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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Labelled Comp Radiopharm. Author manuscript; available in PMC 2010 April 21.
Published in final edited form as:
PMCID: PMC2857661
NIHMSID: NIHMS188796

Synthesis of site-specifically deuterated arachidonic acid derivatives containing a remote tritium radiolabel

Summary

The synthesis of arachidonic acid derivatives containing site-specifically incorporated deuterium atoms and also a remote tritium label are described. Deuterium incorporation at the C11 and/or C15 position was achieved using Wittig chemistry, while the radiolabel was introduced at a remote position using [3H]NaBH4 as the radiolabel source. These compounds can be used to measure secondary kinetic isotope effects for both cyclooxygenase and lipoxygenase enzymes under aerobic turnover with high precision.

Keywords: arachidonic acid, lipoxygenase, isotope effect, radiolabel

Introduction

Prostaglandin H synthase (PGHS), or cyclooxygenase (COX), is a bifunctional enzyme that catalyzes the first committed step in the biosynthesis of prostaglandins and thromboxanes.13 In a first reaction the enzyme converts arachidonic acid to PGG2, followed by reduction of this intermediate to PGH2 in a second active site (Scheme 1). Two different isozymes of PGHS have been identified, PGHS-1 and PGHS-2, and they have been linked to numerous biological pathways including inflammation,4 gastric function5 and cardiovascular processes.6

Scheme 1
PGHS catalyzed reaction of arachidonic acid forming prostaglandin H2

The mechanism of the enzyme as proposed by Hamberg and Samuelsson7 and modified by Ruf and coworkers with the identification of a tyrosyl radical initiator is shown in Scheme 2.810 Although the proposed mechanism is widely accepted, the experimental characterization of the proposed radical intermediates is still incomplete. The reaction is postulated to be initiated by a hydrogen atom abstraction from the C13 position of arachidonic acid to generate a carbon-based radical intermediate I. Characterization of this initial radical intermediate formed under anaerobic conditions has been reported using EPR spectroscopy and suggests that both isozymes proceed through a pentadienyl radical intermediate.11,12 However, it is possible that the radical intermediate observed in these studies is not directly on the reaction pathway, but rather the thermodynamically most stable radical that is only formed when oxygen is not present. For example, the pentadienyl radical may be a thermodynamic sink accessed only when an earlier allyl radical cannot react with oxygen. Structural characterization of the initial radical intermediate during aerobic enzyme turnover would require distinction between an allyl and pentadienyl radical.2 A similar question can be raised for aspirin treated PGHS-2 and PGHS mutants that result in the formation of oxygenation products at C15,1315 and for the lipoxygenases, which also abstract a hydrogen atom from a bisallylic position.1619

Scheme 2
Proposed mechanism for prostaglandin H synthase

Both computationally and experimentally, a small secondary kinetic isotope effect (KIE) has been reported for the formation of an allyl radical when the substrate is isotopically substituted at the vinyl position (Scheme 3).2023 Based on this precedent, the identity of the initial radical intermediate formed during PGHS and lipoxygenase turnover might be determined. If a C11–C13 allyl radical is formed by hydrogen atom abstraction in PGHS, substrate with C11 deuterium labeled would give a normal KIE whereas substrate labeled at C15 would not show a KIE. Conversely, if a C13–C15 allyl radical is formed, substrate with C11 deuterium label would not produce a KIE, whilst C15-labeled substrate would show a normal KIE. If a pentadienyl radical is formed, both compounds would produce a KIE. The predicted magnitude of this secondary isotope effect is only 1.06–1.10,2023 and thus a precise method is necessary for these studies. We opted to utilize the approach of remote radiolabeling, where radionuclei are incorporated into a chemically inert position as reporters for relative reaction rates. For these experiments, a tritium label would be incorporated into the deuterated arachidonic acid (AA), whereas 14C would be used as the remote label in the unlabeled arachidonic acid (Figure 1). In the competition experiment, the enzymatic reaction would be quenched at various extents of conversion, and the starting material and enzymatic product separated by HPLC. The ratio of 14C to 3H radiolabel would then be determined for both, and any isotope effect quantified. The first step of hydrogen atom abstraction from C13 is rate limiting and irreversible and, therefore, the presence of a normal KIE should result in an increase in the ratio of 3H/14C in the recovered starting material and a decrease in this ratio in the enzyme product. The synthesis of the required radiolabeled substrates for these studies, [8-3H]-(11-2H)AA 1 and [8-3H]-(15-2H)AA 2, is described herein.

