InactiVation of GABA
J. Am. Chem. Soc., Vol. 118, No. 6, 1996 1249
M potassium tert-butoxide in THF, N-bromosuccinimide, and HF/
pyridine were purchased from Aldrich. Sodium sulfate, phosphoric
acid, potassium phosphate, and sodium phosphate were acquired from
Fisher. γ-Aminobutyric acid, R-ketoglutarate, â-mercaptoethanol,
3.5, JHF ) 16), 4.54 (dd, 1 H, trans CFCH , JHH ) 3.5, JHF ) 48), 4.2
2
19
(m, 1 H, CHN), 2.4 (m, 4 H, CH
(ddd, 1 F, JHF ) 16, 48, 16); high-resolution electron impact mass
spectrometry calcd for C FNO 129.0590, found 129.0593. Isolation
2 2 3
CH ); F NMR (CDCl ) δ -111
6 8
H
+
NADP , gabaculine, bovine serum albumin (BSA), pyridoxal 5′-
of 9 and 10 is described in the subsequent paper.
phosphate, pyridoxamine 5′-phosphate, and potassium pyrophosphate
After 4 months of storage in a desiccator, the 132 mg of 8 was taken
up in ethyl acetate. A solid precipitated from the solution which was
filtered, leaving, after rotary evaporation of solvent, 95 mg of a yellow
oil, which was further purified by column chromatography with 15 g
of TLC grade silica gel and a 5:1 ethyl acetate/hexane solvent system,
giving 8 (58 mg) as a light yellow oil.
from Sigma were used. Ultrapure urea was a product of ICN
1
4
3
Biomedicals. [5- C]-R-Ketoglutarate and [ H]sodium borohydride
3
i
were obtained from Amersham. [ H]Water (5 C /mL) was a product
of New England Nuclear. Dowex 50 and Dowex 1 resins from Bio-
Rad Laboratories were utilized. GABAse was purchased from Boe-
hringer Mannheim. HPLC grade methanol was obtained from Mallinck-
rodt and was filtered and degassed before use. Doubly distilled
deionized water was also filtered and degassed before use as a HPLC
solvent. Tetrahydrofuran and ether were distilled over sodium with
benzophenone ketyl indicator under nitrogen directly before use.
4-Amino-5-fluoro-5-hexenoic Acid (2). Compound 8 (79 mg, 0.61
mmol) was heated in 1 N HCl to 85 °C for 19 h, and then the water
was evaporated. The residue was dissolved in water, and the water
was evaporated. This dilution/evaporation process was repeated twice
more. The resulting brown semisolid was triturated with ether, leaving
5
-Vinyl-2-pyrrolidinone (5). 4-Amino-5-hexenoic acid (1, 4.5 g,
8
3 mg of a light brown powder. Attempts to recrystallize this solid
3
4.8 mmol) was heated under argon in a pressure tube until it changed
with 2-propanol/ether met with no success. The solid remaining after
from a white solid to a brown oil (180 °C). This oil was taken up in
chloroform and purified on a silica gel column (230-400 mesh, 5 g)
using ethyl acetate as solvent. Fractions containing a compound with
recrystallization attempts was purified on a 5 × 70 mm Dowex 50
column, using 1 N NH
4
OH as eluent. Ninhydrin positive fractions were
10
combined and lyophilized, leaving 29 mg of a light brown solid (33%
an R
f
value of 0.30 (ethyl acetate) were combined, and the solvent was
yield): 1H NMR (D
)
2
O) δ 5.04 (dd, 1 H, cis CH CF, JHF ) 17.3, JHH
4), 4.87 (dd, 1 H, trans CH CF, JHF ) 50, JHH ) 4), 4.07 (dt, 1 H,
NCHCF, JHF ) 23, JHH ) 8), 2.52 (m, 2 H, CH COOH), 2.10 (m, 2 H,
); 19F NMR (D
NCHCH O) δ -114 (ddd, JHF ) 50, 23, 17); high-
resolution electron impact mass spectrometry calcd for C
2
evaporated, yielding 3.66 g (95%) of 5-vinyl-2-pyrrolidinone (5) as a
2
1
clear colorless oil: H NMR (CDCl
ddd, 1 H, CHCH
3
) δ 7.55 (s, 1 H, CONH), 5.75
2
(
2
, JHHcis ) 10.3 Hz, JHHtrans ) 17 Hz, JHH ) 6.7 Hz),
, JHHgem ) 1.0 Hz, JHHtrans ) 17 Hz), 5.05 (dd,
, JHHgem ) 1.0 Hz, JHHcis ) 10.3 Hz), 4.10 (m, 1 H, CHN),
.25 (m, 3 H, CHHCH CO), 1.75 (m, 1 H, CHHCH CO).
