Total synthesis of the lipid mediator PD1n-3 DPA: Configurational assignments and anti-inflammatory and pro-resolving actions

Article abstract of DOI:10.1021/np4009865

The polyunsaturated lipid mediator PD1_{n-3 DPA} (5) was recently isolated from self-resolving inflammatory exudates of 5 and human macrophages. Herein, the first total synthesis of PD1_{n-3 DPA} (5) is reported in 10 steps and 9% overall yield. These efforts, together with NMR data of its methyl ester 6, confirmed the structure of 5 to be (7Z,10R,11E,13E,15Z,17S,19Z)-10,17- dihydroxydocosa-7,11,13,15,19-pentaenoic acid. The proposed biosynthetic pathway, with the involvement of an epoxide intermediate, was supported by results from trapping experiments. In addition, LC-MS/MS data of the free acid 5, obtained from hydrolysis of the synthetic methyl ester 6, matched data for the endogenously produced biological material. The natural product PD1 _{n-3 DPA} (5) demonstrated potent anti-inflammatory properties together with pro-resolving actions stimulating human macrophage phagocytosis and efferocytosis. These results contribute new knowledge on the n-3 DPA structure-function of the growing numbers of specialized pro-resolving lipid mediators and pathways.

Full text of DOI:10.1021/np4009865

Article
pubs.acs.org/jnp
Total Synthesis of the Lipid Mediator PD1n‑3 DPA: Configurational
Assignments and Anti-inflammatory and Pro-resolving Actions
Marius Aursnes,† Jørn E. Tungen,† Anders Vik,† Romain Colas,‡ Chien-Yee C. Cheng,‡ Jesmond Dalli,‡
Charles N. Serhan,‡ and Trond V. Hansen*,†,‡,§
†Department of Pharmaceutical Chemistry, School of Pharmacy, 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 polyunsaturated lipid mediator PD1n‑3 DPA
(5) was recently isolated from self-resolving inflammatory
exudates of 5 and human macrophages. Herein, the first total
synthesis of PD1n‑3 DPA (5) is reported in 10 steps and 9%
overall yield. These efforts, together with NMR data of its
methyl ester 6, confirmed the structure of 5 to be
(7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-
7,11,13,15,19-pentaenoic acid. The proposed biosynthetic
pathway, with the involvement of an epoxide intermediate,
was supported by results from trapping experiments. In addition, LC-MS/MS data of the free acid 5, obtained from hydrolysis of
the synthetic methyl ester 6, matched data for the endogenously produced biological material. The natural product PD1n‑3 DPA
(5) demonstrated potent anti-inflammatory properties together with pro-resolving actions stimulating human macrophage
phagocytosis and efferocytosis. These results contribute new knowledge on the n-3 DPA structure−function of the growing
numbers of specialized pro-resolving lipid mediators and pathways.
produced in neural systems,9 and maresin 1 (4)13 are produced
esolution of inflammation is necessary to re-establish
1−3
Rhomeostasis after injury or infection.
Excessive
inflammatory responses that fail to undergo resolution may
lead to chronic inflammation associated with many diseases,
such as asthma, atherosclerosis, autoimmune diseases, cancer,
and neuropathological disorders, including Alzheimer’s and
Parkinson’s diseases.1,2 Hence, over the last century, inflam-
mation has been the topic of numerous studies at the molecular
and cellular level.3 As of today, several chemical mediators have
been identified that can initiate, modulate, and reduce acute
inflammation and stimulate resolution.4 Continued efforts have
established that the return to homeostasis, catabasis,5 is
mediated by active biosynthesis and termination programs
orchestrated by novel families of natural products coined
specialized pro-resolving mediators (SPMs).6 The SPMs are
derived from polyunsaturated fatty acids (PUFAs) during the
resolution phase of acute inflammation.6,7 These efforts have
provided the fundamentals for the molecular understanding of
the resolution of many inflammatory diseases.7
from DHA.
These mediators are examples of SPMs structurally
elucidated within the past decade showing potent in vivo
anti-inflammatory and pro-resolving activities resulting in
catabasis.5 It is now well established that EPA and DHA are
substrates for the enzymatic formation of the aforementioned
SPMs. The naturally occurring compounds 1−4 have been the
Recent studies have identified several novel SPMs bio-
synthesized from the dietary n-3 PUFAs eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA). These novel di- and
trihydroxy-containing PUFA-derived lipid mediators are termed
resolvins, protectins, and maresins. Resolvin E1 (1) is produced
from EPA,8 while resolvin D1 (2),9,10 protectin D1 (PD1,
3),9−12 also known as neuroprotectin D1 (NPD1) when
Received: November 27, 2013
Published: February 27, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
910
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
subject of many pharmacological studies for the development of
potential new anti-inflammatory drugs,14,15 and some have
already been the subject of clinical trials.15 These autacoids
exhibit stereochemically selective modes of actions, reflecting
their routes of biosynthesis.15 In light of this, we became
interested in investigations using other n-3 PUFAs than EPA
and DHA as potential substrates for the endogenous formation
of novel SPMs. In humans, genome-wide association studies
demonstrate that elevation in circulating levels of n-3
docosapentaenoic acid (n-3 DPA), or (7Z,10Z,13Z,16Z,19Z)-
docosapentaenoic acid, with a concomitant decrease in DHA
levels, are associated with single nucleotide polymorphisms in
the gene encoding for the fatty acid elongase 2 (ELOVL2).16
The levels of circulating n-3 DPA do not appear to be directly
associated with dietary intake of PUFAs.17,18 Recently Dalli et
al. reported that n-3 DPA was converted to novel SPMs by
both human and murine leukocytes.19 One of the novel
products identified was assigned the structure
(7Z,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-
7,11,13,15,19-pentaenoic acid and denoted PD1n‑3 DPA (5),
given its relation to PD1 (3)20 (Figure 1).