Figure 1
Competitive experiment for measuring secondary kinetic isotope effects
Scheme 3
Secondary kinetic isotope effect associated with generation of allyl radical

Results and discussion

In designing the synthetic scheme for these labeled compounds, we had two primary objectives. First, the route should minimize the transformations necessary with radiolabeled intermediates. The incorporation of the tritium label was envisioned using a Wittig reaction of a deuterated phosphonium salt with a labeled aldehyde (Scheme 4). This reaction is known to proceed with good yield and stereocontrol of the alkene product.11,24 Not only is the incorporation of the radiolabel late in the synthesis, but also the methyl arachidonate product requires only a basic hydrolysis to yield the desired product.

Scheme 4
Synthetic approach for radiolabeled arachidonic acids and synthesis of monodeuterated phosphonium salt 9

The labeled aldehyde partner 6 was prepared by reduction of unlabeled aldehyde with [3H]NaBH4 under Luche conditions,25 followed by oxidation with Dess-Martin periodinane.26 This procedure minimized the procedural steps requiring only extraction to isolate the radiolabeled aldehyde. The elimination of silica gel chromatography or distillation greatly reduced the number of laboratory procedures necessary and, therefore, the risk of contamination. After this two-step procedure, the radiolabeled aldehyde 6 was isolated in good purity, with less than 5% isomerization to the α,β-unsaturated aldehyde. This impurity was removed after reaction with phosphonium salt 5.

The second objective of the synthetic route was the generation of phosphonium salts 5 with a high degree of deuterium incorporation. These compounds were synthesized utilizing Wittig reactions for the key transformations. [8-3H]-(15-2H)AA was prepared starting with reaction of previously reported phosphonium salt 727 with (1-2H)hexanal. Deprotection of the substituted alkene 8 with a solution of tetrabutylammonium fluoride provided the alcohol in good yield (Scheme 4). Conversion to the phosphonium salt 9 was accomplished by bromination followed by treatment with triphenylphosphine. At this point, both the substituted phosphonium and the literature reported unlabeled phosphonium salt28 9a were used in a series of Wittig reactions using either protiated24 or deuterated aldehydes 10a, b to generate the unlabeled, monodeuterated, and dideuterated skipped dienes 11 (Scheme 5). The substituted aldehyde 10a was formed by reduction of the corresponding methyl ester with lithium aluminum deuteride followed by pyridinium chlorochromate oxidation.

Scheme 5
Synthesis of substituted phosphonium salts

Deprotection of 11a, b with boron tribromide proceeded in good yield with no isomerization of the skipped double bonds. Similarly, deprotection of 11c, d with a solution of tetrabutylammonium fluoride gave alcohols 12c, d with >98% 2H incorporation for 12c. Subsequent bromination and reaction with triphenylphosphine produced the phosphonium salts 5. A final Wittig reaction utilizing the phosphonium salts 5 and radiolabeled aldehyde 6 yielded methyl arachidonates 13a–d (Scheme 6). Hydrolysis with a 1M solution of LiOH (aq) and THF (1:2) afforded the final arachidonic acid derivatives 1 and 2. These compounds were purified using consecutive C18 and silver-ion reversed phase HPLC to remove the small amount of stereoisomers and the specific radioactivities were determined by liquid scintillation counting. Concentrations of arachidonic acid in stock solutions were determined by complete conversion to HPETE with soybean lipoxygenase and spectrophotometric determination of the HPETE concentration. The methyl ester of [8-3H]-(11,15-2H2)AA 13b will, after hydrolysis, be used to confirm the results obtained with the monodeuterated substrates 1 and 2 and the methyl ester of [8-3H]AA 13d will be used to ensure that the remote tritium label itself does not induce any secondary KIE.

Scheme 6
Synthesis of radiolabeled arachidonic acids

Experimental

Phosphonium salt of 1-bromo-(4-2H)non-3-ene (9)

The synthesis of phosphonium salt 9 and 9a was based on a previously reported synthetic scheme.12

(3-2H)Dodeca-3,6-dienyloxymethyl-benzene (11a)

A dry flask was charged with phosphonium salt 9a (335.2 mg, 0.70 mmol). After purging with Ar, THF (10 ml) was added and the solution was cooled to −78°C. A 1.0M solution of NaHMDS in THF (600 μl, 0.60 mmol) was added and the solution was stirred for 1 h at −78°C. The substituted aldehyde 10a (98.6 mg, 0.60 mmol) in 2ml THF was added very slowly. The reaction was stirred at −78°C for 3 h, and then slowly warmed to room temperature. After 10 h, the reaction was quenched with water and extracted with ether (3 × 30 ml). The combined organic layers were dried with MgSO4, filtered and concentrated. The resulting oil was purified by flash chromatography (100% pentane) yielding 130.6mg (80%) of 11a as a clear oil. 1H NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.1 Hz, 3H), 1.21–1.42 (m, 6 H), 2.07 (q, J = 7.1 Hz, 2H), 2.44 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 6.9 Hz, 2H), 3.52 (t, J = 7.0 Hz, 2H), 4.55 (s, 2H), 5.32–5.51 (m, 3H), 7.25–7.38 (m, 5H). HRMS (CI, M + 1) for C18H36 2HOSi calculated 298.2676, found 298.2668.

