Synthesis of Isotopically Labeled Arachidonic Acids
A R T I C L E S
44%): 1H NMR (500 MHz, CDCl3) δ 5.41-5.37 (8H, m), 3.68 (3H,
s), 2.87-2.81 (5H, m), 2.34 (2H, t, J ) 7.5 Hz), 2.15-2.05 (4H, m),
1.72 (2H, quin, J ) 7.7 Hz), 1.41-1.25 (6H, m), 0.91 (3H, t, J ) 7.0
Hz); 13C NMR (125 MHz) δ 174.31, 130.74, 129.16, 129.11, 128.76,
128.44, 128.40, 128.11, 127.72, 51.70, 33.65, 31.74, 29.54, 27.43, 26.76,
25.85, 25.83, 25.53 (t, J ) 19.3 Hz), 25.00, 22.79, 14.28; FIMS (m/z)
319 (M+, 100). Methyl 13(R)-[2H]-arachidonate (7 mg, 0.022 mmol)
was dissolved in THF (0.37 mL). To this solution was added a 1 M
aqueous solution of LiOH (0.37 mL, 0.47 mmol) at 0 °C, and the
reaction was stirred for 15 h at room temperature. The solution was
acidified to pH 1 with 1 M aqueous HCl, saturated with solid sodium
chloride, and extracted with Et2O (3 × 50 mL). After the combined
organic phases were washed with brine, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure, purification by silica
gel chromatography (Rf 0.2, hexane:EtOAc ) 10:1) gave 13(R)-[2H]-1
(5.4 mg, 81%): 1H NMR (500 MHz, CDCl3) δ 5.46-5.34 (8H, m),
2.87-2.82 (5H, m), 2.39 (2H, t, J ) 7.4 Hz), 2.16 (2H, dt, J ) 6.8,
6.8 Hz), 2.07 (2H, dt, J ) 7.1, 7.1 Hz), 1.74 (2H, quin, J ) 7.5 Hz);
MS (ESI) (m/z) 304 (M+ - 1, 100); HRMS (ESI) (m/z) calcd for C20H30-
DO2 304.2387, found 304.2402; deuterium content 93% as determined
by ESI-MS comparison with unlabeled arachidonic acid.
sulfate, concentrated, and passed through a silica gel column (Rf )
0.5, hexane:EtOAc ) 100:5) to provide methyl 13(S)-[2H]-arachidonate
(15 mg, 40%) as a colorless oil: 1H NMR (500 MHz, CDCl3) δ 5.41-
5.37 (8H, m), 3.68 (3H, S), 2.87-2.81 (5H, m), 2.34 (2H, t, J ) 7.5
Hz), 2.15-2.05 (4H, m), 1.72 (2H, quin, J ) 7.7 Hz), 1.41-1.25 (6H,
m), 0.91 (3H, t, J ) 7.0 Hz). To a solution of methyl 13(S)-[2H]-
arachidonate (9 mg, 0.47 mmol) in THF (0.47 mL) was added a 1 M
aqueous solution of lithium hydroxide (0.47 mL, 0.47 mmol) at 0 °C.
After being stirred at room temperature for 22 h, the mixture was
acidified to pH 1 with 1 M HCl and saturated with solid sodium
chloride. Extraction with Et2O (4 × 20 mL), drying over anhydrous
sodium sulfate, concentration under reduced pressure, and chromatog-
raphy on silica gel (Rf ) 0.2, hexane:EtOAc ) 10:1) gave 13(S)-[2H]-
1 (8 mg, 93%): 1H NMR (500 MHz, CDCl3) δ 5.460-5.335 (8H, m),
2.869-2.819 (5H, m), 2.390 (2H, t, J ) 7.4 Hz), 2.158 (2H, dt, J )
6.8, 6.8 Hz), 2.067 (2H, dt, J ) 7.1, 7.1 Hz), 1.741 (2H, quin, J ) 7.5
Hz); MS (ESI) (m/z) 304 (M+ - 1); deuterium content 93% as
determined by ESI-MS comparison with unlabeled arachidonic acid.