with human polymorphonuclear leukocytes (PMN) followed
by chromatographic purification employing LC-MS/MS
fragmentation. Moreover, the natural product 5 exhibited
identical chromatographic properties with RvD5n‑3 DPA, or
(7S,8E,10Z,13Z,15E,17S,19Z)-7,17-dihydroxydocosa-
8,10,13,15,19-pentaenoic acid, produced under the same
experimental conditions.19 Therefore, the full stereochemical
assignment of the double-bond geometry in the conjugated
triene system as well as the absolute configuration of the C-10
group remained to be established. Also, the assignment of the
S-configuration on carbon atom 17 was based on biosynthetic
considerations. Hence, total synthesis became necessary for
establishing the complete configurational assignment of 5, as
has been necessary for the novel SPMs of interest, but also due
to the pico- to nanogram amounts produced in vivo.15,19 In
addition, the biological actions of 5 remained to be confirmed.
Herein, we report the synthesis and the elucidation of the
absolute configuration of SPM 5 based on matching data from
both LC-MS/MS and GC/MS analyses of synthetic and
endogenous materials, as well as 1D- and 2D-NMR
spectroscopic data of its methyl ester 6. The novel bioactions
of 5 are also reported.
RESULTS AND DISCUSSION
■
The synthesis of PD1n‑3 DPA (5) commenced with the
preparation of the Wittig salt 7 in four steps from cyclo-
heptanone (8). First, a Baeyer−Villiger oxidation23 of 8 yielded
lactone 9, which was reacted with MeOH and catalytic amounts
of H2SO4 to produce methyl-7-hydroxyheptanoate (10). The
alcohol functionality in 10 was converted into the correspond-
ing iodide 11. Reacting triphenylphosphine with 11 afforded
the desired Wittig salt 7 in 62% yield from 8 (Scheme 1). Then
(3R,4E,6E)-7-bromo-3-((tert-butyldimethylsilyl)oxy)hepta-4,6-
dienal (12) was prepared as previously reported from salt 13.24
Aldehyde 12 was reacted with the ylide of 7, the latter obtained
Scheme 1. Synthesis of Wittig Salt 7 and Intermediate 14
Figure 1. Proposed biosynthesis of PD1n‑3 DPA (5).
The only apparent structural difference between DHA and n-
3 DPA is the absence of a cis-double bond at the C-4 position.
This difference is proposed to confer unique biophysical
properties relevant for biological functions.21 With the aim of
providing multimilligram quantities for biological studies, the
synthesis of n-3 DPA was achieved.22
The initial structural assignment of the n-3 DPA-derived
compound PD1n‑3 DPA (5) was based on biosynthetic results
911
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
after reaction with NaHMDS in THF at −78 °C, in a Z-
selective Wittig reaction. After purification using silica gel
chromatography, pure vinylic bromide ester 14 was obtained in
54% yield (Scheme 1). The stereoisomeric purity of 14 was
determined by HPLC and 1H NMR analyses (Supporting
Information).
Scheme 2. Final Steps of the Synthesis of PD1n‑3 DPA (5)
The alkyne 15 was synthesized essentially as previously
reported from 1-butyne (16) and (R)-THP glycidol (17).25
Then vinylic bromide ester 14 and alkyne 15 were reacted in a
Sonogashira reaction26 at ambient temperature in the presence
of catalytic Pd(PPh3)4 and CuI with diethyl amine as solvent.
This afforded the bis-TBS-protected methyl ester 18 in 92%
yield. Deprotection of the two TBS groups in 18 with five
equivalents of TBAF in THF at 0 °C gave an 85% yield of the
diol 19.27 The internal alkyne in 19 was reduced to the
Z,E,E,Z,Z-pentaene ester 6 in 50% yield using a modified
Lindlar hydrogenation reaction28 with high stereoselectivity, as
1
only one diastereomer was detected by HPLC and H NMR
analyses. Finally, hydrolysis of the methyl ester 6 at 0 °C with
dilute aqueous LiOH in MeOH followed by mild acidic workup
with aqueous NaH2PO429 resulted in a 71% yield of PD1n‑3 DPA
(5) after purification by column chromatography (Scheme 2).
The assignment of the Z- or E-geometry for each of the
double bonds was achieved by two-dimensional NMR spec-
troscopy, with MeOH-d4 as the solvent and internal standard.
These experiments revealed the connectivity between adjacent
olefinic hydrogens (H-7/H-8, H-11/H-16, and H-19/H-20),
which in connection with the data from the HMBC spectra
permitted the assignment of all olefinic hydrogens. In particular,
the signals at 5.74 (dd, 1H, J = 14.4 Hz) and 6.07 ppm (t, 1H, J
= 11.0 Hz) were diagnostic for an E- and Z-double bond,
respectively. The COSY spectrum was used for assigning vicinal
signals for both of the two isolated Z-olefins and the E,E,Z-
triene moiety. Moreover, two signals from the hydrogen atoms
attached to the carbinol carbon atoms were observed as
expected with signals at 4.12 (m, 1H) and 4.56 ppm (dt, 1H, J
= 8.9, 6.8 Hz). The HSQC spectrum was used for assigning the
signals from the methylene carbons at 36.36 and 36.38 ppm,
next to the C-19/C-20 and the C-7/C-8 Z-double bonds,
1
respectively. The data from the H and 13C NMR spectra, in
combination with the COSY and the HMBC spectra, allowed
the structural assignment of the rest of the molecule (Table 1).
