A R T I C L E S
Fahmi et al.
a C18 reversed-phase column (250 × 10 mm). The column was washed
with 1% f 63% acetonitrile in 50 mM NH4OAc, pH 4.5, over a period
of 45 min at a flow rate of 3.5 mL/min (monitoring at 260 nm). The
remaining reaction mixture was diluted to a total volume of 600 µL of
1:1 acetonitrile-50 mM NH4OAc, pH 4.5, and purified using the same
was dried (MgSO4), filtered, and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel
column (27 × 3 cm); elution with a gradient of methanol in
dichloromethane (10-30%) afforded N-(4-pentenoyl)-O-[2,3,6-tri-O-
acetyl-4-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopy-
ranosyl]-(S)-tyrosine (23) as a colorless foam: yield 175 mg (34%);
C18 reversed-phase column. Dinucleotide derivative 20a (retention times
17.5 and 18.2 min) was recovered from the appropriate fractions by
lyophilization as a colorless solid: yield 0.8 mg (45%); mass spectrum
(ESI) m/z 1199.4 (M - H)- (theoretical m/z 1199.3).
silica gel TLC Rf 0.6 (7:3 chloroform-methanol); [R]22 +13 (c 1.4,
D
1
CHCl3); H NMR (CDCl3) δ 1.96 (s, 3H), 2.04 (s, 3H), 2.06 (s, 6H),
2.08 (s, 6H), 2.15 (s, 3H), 2.29 (m, 4H), 3.04 (m, 1H), 3.12 (m, 1H),
3.75 (m, 1H), 3.90 (t, 2H, J ) 9.0 Hz), 4.12 (m, 3H), 4.51 (m, 2H),
4.81 (br d, 1H), 4.93-5.18 (m, 6H), 5.27 (t, 1H, J ) 8.9 Hz), 5.65 (m,
1H), 6.53 (br d, 1H), 6.87 (d, 2H, J ) 8.5 Hz), 7.05 (d, 2H, J ) 8.1
Hz); mass spectrum (FAB) m/z 904.2889 (M + Na)+ (C40H51NNaO21
requires m/z 904.2851).
N-(6-Nitroveratryloxycarbonyl)-O-â-D-galactopyranosyl-(S)-ty-
rosine pdCpA Ester (20b). To a conical vial containing 2.3 mg (1.68
µmol) of N-(6-nitroveratryloxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-â-
D-galactopyranosyl)-(S)-tyrosine pdCpA ester (19b) in 350 µL of dry
methanol was added 90 µL of a 54% solution of HBF4 in diethyl ether.
The reaction mixture was stirred at room temperature for 3 h. A 20-
µL aliquot of the reaction mixture was diluted with 30 µL of 1:1
acetonitrile-50 mM NH4OAc, pH 4.5, and was analyzed by HPLC on
a C18 reversed-phase column (250 × 10 mm). The column was washed
with 1% f 63% acetonitrile in 50 mM NH4OAc, pH 4.5, over a period
of 45 min at a flow rate of 3.5 mL/min (monitoring at 260 nm). The
remaining reaction mixture was diluted to a total volume of 700 µL of
1:1 acetonitrile-50 mM NH4OAc, pH 4.5, and purified using the same
N-(4-Pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-
â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine Cyanom-
ethyl Ester (24). To a solution containing 115 mg (0.13 mmol) of
N-(4-pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-â-D-
galactopyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine (23) and 56 mg
(0.52 mmol) of anhydrous Na2CO3 in 4 mL of freshly distilled DMF
was added 66 µL (78 µg, 1.04 mmol) of chloroacetonitrile. The reaction
mixture was stirred at room temperature under argon for 16 h. The
reaction mixture was poured into 4 g of ice, acidified with 4 mL of 1
N aqueous NaHSO4, and extracted with three 10-mL portions of
dichloromethane. The combined organic phase was dried (MgSO4),
filtered, and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column (20 × 3 cm); elution
with a gradient of ethyl acetate in hexane (50-100%) afforded N-(4-
pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-â-D-galac-
topyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine cyanomethyl ester (24)
C18 reversed-phase column. Dinucleotide derivative 20b (retention times
17.5 and 18.2 min) was recovered from the appropriate fractions by
lyophilization as a colorless solid: yield 0.8 mg (40%); mass spectrum
(ESI) m/z 1201.1 (M + H)+ (theoretical m/z 1201.2), 1199.3 (M -
H)- (theoretical m/z 1199.3).