11-[2H]-Arachidonic Acid. (3-[2H]-Dodeca-3,6-cis,cis-dienyl)tri-
phenylphosphonium bromide (21) (200 mg, 0.4 mmol) was thoroughly
dried by azeotropic distillation with anhydrous benzene (4 × 15 mL)
and diluted with THF (4 mL). A 1 M solution of NaHMDS in THF
(0.37 mL, 0.37 mmol) was added at -70 °C, and the bright orange
ylide was stirred for 1 h. After the mixture was cooled to -100 °C, a
solution of 8-oxo-oct-5-cis-enoic acid methyl ester (70 mg, 0.4 mmol)
in THF (1 mL) was added. The reaction mixture was warmed to 0 °C
over 5 h, and saturated aqueous ammonium chloride was added to
quench the reaction. After workup and silica gel chromatography (Rf
0.5, hexane:EtOAc ) 100:5), methyl 11-[2H]-arachidonate (85 mg,
67%) was obtained as a colorless oil: 1H NMR (500 MHz, CDCl3) δ
5.32-5.45 (7H, m), 3.68 (3H, s), 2.82-2.86 (6H, m), 2.34 (2H, t, J )
7.5 Hz), 2.13 (2H, dd, J ) 7.0 Hz, 7.0 Hz), 2.07 (2H, dd, J ) 7.1 Hz,
7.1 Hz), 1.73 (2H, quin, J ) 7.3 Hz), 1.26-1.41 (6H, m), 0.91 (3H, t,
J ) 7.0 Hz); 13C NMR (125 MHz) δ 174.32, 130.715, 129.16, 129.11,
128.67, 128.42, 128.40, 127.78, 51.70, 33.64, 31.74, 30.52, 29.55, 27.44,
26.77, 25.81, 25.73, 24.99, 22.79, 14.29. To a solution of methyl 11-
[2H]-arachidonate (20 mg, 0.06 mmol) in THF (1 mL) was added 1 M
lithium hydroxide in H2O (1 mL, 1 mmol) at 0 °C. After the reaction
was stirred at room temperature for 15 h, HCl (1 N) was added to
adjust the pH to 1. Extraction with Et2O (4 × 30 mL), drying over
Na2SO4, concentration, and chromatography on silica gel (Rf 0.15,
hexane:EtOAc ) 100:10) provided the compound as a colorless oil
(19 mg, 99%): 1H NMR (400 MHz, CDCl3) δ 5.32-5.47 (7H, m),
2.81-2.86 (6H, m), 2.39 (2H, t, J ) 7.5 Hz), 2.15 (2H, dd, J ) 7.0
Hz, 7.0 Hz), 2.07 (2H, dd, J ) 7.0 Hz, 7.0 Hz), 1.73 (2H, quin, J )
7.5 Hz), 1.27-1.42 (6H, m), 0.91 (3H, t, J ) 7.0 Hz); MS (ESI) (m/z)
304 (M+ - 1); HRMS (ESI) (m/z) calcd for C20H30DO2 304.2387, found
304.2361; deuterium content >97% as determined by ESI-MS com-
parison with unlabeled arachidonic acid.
13(R),15-[2H2]-Arachidonic Acid. 5 (116 mg, 0.22 mmol) was
thoroughly dried by four cycles of azeotropic evaporation of anhydrous
benzene and dissolved in THF (2 mL). After the solution was cooled
at -70 °C, a 1 M solution of NaHMDS (0.204 mL, 0.204 mmol) in
THF was added, and the reaction was stirred for 1 h at -70 °C and
then cooled to -90 °C. 2(R),4-[2H]2-Non-3-cis-enal 6c (11 mg, 0.14
mmol) dissolved in THF (1 mL) was added to the solution of the ylide.
