs),4−6 such as protectin D1 (PD1, 1), maresin 1 (2),
resolvin D1 (3), and the protectin conjugate 16R-glutathion-
yl,17S-hydroxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid
(PCTR1, 4).
Protectin D1 (1),7−10 also known as neuroprotectin D1
(NPD1) when produced in neural systems,9 is a 15-
lipoxygenase product of DHA formed in the presence of
molecular oxygen.11 PD1 (1) is a significant example among
the naturally occurring compounds belonging to the SPMs,
exhibiting potent anti-inflammatory properties together with
pro-resolving actions.11 PD1 (1) inhibits polymorphonuclear
leukocyte infiltration and promotes macrophage phagocytosis
and efferocytosis.11,12 The exact biomolecular mechanisms
underlying the resolution of many inflammatory processes have
been investigated with the use of SPMs.5,12 The lipid mediator
PD1 (1) has been the subject of several pharmacological
studies,5 and some clinical trial development programs have
been launched with PD1 (1).5
The exact structure of 1 was established in 2006 by direct
comparison of isomers of 1 obtained by stereoselective
synthesis.11,13 Protectin D1 (1) was disclosed to arise via an
epoxide intermediate based on data from experiments using
Received: June 28, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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both isotopic oxygen incorporation and identification of acid
alcohol trapping products via mass spectrometry (Figure
1).10,11
previously reported data.16 An E-selective Wittig reaction17
between 6 and the commercially available ylide
(triphenylphosphoranylidene)acetaldehyde (7) furnished ster-
eochemically pure aldehyde 8 in 61% yield after chromato-
graphic purification. Next, Wittig salt 9 was prepared from (Z)-
7-methoxy-7-oxohept-3-en-1-yl triphenylphosphonium iodide,
the latter prepared as previously reported from 4-pentynoic
acid.18 Lastly, aldehyde 6 was reacted in a Z-selective Wittig
reaction18−22 with the ylide of 9, which was generated at −78
°C after treatment with NaHMDS in THF and HMPA. This
sequence yielded the epoxide ester 10 in 41% yield (Scheme 1).
Scheme 1. Synthesis of the Methyl Ester 10 of 16S,17S-
Epoxyprotectin (5)
Figure 1. Outline of the biosynthesis of protectin D1 (1) and the
protectin conjugate PCTR1 (4).
Thus, compound 10 was made in 10 steps, starting from
commercially available 4-pentynoic acid, with an overall yield of
6% counting the longest linear sequence (Supporting
Information). The synthesis of the chemically labile 16S,17S-
epoxyprotectin (5) was achieved by a chemoselective basic
aqueous hydrolysis18−22 of the methyl ester in 10 just prior to
incubations (Supporting Information).
The geometrical configurations of the olefins constituting the
conjugated triene moiety of 10 were established using a COSY-
45 NMR experiment. The results thus obtained are in
accordance with a Z,E,E-triene present in 10. The following
chemical shifts and coupling constants (J values) were recorded
for the epoxy methyl ester 10: H12: 6.56 ppm, J = 14.9, 11.2 Hz;
H14: 6.41 ppm, J = 15.2, 10.9 Hz; H13: 6.11 ppm, J = 14.9, 10.8
Hz; H11: 6.03 ppm, J = 11.2 Hz; H4, H5, H7, H8, H10, H15, H19,
H20: 5.54−5.25 ppm. Notably, H11 has a 3J-coupling constant to
H10 with a value of 11.0 Hz, which is consistent with the
assigned Z- rather than an E-configured C10−C11 double
bond.
Identification of Nonenzymatic Aqueous Hydrolysis
Products from Synthetic 16S,17S-Epoxyprotectin (5).
First 16S,17S-epoxyprotectin (5) was added to H2O (pH = 7.0)
at room temperature. Incubations were quenched after 2 min,
and deuterated internal standards were added to facilitate
identification. The products were extracted using solid-phase
extraction on C18 columns, and then the aqueous hydrolysis
product profile of the synthetic material 5 was investigated
using LC/MS-MS-based lipid mediator metabololipidomics.23
The epoxide 5 is most likely also pivotal in the biosynthesis
of novel protectin conjugates in tissue regeneration.14 Earlier
studies of the biosynthesis of 1 yielded insufficient amounts to
confirm the existence and the absolute configuration of this
epoxide biosynthetic intermediate.
