Synthesis, structural confirmation, and biosynthesis of 22-OH-PD1n-3 DPA?

Article abstract of DOI:10.3390/molecules24183228

PD1_{n-3 DPA} belongs to the protectin family of specialized pro-resolving lipid mediators. The protectins are endogenously formed mediators that display potent anti-inflammatory properties and pro-resolving bioactivities and have attracted interest in drug discovery. However, few studies have been reported of the secondary metabolism of the protectins. To investigate the metabolic formation of the putative C22 mono-hydroxylated product, coined 22-OH-PD1_n-3 DPA, a stereoselective synthesis was performed. LC/MS-MS data of synthetic 22-OH-PD1_{n-3 DPA} matched the data for the biosynthetic formed product. Cellular studies revealed that 22-OH-PD1_{n-3 DPA} is formed from n-3 docosapentaenoic acid in human serum, and we confirmed that 22-OH-PD1_{n-3 DPA} is a secondary metabolite produced by ω-oxidation of PD1_{n-3 DPA} in human neutrophils and in human monocytes. The results reported are of interest for enabling future structure–activity relationship studies and provide useful molecular insight of the metabolism of the protectin class of specialized pro-resolving mediators.

Full text of DOI:10.3390/molecules24183228

molecules
Article
Synthesis, Structural Confirmation, and Biosynthesis
†
of 22-OH-PD1n-3 DPA
1
1
1
Jannicke Irina Nesman , Karoline Gangestad Primdahl , Jørn Eivind Tungen ,
Fransesco Palmas 2 , Jesmond Dalli
2,3
and Trond Vidar Hansen *
1,
1
Department of Pharmacy, Section of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068 Blindern,
N-0316 Oslo, Norway
Lipid Mediator Unit, William Harvey Research Institute, Barts and The London School of Medicine,
Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
Centre for Inflammation and Therapeutic Innovation, Queen Mary University of London,
London E1 4NS, UK
2
3
*
†
Correspondence: t.v.hansen@farmasi.uio.no; Tel.: +47-9326-5077
Dedicated to professor Dieter Schinzer on the occasion of his 65th birthday.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Ari Koskinen
Received: 26 August 2019; Accepted: 3 September 2019; Published: 5 September 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: PD1n-3 DPA belongs to the protectin family of specialized pro-resolving lipid mediators.
The protectins are endogenously formed mediators that display potent anti-inflammatory properties
and pro-resolving bioactivities and have attracted interest in drug discovery. However, few studies
have been reported of the secondary metabolism of the protectins. To investigate the metabolic
formation of the putative C22 mono-hydroxylated product, coined 22-OH-PD1n-3 DPA, a stereoselective
synthesis was performed. LC/MS-MS data of synthetic 22-OH-PD1n-3 DPA matched the data for
the biosynthetic formed product. Cellular studies revealed that 22-OH-PD1n-3 DPA is formed
from n-3 docosapentaenoic acid in human serum, and we confirmed that 22-OH-PD1n-3 DPA is
a secondary metabolite produced by
ω-oxidation of PD1n-3 DPA in human neutrophils and in human
monocytes. The results reported are of interest for enabling future structure–activity relationship
studies and provide useful molecular insight of the metabolism of the protectin class of specialized
pro-resolving mediators.
Keywords: natural products; stereoselective synthesis; structural elucidation; biosynthesis; protectins;
2
2-OH-PD1n-3 DPA; specialized pro-resolving mediators; polyunsaturated fatty acids; omega oxidation
1
. Introduction
Inflammation is divided into acute inflammation, which by nature is self-resolving, and chronic
inflammation, which occurs over an extended time period and does not resolve [ ]. Uncontrolled,
1
excessive acute and chronic unresolved inflammation may develop into several diseases, such as
cardiovascular disease, cancer, rheumatoid arthritis, and neurological disorders, e.g., Parkinson‘s
disease and Alzheimer’s disease [
studies at the biomolecular and the cellular levels [
of several chemical mediators, such as peptides, oxygenated polyunsaturated fatty acids (PUFAs),
chemokines, and cytokines that initiate, modulate, and reduce acute inflammatory processes [ ]. Today,
various drugs—mostly inhibitors—that reduce the effects of inflammatory processes are available [ ].
Resolution of inflammation was earlier believed to be a passive process [ ]. However, recent studies
have established that the resolution phase of inflammation and the return to physiology (homeostasis)
are regulated by active and enzymatic formation of several novel families of oxygenated PUFAs [ ].
