Ruhela and Vishwakarma
1
,2,3,6-t et r a -O-a cet yl-r-D-m a n n op yr a n osyl p h osp h a t e]
62.63, 66.55, 67.46, 68.27, 68.64, 69.37, 69.66, 69.84, 70.14,
70.75, 70.88, 71.20, 73.31, 73.76, 74.24, 77.15, 78.95, 90.41,
91.69, 101.08, 101.29, 168-171; 31P NMR δ δ -2.90 (dt, J PH
Tr ieth yla m m on iu m Sa lt (7). A mixture of H-phosphonate
donor 5 (32 mg, 0.036 mmol) and acceptor 6 (23 mg, 0.036
mmol) was dried by evaporation of pyridine (500 µL × 3). The
residue was dissolved in anhyd pyridine (600 µL), and pivaloyl
chloride (15 µL, 0.123 mmol) was added. The reaction mixture
was stirred for 1 h at rt, and a freshly prepared iodine solution
7.5 and 10); ESMS m/z 1290.4 (M - Et
3
N - H); HRMS (ESMS)
-
calcd for (M - Et
1291.3188.
3
N - H)
C H O37P 1291.3177, found
50 68
2
,3,4-Tr i-O-a cet yl-â-D-ga la ct op yr a n osyl-(1f4)-1,2,3,6-
(
600 µL, 18 mg, 0.078 mmol in pyridine-water, 95:5) was
added. After 30 min, CH Cl (10 mL) was added, and the
solution was washed successively with a cold 1 M aqueous
solution of Na
(5 mL × 2) and ice-cold 1 M TEAB buffer
5 mL × 2), dried over Na SO , and concentrated. Column
chromatography on silica gel (3% MeOH in CH Cl with 1%
tetr a-O-acetyl-r-D-m an n opyr an oside 6-[2,3,4-Tr i-O-acetyl-
-O-(ter t-bu tyldim eth ylsilyl)-â-D-ga la ctop yr a n osyl-(1f4)-
,3,6-tr i-O-acetyl-r-D-m an n opyr an osyl ph osph ate 6-[2,3,4-
2
2
6
2
2
2 3
S O
tr i-O-acetyl-â-D-galactopyr an osyl-(1f4)-2,3,6-tr i-O-acetyl-
r-D-m an n opyr an osyl ph osph ate]] Bis-tr ieth ylam m on iu m
Sa lt (10). A mixture of phosphotetrasaccharide acceptor 9
(
2
4
2
2
Et
3
N) afforded product 7 (40 mg, 74%): R
Cl ; [R] ) -6.1 (c 0.18, CHCl
f
) 0.21 in 10% CH
3
3
-
,
(15.6 mg, 0.015 mmol) and H-phosphonate donor 5 (20.8 mg,
1
OH in CH
2
2
D
); H NMR (CDCl
3
0
3
.024 mmol) was dried by evaporation of pyridine (500 µL ×
1
1
3
00 MHz; assignments confirmed by H- H COSY and HMQC
experiments) δ 0.01 (s, 6H), 0.84 (s, 9H), 1.30 (t, 9H), 1.96-
.15 (m, 39H), 3.00 (q, 6H),3.50 (m, 4H), 3.87 (m, 1H), 3.94
). The residue was dissolved in anhydrous pyridine (500 µL),
and pivaloyl chloride (10 µL, 0.083 mmol) was added. The
mixture was stirred for 1 h at rt after which time a freshly
prepared solution of iodine (500 µL, 16 mg, 0.06 mmol in
pyridine/water 95:5) was added. After 30 min, CH Cl was
2 2
added, and the solution was washed successively with cold 1
2
(
m, 1H), 4.07-4.14 (m, 2H), 4.35 (m, 1H), 4.39 (m, 4H), 4.40
(m, 1H), 4.48 (m, 1H), 4.52 (m, 1H), 4.94 (d, J ) 7.7 Hz, 2H),
5
.28 (m, 5H), 5.43 (m, 1H), 5.45 (dd, J HH ) 1.9 and J HP ) 7.0
13
Hz, 1H), 5.46 (m, 1H), 6.01 (d, J ) 2.7 Hz, 1H); C NMR -5.75,
M aq Na S O solution (5 mL × 2) and ice-cold 1 M TEAB
2
2
3
1
6
6
7
7.95, 25.57, 8.5 and 45.50 (TEA ion), 20.48-20.79 (13 peaks),
0.06, 60.42 (d, J CP ) 8 Hz), 62.22, 62.63, 66.55, 67.46, 68.27,
8.64, 69.37, 69.66, 69.84, 70.14, 70.75, 70.88, 71.20, 73.31,
3.76, 74.24, 77.15, 78.95, 90.41 (d), 91.69, 101.08, 101.29, 168;
P NMR: δ -2.90 (dt, J PH 7.5 and 10); ESMS m/z 1405.2 (M
buffer (5 mL × 2), dried over Na SO , and concentrated. The
2 2
2
4
silica column purification using 5% MeOH in CH Cl with 1%
Et N afforded compound 10 (16 mg, 63%): R ) 0.11 in 10%
3
f
1
CH OH in CH Cl ;
3
2
2
H NMR (CDCl ) δ 0.01 (s, 6H), 0.84 (s,
3
3
1
9H), 1.30 (t, 9H, TEA ion), 3.00 (q, 6H, TEA ion), 2.15-1.96
(m, 57H), 3.50 (m, 6H), 3.87-3.94 (m, 3H), 4.14-4.07 (m, 3H),
4.30-4.35 (m, 3H), 4.39 (m, 6H), 4.48 (m, 2H), 4.52 (m, 1H),
4.94 (m, 3H), 5.28 (m, 6H), 5.29 (m, 1H), 5.43 (m, 2H), 5.45
(dd, J HH ) 1.9 and J HP ) 7.0 Hz, 2H), 5.46 (m, 3H), 6.01 (d, J
-
-
-
C
Et
H
3
N - H) ; HRMS (ESMS) calcd for (M - Et
82
O37PSi 1405.4042, found 1405.4105.
