Synthesis, Structural Confirmation, and Biosynthesis of 22-OH-PD1_{n-3 DPA} ^†

Abstract

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.

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 [1]. Uncontrolled, 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 [2]. Over the last century, inflammation has been the topic of numerous studies at the biomolecular and the cellular levels [3]. These efforts have resulted in the identification of several chemical mediators, such as peptides, oxygenated polyunsaturated fatty acids (PUFAs), chemokines, and cytokines that initiate, modulate, and reduce acute inflammatory processes [4]. Today, various drugs—mostly inhibitors—that reduce the effects of inflammatory processes are available [2,5]. Resolution of inflammation was earlier believed to be a passive process [1,2]. 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 [4,6]. These endogenously formed compounds have been coined specialized pro-resolving mediators (SPMs) 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 [5,7] with numerous interesting bioactivities reported [8].

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) [9]. Approximately 35% of all approved non-biological drugs target GPCRs [10]. Moreover, SPMs are excellent biomolecular templates for the development of new, small molecular anti-inflammatory drugs and immunoresolvents [5,9], and some SPMs have entered initial clinical trial programs [9].

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, PD1_{n-3 DPA} (2) has been the topic of detailed biological investigations [11,14,15,16,17,18,19].

The biosynthesis of 2 [11] was recently established [20,21], as presented in Scheme 1. First, a 17-lipoxygenation of n-3 DPA produces 17(S)-hydroperoxy-7Z,10Z,13Z,15E,19Z-docosapentaenoic acid [17(S)-HpDPA, 3] that is converted into the epoxide intermediate 16(S),17(S)-epoxy PD_{n-3 DPA} (4), named ePD_{n-3 DPA}. Epoxide 4 is then hydrolyzed to PD1_{n-3 DPA} (2) by an unknown hydrolase (Scheme 1). Further metabolism of 2 should result in the formation of 22-OH-PD1_{n-3 DPA} (5).

PUFAs and their oxygenated products undergo oxidative metabolism by eicosaoxidoreductase and cytochrome P450 (CYP) enzymes [6,22,23]. The ω-oxidative metabolism has been reported for protectin D1 (PD1) [24,25,26]. Of interest, in contrast to the ω-oxidation product of leukotriene B₄ (LTB₄), 20-OH-LTB₄ [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 5 in a stereoselective manner and utilizing the synthetic material in biosynthetic investigations of the conversion of 1 and 2 into 5.

2. Results and Discussion

2.1. Synthesis of 22-OH-PD1_{n-3 DPA}

To obtain stereochemically pure 5, we first synthesized the Wittig-salt 6 from commercially available (3-bromopropoxy)-tert-butyldimethylsilane. The ylide of 6 was produced in the presence of sodium bis(trimethylsilyl)amide (NaHMDS) in THF/hexamethylphosphoric acid triamide (HMPA) and then reacted with the known aldehyde 7 [27] in a highly Z-selective Wittig reaction [28,29,30,31,32] (Scheme 2). This afforded bis-protected diol 8 in 74% isolated yield. The known vinylic bromide 9 was synthesized as earlier reported [14]. The two fragments 8 and 9 were then reacted in a Sonogashira reaction [(Pd(PPh₃)₄ (3 mol%) in Et₂NH and in the presence of CuI (5 mol%)]. After column chromatography, 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 Information). The geometrical configuration of the E,E,Z-triene moiety in 5 was assigned based on ¹H NMR and UV experiments as well as by analogy with literature [14]. The coupling constants from the ¹H NMR data were determined as 14.3 Hz, 13.9 Hz, and 11.4 Hz. The UV spectra of synthetic material of 5 showed absorbance peaks (λ_max^EtOH) at 262, 271, and 282 nm, which is in agreement with the UV absorption profile of a conjugated triene double bond system [11].

2.2. Matching of Synthetic 22-OH-PD1_{n-3 DPA} with Material Formed in Human Serum

In order to obtain evidence that our synthetic material matched the authentic product of 22-OH-PD1_{n-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-PD1_{n-3 DPA} (5) gave a retention time (T_R-value) of 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_R = 10.4 min (Figure 3C).

