Synthesis and Antioxidant Properties of an Unnatural Plasmalogen Analogue Bearing a trans O-Vinyl Ether Linkage

Abstract

To assess the antioxidant behavior of trans-1, we first synthesized trans-allyl ether 4 by opening an (S)-glycidol derivative with an (E)-alk-2-en-ol, and then produced the unnatural E-enol ether 1 by a stereoselective iridium(I)-catalyzed olefin isomerization. Natural cis-1 was preferentially degraded by HOCl and was more protective than trans-1 against lipid peroxidation induced by a free-radical initiator, demonstrating that the geometry of the 1’-alkenyloxy bond participates in the antioxidant defensive role of 1.

Plasmalogens are a subclass of glycerophospholipids that bear an 1-O-alk-1’-(Z)-enyl chain at the sn-1 position of glycerol (typically a 16-carbon chain), an sn-2 fatty acyl chain (typically unsaturated or polyunsaturated), and a polar head group at the sn-3 position (predominantly a mixture of phosphoethanolamine and phosphocholine).¹ Plasmalogens comprise about 20% of the total phospholipids in humans; they are especially abundant in human cardiac, skeletal muscle, and nervous tissues, which contain high levels of polyunsaturated fatty acids.² Since plasmalogen-deficient cells were more susceptible to free radical mediated oxidative damage than cells with normal plasmalogen levels,³ and because enol ethers are known to be susceptible to oxidation,⁴ it has been proposed that the Z-enol ether functionality, which is unique to plasmalogens, may serve as a trap for reactive oxygen species. The vinyl ether linkage is located in close proximity to the lipid-water interface, and may thus protect the polyunsaturated chains of phospholipids in membranes and lipoproteins from oxidation.⁵ The putative intermediates formed by [2+2] and [2+4] cycloaddition of singlet oxygen to this linkage are dioxetane and ene intermediates, respectively, which subsequently undergo degradation to generate a complex mixture of fatty aldehydes (such as pentadecanal), 1-formyl-2-acyl-sn-glycerophospholipids, and 1-(O-1’-hydroperoxy)-2-acyl-sn-glycero-3-phospholipids (Fig. 2).⁶ Decomposition of the allylic hydroperoxides produces plasmalogen epoxides via a radical process, and 1-formyl- and 1-lyso-phospholipids by hydrolytic processes.^6d,7

Figure 2.

Structures of 1-formyl- and 1-(O-1’-hydroperoxy)plasmalogen derivatives formed on reaction of cis-1 with reactive oxygen species.

Plasmalogens are more susceptible to oxidation than phosphatidylcholine (PC) and sphingomyelin, the other major membrane phospholipids;⁸ however, sphingomyelin has also been found to inhibit PC peroxidation, albeit less efficiently than plasmalogen, but its long-chain base contains a trans double bond near the membrane-water interface.^9a To elucidate the role of the naturally occurring (Z)-O-vinyl linkage of plasmalogen in protecting polyunsaturated lipids from oxidative degradation, we describe herein the first chemical synthesis of an unnatural analogue of plasmalogen with an (E)-O-vinyl linkage at the sn-1 position (trans-1, Figure 1)¹⁰ and a comparison of the antioxidant effects of cis- and trans-1 in two model systems.

Figure 1.

Structures of naturally occurring cis-1 and the unnatural trans-1 analogue.

The first model system we investigated was the reaction of cis- and trans-1 with hypochlorous acid (HOCl), a highly reactive oxidant and chlorinating agent that is produced endogenously when physiological concentrations of chloride ion are oxidized by the myeloperoxidase-catalyzed decomposition of hydrogen peroxide.¹¹ HOCl reacts with many biological molecules,¹² including the double bonds of lipids, to generate chlorinated products.¹³ Recently, Davies and co-workers showed that the kinetics of vinyl ether oxidation is several orders of magnitude greater than that of aliphatic alkenes.¹⁴ The products of plasmalogen oxidation by HOCl are 2-chloro fatty aldehydes and 1-lysophosphatidylcholine (LPC).¹⁵ Phospholipid chlorohydrins in the sn-2 chain of unsaturated LPC molecular species are also formed as secondary reaction products by electrophilic attack of HOCl on alkenyl double bonds.¹⁶ These chlorinated lipid species accumulate in activated neutrophils, monocytes, ischemic myocardium, and human atherosclerotic lesions, and have potential roles in many inflammatory disorders.¹⁷

Peroxidation of phospholipids containing a polyunsaturated fatty acyl chain such as linolenic acid has been studied frequently by using free-radical initiators, such as the water-soluble thermolabile free-radical generator 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH).¹⁸ As a model system to compare the antioxidant capability of cis- and trans-1, we studied the influence of 1 on the rate and extent of peroxidation of 1-palmitoyl-2-linolenoyl-phosphatidylcholine (16:0-18:2-PC) in liposomes exposed to AAPH. The reaction kinetics were monitored by measuring the formation of conjugated diene lipid hydroperoxides, as monitored spectrophotometrically by the absorbance at 234 nm.⁹ A₂₃₄ is diminished when AAPH-induced free-radical chain initiation in a polyunsaturated acyl chain is suppressed.

Previous reports of the preparation of 1-O-alkenyl ether derivatives of glycerol were based on elimination reactions of α-halo cyclic glycerol acetals, which gave low yields and poor stereoselectivity.¹⁹ To construct the glycerol backbone with an O-1’-alkenyl chain at the sn-1 position, we used (S)-glycidol and 1-hexadecanol as the starting materials for the synthesis of trans-1 (see Supporting Information). As shown in Scheme 1, the DPS ether of (S)-glycidol (3) was employed in a regioselective BF₃•OEt₂-mediated ring-opening reaction²⁰ of (E)-octadec-2-en-1-ol (2), prepared by DIBALH reduction of ethyl (E)-octadecanoate. The expected attack at C-3 provided an 8:1 (separable) mixture of allyl ether 4 and the undesired regioisomer 4a.