3
N - H)
56
â-D-Galactopyr an osyl-(1f4)-r-D-m an n opyr an oside 6-[â-
D-Ga la ct op yr a n osyl-(1f4)-r-D-m a n n op yr a n osyl p h os-
p h a te] Tr ieth yla m m on iu m Sa lt (8). A solution of 48% aq
HF in CH
3
1
) 1.9 Hz, 1H); P NMR δ δ -1.94; ESMS m/z 2061.44 (M -
2Et N - H), 2062.35 (M - 2Et N); HRMS (ESMS) calcd for
(M - Et Si 2061.5396, found 2061.5399.
3
CN (5:95, 1.5 mL) was added to compound 7 (15
3
3
-
mg, 0.01 mmol) at 0 °C. The solution was stirred at 0 °C for 2
3
115 56 2
N - H) C80H O P
h. The reaction was quenched by the addition of aq NaHCO
solution until effervescence ceased and diluted with CH Cl
3
2
â-D-Galactopyr an osyl-(1f4)-r-D-m an n opyr an oside [6-â-
D-Ga la ct op yr a n osyl-(1f4)-r-D-m a n n op yr a n osyl p h os-
p h a te 6-[â-D-ga la ctop yr a n osyl-(1 f 4)-r-D-m a n n op yr a -
n osyl p h osp h a te]] Bis-tr ieth yla m m on iu m Sa lt (11). The
global deprotection of fully protected phosphohexasaccharide
10 was carried out by same method as given for the prepara-
2
(
5 mL). The organic layer was washed with water, dried over
Na SO , and concentrated. The residue was dissolved in anhyd
CH OH (500 µL), NaOMe (15 mg) was added, the solution was
stirred overnight at rt, deionized with AG-X8 resin (H ),
filtered, and immediately neutralized with Et N. After con-
2
4
3
+
1
3
tion of compound 8 earlier: H NMR (D O) δ 1.38 (t, 9H), 3.30
2
centration, water (500 µL × 3) was evaporated off from the
(q, 6H),3.45 (m, 3H), 3.46 (m, 2H), 3.55 (m, 1H), 3.56-3.53
(m, 3H), 3.60 (m, 3H), 3.68 (m, 6H), 3.76 (m, 3H), 3.80 (m,
6H), 3.83 (m, 3H), 3.85 (m, 1H), 3.94 (m, 2H), 3.90-4.20 (m,
residue to afford tetrasaccharide phosphodiester 8 (7.9 mg,
1
1
1
9
4%): [R]
D
2 2
) 34 (c 0.15, H O); H NMR (D O; H- H COSY
3
1
assignments) δ 1.30 (t, 9H), 3.00 (q, 6H), 3.45 (m, 2H), 3.46
4H), 5.09 (d, J ) 1.8 Hz, 1H, H-1), 5.29 (brd, 2H); P NMR δ
2
-
(
(
m, 1H), 3.55 (m, 1H), 3.56-3.53 (m, 2H), 3.60 (m, 2H), 3.68
-1.29; ESMS m/z 574.12 ([M - 2Et N - 2H] ; HRMS
3
-
m, 4H), 3.76 (m, 2H), 3.80 (m, 4H), 3.83 (m, 2H), 3.85 (m,
(ESMS)t calcd for (M - Et N - H) C H O P 574.1222,
3
36 62 37
2
1
H), 3.94 (m, 1H), 4.32 (m, 1H), 4.37 (d, J ) 7.6 Hz, 1H), 4.35
found 574.1234.
