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

Article abstract of DOI:10.1002/jlcr.1073

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 ra

Full text of DOI:10.1002/jlcr.1073

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 545–558.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1073
Research Article
Synthesis of site-specifically deuterated arachidonic
acid derivatives containing a remote tritium radiolabel
Chris M. McGinley and Wilfred A. van der Donk*
Roger Adams Laboratory, Department of Chemistry, University of Illinois, 600
S. Mathews Avenue, Urbana, IL 61801, USA
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. Copyright # 2006 John Wiley & Sons, Ltd.
Received 21 January 2006; Revised 28 March 2006; Accepted 28 March 2006
Key Words: 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.1–3 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 cardio-
vascular processes.6
*Correspondence to: W. van der Donk, Roger Adams Laboratory, Department of Chemistry, University of
Illinois, 600 S. Mathews Avenue, Urbana, Illinois, USA. E-mail: vddonk@uiuc.edu
Contract/grant sponsor: National Institutes of Health; contract/grant number: GM44911
Contract/grant sponsor: American Heart Association; contract/grant number: 0310006Z
Copyright # 2006 John Wiley & Sons, Ltd.

546
C. M. MCGINLEY AND W. A. VAN DER DONK
O
O
O
O
11
9
11
9
13
O2
[R]
15
HOO
HO
15
HO2C
HO2C
CO2H
Prostaglandin G2
(PGG2)
Prostaglandin H2
(PGH2)
Arachidonic acid
(AA)
Scheme 1. PGHS catalyzed reaction of arachidonic acid forming prostaglandin
H2
O
SHO
RH
9
RH HS
8
O
O
11
13
n-Bu
n-Bu
n-Bu
O2
3 COOH
3 COOH
COOH
3
I
PGG2
AA
•
O
O
O
O
O
n-Bu
O
O
O
n-Bu
n-Bu
O2
15
12
3 COOH
3 COOH
3
COOH
Scheme 2. Proposed mechanism for prostaglandin H synthase
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 2.8–10 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
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
547
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 C15,13–15 and for the lipoxygenases, which also abstract a
hydrogen atom from a bisallylic position.16–19
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).20–23 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 1.06–1.10,20–23 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
3
enzymatic product separated by HPLC. The ratio of 14C to H 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
D/H
kH/kD = 1.06-1.10
D/H
X
Scheme 3. Secondary kinetic isotope effect associated with generation of allyl
radical
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

548
C. M. MCGINLEY AND W. A. VAN DER DONK
O
14C
O
H [T]
OH
OH
vs
H/D
H/D
Figure 1. Competitive experiment for measuring secondary kinetic isotope
effects
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.
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.
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.
CO2Me
1) NaHMDS
O
PPh3Br
C5H12
H/D
C5H12
H/D
D/H
D/H
CO2Me
[T]H
5
6
1) NaHMDS
2) (1-2H)Hexanal
D
BrPh3P
OTBDMS
TBDMSO
C5H11
97%
8
7
1) TBAF
2) PPh3, Br2
3) PPh3
D
BrPh3P
C5H11
9
68%
Scheme 4. Synthetic approach for radiolabeled arachidonic acids and synthesis
of monodeuterated phosphonium salt 9
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
549
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 a,b-
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 transforma-
tions. [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 triphenylpho-
sphine. 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 correspond-
ing methyl ester with lithium aluminum deuteride followed by pyridinium
chlorochromate oxidation.
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
2
>98% H incorporation for 12c. Subsequent bromination and reaction with
triphenylphosphine produced the phosphonium salts 5. A final Wittig reaction
R1
R3
1) NaHMDS
O
O
BBr3 or TBAF
BrPh3P
R2
R1
R3
2)
R2
O
10a R2 = D, R3 = Bn
10b R2 = H, R3 = TBDMS
9a R1 = H
9 R2 = D
11a R1 = H, R2 = D, R3 = Bn, 80%
11b R1 = D, R2 = D, R3 = Bn, 97%
11c R1 = D, R2 = H, R3 = TBDMS, 92%
11d R1 = H, R2 = H, R3 = TBDMS, 89%
1) PPh3, Br2, Pyr
2) PPh3
PPh3Br
OH
R2
R1
R2
R1
5a R1 = H, R2 = D, 66%
5b R1 = D, R2 = D, 70%
5c R1 = D, R2 = H, 88%
5d R1 = H, R2 = H, 58%
12a R1 = H, R2 = D, 91%
12b R1 = D, R2 = D, 57%
12c R1 = D, R2 = H, 98%
12d R1 = H, R2 = H, >98%
Scheme 5. Synthesis of substituted phosphonium salts
Copyright # 2006 John Wiley & Sons, Ltd.
