H. Ochiai et al. / Carbohydrate Research 344 (2009) 592–598
597
molecular sieves (905 mg) in CH2Cl2 (7.5 mL) was added AgOTf
(255 mg 0.99 mmol) at 0 °C. After stirring at room temperature
overnight, the mixture was filtered through a Celite pad and the fil-
trate was concentrated. The resulting residue was subjected to sil-
ica gel column chromatography (hexanes–EtOAc 5:1) to afford 3
(304 mg, 69%) as a white amorphous powder. 1H NMR (400 MHz,
CDCl3, TMS) d 7.96–7.94 (2H, m, Ar), 7.90–7.86 (4H, m, Ar), 7.81–
7.78 (2H, m, Ar), 7.56–7.18 (27H, m, Ar), 5.68 (1H, t, J = 9.6 Hz, H-
3II), 5.55 (1H, t, J = 9.6 Hz, H-4II), 5.48 (1H, dd, J = 9.7, 8.3 Hz, H-
2II), 5.04 (1H, d, J = 11.0 Hz, CH2Ph), 4.90 (1H, d, J = 8.2 Hz, H-1II),
4.85 (1H, d, J = 12.4 Hz, CH2Ph), 4.76–4.73 (2H, m, CH2Ph ꢂ 2),
4.58 (1H, d, J = 11.9 Hz, CH2Ph), 4.42 (1H, dd, J = 12.4, 3.2 Hz, H-
6IIa), 4.40 (1H, d, J = 11.9 Hz, CH2Ph), 4.25 (1H, dd, J = 12.1,
5.3 Hz, H-6IIb), 4.19 (1H, d, J = 7.8 Hz, H-1I), 4.06 (1H, t, J = 9.2 Hz,
H-4I), 3.75 (1H, m, H-5II), 3.69 (1H, dd, J = 11.5, 3.2 Hz, H-6Ia),
3.54 (1H, m, H-6Ib), 3.42–3.34 (2H, m, H-2I, H-3I), 3.14 (1H, m,
H-5I); 13C NMR (100 MHz, CDCl3) d 165.90, 165.61, 165.03 and
164.72 (COPh), 138.18, 137.84, 136.66, 133.37, 133.14, 132.98
and 129.66–127.45 (Ar), 100.31 (C-1I), 100.26 (C-1II), 80.66 (C-3I),
76.69 (C-4I), 75.08 (CH2Ph), 74.38 (C-5I), 73.52 (CH2Ph), 72.91 (C-
3II), 72.09 (C-2II), 71.92 (C-5II), 70.77 (CH2Ph), 69.72 (C-4II), 67.31
(C-6I), 65.85 (C-2I), 62.96 (C-6II).
concentrated. Silica gel column chromatography (CH2Cl2–MeOH
40:1) of the residue afforded 2,3,4,6-tetra-O-acetyl-b- -glucopyr-
anosyl-(1?4)-2-acetamido-1,3,6-tri-O-acetyl-2-deoxy- -gluco-
pyranose 5 (152 mg, 95% from 4) as a white amorphous powder.