(3-2H-7-2H)Dodeca-3,6-dienyloxymethyl-benzene (11b)

The procedure described for the synthesis of 11a was followed using phosphonium salt 9 (246 mg, 0.52 mmol) and aldehyde 10a (90.2 mg, 0.55 mmol). After purification by column chromatography (5% ether in pentane), 11b (145.5 mg, 97%) was obtained as a clear oil. 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 3H), 1.22–1.39 (m, 6H), 2.04 (bt, J = 7.1 Hz, 2H), 2.41 (d, J = 7.0 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H), 3.49 (t, J = 7.1 Hz, 2H), 4.53 (s, 2H), 5.33 (tt, J = 1.5 Hz, 7.3 Hz, 1H), 5.45 (tt, J = 1.5 Hz, 7.3 Hz, 1H), 7.25–7.38 (m, 5H). 13C NMR (100.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.3 (CH2), 28.1 (CH2), 29.5 (CH2), 31.8 (CH2), 70.1 (CH2), 73.1 (CH2), 127.7 (CH), 127.8 (CH), 127.9 (CH), 128.6 (CH), 130.3 (CH), 138.7 (Cq). IR (cm−1): 3029, 2928, 2855, 2355, 2323, 2221, 1633, 1454, 1362. HRMS (EI, M +) for C19H26 2H2O calculated 274.2266, found 274.2270.

tert-Butyl-dodeca-3,6-(7-2H)dienyloxy-dimethylsilane (11c)

A dry flask was charged with phosphonium salt 9 (521.3 mg, 1.11 mmol) and purged with N2. THF (25 ml) was added and the reaction was cooled to −78°C. A 1.0M solution of NaHMDS (1.0 ml, 1.00 mmol) was added dropwise and the reaction was stirred for 1 h. A solution of the aldehyde 10b (291.2 mg, 1.55 mmol) in THF (2 ml) was added slowly over 30 min. The reaction was stirred at −78°C for 3 h, and then slowly warmed to room temperature. After an additional 10 h, the reaction was quenched with water (40 ml) and extracted with ether (3 × 50 ml). The combined organic layers were washed with water (1× 10 ml), dried with MgSO4, filtered and concentrated. The resulting oil was purified by flash chromatography (3% ether in pentane) yielding 11c (274.0 mg, 92%) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.06 (s, 6H), 0.90 (s, 9H), 1.24–1.39 (m, 6H), 2.04 (t, J = 7.1 Hz, 2H), 2.30 (q, J = 6.8 Hz, 2H), 2.79 (t, J = 6.9 Hz, 2 H), 3.62 (t, J = 7.0 Hz, 2H), 5.33 (t, J = 7.2 Hz, 1H), 5.36–5.46 (m, 2H). 13C NMR (125.6 MHz, CDCl3) δ −5.1 (CH3), 14.3 (CH3), 18.6 (Cq), 22.8 (CH2), 25.9 (CH2), 26.2 (CH3), 27.3 (CH2), 29.5 (CH2), 31.3 (CH2), 31.7 (CH2), 63.1 (CH2), 126.0 (CH), 127.8 (CH), 130.3 (CH). IR (cm−1): 3014, 2957, 2929, 2858, 1472. HRMS (CI, M + 1) for C18H36 2HOSi calculated 298.2676, found 298.2668.

tert-Butyl-dodeca-3,6-dienyloxy-dimethylsilane (11d)

The procedure described for the synthesis of 11c was followed using phosphonium salt 9a (819.4 mg, 1.75 mmol) and aldehyde 10b (291.2 mg, 1.55 mmol). After purification by column chromatography (3% ether in pentane), 11d (345.1 mg, 89%) was obtained as a clear oil. 1H NMR (400 MHz, CDCl3) δ 0.06 (s, 6H), 0.88–0.91 (m, 12H), 1.24–1.39 (m, 6H), 2.05 (q, J = 7.0 Hz, 2H), 2.30 (q, J = 6.8 Hz, 2H), 2.80 (t, J = 6.4 Hz, 2H), 3.61 (t, J = 7.0 Hz, 2H), 5.29–5.47 (m, 4H). 13C NMR (100.6 MHz, CDCl3) δ −5.0 (CH3), 14.3 (CH3), 18.6 (Cq), 22.8 (CH2), 26.0 (CH2), 26.2 (CH3), 27.4 (CH2), 29.6 (CH2), 31.4 (CH2), 31.8 (CH2), 63.1 (CH2), 126.0 (CH), 127.9 (CH), 130.2 (CH), 130.6 (CH).