The MS-MS spectra obtained from both endogenous 5 and synthetic 5 displayed essentially identical MS-MS fragmentation spectra with the following fragments assigned: m/z 377 = M − H, m/z 359 = M − H − H₂O, m/z 341 = M − H − 2H₂O, m/z 323 = M − H − 3H₂O, m/z 297 = M − H − 2H₂O − CO₂, m/z 245 = 263 − H₂O, m/z 165 = 183 − H₂O and m/z 139 = 183 − CO₂ (Supporting Information).

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. PD1_{n-3 DPA} is A Precursor in the Biosynthesis of 22-OH-PD1_{n-3 DPA} in Human Neutrophils

First, we investigated if human neutrophils produced 22-OH-PD1_{n-3 DPA} (5) (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_R = 10.4 min (Figure 4A), and an MS/MS spectrum for the product under this peak matched that of the material identified in human serum and synthetic 5 (Supporting Information). Next, we determined whether PD1_{n-3 DPA} (2) was a precursor of 22-OH-PD1_{n-3 DPA} (5). The latter was obtained from earlier synthetic work [14]. For this purpose, we incubated 2 (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_R = 10.4 min corresponding to 22-OH-PD1_{n-3 DPA} was 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_R = 10.4 min (Figure 4C). Overall, these experiments proved that PD1_{n-3 DPA} (2) was converted to 22-OH-PD1_{n-3 DPA} (5) by human neutrophils.

2.4. Biosynthesis of 22-OH-PD1_{n-3 DPA} from PD1_{n-3 DPA} in Human Monocytes

In order to gain additional confidence for the direct biosynthetic formation of 22-OH-PD1_{n-3 DPA} (5) from PD1_{n-3 DPA} (2), SPM 2 was incubated (10 nM) with human monocytes. Again, data from LC/MS-MS experiments showed that 22-OH-PD1_{n-3 DPA} (5) was indeed formed from PD1_{n-3 DPA} (2) by human monocytes, since identical retention times were observed (Figure 5). In addition, the MS/MS-data of 5 from this experiment were in agreement with the data obtained from synthetic material of 5 (Supporting Information).

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 60 (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, respectively, for ¹H NMR and at 150 MHz, 100 MHz, or 75 MHz, respectively, for ¹³C NMR. Coupling constants (J) are reported in hertz, and chemical shifts are reported in parts per million (δ) relative to the central residual protium solvent resonance in ¹H NMR (CDCl₃ = δ 7.26, DMSO-d₆ = δ 2.50, and MeOD = δ 3.31) and the central carbon solvent resonance in ¹³C NMR (CDCl₃ = δ 77.00 ppm, DMSO-d₆ = δ 39.43, and MeOD = δ 49.00). Optical rotations were measured using a 1 mL cell with a 1.0 dm path length on a Perkin Elmer 341 polarimeter by Perkin Elmer, Waltham, MA, USA. Mass 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 × 250 mm, particle size 5 μm, from Agilent Technologies, Santa Clara, CA, USA), applying the conditions stated. The UV/VIS spectra were recorded using an Agilent Technologies Cary 8485 UV-VIS spectrophotometer using quartz cuvettes.

3.1. Synthesis of Compounds

3.1.1. Bromo(3-((tert-butyldimethylsilyl)oxy)propyl)triphenyl-^λl5-phosphane (6)

Commercially available (3-bromopropoxy)-tert-butyldimethylsilane (1.09 g, 4.32 mmol, 1.00 eq.) and PPh₃ (1.25 g, 4.75 mmol, 1.10 eq.) in toluene (5.0 mL) were heated to reflux for 16 h. The reaction mixture was cooled to rt and extracted with Et₂O (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 PPh₃ was eluted and there was 7% MeOH in CH₂Cl₂. 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 literature [38]. ¹H NMR (400 MHz, CDCl₃) δ 7.89–7.75 (m, 9H), 7.74–7.64 (m, 6H), 3.96–3.80 (m, 4H), 1.97–1.83 (m, 2H), 0.85 (s, 9H), 0.03 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 135.1 (d, ^4′J_CP = 3.0 Hz), 133.9 (d, ^3′J_CP = 9.9 Hz), 130.6 (d, ^2′J_CP = 12.5 Hz), 118.6 (d, ^1′J_CP = 86.1 Hz), 61.9 (d, ³J_CP = 16.9 Hz), 26.2 (d, ²J_CP = 4.1 Hz), 26.1 (3C), 19.1 (d, ¹J_CP = 52.6 Hz), 18.3, 5.2 (2C); thin layer chromatography (TLC) (MeOH/CH₂Cl₂ 1:20, KMnO₄ stain) R_f = 0.18; Mp: 137–139 °C.

