1056
CLARKE ET AL.
ꢀ24 mL of ethanol-d and 1 mL of D2O)and then
placed under an atmosphere of D2 ꢀusing a balloon
®lled with D2 gas). A freshly prepared solution
of 1 M NaBD4 in CH3CH2OD ꢀ4 mL)was added
quickly via syringe to the nickel acetate solution
resulting in the formation of the catalyst ꢀnickel
boride)as a black suspension. To this mixture
was added 4-phenylbut-2-ynoic acid ethyl ester
ꢀ32 mmol, 6.0 g). The ¯ask and syringe used
in the transfer of the alkyne were rinsed with
CH3CH2OD ꢀ2 Â 2 mL)and these washings were
added to the reaction mixture. The reaction ¯ask
was evacuated ꢀꢀ150 mmHg)and re®lled ꢀ3 Â)
with D2 gas using a balloon. Finally, the reaction
mixture was stirred vigorously under D2 ꢀballoon
pressure)for 16 h. The mixture was then vacuum
®ltered through Celite, and the ®ltrate was con-
centrated on a rotary evaporator. The residual
material was ¯ash chromatographed on silica gel
ꢀ230±400 mesh, 50 mL, 6-in. column height)using
ether±hexanes ꢀ3:7)as eluent. The crude product
was again ¯ash chromatographed on silica gel
ꢀ230±400 mesh, 100 mL, 6-in. column height)
using ether±hexanes ꢀ1:9)as eluent. The product
ꢀC6H5CH2CD2CD2CO2CH2CH3)was obtained
as a yellow oil in 90% yield ꢀ5.6 g, Rf 0.4 in
ether±hexanes, 1:9): 1H NMR ꢀCDCl3) d 7.14±
7.25 ꢀm, 5H, aromatic), 4.10 ꢀq, J 7.1 Hz, 2H,
OCH2), 2.61 ꢀbr s, 2H, C6H5CH2), and 1.22
ꢀt, J 7.1 Hz, 3H, CH3); 13C NMR ꢀCDCl3) d
standing ꢀ26 mmol, 4.3 g, 90% yield, mp 48±
508C): 1H NMR ꢀCDCl3) d 11.78 ꢀbr s, 1H, CO2H),
7.17±7.30 ꢀm, 5H, aromatic), and 2.65 ꢀs, 2H,
13
±
CH2); C NMR ꢀCDCl3) d 180.1 ꢀC±O), 141.2,
128.4, 128.4, and 126.0 ꢀaromatic), 34.72 ꢀCH2).
Anal. calcd. for C10HꢀD)12O2 ꢀdeuterium ana-
lyzed as hydrogen): C, 71.42; HꢀD), 7.19. Found:
C, 71.18; HꢀD), 7.17.
[3H]Thymidine
HT-29 cells were seeded in 96 well plates and
treated for 24 and 48 h with butyrate, PB, and
D4PB [0 ꢀcontrol), 0.5 mM, 1 mM, and 3 mM].
After treatment, [Methyl-3H]thymidine ꢀ1.0 mCi,
NEN)was added to each well and incubated for
4 h. Cells were harvested on ®lter paper and
[3H]thymidine incorporation was measured by a
scintillation counter ꢀBeckman LS 5000TD).
Flow Cytometry
For annexin V staining, FITC-conjugated annexin
and propidium iodide ꢀPI)were added to cells and
¯uorescence intensity was determined using a
FACScan ¯ow cytometer ꢀBecton Dickinson, San
Jose, CA)and analyzed by CellQuest software
ꢀBecton Dickinson, San Jose, CA). Mitochondrial
membrane potential ꢀDCmt)was determined by
¯ow cytometry using the dye, JC-1 ꢀ5,506,60-
tetrachloro-1,10,3,30-tetraethylbenzimadozolcar-
bocyanine iodide, Molecular Probes, Eugene, OR).
Brie¯y, cells were harvested, washed with PBS,
and incubated with 10 mM JC-1 at 378C for 15 min.
Fluorescence intensity was determined using a
FACScan ¯ow cytometer ꢀBecton Dickinson, San
Jose, CA)and analyzed by CellQuest software
ꢀBecton Dickinson, San Jose, CA). Cell cycle dis-
tribution was determined by ®xing 106 cells in
75% ethanol and staining with PI ꢀ10 mg/mL)in
the presence of 100 U/L RNase ꢀBoehringer-
Mannheim, Indianapolis, IN). The DNA content
distribution was determined using a FACScan
¯ow cytometer ꢀBecton Dickinson)and analyzed
using ModFIT ꢀBecton Dickinson).
±
173.1 ꢀC±O), 141.1, 128.2, 128.1, and 125.7
ꢀaromatic), 60.01 ꢀOCH2), 34.81 ꢀC6H5CH2), and
14.18 ꢀCH3). ꢀNote: The 13C signals for the CD2
moieties were only visible with long accumulation
times).
2,2,3,3-Tetradeutero-4-phenylbutanoic
acid ꢀ``D4-PB'')
A
turbid mixture of 2,2,3,3-tetradeutero-4-
phenylbutanoic acid ethyl ester ꢀ29 mmol, 5.6 g)
and 2 M NaOD/D2O ꢀ30 mL, 60 mmol)was
re¯uxed ꢀ2.5 h), and the resultant clear solution
was washed with ether ꢀ3 Â 30 mL)to remove un-
reacted starting material. The pH of the reaction
mixture was adjusted to 7 using conc. HCl, and
the aqueous layer was washed again with ether
to remove impurities ꢀ1 Â 30 mL). The pH of the
aqueous layer was then adjusted to 2 ꢀconc. HCl)
and the product was extracted with ether ꢀ2 Â
30 mL). The ®nal ether extracts were com-
bined, dried ꢀMgSO4), ®ltered, and concentrated
on a rotary evaporator to give the product
ꢀC6H5CH2CD2CD2CO2H), which solidi®ed on
Western Blot Analysis
Whole cell extracts were prepared in lysis buffer
ꢀ50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40,
50 mM NaF, 0.2 mM NaVO4, 1 mM DTT, 1 mM
phenylmethylsulfonyl ¯uoride, 25 mg/mL leupep-
tin, 25 mg/mL aprotonin, 25 mg/mL pepstatin A).
Cell debris was removed by centrifugation, and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002