784
R. V. Stick, K. A. Stubbs and A. G. Watts
was added, and the solution was kept at 25◦C for 7 days. The solvent
was then removed under reduced pressure, the residue was dissolved
in Ac2O (5 mL) that contained NaOAc (50 mg), and the solution was
heated at reflux for 10 min. The reaction was quenched by the addition
of ice/water. Usual workup (CH2Cl2) followed by flash chromatography
(EtOAc/lightpetroleum, 1 : 4to1 : 1)gavetheappropriateper-acetylated
disaccharides.
Next to elute was 1,2,4-tri-O-acetyl-6-O-(4-nitrobenzyl)-3-O-(tetra-
O-acetyl-β-d-galactopyranosyl)-β-d-glucose (115 mg, 29%), which
was isolated as an oil (Found: m/z 772.2276. C33H41NO20 requires
[M + H]+• 772.2300). δH (600 MHz) 8.16 and 7.47 (4H, AAꢀBBꢀ, Ar),
5.59 (d, J1,2 8.4, H1), 5.33 (dd, J3 ,4 3.5, J4 ,5 1.0, H4ꢀ), 5.08–5.05 (m,
ꢀ
ꢀ
ꢀ
ꢀ
H2 and H2ꢀ), 5.00 (dd, J3,4 9.5, J4,5 9.9, H4), 4.94 (dd, J2 ,3 10.4, H3ꢀ),
ꢀ
ꢀ
4.60 and 4.58 (AB, J 13.4, CH2Ar), 4.55 (d, J1 ,2 8.0, H1ꢀ), 4.15 (dd,
ꢀ
ꢀ
J5 ,6 3.9, J6 ,6 11.1, H6ꢀ), 4.04 (dd, J5 ,6 6.4, H6ꢀ), 3.95 (dd, J2,3 9.4,
H3), 3.90 (ddd, H5ꢀ), 3.76 (ddd, J5,6 2.6 and 2.5, J6,6 11.3, H5), 3.62
(dd, H6), 3.56 (dd, H6), 2.16, 2.11, 2.08, 2.02, 2.00, 1.99, and 1.94 (21H,
7 s, Me). δC (150.8 MHz) 170.3, 170.1, 170.1, 170.0, 169.3, 169.1, and
168.9 (7C, C=O), 147.3, 145.3, 127.8, and 123.5 (6C, Ar), 101.1 (C1ꢀ),
91.8 (C1), 78.3 (C3), 74.2 (C5), 72.3 (CH2Ar), 70.9 (C3ꢀ), 70.5 (C5ꢀ),
69.0 (C6), 71.3, 68.7, and 68.3 (C2, C4, and C2ꢀ), 66.7 (C4ꢀ), 60.9 (C6ꢀ),
20.8, 20.7, 20.7, 20.5, 20.4, 20.4, and 20.3 (7C, Me).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1,3,4-Tri-O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
Utilizing the above general procedure, tetra-O-acetyl-α-d-galactopyra-
nosyl fluoride (275 mg, 0.79 mmol) and tetra-O-acetyl-6-O-benzyl-
d-glucopyranose
4 (230 mg, 0.52 mmol) firstly gave 1,3,4-tri-
O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-β-d-galactopyranosyl)-β-d-
glucose (200 mg, 53%) as an oil (Found: m/z 727.2385. C33H42O18
requires [M + H]+• 727.2449). δH (600 MHz) 7.31–7.25 (m, Ph), 5.63
1,3,4-Tri-O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