(3-2H)Dodeca-3,6-dien-1-ol (12a)

A dry flask was charged with the protected alcohol 11a (106.8 mg, 0.390 mmol) and purged with N2. CH2Cl2 (8 ml) was added and the solution was cooled to −78°C. A 1.0M solution of boron tribromide in CH2Cl2 (390 μl, 0.390 mmol) was added dropwise. After 15 min, an additional 100 μl of the 1.0M solution of BBr3 was added, and the reaction stirred at −78°C. After 10 min, the reaction was quenched with methanol (1 ml) and the reaction warmed to room temperature. A saturated solution of NaHCO3 in water (5 ml, aq) was added and the aqueous layer extracted with ether (4 × 30 ml). The combined organic layers were dried with MgSO4, filtered and concentrated. The resulting oil was purified by flash chromatography (25% ether in pentane) yielding 12a (65.0 mg, 91%) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.22–1.38 (m, 6H), 1.59 (s, 1H), 2.04 (q, J = 7.2 Hz, 2H), 2.35 (t, J = 6.5 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 3.65 (t, J = 6.5 Hz, 2H), 5.28–5.34 (m, 1H), 5.37–5.43 (m, 1H), 5.53 (t, J = 7.4 Hz, 1H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.4 (CH2), 29.5 (CH2), 30.9 (CH2), 31.7 (CH2), 62.4 (CH2), 125.2 (t, J = 23.5 Hz, CD), 127.6 (CH), 130.9 (CH), 131.6 (CH). IR (cm−1): 3339 (b), 3012, 2927, 2857, 1654, 1636, 1458. HRMS (CI, M + 1) for C12H21DO calculated 183.1733, found 183.1735.

(3-2H-7-2H)Dodeca-3,6-dien-1-ol (12b)

The procedure described for the synthesis of 12a was followed using compound 11b. Purification by column chromatography (30% ether in pentane), yielded 12b (40.0 mg, 57%) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.24–1.37 (m, 6H), 1.51 (bs, 1H), 2.04 (t, J = 7.2 Hz, 2H), 2.35 (t, J = 6.4 Hz, 2H), 2.81 (t, J = 7.1 Hz, 2H), 3.65 (t, J = 6.5 Hz, 2H), 5.32 (tt, J = 1.4 Hz, 7.3 Hz, 1H), 5.54 (tt, J = 1.4 Hz, 7.4 Hz, 1H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.3 (CH2), 29.5 (CH2), 30.9 (CH2), 31.7 (CH2), 62.4 (CH2), 125.2 (t, J = 23.0 Hz, CD), 127.5 (s, CH), 130.5 (t, J = 23.5 Hz, CD), 131.7 (s, CH). IR (cm−1): 3339 (b), 2957, 2929, 2858, 1458. HRMS (EI, M+) for C12H20D2O calculated 184.1796, found 184.1794.

(7-2H)Dodeca-3,6-dien-1-ol (12c)

A dry flask was charged with protected alcohol 11c (258.1 mg, 0.86 mmol) and purged with N2. CH2Cl2 (5 ml) was added and the reaction was cooled to 0°C. A 1.0M solution of TBAF in THF (1.20 ml, 1.20 mmol) was then added dropwise. The reaction was stirred at 0°C for 15 min, then warmed to room temperature. After an additional 24 h, the solvent was removed under vacuum. The resulting oil was purified by flash chromatography (25% ether in pentane) yielding 12c (155.0 mg, 98%) as a clear oil. 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 3H), 1.24–1.41 (m, 6H), 2.04 (t, J = 7.1 Hz, 2H), 2.36 (q, J = 6.9 Hz, 2H), 2.82 (t, J = 7.3 Hz, 2H), 3.66 (q, J = 6.2 Hz, 2H), 5.32 (tt, J = 1.4 Hz, 7.3 Hz, 1H), 5.35–5.43 (m, 1H), 5.52–5.58 (m, 1H). 13C NMR (100.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.3 (CH2), 29.5 (CH2), 31.0 (CH2), 31.7 (CH2), 62.5 (CH2), 125.5 (CH), 127.4 (CH), 131.8 (CH).

Dodeca-3,6-dien-1-ol (12d)

The procedure described for the synthesis of 12c was followed using compound 11d. After purification by column chromatography (30% ether in pentane), 12d was obtained as a clear oil (quant., 127.7 mg). 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.23–1.46 (m, 6H), 2.05 (q, J = 7.1 Hz, 2H), 2.36 (q, J = 6.9 Hz, 2H), 2.82 (t, J = 7.1 Hz, 2H), 3.65 (q, J = 6.3 Hz, 2H), 5.28–5.43 (m, 3H), 5.51–5.57 (m, 1H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.4 (CH2), 29.5 (CH2), 31.0 (CH2), 31.7 (CH2), 62.4 (CH2), 125.5 (CH), 127.6 (CH), 130.9 (CH), 131.8 (CH).