Due to the potent bioactions reported for protectin D1 (1)5
and the protectin glutathione conjugate PCTR1 (4),14 it is of
considerable interest to investigate the involvement of the
epoxide intermediate 5 in the biosynthesis of 1. Herein, support
for the existence of such an intermediate, named 16S,17S-
epoxyprotectin (5), is provided. In addition, data on the
aqueous stability, the nonenzymatic aqueous hydrolysis
products, and the enzymatic product formed from 5, but also
the half-life of 16S,17S-epoxyprotectin (5) under aqueous
conditions, are reported. Together, these efforts render support
for the involvement of 5 in the biosynthesis of protectin D1 (1)
in human macrophages.
RESULTS AND DISCUSSION
■
To establish evidence of the existence of an epoxide
intermediate and its role as a biosynthetic precursor to
bioactive protectins, stereochemically pure epoxide 5 was
obtained by total synthesis. First, aldehyde 6 was prepared from
commercially available cis-hex-3-en-1-ol in several steps,
including a Katsuki−Sharpless asymmetric epoxidation reaction
(Supporting Information).15 The absolute configuration set in
the epoxidation process was assured via correlation with
B
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Figure 2. LC/MS-MS profile of synthetic 16S,17S-epoxyprotectin (5) derived nonenzymatic aqueous hydrolysis products. Synthetic 16S,17S-
epoxyprotectin (5, 200 ng/200 μL) was placed in doubly distilled H2O (pH = 7.0), and products were extracted and profiled using lipid mediator
metabololipidomics. (A) MRM chromatogram of products m/z 359 > 217. Arrow denotes retention time of synthetic and authentic protectin D1
(1). (B, C) MS-MS spectra employed for identification of products under peaks (B) I and (C) III. Results are representative of n = 4 incubations.
Chromatographic analysis using multiple reaction monitoring
gave four distinct peaks in incubations of synthetic 16S,17S-
epoxyprotectin (5) with pH = 7.0 H2O (Figure 2A). Their
chromatographic behavior and respective MS-MS spectra
indicated the presence of two vicinal diol epoxide hydrolysis
products (tR ≈ 9.8 min and tR ≈ 10.3 min) as well as two
products (tR ≈ 11.2 min and tR ≈ 11.8 min) that arose from
addition of H2O to the least sterically hindered end of the
protectin carbocation intermediate.11,24 We next determined
the identity of each of the products in these incubations,
assessing their respective MS-MS spectra and matching these
with authentic and/or synthetic standards.24 These results
demonstrated that the four products were isomers of protectin
D1 (1), as authentic protectin D1 (1) eluted at a different
retention time in this system (Figure 2A). The products
beneath peaks I and II gave essentially identical MS-MS
fragmentation spectra (Figure 2B) and were consistent with
10,17S-dihydroxydocosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic
acid, with the double bonds at positions 4, 7, and 19 being
uninvolved in the aqueous hydrolysis reaction, and therefore
their configurations are proposed to be retained in the cis
configuration.25,26 Products under peaks III and IV also gave
essentially identical MS-MS fragmentation spectra to each other
(Figure 2C) and were consistent with the vicinal diol structure
of 16,17S-dihydroxydocosa-4Z,7Z,10Z,12E,14E,19Z-hexaenoic
acid. The double bonds at positions 4, 7, and 19 are also
thought to retain their cis configuration given their noninvolve-
ment in the aqueous hydrolysis reaction. Because authentic
PD1 (1) eluted at a different retention time from these four
products, this indicated that the aqueous hydrolysis of this
protectin intermediate 5 is analogous27 to that of leukotriene A4
(LTA4)28,29 and 13S,14S-epoxymaresin.30
Matching of Products from Synthetic 16S,17S-Epox-
yprotectin (5) and Products from Macrophage Epoxide
Intermediate. To assess the production of this protectin
epoxide intermediate by human macrophages, we sought
evidence for the formation of methoxy-trapping products
following acidic MeOH trapping. This approach was taken
because the amounts of this intermediate produced by human
cells are less than what is required for direct NMR analysis.