(d, J1,2 8.0, H1), 5.33 (dd, J4 ,5 1.1, J3 ,4 3.5, H4ꢀ), 5.17 (dd, J2,3 9.3,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J3,4 10.0, H3), 5.10 (dd, J4,5 9.8, H4), 5.08 (dd, J1 ,2 8.0, J2 ,3 10.4,
H2ꢀ), 4.91 (dd, H3ꢀ), 4.58 (d, H1ꢀ), 4.55 and 4.40 (AB, J 12.2, CH2Ph),
Utilizing the above general procedure, tetra-O-acetyl-α-d-glucopyra-
nosyl fluoride (180 mg, 0.52 mmol) and tetra-O-acetyl-6-O-benzyl-
d-glucopyranose 4 (230 mg, 0.52 mmol) firstly gave 1,3,4-tri-O-acetyl-
6-O-benzyl-2-O-(tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucose
(147 mg, 39%) as an oil (Found: m/z 727.2455. C33H42O18 requires
[M + H]+• 727.2449). δH (600 MHz) 7.31–7.25 (m, Ph), 5.61 (d, J1,2
8.1, H1), 5.17–5.16 (m, H3 and H3ꢀ), 5.10–5.07 (m, H4 and H4ꢀ), 4.89
4.11 (dd, J5 ,6 6.1, J6 ,6 11.8, H6ꢀ), 4.06 (dd, J5 ,6 9.9, H6ꢀ), 3.92 (ddd,
H5ꢀ), 3.81 (dd, H2), 3.70 (ddd, J5,6 3.9 and 2.6, H5), 3.56 (dd, J6,6 11.1,
H6), 3.47 (dd, H6), 2.12, 2.09, 2.06, 2.02, 1.99, 1.94, and 1.84 (21H,
7 s, Me). δC (150.8 MHz) 170.3, 170.1, 170.1, 169.9, 169.6, 169.3, and
168.8 (7C, C=O), 137.4, 128.3, 127.9, and 127.7 (Ph), 101.1 (C1ꢀ), 91.7
(C1), 76.9 (C2), 75.0 (C5ꢀ), 74.7 (C3), 73.5 (C5), 73.4 (CH2Ph), 70.9
(C3ꢀ), 68.6 and 68.5 (C4 and C2ꢀ), 67.3 (C6), 66.8 (C4ꢀ), 61.2 (C6ꢀ),
20.9, 20.7, 20.7, 20.5, 20.4, 20.4, and 20.4 (7C, Me).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J1 ,2 8.0, J2 ,3 9.9, H2ꢀ), 4.55 (d, H1ꢀ), 4.45 and 4.40 (AB, J 12.5,
ꢀ
ꢀ
ꢀ
ꢀ
CH2Ph), 4.12 (dd, J5 ,6 2.7, J6 ,6 11.9, H6ꢀ), 4.06 (dd, J5 ,6 4.4, H6ꢀ),
3.83 (dd, J2,3 9.9, H2), 3.71 (ddd, J4,5 10.0, J5,6 3.6 and 2.8, H5), 3.64
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-
acetyl-β-d-galactopyranosyl)-β-d-glucose (120 mg, 33%), which was
isolated as an oil (Found: m/z 727.2445. C33H42O18 requires [M + H]+•
727.2449). δH (600 MHz) 7.31–7.23 (m, Ph), 5.60 (d, J1,2 8.2, H1), 5.32
(ddd, J4 ,5 8.2, H5ꢀ), 3.58 (dd, J6,6 11.0, H6), 3.46 (dd, H6), 2.14, 2.11,
2.07, 2.02, 1.99, 1.96, and 1.94 (21H, 7 s, Me). δC (150.8 MHz) 170.3,
170.1, 169.9, 169.2, and 168.6 (7C, C=O), 137.2, 128.1, 127.5, and
127.3 (Ph), 100.3 (C1ꢀ), 91.9 (C1), 76.8 (C2), 75.2, 68.9, 68.2, and
68.1 (C3, C4, C3ꢀ, and C4ꢀ), 73.9 (C5), 73.1 (CH2Ph), 71.8 (C5ꢀ), 71.0
(C2ꢀ), 67.1 (C6), 61.6 (C6ꢀ), 20.7, 20.7, 20.6, 20.5, 20.4, 20.4, and 20.2
(7C, Me).
