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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Bioorg Med Chem Lett. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2783685

A new approach to the synthesis of polyunsaturated deuterated isoprostanes: Total synthesis of d4-5-epi-8,12-iso-iPF-VI and d4-8,12-iso-iPF-VI


The total and stereospecific synthesis of d4-5-epi-8,12-iso-iPF-VI 55 and d4-8,12-iso-iPF-VI 64, EPA-derived all-syn-isoprostanes (iPs), has been accomplished. Because of issues related to volatility and yield with some of the primary deuterated synthons an improved synthesis is presented. These two deuterated analogs were used to discover and quantify the presence of the corresponding endogenous isoprostanes in human urine. These assays may serve as a valuable index of oxidative stress in population with omega-3 fatty acid enriched diets containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and may also be useful as an index of the severity of inflammatory diseases such as atherosclerosis and Alzheimer's disease.

Isoprostanes (iPs) are natural products of non-enzymatic free radical peroxidation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) and eicosapentaenoic acid (EPA).1,2 Isoprostanes are isomeric with prostaglandins (PGs), which are natural products generated by the action of enzymes. The mechanisms of formation of isoprostanes have been discussed in some detail.3-5 Several syntheses of iPs have been reported.3,6 These compounds have been widely used as markers of oxidative stress in various diseases. We and others have shown that the measurement of iPs in urine can be used as an index of the severity of inflammatory diseases such as Alzheimer's disease (AD) and atherosclerosis.2,7

Isoprostanes generated from EPA have received less attention than AA-derived isoprostanes.8,9 However, the AA-derived iPs we and others have measured so far as an index of oxidative stress10-12 may not be appropriate for individuals in which the major PUFAs in the phospholipids are EPA and DHA. A more appropriate index in these cases might be the measurement of EPA- and/or DHA-derived iPs, e.g. 24 and 25 (Scheme 2A). For these reasons we have synthesized these two all-syn Group VI isoprostanes and identified their occurrence in human urine.13 The reason for choosing Group VI isoprostanes derived from EPA stems from our experience that the presence of a hydroxyl group in the 5-position of these iPs causes them to be resistant to β-oxidation,8 facilitating their detection in urine. For example, we have shown that iPF-VI is highly resistant to β-oxidation and we have been able to detect appreciable amounts of this substance in human urine.8 Recent studies from our laboratory suggest that 5-epi-8,12-iso-iPF-VI 24 and 8,12-iso-iPF-VI 25 are considerably more abundant in urine than iPF-VI and may therefore serve as excellent markers for oxidative stress involving degenerative diseases of the brain, which contains high levels of DHA, as well as in individuals who consume large amounts of ω3-PUFA. To provide the deuterium-labeled standards required for the quantitation of these major EPA-derived iPs in biological fluids14 we are reporting here the total synthesis of deuterated analogs of 24 and 25, namely d4-5-epi-8,12-iso-iPF-VI 55 and d4-8,12-iso-iPF-VI 64 (Scheme 4).

Scheme 2
Structures of the major EPA-derived iPs 5-epi-8,12-iso-iPF-VI and 8,12-iso-iPF-VI.
Scheme 4
Stereospecific synthesis of d4-5-epi-8,12-iso-iPF-VI 55 and d4-8,12-iso-iPF-VI 64

Site of deuteration

We have previously developed methods to introduce four deuterium atoms into the lower side chains of iPs, as this would avoid the loss of deuterium as a result of β-oxidation. Although this is much less of an issue with Group VI iPs, as discussed above, there is an additional advantage in avoiding the addition of deuterium to the upper side chain due to the risk of scrambling in the synthetic oxo precursors of this side chain. Because of the presence of the ω3-double bond in 24 and 25, we elected to introduce four deuterium atoms in positions 19 and 20 of these molecules.


The most difficult step in our prior synthesis of tetradeutero iPF-VI was the isolation of deuterated propanol 32 (Scheme 3).15 The continuation of the synthesis to obtain derivative 38 was impractical at that point because of the dwindling yields due to the multistep synthesis. We elected at that time to redesign the synthesis by extending the isoprostane synthon by three additional carbons and reacting it with deuterated synthon 35.15 This was hardly a fruitful alternative since the isoprostane synthon requires a multistep synthesis, making the synthesis completely linear rather than convergent, which was our original intention.