These endogenously formed compounds have been coined specialized pro-resolving mediators (SPMs)
2]. Over the last century, inflammation has been the topic of numerous
3]. These efforts have resulted in the identification
4
2,5
1,2
4,6
Molecules 2019, 24, 3228; doi:10.3390/molecules24183228
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3228
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and are biosynthesized in the presence of cyclooxygenase and lipoxygenase enzymes from the dietary
n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1) [6]. The E-series
resolvins origin from EPA, the D-series resolvins, protectins and maresins, as well as the recently
described sulfido-conjugates of resolvins (RCTRs), protectins (PCTRs), and maresins (MCTRs), are all
biosynthesized from DHA [
4]. Lipoxins are biosynthetically formed from arachidonic acid (AA) [
3,5
].
SPMs and their bioactions are considered to constitute a biomedical paradigm shift [
interesting bioactivities reported [8].
5,7] with numerous
Figure 1. Overview of families of specialized pro-resolving mediators (SPMs) derived from
eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), and n-3
docosapentaenoic acid (DPA).
SPMs display highly potent agonist effects in vivo, often in the low nanomolar range, by acting as
ligands on individual G-protein coupled receptors (GPCRs) [ ]. Approximately 35% of all approved
non-biological drugs target GPCRs [10]. Moreover, SPMs are excellent biomolecular templates for the
9
development of new, small molecular anti-inflammatory drugs and immunoresolvents [
SPMs have entered initial clinical trial programs [9].
5,9], and some
New SPMs biosynthesized from the PUFA n-3 docosapentaenoic acid (n-3 DPA,
1) were recently
reported (Figure 2) [11 12]. N-3 DPA is formed from EPA and is also an intermediate in the biosynthesis
,
of DHA [13]. Among these novel SPMs, PD1n-3 DPA
investigations [11,14–19].
(2) has been the topic of detailed biological
The biosynthesis of 2 [11] was recently established [20
a 17-lipoxygenation of n-3 DPA produces 17(S)-hydroperoxy-7Z,10Z,13Z,15E,19Z-docosapentaenoic
acid [17(S)-HpDPA, ] that is converted into the epoxide intermediate 16(S),17(S)-epoxy PDn-3 DPA
), named ePDn-3 DPA. Epoxide is then hydrolyzed to PD1n-3 DPA ) by an unknown hydrolase
,21], as presented in Scheme 1. First,
3
(4
4
(2
(Scheme 1). Further metabolism of 2 should result in the formation of 22-OH-PD1n-3 DPA (5).
PUFAs and their oxygenated products undergo oxidative metabolism by eicosaoxidoreductase
and cytochrome P450 (CYP) enzymes [
protectin D1 (PD1) [24 26]. Of interest, in contrast to the
LTB ), 20-OH-LTB [22 23], the PD1 metabolite 22-OH-PD1 [24] exhibited potent pro-resolving and
anti-inflammatory bioactions in the nanomolar range [25]. Hence, we became interested in synthesizing
in a stereoselective manner and utilizing the synthetic material in biosynthetic investigations of the
conversion of 1 and 2 into 5.
6
,22
,23]. The
ω-oxidative metabolism has been reported for
–
ω-oxidation product of leukotriene B4
(
,
4
4
5

Molecules 2019, 24, 3228
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Figure 2. Chemical structures of some n-3 DPA derived SPMs. Where known, the absolute configuration
is included.
Scheme 1. Established biosynthesis of 2 [20,21] and putative formation of 22-OH-PD1n-3 DPA (5).
2
. Results and Discussion
2
.1. Synthesis of 22-OH-PD1n-3 DPA
To obtain stereochemically pure
available (3-bromopropoxy)-tert-butyldimethylsilane. The ylide of
sodium bis(trimethylsilyl)amide (NaHMDS) in THF/hexamethylphosphoric acid triamide (HMPA) and
then reacted with the known aldehyde 27] in a highly Z-selective Wittig reaction [28 32] (Scheme 2).
This afforded bis-protected diol in 74% isolated yield. The known vinylic bromide was synthesized
as earlier reported [14]. The two fragments and were then reacted in a Sonogashira reaction
5, we first synthesized the Wittig-salt
6 from commercially
6
was produced in the presence of
7
[
–
8
9
8
9

Molecules 2019, 24, 3228
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[
(Pd(PPh ) (3 mol%) in Et NH and in the presence of CuI (5 mol%)]. After column chromatography,
3 4 2
isomeric pure 10 was obtained in 84% yield. Global deprotection using tetrabutylammonium fluoride
◦
(
TBAF) (7.5 eq.) in THF at
−
78 C afforded the triol 11 in 91% isolated yield. The Z-selective reduction
of the internal alkyne in 11 proved challenging compared to similar systems [14,33,34]. Gratifyingly,
the Boland reduction method [35] afforded the methyl ester 12 in 46% isolated yield after careful
and repeated purifications by column chromatography. The chemical purity and the stereochemical
integrity of 12 were validated using NMR and HPLC analyses, reaching > 95% purity (Supporting
Information). Finally, mild saponification of the methyl ester in 12 afforded the target compound
5
in 90% isolated yield and in > 94% chemical purity based on NMR and HPLC analyses (Supporting
1
Information). The geometrical configuration of the E,E,Z-triene moiety in
5
was assigned based on H
NMR and UV experiments as well as by analogy with literature [14]. The coupling constants from the
1
H NMR data were determined as 14.3 Hz, 13.9 Hz, and 11.4 Hz. The UV spectra of synthetic material
EtOH
of
5
showed absorbance peaks (λmax
) at 262, 271, and 282 nm, which is in agreement with the UV
absorption profile of a conjugated triene double bond system [11].
