Stereoselective Synthesis and Structural Confirmation of the Specialized Pro-Resolving Mediator Resolvin E4

Herein, we report the stereoselective and convergent synthesis of resolvin E4, a newly identified specialized pro-resolving mediator. This synthesis proves the absolute configuration and exact olefin geometry. Key elements of the successful strategy include a highly stereoselective MacMillan organocatalytic oxyamination, a Midland Alpine borane reduction, and the use of a 1,4-pentadiyne unit as a linchpin building block. The application of reaction telescoping in several of the synthetic transformations enabled the preparation of the resolvin E4 methyl ester in 10% yield over 10 steps (longest linear sequence). The physical property (UV–Vis and LC–MS/MS) data of synthetic resolvin E4 matched those obtained from biologically produced material.


■ INTRODUCTION
Inflammation is a consequence of the immune system responding to injurious stimuli and constitutes an essential, protective strategy with the aim of restoring cellular homeostasis. Recent efforts concerning the mechanisms involved in the resolution of acute inflammation have provided evidence for a new superfamily of endogenous lipid mediators named specialized pro-resolving mediators (SPMs). 1 These oxygenated polyunsaturated fatty acids are biosynthesized in the presence of lipoxygenase and cyclooxygenase enzymes. 2 SPMs are chemically labile molecules formed in nano-to picogram amounts in vivo 3 and exhibit anti-inflammatory and proresolving bioactions, often in the low nano-to picomolar range. 2,3 Additionally, SPMs are important in the process of clearing bacterial infections and participate in host defense, organ protection, pain reduction and also play a role in tissue remodeling. 3 The E-series resolvins, derived from eicosapentaenoic acid (EPA), were among the first SPMs to be reported ( Figure 1). 4 RvE1 and RvE2 have been subjected to clinical trial development programs 5 as well as drug discovery efforts with the aim of establishing new pro-resolution agonists. 6 The active resolution processes governed by SPMs are considered a biomedical paradigm shift. 7 In 2019, Serhan and co-workers reported a new SPM and named it resolvin E4 (RvE4) based on its potent physiologic actions. 8 This SPM is produced by human macrophages and neutrophils during physiologic hypoxic conditions (1-5% O 2 ). In contrast to the three earlier reported E-series resolvins, this SPM is formed after two consecutive lipoxygenation reactions (Scheme 1). 8 Earlier, 18S-configured epimers of RvE1, RvE2, and RvE3 have been identified. 9 In the first step of the biosynthesis of RvE4, 15S-HpEPE is formed by 15-LOX, while the second lipoxygenation step is catalyzed by 5-LOX. Reductions of the hydroperoxide intermediates 15S-HpEPE and 5S-HpEPE are facilitated by peroxidase activity (Scheme 1).

■ RESULTS AND DISCUSSION
An overview of the retrosynthetic analysis applied to the tentatively assigned structure of RvE4 (1) is shown in Scheme 2. The observation of a central (Z,Z)-1,4-pentadiene structural motif contained within the C 2 -symmetric C4−C16-domain of the molecule resulted in the first two disconnections being based on the Sonogashira cross-coupling reaction 12 followed by Z-selective hydrogenation. This analysis identified three key fragments 3, 5, and 1-trimethylsilyl-1,4-pentadiyne (4), the latter serving the role of a linchpin, to be convergently assembled in the synthesis.
Fragment 3 was disconnected back to cis-4-heptenal (6) with an enantioselective, organocatalytic oxyamination 13 as well as a Takai olefination 14 planned as the two pivotal steps in the forward direction. The Carreira alkynylation was chosen as the key transformation for furnishing fragment 5, with the intent of later transforming the acetylene moiety into the corresponding E-vinyl halide functionality needed for the planned palladium cross-coupling chemistry.
The project commenced with the construction of ω-3 fragment 3, starting from commercially available and affordable cis-4-heptenal (6). To this end, different α-oxidation protocols were first examined based on literature protocols (Table 1).