UV spectra for both synthetic and endogenously produced
MeOH
PD1n‑3 DPA (5) were compared. For synthetic 5, λmax
proposed biosynthetic pathway19 as depicted in Figure 2,
involving an acid-catalyzed ring-opening of the 16,17-
epoxyPDn‑3 DPA. In addition, these observations are in
accordance with the reaction pathway reported by Corey and
Mehrotra33 as well as enzyme-catalyzed mechanisms in the
biosynthesis of other pro-resolving mediators.34
In order to determine whether synthetic 5 matched
endogenous PD1n‑3 DPA (5), authentic 5 from murine self-
resolving exudates as well as human macrophages was
employed.19 Figure 2A shows human macrophage PD1n‑3 DPA
(5) from n-3 DPA with a retention time (tR) of 12.4 min,
together with its natural isomers. Figure 2B depicts endogenous
PD1n‑3 DPA (5) from mouse peritoneal exudates that also
displayed tR = 12.4 min. This is shown together with its natural
isomers produced in mouse peritoneal exudates. The chromato-
graphic behavior of synthetic 5 (tR = 12.4 min), obtained from
hydrolysis of synthetic methyl ester 6, is shown in Figure 2C.
Figure 2D reports the co-injection of synthetic and
endogenously obtained material added at essentially equal
absorbances were observed at 262, 271, and 282 nm; all in
excellent agreement with the endogenous product18 (261, 271,
282 nm, λmaxMeOH), as well as with literature values.30 Synthetic
5 was treated with excess diazomethane followed by N,O-
bis(trimethylsilyl)trifluoroacetamide (BSTFA) to produce the
bis-TMS ether of methyl ester 6, which was subjected to GC/
MS (Supporting Information, Figure S-25). The following
characteristic m/z values were observed: 520 = M, 505 = M −
CH3, 489 = M − OCH3, 430 = M − OTMS, 340 = M −
2×OTMS, and 73 = TMS.
Experiments supporting the proposed biosynthetic pathway
in Figure 2 were then performed. Soybean lipoxygenase was
incubated with n-3 DPA in borate buffer (pH = 8.0) at ambient
temperature essentially as described.9 Excess acidic MeOH was
added to quench the reaction and promote opening of the
epoxide. Then the product mixture was assessed by lipid
mediator metabololipidomics.31,32 From the MS/MS fragmen-
tation patterns of the formed products, four structures were
assigned (Figure S-26). These results render support for the
912
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
1
Table 1. Compilation of H and 13C NMR Data of the
a
Methyl Ester 6
b
c
d
position
δC, mult.
δH, mult. (J in Hz)
HMBC
COSY
1
175.9, C
2
34.8, CH2
25.9, CH2
29.8, CH2
30.3, CH2
28.2, CH2
132.8, CH
126.2, CH
36.4, CH2
73.1, CH
2.31, m
1, 3, 4
3
3
1.60, quint (7.3)
1.33, m
1, 2, 4, 5
2, 3, 5, 6
3, 4, 6, 7
4, 5, 7, 8
5, 6, 9
2, 4
4
3, 5
5
1.37, m
4, 6
6
2.04, m
5, 7
7
5.47, m
6, 8
8
5.40, m
6, 9, 10
7, 9
9
2.26 + 2.32, m
4.12, m
7, 8, 10, 11
8, 9, 11, 12
9, 10, 13
8, 10
9, 11
10, 12
11, 13
12, 14
13, 15
14, 16
15, 17
16, 18
17, 19
18, 20
19, 21
20, 22
21
10
11
12
13
14
15
16
17
18
19
20
21
22
23
138.0, CH
131.3, CH
134.9, CH
128.9, CH
130.5, CH
134.8, CH
68.6, CH
5.74, dd (14.4, 6.7)
6.26, m
10, 11, 13, 14
11, 12, 15
12, 13, 15, 16
13, 14, 16, 17
14, 18
6.24, m
6.52, dd (13.8, 11.2)
6.07, t
5.38, m
4.56, dt (8.9, 6.8)
2.36 + 2.21, m
5.34, m
15, 16, 18, 19
16, 17, 19, 20
17, 18, 20, 21
18, 19, 21, 22
19, 20, 22
20, 21
36.4, CH2
125.3, CH
134.7, CH
21.7, CH2
14.6, CH3
52.0, CH3
5.45, m
2.07, m
0.97, t (7.5)
3.65, s
1
a
MeOH-d4 was used as solvent. See Figure 1 for atom labeling.
Measured at 125 MHz. Measured at 500 MHz. HMBC correlations
b
c
d
are from proton(s) stated to the indicated carbon(s). The ppm values
listed above for δH were assigned using the center of the COSY and
HSQC peak intensities.
Figure 2. Endogenous PD1n‑3 DPA (5) from human macrophages and
resolving inflammatory exudates match synthetic material. MRM
chromatograms for selected ion pair m/z 361−183 depicting (A)
PD1n‑3 DPA (5) from human macrophages (5 × 107 cells/mL)
incubated with 0.1 mg of opsonized zymosan and n-3 DPA (1 μM,
37 °C, 30 min, DPBS+/+, pH = 7.45). Results are representative of n =
3 human macrophage preparations. (B) Endogenous PD1n‑3 DPA (5)
obtained from mice injected with zymosan (1 mg/mouse) and
exudates collected at 4 h. Results are representative of n = 4 mice
amounts. Altogether, Figure 2 demonstrates that synthetic 5
coelutes with endogenously obtained 5. In addition, the MS/
MS spectra for both natural and synthetic 5 were essentially
identical (Figure S-24).