To a solution of this disaccharide (72 mg, 0.11 mmol) in dichlo-
roethane (11 mL) containing activated 4 Å molecular sieves (1.3 g)
D
D
were added 2,4,6-collidine (70
lL, 0.53 mmol), TMSBr (206 lL,
1.6 mmol), and BF3ꢁOEt2 (200 L, 1.6 mmol). The reaction mixture
l
was stirred at room temperature overnight. The mixture was di-
luted with CH2Cl2, filtered through a Celite pad, washed sequen-
tially with saturated NaHCO3 and brine, dried over MgSO4,
filtered, and concentrated. The residue was chromatographed
(CH2Cl2–MeOH 90:1) to give 6 (42 mg, 64%) as a white amorphous
powder. 1H NMR (400 MHz, CDCl3, TMS) d 5.92 (1H, d, J = 7.3 Hz, H-
1I), 5.66 (1H, d, J = 2.3 Hz, H-3I), 5.19 (1H, t, J = 9.4 Hz, H-3II), 5.10
(1H, t, J = 9.6 Hz, H-4II), 5.00 (1H, t, J = 9.0 Hz, H-2II), 4.72 (1H, d,
J = 7.8 Hz, H-1II), 4.29 (1H, dd, J = 12.2, 4.4 Hz, H-6IIa), 4.20 (1H,
dd, J = 11.9, 2.3 Hz, H-6Ia), 4.14 (1H, dd, J = 12.3, 2.3 Hz, H-6IIb),
4.12 (1H, m, H-2I), 4.04 (1H, dd, J = 12.2, 5.7 Hz, H-6Ib), 3.79 (1H,
ddd, J = 9.9, 4.3, 2.5 Hz, H-5II), 3.64 (1H, d, J = 9.2 Hz, H-4I), 3.48
(1H, m, H-5I), 2.11–2.00 (21H, m, COCH3, CH3 of oxazoline); 13C
NMR (100 MHz, CDCl3) d 170.68, 170.54, 170.24 and 169.34
(COCH3), 166.82 (O–C@N of oxazoline), 102.05 (C-1II), 98.99 (C-
1I), 78.12 (C-4I), 72.99 (C-3II),71.97 (C-5II), 71.27 (C-2II), 70.14 (C-
3I), 68.05 (C-4II), 67.44 (C-5I), 64.81 (C-2I), 63.50 (C-6I), 61.80 (C-
6II), 20.96, 20.75, 20.69, 20.55 and 20.50 (COCH3), 13.92 (CH3 of
oxazoline).
4.4. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
D-glucopyranosyl-(1?4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-
D
-glucopyranoside (4)
To a solution of 3 (294 mg, 0.28 mmol) in CHCl3 (3.0 mL) were
added pyridine (3.0 mL) and thioacetic acid (3.0 mL) at room tem-
perature. After stirring for 48 h, the mixture was concentrated and
purified by silica gel column chromatography (hexanes–EtOAc 1:1)
to afford 4 (265 mg, 89%) as a white amorphous powder. 1H NMR
(400 MHz, CDCl3, TMS) d 7.99–7.97 (2H, m, Ar), 7.93–7.88 (4H,
m, Ar), 7.83–7.81 (2H, m, Ar), 7.56–7.19 (27H, m, Ar), 5.78 (1H, t,
J = 9.6 Hz, H-3II), 5.68 (1H, d, J = 8.7 Hz, NHI), 5.60 (1H, t, J =
9.9 Hz, H-4II), 5.49 (1H, dd, J = 9.9, 8.1 Hz, H-2II), 4.88 (1H, d,
J = 8.2 Hz, H-1II), 4.83 (1H, d, J = 11.9 Hz, CH2Ph), 4.73 (1H, d,
J = 11.9 Hz, CH2Ph), 4.67–4.63 (3H, m, CH2Ph ꢂ 2, H-1I), 4.51 (1H,
dd, J = 12.3, 3.2 Hz, H-6IIa), 4.39 (1H, d, J = 11.9 Hz, CH2Ph), 4.37–
4.29 (2H, m, H-6IIb, CH2Ph), 4.11 (1H, t, J = 6.9 Hz, H-4I), 3.90 (1H,
t, J = 7.1 Hz, H-3I), 3.82 (1H, m, H-5II), 3.74–3.68 (2H, m, H-2I, H-
6Ia), 3.63 (1H, dd, J = 10.1, 4.1 Hz, H-6Ib), 3.44 (1H, m, H-5I), 1.86
(3H, s, COCH3); 13C NMR (100 MHz, CDCl3) d 170.04 (COCH3),
165.97, 165.63, 165.19 and 165.03 (COPh), 138.52, 137.97,
137.43, 133.44, 133.39 and 133.19–127.35 (Ar), 99.78 (C-1II),
99.62 (C-1I), 77.78 (C-3I), 76.02 (C-4I), 74.40 (C-5I), 73.44 (CH2Ph),
72.65 (C-3II), 72.06 (C-2II), 71.99 (C-5II), 70.58 (CH2Ph), 69.57 (C-
4II), 68.33 (C-6I), 62.84 (C-6II), 54.18 (C-2I), 23.28 (COCH3).
