The First Total Synthesis of the Lipid Mediator PD2n-3 DPA

The resolution of inflammation is governed by the active biosynthesis of specialized pro-resolving mediators using ω-6 and ω-3 polyunsaturated fatty acids as substrates. These mediators act as resolution agonists and display several interesting bioactivities. PD2n-3 DPA is an oxygenated polyunsaturated fatty acid biosynthesized from n-3 docosapentaenoic acid belonging to the specialized pro-resolving lipid mediator family named protectins. The protectins exhibit anti-inflammatory properties and pro-resolving bioactivities. These endogenously produced compounds are of interest as leads in resolution pharmacology and drug development. Herein, together with its NMR, MS, and UV data, a stereoselective total synthesis of PD2n-3 DPA is presented.

* sı Supporting Information ABSTRACT: The resolution of inflammation is governed by the active biosynthesis of specialized pro-resolving mediators using ω-6 and ω-3 polyunsaturated fatty acids as substrates. These mediators act as resolution agonists and display several interesting bioactivities. PD2 n-3 DPA is an oxygenated polyunsaturated fatty acid biosynthesized from n-3 docosapentaenoic acid belonging to the specialized pro-resolving lipid mediator family named protectins. The protectins exhibit anti-inflammatory properties and proresolving bioactivities. These endogenously produced compounds are of interest as leads in resolution pharmacology and drug development. Herein, together with its NMR, MS, and UV data, a stereoselective total synthesis of PD2 n-3 DPA is presented. E ndogenous mechanisms that control resolution programs during acute inflammation are essential in maintaining health. 1 If uncontrolled, chronic inflammation may result in the development of several human diseases. 1,2 Individual families of specialized pro-resolving mediators (SPMs) are the lipoxins, the resolvins, the maresins, and the protectins. SPMs are oxygenated polyunsaturated fatty acids (PUFAs) that stimulate the resolution of inflammation and fight infections. 2 The ω-3 PUFAs eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and n-3 docosapentaenoic acid (n-3 DPA) are, in the presence of cyclooxygenase-2 and various lipoxygenases, converted into SPMs. 3 SPMs exhibit antiinflammatory and pro-resolving bioactions in the low nanomolar range due to their agonist effects on G-protein coupled receptors (GPCRs). 4 Such resolution processes have attracted interest in drug development research programs, given that approximately 30% of all approved drugs act on this receptor family. 5 Because SPMs are endogenously biosynthesized in minute amounts, stereoselective total synthesis becomes necessary for configurational assignment and extensive biological evaluations facilitating drug development efforts. 3 In 2013 and 2015, new SPMs biosynthesized from n-3 DPA were reported. 6 N-3 DPA is a biochemical intermediate in the formation of DHA from EPA. 7 Some examples of n-3 DPA derived SPMs are shown in Figure 1. 6a Stereoselective total synthesis of 1 confirmed its structure as shown in Figure 1. 8,9 In collaboration with the Dalli group, we elucidated the biosynthetic pathway of PD1 n-3 DPA (1) and PD2 n-3 DPA (2) as depicted in Scheme 1. 10 Oxygenation of n-3 DPA by 15-LOX gives the hydroperoxide 5, which is transformed into the epoxide ePD n-3 DPA (6). Hydrolysis of 6 in the presence of an unknown enzyme provides 1 and 2. 10 The SPM 1 displayed potent anti-inflammatory and proresolving bioactions together with stimulation of human macrophage phagocytosis and efferocytosis. 8 In the resolution of inflammation these bioactions are distinct features. 2−4 As of today, no total synthesis of PD2 n-3 DPA (2) has been reported. Against this, and the interesting bioactions of the n-3 DPA derived SPMs, 11 the first total synthesis of 2 is presented.