Scheme 3
Synthesis of deuterated side chain.

In the present study we circumvented the volatility issue in two ways. We used a non-volatile tosylate intermediate and a very efficient procedure to convert a tosylate phosphonium salt into an iodide derivative, as the use of the phosphonium tosylate by itself results in low yields in a Wittig reaction. Transformation of tolylate salts 34 and 40 to iodides 35 and 41, respectively, proceeds in quantitative yields. We were surprised to note that the literature does not contain examples of such transformations. This can be a useful general procedure to deal with deuterium labeled small molecules.

Scheme 3 describes the synthesis of the deuterated side chain, which was subsequently connected to the 5-membered all-syn isoprostane ring. Using commercial propargyl alcohol as our starting material, the alcohol was protected using THP. The acetylene derivative 30 (Scheme 3) was then treated with Wilkinson's catalyst in the presence of deuterium gas to generate the deuterated intermediate 31, which on further treatment with dimethyl aluminum chloride generated the low-boiling volatile three-carbon alcohol 32. Attempts to isolate this alcohol resulted in very low recovery. The crude 32, which was used without purification or workup in the next step, was treated with TsCl to generate tosylate 33. After workup and purification, 33 was treated with triphenyl phosphine in the presence of CH3CN to generate the phosphonium salt 34, which was purified by column chromatography. We attempted to react 34 with aldehyde 36 and obtained a low yield of the desired product 37 (22-38%). By changing the tosylate to an iodide using sodium iodide, we obtained the iodo phosphonium salt 35.16 This was then used in a Wittig reaction with aldehyde 36 to obtain the desired product in 85% isolated yield. A cursory look at the literature reveals that tosylate salts, when used in Wittig reactions, afford low yields of the desired products. It is worth mentioning that an attempt to prepare the tetradeutero propyl iodide from 32 resulted in very low yield of the iodo derivative, probably due to volatility problems. As shown in Scheme 3, phosphonium salt 41 was prepared in good yield by a similar sequence (Panel B). Scheme 4 describes the stereospecific synthesis of d4-5-epi-8,12-iso-iPF-VI 55 and d4-8,12-iso-iPF-VI 64.17 For reasons of clarity we have included the first few structures, which we have previously disclosed.15 Step u, Scheme 4B afforded in addition to the desired product 60, 8-9% of the cis derivative 59. The isomerization procedure works well also on the crude mixture and no special purification and isolation of 59 is necessary. The transformation of bis-TES 56 to a TES aldehyde 57 was accomplished in high yields using Spur's procedure.18


The iPs 24 and 25 (Scheme 2) can be formed by the direct autooxidation of EPA as shown in Scheme 1. Alternatively, these iPs can be formed as a result of β-oxidation of DHA-derived neuroprostanes (nPs) as shown in Scheme 5, which we have previously demonstrated in the case of isomers of 65.8 Furthermore we and others have shown the DHA-derived nPs could not be detected in urine, a fact we interpreted as being due to β-oxidation.8,9 Since iPs such as 24 and 25 are resistant to β-oxidation we were readily able to measure them in urine. Hence the measurement of 24 and 25, which we have accomplished in human and murine urine,14 is an indication of the in vivo formation of either or both EPA-derived iPs and DHA-derived nPs that have been converted to the corresponding C20 derivatives by endogenous β-oxidation.

Scheme 1
iPs and nPs derived from AA, EPA, and DHA by free radical peroxidation.
Scheme 5
β-oxidation of neuroprostane to EPA-derived isoprostane.
Figure 1
LC/MS/MS chromatograms of d4-5-epi-8,12-iso-iPF-VI 55 and d4-8,12-iso-iPF-VI 64.


We wish to acknowledge the National Institutes of Health for support under Grants HL-81873 (J.R.) and HL-62250 (G.A.F.). J.R. also wishes to acknowledge the National Science Foundation for the AMX-360 (CHE-90-13145) and Bruker 400 MHz (CHE-03-42251) NMR instruments. G.A.F. is the McNeil Professor of Translational Medicine and Therapeutics. W.S.P. wishes to acknowledge the Canadian Institutes of Health Research, grant number MOP-6254, the Heart and Stroke Foundation of Quebec, and the J.R. Costello Memorial Research Fund. W.S. wishes to acknowledge the AHA grant 09CRP2260567.