Scheme 2. Synthesis of 22-OH-PD1n-3 DPA (5).
2
.2. Matching of Synthetic 22-OH-PD1n-3 DPA with Material Formed in Human Serum
In order to obtain evidence that our synthetic material matched the authentic product of
2
2-OH-PD1n-3 DPA (5), we assessed whether the physical properties of synthetic (5) matched with those
of the product found in human serum [36]. Using liquid chromatography tandem mass spectrometry
LC/MS-MS) [37], we found that endogenous 22-OH-PD1n-3 DPA ) gave a retention time (T -value) of
(
(5
R

Molecules 2019, 24, 3228
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10.4 min using multiple reaction monitoring (MRM) (Figure 3A). Of note, synthetic
5
displayed the
similar chromatographic behavior as the endogenous material (Figure 3B), and when these products
were co-injected, they gave one peak in the MRM chromatogram with T = 10.4 min (Figure 3C).
R
Figure 3. Multiple reaction monitoring (MRM) chromatogram of the products from (
2-OH-PD1n-3 DPA ) produced in human serum; ( ) synthetic material of ; ( ) co-injection of
endogenous and synthetic material.
A) endogenous
2
(5
B
5
C
The MS-MS spectra obtained from both endogenous
identical MS-MS fragmentation spectra with the following fragments assigned: m/z 377 = M
59 = M H O, m/z 341 = M 2H O, m/z 323 = M 3H O, m/z 297 = M
CO , m/z 245 = 263 − H O, m/z 165 = 183 − H O and m/z 139 = 183 − CO (Supporting Information).
5
and synthetic
5
displayed essentially
H, m/z
2H O
−
3
−
H
−
−
H
−
−
H
−
−
H
−
−
2
2
2
2
2
2
2
2
Overall, these results confirmed the structure of 5, as depicted in Scheme 2, to be
7Z,10R,11E,13E,15Z,17S,19Z)-10,17,22-trihydroxydocosa-7,11,13,15,19-pentaenoic acid.
(
2
.3. PD1n-3 DPA is A Precursor in the Biosynthesis of 22-OH-PD1n-3 DPA in Human Neutrophils
First, we investigated if human neutrophils produced 22-OH-PD1n-3 DPA ) (Figure 4A).
Human neutrophils were isolated from whole blood and profiled by means of LC/MS-MS.
The MRM-chromatogram displayed a peak with T = 10.4 min (Figure 4A), and an MS/MS spectrum
(
5
R

Molecules 2019, 24, 3228
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for the product under this peak matched that of the material identified in human serum and
synthetic (Supporting Information). Next, we determined whether PD1n-3 DPA ) was a precursor
of 22-OH-PD1n-3 DPA ). The latter was obtained from earlier synthetic work [14]. For this purpose,
we incubated (10 nM) with human neutrophils, and the resulting product(s) profile was assessed
using LC-MS/MS, searching targeting ion pairs with m/z 377 > 261 [36] in the MRM chromatogram.
Here, we found that the intensity of the peak at T = 10.4 min corresponding to 22-OH-PD1 was
5
(2
(5
2
R
n-3 DPA
markedly increased when compared to the intensity of the peak obtained with human neutrophils
alone (Figure 4B). Furthermore, the co-injection of samples from both experiments gave one single
sharp peak at T = 10.4 min (Figure 4C). Overall, these experiments proved that PD1
(2) was
R
n-3 DPA
converted to 22-OH-PD1n-3 DPA (5) by human neutrophils.
Figure 4. MRM chromatogram of the products from (
A
C
) human neutrophils incubated with vehicle;
) co-injection of samples obtained from ( ).
(B
) human neutrophils incubated with PD1n-3 DPA ); (
(
2
A,B
2
.4. Biosynthesis of 22-OH-PD1n-3 DPA from PD1n-3 DPA in Human Monocytes
In order to gain additional confidence for the direct biosynthetic formation of 22-OH-PD1n-3 DPA
from PD1n-3 DPA ), SPM was incubated (10 nM) with human monocytes. Again, data from LC/MS-MS
experiments showed that 22-OH-PD1n-3 DPA ) was indeed formed from PD1n-3 DPA ) by human
monocytes, since identical retention times were observed (Figure 5). In addition, the MS/MS-data
(5)
(2
2
(
5
(2

Molecules 2019, 24, 3228
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of
5
from this experiment were in agreement with the data obtained from synthetic material of
5
(Supporting Information).