Having established that the physical properties of synthetic 5
matched those of endogenous 5, we next required the
confirmation of its anti-inflammatory and pro-resolving actions.
Administration of synthetic 5 at a dose as low as 10 ng per
mouse significantly reduced neutrophil recruitment during
peritonitis, giving a significant reduction in the number of
neutrophils recovered from peritoneal exudates following
zymosan (1 mg/mouse) challenge determined by light
microscopy and flow cytometry (Figure 3A). These actions
were comparable to those displayed by the potent DHA-
derived pro-resolving mediator protectin D1 (3).20 Human
macrophage phagocytosis and efferocytosis (clearance of
apoptotic neutrophils) are key processes in the resolution of
inflammation5 and defining actions of SPMs.6 The ability of
synthetic 5 in this system to stimulate human macrophage
phagocytosis and efferocytosis was investigated.13 Incubation of
synthetic 5 with human macrophages and increasing concen-
trations of 5 led to an increase in macrophage phagocytosis of
both fluorescence-labeled yeast cell wall particles (zymosan)
and apoptotic neutrophils. These actions were shared with the
DHA-derived protectin D1 (3) (Figure 3B). In addition,
PD1n‑3 DPA (5) displayed potent efferocytosis effects against
apoptotic human neutrophils (Figure 3C). PD1n‑3 DPA stimu-
lated proresolving actions with macrophages at picomolar
concentrations and appeared to give a trend toward a bell-
shaped dose−response with these isolated human cells. These
exudates. (C) Synthetic material (inset: characteristic UV-absorption
MeOH
spectrum, λmax
1 nm). (D) Co-injection of resolving exudate
endogenous PD1n‑3 DPA (5) with synthetic material. Results are
representative of n = 4.
findings demonstrate that 5 displayed both potent anti-
inflammatory and pro-resolving actions, confirming the potent
immunoresolvent properties of PD1n‑3 DPA (5).
The complete structure elucidation and a stereocontrolled
total synthesis of PD1n‑3 DPA have been reported. These efforts
unambiguously confirmed the structure of PD1n‑3 DPA (5) to be
(7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-
7,11,13,15,19-pentaenoic acid. The synthetic material was
matched to that of endogenously produced PD1n‑3 DPA (5).
Compound 5 displayed potent anti-inflammatory and pro-
resolving activities characteristic for members of the SPM class
of natural products. The dihydroxy-polyunsaturated fatty acid 5
is a congener with the DHA-derived protectin D1 (3).7−11,20
Protectin D1 (3) exhibits potent protective activity in vivo in
models of inflammatory diseases5,20 as well as other interesting
biological actions in organ protection30 and reduces pain.35
Further biological evaluations of PD1n‑3 DPA (5) are ongoing
and will be reported separately.
913
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
spectrophotometer. NMR spectra were recorded on a Bruker DRX500
or a Bruker AVII400 spectrometer at 500 or 400 MHz, respectively,
1
for H NMR and at 126 or 101 MHz, respectively, for 13C NMR.
Spectra are referenced relative to the central residual protium solvent
resonance in 1H NMR (CDCl3 δ = 7.27 and MeOH-d4 δ = 3.31) and
the central carbon solvent resonance in 13C NMR (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 Merck. Flash column
chromatography was performed on silica gel 60 (40−63 μm) produced
by Merck. HPLC analyses were performed on an Agilent Technologies
1200 Series instrument with 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. Diastereomeric ratios
reported in this paper have not been validated by calibration.36
M e th y l ( 1 0R, 7Z, 11E , 13 E) -1 4 -B ro mo - 10 -(( t e r t-
butyldimethylsilyl)oxy)tetradeca-7,11,13-trienoate (14). To
the Wittig salt 7 (1.67 g, 3.13 mmol, 1.0 equiv) in THF (47 mL)
was added NaHMDS (0.6 M in toluene, 1.0 equiv) at −78 °C, and the
mixture was stirred for 60 min at that temperature. Aldehyde 12 was
added. The solution was allowed to warm slowly to room temperature
in a dry ice/acetone bath and stirred for 24 h before it was quenched
with phosphate buffer (45 mL, pH = 7.2). Et2O (60 mL) was added,
and the phases were separated. The H2O phase was extracted with
Et2O (2 × 60 mL) and the combined organic layers were dried
(Na2SO4), before the solvent was evaporated. The crude product was
purified by column chromatography on silica (hexanes/EtOAc, 97:3)
to afford the title compound 14 as a colorless oil. Yield: 754 mg (54%
1
over two steps). [α]20 −18 (c 0.09, MeOH); H NMR (400 MHz,
D
CDCl3) δ 6.68 (dd, J = 13.4, 10.9 Hz, 1H), 6.27 (d, J = 13.5 Hz, 1H),
6.09 (dd, J = 15.2, 10.9 Hz, 1H), 5.71 (dd, J = 15.3, 9.6 Hz, 1H), 5.48−
5.29 (m, 1H), 4.13 (dd, J = 6.0, 1.4 Hz, 1H), 3.66 (s, 3H), 2.30 (t, J =
7.5 Hz, 2H), 2.28−2.17 (m, 2H), 2.05−1.95 (m, 2H), 1.62 (p, J = 7.4
Hz, 2H), 1.40−1.25 (m, 4H), 0.89 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H);
13C NMR (101 MHz, CDCl3) δ 174.3, 138.1, 137.2, 131.9, 126.6,
125.2, 108.2, 72.7, 51.6, 36.3, 34.2, 29.4, 29.0, 27.4, 26.0 (3C), 25.0,
18.4, −4.4, −4.6; HREIMS m/z 447.1751 [M + H]+ (calcd for
C21H3881BrO3Si, 447.1753); TLC (hexanes/EtOAc, 95:5, CAM stain)
Rf = 0.21; diastereomeric ratio (>98%) was determined by HPLC
analysis (Eclipse XDB-C18, MeOH/H2O, 92:8, 1.1 mL/min);
tr(minor) = 8.41 and 8.74 min and tr(major) = 9.30 min.