-(1(or 2)-Bromo-2(or 1)-fluoroethyl)-2-pyrrolidinone (6 or 7).
2
2
5
1
2
.17 (dd, 1 H, CHCH
H, CHCH
2
6 2
H11FNO
2
1
47.0696, found 147.0699.
2
2
[3
H]-5-(1-Fluorovinyl)-2-pyrrolidinone. To 47.5 mg of 5-(1-
5
fluorovinyl)-2-pyrrolidinone (8) in THF under nitrogen was added 17
N-Bromosuccinimide (6.8 g, 38.2 mmol) was dissolved under nitrogen
in 30 mL of ether in a 50 mL plastic centrifuge tube equipped with a
septum and a line to N . To this stirred solution was added 33 mL of
2
HF/pyridine (70%, 1.16 mol). The mixture was stirred in an ice bath
for 15 min, at which time 3.66 g (32.9 mmol) of 5 in 3 mL of ether
was added slowly. The reaction was allowed to warm to room
temperature over 30 min. After being stirred at room temperature for
3
2
µL of H O (5 Ci/mL, corrected for decay, 2 equiv). Potassium tert-
butoxide in THF (1.1 mL, 1.0 M, 3 equiv) was added, which turned
the reaction solution cloudy yellow. The reaction mixture was allowed
3
to stir for 8 days, and then another 17 µL of H
2
O was added. The
solution was stirred for another hour, and then it was quenched with 3
drops of glacial acetic acid. The solid was removed by filtration and
rinsed with THF (1 mL). Solvent was evaporated by bulb-to-bulb
distillation in a closed system. The remaining semisolid was dissolved
in water and evaporated by bulb-to-bulb distillation two times. The
result was 7.1 mg of 11 as a gold solid in a 15% yield. TLC on silica
gel utilizing ethyl acetate as the mobile phase showed that the
2
0
3
2 3
h, the mixture was poured into ice-water containing K CO (79.8 g,
.578 mol). The reaction mixture was extracted four times, each with
00 mL of ether, and then the water layer was subjected to continuous
extraction with ether for 6 h. The combined ether layers from these
extractions were dried over Na SO . The ether was evaporated on a
2
4
radioactivity comigrated with 5-(1-fluorovinyl)-2-pyrrolidinone.
rotary evaporator and the remaining yellow oil was chromatographed
on a 15 g silica gel column (230-400 mesh) to separate the succinimide.
The product (3.30 g, 15.7 mmol, 48% yield) was obtained as a 1:3
mixture of 5-(2-bromo-1-fluoroethyl)-2-pyrrolidinone (6) and 5-(1-
[3
H]-4-Amino-5-fluoro-5-hexenoic Acid (12). [ H]-5-(1-Fluorovi-
3
nyl)-2-pyrrolidinone (7.1 mg, 0.055 mmol, 11) was dissolved in 5 mL
of 1 N HCl and was stirred at 84 °C for 17 h. The residue was dissolved
in 2 mL of water and the water was evaporated. Additional water was
added and evaporation was repeated. The product was then dissolved
in 1 mL of water, 1 mL of benzene was added, and the solvent was
evaporated again. The resulting brown semisolid was purified on a 5
1
0 1
bromo-2-fluoroethyl)-2-pyrrolidinone (7): H NMR (CDCl
3
) δ 4.65
(m, 2 H from 7), 4.1 (m, 2 H from 6, 2 H from 7), 3.5 (m, 2 H from
6
), 2.4 (m, 3 H from 6, 3 H from 7), 2.0 (m, 1 H from 6, 1 H from 7).