Figure 5. PD1n-3 DPA
(2) was converted to 22-OH-PD1n-3 DPA
(
5) by human monocytes. Multiple
reaction monitoring chromatograms for m/z 377 > 361 of the products obtained from (
A
) human
monocytes incubated with PD1n-3 DPA; (B) synthetic 22-OH-PD1n-3 DPA
.
3
. Materials and Methods
General: Unless otherwise stated, all commercially available reagents and solvents were used
in the form they were supplied without any further purification. The stated yields were based on
isolated material. All reactions were performed under an argon atmosphere using Schlenk techniques.
Reaction flasks were covered with aluminum foil during reactions and storage to minimize exposure
to light. Thin layer chromatography was performed on silica gel 60 F254 aluminum-backed plates
fabricated by Merck, Darmstadt, Germany. Flash column chromatography was performed on silica gel
6
0 (40–63
µm) produced by Merck. NMR spectra were recorded on a Bruker AVI600 by Bruker, Billerica,
MA, USA, a Bruker AVII400, or a Bruker DPX300 spectrometer at 600 MHz, 400 MHz, or 300 MHz,
1
13
respectively, for H NMR and at 150 MHz, 100 MHz, or 75 MHz, respectively, for C NMR. Coupling
constants (J) are reported in hertz, and chemical shifts are reported in parts per million (
δ
) relative
2.50,
77.00 ppm,
49.00). Optical rotations were measured using a 1 mL cell with a
.0 dm path length on a Perkin Elmer 341 polarimeter by Perkin Elmer, Waltham, MA, USA. Mass
1
to the central residual protium solvent resonance in H NMR (CDCl =
δ 7.26, DMSO-d = δ
6
3
and MeOD =
DMSO-d = δ 39.43, and MeOD =
δ
3.31) and the central carbon solvent resonance in 13C NMR (CDCl =
δ
3
δ
6
1
spectra were recorded at 70 eV on Micromass Prospec Q or Micromass QTOF 2 W spectrometer by
Miltham, MA, USA, using electrospray ionization (ESI) as the method of ionization. High-resolution
mass spectra were recorded at 70 eV on Micromass Prospec Q or Micromass QTOF 2W spectrometer
using ESI as the method of ionization. HPLC analyses were performed using a C18 stationary phase
(
Eclipse XDB-C18, 4.6
applying the conditions stated. The UV/VIS spectra were recorded using an Agilent Technologies Cary
485 UV-VIS spectrophotometer using quartz cuvettes.
×
250 mm, particle size 5 µm, from Agilent Technologies, Santa Clara, CA, USA),
8

Molecules 2019, 24, 3228
8 of 13
3
.1. Synthesis of Compounds
λ
3
.1.1. Bromo(3-((tert-butyldimethylsilyl)oxy)propyl)triphenyl- l5-phosphane (6)
Commercially available (3-bromopropoxy)-tert-butyldimethylsilane (1.09 g, 4.32 mmol, 1.00 eq.)
and PPh (1.25 g, 4.75 mmol, 1.10 eq.) in toluene (5.0 mL) were heated to reflux for 16 h. The reaction
3
mixture was cooled to rt and extracted with Et O (3
×
25 mL). The solvent was removed in vacuo
to give a white, cloudy liquid. This liquid was purified by column chromatography on silica using
pure dichloromethane as eluent until all excess PPh was eluted and there was 7% MeOH in CH Cl .
2
3
2
2
The purified product was concentrated in vacuo to afford the title compound as a white solid in 76%
yield (1.70 g). All spectroscopic and physical data were in agreement with those reported in the
1
literature [38]. H NMR (400 MHz, CDCl )
δ
1
7.89–7.75 (m, 9H), 7.74–7.64 (m, 6H), 3.96–3.80 (m, 4H),
3
0
3
4
1
1
2
.97–1.83 (m, 2H), 0.85 (s, 9H), 0.03 (s, 6H); C NMR (101 MHz, CDCl )
33.9 (d, J = 9.9 Hz), 130.6 (d, J = 12.5 Hz), 118.6 (d, J = 86.1 Hz), 61.9 (d, J = 16.9 Hz),
δ
135.1 (d, JCP = 3.0 Hz),
3
0
0
0
1
3
2
3
CP
CP
CP
CP
2
1
6.2 (d, J = 4.1 Hz), 26.1 (3C), 19.1 (d, J = 52.6 Hz), 18.3, 5.2 (2C); thin layer chromatography
CP
CP
◦
TLC) (MeOH/CH Cl 1:20, KMnO stain) R = 0.18; Mp: 137–139 C.