Figure 3. PD1n‑3 DPA (5) displays potent anti-inflammatory and pro-
resolving actions. PD1n‑3 DPA, (5), PD1 (3), or vehicle (saline
containing 0.01% EtOH) were administered iv 5 min prior to ip
administration of zymosan (1 mg). Exudates were collected at 4 h, and
the number of infiltrated neutrophils was determined by flow
cytometry (top right inset) and light microscopy. Results are mean
SEM. n = 4 mice per treatment (**p < 0.01 vs vehicle group). (B,
C) Macrophages were incubated with vehicle (0.1% EtOH in PBS),
PD1n‑3 DPA (5) (100 nM to 10 pM), or PD1 (3) (100 nM to 10 pM;
15 min, 37 °C, pH = 7.45) prior to addition of (B) fluorescently
labeled zymosan (1:10 macrophages to zymosan) or (C) fluorescently
labeled apoptotic human neutrophils. After 60 min (37 °C, pH =
7.45), the incubation was stopped, extracellular fluorescence quenched
using trypan blue, and phagocytosis assessed using a SpectraMax M3
plate reader. Results are mean SEM. n = 4 macrophage preparations
(*p < 0.05 vs PBS-incubated macrophages).
Methyl (7Z,10R,11E,13E,17S,19Z)-10,17-Bis((tert-
butyldimethylsilyl)oxy)docosa-7,11,13,19-tetraen-15-ynoate
(18). To a solution of vinyl bromide 14 (285 mg, 0.64 mmol, 1.0
equiv) in Et2NH (1.5 mL) and benzene (0.6 mL) was added
Pd(PPh3)4 (22 mg, 0.019 mmol, 3 mol %), and the reaction was
stirred for 45 min in the dark. CuI (6 mg, 0.032 mmol, 5 mol %) in a
minimum amount of Et2NH was added, followed by dropwise addition
of alkyne 15 (153 mg, 0.64 mmol, 1.0 equiv) in Et3N (1.5 mL). After
stirring at ambient temperature for 20 h, the reaction was quenched by
saturated NH4Cl (15 mL). Et2O (15 mL) was added, and the phases
were separated. The H2O phase was extracted with Et2O (2 × 15 mL)
and the combined organic layers were dried (Na2SO4), before the
solvent was evaporated. The crude product was purified by column
chromatography on silica (hexanes/EtOAc, 9:1) to afford the title
compound 18 as a pale yellow oil. Yield: 387 mg (92%); [α]20D −21 (c
0.08, MeOH); 1H NMR (400 MHz, CDCl3) δ 6.51 (dd, J = 15.5, 10.9
Hz, 1H), 6.18 (ddd, J = 14.2, 10.3, 1.0 Hz, 1H), 5.75 (dd, J = 15.2, 5.9
Hz, 1H), 5.62−5.31 (m, 5H), 4.47 (td, J = 6.6, 1.8 Hz, 1H), 4.21−4.12
(m, 1H), 3.66 (t, 3H), 2.44 (t, J = 7.9 Hz, 2H), 2.30 (t, J = 7.6 Hz,
2H), 2.28−2.15 (m, 2H), 2.11−1.94 (m, 4H), 1.63 (q, J = 7.4 Hz,
2H), 1.40−1.25 (m, 4H), 0.96 (t, J = 7.5 Hz, 3H), 0.91 (s, 9H), 0.89
(s, 9H), 0.12 (d, J = 8.3 Hz, 6H), 0.03 (d, J = 8.3 Hz, 6H); 13C NMR
(101 MHz, CDCl3) δ 174.3, 141.2, 139.3, 134.4, 131.9, 128.6, 125.4,
124.1, 110.7, 93.4, 83.6, 72.9, 63.7, 51.6, 36.8, 36.4, 34.2, 29.4, 29.0,
27.4, 26.0 (3C), 26.0 (3C), 25.0, 20.9, 18.5, 18.4, 14.4, −4.3 (2C),
−4.6, −4.8; HRESTOFMS m/z 625.4084 [M + Na]+ (calcd for
EXPERIMENTAL SECTION
■
General Experimental Procedures. 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. Zymosan was purchased from Sigma-
Aldrich. Optical rotations were measured using a 1 mL cell with a 1.0
dm path length on a Perkin-Elmer 341 polarimeter. The UV/vis
spectra from 190 to 900 nm were recorded using a Biochrom Libra
S32PC spectrometer using quartz cuvettes. IR spectra (4000−600
cm−1) were obtained on a Perkin-Elmer Spectrum BX series FT-IR
914
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
C35H62O4Si2Na, 625.4085); TLC (hexanes/EtOAc, 9:1, CAM stain) Rf
= 0.63.