Phosphonium salt of 1-bromo-(3-2H)dodeca-3,6-diene (5a)

The synthesis of phosphonium salt 5a was based on a previously reported synthetic scheme.12

Phosphonium salt of 1-bromo-(3-2H-7-2H)non-3-ene (5b)

The procedure described for the synthesis of (3-2H)-1-bromo-dodeca-3,6-diene12 was followed using compound (12b). After purification by column chromatography (100% pentane), 1-bromo-(3-2H-7-2H)dodeca-3,6-diene was obtained in 83% yield (41.7 mg) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.89 (t, J = 6.9 Hz, 3H), 1.24–1.39 (m, 6H), 2.04 (t, J = 7.1 Hz, 2H), 2.65 (t, J = 7.1 Hz, 2H), 2.79 (t, J = 7.3 Hz, 2H), 3.37 (t, J = 7.2 Hz, 2H), 5.32 (tt, J = 1.4 Hz, 7.3 Hz, 1H), 5.52 (tt, J = 1.4 Hz, 7.4 Hz, 1H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.9 (CH2), 27.3 (CH2), 29.5 (CH2), 30.9 (CH2), 31.7 (CH2), 32.6 (CH2), 125.9 (t, J = 24.0 Hz, CD), 127.2 (s, CH), 130.7 (t, J = 23.0 Hz, CD), 131.4 (s, CH).

1-Bromo-(3-2H-7-2H)dodeca-3,6-diene was converted to phosphonium salt 5b as reported previously.12 After purification by column chromatography, 5b was obtained in 84% yield (65.9 mg) as a gummy oil. 1H NMR (500 MHz, CDCl3) δ 0.80 (t, J = 7.2 Hz, 3H), 1.10–1.24 (m, 6H), 1.80 (t, J = 7.1 Hz, 2H), 2.37–2.43 (m, 2H), 2.47 (t, J = 7.4 Hz, 2H), 3.76–3.83 (m, 2H), 5.08 (t, J = 7.1 Hz, 1H), 5.30 (t, J = 7.4 Hz, 1H), 7.63–7.67 (m, 6H), 7.72–7.82 (m, 9H). 13C NMR (125.6 MHz, CDCl3) δ 14.2 (CH3), 20.5 (d, J = 3.8 Hz, CH2), 22.7 (CH2), 23.1 (d, J = 47.9 Hz, CH2), 25.6 (CH2), δ 27.2 (CH2), 29.3 (CH2), 31.6 (CH2), 118.3 (d, J = 85.6 Hz, Cq), 126.7 (CH), 130.7 (CH), 130.8 (d, J = 12.8 Hz, CH), 133.9 (d, J = 10.2 Hz, CH), 135.3 (d, J = 2.8 Hz, CH). 31P NMR (202.3 MHz, CDCl3) δ 25.2 (s).

Phosphonium salt of 1-bromo-(7-2H)dodeca-3,6-diene (5c)

The procedure described for the synthesis of 1-bromo-(3-2H)dodeca-3,6-diene12 was followed using compound 12c. After purification by column chromatography (100% pentane), 1-bromo-(7-2H)dodeca-3,6-diene was obtained in 88% yield (71.5 mg) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 3H), 1.24–1.39 (m, 6H), 2.04 (t, J = 7.2 Hz, 2H), 2.65 (q, J = 7.2 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H), 3.38 (t, J = 7.1 Hz, 2H), 5.32 (t, J = 7.3 Hz, 1H), 5.35–5.42 (m, 1H), 5.48–5.55 (m, 1H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 26.0 (CH2), 27.3 (CH2), 29.5 (CH2), 31.0 (CH2), 31.7 (CH2), 32.6 (CH2), 126.3 (CH), 127.2 (CH), 130.7 (t, J = 23.5 Hz, CD), 131.5 (CH). IR (cm−1): 3015, 2958, 2928, 2856, 1458, 1435. HRMS (CI, M + 1) for C12H20DBr calculated 245.0889, found 245.0884.