2 2 4 f
(
3
.1.2. (S,Z)-5-Ethynyl-2,2,3,3,12,12,13,13-octamethyl-4,11-dioxa-3,12-disilatetradec-7-ene (8)
Wittig salt
The solution was cooled to
.95 mL, 0.986 eq.). The reaction mixture was stirred for 50 min, upon which it was warmed to rt
and re-cooled to
6
(298 mg, 0.578 mmol, 1.00 eq.) was dissolved in THF (8.0 mL) and HMPA (0.45 mL).
◦
−
78 C followed by dropwise addition of NaHMDS (0.6 M in toluene,
0
◦
−
78 C. Aldehyde
7
(145 mg, 0.683 mmol, 1.20 eq.) in THF (1.0 mL) was added
dropwise to the reaction mixture. After two hours, the reaction mixture was slowly allowed to warm
to rt and then quenched with phosphate buffer (4.7 mL, pH = 7.2). The phases were separated, and
the aqueous layer was extracted with Et O (2
×
4.0 mL). The combined organic layers were dried
2
(
Na SO ), and the solvent was removed by rotary evaporation. The crude product was filtered through
2
4
a silica plug (hexane/EtOAc, 9:1) and concentrated in vacuo to afford compound
8
as a clear oil in
= 15.0 (c = 1.33, CHCl ); H NMR (400 MHz, CDCl ) δ 5.59–5.49 (m, 2H),
3 3
20
1
7
4
2
4% yield (157 mg); [
α
]
−
D
.34 (td, J = 6.5, 2.1 Hz, 1H), 3.61 (t, J = 7.0 Hz, 2H), 2.45 (t, J = 6.0 Hz, 2H), 2.38 (d, J = 2.1 Hz, 1H),
.30 (q, J = 6.7 Hz, 2H), 0.90 (d, J = 3.8 Hz, 18H), 0.13 (s, 3H), 0.11 (s, 3H), 0.05 (s, 6H); 13C NMR
(
101 MHz, CDCl3)
δ
128.7, 126.3, 85.4, 72.3, 63.0, 62.8, 36.8, 31.5, 26.1, 25.9, 18.5, 18.4,
−
4.5,
−
4.9, 5.1;
−
TLC (hexane/EtOAc 95:5, KMnO stain) R = 0.75; HRMS: exact mass calculated for C H O Si Na
[
4
f
20 40
2
2
+
M + Na] : 391.2459, found: 391.2459.
3
.1.3. Methyl (7Z,10R,11E,13E,17S,19Z)-10,17,22-tris((tert-butyldimethylsilyl)oxy)
docosa-7,11,13,19-tetraen-15-ynoate (10)
To a solution of vinyl bromide
9 (182 mg, 0.409 mmol, 1.00 eq.) in Et NH (0.8 mL) and benzene
2
(
0.3 mL) was added Pd(PPh ) (16.0 mg, 0.0140 mmol, 3.00 mol%), and the reaction was stirred for
3
4
4
5 min in the dark. CuI (4.00 mg, 0.0210 mmol, 5.00 mol%) dissolved in a minimal amount of Et NH
2
was added, followed by dropwise addition of alkyne
0.8 mL). After 20 h of stirring at ambient temperature, the reaction was quenched with saturated
aqueous NH Cl (10 mL). Et O (15 mL) was added, and the phases were separated. The aqueous phase
8 (160 mg, 0.434 mmol, 1.06 eq.) in Et NH
2
(
4
2
was extracted with Et O (2
being concentrated in vacuo. The crude product was purified by column chromatography on silica
×
15 mL), and the combined organic layers were dried (Na SO ) before
2 4
2
2
5
(
(
hexane/EtOAc, 95:5) to afford the title compound 10 as a clear oil in 84% yield (239 mg); [
c = 0.95, MeOH); H NMR (400 MHz, CDCl )
α
]
= 11.6
−
D
1
δ 6.50 (dd, J = 10.9, 15.5 Hz, 1H), 6.18 (dd, J = 10.9,
3
1
1
5.5 Hz, 1H), 5.76 (dd, J = 6.0, 15.1 Hz, 1H), 5.61–5.49 (m, 3H), 5.47–5.30 (m, 2H), 4.47 (td, J = 6.5, 1.8 Hz,
H), 4.20–4.12 (m, 1H), 3.66 (s, 3H), 3.61 (t, J = 7.0 Hz, 2H), 2.49–2.41 (m, 2H), 2.32–2.16 (m, 6H), 2.01
(
0
1
q, J = 7.1 Hz, 2H), 1.68–1.53 (m, 2H), 1.39–1.24 (m, 5H), 0.91–0.88 (m, 27H), 0.13 (s, 3H), 0.11 (s, 3H),
.06–0.01 (m, 12H); 13C NMR (101 MHz, CDCl3)
174.4, 141.2, 139.3, 131.9, 128.6, 128.5, 126.6, 125.3,
10.6, 93.3, 83.2, 72.9, 63.6, 63.0, 51.6, 37.0, 36.4, 34.2, 31.5, 29.4, 29.0, 27.4, 26.1, 26.0, 26.0, 25.0, 18.5,
δ

Molecules 2019, 24, 3228
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1
8.4, 18.4,
−
4.3,
−
4.3,
−
4.6,
−
4.8,
−
5.1; TLC (hexane/EtOAc 95:5, UV-VIS) R = 0.33; HRMS: exact mass
f
+
calculated for C H O Si Na [M + Na] : 755.4891, found: 755.4893.