Briefly, as developed for other lipid mediators, all samples for LC-MS/
MS-based lipidomics were subject to solid-phase extraction. Prior to
sample extraction, d4-LTB4 (500 pg) was added. Extracted samples
were analyzed with an LC-MS system, a QTrap 5500 (ABSciex)
equipped with a Shimadzu SIL-20AC HT autosampler and LC-20AD
LC pumps. An Agilent Eclipse Plus C18 column (100 mm × 4.6 mm
× 1.8 μm) was used with a gradient of MeOH/H2O/acetic acid of
55:45:0.01 (v/v/v) to 100:0:0.01 at a 0.4 mL/min flow rate. To
monitor targeted SPMs, we used multiple reaction monitoring
(MRM) with signature ion fragments for each molecule (at least six
diagnostic ions).32 GC-MS analyses were carried out as previously
reported.9
Methyl (7Z,10R,11E,13E,17S,19Z)-10,17-Dihydroxydocosa-
7,11,13,19-tetraen-15-ynoate (19). TBAF (704 mg, 2.70 mmol,
5.0 equiv, 1.0 M in THF) was added to a solution of TBS-protected
alcohol 18 (325 mg, 0.54 mmol, 1.0 equiv) in THF (6.9 mL) at 0 °C.
The reaction was stirred for 2.5 h before it was quenched with
phosphate buffer (pH = 7.2, 2.8 mL). Brine (30 mL) and EtOAc (30
mL) were added, and the phases were separated. The H2O phase was
extracted with EtOAc (2 × 30 mL) and the combined organic layers
were dried (Na2SO4) before the solvent was evaporated. The crude
product was purified by column chromatography on silica (hexanes/
EtOAc, 7:3) to afford the title compound 19 as a pale yellow oil. Yield:
Preparation of Naturally Occurring PD1n‑3 DPA (5). Male FVB
mice (6 to 8 weeks old) purchased from Charles River Laboratories
were fed ad libitum laboratory rodent diet 20-5058 (Lab Diet, Purina
Mills). All animal experimental procedures were approved by the
Standing Committee on Animals of Harvard Medical School (protocol
no. 02570) and complied with institutional and U.S. National
Institutes of Health (NIH) guidelines. Peritonitis was induced by
zymosan injection (1 mg/mL) intraperitoneally (ip), and exudates
were obtained 4 h later. Human macrophages were prepared from
peripheral blood mononuclear cells following literature protocols.31
Macrophages were suspended in DPBS+/+ (5 × 107 cells/mL) and
incubated with n-3 DPA (1 μM) and 0.1 mg of serum-treated zymosan
(37 °C, 30 min, pH = 7.45). The incubations were stopped with 2 mL
of ice-cold MeOH, and mediators extracted over C18 columns as
described above.
Anti-inflammatory and Pro-resolving Actions. Mice were
administered intravenously (iv) vehicle (saline containing 0.1%
EtOH), PD1n‑3 DPA (5) (10 ng/mouse), or protectin D1 (3) for the
purpose of direct comparison (10 ng/mouse) 5 min prior to ip
zymosan administration (1 mg). After 4 h the exudates were collected
and the number of extravasated neutrophils was determined using
Turks solution and flow cytometry.19 Human macrophages and
peripheral blood neutrophils were prepared; then phagocytosis and
efferocytosis were assessed as described in ref 19. Briefly, cells were
incubated with vehicle (0.1% EtOH in DPBS), PD1n3 DPA (5), or
protectin D1 (5) at the indicated concentrations for 15 min at 37 °C;
then FITC-labeled zymosan- or bisbenzimide-labeled apoptotic
neutrophils were added and cells incubated for 60 min at 37 °C.
Phagocytosis was assessed using an M3 SpectraMax plate reader.
1
202 mg (85%); [α]20 −16 (c 0.06, MeOH); H NMR (400 MHz,
D
CDCl3) δ 6.55 (dd, J = 15.6, 11.0 Hz, 1H), 6.27 (dd, J = 14.9, 11.6 Hz,
1H), 5.81 (dd, J = 15.2, 6.1 Hz, 1H), 5.67−5.49 (m, 3H), 5.48−5.31
(m, 2H), 4.52 (td, J = 6.4, 1.9 Hz, 1H), 4.25−4.16 (m, 1H), 3.68 (t,
3H), 2.49 (t, J = 6.9 Hz, 2H), 2.35−2.26 (m, 4H), 2.14−1.99 (m, 4H),
1.98 (d, J = 5.4 Hz, 1H), 1.71 (bs, 1H); 1.62 (p, J = 7.5 Hz, 2H),
1.43−1.27 (m, 4H), 0.97 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 174.4, 141.5, 138.4, 136.1, 133.8, 129.4, 124.3, 122.9, 110.9,
92.6, 84.1, 71.7, 62.7, 51.7, 35.8, 35.5, 34.2, 29.3, 28.9, 27.4, 25.0, 21.0,
14.4; HRESTOFMS m/z 397.2354 [M + Na]+ (calcd for C23H34O4Na,
397.2355); TLC (hexanes/EtOAc, 7:3, CAM stain) Rf = 0.26.
Methyl (7Z,10R,11E,13E,15Z,17S,19Z)-10,17-Dihydroxydoco-
sa-7,11,13,15,19-pentaenoate (6). To a solution of alkyne 19 (30
mg, 0.08 mmol) in EtOAc/pyridine/1-octene (1.65 mL, 10:1:1) under
argon) was added Lindlar’s catalyst (45 mg), and the flask was
evacuated and filled with argon. The reaction was stirred for 60 h at
ambient temperature under a balloon of hydrogen gas until
completion. The reaction mixture was loaded directly onto a silica
gel column and purified by chromatography (hexanes/EtOAc, 9:1) to
afford the title compound 6 as a pale yellow oil. Yield: 15 mg (50%).
[α]20 = −19 (c 0.08, MeOH); UV (MeOH) λmax 262 (log ε 4.52),
D
1
271 (log ε 4.60) and 282 nm (log ε 4.53); H NMR (500 MHz,
MeOH-d4) and 13C NMR (126 MHz, MeOH-d4), see Table 1;
HRESTOFMS m/z 399.2511 [M + Na]+ (calcd for C23H36O4Na,
399.2506); TLC (hexanes/EtOAc, 6:4, CAM stain) Rf = 0.16;
diastereomeric ratio was determined by HPLC (Eclipse XDB-C18,
MeOH/H2O, 8:2, 0.8 mL/min); tr(minor) = 6.67 min and tr(major) =
8.31 min.
