Cellular Metabolism of Synthetic Azidosugars
A R T I C L E S
saline (PBS), FITC-labeled avidin (stock concentration 2.8 mg/mL),
and penicillin-streptomycin (P-S) were from Sigma. ManNAc was
from Pfanstiehl. PE-labeled avidin (stock concentration 0.6 mg/mL)
was from Caltag Laboratories. Sulfo-NHS-LC-LC-biotin and tricarboxy-
ethylphosphine (TCEP) were from Pierce. FITC-labeled anti-FLAG M2
(stock concentration 1.8 mg/mL) was prepared according to a literature
protocol37 from anti-FLAG M2 (Sigma) and fluorescein isothiocyanate
(Aldrich). RPMI medium 1640 was from Life Technologies, Inc.,
Dulbecco’s modified Eagle’s medium (DMEM) was from CellGro, and
fetal bovine serum (FBS) was from HyClone Laboratory. Iscoves MEM
medium and Ham F12 nutrient mixture were from Invitrogen. Cell
densities were determined on a Coulter Counter-ZM. Flow cytometry
analysis was performed on a Coulter Epics XL-MCL cytometer using
a 488 nm argon laser. At least 104 viable cells were analyzed from
each sample. Cell viability was ascertained by gating the samples on
the basis of forward scatter (to sort by size) and side scatter (to sort by
granularity). All cell-based experiments were performed in triplicate.
The average of the mean fluorescence intensity obtained from each of
the three replicate experiments was calculated to obtain a representative
value in arbitrary units.
and extracting with pentane. Evaporation of the pentane produced 3.5
1
g (91%) of a clear oil. H NMR (300 MHz, CDCl3): δ 4.35 (s, 2H),
7.31-7.42 (m, 5H). Lit. 1H NMR (300 MHz, CDCl3): δ 4.35 (s, 2H),
7.26-7.45 (m, 5H).
Compound 5. Methyl 2-(diphenylphosphanyl)benzoate (1, 32 mg,
0.099 mmol) and benzyl azide (13 mg, 0.099 mmol) were dissolved in
CH3CN/H2O (3:1, 1 mL). The evolution of N2 gas was observed
immediately upon reactant mixing. The solution was stirred at room
temperature for 2 h. Removal of the solvent provided the crude ligation
product which was purified by SiO2 column chromatography eluting
with a gradient of 20-50% EtOAc in hexanes to provide 38 mg of a
1
white solid (92%). H NMR (400 MHz, CDCl3): δ 4.06 (d, 2H, J )
5.4), 7.06 (dd, 1H, J ) 7.7, 14.4), 7.19-7.24 (m, 5H), 7.36 (t, 1H, J
) 7.6), 7.45-7.48 (m, 4H), 7.54-7.66 (m, 7H), 8.00 (dd, 1H, J )
4.0, 7.6), 9.04 (br s, 1H). 13C NMR (125 MHz, CDCl3): δ 44.1, 127.2,
128.0, 128.6, 128.7, 128.8, 129.8, 130.0, 130.5, 131.5, 131.7, 131.8,
131.8, 132.4, 132.4, 132.7, 132.7, 133.3, 133.4, 137.6, 141.0, 141.0,
167.3 (d, JC-P ) 3.92). 31P NMR (160 MHz, CDCl3): δ 39.56. HRMS
calcd for C26H23NO2P (MH+) 412.1466, found 412.1467.
31P NMR Analysis of Reaction Intermediates. Methyl 2-(diphenyl-
phosphanyl)benzoate (1, 32 mg, 0.099 mmol) and benzyl azide (13
mg, 0.099 mmol) were dissolved in wet CD3CN (1 mL) and transferred
to an NMR tube. The evolution of N2 gas was observed immediately
upon reactant mixing. The appearance of new 31P resonances was
monitored on a Bruker AMX-400 spectrophotometer. After 50 min of
reaction time, water (10 µL) was added.
Cell Culture Conditions. All cells were cultured in media containing
2% P-S. Jurkat cells were grown in RPMI medium 1640 supplemented
with L-glutamine and 5-10% FBS. HL-60 cells were grown in Iscoves
MEM supplemented with L-glutamine, sodium pyruvate, and 20% FBS.
CHO cells were grown in Ham F12 nutrient mixture supplemented
with L-glutamine and 10% FBS. HeLa and COS-7 cells were grown in
DMEM supplemented with 10% FBS. S49 cells were grown in RPMI
medium 1640 supplemented with 1% sodium pyruvate and 10% FBS.
Treatment of Cells with ManNAc and GlcNAc Analogues. The
appropriate amount of peracetylated compound in a solution of ethanol
was added to 6-well tissue culture plates. After evaporation of the
ethanol, cultures of cells were seeded at a density of 1.5 × 105 cells/
mL in a total volume of 2 mL of culture medium. The cells were
incubated for 3 days. Control wells containing no added sugar were
also seeded with cells and incubated.