-(1-Fluorovinyl)-2-pyrrolidinone (8). The mixture of 6 and 7
3.30 g, 15.7 mmol) in 20 mL of THF was cooled to -78 °C in a dry
5
×
70 mm Dowex 50 column, eluting with 1 N NH
4
OH. Fractions
(
were counted, and those containing radioactivity were combined and
evaporated, leaving 4.4 mg of an off-white solid (12, 54% yield). The
compound was found to have a specific activity of 9.7 mCi/mmol and
a radiopurity of 97%. The radioactivity comigrates with 4-amino-5-
ice/acetone bath, and 63 mL of 1.0 M potassium tert-butoxide in THF
was added dropwise. The reaction was allowed to warm to -30 °C
and was kept between -30 and -20 °C for 2 h. It was then cooled
back down to -78 °C, and 2.7 mL of acetic acid was added followed
by 15 mL of ether. The precipitate was removed by filtration and
washed with ether. Solvents were evaporated, leaving 1.93 g (95%
yield) of a 2:5:1 mixture of 5-(1-fluorovinyl)-2-pyrrolidinone (8), (Z)-
2
fluoro-5-hexenoic acid on silica gel with a 3:1:1 n-BuOH/HOAc/H O
solvent system. Some unlabeled 2 was added to increase the amount
of product and to lower the specific activity. This mixture was
dissolved in water and run over a 5 × 70 mm Dowex 50 column,
5
-(fluorovinyl)-2-pyrrolidinone (9), and (E)-5-(fluorovinyl)-2-pyrroli-
3
resulting in [ H]-2 (12), which is 97% radiopure and has a specific
dinone (10). The mixture was column chromatographed using 15 g of
TLC grade silica gel and a 5:1 ethyl acetate/hexane solvent system.
activity of 3.3 mCi/mmol.
3
[
7- H]Pyridoxal 5′-phosphate. Tritiated pyridoxal 5′-phosphate
The first compound to elute off the column, with an R
= 0.30, was 8.
f
3
(
[ H]PLP) was synthesized using a variation on the method of Stock
Several of these columns were necessary, with 100 mg of the mixture
of (fluorovinyl)pyrrolidinones applied each time, and then repeat
columns were run on the fractions that contained mixtures of products.
21
et al. PLP (140 mg) was dissolved in 1.8 mL of water and 20 drops
of 1 M sodium hydroxide. The solution was protected from light and
3
cooled to 0 °C. Sodium [ H]borohydride (100 mCi, 11.6 Ci/mmol)
f
The three compounds have R values very close to one another, and
was dissolved in 450 µL of a 0.3 M solution of sodium borohydride in
analysis of the column fractions was done by removing the solvent by
rotary evaporation and checking for purity by NMR. At the end of
0.1 M NaOH. This was added to the cold PLP solution and was stirred
10
for 20 min. Another 2.5 mg of unlabeled sodium borohydride was
added in an attempt to remove all of the yellow color from the solution.
This solution was then stirred at 0 °C for another 20 min. The solution
the chromatography, 132 mg of pure 8, a light yellow oil, was obtained
1
(
27% yield): H NMR (CDCl
3
) δ 4.71 (dd, 1 H, cis CFCH
2
, JHH )
(
20) Condon, M. E.; Petrillo, E. W.; Ryono, D. E.; Reid, J. A.; Neubeck,
R.; Puar, M.; Heikes, J. E.; Sabo, E. F.; Losee, K. A.; Cushman, D. W.;
Ondetti, M. A. J. Med. Chem. 1982, 25, 250-258.
(21) Stock, A.; Ortanderl, F.; Pfleiderer, G. Biochem. Z. 1966, 344, 353-
360.