4
1
76
5
3
3
.1.4. Methyl (7Z,10R,11E,13E,17S,19Z)-10,17,22-trihydroxydocosa-7,11,13,19-tetraen-15-ynoate (11)
TBAF (1.0 M in THF, 1.07 mL, 1.07 mmol, 7.48 eq.) was added to a solution of TBS-protected
◦
alcohol 10 (105 mg, 0.143 mmol, 1.00 eq.) in THF (1.5 mL) at
2
−
78 C. The reaction was stirred for
1 h before it was quenched with phosphate buffer (pH = 7.2, 3.5 mL). Brine (15 mL) and EtOAc
(
(
15 mL) were added, and the phases were separated. The aqueous phase was extracted with EtOAc
2 × 10 mL), and the combined organic layer was dried (Na SO ) before being concentrated in vacuo.
2
4
The crude product was purified by column chromatography on silica (hexane/EtOAc, 4:6) to afford the
2
5
1
title compound as a clear oil. Yield: 51 mg (91%); [
MeOD) 6.55 (dd, J = 15.5, 10.8 Hz, 1H), 6.27 (dd, J = 15.5, 10.8 Hz, 1H), 5.81 (dd, J = 15.3, 6.4 Hz, 1H),
.69–5.63 (m, 1H), 5.60–5.53 (m, 2H), 5.51–5.36 (m, 2H), 4.44 (td, J = 6.6, 1.9 Hz, 1H), 4.12 (q, J = 7.0,
.5 Hz, 1H), 3.65 (s, 3H), 3.56 (t, J = 6.8 Hz, 2H), 2.49–2.44 (m, 2H), 2.36–2.22 (m, 6H), 2.05 (q, J = 6.9 Hz,
H), 1.61 (p, J = 7.4 Hz, 2H), 1.42–1.28 (m, 4H); 13C NMR (101 MHz, MeOD)
176.0, 142.5, 139.9, 132.9,
α
]
=
−
24.4 (c = 0.67, MeOH); H NMR (400 MHz,
D
δ
5
6
2
δ
1
30.3, 129.7, 127.5, 126.1, 111.7, 93.8, 84.3, 72.8, 63.1, 62.6, 52.0, 37.0, 36.2, 34.8, 32.0, 30.3, 29.80, 28.2, 25.9;
+
TLC (hexane/EtOAc 2:3, UV-VIS) R = 0.22; HRMS: exact mass calculated for C H O Na [M + Na] :
f
23 34
5
4
13.2299, found: 413.2298.