Protectin D1 (1) is produced during self-resolving Escherichia
coli (E. coli) infections,31 and given the fact that the lipid
mediator 1 accelerates the resolution of E. coli infections as well
as stimulates the phagocytosis of E. coli by macrophages, we
tested if these human macrophages would produce 16S,17S-
epoxyprotectin (5). E. coli does not form protectin D1 (1).31
Using multiple reaction monitoring (MRM) of products
obtained with human macrophage incubations gave two distinct
peaks (Figure 3A); the major peak I gave a retention time (tR)
of 12.0 min, and peak II gave tR 13.4 min. Incubation of
synthetic 16S,17S-epoxyprotectin (5) with acidified methanol
also gave two peaks, with tR 12.0 and 13.4 min, respectively
(Figure 3B). Assessment of the MS-MS fragmentation spectra
for the product beneath peak I gave characteristic fragmenta-
t i o n o f 1 0 - m e t h o x y - 1 7 S - h y d r o x y d o c o s a -
4Z,7Z,11E,13E,15E,19Z-hexaenoic acid (likely a pair of
C
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Figure 3. Evidence for the production of 16S,17S-epoxyprotectin by human macrophages. Human macrophages (5 × 106 cells/mL) were incubated
(PBS, pH = 7.45, 37 °C) and quenched after 2 min with acidified (apparent pH = ∼3) MeOH, and products were extracted and profiled using lipid
mediator metabololipidomics. (A) Synthetic 16S,17S-epoxyprotectin (5, 200 ng/200 μL) was placed in acidified methanol (apparent pH = ∼3), and
products were profiled using lipid mediator metabololipidomics. MRM chromatogram for transition m/z 373 > 246. (B) MRM chromatogram of
acid alcohol trapping products of the protectin intermediate from macrophages, transition m/z 373 − 246. Synthetic 16S,17S-epoxyprotectin (5, 200
ng/200 μL) was employed. (C, D) MS-MS spectra employed for the identification of products under peaks (C) I and (D) II. Results are
representative of n = 3 separate healthy donors for A, C, and D. For B, results are representative of n = 3 incubations.
diastereomers that coelute) and displayed m/z 373 = M − H,
m/z 355 = M − H − H2O, m/z 342 = M − H − OCH3, m/z
329 = M − H − CO2, m/z 324 = M − H − H2O − OCH3, m/z
311 = M − H − H2O − CO2, and m/z 246 = 275 − OCH3
(Figure 3C). Assessment of the MS-MS fragmentation spectra
for the product under peak II gave characteristic fragmentation
of 16-methoxy-17S-hydroxydocosa-4Z,7Z,10Z,12E,14E,19Z-
containing products (also likely a pair of diastereomers that
coelute) (Figure 3D). The configurations of the double bonds
at carbon positions 4, 7, and 19 are likely to be retained in the
cis configuration since they are uninvolved in the reaction with
MeOH. These results provide evidence that the acid alcohol
trapping products of synthetic 16S,17S-epoxyprotectin (5)
match those derived from human macrophage incubations and
are in accord with previous findings.8 In both experiments using
acidified MeOH, the formation of the two 10-methoxy-17S-
hydroxy isomeric products dominated.
Conversion of 16S,17S-Epoxyprotectin (5) to Protec-
tin D1 and Isomers (1) by Human Macrophages. We next
investigated the conversion of 16S,17S-epoxyprotectin (5) to
protectin D1 (1) and isomers of 1. To this end, we incubated
human macrophages with synthetic 5 and profiled the products
using LC-MS-MS-based lipid mediator profiling.23 Liquid
chromatographic analysis of the products obtained using
MRM for the ion pair m/z 359 > 153 gave a sharp peak with
tR = 10.5 min (Figure 4A). Moreover, an MS-MS fragmentation
pattern with m/z 359 = M − H, m/z 341 = M − H − H2O −
CO2, m/z 323 = M − H − 2H2O, m/z 315 = M − H − CO2,
m/z 297 = M − H − H2O − CO2, m/z 279 = M − H − 2H2O
− CO2, m/z 243 = 261 − H2O, m/z 217 = 261 − CO2, m/z
199 = 261 − H2O − CO2, and m/z 137 = 181 − CO2,
D
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products using denatured macrophages prior to addition of 5,
as reported in Figure 2B and C, consistent with isomers of
10,17S-dihydroxydocosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic
acid13,32 and two vicinal diol isomers of 16,17S-dihydroxydo-
cosa-4Z,7Z,10Z,12E,14E,19Z-hexaenoic acid, respectively. Alto-
gether, data from these experiments reveal that stereochemi-
cally pure epoxide 5 was converted to protectin D1 (1). In the
absence of enzymatic activity, epoxide 5 was also converted to
the same isomers of protectin D1 (1) shown in Figure 2.