After 0.5 h at -90 °C, the reaction was allowed to warm to 0 °C over
5 h. Saturated aqueous sodium bicarbonate was added, and the aqueous
layer was extracted with Et2O (3 × 30 mL). The combined organic
phases were washed with brine, dried over anhydrous sodium sulfate,
concentrated, and purified by silica gel chromatography (Rf 0.5, hexane:
EtOAc ) 100:5) to provide methyl 13(R),15-[2H]2-arachidonate as a
light yellow oil (10 mg, 40%): 1H NMR (500 MHz, CDCl3) δ 5.41-
5.37 (7H, m), 3.69(3H, s), 2.86-2.81 (5H, m), 2.34 (2H, t, J ) 6.7
Hz), 2.15-2.11 (2H, m), 2.07 (2H, t, J ) 7.0 Hz), 1.73 (2H, quin, J )
7.7 Hz), 1.41-1.24 (6H, m), 0.90 (3H, t, J ) 7.2 Hz); 13C NMR (125
MHz, CDCl3) δ 174.33, 129.17, 129.11, 128.77, 128.45, 128.39, 128.11,
127.59, 51.72, 33.65, 31.74, 29.52, 27.33, 26.76, 25.84, 25.82, 24.99,
22.79, 14.29. To a solution of methyl 13(R),15-[2H]2-arachidonate (10
mg, 0.031 mmol) in THF (0.57 mL) was added a 1 M aqueous solution
of LiOH (0.57 mL, 0.57 mmol) at 0 °C. After being stirred for 18 h at
room temperature, the solution was acidified to pH 1 with 1 M HCl,
saturated with solid NaCl, and extracted with Et2O (3 × 50 mL). After
the combined organic phases were washed with brine, dried over
anhydrous sodium sulfate, and concentrated under reduced pressure,
silica gel chromatography (Rf 0.2, hexane:EtOAc ) 100:10) gave 13-
(R),15-[2H]2-1 (7 mg, 73%): 1H NMR (500 MHz, CDCl3) δ 5.46-
5.35 (7H, m), 2.88-2.79 (5H, m), 2.40 (2H, t, J ) 7.2 Hz), 2.17-2.13
(2H, m), 2.70 (2H, t, J ) 7.2 Hz), 1.74 (2H, quin, J ) 7.3 Hz), 1.39-
1.27 (6H, m), 0.90 (3H, t, J ) 7.0 Hz); ESIMS (m/z) 305 (M+ - 1,
Determination of the Stereochemical Purity of Labeled Arachi-
donic Acids. The assays were carried out at 4 °C in 2 mL of 0.1 M
Tris-HCl buffer (pH 8.5) containing 1 mM phenol.58 SLO-1 (60 ×
103 units) was added to the buffer, followed by dropwise addition of
arachidonic acid (1 mg, 0.0033 mmol) dissolved in 80 µL of 0.01 M
aqueous NH4OH. A relatively large amount of SLO-1 was used to avoid
any artificial enrichment of 13-[2H]-HPETE because of a possible large
selection against 13(S)-[2H]-1 as reported for the reaction of linoleic
acid and SLO-1.33 After completion of the enzymatic reaction, the assay
mixture was acidified with 1 M HCl to pH 3. The lipid product was
extracted into Et2O (2 × 20 mL), and the pooled extracts were washed
three times with ice-cold water, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure to give crude HPETE, a sample
of which was submitted to MS directly without further purification.
Subsequently, 2 mg of triphenylphosphine was added to the remaining
2
100); deuterium content (for two H) 93% as determined by ESI-MS
comparison with unlabeled arachidonic acid.
13(S)-[2H]-Arachidonic Acid. Hexyltriphenylphosphonium bromide
(103 mg, 0.24 mmol) prepared by reflux of 1-bromohexane with
triphenylphosphine was thoroughly dried by four cycles of azeotropic
distillation of anhydrous benzene and diluted in THF (3 mL). A 1 M
solution of NaHMDS in THF (0.23 mL, 0.23 mmol) was added at -10
°C, and the bright orange ylide was stirred at room temperature for 1
h. Then the mixture was cooled to -90 °C, and a solution of aldehyde
13a (30 mg, 0.071 mmol) in THF (1 mL) was added dropwise. After
0.5 h at -90 °C, the reaction was allowed to warm to room temperature
over 5 h. Saturated aqueous sodium bicarbonate was added, and the
aqueous layer was extracted with Et2O (3 × 50 mL). The combined
organic phase was washed with brine, dried over anhydrous sodium
(58) Fogh, K.; Kragballe, K.; Larsen, E.; Egsgaard, H.; Shukla, V. K. S. Biomed.
EnViron. Mass Spectrom. 1988, 17, 459-61.
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J. AM. CHEM. SOC. VOL. 124, NO. 36, 2002 10795