ChemBioChem
10.1002/cbic.201800248
FULL PAPER
better results with the unprotected hydroxyl groups of 1a,
although we did not study partially acylated probes. In addition
to this factor, variations may result from differences in transport
or esterase activity of the different probes, particularly since
different cell lines were used, and thus cell wall/membrane
composition and transport machinery differ.
4.29 (m, 2H), 4.37 (t, 2H, J = 3.2 Hz), 4.45 (m, 1H), 4.63 (dd, 4H, J =
6
2
1
9.2, 11.7 Hz), 5.51 (s, 1H), 7.31 (m, 10H). 13C NMR (CDCl
3
, 125 MHz) δ
9.27, 48.32, 65.86, 68.07, 68.36, 70.30, 71.95, 74.10, 103.20, 127.77,
27.99, 128.51, 137.61. MS (DART-TOF, m/z): calcd for C22 (M –
)+ 426.1838. Found 426.1858.
23 3 6
H N O
N
2
2-O-(3-azidopropyl)-4,6-di-O-benzyl-myo-inositol (5): Compound
4
One issue with the current approach is that the presence of
numerous inositol-containing products may complicate the
differentiation of labeled products. In addition, modification of
any of the 6-hydroxyl groups of myo-inositol will inherently
abrogate certain products, such as the MIPC pathway in yeast
(
0.33 g, 0.77 mmol), and para-toluene sulfonic acid (0.12 g, 0.72 mmol)
were combined together in methanol (2 mL) in a 25 mL round-bottomed
flask and allowed to stir at rt for 24 h. All volatiles were removed under
reduced pressure and the crude mixture was subjected to flash
chromatography using 50% EtOAc/hexanes as eluant to afford 5 (0.23 g,
7
J = 5.51 Hz), 3.38 (t, 2H, J = 6.6 Hz), 3.55 (m, 5H), 3.81 (t, 1H, J = 2.6
Hz), 3.87 (t, 2H, J = 5.8 Hz), 4.86 (q, 4H, J = 11.4 Hz), 7.28-7.40 (m,
1
8
1
3%) as a clear oil. H NMR (CDCl
3
, 500 MHz) δ 1.85 (m, 2H), 2.49 (t, 2H,
6
or other metabolites including inositol hexakisphosphate (IP ). In
future work, the combination of varying probe structures to
differentially label lipid products with mutant cell lines deficient in
certain biosynthetic pathways will be pursued to differentiate
labeled products.
0H). 13C NMR (CDCl
0.21, 81.86, 127.98, 128.08, 128.63, 138.46. MS (DART-TOF, m/z):
(M + H) 416.1743. Found 416.2085.
3
, 125 MHz) δ 29.32, 48.90, 70.92, 72.39, 75.05,
25 3 6
calcd for C21H N O
1,3,5-tri-O-acetyl-2-O-(3-azidopropyl)-4,6-di-O-benzyl-myo-inositol
Experimental Section
(6): Compound 5 (0.297 g, 0.671 mmol), DMAP (0.041 g, .336 mmol)
and acetic anhydride (10 mL, 110 mmol) were dissolved in pyridine (30
o
mL) in a 100 mL round-bottomed flask and stirred at 65 C for 12 h. The
General Experimental: All non-aqueous reactions were performed in
flame dried glassware under nitrogen or argon atmosphere unless stated
otherwise. All solvents used in the reactions were as is unless stated
otherwise. Dichloromethane (DCM), N,N-dimethylformamide (DMF), and
tetrahydrofuran (THF) were obtained from an in–house dry solvent
system and stored over activated 4 Å molecular sieves under nitrogen
atmosphere. All commercially available compounds were used as
received without further purification. Reactions were monitored by thin–
layer chromatography (TLC) carried out on 0.25 mm Sorbtech silica gel
plates (60F-254) under 254 nm UV lamp and stained by potassium
3
reaction was quenched with saturated NaHCO (50 mL) and extracted
with DCM (3 x 15 mL). The organic layers were washed with brine, dried
over sodium sulfate and the solvent evaporated was under reduced
pressure. The crude product was subjected to flash chromatography
using 25% EtOAc/hexanes to afford compound 6 (0.291 g, 74%) as an
off-white solid. 1H NMR (CDCl
, 500 MHz) δ 1.80-1.85 (m, 2H), 1.87 (s,
H), 2.00 (s, 6H), 3.43 (t, 2H, J = 6.6 Hz), 3.64 (t, 2H, J = 5.7 Hz), 3.91 (t,
H, J = 10.2 Hz), 4.59 (q, 4H, J = 11.7 Hz), 4.91 (dd, 2H, J = 10.2, 2.6
, 125
MHz) δ 20.78, 29.51, 48.11, 70.26, 72.96, 73.52, 74.66, 127.56, 127.66,
128.36, 138.08, 169.60. MS (DART-TOF, m/z): calcd for C (M
– N
)+ 542.2239. Found 542.2362.