Investigations on Aqueous Stability and Determina-
tion of Half-Life. We next tested the aqueous stability of
synthetic 16S,17S-epoxyprotectin (5); see Supporting Informa-
tion for details. Assessment of UV absorbance for this synthetic
product in phosphate-buffered saline (PBS, pH = 7.45)
demonstrated a rapid shift in the λmax from 280 nm,
characteristic of a conjugated triene allylic to an auxochrome,
to 270 nm (Figure 5). For comparison, LTA4 also gave a shift
in λmax from 280 nm to 270 nm (Figure 5). In these
incubations, we also found a reduction in absorbance at 293
nm, giving an estimated half-life of ∼19 s for 16S,17S-
epoxyprotectin (5) and ∼24 s for LTA4 (Figure 5). Together
these results establish the identity of the nonenzymatic
hydrolysis products for the protectin epoxide biosynthetic
intermediate and the configuration of the conjugated triene in
this human macrophage intermediate 5. The time course of
16S,17S-epoxyprotectin (5) stability was similar to that of
LTA4, and this suggests also that the protectin epoxide 5, like
that of LTA4 in the biosynthesis of for example LTB4, can be a
key intermediate in transcellular biosynthesis during cell−cell
interactions in complex tissues.27−29
The first step in the biosynthesis of protectin D1 (1)
depicted in Scheme 1 is a stereoselective abstraction of the pro-
S hydrogen atom at C-17 of DHA to form 17S-HpDHA
mediated by 15-lipoxygenase type I. This hydroperoxide is
converted to the 16S,17S-configurated epoxide 5 with a
concurrent second hydrogen abstraction at C-12 mediated by
the same lipoxygenase.5,24 These processes result in the
formation of the 10Z,12E,14E-configured triene in 5. Then 5,
under enzymatic hydrolysis conditions, is regio- and stereo-
selectively attacked by water at the C-10 position, via a
transient allylic carbocation species, which results in the
formation of the 10R-configured allylic alcohol and the
11E,13E,15Z-configured triene in protectin D1 (1). During
these enzymatically controlled steps the S-configuration at C-17
is not altered.5 This is consistent with the accepted biosynthesis
of other known pro-resolving mediators25,26 and the leuko-
trienes, such as LTB4, the latter class of mediators via the LTA4
epoxide.28,29 On the basis of these observations, and by
comparison of data obtained from LC-MS/MS experiments
using synthetic isomers of 1,13,32,33 it is unlikely that the
enantiomeric 16R,17R-configurated epoxide of 5 is formed in
human macrophages. Moreover, the formation of the 17R-
HpDHA stereoisomer has been observed in the presence of
aspirin and recombinant isolated COX-2 enzyme,7 which
results in the formation of the 17R-epimer of protectin D1 (1),
coined AT-protectin D1, most likely via a 16R,17R-configurated
epoxide intermediate. The absolute configuration of AT-
protectin D1 was determined on the basis of results from
LC/MS-MS metabololipidomics experiments32 using synthetic
standards.33
Figure 4. Stereochemically pure 16S,17S-epoxyprotectin (5) is
converted to protectin D1 (1) and isomers of 1 by human
macrophages. Human macrophages (5 × 107 cells/mL) obtained
from peripheral blood mononuclear cells were incubated with 16S,17S-
epoxyprotectin (5) (1.0 μM) and E. coli (2.5 × 108 cells/mL; DPBS
containing 10% BSA; pH = 7.45; 37 °C). Incubations were quenched
after 30 min using ice-cold methanol, and products were assessed
using metabololipidomics. (A) Selected ion chromatogram (m/z 359 >
153) depicting protectin D1 (1). (B) MS-MS spectrum employed for
identification of protectin D1 (1). (C) Human macrophages were
incubated at 100 °C for 1 h, then with 16S,17S-epoxyprotectin (5)
(1.0 μM) and E. coli (2.5 × 108 cells/mL; DPBS containing 10% BSA;
pH = 7.45; 37 °C), incubations were quenched after 60 min, and
products were assessed using metabololipidomics. Results are
representative of n = 4 separate cell preparations.