Protectin D1n‑3 DPA (5). Methyl ester 6 (14 mg, 0.04 mmol) was
dissolved in MeOH/H2O, 1:1 (35 mL), and cooled to 0 °C. LiOH
(1.0 M, 2.2 mL) was added dropwise. The reaction mixture was stirred
at the above-mentioned temperature for 48 h, after which a saturated
solution of NaH2PO4 (3.3 mL) was added. Next, NaCl (10.0 g) was
added followed by EtOAc (50 mL). The organic phase was decanted,
dried (Na2SO4), and concentrated in vacuo, affording the title
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization data of synthetic
intermediates 7−19, PD1n‑3 DPA (5), and its methyl ester 6, 1H,
13C, and 2D-NMR spectra data, HRMS and UV/vis spectra,
HPLC analyses of synthetic compounds as well as LC/MS/MS
data and GC/MS chromatograms of endogenous PD1n‑3DPA (5)
and its bis-TMS ether of methyl ester 6 are available free of
charge via the Internet at http://pubs.acs.org.
compound 5 as a colorless oil. Yield: 9.5 mg (71%); [α]20 −28 (c
D
0.1, MeOH); UV (MeOH) λmax 262 (log ε 4.53), 271 (log ε 4.60),
282 nm (log ε 4.54); 1H NMR (400 MHz, MeOH-d4) δ 6.52 (dd, J =
13.7, 11.1 Hz, 1H), 6.31−6.20 (m, 2H), 6.08 (t, J = 11.0 Hz, 1H), 5.74
(dd, J = 14.5, 6.6 Hz, 1H), 5.50−5.30 (m, 5H), 4.56 (dt, J = 8.6, 6.3
Hz, 1H), 4.15−4.09 (m, 1H), 2.40−2.17 (m, 6H), 2.10−2.02 (m, 4H),
1.60 (p, J = 7.4 Hz, 2H), 1.42−1.30 (m, 4H), 0.97 (t, J = 7.5 Hz, 3H);
13C NMR (101 MHz, MeOH-d4) δ 178.2, 138.0, 134.9, 134.8, 134.7,
132.9, 131.3, 130.6, 128.9, 126.2, 125.3, 73.1, 68.6, 36.4, 36.4, 35.4,
30.4, 29.9, 28.3, 26.2, 21.7, 14.6; HRESTOFMS m/z 361.2378 [M −
H]+ (calcd for C22H33O4 361.2385); TLC (Et2O with a drop of
AcOH, CAM stain) Rf = 0.34. The purity (>98%) was determined by
HPLC analysis (Eclipse XDB-C18, MeOH/3.3 mM HCOOH in H2O,
7:3, 1.0 mL/min); tr(major) = 9.52 min and tr(minor) = 13.49 min.
Biogenic Synthesis and Identification of 16,17-Epoxy
PDn‑3 DPA. In brief, soybean lipoxygenase (50 U/100 mL) was
incubated with n-3 DPA (0.2 μM in 200 μL of borate buffer (pH =
8.2)) at 4 °C. After 20 s, eight volumes of acidified MeOH (apparent
pH ∼3.5) were added. The resulting products were investigated by
target lipid mediator metabololipidomics as previously described.32
Lipid Mediator Metabololipidomics. Matching of synthetic 5
with endogenous products was conducted as previously reported.31
AUTHOR INFORMATION
Corresponding Author
*E-mail: t.v.hansen@farmasi.uio.no.
■
Present Address
§On leave from the Department of Pharmaceutical Chemistry,
School of Pharmacy, University of Oslo.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): J.D. and C.N.S. have filed patents on PD1n‑3 DPA
(5) and related compounds. C.N.S.’s interests are reviewed and
915
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916

Journal of Natural Products
Article
(24) Tello-Aburto, R.; Ochoa-Teran, A.; Olivo, H. F. Tetrahedron
Lett. 2006, 47, 5915−5917.
are managed by BWH and Partners HealthCare in accordance
with their conflict of interest policies.
(25) Nicolaou, K. C.; Webber, S. E.; Ramphal, J.; Abe, Y. Angew.
Chem. 1987, 99, 1077−1079.
ACKNOWLEDGMENTS
(26) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
16, 4467−4470.
■
We are indebted to Dr. P. P. Molesworth, Department of
Chemistry, University of Oslo, for skillful technical assistance
with acquisition of 2D-NMR spectra. The Norwegian Research
Council (KOSK II) and the School of Pharmacy, University of
Oslo, are gratefully acknowledged for Ph.D. scholarships to
M.A. and J.E.T., respectively. T.V.H. is grateful for a Leiv
Eriksson travel grant from The Norwegian Research Council.
J.D., R.C., and C.Y.C. are supported by the National Institutes
of Health GM Grant PO1GM095467 (C.N.S.).
(27) Corey, E. J.; Snider, B. B. J. Am. Chem. Soc. 1972, 94, 6190−
6192.
(28) Nicolaou, K. C.; Ladduwahetty, T.; Taffer, I. M.; Zipkin, R. E.
Synthesis 1986, 344−347.
(29) Aursnes, M.; Tungen, J. T.; Vik, A.; Dalli, J.; Hansen, T. V. Org.
Biomol. Chem. 2014, 12, 432−437.