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550
C. M. MCGINLEY AND W. A. VAN DER DONK
(T)H
CO2Me
PPh3Br
1) NaHMDS
C5H12
2) 6
R2
R1
R2
R1
13a R1 = H, R2 = D, 35%
13b R1 = D, R2 = D, 54%
13c R1 = D, R2 = H, 53%
13d R1 = H, R2 = H, 56%
5a R1 = H, R2 = D
5b R1 = D, R2 = D
5c R1 = D, R2 = H
5d R1 = H, R2 = H
O
(T)H
1M LiOH
OH
R2
R1
1 R1 = H, R2 = D, >95%
2 R1 = D, R2 = H, >95%
Scheme 6. Synthesis of radiolabeled arachidonic acids
utilizing the phosphonium salts 5 and radiolabeled aldehyde 6 yielded methyl
arachidonates 13a-d (Scheme 6). Hydrolysis with a 1 M 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. Concentra-
tions 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.
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
ꢀ788C. A 1.0 M solution of NaHMDS in THF (600 ml, 0.60 mmol) was added
and the solution was stirred for 1 h at ꢀ788C. The substituted aldehyde 10a
(98.6 mg, 0.60 mmol) in 2 ml THF was added very slowly. The reaction was
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SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
551
stirred at ꢀ788C 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
1
(100% pentane) yielding 130.6 mg (80%) of 11a as a clear oil. H NMR
(400 MHz, CDCl3) d 0.91 (t, J ¼ 7:1 Hz, 3H), 1.21–1.42 (m, 6 H), 2.07 (q,
J ¼ 7:1 Hz, 2H), 2.44 (t, J ¼ 7:0 Hz, 2H), 2.83 (t, J ¼ 6:9 Hz, 2H), 3.52 (t,
J ¼ 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 C18H362HOSi 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) d 0.89 (t, J ¼ 7:0 Hz, 3H), 1.22–1.39 (m, 6H), 2.04 (bt,
J ¼ 7:1 Hz, 2H), 2.41 (d, J ¼ 7:0 Hz, 2H), 2.80 (t, J ¼ 7:3 Hz, 2H), 3.49 (t,
J ¼ 7:1 Hz, 2H), 4.53 (s, 2H), 5.33 (tt, J ¼ 1:5 Hz, 7.3 Hz, 1H), 5.45 (tt,
J ¼ 1:5 Hz, 7.3 Hz, 1H), 7.25–7.38 (m, 5H). 13C NMR (100.6 MHz, CDCl3) d
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 (cmꢀ1): 3029, 2928, 2855, 2355, 2323, 2221,
1633, 1454, 1362. HRMS (EI, M+) for C19H262H2O 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
ꢀ788C. A 1.0 M 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 ꢀ788C 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) d 0.06
(s, 6H), 0.90 (s, 9H), 1.24–1.39 (m, 6H), 2.04 (t, J ¼ 7:1 Hz, 2H), 2.30 (q,
J ¼ 6:8 Hz, 2H), 2.79 (t, J ¼ 6:9 Hz, 2 H), 3.62 (t, J ¼ 7:0 Hz, 2H), 5.33 (t,
J ¼ 7:2 Hz, 1H), 5.36–5.46 (m, 2H). 13C NMR (125.6 MHz, CDCl3) d ꢀ5.1
(CH3), 14.3 (CH3), 18.6 (Cq), 22.8 (CH2), 25.9 (CH2), 26.2 (CH3), 27.3 (CH2),
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C. M. MCGINLEY AND W. A. VAN DER DONK
29.5 (CH2), 31.3 (CH2), 31.7 (CH2), 63.1 (CH2), 126.0 (CH), 127.8 (CH), 130.3
(CH). IR (cmꢀ1): 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) d 0.06 (s, 6H), 0.88–0.91 (m, 12H), 1.24–1.39 (m, 6H), 2.05