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 6 (298 mg, 0.578 mmol, 1.00 eq.) was dissolved in THF (8.0 mL) and HMPA (0.45 mL). The solution was cooled to −78 °C followed by dropwise addition of NaHMDS (0.6 M in toluene, 0.95 mL, 0.986 eq.). The reaction mixture was stirred for 50 min, upon which it was warmed to rt and re-cooled to −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 Et₂O (2 × 4.0 mL). The combined organic layers were dried (Na₂SO₄), and the solvent was removed by rotary evaporation. The crude product was filtered through a silica plug (hexane/EtOAc, 9:1) and concentrated in vacuo to afford compound 8 as a clear oil in 74% yield (157 mg); [α]${}_{\mathrm{D}}^{20}$ = −15.0 (c = 1.33, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.59–5.49 (m, 2H), 4.34 (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), 2.30 (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, KMnO₄ stain) R_f = 0.75; HRMS: exact mass calculated for C₂₀H₄₀O₂Si₂Na [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 Et₂NH (0.8 mL) and benzene (0.3 mL) was added Pd(PPh₃)₄ (16.0 mg, 0.0140 mmol, 3.00 mol%), and the reaction was stirred for 45 min in the dark. CuI (4.00 mg, 0.0210 mmol, 5.00 mol%) dissolved in a minimal amount of Et₂NH was added, followed by dropwise addition of alkyne 8 (160 mg, 0.434 mmol, 1.06 eq.) in Et₂NH (0.8 mL). After 20 h of stirring at ambient temperature, the reaction was quenched with saturated aqueous NH₄Cl (10 mL). Et₂O (15 mL) was added, and the phases were separated. The aqueous phase was extracted with Et₂O (2 × 15 mL), and the combined organic layers were dried (Na₂SO₄) before being concentrated in vacuo. The crude product was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the title compound 10 as a clear oil in 84% yield (239 mg); [α]${}_{\mathrm{D}}^{25}$ = −11.6 (c = 0.95, MeOH); ¹H NMR (400 MHz, CDCl₃) δ 6.50 (dd, J = 10.9, 15.5 Hz, 1H), 6.18 (dd, J = 10.9, 15.5 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, 1H), 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 (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), 0.06–0.01 (m, 12H); ¹³C NMR (101 MHz, CDCl₃) δ 174.4, 141.2, 139.3, 131.9, 128.6, 128.5, 126.6, 125.3, 110.6, 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, 18.4, 18.4, −4.3, −4.3, −4.6, −4.8, −5.1; TLC (hexane/EtOAc 95:5, UV-VIS) R_f = 0.33; HRMS: exact mass calculated for C₄₁H₇₆O₅Si₃Na [M + Na]⁺: 755.4891, found: 755.4893.

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 −78 °C. The reaction was stirred for 21 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 (Na₂SO₄) before being concentrated in vacuo. The crude product was purified by column chromatography on silica (hexane/EtOAc, 4:6) to afford the title compound as a clear oil. Yield: 51 mg (91%); [α]${}_{\mathrm{D}}^{25}$ = −24.4 (c = 0.67, MeOH); ¹H NMR (400 MHz, MeOD) δ 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), 5.69–5.63 (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, 6.5 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, 2H), 1.61 (p, J = 7.4 Hz, 2H), 1.42–1.28 (m, 4H); ¹³C NMR (101 MHz, MeOD) δ 176.0, 142.5, 139.9, 132.9, 130.3, 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_f = 0.22; HRMS: exact mass calculated for C₂₃H₃₄O₅Na [M + Na]⁺: 413.2299, found: 413.2298.