characteristic of protectin D1 (1), was observed (Figure
4B).7,11,18 Hence, enzymatic conversion of 5 to protectin D1
(1) was detected. Moreover, when the 16S,17S-epoxyprotectin
(5) was incubated with macrophages that were incubated for 1
h at 100 °C prior to addition of 5, four isomeric products were
observed (Figure 4C). The peaks I and II gave characteristic
retention times and MS-MS spectra corresponding with 10,17S-
dihydroxydocosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic acid iso-
mers. In addition, peaks III and IV gave retention times and
MS-MS products consistent with 16,17S-dihydroxydocosa-
4Z,7Z,10Z,12E,14E,19Z-hexaenoic acid isomers. The same
MS-MS fragmentation patterns were observed for these four
To summarize, we have now provided additional support for
the biosynthesis of protectin D1 (1) in human macrophages
occurring as depicted in Scheme 1 via the intermediate
E
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Figure 5. Aqueous stability of 16S,17S-epoxyprotectin (5). UV chromophores of (A) LTA4 and (B) 16S,17S-epoxyprotectin (5) and their
nonenzymatic hydrolysis products (PBS, pH = 7.45, room temperature) at indicated intervals. UV absorbance at 293 nm for (C) synthetic 16S,17S-
epoxyprotectin (5, 200 ng/200 μL, PBS, pH = 7.45, room temperature) and (D) LTA4 (200 ng/200 μL, PBS, pH = 7.45, room temperature). A and
B are representative of n = 3 experiments. C and D are mean of n = 3 experiments. The methyl ester of LTA4 was purchased from Cayman Chemical
Company and hydrolyzed just prior to use; see Supporting Information.
16S,17S-epoxyprotectin (5). Recently Rodriguez and Spur
reported a stereoselective total synthesis of PCTR1 (4) that
established the absolute configuration of 4.35,36 Notably, the
SPM 4 contains the 10Z,12E,14E-triene and an anti-
disubstituted thioether alcohol moiety with R- and S-
configurations at the C16 and C17 carbon atoms, respectively.
These structural features also render support for the existence
and involvement of 16S,17S-epoxyprotectin (5) as an
intermediate in the biosynthesis of the protectins.14 The
biosynthesis of the protectin members of the SPMs involved in
human physiological processes differs from other oxygenated
natural products, such as the oxylipins.37 For the latter class of
natural products, lipoxygenase and allene oxide synthase are the
enzymes involved in the formation of epoxy allylic cation
derived intermediates.38 In human physiological systems the
biosynthesis of the protectin class of SPMs results in natural
products, with an E,E,Z-triene moiety, exhibiting potent pro-
resolving actions. Altogether, these features distinguish the
SPMs from other oxygenated natural products of nonhuman
origin. Further investigations are ongoing toward elucidating
the role of the intermediate 5 in the biosynthesis of novel SPMs
and protectin conjugates in tissue regeneration.14,34 Since the
SPMs are of interest as agonists in resolution physiology, these
natural products will continue to attract interest from the
research community.4
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a 1 mL cell with a 1.0 dm path length on a
PerkinElmer 341 polarimeter. The UV/vis spectra from 190 to 900 nm
were recorded using a Biochrom Libra S32PC spectrometer using
quartz cuvettes. NMR spectra were recorded on a Bruker AVII400
spectrometer at 400 MHz or a Bruker AVII600 spectrometer at 600
MHz, for 1H NMR, and at 101 or 150 MHz for 13C NMR. Spectra are
referenced relative to the central residual protium solvent resonance in
1H NMR (CDCl3 δ = 7.27, C6D6 δ = 7.16, and MeOH-d4 δ = 3.31)
and the central carbon solvent resonance in 13C NMR (C6D6 δ =
128.06 ppm, CDCl3 δ = 77.00 ppm, and MeOH-d4 = δ 49.00). Mass