(q, J ¼ 7:0 Hz, 2H), 2.30 (q, J ¼ 6:8 Hz, 2H), 2.80 (t, J ¼ 6:4 Hz, 2H), 3.61 (t,
J ¼ 7:0 Hz, 2H), 5.29–5.47 (m, 4H). 13C NMR (100.6 MHz, CDCl3) d ꢀ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
ꢀ788C. A 1.0 M solution of boron tribromide in CH2Cl2 (390 ml, 0.390 mmol)
was added dropwise. After 15 min, an additional 100 ml of the 1.0 M solution
of BBr3 was added, and the reaction stirred at ꢀ788C. 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) d 0.88 (t,
J ¼ 7:0 Hz, 3H), 1.22–1.38 (m, 6H), 1.59 (s, 1H), 2.04 (q, J ¼ 7:2 Hz, 2H), 2.35
(t, J ¼ 6:5 Hz, 2H), 2.81 (t, J ¼ 7:2 Hz, 2H), 3.65 (t, J ¼ 6:5 Hz, 2H), 5.28–
5.34 (m, 1H), 5.37–5.43 (m, 1H), 5.53 (t, J ¼ 7:4 Hz, 1H). 13C NMR
(125.6 MHz, CDCl3) d 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 ¼ 23:5 Hz, CD), 127.6
(CH), 130.9 (CH), 131.6 (CH). IR (cmꢀ1): 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
1
pentane), yielded 12b (40.0 mg, 57%) as a clear oil. H NMR (500 MHz,
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SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
553
CDCl3) d 0.88 (t, J ¼ 7:0 Hz, 3H), 1.24–1.37 (m, 6H), 1.51 (bs, 1H), 2.04 (t,
J ¼ 7:2 Hz, 2H), 2.35 (t, J ¼ 6:4 Hz, 2H), 2.81 (t, J ¼ 7:1 Hz, 2H), 3.65 (t,
J ¼ 6:5 Hz, 2H), 5.32 (tt, J ¼ 1:4 Hz, 7.3 Hz, 1H), 5.54 (tt, J ¼ 1:4 Hz, 7.4 Hz,
1H). 13C NMR (125.6 MHz, CDCl3) d 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 ¼ 23:0 Hz, CD), 127.5 (s, CH), 130.5 (t, J ¼ 23:5 Hz, CD), 131.7 (s, CH). IR
(cmꢀ1): 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 08C.
A 1.0 M solution of TBAF in THF (1.20 ml, 1.20 mmol) was then added
dropwise. The reaction was stirred at 08C 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) d 0.89
(t, J ¼ 7:0 Hz, 3H), 1.24–1.41 (m, 6H), 2.04 (t, J ¼ 7:1 Hz, 2H), 2.36 (q,
J ¼ 6:9 Hz, 2H), 2.82 (t, J ¼ 7:3 Hz, 2H), 3.66 (q, J ¼ 6:2 Hz, 2H), 5.32 (tt,
J ¼ 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) d 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) d 0.88 (t, J ¼ 7:0 Hz, 3H), 1.23–1.46 (m, 6H), 2.05 (q,
J ¼ 7:1 Hz, 2H), 2.36 (q, J ¼ 6:9 Hz, 2H), 2.82 (t, J ¼ 7:1 Hz, 2H), 3.65 (q,
J ¼ 6:3 Hz, 2H), 5.28–5.43 (m, 3H), 5.51–5.57 (m, 1H). 13C NMR (125.6 MHz,
CDCl3) d 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 ð12bÞ. After purification by column
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C. M. MCGINLEY AND W. A. VAN DER DONK
chromatography (100% pentane), 1-bromo-(3-2H-7-2H)dodeca-3,6-diene was
1
obtained in 83% yield (41.7 mg) as a clear oil. H NMR (500 MHz, CDCl3) d
0.89 (t, J ¼ 6:9 Hz, 3H), 1.24–1.39 (m, 6H), 2.04 (t, J ¼ 7:1 Hz, 2H), 2.65 (t,
J ¼ 7:1 Hz, 2H), 2.79 (t, J ¼ 7:3 Hz, 2H), 3.37 (t, J ¼ 7:2 Hz, 2H), 5.32 (tt,