(7-2H)-1-Bromo-dodeca-3,6-diene was converted to phosphonium salt 5c as reported previously.12 After purification by column chromatography, 5c (132.2 mg, 92%) was obtained as a gummy oil. 1H NMR (400 MHz, CDCl3) δ 0.79 (t, J = 7.1 Hz, 3H), 1.10–1.25 (m, 6H), 1.80 (q, J = 7.1 Hz, 2H), 2.36–2.43 (m, 2H), 2.47 (t, J = 7.3 Hz, 2H), 3.65–3.73 (m, 2H), 5.04–5.12 (m, 1H), 5.22–5.28 (m, 1H), 5.31 (t, J = 7.4 Hz, 1H), 7.63–7.81 (m, 15H). 13C NMR (100.6 MHz, CDCl3) δ 14.3 (CH3), 20.4 (d, J = 3.7 Hz, CH2), 22.7 (CH2), 23.0 (d, J = 48.8 Hz, CH2), 25.7 (CH2), 27.3 (CH2), 29.3 (CH2), 31.6 (CH2), 118.2 (d, J = 86.2 Hz, CH), 126.8 (CH), 130.79 (d, J = 12.9 Hz, CH), 130.81 (CH), 131.1 (CH), 133.8 (d, J = 10.8 Hz, CH), 135.4 (d, J = 3.1 Hz, CH). 31P NMR (161.9 MHz, CDCl3) δ 25.0 (s). HRMS (ESI, M-Br) for C30H35DP calculated 428.2617, found 428.2639.

Phosphonium salt of 1-bromo-dodeca-3,6-diene (5d)

The synthesis of phosphonium salt 5d was based on a previously reported synthetic scheme.29

Methyl [1-3H]8-oxo-oct-5-enoate (6)

A dry flask was charged with methyl 8-oxo-oct-5-enoate11,12 (54.6 mg, 0.32 mmol) and purged with N2. THF (10 ml) was added and the solution was cooled to 0°C. Cerium chloride (97.4 mg, 0.38 mmol) was added in one portion and the reaction was stirred for 45 min, then the [T]NaBH4 (6.1 mg, 0.16 mmol, ~200 mCi/mmol) was added. The reaction was stirred at 0°C for 4 h, and then slowly warmed to room temperature overnight. After 10 h, the reaction was quenched with water (10 ml) and extracted with ether (6 × 30 ml). The combined organic layers were dried with MgSO4, filtered and concentrated. The resulting oil was used without further purification.

The labeled alcohol was purged with N2, and CH2Cl2 (10 ml) was added. The solution was cooled to 0°C and then Dess-Martin periodinane (275.6 mg, 0.64 mmol) was added in one portion. The reaction was stirred at 0°C for 15 min, and then slowly warmed to room temperature. After an additional 1 h, the reaction was quenched with water (20 ml) and extracted with pentane (6 × 30 ml). The combined organic layers were washed with water (4 × 20 ml), dried with MgSO4, filtered and concentrated yielding 37.9mg (70%) of 6 as a clear oil. The labeled aldehyde was used without further purification. 1H NMR (100.6 MHz, CDCl3) δ 1.71 (quintet, J = 7.3 Hz, 2H), 2.09 (q, J = 7.3 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 3.18–3.21 (m, 2H), 3.67 (s, 3H), 5.56–5.72 (m, 2H), 9.67 (t, J = 1.8 Hz, 1H). 1H NMR spectroscopy indicates only trace α,β-unsaturated aldehyde present (<5%).

Methyl [8-3H]-(11-2H)arachidonate (13a)

A dry flask was charged with phosphonium salt 5a (39.0 mg, 0.077 mmol) and dried by azeotroping with benzene (3 × 10 ml). THF (5 ml) was added and the solution was cooled to −78°C. A 1.0M solution of NaHMDS in THF (65 μl, 0.065 mmol) was added dropwise and the reaction was stirred for 1 h. A solution of aldehyde 6 (10.1 mg, 0.059 mmol) in THF (500 μl) was added dropwise over ~15 min. The reaction was stirred at −78°C for 3 h, and then slowly warmed to room temperature. After an additional 9 h, the reaction was quenched with water (10 ml) and extracted with ether (5 × 30 ml). The combined organic layers were dried with MgSO4, filtered and concentrated. The resulting oil was purified by flash chromatography (3% ether in pentane) yielding 13a (7.3 mg, 35%) as a clear oil. 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.24–1.39 (m, 6H), 1.70 (p, J = 7.4 Hz, 2H), 2.05 (q, J = 7.0Hz, 2H), 2.11 (q, J = 6.8Hz, 2H), 2.32 (t, J = 7.5Hz, 2H), 2.77–2.85 (m, 6H), 3.66 (s, 3H), 5.29–5.44 (m, 7H). 13C NMR (100.6MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.0 (CH2), 25.82 (CH2), 25.83 (CH2), 26.8 (CH2), 27.4 (CH2), 29.6 (CH2), 31.7 (CH2), 33.7 (CH2), 51.7 (CH3), 127.8 (CH), 128.38 (CH), 128.41 (CH), 128.7 (CH), 129.09 (CH), 129.15 (CH), 130.7 (CH), 174.3 (Cq).