ꢀ
ꢀ
(dd, J3 ,4 3.5, J4 ,5 1.0, H4ꢀ), 5.09 (dd, J2,3 7.9, H2), 5.05 (dd, J1 ,2 8.0,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,3 10.4, H2ꢀ), 4.99 (dd, J3,4 7.9, J4,5 9.8, H4), 4.91 (dd, H3ꢀ), 4.54
ꢀ
ꢀ
(d, H1ꢀ), 4.51 and 4.49 (AB, J 12.0, CH2Ph), 4.11 (dd, J5 ,6 6.8, J6 ,6
ꢀ
ꢀ
ꢀ
ꢀ
11.0, H6ꢀ), 4.06 (dd, J5 ,6 8.8, H6ꢀ), 4.00 (ddd, H5ꢀ), 3.86 (dd, H3), 3.75
(ddd, J5,6 4.9 and 2.9, H5), 3.56 (dd, J6,6 11.0, H6), 3.49 (dd, H6), 2.11,
2.07, 2.05, 2.03, 1.99, 1.94, and 1.93 (21H, 7 s, Me). δC (150.8 MHz)
170.3, 170.1, 170.1, 170.1, and 168.7 (7C, C=O), 137.5, 128.2, 127.8,
and 127.6 (Ph), 101.1 (C1ꢀ), 91.8 (C1), 78.4 (C5ꢀ), 74.1 (C5), 73.4
(CH2Ph), 72.0 (C2), 70.9 (C3ꢀ), 70.4 (C3), 68.5 and 68.5 (C4 and C2ꢀ),
68.3 (C6), 66.7 (C4ꢀ), 60.9 (C6ꢀ), 20.9, 20.7, 20.7, 20.5, 20.4, 20.4, and
20.3 (7C, Me).
ꢀ
ꢀ
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-
acetyl-β-d-glucopyranosyl)-β-d-glucose (120 mg, 32%), which was
isolated as an oil (Found: m/z 727.2445. C33H42O18 requires [M + H]+•
727.2449). δH (600 MHz) 7.38–7.25 (m, Ph), 5.62 (d, J1,2 8.2, H1), 5.14
(dd, J2 ,3 10.0, J3 ,4 10.0, H3ꢀ), 5.10–5.08 (m, H2 and H4ꢀ), 4.99 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
J3,4 7.9, J4,5 9.9, H4), 4.88 (dd, J1 ,2 7.9, H2ꢀ), 4.59 (d, H1ꢀꢀ), 4.48 and
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4.44 (AB, J 12.3, CH2Ph), 4.19 (dd, J5 ,6 2.9, J6 ,6 11.3, H6 ), 4.05 (dd,
J5 ,6 4.3, H6ꢀ), 3.84 (dd, J2,3 8.2, H3), 3.73 (ddd, J5,6 4.9 and 2.7, H5),
ꢀ
ꢀ
3.65 (ddd, J4 ,5 8.8, H5ꢀ), 3.53 (dd, J6,6 11.2, H6), 3.51 (dd, H6), 2.09,
2.06, 2.05, 2.01, 1.99, 1.96, and 1.94 (21H, 7 s, Me). δC (150.8 MHz)
170.8, 170.4, 170.2, 169.9, 169.6, 169.0, and 168.6 (7C, C=O), 137.4,
128.8, 128.0, and 127.1 (Ph), 100.7 (C1ꢀ), 91.9 (C1), 74.3 (C5), 73.3
(CH2Ph), 70.9 (C3), 70.5 (C2ꢀ), 68.6 (C4), 68.4 (C6), 68.2 (C3ꢀ), 72.2
and 67.9 (C2, C4ꢀ, and C5ꢀ), 61.8 (C6ꢀ), 20.8, 20.7, 20.6, 20.5, 20.4,
20.3, and 20.2 (7C, Me).