In light of these results, we settled on an enantioselective, organocatalytic α-oxyamination using 10 mol % D-proline and nitrosobenzene in CHCl 3 based on the procedure developed by the MacMillan group. 13d A solvent switch to ethanol preceded the NaBH 4 -based reduction of the in situ masked aldehyde functionality, and then the comparatively weak O−N bond was cleaved using zinc and acetic acid. After this sequence, a chromatographic purification step was introduced. The overall yield obtained for the described synthetic sequence was 80%, and chiral HPLC analysis of the α-aminoxylated alcohol intermediate 21 before zinc reduction to 7 showed an enantiomeric excess of 98% (Supporting Information).
The next objective was the regioselective TBS-protection of the secondary alcohol present in the 1,2-diol system in 7, and this was achieved by first masking the primary alcohol as the corresponding bulky pivaloyl ester and then adding a catalytic amount of 4-dimethylaminopyridine (DMAP) together with an excess of TBS triflate to the reaction mixture, yielding bisprotected 8 in 81% after column chromatography. A DIBAL-H reduction then cleanly did away with the pivaloyl moiety, and the primary alcohol was obtained in a crude form after work up and removal of volatiles under high-vacuum. This material was directly subjected to a Dess−Martin oxidation 15 to give the corresponding aldehyde. Passing the crude material through a short plug of silica gel to remove periodinane-related residues was found beneficial before the next reaction. Finally, the vinyl Scheme 1. Proposed Biosynthetic Pathway for RvE4 The synthetic sequence depicted in Scheme 3 is shorter than our previously reported preparation 16 of 3 when the step count for one-pot reactions is taken into account. Furthermore, this approach comes with other benefits: for example, (i) the catalytic, highly enantioselective oxyamination replaces the rather expensive use of chiral pool starting materials of unreliable supply, (ii) the thoughtful use of reaction telescoping 17 allows for the conduction of several transformations without the need to isolate, purify, and handle sensitive intermediates, and (iii) cryogenic conditions combined with an array of hazardous reagents and additives have been avoided.
Turning our attention to the preparation of α-fragment 5, the first step was the straight-forward esterification of lactone 9 in basic methanol and a subsequent copper-catalyzed Stahl aerobic oxidation 18 of the resulting primary alcohol 10, affording 11 in good yield. The Carreira alkynylation 19 between aldehyde 11 and 2-methylbut-3-yn-2-ol was studied next, and we found that a yield of 50% could be achieved if a solution of the aldehyde in toluene was added dropwise with the aid of a syringe pump, over a 24 h period, to two equivalents of the corresponding alkynylzinc species of said alkyne (Scheme 4). Slow addition is often needed for αunbranched aliphatic aldehydes in order to minimize the competing aldol self-condensation pathway. 20 Surprisingly, however, chiral HPLC analysis of the 2naphthoate derivative of 12 revealed that the obtained enantiomeric excess was only 34% in this case (Supporting Information), which is significantly lower than what we have previously obtained for other structurally similar substrates in hitherto unpublished work. Hence, in light of this outcome, the alkynylation sequence was put to the side in favor of an alternative approach (Scheme 5).
Capitalizing on the β-silicon effect, an aliphatic Friedel− Crafts acylation between acid chloride 13 and bis-(trimethylsilyl)acetylene in the presence of Lewis acidic AlCl 3 , gave ketone 14 in 72% yield. 21 Gram-scale asymmetric reduction of the alkynyl ketone was achieved by the addition of the Midland (S)-Alpine borane reagent 22 in tetrahydrofuran (THF) at 0°C, followed by swift removal of the solvent to give essentially neat conditions, ultimately furnishing the desired propargylic alcohol 15 in 96% enantiomeric excess and 89% yield after workup and purification (Supporting Information). The secondary alcohol in 15 was then protected using TBS chloride and imidazole in dichloromethane, followed by a solvent switch to methanol and addition of K 2 CO 3 , effectively removing the TMS-group attached to alkyne 5 in 91% overall yield.
At this stage, it was necessary to convert the terminal acetylene into the corresponding E-vinyl iodide, and this was achieved by a two-step process: first, free radical hydrostannation was initiated using a catalytic amount of azobisisobutyronitrile (AIBN), with excess tributyltin hydride added to ensure complete equilibration to the desired geometrical isomer, and then, iododestannylation was performed, yielding 16 in 74% over two steps.
