10978 J. Am. Chem. Soc., Vol. 118, No. 45, 1996
Dakoji et al.
2.85 (1H, m, 2-H), 1.30 (3H, s, Me); 13C NMR (CDCl3) δ 61.2 (C-1),
59.0 (C-2), 16.6 (C-4).
gel column and purified by flash chromatography (4:1 to 1:2 hexane/
EtOAc) to give the desired product 14 in 20% yield. 1H NMR (CDCl3)
δ 5.86 (1H, ddd, J ) 17.1, 10.6, 5.3, 5-H), 5.38 (1H, d, J ) 17.1,
6-H), 5.28 (1H, d, J ) 10.6, 6-H), 4.85-4.83 (1H, m, 4-H), 4.35 (1H,
dt, J ) 6.2, 3.2, 3-H), 3.15 (1H, br s, COOH), 2.77 (1H, dd, J ) 17.8,
6.2, 2-H), 2.53 (1H, dd, J ) 17.8, 3.2, 2-H); 13C NMR (CDCl3) δ 175.8
(C-1), 132.5 (C-5), 117.9 (C-6), 87.4 (C-4), 71.4 (C-3), 36.4 (C-2).
trans-3,4-Epoxy-5-hexenoyl-CoA (9). Acid 14 was coupled with
coenzyme A to give the CoA product 9 in 70% yield. 1H NMR (D2O,
signals of the oxiranyl moiety shown in italics) δ 8.74, 8.51 (1H each,
s, adenine H’s), 6.27 (1H, d, J ) 5.1, ribose anomeric H), 6.01 (1H,
ddd, J ) 18.5, 11.0, 6.0, 5-H), 5.49 (1H, d, J ) 18.5, 6-H), 5.44 (1H,
d, J ) 11.0, 6-H), 5.05 (1H, m, 4-H), 4.92 (2H, m), 4.66 (1H, s, ribose
H), 4.54 (1H, m, 3-H), 4.38 (2H, bs, C(Me)2CH2O), 4.13 (1H, s,
HOCHC(Me)2), 3.96, 3.73 (1H each, m, ribose H), 3.58 (2H, t, J )
6.1), 3.42 (2H, t, J ) 6.2), 3.09 (1H, dd, J ) 18.2, 6.4, 2-H), 2.70 (2H,
t, J ) 6.2), 2.67 (1H, dd, J ) 18.2, 3.2, 2-H), 2.56 (2H, t, J ) 6.2),
1.04, 0.91 (3H each, s, 2 × Me).
trans-[3-3H]-2,3-Epoxybutanoic Acid (8). To a solution of 7 (0.15
g, 1.68 mmol) in 4.0 mL of acetonitrile was added a mixture of NaIO4
(1.5 g, 7.1 mmol), RuCl3‚3H2O (13.6 mg, 51.9 µmol) and water (106
µL, 5.9 mmol) in one portion. The reaction mixture was vigorously
stirred at room temperature overnight. The reaction was complete after
12 h, monitored by TLC (1:1 hexane/EtOAc), and the solution turned
greenish black over the course of the reaction. The crude mixture was
chromatographed directly on a silica gel column (4:1 ether/pentane) to
give 8 as a white solid in 74% yield. Spectral data of the C-3 deuterated
sample obtained from a test experiment: 1H NMR (CDCl3) δ 3.20 (1H,
s, 2-H), 1.39 (3H, s, Me).
trans-[3-3H]-2,3-Epoxybutyryl-CoA (4). Acid 8 was coupled with
coenzyme A to furnish the final product 4 as a white solid (specific
activity ) 0.16 mCi/mmol). The labeled product eluted with an
identical retention time as the unlabeled standard 1: HPLC (Partisil-
C18 column) tR ) 7.8 min (see General Procedures for separation
conditions).
4,5-Epoxypentanoic Acid (18). To a solution of the commercially
available 4-pentenoic acid 17 (1 g, 10 mmol) in 80 mL of methylene
chloride was added 57-86% m-CPBA (3.4 g, 20.0 mmol) at room
temperature. After being stirred overnight, the solution was directly
loaded on the silica gel and purified by flash column chromatography
(2:1 to 1:2 hexane/EtOAc) to give the desired product 18 in 90% yield.