ꢀ
ꢀ
1,3,4-Tri-O-acetyl-6-O-(4-nitrobenzyl)-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-(4-nitrobenzyl)-3-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
Utilizing the above general procedure, tetra-O-acetyl-α-d-
galactopyranosyl fluoride (275 mg, 0.79 mmol) and tetra-O-acetyl-
6-O-(4-nitrobenzyl)-d-glucopyranose 5 (250 mg, 0.52 mmol) firstly
gave 1,3,4-tri-O-acetyl-6-O-(4-nitrobenzyl)-2-O-(tetra-O-acetyl-β-d-
galactopyranosyl)-β-d-glucose (190 mg, 47%) as an oil (Found: m/z
772.2224. C33H41NO20 requires [M + H]+• 772.2300). δH (600 MHz)
General Procedure for the Glycosynthase (E358S)-Mediated
Glycosylation of Hemiacetals 2 and 11
8.19 and 7.46 (4H, AAꢀBBꢀ, Ar), 5.65 (d, J1,2 8.0, H1), 5.35 (dd, J3 ,4
A few drops of NaOMe in MeOH (1.5 M) were added to tetra-O-acetyl-
α-d-galactopyranosyl or -glucopyranosyl fluoride in MeOH (5 mL) and
the resultant solution was stirred for 30 min. The mixture was quenched
with resin (Amberlite IR-120, H+), filtered, and the filtrate was con-
centrated. The residue was dissolved in NH4HCO3 solution (3 mL of
150 mM), the hemiacetal and Abg E358S (2 mg) were added, and the
solution was kept at 25◦C for 7 days. The solution was then concen-
trated, the residue was dissolved in Ac2O (5 mL) that contained NaOAc
(50 mg), and the solution was heated at reflux for 10 min. The reaction
was quenched by the addition of ice/water. Usual workup (CH2Cl2) fol-
lowed by flash chromatography (EtOAc/light petroleum, 1 : 4 to 1 : 1)
gave the appropriate per-acetylated disaccharide(s).
ꢀ
ꢀ
ꢀ
ꢀ
3.5, J4 ,5 1.1, H4ꢀ), 5.24 (dd, J2,3 8.9, J3,4 9.3, H3), 5.14 (dd, J1 ,2
ꢀ
ꢀ
8.0, J2 ,3 9.9, H2ꢀ), 5.06 (dd, J4,5 8.4, H4), 4.94 (dd, H3ꢀ), 4.65 and
ꢀ
ꢀ
4.63 (AB, J 13.2, CH2Ar), 4.59 (d, H1ꢀ), 4.13 (dd, J5 ,6 3.4, J6 ,6 10.9,
ꢀ
ꢀ
ꢀ
ꢀ
H6ꢀ), 4.05 (dd, J5 ,6 6.4, H6ꢀ), 3.98 (ddd, H5ꢀ), 3.84 (dd, H2), 3.68 (dd,
J5,6 2.4, J6,6 11.1, H6), 3.65 (ddd, J5,6 4.1, H5), 3.56 (dd, H6), 2.11,
2.09, 2.08, 2.05, 2.01, 1.99, and 1.95 (21H, 7 s, Me). δC (150.8 MHz)
170.4, 170.3, 170.1, 170.1, 169.9, 169.3, and 168.8 (7C, C=O), 147.4,
145.2, 127.8, and 123.6 (6C,Ar), 101.3 (C1ꢀ), 91.8 (C1), 75.3 (C5), 74.4
(C5ꢀ), 73.5 (C3), 72.3 (CH2Ar), 70.9 (C3ꢀ), 69.1 (C4), 68.6 (C2ꢀ), 68.5
(C6), 67.8 (C2), 66.8 (C4ꢀ), 61.1 (C6ꢀ), 20.8, 20.7, 20.6, 20.5, and 20.4
(7C, Me).
ꢀ
ꢀ