(30) Duffield, J. S.; Hong, S.; Vaidya, V. S.; Lu, Y.; Fredman, G.;
Serhan, C. N.; Bonventre, J. V. J. Immunol. 2006, 177, 5902−5911.
(31) Dalli, J.; Winkler, W.; Colas, R. A.; Arnardottir, H.; Cheng, C.
Y.; Chiang, N.; Petasis, N. A.; Serhan, C. N. Chem. Biol. 2013, 20,
188−201.
(32) Dalli, J.; Serhan, C. N. Blood 2012, 120, e60−72.
(33) Corey, E. J.; Mehrota, M. M. Tetrahedron Lett. 1986, 27, 5173−
5176.
REFERENCES
■
(1) Tabas, I.; Glass, C. K. Science 2013, 339, 166−172.
(2) Majno, G.; Joris, I. Cells, Tissues and Disease. Principles of General
Pathology; Blackwell Science: Cambridge, 2004; pp 307−382.
(3) Ward, P. A. Acute and Chronic Inflammation. In Fundamentals of
Inflammation; Serhan, C. N.; Ward, P. A.; Gilroy, D. W., Eds.;
Cambridge University Press: Cambridge, 2010; pp 1−16.
(4) Serhan, C. N.; Savill, J. Nat. J. Immunol. 2005, 6, 1191−1197.
(5) Serhan, C. N.; Gotlinger, K.; Hong, S.; Arita, M. Prostaglandins
Other Lipid Mediators 2004, 73, 155−172.
(34) Serhan, C. N. Biochim. Biophys. Acta 1994, 1212, 1−25.
(35) Xu, Z.-Z.; Liu, X.-J.; Berta, T.; Park, C.-K.; Lu, N.; Serhan, C. N.;
̈
Ji, R.-R. Ann. Neurol. 2013, 74, 490−495.
(36) Wernerova, M.; Hudlicky, T. Synlett 2010, 18, 2701−2707.
(6) Serhan, C. N. Annu. Rev. Immunol. 2007, 25, 101−137.
(7) Serhan, C. N.; Chiang, N. Br. J. Pharmacol. 2008, 153, S200−
S215.
(8) Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.;
Gronert, K. J. Exp. Med. 2000, 192, 1197−1204.
(9) Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P.
R.; Mirick, G.; Moussignac, R. L. J. Exp. Med. 2002, 196, 1025−1037.
(10) Hong, S.; Gronert, K.; Devchand, P. R.; Moussignac, R.-L.;
Serhan, C. N. J. Biol. Chem. 2003, 278, 14677−14687.
(11) Mukherjee, P. K.; Marcheselli, V. L.; Serhan, C. N.; Bazan, N. G.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8491−8496.
(12) Ariel, A.; Pin-Lan, L.; Wang, W.; Tang, W. X.; Fredman, G.;
Hong, S.; Gotlinger, K. H.; Serhan, C. N. J. Biol. Chem. 2005, 280,
43079−43086.
(13) Serhan, C. N.; Yang, R.; Martinod, K.; Kasuga, K.; Pillai, P. S.;
Porter, T. F.; Oh, S. F.; Spite, M. J. Exp. Med. 2009, 206, 15−23.
(14) Serhan, C. N.; Chiang, N. Curr. Opin. Pharmacol. 2013, 13,
632−640.
(15) Serhan, C. N.; Petasis, N. A. Chem. Rev. 2011, 111, 5922−5943
and references therein.
(16) Lemaitre, R. N.; Tanaka, T.; Tang, W.; Manichaikul, A.; Foy,
M.; Kabagambe, E. K.; Nettleton, J. A.; King, I. A.; Weng, L.-C.;
Bhattacharya, S.; Bandinelli, S.; Bis, J. C.; Rich, S. S.; Jacobs, D. R., Jr.;
Cherubini, A.; McKnight, B.; Liang, S.; Gu5, X.; Rice, K.; Laurie, C. C.;
Lumley, T.; Browning, B. L.; Psaty, B. M.; Chen, Y.-D. I.; Friedlander,
Y.; Djousse, L.; Wu, J. H. Y.; Siscovick, D. S.; Uitterlinden, A. G.;
Arnett, D. K.; Ferrucci, L.; Fornage, M.; Tsai, M. Y.; Mozaffarian, D.;
Steffen, L. M. PLoS Genet. 2011, 7, e1002193.
(17) Kaur, G.; Cameron-Smith, D.; Garg, M.; Sinclair, A. J. Prog.
Lipid Res. 2011, 50, 28−34 and references therein.
(18) Hussein, N.; Fedorova, I.; Moriguchi, T.; Hamazaki, K.; Kim,
H.-Y.; Hoshiba, J.; Salem, N., Jr. Lipids 2009, 44, 685−702.
(19) Dalli, J.; Colas, R. A.; Serhan, C. N. Sci. Rep. 2013, 3, 1940.
(20) Serhan, C. N.; Gotlinger, K.; Hong, S.; Lu, Y.; Siegelman, J.;
Baer, T.; Yang, R.; Colgan, S. P.; Petasis, N. A. J. Immunol. 2006, 176,
1848−1859.
(21) Crawford, M. A.; Broadhurst, C. L.; Guest, M.; Nagar, A.; Wang,
Y.; Ghebremeskel, K. A.; Schmidt, W. Prostaglandins Leukot. Essent.
Fatty Acids 2013, 88, 5−13.
(22) Jakobsen, M. G.; Vik, A.; Hansen, T. V. Tetrahedron Lett. 2012,
53, 5837−5839.
(23) Krow, G. R. Org. React. 1993, 43, 251−265.
916
dx.doi.org/10.1021/np4009865 | J. Nat. Prod. 2014, 77, 910−916