■ RESULTS AND DISCUSSION
Based on the biosynthesis presented in Scheme 1, we anticipate the diol moiety in PD2 n-3 DPA (2) to be anti and 16R,17S configured. Hence, our synthesis of 2 was planned as outlined in Scheme 2, leading back to the triene-aldehyde 7 and the known Wittig salt 8; the latter has been prepared from 11 and 12. 12 Compound 9 may be prepared from the Wittig reagent 13 and 2-deoxy-D-ribose (14), both commercially available.
Aldehyde 15 was prepared from 2-deoxy-D-ribose (14) over three steps as previously reported 11c (Scheme 3). Aldehyde 15 was reacted with the ylide of commercially available triphenyl-(propyl)phosphonium bromide (13) in a Z-selective Wittig reaction. The ylide of 13 was formed after reaction with NaHMDS in CH 2 Cl 2 at −78°C. After column chromatography, 16 was obtained as one stereoisomer in 86% yield (Scheme 3). Next, we used our monodeprotection protocol 11c in order to obtain alcohol 17, which was oxidized to the aldehyde 9. Reacting 9 with an excess of 10 afforded the desired aldehyde 7 in 28% isolated yield over the three steps. The desired product was readily separated from substantial quantities of the mono Wittig product also produced, by column chromatography. Of notice, ylide 10 had to be added in several portions over the course of the reaction due to the instability of 10 at elevated temperatures.
The COSY spectrum was used for assigning vicinal signals for each of the two isolated Z-olefins and the Z,E,E-moiety. Moreover, two signals from the hydrogen atoms attached to the carbinol carbon atoms were observed as expected with signals at 4.01 ppm (ddd, 1H, J = 7.0, 4.9, 1.1 Hz) and 3.55 ppm (dt, 1H, J = 8.1, 4.9 Hz). MaR2 n-3 DPA (3) 11a and RvD1 n-3 DPA (4) 11c displayed similar chemical shift values and coupling pattern. Of note, these SPMs were matched against authentic materials using LC/MS-MS analysis.
The HSQC spectrum was used for assigning the signals from the methylene carbons at 27.1 and 31.8 ppm, next to the C-19/ C-20 and the C-7/C-8 Z-double bonds, respectively. The data from the 1 H and 13 C NMR spectra, in combination with the COSY and the HMBC spectra, allowed the structural assignment of the rest of the molecule (Table 1).
Moreover, the ultraviolet absorbance profile of 2 gave absorbance characteristics of a triene chromophore that is allylic to an auxochrome with λ max (MeOH) = 272 nm with shoulders at 262 and 283 nm.

■ CONCLUSIONS
To conclude, the first total synthesis of the oxygenated PUFA product PD2 n-3 DPA (2) is reported, enabling its exact structural assignment. The key synthetic reactions were E-and Zselective Wittig reactions, avoiding the use of challenging Zselective reduction protocols of internal alkynes. 19 Multimilligrams of material are now available for biological testing.

■ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a 0.7 mL cell with a 1.0 dm path length on an Anton Paar MCP 100 polarimeter. The UV/vis spectra from 190 to 900 nm were recorded using an Agilent Technologies Cary 8485 UV−vis spectrophotometer using quartz cuvettes. NMR spectra were recorded on a Bruker AVII400 or a Bruker AVIII HD 400 spectrometer at 400 MHz or a Bruker AVII600 spectrometer at 600 MHz for 1 H NMR and at 100 or 150 MHz for 13 C NMR. Spectra are referenced relative to the central residual protium solvent resonance in 1 H NMR (CDCl 3 δ H 7.26, DMSO-d 6 δ H 2.50 and methanol-d 4 δ H 3.31) and the central carbon solvent resonance in 13 C NMR (CDCl 3 δ C 77.00, DMSO-d 6 δ C 39.43 and methanol-d 4 δ C 49.00). Mass spectra were recorded at 70 eV on a Waters Prospec Q or Micromass QTOF 2W spectrometer using ESI as the method of ionization. Highresolution mass spectra were recorded at 70 eV on a Waters Prospec Q or Micromass QTOF 2W spectrometer using ESI as the method 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 using a C 18 stationary phase (Eclipse XDB-C18, 4.6 × 250 mm, particle size 5 μm, from Agilent Technologies), applying the conditions stated. Separation of diastereomers of 21 were conducted using a Biotage Select purification system (Biotage Sfar C18) applying the conditions stated. Diastereomeric ratios reported have not been validated by calibration; see Wernerova and Hudlicky for discussions and guidelines. 20 Unless stated otherwise, all commercially available reagents and solvents were used in the form they were supplied without any further purification. All reactions were performed under an argon atmosphere, unless otherwise stated. The stated yields are based on isolated material. Liquid chromatography (LC)-grade solvents were purchased from Fisher Scientific.