3
.1.5. 22-OH-PD1n-3 DPA Methyl Ester (12)
The Zn(Cu/Ag) mixture was prepared as described by Boland et al. [35]. Zinc dust (3.12 g) in
degassed H O (18.8 mL, pH = 7.0) was stirred under argon for 15 min before Cu(OAc) (312 mg)
2
2
was added and stirred for an additional 15 min. AgNO (312 mg) was then added, and the reaction
3
mixture was stirred for 30 min. The mixture was filtered and washed successively with H O, MeOH,
2
acetone, and Et O before it was transferred to a flask containing alkyne 11 (50.0 mg, 0.128 mmol) in
2
MeOH/H O (1:3, 9.2 mL). The mixture was stirred at rt and monitored by TLC analysis. After 2 h, the
2
®
reaction was judged complete, and the reaction mixture was filtered through a pad of Celite by Merck,
Darmstadt, Germany, with Et O. Water was added to the filtrate, and the layers were separated. The
2
aqueous layer was extracted with Et O (2
×
10 mL). The combined organic layers were washed with
2
brine and dried (Na SO ). The solvent was removed in vacuo, and the crude product was purified by
2
4
column chromatography on silica gel (hexane/EtOAc/MeOH, 49:50:1) to afford the methyl ester 12 as a
2
5
1
pale yellow oil in 46% yield (23 mg); [
.58–6.48 (m, 1H), 6.32–6.20 (m, 2H), 6.08 (t, J = 11.2 Hz, 1H), 5.75 (dd, J = 6.6, 14.5 Hz, 1H), 5.53–5.36
m, 5H), 4.62–4.55 (m, 1H), 4.15–4.08 (m, 1H), 3.65 (s, 3H), 3.55 (t, J = 6.8 Hz, 2H), 2.43–2.36 (m, 1H),
.35–2.18 (m, 7H), 2.09–1.99 (m, 2H), 1.61 (p, J = 7.4 Hz, 2H), 1.42–1.27 (m, 4H); 13C NMR (151 MHz,
MeOD) 176.0, 138.1, 135.0, 134.8, 132.8, 131.4, 130.6, 129.1, 128.8, 128.1, 126.2, 73.1, 68.5, 62.6, 52.0,
6.6, 36.4, 34.8, 32.0, 30.3, 29.8, 28.2, 25.9; TLC (hexane/EtOAc/MeOH 49:50:1) R = 0.22; HRMS: exact
α
]
=
−
20.0 (c = 0.20, MeOH); H NMR (600 MHz, MeOD) δ
D
6
(
2
δ
3
f
+
mass calculated for C H O Na [M + Na] : 415.2455, found: 415.2455; UV-VIS: λmax (EtOH) = 269,
23
36
5
2
71, 282 nm. The purity (96%) was determined by HPLC analysis (Eclipse XDB-C18, MeOH/H O 3:1,
2
1
.0 mL/min); tr = 32.6.
3
.1.6. 22-OH-PD1n-3 DPA (5)
Solid LiOH (17.0 mg, 0.710 mmol, 31.0 eq.) was added to a solution of methyl ester 12 (9 mg,
◦
0
.0229 mmol, 1.00 eq.) dissolved in THF-MeOH-H O (2:2:1, 2.8 mL) at 0 C. The reaction mixture was
2
◦
stirred at 0 C for three hours and then allowed to warm to rt. After 7.5 h, the solution was cooled
to 0 C, acidified with saturated aqueous NaH PO (4.0 mL), and then EtOAc (4.0 mL) was added.
◦
2
4
The layers were separated, and the water phase was extracted with EtOAc (2
organic layers were dried (Na SO ) before the solvent was removed in vacuo. Then, 22-OH-PD1
n-3 DPA
×
4.0 mL). The combined
2
4
(5) was obtained as a colorless oil after purification by column chromatography (MeOH/CH Cl , 1:9)
2 2
2
5
1
R = 0.22; yield: 10 mg (90%); [
α
]
= 25.4 (c = 0.35, MeOH); H NMR δ 6.53 (dd, J = 13.9, 11.4 Hz, 1H),
−
f
D

Molecules 2019, 24, 3228
10 of 13
6
.32–6.21 (m, 2H), 6.08 (t, J = 11.4 Hz, 1H), 5.74 (dd, J = 14.3, 6.5 Hz, 1H), 5.56–5.44 (m, 3H), 5.43–5.36
(
(
m, 2H), 4.63–4.55 (m, 1H), 4.12 (q, J = 6.6 Hz, 1H), 3.55 (t, J = 6.8 Hz, 2H), 2.44–2.36 (m, 1H), 2.36–2.20
m, 7H), 2.06 (q, J = 7.0 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.41–1.32 (m, 4H); 1 C NMR (151 MHz,
3
MeOD)
δ
178.0, 138.1, 135.0, 134.8, 132.9, 131.4, 130.6, 129.1, 128.8, 128.1, 126.2, 73.1, 68.5, 62.6, 36.6, 36.4,
+
35.3, 32.0, 30.4, 29.9, 28.3, 26.1; HRMS: exact mass calculated for C H O Na [M + Na] : 401.2298,
22
34
5
found:401.2298; UV-VIS: λmax (EtOH) = 269, 271, 282 nm. The purity (94%) was determined by HPLC
analysis (Eclipse XDB-C18, MeOH/H O/10mM acetic acid 65:20:20, 1.0 mL/min); Tr = 18.1.
2
3
.2. Lipid Mediator Profiling
MeOH—two and four volumes, respectively—containing deuterium-labeled synthetic internal
standards of d -LTB (500 pg) and d -RvE1 (100 pg) were added to cell incubations and human serum.
4
4
5
◦
Samples were stored at
−
40 C until extraction. Prior to extraction, samples were centrifuged at 2500
◦
rpm, 4 C, for 10 min. Supernatants were then collected and concentrated to ~1.0 mL of MeOH content
using a gentle stream of nitrogen gas (TurboVap LV system, Biotage, Uppsala, Sweden). Solid phase
extraction was performed by means of the ExtraHera (Biotage) automated extraction system as follows.