Azide Labeling Procedures. After growth in the presence of the
appropriate monosaccharides, cells from each well were distributed
among three wells of a 96-well V-bottom tissue culture plate. The cells
were pelleted (3500 rpm, 3 min) and washed twice with 200 µL of
labeling buffer (1% FBS in PBS, pH ) 7.4). After the second wash,
cells were typically resuspended in a volume of 50 µL of labeling buffer
and 50 µL of 2 in solution (0.5 mM in PBS, pH ) 7.4). After incubation
at room temperature for 1 h, the cells were pelleted (3500 rpm, 3 min)
and washed three times with ice-cold labeling buffer. Cells were then
resuspended in 100 µL of FITC-anti-FLAG staining solution (1:900
dilution in labeling buffer). After a 30-min incubation in the dark at 4
°C, the cells were pelleted, washed with 200 µL of ice-cold labeling
buffer, and then diluted to a volume of 400 µL for flow cytometry
analysis. To determine optimal conditions, the concentration of 2,
incubation time, and pH of the reaction buffer were varied as described
under Results and Discussion.
All chemical reagents were of analytical grade, obtained from
commercial suppliers and used without further purification unless
otherwise noted. Thin-layer chromatography was performed on Analtech
Uniplate silica gel plates. Compounds were visualized by staining with
ceric ammonium molybdate, by triphenylphosphine followed by
1
ninhydrin (for azides), and/or by the absorbance of UV light. All H
and 13C NMR spectra were measured with a Bruker AMX-300, AMX-
400, or DRX-500 MHz spectrometer as noted. Chemical shifts are
1
reported as δ relative to tetramethylsilane for H and 13C spectra and
relative to H3PO4 for 31P spectra. Coupling constants (J) are reported
in hertz. Fast atom bombardment (FAB), chemical ionization (CI), and
electrospray (ES) mass spectra were obtained at the UC Irvine and UC
Berkeley Mass Spectrometry Laboratories. Elemental analyses were
obtained at the UC Berkeley Microanalytical Laboratory. Infrared
spectra were acquired on a Perkin-Elmer series Fourier transform
infrared spectrometer.
Synthesis of Model Compounds. Methyl 2-(Diphenylphosphanyl)-
benzoate (1).38 To a flame-dried flask was added CH3CN (21 mL),
TEA (1.4 mL, 9.4 mmol), methyl 2-iodobenzoate (1.4 mL, 9.4 mmol),
and palladium acetate (2.2 mg, 0.01 mmol). The mixture was degassed
in vacuo before diphenylphosphine (1.6 mL, 9.4 mmol) was added to
the flask via syringe under Ar. The resulting solution was heated at
reflux for 4 h, at which point it was cooled to room temperature and
concentrated. The residue was partitioned between 250 mL of 1:1 Et2O/
H2O and the layers were separated. The organic layer was concentrated
and dried over Na2SO4, and the product was purified via silica gel
chromatography with elution by 50:1 EtOAc/hexanes. The pure product
was obtained as 2.0 g (66%) of a white crystalline solid, mp 95-96
°C (lit. 96°C). IR (thin film): 3052, 3000, 2648, 2839, 1719, 1584
1
cm-1. H NMR (400 MHz, CDCl3): δ 3.73 (s, 3H), 6.90-6.94 (m,
1H), 7.26-7.84 (m, 12H), 8.03-8.05 (m, 1H). 13C NMR (125 MHz,
CDCl3): δ 51.9, 128.2, 128.4, 128.5, 128.6, 130.7, 131.9, 133.8, 133.9,
134.2, 134.3, 137.8, 137.9, 140.3, 140.5, 167.2, 167.2. 31P NMR (160
MHz, CDCl3): δ -0.73. MS (CI) m/z 321.1 (MH+). Anal. Calcd for
C20H17O2P: C, 74.99; H, 5.35. Found: C, 74.75; H, 5.42.
Benzyl Azide.39 Benzylbromide (3.5 mL, 29 mmol) was dissolved
in DMF (70 mL), and NaN3 (2.9 g, 44 mmol) was added. After the
solution was stirred at room temperature for 12 h, EtOAc (150 mL)
was added. The solution was washed with H2O (4 × 30 mL) followed
by brine (2 × 30 mL), dried over Na2SO4, and concentrated. Remaining
traces of DMF were removed by dissolving the crude product in H2O
Labeling of Cell-Surface Amines. Cells were grown in the presence
of the appropriate sugars, transferred to a 96-well V-bottom tissue
culture plate, pelleted, and washed as described above. Reduction of
cell-surface azides was accomplished by resuspending cells in 100 µL
of TCEP solution (1 mM in labeling buffer) and incubating at room
temperature for 1 h. Control populations of cells were exposed to either
the normal azide labeling conditions or labeling buffer alone. After
incubation, the cells were pelleted and washed twice with 200 µL of
labeling buffer. Cells were then resuspended in 90 µL of labeling buffer,
(37) Harlow, E., Lane, D., Eds. Antibodies: a laboratory manual; Cold Spring
Harbor Laboratory: Cold Spring Harbor, NY, 1988.
(38) Stelzer, O.; Tepper, M.; Hingst, M.; Hebler, A.; Herd, O. J. Organomet.
Chem. 1996, 522, 69.
(39) Alvarez, S. G.; Alvarez, M. T. Synthesis-Stuttgart 1997, 413.
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J. AM. CHEM. SOC. VOL. 124, NO. 50, 2002 14901