2
192
D. Lazare6ic, J. Thiem / Carbohydrate Research 337 (2002) 2187–2194
mg of the starting material. The reaction was therefore
carried out with 450 mg GalN-1-P, and was done in 40
parallel reactions with an absolute volume of 0.5 mL
each. Thus UDP-GalN-1-P (9) was obtained in modest
(0.5 mL). The pH was adjusted to 7.0 using a 0.4-M
solution of potassium hydroxide in water. After stirring
over night at rt, the resulting solution was, if necessary,
treated with some more N-acyloxysuccinimide (0.1
mmol) in 1:4 THF–water (0.5 mL) with subsequent pH
adjustment to 7.0 and stirring over night. The resulting
solution was purified on Biogel P2 with water as eluent,
and after freeze drying the product containing frac-
tions, yielded 80–90% of the desired acylamido hexosyl
phosphate.
Procedure C: morpholidate coupling of hexosyl phos-
phates.—The phosphate (0.18 mmol) obtained by pro-
cedure B was dissolved in water (1 mL) and passed
through a column (1×5.5 cm) of Dowex 50W-X8
(triethylammonium form) to give the phosphate in the
form of the corresponding triethylammonium salt in
quantitative yield. The phosphate was dissolved to-
gether with uridine-5%-monophosphomorpholidate (4-
morpholine-N,N%-dicyclohexyl carboxamidinium salt,
1
3
yields. The C NMR chemical shifts for both the
phosphates 4 and 8, as well as their corresponding
UDP derivatives 9 and 13, were almost identical apart
from C-2 being 10 ppm shifted downfield when at-
tached to an azido group compared to an amino group
1
13
in both cases. H and C NMR data of the UDP
hexoses 9–13 are summarized in Tables 1 and 2 (lower
parts). Attempts to convert the UDP-azide 13 to the
UDP-amine 9 by reduction methods were not yet suc-
cessful. Further work in this direction and additional
syntheses of novel N-acylamido-UDP-hexoses are in
progress.
3. Experimental
1
.6 equiv, 0.22–0.29 mmol) in anhyd pyridine (10 mL)
General methods.—TLC was performed on Silica Gel
0-coated aluminium sheets (E. Merck) using the given
and concentrated to dryness without heating under
reduced pressure. Ventilation to normal pressure was
carried out with dry argon. After repeating this proce-
dure three times, the resulting syrup-like residue was
dissolved in 1:1 anhyd pyridine–anhyd DMF (3 mL)
and stirred for 5–7 days at rt, sealed under an argon
atmosphere. Removal of the solvents without heating
under reduced pressure, followed by dissolving the
residue in water, filtration and subsequent separation
on Biogel P2 with first 0.25 M NH HCO solution, and
6
eluent mixtures. Spots were visualized under UV light
at 366 nm and by spraying with 10% H SO in EtOH
2
4
and subsequent heating. Column chromatography was
performed on Silica Gel 60 (230–240 mesh, grain size
.040–0.063 nm, E. Merck). Petroleum ether refers to
the fraction with distillation range 5–-70 °C. Biogel
0
separation was performed on Biogel P2 (Bio-Rad) ei-
ther with 0.25 M NH HCO3 solution or water as
4
4
3
eluent. Optical rotations were measured on a Perkin–
afterwards for desalting purpose with water as eluent,
yielded the uridine diphosphohexose in form of its
ammonium salt.
Elmer Polarimeter 243, with [h] values given in units
D
−
1
2
−1
of 10
deg cm g . Elemental analyses were per-
formed by the microanalytical laboratory of the Uni-
versity of Hamburg. IR absorptions were recorded on a
ATI Matteson FT-IR (Genesis Series). NMR spectra
were recorded on a Bruker AMX-400 NMR spectrome-
ter. Chemical shifts are referred to the solvents used.
MALDI-TOF spectra were measured on a Bruker
Biflex-II spectrometer with DHB as matrix.
(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-galactopy-
ranosyl) dibenzyl phosphate (3).—1H-Tetrazole (368
mg, 5.25 mmol) was suspended in dry CH Cl (3 mL)
2
2
under argon and stirring at rt, followed by the addition
of dibenzyl-N,N%-diisopropylphosphoramidite (0.87
mL, 894 mg, 2.59 mmol). Within 15 min the suspension
became a clear solution and 2-azido-3,4,5-tri-O-benzyl-
16
Procedure A: preparation of triflic azide in
2-deoxy-a/b-D-galactopyranose in dry CH Cl (3 mL)
2 2
dichloromethane.—NaN (960 mg, 14.8 mmol) was dis-
was added rapidly. After stirring for 5 h at rt, the
solution was cooled to 0 °C and 3-chloro-perbenzoic
acid (740 mg, 3 mmol) was added in small doses,
followed by stirring for 1 h at rt. Removal of the
solvent under reduced pressure at 30 °C bath tempera-
ture and purification by column chromatography
yielded compound 3 (493 mg, 62%) as a colorless syrup.
3
solved in water (2.7 mL) and after addition of CH Cl2
2
(
2 mL) cooled to ca. 2 °C. Under vigorous stirring,
trifluoromethanesulfonic anhydride (0.49 mL) was
added dropwise within 5 min. The mixture was stirred
another 2 h, followed by separation of the organic layer
and twice extracting the aqueous layer with CH Cl (2
2
2
2
0
mL), thus obtaining triflic azide in CH Cl (8 mL). This
[h] +76° (c 1.0, CHCl ); MALDI-TOF (DHB, posi-
2
2
D 3
+
+
solution was washed once with a solution of satd
Na CO (2 mL), and after separation, used without
tive mode): m/z 774 [M+K] , 758 [M+Na] , 746
+
+
[M−N +K] , 730 [M−N +Na] , 684 [M−
2
3
2
2
+
7 7 7 7
+
further purification.
C H +K] , 668 [M−C H +Na] , IR (KBr): w
−
1
1
Procedure B: selecti6e N-acylation.—
D-Galac-
2114.1 cm (N ); H NMR (acetone-d ): l 7.20 (m, 25
3 6
tosamine-1-phosphate (0.1 mmol) was dissolved in wa-
ter (0.5 mL) and treated with a solution of the
N-acyloxysuccinimide (0.15 mmol) in 1:4 THF–water
H, 5×Ph), 5.75 (dd, 1 H, H-1, J1,2 3.1, J1,P 6.1 Hz),
4.99, 4.95 (2×d, 4 H, 2×POCH Ph), 4.80, 4.77, 4.62,
2
4.47, 4.36, 4.29 (6×d, 6 H, 3×OCH Ph), 4.18 (d, 1 H,
2