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References and Notes

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16. Experimental procedure for Scheme 3a: (Step a) To the solution of acetylene 30 (4 g, 28.54 mmol) in 70 mL benzene was added 1 g of Wilkinson's catalyst (25 wt %) at room temperature. The mixture was purged with argon gas. The deuterium gas was passed in the flask until deuterium gas absorption stopped. The crude mixture was filtered through celite and florisil layers to afford the desired deuterium product 31 in quantitative yields. Sometimes the deuteration did not go to completion. In this case, the crude mixture was filtered and the deuteration process repeated.
(Steps b and c) To the solution of deuterium 31 (311 mg, 2.10 mmol) in CH2Cl2 (15 mL), Me2AlCl (6.29 mL, 6.29 mmol) was added dropwise at −25 °C and stirred for 30 min at −25 °C. The reaction mixture was gradually allowed to reach room temperature and the reaction continued for 12 h. After all the starting material was consumed the reaction was quenched using aqueous saturated NaHCO3 solution at 0 °C. The aqueous layer was extracted with CH2Cl2 and the organic layers combined and dried using Na2SO4. The solvent was cautiously evaporated up to the point where approximately 2 mL of CH2Cl2 was left inside the flask. The deuterium compound 32 was used without further purification in the next step. To the solution of crude alcohol 32, TsCl was added at 0 °C followed by pyridine and stirred for 10 min. The reaction mixture was gradually allowed to reach room temperature and the stirring continued for 12 h. The reaction was quenched by adding water and extracted using CH2Cl2, then dried and purified by silica gel chromatography to afford tosylate 33 in 75% combined yield.
(Step d) To the solution of tosylate 33 (660 mg, 3 mmol) in 5 mL of CH3CN was added Ph3P (3 g, 11.4 mmol) and the reaction was continued at 70 °C for 24 h. The solvent was evaporated and the crude compound purified using silica gel column chromatography to afford 34 in quantitative yields.
(Step e) To the solution of the salt 34 (774 mg, 1.60 mmol) in 15 mL of CH3CN was added NaI (1.2 g, 8.05 mmol) at room temperature and stirred for 12 h. The solvent was evaporated and the crude mixture purified using column chromatography to afford the desired iodo salt 35 (683 mg) in 97% yield.
17. Spectral data of d4-5-epi-8,12-iso-iPF-VI 55: 1H NMR (Methanol-d4, 400 MHz) δ 5.75 (1H, dd, J=15.3, 10.6), 5.44-5.32 (2H, m), 5.28-5.13 (3H, m), 4.09-3.92 (3H, m), 2.72-2.64 (2H, m), 2.57-2.49 (1H, m), 2.36-2.27 (1H, m), 2.26-2.02 (4H, m), 1.77-1.68 (1H, m), 1.68-1.42 (5H, m), 0.81 (1H, s); 13C NMR (Methanol-d4, 100 MHz) δ 177.59, 137.46, 132.48, 130.08, 129.60, 129.41, 128.69, 74.83, 73.09, 72.80, 52.03, 49.86, 43.49, 37.83, 35.13, 26.64, 25.10, 22.37, 13.90(m); spectral data of d4-8,12-iso-iPF-VI 64: 1H NMR (Methanol-d4, 400 MHz) δ 5.72 (1H, dd, J= 15.4, 10.6), 5.47-5.132 (2H, m), 5.27-5.16 (3H, m), 4.05-3.96 (3H, m), 2.75-2.67 (2H, m), 2.56-2.52 (1H, m), 2.38-2.30 (1H, m), 2.22-1.99 (4H, m), 1.78-1.68 (1H, m), 1.63-1.36 (5H, m), 0.81 (1H, s); 13C NMR (Methanol-d4, 100 MHz) δ 177.58, 137.62, 132.65, 131.11, 130.10, 129.59, 128.75, 74.97, 73.96, 72.93, 52.09, 48.63, 43.70, 37.74, 35.06, 26.81, 25.26, 22.41, 13.92(m).
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