Methyl [8-3H]-(11,15-2H2)arachidonate (13b)

The procedure described for the synthesis of 13a was followed using phosphonium salt 5b (140.3 mg, 0.275 mmol) and aldehyde 6 (25.3 mg, 0.148 mmol). After purification by column chromatography (3% ether in pentane), 13b (25.8 mg, 54%) was obtained as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H), 1.24–1.38 (m, 6H), 1.71 (p, J = 7.4 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 2.11 (q, J = 7.0 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 2.78–2.85 (m, 6H), 3.67 (s, 3H), 5.31–5.43 (m, 6H). Specific radioactivity 44.8 mCi/mmol.

Methyl [8-3H]-(15-2H)arachidonate (13c)

The procedure described for the synthesis of 13a was followed using phosphonium salt 5c (41.6 mg, 0.081 mmol) and aldehyde 6 (10.1 mg, 0.059 mmol). After purification by column chromatography (3% ether in pentane), 13c (10.0 mg, 53%) was obtained as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 3H), 1.24–1.38 (m, 6H), 1.70 (p, J = 7.4 Hz, 2H), 2.05 (t, J = 7.3 Hz, 2H), 2.11 (q, J = 7.0 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 2.76–2.85 (m, 6H), 3.67 (s, 3H), 5.31–5.43 (m, 7H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 25.0 (CH2), 25.80 (CH2), 25.81 (CH2), 26.7 (CH2), 27.3 (CH2), 29.5 (CH2), 31.7 (CH2), 33.6 (CH2), 51.7 (CH3), 127.6 (CH), 128.1 (CH), 128.40 (CH), 128.43 (CH), 128.8 (CH), 129.1 (CH), 129.2 (CH), 174.3 (Cq).

Methyl [8-3H]arachidonate (13d)

The procedure described for the synthesis of 13a was followed using phosphonium salt 5d (68.9 mg, 0.135 mmol) and aldehyde 6 (12.6 mg, 0.074 mmol). After purification by column chromatography (3% ether in pentane), 13d (13.3 mg, 56%) was obtained as a clear oil. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.22–1.39 (m, 6H), 1.71 (p, J = 7.4 Hz, 2H), 2.05 (q, J = 7.2 Hz, 2H), 2.11 (q, J = 7.0 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 2.78–2.86 (m, 6H), 3.67 (s, 3H), 5.30–5.43 (m, 8H). Specific activity 45 mCi/mmol.

[8-3H]-(11-2H)Arachidonic acid (1)

A dry flask was charged with methyl arachidonate 13a (3.6 mg, 0.011 mmol) and purged with N2. THF (2 ml) was added and the solution was cooled to 0°C. A 1.0M solution of LiOH (aq) (500 μl) was added and the reaction was stirred for 1 h. The reaction was slowly warmed to room temperature and stirred for an additional 25 h. The reaction was quenched with a 1M HCl (aq) solution (2 ml) and extracted with ether (6 × 20 ml). The combined organic layers were dried with MgSO4, filtered and concentrated. The resulting oil was purified by flash chromatography (40% ether in pentane) yielding 1 (~4.0 mg, quant.) as a clear oil. The final compound was then further purified by HPLC using first a Varian Microsorb C18 column followed by a Chromsper 5 Lipids silver-ion column (both 250 × 4.6 mm). 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.24–1.38 (m, 6H), 1.72 (p, J = 7.4 Hz, 2H), 2.05 (q, J = 7.1 Hz, 2H), 2.14 (q, J = 7.1 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 2.79–2.85 (m, 6H), 5.30–5.44 (m, 6H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 24.7 (CH2), 25.7 (CH2), 25.8 (CH2), 26.6 (CH2), 27.4 (CH2), 29.5 (CH2), 30.5 (CH2), 31.7 (CH2), 33.2 (CH2), 127.8 (CH), 128.3 (CH), 128.5 (CH), 128.7 (CH), 129.0 (CH), 129.3 (CH), 130.7 (CH). Specific activity 96.9 mCi/mmol.

[8-3H]-(15-2H)Arachidonic acid (2)

The procedure described for the synthesis of 1 was followed using compound 13c (4.3 mg). After purification by column chromatography (40% ether in pentane), 2 (quant., ~5.2 mg) was obtained as a clear oil. The final compound was then purified by HPLC as described for 1. 1H NMR (500 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, CH3), 1.25–1.39 (m, 6H), 1.72 (p, J = 7.4 Hz, 2H), 2.05 (t, J = 7.2 Hz, 2H), 2.13 (q, J = 7.0 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 2.79–2.86 (m, 6H), 5.31–5.46 (m, 7H). 13C NMR (125.6 MHz, CDCl3) δ 14.3 (CH3), 22.8 (CH2), 24.7 (CH2), 25.8 (CH2), 25.8 (CH2), 26.6 (CH2), 27.3 (CH2), 29.5 (CH2), 30.5 (CH2), 31.7 (CH2), 33.2 (CH2), 127.6 (CH), 128.1 (CH), 128.3 (CH), 128.5 (CH), 128.8 (CH), 129.0 (CH), 129.3 (CH). Specific activity 97 mCi/mmol.