1H NMR (CDCl3) δ 4.72-4.60 (1H, m, 4-H), 3.90 (1H, dd, J ) 12.3,
2.7, 5-H), 3.64 (1H, dd, J ) 12.3, 4.5, 5-H), 2.70-2.45 (2H, m, 2-H’s),
2.50-2.32 (2H, m, 3-H’s); 13C NMR (CDCl3) δ 178.8 (C-1), 80.9 (C-
4), 64.1 (C-5), 28.7 (C-2), 23.2 (C-3).
4,5-Epoxypentanoyl-CoA (15). Acid 18 was coupled with coen-
zyme A to give the CoA product 15 in 80% yield. 1H NMR (D2O) δ
8.37, 8.04 (1H each, s, adenine H’s), 5.98 (1H, d, J ) 6.0, ribose
anomeric H), 4.88-4.83 (2H, m), 4.42 (1H, br s), 4.10 (2H, br s,
C(Me)2CH2O), 3.85 (1H, s, HOCHC(Me)2), 3.70-3.65 (2H, m), 3.48
(1H, dd, J ) 12.6, 5.1, 5-H), 3.40-3.36 (1H, m), 3.16-3.10 (2H, m),
3.25-3.15 (2H, burried under triethylamine.HCl salt), 3.01-2.95 (2H,
m), 2.57 (2H, t, J ) 6.0), 2.50-2.45 (2H, m, 2-Hs), 2.28 (2H, t, J )
6.0), 2.28-2.10 (1H, m, 3-H), 1.95-1.60 (1H, m, 3-H), 0.72, 0.58
(3H each, s, 2 × Me): High-resolution FABMS calcd for C26H43N7O18P3S
(M + H)+ 866.1755; found 866.1743.
Ethyl (E)-3,5-Hexadienoate (11). To a solution of diisopropylamine
(12.4 mL, 90 mmol) in 60 mL of dry THF was added a solution of
n-butyllithium in hexane (37 mL, 90 mmol, 2.5 M) at -10 °C under
argon. After the solution was stirred for 30 min, the reaction mixture
was cooled to -78 °C. To this solution was added 20 mL of
hexamethylphosphoramide (HMPA), and stirring was continued for 20
min. Ethyl sorbate (10, 7 g, 50 mmol) in dry THF (20 mL) was then
added dropwise at -78 °C. After the addition was complete, the
reaction mixture was stirred for an additional 30 min at the same
temperature. The resulting dark red solution was poured into a well-
stirred solution of 18 mL of acetic acid and 100 mL of ice water. The
reaction mixture was extracted with pentane (3 × 50 mL), and the
combined extracts were washed with NaHCO3 and water, dried, filtered,
and concentrated. The residue (6 g, 86%), which was essentially pure,
was used for the next reaction without further purification. 1H NMR
(CDCl3) δ 6.32 (1H, dt, J ) 16.9, 10.4, 5-H), 6.12 (1H, dd, J ) 15.2,
10.4, 4-H), 5.77 (1H, dt, J ) 15.2, 7.2, 3-H), 5.14 (1H, d, J ) 16.9,
6-H), 5.04 (1H, d, J ) 10.1, 6-H), 4.12 (2H, q, J ) 7.2, OCH2), 3.09
(2H, d, J ) 7.2, 2-Hs), 1.23 (3H, t, J ) 7.2, Me); 13C NMR (CDCl3)
δ 171.1 (CdO), 136.1, 133.9, 125.4, 116.5, 60.4 (OCH2), 37.7 (C-2),
13.8 (Me).
(E)-3,5-Hexadien-1-ol (12). To a well-stirred suspension of lithium
aluminum hydride (2.0 g, 52.6 mmol) in 100 mL of anhydrous ether
was added dropwise a solution of ester 11 (5.4 g, 38.6 mmol) in 5 mL
of anhydrous ether at 0 °C. The ice bath was removed, and the mixture
was allowed to warm to room temperature. After being stirred for 30
min, the reaction was quenched by sequential addition of 2 mL of water,
2 mL of 15% NaOH, and 6 mL of water. The mixture was filtered,
and the filtrate was washed with brine, dried, filtered, and concentrated
in Vacuo to give crude 12. Without further purification the title
compound was subjected to oxidation by the Jones reagent.