spectra were recorded at 70 eV on a Waters Prospec Q spectrometer
using EI, ES, or CI as the method of ionization. High-resolution mass
spectra were recorded on a Waters Prospec Q spectrometer using EI
or ES as the methods of ionization. Thin-layer chromatography was
performed on silica gel 60 F254 aluminum-backed plates fabricated by
F
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Merck. Flash column chromatography was performed on silica gel 60
(40−63 μm) produced by Merck. HPLC analyses for chemical purities
were performed on an Agilent Technologies 1200 Series instrument
with a diode array detector set at 254 nm and equipped with a C18
stationary phase (Eclipse XDB-C18 5 μm 4.6 × 150 mm), applying the
conditions stated. Unless stated otherwise, all commercially available
reagents and solvents were used in the form they were supplied
without any further purification. The stated yields are based on
isolated material. Diastereomeric ratios reported in this paper have not
been validated by calibration; see Wernerova and Hudlicky for
discussions and guidelines.39 Liquid chromatography (LC)-grade
solvents were purchased from Fisher Scientific. The Eclipse Plus C18
column (100 × 4.6 mm × 1.8 μm) was obtained from Agilent, and C18
SPE columns were from Waters. The synthetic standards for LC-
tandem mass spectrometry (MS-MS) quantitation and deuterated (d)
internal standards (d4-PGE2 and d4-LTB4) were purchased from
Cayman Chemicals. Zymosan A was purchased from Sigma-Aldrich;
anti-mouse CD11b and anti-mouse Ly6G were purchased from
Biolegend.
spectral data were in accord by those reported by Rodriguez and
Spur.35
Lipid Mediator Metabololipidomics.23 The LC-MS-MS system
included a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC
autoinjector (Shimadzu) paired with a QTrap 5500 (ABSciex) coupled
to a Poroshell 120 EC column (100 mm × 4.6 mm × 2.7 μm; Agilent)
maintained at 50 °C using a column oven (ThermaSphere TS-130;
Phenomenex) employed for identification and quantification. For
product profiling the following mobile phase was employed: MeOH/
H2O/acetic acid of 55:45:0.1 (v/v/v) with a gradient ramped to
80:20:0.1 (v/v/v) for 8 min, which was held for 3 min and then
ramped to 98:2:0.1 (v/v/v) for the next 0.1 min and maintained at
98:2:0.1 (v/v/v) for 4 min, with a flow rate maintained at 0.6 mL/min.
The QTrap 5500 was operated in negative ionization mode using
scheduled multiple reaction monitoring coupled with information-
dependent acquisition and an enhanced product ion scan.
Incubations with Human Macrophages. Human peripheral
blood mononuclear cells (PBMCs) were isolated by density-gradient
Ficoll-Histopaque isolation as described earlier.40 Isolated PBMCs
were washed three times to remove platelets, and monocytes purified
by adhesion to tissue culture plates for 30 min (37 °C, DPBS+/+, pH =
7.45). These were then cultured for 7 days in RPMI-1640 medium
supplemented with 10% FCS and 20 ng/mL GM-CSF. Macrophages
(∼1 × 106 cells/mL) were suspended in PBS+/+ containing DHA (1
μM), and E. coli (5 × 107 cfu) was added. After 2 min (37 °C, pH =
7.45) the incubation was stopped with 2 volumes of acidified ice-cold
MeOH (apparent pH = ∼3), deuterated internal standards were added
to facilitate identification, and products were extracted using C18 solid-
phase extraction and profiled using LC/MS-MS-based lipid mediator
metabololipidomics.23 In separate experiments human macrophages
(∼1 × 106 cells/mL) were suspended in PBS+/+, then incubated with 5
(200 ng/200 μL) and E. coli (5 × 107 cfu). After 5 min the incubations
were quenched and products profiled as above. The solution of 5 was
kept on solid dry ice in a closed container prior to use.