Conclusion

The syntheses of four deuterium-labeled arachidonic acid derivatives containing a C8 remote tritium label are described. These substrates can be used to measure the secondary kinetic isotope effect of both soybean lipoxygenase and PGHS to provide evidence for the structure of the initial radical intermediate formed during enzyme turnover.

Acknowledgments

Contract/grant sponsor: National Institutes of Health; contract/grant number: GM44911

Contract/grant sponsor: American Heart Association; contract/grant number: 0310006Z

This work was supported in part by grant GM44911 from the National Institutes of Health and by a predoctoral fellowship to CMM from the American Heart Association (#0310006Z). We thank Andrew LaFrate and Professor John Katzenellenbogen for their helpful suggestions and advice on the synthesis of radiolabeled compounds.

References

1. Smith WL, DeWitt DL, Garavito RM. Annu Rev Biochem. 2000;69:145–182. [PubMed]
2. van der Donk WA, Tsai AL, Kulmacz RJ. Biochemistry. 2002;41:15451–15458. [PubMed]
3. Rouzer CA, Marnett LJ. Chem Rev. 2003;103:2239–2304. [PubMed]
4. Smith WL, Langenbach R. J Clin Invest. 2001;107:1491–1495. [PMC free article] [PubMed]
5. Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Proc Natl Acad Sci USA. 1999;96:9966. [PubMed]
6. Pratico D, Tillmann C, Zhang Z-B, Li H, FitzGerald GA. Proc Natl Acad Sci USA. 2001;98:3358–3363. [PubMed]
7. Hamberg M, Samuelsson B. J Biol Chem. 1967;242:5336–5343. [PubMed]
8. Tsai AL, Hsi LC, Kulmacz RJ, Palmer G, Smith WL. J Biol Chem. 1994;269:5085–5091. [PubMed]
9. Tsai A-l, Kulmacz RJ, Palmer G. J Biol Chem. 1995;270:10503–10508. [PubMed]
10. Karthein R, Dietz R, Nastainczyk W, Ruf HH. Eur J Biochem. 1988;171:313–320. [PubMed]
11. Peng S, Okeley NM, Tsai A-L, Wu G, Kulmacz RJ, van der Donk WA. J Am Chem Soc. 2001;123:3609–3610. [PubMed]
12. Peng S, Okeley NM, Tsai A-L, Wu G, Kulmacz RJ, van der Donk WA. J Am Chem Soc. 2002;124:10785–10796. [PubMed]
13. Thuresson ED, Lakkides KM, Smith WL. J Biol Chem. 2000;275:8501–8507. [PubMed]
14. Schneider C, Brash AR. J Biol Chem. 2000;275:4743–4746. [PubMed]
15. Schneider C, Boeglin WE, Prusakiewicz JJ, Rowlinson SW, Marnett LJ, Samel N, Brash AR. J Biol Chem. 2002;277:478–485. [PubMed]
16. McGinley CM, van der Donk WA. Chem Commun. 2003:2843–2846. [PubMed]
17. Knapp MJ, Seebeck FP, Klinman JP. J Am Chem Soc. 2001;123:2931–2932. [PubMed]
18. Nelson MJ, Seitz SP, Cowling RA. Biochemistry. 1990;29:6897–6903. [PubMed]
19. Nelson MJ, Cowling RA, Seitz SP. Biochemistry. 1994;33:4966–4973. [PubMed]
20. Houk KN, Gustafson SM, Black KA. J Am Chem Soc. 1992;114:8565–8572.
21. Takada T, Dupuis M. J Am Chem Soc. 1983;105:1713–1716.
22. Gajewski JJ, Olson LP, Tupper KJ. J Am Chem Soc. 1993;115:4548–4553.
23. Wiseman JS. Biochemistry. 1989;28:2106–2111. [PubMed]
24. Peng S, McGinley CM, van der Donk WA. Org Lett. 2004;6:349–352. [PubMed]
25. Luche JL. J Am Chem Soc. 1978;100:2226–2227.
26. Wavrin L, Viala J. Synthesis. 2002:326–330.
27. Morikawa T, Nishiwaki T, Nakamura K, Kobayashi Y. Chem Pharm Bull. 1989;37:813–815.
28. Pommier A, Pons J-M, Kocienski PJ. J Org Chem. 1995;60:7334–7339.
29. Khanapure SP, Shi XX, Powell WS, Rokach J. J Org Chem. 1998;63:337–342.