The first of the two planned Sonogashira cross-coupling reactions was performed using catalytic amounts of Pd-Scheme 3. Organocatalytic Approach to the Construction of ω-3 Fragment 3 (PPh 3 ) 2 Cl 2 /CuI, which cleanly effected the union between vinyl iodide 16 and linchpin 4 in 98% yield. Given the inherent lability of the resulting diyne system in 17, especially to basic reaction conditions, protiodesilylation was performed in a mild manner by the employment of AgNO 3 and KCN, 23 affording the terminal alkyne 18 in 65% yield. The same catalyst system was then used again for the final Sonogashira carbon−carbon bond-forming reaction between alkyne 18 and vinyl iodide 3, giving the complete carbon skeleton 19 in 78%. The two internal, conjugated triple bonds were reduced in 70% yield using the tried-and-tested Lindlar hydrogenation protocol which involves the utilization of a mixed solvent system consisting of EtOAc/pyridine/1octene. 24 The inclusion of pyridine helps to modulate and control the activity of the heterogeneous catalyst, and 1-octene serves as a sacrificial olefin, the presence of which aids in minimizing competing over-reduction as the reaction nears completion. Removal of the two TBS-groups in 20 was first attempted using tetra-n-butylammonium fluoride (TBAF) in THF; however, significant byproduct formation was observed, leading to a diminished yield and difficulties during the purification process. A different deprotection approach was thus sought and found. Subjecting 20 instead to a catalytic amount of acetic chloride in methanol 25 afforded RvE4 methyl ester (2) in 66% yield (Scheme 6) and chemical purity >97% (Supporting Information). The NMR-( 1 H, 13 C, and COSY), MS-, and UV-data were all in accordance with the structure of 2 (Supporting Information).
MRM LC−MS/MS Matching Experiments. Since SPMs are formed in the nano-to picogram range in vivo, direct NMR analyses for structural verification are not viable. In order to ascertain that our synthetically prepared material was identical to that of authentic RvE4 (1) produced in vitro, matching experiments were conducted. Due to the chemically sensitive nature of this and other SPMs, 26 hydrolysis was performed just prior to the LC−MS/MS experiments, as earlier reported. 8 In Figure  and m/z 115). In the middle panel, the chromatographic behavior of synthetically produced RvE4 (1), with an identical observed retention time (12.9 min) to that of the authentic material, is shown. Next, the result from coinjection of the biologically produced material and synthetically produced RvE4 (1) appears in the bottom panel, resulting in both coelution as well as an overall matching MS/MS fragmentation fingerprint. Additionally, the UV−Vis spectrum was in agreement with the original isolation of RvE4 (1). 8 Overall, these results confirm that the synthetic material matched the biogenic material.

■ CONCLUSIONS
A total synthesis providing multi-milligram quantities of the methyl ester 2 of the SPM RvE4 (1) has been reported in 10% yield over 10 steps (longest linear sequence). Several of the reactions were performed using telescoping techniques, establishing the basis for an efficient total synthesis. Moreover, the successful use of the organocatalytic MacMillian oxyamination reaction is presented. The application of stereo-selective organocatalytic protocols offers many advantages in the total synthesis of natural products. 27 The integrity of the synthetically prepared material was demonstrated through matching experiments with authentic material obtained from human macrophages and neutrophils during hypoxic conditions. These results showed that synthetic and biologically produced RvE4 (1) matched, thus establishing both the absolute configurations of the carbinol atoms as well as an overall alkene geometry. Collectively, this provided evidence for the complete stereochemical assignment as (5S,6E,8Z,11-Z,13E,15S,17Z)-5,15-dihydroxyicosa-6,8,11,13,17-pentaenoic acid.