4.6. 2-Methyl-4,5-dihydro-[4-O-(b-
D-glucopyranosyl)-1,2-
dideoxy- -glucopyranoso][2,1-d]-1,3-oxazole (7)
a-D
To a solution of 6 (37 mg, 59
lmol) in MeOH (3 mL) was added
NaOMe in MeOH (0.5 M, 11.8 L, 5.9
l
l
mol). After stirring at room
temperature for 2 h, the reaction mixture was concentrated to dry-
ness. The residue was dissolved in water and lyophilized to give
the oxazoline 7 (22 mg, quantitative). 1H NMR (400 MHz, D2O) d
5.97 (1H, d, J = 7.3 Hz, H-1I), 4.37 (1H, d, J = 7.8 Hz, H-1II), 4.28
(1H, m, H-3I), 4.08 (1H, m, H-2I), 3.80 (1H, m, H-6Ia), 3.70 (1H,
m, H-6aII), 3.64–3.53 (3H, m, H-6Ib, H-4I, H-6bII), 3.37–3.22 (4H,
m, H-3II, H-5II, H-5, H-4II), 3.16 (1H, dd, J = 8.5 Hz, H-2II), 1.94
(3H, s, CH3 of oxazoline); 13C NMR (100 MHz, D2O) d 168.33 (O–
C@N of oxazoline), 104.10 (C-1II), 99.83 (C-1I), 78.38 (C-4I), 75.99
(C-5II), 75.55 (C-3II), 73.16 (C-2II), 70.90 (C-3I), 69.50 (C-5I), 69.07
(C-4II) 65.23 (C-2I), 61.66 (C-6II), 60.65 (C-6I), 12.96 (CH3 of
oxazoline).
4.7. Endo-A-catalyzed transglycosylation with Glc-b-(1?4)-
GlcNAc oxazoline (7)
4.5. 2-Methyl-4,5-dihydro-[4-O-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)-3,6-di-O-acetyl-1,2-dideoxy-
glucopyranoso][2,1-d]-1,3-oxazole (6)
a-
D-
A solution of the disaccharide oxazoline 7 (625
and the GlcNAc-Asn-Fmoc 8 (318 g, 0.57 mol) in a phosphate
buffer (50 mM, pH 7.5, 125 L) was incubated at 30 °C with
lg, 1.7 lmol)
l
l
l
Compound 4 (255 mg, 0.24
l
mol) was dissolved in 1:1 (v/v)
Endo-A (500 mU). After 48 h, the reaction was terminated by 10%
TFA and the mixture was analyzed by RP-HPLC. The new peak
was isolated and subject to MS analysis. MALDI-TOF MS of the
products; m/z 923.70 [M+H]+ (9a), 1289.40 [M+H]+ (9b), 1653.28
[M+H]+ (9c).
CH2Cl2–MeOH (16 mL), and a solution of 0.5 M NaOMe in MeOH
(0.7 mL, 0.35 mmol) was added. After being stirred at room tem-
perature overnight, the solution was neutralized with Dowex
50W (H+), filtered, and concentrated. To a solution of the residue
in 100:1 (v/v) MeOH–AcOH (8.1 mL) was added 20% palladium(II)
hydroxide on activated carbon (80 mg), and the reaction mixture
was vigorously stirred at room temperature under hydrogen atmo-
sphere for 4 h. The mixture was filtered through a Celite pad and
the filtrate was concentrated in vacuo. Pyridine (10 mL) and Ac2O
(10 mL) were added, and the mixture was stirred at room temper-
ature overnight. The solution was concentrated, diluted with
CH2Cl2, and washed sequentially with 1 M HCl, saturated NaHCO3,
and brine. The organic layer was dried over MgSO4, filtered, and
4.8. A typical procedure for the Endo-A-catalyzed
polymerization of Glc-b-(1?4)-GlcNAc oxazoline (7)
A solution of oxazoline 7 (2.28 mg, 6.24
lmol) in a phosphate
buffer (50 mM, pH 7.5, 31.2 L) was incubated at 30 °C with
l
Endo-A (624 mU) for 48 h. A white precipitate that was formed dur-
ing the reaction was separated by centrifugation and washed with
distilled water (200 lL) three times to give the high-molecular