3
3
3
Hz), 5.16 (t, 1H, J = 9.7 Hz), 7.21-7.34 (m, 10H). 13C NMR (CDCl
3
permanganate solution or other functional group-specific stains. Flash
chromatography was performed on silica gel 60 (0.040–0.063 mm). 1H,
29 35 3 9
H N O
13C, and 2D NMR spectra were recorded on liquid state Varian VNMRS
2
spectrometer (500 MHz). Chemical shifts are reported in δ (ppm) and
calibrated using residual undeuterated solvents as an internal reference.
The following abbreviations were used to explain the multiplicities:
s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublet,
1,3,5-tri-O-acetyl-2-O-(3-azidopropyl)-myo-inositol (7): To a solution
of 6 (0.100 g, 0.176 mmol) in EtOAc (2.5 mL) in a 50 mL round-bottomed
flask was added a solution of sodium bromate (0.265 g, 1.73 mmol) in
water (2 mL). A solution of sodium hydrosulfite (0.275 g, 1.58 mmol) in
water (5 mL) was added dropwise over 10 min and the reaction was
vigorously stirred for 24 h at rt. The reaction was then diluted with EtOAc
(10 mL), quenched with 10% sodium thiosulfate (2 mL) and extracted
with water. The combined organic layers were dried over MgSO4 and
evaporated under reduced pressure. The crude product was then
subjected to flash chromatography with a 50%-75% EtOAc/hexanes
1
dt=doublet of triplet, td=triplet of doublet, m=multiplet, br=broad. H NMR
coupling constants (J) are reported in Hertz (Hz), Mass spectra were
obtained using Applied Biosystems/ QStar Elite HPLC—QTOF mass
spectrometers. 4,6-di-O-benzyl-1,3,5-methylidyne-myo-inositol (3) was
synthesized as previously described.30
2-O-(3-azidopropyl)-4,6-di-O-benzyl-1,3,5-methylidyne-myo-inositol
gradient to afford 7 (0.0435 g, 64%) as a clear oil. 1H NMR (CDCl
3
, 500
(4): To dichloromethane (10 mL) in a 50 mL round-bottomed flask at -78
o
MHz) δ 1.78-1.83 (m, 2H), 2.12 (s, 6H), 2.14 (s, 3H), 3.43 (t, 2H, J = 6.6
Hz), 3.67 (t, 2H, J = 5.7 Hz), 3.88 (s, 1H), 3.96 (t, 2H, J = 9.9 Hz), 4.90
C were added 3-azido-1-propanol (0.650 g, 6.42 mmol), 2,6-lutidene
0.488 mL, 4.21 mmol) and triflic anhydride (0.712 mL, 4.21 mmol). The
reaction was stirred at this temperature for 1 h and then quenched with
O (10 mL) and extracted (3 x 5 mL) with dichloromethane. The organic
layer was collected and dried over Na SO . The solvent was removed
under reduced pressure, and the crude triflate was capped. Compound 3
0.500 g, 1.35 mmol) and proton sponge (0.694 g, 3.24 mmol) were
(
(
dd, 2H, J = 10.3, 2.5 Hz), 4.95 (t, 1H, J = 9.6 Hz). 13C NMR (CDCl
3
, 125
MHz) δ 20.89, 21.01, 29.51, 48.02, 69.86, 70.12, 73.44, 75.98, 170.71,
71.89. MS (DART-TOF, m/z): calcd for C15 (M + H) 390.1434.
Found 390.1453.
H
2
1
23 3 9
H N O
2
4
(
added to a glass sealed tube. Dichloromethane (4 mL) was added to the
tube along with a small stirbar and the crude azido-triflate. The tube was
sealed tightly, and the reaction mixture was then allowed to stir and
heated to 85 oC for 12 h. The reaction mixture was allowed to cool to rt
2-O-(3-azidopropyl)-myo-inositol (1a): Compound 7 (0.0223 g, 0.0573
mmol) was dissolved in dry methanol (4 mL) in a 25 mL round-bottomed
flask and sodium methoxide (0.0092 g, 0.1719 mmol) was added. The
o
mixture was heated at 70
methoxide was quenched with H
then reconstituted in 1 mL of H O. Cation-exchange resin Amberlite IR-
120 (pretreated with 2M HCl and H O) was added and the mixture was
C for 6 h, after which the excess sodium
and then partitioned between H
2
O (15 mL) and dichloromethane (3 x 15
2
O. The contents were lyophilized and
mL). The organic layer was collected and dried over Na
2
SO . The solvent
4
2
was removed under reduced pressure and the crude oil was subjected to
2
flash chromatography using 10% EtOAc/hexanes as eluent to afford 4 as
vortexted for 5 min. The resin was removed by filtration with methanol
1
and the filtrate was lyophilized to afford 1a quantitatively (0.0134 g) as a
a colorless oil (0.329 g, 58%) . H NMR (CDCl
3
, 500 MHz) δ 1.90 (m, 2H),
1
3.44 (t, 2H, J = 6.6 Hz), 3.58 (t, 2H, J = 6.1 Hz), 3.90 (q, 1H, J = 1.69 Hz),
white solid. H NMR (CDCl
3
, 500 MHz) δ 1.84 (m, 2H), 3.12 (t, 1H, J =
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