J ¼ 1:4 Hz, 7.3 Hz, 1H), 5.52 (tt, J ¼ 1:4 Hz, 7.4 Hz, 1H). 13C NMR
(125.6 MHz, CDCl3) d 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 ¼ 24:0 Hz, CD), 127.2
(s, CH), 130.7 (t, J ¼ 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
1
was obtained in 84% yield (65.9 mg) as a gummy oil. H NMR (500 MHz,
CDCl3) d 0.80 (t, J ¼ 7:2 Hz, 3H), 1.10–1.24 (m, 6H), 1.80 (t, J ¼ 7:1 Hz, 2H),
2.37–2.43 (m, 2H), 2.47 (t, J ¼ 7:4 Hz, 2H), 3.76–3.83 (m, 2H), 5.08
(t, J ¼ 7:1 Hz, 1H), 5.30 (t, J ¼ 7:4 Hz, 1H), 7.63–7.67 (m, 6H), 7.72–7.82
(m, 9H). 13C NMR (125.6 MHz, CDCl3) d 14.2 (CH3), 20.5 (d, J ¼ 3:8 Hz,
CH2), 22.7 (CH2), 23.1 (d, J ¼ 47:9 Hz, CH2), 25.6 (CH2), d 27.2 (CH2), 29.3
(CH2), 31.6 (CH2), 118.3 (d, J ¼ 85:6 Hz, Cq), 126.7 (CH), 130.7 (CH), 130.8
(d, J ¼ 12:8 Hz, CH), 133.9 (d, J ¼ 10:2 Hz, CH), 135.3 (d, J ¼ 2:8 Hz, CH).
31P NMR (202.3 MHz, CDCl3) d 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 ob-
tained in 88% yield (71.5 mg) as a clear oil. 1H NMR (500 MHz, CDCl3) d 0.89
(t, J ¼ 7:0 Hz, 3H), 1.24–1.39 (m, 6H), 2.04 (t, J ¼ 7:2 Hz, 2H), 2.65 (q,
J ¼ 7:2 Hz, 2H), 2.80 (t, J ¼ 7:3 Hz, 2H), 3.38 (t, J ¼ 7:1 Hz, 2H), 5.32 (t,
J ¼ 7:3 Hz, 1H), 5.35–5.42 (m, 1H), 5.48–5.55 (m, 1H). 13C NMR (125.6 MHz,
CDCl3) d 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 ¼ 23:5 Hz,
CD), 131.5 (CH). IR (cmꢀ1): 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) d
0.79 (t, J ¼ 7:1 Hz, 3H), 1.10–1.25 (m, 6H), 1.80 (q, J ¼ 7:1 Hz, 2H), 2.36–2.43
(m, 2H), 2.47 (t, J ¼ 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 ¼ 7:4 Hz, 1H), 7.63–7.81 (m, 15H). 13C NMR
(100.6 MHz, CDCl3) d 14.3 (CH3), 20.4 (d, J ¼ 3:7 Hz, CH2), 22.7 (CH2), 23.0
(d, J ¼ 48:8 Hz, CH2), 25.7 (CH2), 27.3 (CH2), 29.3 (CH2), 31.6 (CH2), 118.2
(d, J ¼ 86:2 Hz, CH), 126.8 (CH), 130.79 (d, J ¼ 12:9 Hz, CH), 130.81 (CH),
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
555
131.1 (CH), 133.8 (d, J ¼ 10:8 Hz, CH), 135.4 (d, J ¼ 3:1 Hz, CH). 31P NMR
(161.9 MHz, CDCl3) d 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 08C. 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, ꢂ200 mCi/mmol) was added. The reaction was stirred at 08C 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 08C and then Dess-Martin periodinane (275.6 mg,
0.64 mmol) was added in one portion. The reaction was stirred at 08C 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 37.9 mg (70%) of 6 as a
clear oil. The labeled aldehyde was used without further purification. 1H NMR
(100.6 MHz, CDCl3) d 1.71 (quintet, J ¼ 7:3 Hz, 2H), 2.09 (q, J ¼ 7:3 Hz,
2H), 2.32 (t, J ¼ 7:4 Hz, 2H), 3.18–3.21 (m, 2H), 3.67 (s, 3H), 5.56–5.72 (m,
2H), 9.67 (t, J ¼ 1:8 Hz, 1H). 1H NMR spectroscopy indicates only trace a,b-
unsaturated aldehyde present (55%).
