R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
10.1, H-6a), 4.39 (d, 1H, J1′,2′ 7.9, H-1′), 4.23 (dd, 1H, J2,3 8.4, H-2),
3.99-3.79 (m, 1H, H-4), 3.97 (d, 1H, J3′-4′ 2.6, H-4′), 3.93 (m, 1H,
H-5), 3.82 (m, 1H, H-3), 3.61-3.46 (m, 3H, H-5′, H-6a′, H-6b′), 3.53
(dd, 1H, J2′-3′ 10.1, H-3′), 3.44 (s, 3H, OCH3), 2.82 (s, 3H, CH3SO2),
2.07 (s, 3H, CH3C(O)O), 1.99 (s, 3H, CH3C(O)NH). 13C NMR (CDCl3,
125 MHz): δ 170.78, 170.51, 138.47, 138.10, 137.99, 128.84, 128.70,
128.58, 128.55, 128.48, 128.28, 128.15, 128.12, 127.95, 127.73, 101.77,
100.19, 80.00, 77.66, 77.43, 77.23, 76.81, 75.50, 74.93, 74.08, 73.78,
72.89, 72.63, 72.43, 71.90, 69.42, 68.39, 56.92, 48.41, 37.26, 29.92,
23.34, 21.27. MALDI-TOF: m/z 900.5 [M + Na]+. Anal. Calcd for
C46H55NO14: C, 62.93; H, 6.31; N, 1.60; O, 25.51; S, 3.65. Found: C,
63.05; H, 6.36; N, 1.75. [R]D ) -24.8 (c 0.13, CHCl3).
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-6-O-methanesulfonyl-
4-O-(3,4,6-tri-O-benzyl-â-D-galactopyranosyl)-â-D-glucopyrano-
side (13). Sodium methoxide (5.4 mg, 0.1 mmol) was added to a
solution of 12 (447 mg, 0.51 mmol) in methanol (10 mL). After stirring
at room temperature for 48 h, TLC (hexane/acetone, 1/1, v/v) indicated
completion of the reaction; the mixture was then neutralized with
Dowex 50H+ until pH ) 7.0, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (gradient hexane/acetone,
3/1 to 1/1, v/v) to afford compound 13 (418 mg, 98%) as a white foam.
1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.88 (d,
1H, JNH,2 7.7, NH), 4.86, 4.72 (AB q, 2H, JAB 11.5, OCH2Ph), 4.81,
4.54 (AB q, 2H, JAB 11.3, OCH2Ph), 4.72, 4.57 (AB q, 2H, JAB 11.8,
OCH2Ph), 4.60, 4.40 (AB q, 2H, JAB 12.3, OCH2Ph), 4.62 (d, 1H, J1,2
7.4, H-1), 4.60 (m, 1H, H-6b), 4.47 (m, 1H, H-6a), 4.44 (d, 1H, J1′,2′
7.7, H-1′), 4.00 (dd, 1H, J3,4 8.5, H-3), 3.99 (dd, 1H, J3′,4′ 3.8, H-3′),
3.96 (dd, 1H, J2′,3′ 9.6, H-2′), 3.86 (m, 2H, H-4, H-5), 3.80 (m, 1H,
H-4′), 3.64 (m, 1H, H-2), 3.60 (dd, 1H, J6a′-6b′ 11.8, H-6b′), 3.57 (dd,
1H, J6a′-5 2.2, H-6a′), 3.48-3.30 (m, 1H, H-5′), 3.46 (s, 3H, OCH3),
2.96 (s, 3H, CH3SO2), 1.85 (s, 3H, CH3C(O)NH). MALDI-TOF: m/z
858.0 [M + Na]+. Analytical data are in agreement with Lemieux et
al.25
tion of the filtrate, the crude material was purified by column
chromatography (Iatrobeads, chloroform/methanol/water, 74/24/2, v/v/
v) to afford compound 3 (21 mg, 95%) as a white amorphous solid.
MALDI-TOF: m/z 402.3 [M + Na]+. [R]D ) -15.6 (c 0.79, D2O).
1
For H NMR and 13C NMR data, see Supporting Information.
1-O-tert-Butyldimethylsilyl-2-O-methanesulfonyloxy-ethane (15).