Scheme 1.

Preparation of Allyl Ether 4

Next, isomerization of the double bond was investigated using transition metal complexes as catalysts.²¹ An initial attempt using RhCl₃•3H₂O²² as a catalyst resulted in no reaction. Fortunately, we found that use of (1,5-cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (Ir(COD)[PMePh₂]₂PF₆),²³ after activation with hydrogen for 5 min, resulted in the stereoselective isomerization of allylic ether 4 to enol ether 5 in 2 h at rt, and with the desired E double bond as the only product (Scheme 2). The coupling constant (J_{H1’–H2’} = 12.6 Hz at δ 6.24 ppm) in the ¹H-NMR spectrum of 5 is indicative of the desired E isomer, which was obtained in 96% yield.²⁴ sec-Alcohol 5 was then esterified with oleic acid, using DCC in the presence of DMAP, affording ester 6. The final steps in the synthesis of trans-1 require conditions that do not cleave the acid-labile and oxidizable vinyl ether moiety or the alkaline-labile acyl ester bond. After the silyl ether in 6 was removed using TBAF (6 equiv) and imidazole (2.5 equiv) in THF at −23 °C, the phosphocholine head group was installed at the sn-3 position in 54% overall yield without accompanying acyl migration by opening of a cyclic phospholane intermediate in a pressure tube with anhydrous trimethylamine (200 equiv, condensed at −10 °C) in MeCN/C₆H₆ (3:1) in the presence of pyridine.²⁵

Scheme 2.

Ir(I)-mediated Olefin Isomerization and Final Conversion to trans-1

We analyzed the HOCl oxidation reaction of cis-and trans-1 using increasing concentrations of HOCl. At equimolar concentrations of HOCl and 1, the extent of plasmalogen oxidation was minimal with both species (data not shown). Figure 3 shows the loss of both cis- and trans-1 as the HOCl concentration was increased. At both a 2- and 5-fold molar ratio of [HOCl]/[1], the cis-vinyl ether linkage was oxidized faster than the trans linkage. This selectivity was not observed at a higher HOCl concentration (1.0 mM).

Figure 3.

Disparate cis- and trans-1 oxidation by HOCl.

The bars show the amounts of enol ether remaining (left panel) and the amount of 2-chloro-aldehyde produced (right panel) (both in nmoles) after treatment with various HOCl concentrations (in mM) for 5 min at 37 °C. Data are the mean ± SD of 3 experiments. See S.I. for experimental details.

ESI-MS analyses of the reaction mixture with 1 mM HOCl demonstrated that the other product of HOCl oxidation is LPC containing an oleic acid moiety (m/z 544.24) (Figure 4). The chlorohydrin of this material, formed by reaction with HOCl,¹⁵ is a by-product with m/z 596.18. In these spectra, m/z 794.35 is the unreacted cis- or trans-1. These spectra were acquired in the presence of dilute NaOH, which facilitates the production of the observed sodiated adduct ions.

Figure 4.

ESI-MS of the HOCl oxidation products.

cis- or trans-1 (50 nmol) was incubated in 500 µL of 20 mM phosphate-buffered saline containing 0.1 mM diethylenetriaminepentaacetic acid (pH 7.0) in the presence or absence of 1 mM chloride-free NaOCl for 5 min at 37 °C. After the reactions were terminated by addition of MeOH, products were extracted into CHCl₃. Reaction products and precursor cis-or trans-1 were resuspended in 500 µL of MeOH/CHCl₃ (4:1) containing 1 µM NaOH and analyzed by ESI-MS in the direct infusion mode at a flow rate of 3 µL/min. Samples were analyzed in the positive ion mode with detection of their sodiated adducts.

Plasmalogens are known to protect membrane phospholipids against radical-induced damage.^1a Therefore, we next evaluated the ability of cis- and trans-1 to protect 16:0-18:2-PC from radical-mediated peroxidation in liposomes. The liposomes, which were prepared with 80 mol% 16:0-18:2-PC and 20 mol% cis- or trans-1, were subjected to oxidation by 3.3 mM AAPH. The oxidation of 16:0-18:2-PC²⁶ was monitored by the increase in absorbance at 234 nm, a measure of conjugated diene hydroperoxide formation in the sn-2 fatty acyl group of PC.²⁷ Figure 5 shows that cis-1 inhibited the rate of AAPH-mediated oxidation of 16:0-18:2-PC by 33% whereas trans-1 inhibited the rate by only 12%, based on the slopes of the reaction curves in the linear range (0–100 min). Similar data were obtained when 1 mM AAPH was used to induce oxidation (data not shown).

Figure 5.

Effect of cis- and trans-1 on the oxidation of 16:0-18:2 PC induced by AAPH.

We also determined the effect of cis- and trans-1 on 16:0-18:2-PC oxidation in liposomes in the presence of 50 µM Cu²⁺ as the oxidizing agent, which facilitates free-radical chain propagation.²⁸ We found that trans-1 was completely ineffective in protecting 16:0-18:2-PC oxidation whereas cis-1 inhibited PC oxidation by 45% (results not shown).

In summary, the first chemical synthesis of trans-1, an unnatural analog of plasmalogen bearing a trans O-vinyl linkage at the sn-1 position, has been achieved. A key step in the synthesis involves E stereoselective enol ether formation of 5 via an iridium(I)-mediated olefin isomerization of O-allyl ether 4. The kinetic data (Figure 3 and Figure 5) indicate that the geometry of the alkenyl ether linkage of plasmalogen plays a significant role in plasmalogen’s antioxidant properties.