■ EXPERIMENTAL SECTION General Information. Unless otherwise stated, all commercially available reagents and solvents were used in the form they were supplied without any further purification. The stated yields are based on the isolated material. All sensitive reactions were performed under an argon or nitrogen atmosphere using Schlenk techniques. Reaction flasks were covered with aluminum foil during sensitive reactions and storage to minimize exposure to light. Thin layer chromatography was performed on silica gel 60 F 254 aluminum-backed plates fabricated by Merck. Flash column chromatography was performed on silica gel 60 (40−63 μm) produced by Merck. NMR spectra were recorded on a Scheme 6. Sonogashira Cross-Coupling Reactions and Z-Selective Hydrogenation to Complete the Synthesis of RvE4 Methyl Ester (2) The Journal of Organic Chemistry The UV−Vis spectrum was recorded using an Agilent Technologies Cary 8485 UV−Vis spectrophotometer using quartz cuvettes.
The reaction mixture was allowed to warm up to room temperature and stirred overnight. The solvent was removed under a gentle stream of argon, and the material thus obtained was purified by flash column chromatography (SiO 2 , gradient elution, 10−30% EtOAc in hexane) to give the desired naphthalate 23 (31.5 mg, 85.6 μmol, 92%) as a white solid. The enantiomeric excess (34%) was determined by HPLC analysis using a chiral column (AD-H, i-PrOH/hexane, 15 175.4, 166.9, 137.1, 133.9,  132.2, 130.4, 129.7, 129.4, 128.9, 128.3, 128.0, 126.0, 92.2, 79.7, 65.6 The exact same procedure was followed for the preparation of racemic naphthalate. Yield: 30.8 mg, 0.0837 mmol, 90%. The obtained experimental data matched that given for compound 23. Methyl 5-Oxo-7-(trimethylsilyl)hept-6-ynoate (14). A flamedried flask under argon was charged with AlCl 3 (10.5 g, 78.8 mmol, 1.30 equiv) and CH 2 Cl 2 (75 mL) at 0°C. A solution of bis(trimethylsilyl)acetylene (10.4 g, 60.8 mmol, 1.00 equiv) and methyl 4-(chloroformyl)butyrate (13, 10.0 g, 60.8 mmol, 1.00 equiv) in CH 2 Cl 2 (75 mL) was then added in a dropwise manner over 15 min. The reaction mixture was stirred at 0°C for 30 min, warmed up to room temperature over a period of 45 min, and then cooled back down to 0°C. The reaction was quenched by the addition of 1 M HCl (80 mL) and stirred for 10 min. The resulting thick suspension was vacuum filtrated through a short plug of silica gel directly into a separatory funnel, and the plug was washed with additional fresh CH 2 Cl 2 (50 mL). The phases were separated, and the aqueous phase was extracted with CH 2 Cl 2 (3 × 50 mL). The combined organic phase was dried (Na 2 SO 4 ), filtrated, and concentrated in vacuo. The crude product thus obtained was purified by flash column chromatography (SiO 2 , 10% EtOAc in hexane) to yield the desired product 14 (9.90 g, 43.7 mmol 72%) as a yellow oil. The spectroscopic data was in agreement with previously reported data. 31  Methyl (S)-5-Hydroxy-7-(trimethylsilyl)hept-6-ynoate (15). Ketone 14 (5.66 g, 25.0 mmol, 1.00 equiv) was azeotropically dried with 2-MeTHF (2 × 15 mL) and then placed under high vacuum for 30 min. The flask was vented with argon and cooled to −10°C, and (S)-Alpine-borane solution (0.5 M in THF, 100 mL, 50.0 mmol, 2.00 equiv) was added over a period of 15 min. Most of the THF solvent was immediately removed under vacuum with efficient stirring while warming up to 0°C. The resulting, highly viscous reaction mixture was then allowed to warm to room temperature and stirred overnight. Next, the reaction mixture was cooled to 0°C, and acetaldehyde (1.40 mL, 1.10 g, 25.0 mmol, 1.00 equiv) was added in a dropwise manner. After 15 min, diethyl ether (100 mL) was added, followed by the dropwise addition of ethanolamine (3.00 mL, 3.00 g, 50.0 mmol, 2.00 equiv). The reaction mixture was