Sodium hydride (60% in oil suspension, 28.2 mg, 0.71 mmol) was
added to a solution of ethylene glycol (2.05 g, 33 mmol) in dry N,N-
dimethylformamide (25 mL). After stirring at room temperature for
30 min, tert-butyldimethylsilyl chloride (5.0 g, 33 mmol) was added
dropwise. The mixture was left stirring at room temperature for 16 h.
Triethylamine (6.91 mL, 50 mmol) was then added, followed by slow
addition of methanesulfonyl chloride (3.6 mL, 46 mmol). TLC (hexane/
ethyl acetate, 3/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol, diluted with ethyl acetate (100
mL), and washed successively with water (5 × 20 mL), brine (20 mL),
followed by drying over MgSO4. After evaporation of the solvent, the
residue was then purified by flash chromatography (gradient hexane/
ethyl acetate, 5/1 to 3/2, v/v) to afford compound 15 (5.9 g, 70%) as
1
a white foam. H NMR (CDCl3, 300 MHz): 4.58 (dd, 2H, J 4.7, 9.1,
MsOCH2), 3.88 (dd, 2H, J 4.7, 9.1, TBDMSOCH2), 3.02 (s, 3H, CH3-
SO2), 0.90 (s, 9H, Si(CH3)3), 0.09 (2s, 6H, Si(CH3)2). 13C NMR (CDCl3,
125 MHz): δ 71.22, 61.28, 37.60, 25.96, 25.87, 18.37, -5.27. MALDI-
TOF: m/z 277.1 [M + Na]+. Anal. Calcd for C9H22O4SSi: C, 42.49;
H, 8.72; O, 25.15; S, 12.60; Si, 11.04. Found: C, 42.65; H, 8.76; O,
25.26.
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-
â-D-galactopyranosyl)-6-di-O-(2-O-methanesulfonyl-ethane)-â-D-glu-
copyranoside (18). Sodium hydride (60% in oil suspension, 28.2 mg,
0.71 mmol) was added to a solution of 10 (67 mg, 0.09 mmol) in dry
N,N-dimethylformamide (10 mL). After stirring for 30 min at 50 °C,
15 (336 mg, 1.32 mmol) was added dropwise. The mixture was stirred
at 50 °C for 16 h. TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. The reaction mixture was quenched with
methanol, diluted in ethyl acetate (30 mL), washed successively with
water (5 × 10 mL), brine (10 mL), followed by drying over MgSO4.
After evaporation of the solvent, the residue was dissolved in acetonitrile
(20 mL), and tetrafluoroboric acid (48% in water, 21.3 µL, 0.16 mmol)
was added. The reaction mixture was stirred at room temperature for
5 min after which TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. The mixture was neutralized with triethyl-
amine (23.0 µL, 0.16 mmol) and concentrated in vacuo. The residue
was then diluted in dichloromethane (10 mL), successively washed with
a saturated solution of NaHCO3 (5 mL), water (2 × 5 mL), brine (5
mL), followed by drying over MgSO4. After concentration of the filtrate,
the crude mixture was purified by flash chromatography (gradient
hexane/acetone, 3/1 to 1/1, v/v). The dried product was then dissolved
in pyridine (5 mL), and methanesulfonyl chloride (7.0 µL, 0.09 mmol)
was added to the stirred mixture at -20 °C. The reaction mixture was
then left stirring at room temperature for 2 h. TLC (chloroform/
methanol, 9/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol. Toluene was then added to the
crude mixture which was concentrated in vacuo. The reaction mixture
was diluted with dichloromethane (10 mL) and then washed succes-
sively with water (2 × 5 mL), brine (5 mL), followed by drying over
MgSO4. After evaporation of the solvent, the residue was purified by
flash silica gel chromatography (gradient hexane/acetone, 3/1 to 1/1,
v/v) to afford compound 18 (37 mg, 47%) as a white amorphous solid.