(d, J ) 7.6 Hz, 1H), 5.09 (d, J ) 1.8 Hz, 1H), 5.36 (dd, J HH )
2
,3,4-Tr i-O-a cet yl-â-D-ga la ct op yr a n osyl-(1f4)-1,2,3,6-
13
1
6
7
.9 Hz, J HP ) 6.8 Hz, 1H); C NMR δ 8.5 and 45 (TEA ion),
tetr a-O-acetyl-r-D-m an n opyr an oside 6-[2,3,4-Tr i-O-acetyl-
1.37, 61.37, 62.30, 65.53, 69.28, 69.83, 70.84, 71.08, 72.13,
6
2
-O-(ter t-bu tyldim eth ylsilyl)-â-D-ga la ctop yr a n osyl-(1f4)-
,3,6-tr i-O-a cetyl-r-D-m a n n op yr a n osyl h yd r ogen p h os-
3
1
2.34, 73.69, 74.89, 76.52, 77.05, 78.14, 97.0, 102.9; P NMR
-
δ -1.29; ESMS m/z 745.38 (M - Et
3
N - H) ; HRMS (ESMS)
24 42
p h on a te] Tr ieth yla m m on iu m Sa lt (12). Compound 7 (50
-
calcd for (M - Et
45.1830.
,3,4-Tr i-O-a cet yl-â-D-ga la ct op yr a n osyl-(1f4)-1,2,3,6-
3
N - H)
C H O24P 745.1804, found
mg, 0.03 mmol) was dissolved in a saturated solution of Me
NH in anhyd CH CN (2 mL) at -20 °C, and the solution was
stirred for 3 h after which time TLC confirmed disappearance
of the starting material. Excess Me NH was removed under
2
-
7
3
2
tetr a-O-acetyl-r-D-m an n opyr an oside 6-(2,3,4-Tr i-O-acetyl-
â-D-ga la ct op yr a n osyl-(1f4)-2,3,6-t r i-O-a cet yl-r-D-m a n -
n op yr a n osyl p h osp h a te) Tr ieth yla m m on iu m Sa lt (9). A
2
reduced pressure below 30 °C, and the reaction mixture was
concentrated to give the anomeric deprotected product. To a
solution of 48% aq HF in CH
compound 7 (20 mg, 0.015 mmol) at 0 °C and stirred at 0 °C
for 2 h. The reaction was quenched by the addition of aq
3
CN (5:95, 5 mL) was added to
stirred solution of imidazole (60 mg, 8.7 mmol) in anhyd CH
CN (2.50 mL) at 0 °C were added PCl (100 µL, 1.12 mmol)
and Et N (300 µL, 2.15 mmol). The mixture was stirred for 20
min, after which time a solution of the above anomeric
deprotected compound dissolved in anhyd CH
added dropwise. The mixture was stirred at 0 °C for 2 h and
quenched with 1 M TEAB solution (pH ) 7, 1 mL). The clear
solution was stirred for 15 min, after which time CH Cl was
2 2
3
-
3
3
NaHCO
CH Cl
dried over Na
to give compound 9 (15.6 mg, 85%): H NMR (CDCl
MHz) δ 1.30 (t, 9H), 3.00 (q, 6H), 1.96-2.15 (m, 39H), 3.50
m, 4H), 3.87 (m, 1H), 3.94 (m, 1H), 4.07-4.10 (m, 1H), 4.07-
.14 (m, 1H), 4.35 (m, 1H), 4.39 (m, 4H), 4.40 (m, 1H), 4.48
m, 1H), 4.52 (m, 1H), 4.94 (d, 1H), 5.28 (m, 4H), 5.29 (m, 1H),
.43 (m, 1H), 5.45 (dd, 1H), 5.46 (m, 1H), 6.01 (d, 1H); 13C NMR
δ 8.00 and 45.50 (TEA ion), 20.48-20.7, 60.06, 60.42, 62.22,
3
solution until effervescence ceased and diluted with
(5 mL). The organic layer was washed with water,
SO , and filtered through a small silica column
2
2
3
CN (500 µL) was
2
4
1
3
, 300
(
4
(
5
added and the organic layer was washed with ice-cold water
(5 mL × 2) and cold 1 M TEAB buffer (5 mL × 2), dried over
Na
2
SO
4
1
, and concentrated to yield compound 12 (51 mg,
86%): H NMR δ 0.01 (s, 6H), 0.84 (s, 9H), 1.30 (t, 9H), 1.96-
2.15 (m, 36H, 3.10 (q, 6H),3.50 (m, 4H), 3.87 (m, 1H), 3.94 (m,
4
454 J . Org. Chem., Vol. 68, No. 11, 2003