Human PBMCs were isolated by density-gradient Ficoll-Histopaque
isolation.30 Isolated PBMCs were washed three times to remove
platelets, and monocytes were purified by adhesion to tissue culture
plates for 30 min (37 °C, DPBS+/+, pH = 7.45). Cells were then
cultured for 7 days in RPMI 1640 medium supplemented with 10%
FCS and 20 ng/mL GM-CSF. Macrophages (5 × 107 cells) were
suspended in PBS+/+ containing 10% BSA and incubated for 10 min
(37 °C, pH 7.45) prior to the addition of 16S,17S-epoxyprotectin (5)
(∼1.0 μM) and E. coli (2.5 × 108 cells/mL). The solution of 5 was
kept on solid dry ice in a closed container prior to use. After 30 min,
incubations were stopped with 2 volumes of ice-cold methanol, d4-
LTB4 was added to facilitate quantification and identification, and
products were extracted using C18 columns and quantified by LC-MS-
MS metabololipidomics.23 In select experiments macrophages were
incubated at 100 °C for 1 h prior to addition of 16S,17S-
epoxyprotectin (5) and E. coli, and incubations conducted as detailed
above.
(2E,4E)-5-((2S,3S)-3-((Z)-Pent-2-en-1-yl)oxiran-2-yl)penta-
2,4-dienal (8). Aldehyde 6 (100 mg, 0.71 mmol, 1.0 equiv) was
dissolved in anhydrous toluene (4 mL), and solid
(triphenylphosphoranylidene)acetaldehyde (7) (446 mg, 1.50 mmol,
2.1 equiv) was added in one portion. The reaction setup was
thoroughly evacuated, refilled with argon (3×), and then heated to 90
°C for 4.5 h. The resulting deep red solution was allowed to cool to
room temperature and concentrated in vacuo. The residue was loaded
onto a silica gel column and eluted with 26% Et2O in heptane to yield
product 8 (84 mg, 0.44 mmol, 61%) as a colorless oil: [α]2D0 −57 (c
1
0.10, CHCl3); H NMR (600 MHz, CDCl3) δH 9.57 (d, J = 7.9 Hz,
1H), 7.07 (dd, J = 15.3, 11.0 Hz, 1H), 6.62 (dd, J = 15.3, 11.0 Hz, 1H),
6.16 (dd, J = 15.3, 7.9 Hz, 1H), 5.98 (dd, J = 15.3, 7.4 Hz, 1H), 5.61−
5.49 (m, 1H), 5.40−5.30 (m, 1H), 3.25 (dd, J = 7.4, 2.0 Hz, 1H), 2.95
(td, J = 5.3, 2.0 Hz, 1H), 2.49−2.41 (m, 1H), 2.41−2.32 (m, 1H), 2.06
(p, J = 6.8, 6.2 Hz, 2H), 0.98 (t, J = 7.5 Hz, 3H); 13C NMR (151 MHz,
CDCl3) δC 193.7, 150.1, 141.4, 135.5, 132.3, 131.0, 121.9, 61.0, 56.9,
29.7, 20.9, 14.3; HRESITOFMS m/z 215.1050 [M + Na]+ (calcd for
C12H16O2Na, 215.1047); TLC (heptane/Et2O, 74:26, KMnO4 stain)
Rf = 0.12. Note: A significant amount of the α,β-unsaturated aldehyde
resulting from a single Wittig reaction between 6 and 7 pre-elutes
product 8 under these purification conditions.