3.1.5. 22-OH-PD1_{n-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 H₂O (18.8 mL, pH = 7.0) was stirred under argon for 15 min before Cu(OAc)₂ (312 mg) was added and stirred for an additional 15 min. AgNO₃ (312 mg) was then added, and the reaction mixture was stirred for 30 min. The mixture was filtered and washed successively with H₂O, MeOH, acetone, and Et₂O before it was transferred to a flask containing alkyne 11 (50.0 mg, 0.128 mmol) in MeOH/H₂O (1:3, 9.2 mL). The mixture was stirred at rt and monitored by TLC analysis. After 2 h, the reaction was judged complete, and the reaction mixture was filtered through a pad of Celite^® by Merck, Darmstadt, Germany, with Et₂O. Water was added to the filtrate, and the layers were separated. The aqueous layer was extracted with Et₂O (2 × 10 mL). The combined organic layers were washed with brine and dried (Na₂SO₄). The solvent was removed in vacuo, and the crude product was purified by column chromatography on silica gel (hexane/EtOAc/MeOH, 49:50:1) to afford the methyl ester 12 as a pale yellow oil in 46% yield (23 mg); [α]${}_{\mathrm{D}}^{25}$ = −20.0 (c = 0.20, MeOH); ¹H NMR (600 MHz, MeOD) δ 6.58–6.48 (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), 2.35–2.18 (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) δ 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, 36.6, 36.4, 34.8, 32.0, 30.3, 29.8, 28.2, 25.9; TLC (hexane/EtOAc/MeOH 49:50:1) R_f = 0.22; HRMS: exact mass calculated for C₂₃H₃₆O₅Na [M + Na]⁺: 415.2455, found: 415.2455; UV-VIS: λ_max (EtOH) = 269, 271, 282 nm. The purity (96%) was determined by HPLC analysis (Eclipse XDB-C18, MeOH/H₂O 3:1, 1.0 mL/min); t_r = 32.6.

3.1.6. 22-OH-PD1_{n-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-H₂O (2:2:1, 2.8 mL) at 0 °C. The reaction mixture was 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 NaH₂PO₄ (4.0 mL), and then EtOAc (4.0 mL) was added. The layers were separated, and the water phase was extracted with EtOAc (2 × 4.0 mL). The combined organic layers were dried (Na₂SO₄) before the solvent was removed in vacuo. Then, 22-OH-PD1_{n-3 DPA} (5) was obtained as a colorless oil after purification by column chromatography (MeOH/CH₂Cl₂, 1:9) R_f = 0.22; yield: 10 mg (90%); [α]${}_{\mathrm{D}}^{25}$ = −25.4 (c = 0.35, MeOH); ¹H NMR δ 6.53 (dd, J = 13.9, 11.4 Hz, 1H), 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); ¹³C NMR (151 MHz, 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 C₂₂H₃₄O₅Na [M + Na]⁺: 401.2298, found:401.2298; UV-VIS: λ_max (EtOH) = 269, 271, 282 nm. The purity (94%) was determined by HPLC analysis (Eclipse XDB-C18, MeOH/H₂O/10mM acetic acid 65:20:20, 1.0 mL/min); T_r = 18.1.

3.2. Lipid Mediator Profiling

MeOH—two and four volumes, respectively—containing deuterium-labeled synthetic internal standards of d₄-LTB₄ (500 pg) and d₅-RvE1 (100 pg) were added to cell incubations and human serum. 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 H₂O (6.0 mL). The extraction products were washed with H₂O (4.0 mL) and hexane (5.0 mL) followed by product elution with 4.0 mL of methyl formate. Products were brought to dryness with a gentle stream of nitrogen (TurboVap LV, Biotage) and suspended in MeOH:H₂O (50:50, v/v). Samples were centrifuged (2500 rpm, 4.0 °C, 5 min), and 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 to the addition of the indicated concentrations of n-3 DPA (1) and Escherichia coli (2.5 × 10⁸ 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-PD1_{n-3 DPA} (5) was stereoselectively synthesized in six steps and with 38% yield from commercially available (3-bromopropoxy)-tert-butyldimethylsilane and known (S)-3-((tert-butyldimethylsilyl)oxy)pent-4-ynal (7). We herein established the exact configuration of the biomolecule 5, and that 5 is an ω-oxidation metabolic product of the SPM PD1_{n-3 DPA} (2). In addition, we showed that 22-OH-PD1_{n-3 DPA} (5) is formed in human serum, human neutrophils, 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].