stirred for 30 min at 0°C, warmed to room temperature, and then stirred an additional hour. The white, solid 9-BBN-ethanolamine complex was removed by filtration, and the filtrate was washed with water (2 × 30 mL). The organic phase was dried (Na 2 SO 4 ), filtrated, and concentrated in vacuo. The crude product thus obtained was purified by flash column chromatography (SiO 2 , gradient elution, 10−20% EtOAc in hexane) to give the desired product 15 (5. Methyl 5-Hydroxy-7-(trimethylsilyl)hept-6-ynoate (rac-15). Ketone 14 (200 mg, 0.884 mmol, 1.00 equiv) was azeotropically dried with 2-MeTHF (2 × 1 mL) and then placed under high vacuum for 30 min. The flask was cooled to 0°C, and 9-BBN-H (0.5 M in THF, 3.53 mL, 1.77 mmol, 2.00 equiv) was added, and approximately half the solvent volume was removed under vacuum at room temperature. The reaction mixture was stirred for 72 h before acetaldehyde (0.05 mL, 0.884 mmol, 1.00 equiv) was added dropwise, and the reaction mixture was stirred for an additional hour. The reaction mixture was diluted with Et 2 O (5 mL), and ethanolamine (53.0 μL, 0.884 mmol, 1.00 equiv) was added in a dropwise manner. After 30 min, the reaction mixture was concentrated in vacuo to give a yellow oil together with some solid material. Water (5 mL) was added, and the aqueous phase was extracted with Et 2 O (3 × 3 mL). The organic phase was dried (Na 2 SO 4 ), filtrated, and concentrated in vacuo. The crude material thus obtained was purified by flash column chromatography (SiO 2 , gradient elution, 10−20% EtOAc in hexane) to give the desired racemic product rac-15 (109 mg, 0.477 mmol, 54%) as a clear oil. The obtained experimental data matched that given for compound 15.
Methyl (S)-5-((tert-Butyldimethylsilyl)oxy)hept-6-ynoate (5). Propargylic alcohol 15 (3.90 g, 17.1 mmol, 1.00 equiv) was dissolved in CH 2 Cl 2 (45 mL). Imidazole (2.33 g, 34.2 mmol, 2.00 equiv) and tert-butyldimethylsilyl chloride (3.86 g, 25.6 mmol, 1.50 equiv) were added in a successive manner at room temperature. The reaction mixture was stirred overnight and then the solvent was removed in vacuo. The material was dissolved in methanol (172 mL) and then cooled to 0°C. Next, K 2 CO 3 (4.74 g, 34.2 mmol, 2.00 equiv) was added in one portion, and the reaction mixture was allowed to warm to room temperature. The reaction was followed by TLC analysis (product is observed just below the starting material with 5% EtOAc in hexane as the eluent), and when deemed complete by TLC analysis (∼1 h), the flask was cooled back down to 0°C. The reaction was quenched by the addition of phosphate buffer (132 mL, pH = 7), and the reaction mixture was stirred for 5 min. NaCl (∼10 g) was added, and the aqueous phase was extracted with hexane (5 × 50 mL). The combined organic phase was dried (Na 2 SO 4 ), filtrated, and concentrated in vacuo. The crude material thus obtained was purified by flash column chromatography (SiO 2 , 5% EtOAc in hexane) to give the desired product 5 (4.21 g, 15.6 mmol, 91%) as a clear oil. The spectroscopic data was in agreement with previously reported data. 33  Methyl (S,E)-5-((tert-Butyldimethylsilyl)oxy)-7-iodohept-6enoate (16). Vinyl iodide 16 was prepared following the procedure by Sulikowski et al. 35 Alkyne 5 (100 mg, 0.370 mmol, 1.00 equiv) was dissolved in benzene (7.5 mL), and then, nBu 3 SnH (0.30 mL, 1.11 mmol, 3.00 equiv) and AIBN (10.0 mg, 60.9 μmol, 16.5 mol %) were added. The reaction was heated to 80°C (oil bath) for 2 h. The reaction mixture was then cooled to room temperature, and the solvent was removed in vacuo. The crude product thus obtained was purified by flash column chromatography (SiO 2 , 1% Et 2 O in hexane) to give the desired product as a clear oil which was used directly in the next reaction. R f (5% Et 2 O in hexane, visualized with KMnO 4 stain) = 0.35.