Aqueous HCl solution (pH = 3.5, 9.0 mL) was added to the samples, and the acidified solutions were
loaded onto conditioned C18 500 mg 200-0050-B cartridges (Biotage). Prior to extraction, solid phase
C18 cartridges were equilibrated with MeOH (3.0 mL) and H O (6.0 mL). The extraction products were
2
washed with H O (4.0 mL) and hexane (5.0 mL) followed by product elution with 4.0 mL of methyl
2
formate. Products were brought to dryness with a gentle stream of nitrogen (TurboVap LV, Biotage)
◦
and suspended in MeOH:H O (50:50, v/v). Samples were centrifuged (2500 rpm, 4.0 C, 5 min), and
2
◦
the supernatant was collected and centrifuged (9900 rpm, 4.0 C, 10 sec). The collected supernatant
was then subjected to LC/MS-MS, as described in reference [39].
3
.3. Human Neutrophil Incubations
Human peripheral blood neutrophils were isolated from healthy volunteers using density-gradient
Ficoll-Histopaque isolation. Volunteers gave written consent in accordance with a Queen Mary Research
Ethics Committee (QMREC 2014:61) and the Helsinki declaration. Isolated neutrophils were suspended
in PBS+ containing 10% bovine serum albumin (BSA) and incubated for 10 min (37 C, pH = 7.45) prior
/+
◦
8
to the addition of the indicated concentrations of n-3 DPA (
1
) and Escherichia coli (2.5
×
10 cells/mL).
Incubations were quenched using two volumes ice-cold methanol containing deuterium-labeled
internal standards to facilitate quantification and identification. Products were extracted using C18
columns and quantified by LC/MS-MS metabololipidomics, as detailed above. The same experiment
was repeated with synthetic 2.
3
.4. Human Serum Incubations
Human pool serum was purchased from Sigma-Aldrich (Poole, UK). Four volumes of methanol
containing deuterium-labeled standards were added, and products were extracted using C18 column
and profiled using LC/MS-MS metabololipidomics, as detailed above [39].
4
. Conclusions
To summarize, 22-OH-PD1n-3 DPA
8% yield from commercially available (3-bromopropoxy)-tert-butyldimethylsilane and known
). We herein established the exact configuration
-oxidation metabolic product of the SPM PD1n-3 DPA ).
) is formed in human serum, human neutrophils,
(5) was stereoselectively synthesized in six steps and with
3
(S)-3-((tert-butyldimethylsilyl)oxy)pent-4-ynal (7
of the biomolecule
In addition, we showed that 22-OH-PD1n-3 DPA (5
5, and that
5
is an
ω
(2
and by human monocytes. The results reported contribute to additional information and knowledge
of the growing number of members of the SPM family of endogenously formed lipid mediators
and their metabolism, which is of interest in regard to developing new immunoresolvents without
immunosuppressive effects [5,37].

Molecules 2019, 24, 3228
11 of 13
Supplementary Materials:
HPLC-chromatograms, UV-VIS, MS/MS and HRMS data of
See http://www.mdpi.com/1420-3049/24/18/3228/s1.
Electronic Supporting Information is available with 1H-, 13C-NMR,
5 and intermediates 6–11 and methyl ester 12.
Author Contributions: T.V.H., J.E.T. and J.D. designed the concept of the study. J.I.N. performed all the synthetic
experiments with supervision by K.G.P. and J.E.T. K.G.P. performed purification of compounds and analyzed
data with J.I.N., F.P and J.I.N. performed lipid mediator profiling. J.I.N performed human serum- and human
neutrophil incubations. J.I.N. and T.V.H. wrote the paper. K.G.P., J.E.T., J.D. and F.P. reviewed and edited the paper.
Funding: The School of Pharmacy, University of Oslo is gratefully acknowledged for a Ph.D.-scholarship to
J.I.N. We appreciate the funding for a travel grant to J.I.N. from The Norwegian Research School in Pharmacy
(
N.F.I.F.). The Norwegian Research Council is gratefully acknowledged for funding to T.V.H. (BIOTEK2021 224811
and FRIPRO-FRINATEK 230470). J.D. received funding from the European Research Council (ERC) under the
European Union’s Horizon 2020 research and innovation program (grant no: 677542) and the Barts Charity (grant
no: MGU0343). J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Welcome Trust and
the Royal Society (grant 107613/Z/15/Z).
Acknowledgments: T.V.H. is most grateful towards Dieter Schinzer, University of Magdeburg, Germany, for all
discussions, interactions and fun, socially as well as professionally. Let the “Dolce Vita” live on!
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(