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-ty-
rosine (22). To a solution of 1.35 g (2.0 mmol) of â-D-lactose
octaacetate (21) and 0.97 g (2.4 mmol) of N-Fmoc-(S)-tyrosine in 40
mL of dry dichloromethane was added 0.76 mL (0.85 g, 6.0 mmol) of
BF3‚OEt2. The reaction mixture was stirred at room temperature under
argon for 24 h, after which another 0.76 mL (0.85 g, 6.0 mmol) of
BF3‚OEt2 was added and the reaction mixture was stirred for an
additional 16 h. The reaction was quenched by addition of 50 mL of
0.5 N aqueous NaHSO4 and extracted with two 50-mL portions of
dichloromethane. The combined organic phase was dried (MgSO4),
filtered, and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column (30 × 3 cm); elution
with a step gradient of methanol in dichloromethane (2-16%) afforded
N-(9-fluorenylmethoxycarbonyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-ty-
rosine (22), slightly contaminated with unreacted Fmoc-(S)-tyrosine
as a colorless foam: yield 720 mg (35%); silica gel TLC Rf 0.76 (7:3
chloroform-methanol); [R]22D +30 (c 1.2, CHCl3); 13C NMR (CDCl3)
δ 20.15, 20.25, 20.33, 20.37, 20.43, 46.67, 60.48, 61.62, 66.30, 68.73,
70.28, 70.47, 70.60, 71.07, 72.28, 72.44, 75.81, 79.84, 95.16, 98.40,
100.72, 101.32, 116.17, 116.87, 119.70, 124.63, 126.69, 127.44, 130.09,
140.86, 143.23, 143.28, 155.48, 155.53, 168.82, 169.35, 169.49, 169.71,
169.79, 169.85, 169.89, 170.11, 170.28; mass spectrum (CI) m/z 1021.7
(M)+ (theoretical m/z 1021.3).
as a colorless foam: yield 28 mg (53%); silica gel TLC Rf 0.69 (ethyl
1
acetate); [R]23 +5 (c 1.3, CHCl3); H NMR (CDCl3) δ 1.94, 2.03,
D
2.04, 2.05, 2.06, 2.13 (6s, 21H), 2.27 (m, 4H), 3.06 (d, 1H, J ) 6.2
Hz), 3.77 (m, 1H), 3.87 (m, 2H), 4.09 (m, 3H), 4.48 (m, 2H), 4.64 (d,
1H, J ) 5.8 Hz), 4.80 (d, 1H, J ) 5.8 Hz), 4.86 (d, 1H, J ) 7.3 Hz),
4.92-5.16 (m, 8H), 5.25 (t, 1H, J ) 8.8 Hz), 5.33 (d, 1H, J ) 3.1
Hz), 5.67 (m, 1H), 5.93 (d, 1H, J ) 7.7 Hz); 13C NMR (CDCl3) δ
19.76, 20.14, 20.27, 20.42, 28.80, 29.29, 34.92, 36.43, 48.50, 52.44,
60.42, 61.56, 66.19, 68.65, 70.30, 70.54, 70.99, 72.35, 72.41, 75.73,
98.26, 100.72, 113.42, 115.47, 116.90, 129.97, 136.24, 168.71, 169.24,
169.37, 169.70, 169.77, 169.95, 170.01, 171.72; mass spectrum (FAB)
m/z 921.3195 (M + H)+ (C42H53N2O21 requires m/z 921.3141).
N-(4-Pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-
â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine pdCpA Es-
ter (25). To a conical vial containing 17.2 mg (18.7 µmol) of N-(4-
pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-â-D-
galactopyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine cyanomethyl ester
(24) was added a solution of 5.1 mg (3.7 µmol) of the tris-
(tetrabutylammonium) salt of pdCpA11i in 68 µL of freshly distilled
acetonitrile. The reaction mixture was stirred at room temperature for
24 h. A 2-µL aliquot of the reaction mixture was diluted with 58 µL
of 1:1 acetonitrile-50 mM NH4OAc, pH 4.5, and was analyzed by
HPLC on a C18 reversed-phase column (250 × 10 mm). The column
was washed with 1% f 63% acetonitrile in 50 mM NH4OAc, pH 4.5,
over a period of 45 min at a flow rate of 3.5 mL/min (monitoring at
260 nm). The remaining reaction mixture was diluted to a total volume
of 600 µL of 1:1 acetonitrile-50 mM NH4OAc, pH 4.5, and purified
using the same C18 reversed-phase column. Dinucleotide derivative 25
(retention time 27-28 min) was recovered from the appropriate
fractions by lyophilization as a colorless solid: yield 2.7 mg (48%);
mass spectrum (FAB) m/z 1500.4049 (M + H)+ (C59H76N9O33P2
requires m/z 1500.4020).
N-(4-Pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-
â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-tyrosine (23). To 0.6
g (0.58 mmol) of N-(9-fluorenylmethoxycarbonyl)-O-[2,3,6-tri-O-acetyl-
4-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranosyl]-
(S)-tyrosine (22) in 4.8 mL of dichloromethane was added 1.2 mL of
piperidine (1.03 g, 12.1 mmol) at 25 °C for 30 min. The reaction mixture
was diluted with 20 mL of toluene, and the solvent was concentrated
under diminished pressure (T < 30 °C). The residue was dissolved in
8 mL of DMF, and 174 mg (0.88 mmol) of 4-pentenoic acid
succinimide ester was added, followed by a solution of 74 mg (0.88
mmol) of NaHCO3 in 3 mL of water. The reaction mixture was stirred
vigorously at room temperature for 24 h. The mixture was poured into
12 g of ice, acidified with 12 mL of 1 N NaHSO4, and extracted with
three 15-mL portions of dichloromethane. The combined organic phase
N-(4-Pentenoyl)-O-[4-O-(â-D-galactopyranosyl)-â-D-glucopyrano-
syl]-(S)-tyrosine pdCpA Ester (26). To a conical vial containing 1.4
mg (0.9 µmol) of N-(4-pentenoyl)-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranosyl]-(S)-ty-
9
3596 J. AM. CHEM. SOC. VOL. 129, NO. 12, 2007