(E)-3,5-Hexadienoic Acid (13). Alcohol 12 (307 mg, 3.06 mmol)
was dissolved in acetone and treated at 0 °C with Jones oxidant
(prepared by mixing 26.7 g of chromium trioxide and 23 mL of
concentrated sulfuric acid followed by water dilution to a final volume
of 100 mL) until the red orange color persisted for at least 1 min. The
resulting mixture was stirred at room temperature for 30 min to ensure
complete oxidation of 12. Excess oxidizing reagent was quenched with
2-propanol at 0 °C. The reaction mixture was then diluted with water,
and the product was extracted into the organic phase with ether (5 ×
50 mL). The combined organic extracts were dried, filtered, and
concentrated in Vacuo. The crude product was purified by flash column
chromatography (4:1 ether/pentane) to give the desired acid in 31%
yield. 1H NMR (CDCl3) δ 11.6 (1H, bs, COOH), 6.21 (1H, dt, J )
16.5, 10.2, 5-H), 6.15 (1H, dd, J ) 15.2, 10.2, 4-H), 5.76 (1H, dt, J )
15.2, 7.1, 3-H), 5.17 (1H, d, J ) 16.5, 6-H), 5.07 (1H, d, J ) 10.0,
6-H), 3.16 (2H, d, J ) 7.1, 2-Hs); 13C NMR (CDCl3) δ 178.1 (C-1),
136.2, 135.0, 124.7, 117.4, 37.5 (C-2).
[4-3H]-2-Butynoic Acid (21). To a solution of anhydrous ammonia
(10 mL) at -40 °C was added a small piece of Li metal. The
subsequent addition of a few crystals of Fe(NO3)3 changed the color
of the reaction solution from blue to gray, which is indicative of the
formation of LiNH2. More Li metal (78.7 mg, 11.4 mmol) was added,
and the resulting mixture was vigorously stirred for 20 min. Propiolic
acid (20, 176 µL, 2.85 mmol) in 3 mL of anhydrous ether was added
dropwise to this solution, and stirring was continued for 10 min prior
to the addition of [3H]CH3I (70 µmol in 0.69 mL of toluene, specific
activity ) 75 mCi/mmol) in 3 mL of dry ether. Twenty minutes later,
a solution of iodomethane (142 µL, 2.28 mmol) in 5 mL of dry ether
was added dropwise, and the reaction mixture was stirred at -40 °C
for 6 h and then at room temperature for an additional 6 h. After
quenching with saturated NH4Cl solution (20 mL) at 0 °C, the resulting
mixture was acidified with concentrated sulfuric acid and stirred for
30 min. The product was extracted into ether (3 × 75 mL), and the
combined organic layers were dried and concentrated. The crude
product was purified by flash column chromatography on silica gel
(1:1 ether/pentane) to provide 21 as a white solid in 46% yield. 1H
NMR (CDCl3) of the unlabeled 21: δ 2.01 (3H, s, Me).
[4-3H]-2-Butynoyl-CoA (19). The labeled acid 21 and the unlabeled
2-butynoic acid were each coupled with coenzyme A to afford the
corresponding acyl-CoA product as a white solid in 67% yield. Both
unlabeled and labeled products were eluted with identical retention
times: HPLC (Partisil-C18 column) tR ) 9.4 min. The specific activity
of the labeled product ) 1.51 mCi/mmol. Spectral data of the unlabeled
19: 1H NMR (D2O, signal of the acyl moiety given in italics) δ 8.60,
8.31 (1H each, s, adenine H’s), 6.21 (1H, d, J ) 6.0, ribose anomeric
H), 4.96 (2H, m), 4.63 (1H, s, ribose H), 4.29 (2H, br s, C(Me)2CH2O),
4.07 (1H, s, HOCHCMe2), 3.90, 3.61 (1H each, m, ribose H), 3.51
(2H, t), 3.39 (2H, m), 3.11 (2H, t, J ) 6.5), 2.47 (2H, t, J ) 6.4), 2.08
(3H, s, 4-Hs), 0.95, 0.81 (3H each, s, 2 × Me).
trans-3,4-Epoxy-5-hexenoic Acid (14). To a solution of acid 13
(1 g, 9 mmol) in 100 mL of methylene chloride was added 57-86%
m-CPBA (3 g, 22.5 mmol) at room temperature. After being stirred
overnight, the reaction mixture was filtered through silica gel and
washed with hexane. The filtrate was directly loaded onto the silica
[2,3-13C2]-1,1-Dibromo-1-propene (24). To a solution of 100 mL