1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.64 (d,
1H, JNH,2 8.3, NH), 4.92, 4.38 (AB q, 2H, JAB 12.3, OCH2Ph), 4.88,
4.56 (AB q, 2H, JAB 12.3, OCH2Ph), 4.75, 4.61 (AB q, 2H, JAB 11.8,
OCH2Ph), 4.67 (d, 1H, J1,2 7.4, H-1), 4.50 (m, 2H, H-2, H-5′), 4.36,
4.28 (AB q, 2H, JAB 11.4, OCH2Ph), 4.33 (d, 1H, J1′,2′ 8.0, H-1′), 4.1-
4.0 (m, 1H, H-4), 4.0-3.88 (m, 1H, H-5), 3.92 (dd, 1H, J3,4 8.4 H-3),
3.84-3.44 (m, 2H, H-6b, H-6a), 3.80 (d, 1H, J3′,4′ 3.0, H-4′), 3.72 (m,
1H, H-3′), 3.58 (m, 1H, H-6b′), 3.45 (s, 3H, OCH3), 3.42 (dd, 1H, J2,3
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-2′,6-anhydro-4-O-(3,4,6-
tri-O-benzyl-â-D-galactopyranosyl)-â-D-glucopyranoside (14). So-
dium hydride (60% in oil suspension, 19.3 mg, 0.48 mmol) was added
to a solution of 13 (134 mg, 0.16 mmol) in dry N,N-dimethylformamide
(20 mL). After stirring at room temperature for 16 h, TLC (chloroform/
methanol, 9/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol, diluted in ethyl acetate (100 mL),
and successively washed with water (2 × 20 mL) and brine (20 mL),
followed by drying over MgSO4. After evaporation of the solvent, the
residue was then purified by flash silica gel chromatography (hexane/
acetone, gradient 3/1, 3/2 followed by 1/1, v/v) to afford compound
1
14 (83 mg, 70%) as a white foam. H NMR (CDCl3, 300 MHz): δ
7.40-7.10 (m, 20H, arom), 5.40 (d, 1H, JNH,2 7.4, NH), 4.96, 4.62
(AB q, 2H, JAB 11.3, OCH2Ph), 4.93, 4.60 (AB q, 2H, JAB 11.6, OCH2-
Ph), 4.81, 4.70 (AB q, 2H, JAB 12.1, OCH2Ph), 4.70 (d, 1H, J1,2 7.2,
H-1), 4.48, 4.42 (AB q, 2H, JAB 11.8, OCH2Ph), 4.37 (d, 1H, J1′,2′ 7.4,
H-1′), 4.20 (dd, 1H, J6b,5 6.9, J6b,6a 13.5, H-6b), 4.09 (m, 1H, H-3),
4.02 (dd, 1H, J6a,5 3.3, H-6a), 3.89 (d, 1H, J3′,4′ 2.5, H-4′), 3.82 (dd,
1H, J3,4 8.5, J4,5 9.6, H-4), 3.75 (m, 1H, H-5), 3.68-3.62 (m, 3H, H6b′,
H-6a′, H-5′), 3.61 (m, 1H, H-2′), 3.49 (m, 1H, H-3′), 3.46 (s, 3H,
OCH3), 3.28 (m, 1H, H-2), 1.82 (s, 3H, CH3C(O)NH). 13C NMR
(CDCl3, 125 MHz): δ 170.39, 138.79, 138.63, 137.92, 128.47, 128.23,
127.88, 128.68, 127.59, 127.50, 103.75, 101.03, 85.83, 80.19, 78.83,
78.66, 75.27, 75.08, 74.98, 74.78, 74.31, 73.70, 73.49, 68.74, 57.17,
56.86, 29.95, 23.86. MALDI-TOF: m/z 762.2 [M + Na]+. Anal. Calcd
for C43H49NO10: C, 69.81; H, 6.68; N, 1.89; O, 21.63. Found: C, 69.95;
H, 6.76; N, 1.92. [R]D ) -20.8 (c 0.27, CH2Cl2).
Methyl 2-Acetamido-2-deoxy-2′,6-anhydro-4-O-(â-D-galactopy-
ranosyl)-â-D-glucopyranoside (3). 10% palladium on charcoal (55.0
mg) was added to a solution of 14 (43 mg, 0.058 mmol) in ethanol (3
mL). The mixture was vigorously stirred under an atmosphere of
hydrogen for 16 h. TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. After filtration using Celite and concentra-
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5971