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 ꢀ788C. A 1.0 M solution of NaHMDS in THF (65 ml,
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 ml) was added
dropwise over ꢂ15 min. The reaction was stirred at ꢀ788C 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.
Copyright # 2006 John Wiley & Sons, Ltd.
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556
C. M. MCGINLEY AND W. A. VAN DER DONK
The resulting oil was purified by flash chromatography (3% ether in pentane)
1
yielding 13a (7.3 mg, 35%) as a clear oil. H NMR (400 MHz, CDCl3) d 0.88
(t, J ¼ 7:0 Hz, 3H), 1.24–1.39 (m, 6H), 1.70 (p, J ¼ 7:4 Hz, 2H), 2.05 (q, J ¼
7:0 Hz, 2H), 2.11 (q, J ¼ 6:8 Hz, 2H), 2.32 (t, J ¼ 7:5 Hz, 2H), 2.77–2.85 (m,
6H), 3.66 (s, 3H), 5.29–5.44 (m, 7H). 13C NMR (100.6 MHz, CDCl3) d 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) d 0.88 (t, J ¼ 7:1 Hz, 3H), 1.24–1.38 (m, 6H), 1.71 (p, J ¼ 7:4 Hz, 2H),
2.05 (t, J ¼ 7:4 Hz, 2H), 2.11 (q, J ¼ 7:0 Hz, 2H), 2.32 (t, J ¼ 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) d 0.89 (t, J ¼ 7:0 Hz, 3H), 1.24–1.38 (m, 6H), 1.70 (p, J ¼ 7:4 Hz, 2H),
2.05 (t, J ¼ 7:3 Hz, 2H), 2.11 (q, J ¼ 7:0 Hz, 2H), 2.32 (t, J ¼ 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) d 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) d 0.88 (t, J ¼ 7:0 Hz, 3H), 1.22–1.39 (m, 6H), 1.71 (p, J ¼ 7:4 Hz, 2H),
2.05 (q, J ¼ 7:2 Hz, 2H), 2.11 (q, J ¼ 7:0 Hz, 2H), 2.32 (t, J ¼ 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.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF TRITIUM-LABELED ARACHIDONIC ACIDS
557
[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
08C. A 1.0 M solution of LiOH (aq) (500 ml) 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 1 M 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 (ꢂ4.0 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) d 0.88 (t,
J ¼ 7:0 Hz, 3H), 1.24–1.38 (m, 6H), 1.72 (p, J ¼ 7:4 Hz, 2H), 2.05 (q,
J ¼ 7:1 Hz, 2H), 2.14 (q, J ¼ 7:1 Hz, 2H), 2.37 (t, J ¼ 7:5 Hz, 2H), 2.79–2.85
(m, 6H), 5.30–5.44 (m, 6H). 13C NMR (125.6 MHz, CDCl3) d 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., ꢂ5.2 mg) was obtained as a clear oil. The final compound
was then purified by HPLC as described for 1. 1H NMR (500 MHz, CDCl3) d
0.89 (t, J ¼ 7:0 Hz, CH3), 1.25–1.39 (m, 6H), 1.72 (p, J ¼ 7:4 Hz, 2H), 2.05 (t,
J ¼ 7:2 Hz, 2H), 2.13 (q, J ¼ 7:0 Hz, 2H), 2.37 (t, J ¼ 7:5 Hz, 2H), 2.79–2.86
(m, 6H), 5.31–5.46 (m, 7H). 13C NMR (125.6 MHz, CDCl3) d 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 contain-
ing 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.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 545–558
DOI: 10.1002/jlcr

558
C. M. MCGINLEY AND W. A. VAN DER DONK
Acknowledgements
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.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 545–558
DOI: 10.1002/jlcr