Methyl (4Z,7Z,10Z,12E,14E)-15-((2S,3S)-3-((Z)-Pent-2-en-1-
yl)oxiran-2-yl)pentadeca-4,7,10,12,14-pentaenoate (10). Wittig
salt 9 (50 mg, 0.088 mmol, 1.3 equiv) was dissolved in THF (1 mL)
and HMPA (0.15 mL) and cooled to −78 °C. NaHMDS (0.6 M in
toluene, 0.15 mL, 0.088 mmol, 1.3 equiv) was added dropwise, and the
reaction mixture was stirred for 30 min, giving an orange, completely
homogeneous solution. Aldehyde 8 (13 mg, 0.067 mmol, 1.0 equiv)
was azeotroped twice with 2-methyltetrahydrofuran, dissolved in THF
(0.5 mL), cooled to −78 °C, and added dropwise to the reaction flask
via cannula. After 1 h, the flask was rapidly warmed to −20 °C for a
few minutes and then quenched by the addition of phosphate buffer
(pH = 7, 2 mL). The phases were separated, and the aqueous phase
was extracted with Et2O (3 × 1 mL). The combined organic layers
were dried (Na2SO4), filtrated, and concentrated in vacuo. The silica
gel to which was added as a slurry prepared using 3% Et3N, 20% Et2O
in heptane, and the column was eluted with 25% Et2O in heptane to
provide the epoxy methyl ester 10 in 42% yield (10 mg) as a colorless
oil: [α]2D0 −44 (c 0.30, CHCl3); UV (hexane) λmax 272 (log ε 3.61),
280 (log ε 4.05), 291 (log ε 2.79) nm; 1H NMR (400 MHz, C6D6) δH
6.56 (dd, J = 14.9, 11.3 Hz, 1H), 6.41 (dd, J = 15.2, 10.9 Hz, 1H), 6.11
(dd, J = 14.9, 10.8 Hz, 1H), 6.09−5.98 (m, 1H), 5.54−5.25 (m, 8H),
3.34 (s, 3H), 3.06 (dd, J = 7.8, 2.0 Hz, 1H), 2.96−2.86 (m, 2H), 2.85−
2.76 (m, 2H), 2.72 (td, J = 5.2, 2.0 Hz, 1H), 2.31 (q, J = 7.6 Hz, 2H),
2.26−2.16 (m, 2H), 2.18−2.07 (m, 2H), 1.92 (p, J = 7.3 Hz, 2H), 0.88
(t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, C6D6) δC 172.8, 134.7,
134.2, 132.4, 131.4, 131.1, 129.3, 129.2, 129.0, 128.8, 128.5, 128.5,
123.2, 60.3, 57.8, 51.1, 34.0, 30.1, 26.6, 26.0, 23.2, 21.0, 14.4;
HRESITOFMS m/z 379.2252 [M + Na]+ (calcd for C23H32O3Na,
379.2249); TLC (heptanes/Et2O, 74:26, CAM stain) Rf = 0.23. The
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00574.
Experimental procedures for the synthesis and character-
1
ization data, i.e., H and 13C NMR spectral data, HRMS
and UV/vis spectra, and chromatograms from GLC and
HPLC analyses of intermediates 6, 8, and 9 and the
epoxy methyl ester 10, as well as for the intermediates
employed in the preparation of 6 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: cnserhan@zeus.bwh.harvard.edu.
■
G
DOI: 10.1021/acs.jnatprod.5b00574
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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*E-mail: t.v.hansen@farmasi.uio.no.
Notes
The authors declare the following competing financial
interest(s): Charles N. Serhan has filed patents on protectin
D1 (PD1, 1) and related compounds. C.N.S.’s interests are
reviewed and are managed by BWH and Partners HealthCare
in accordance with their conflict of interest policies.
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inflammatory mediators: eicosanoids, cytokines, and adhesion; Plenum
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D1 (1)); 10R,17R-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexae-
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4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid; 10R,17R-dihydroxydocosa-
4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid; 10S,17R-dihydroxydocosa-
4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid; 10R,17R-dihydroxydocosa-
4Z,7Z,11E,13E,15E,19Z-hexaenoic acid; and 10S,17R-dihydroxy-
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chromatographic properties and MS-MS fragmentation spectra with
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ACKNOWLEDGMENTS
■
The Research Council of Norway (KOSK-II 197704/V30) and
the School of Pharmacy, University of Oslo, are gratefully
acknowledged for Ph.D. scholarships to M.A. and J.E.T.,
respectively. I.V., R.A.C., and J.D. are supported by the National
Institutes of Health GM Grant PO1GM095467 (C.N.S.).
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DOI: 10.1021/acs.jnatprod.5b00574
J. Nat. Prod. XXXX, XXX, XXX−XXX