J. Brabcova et al. / Journal of Molecular Catalysis B: Enzymatic 107 (2014) 31–38
33
4.49 (d, J = 7.9 Hz, 1H, H-1ꢀ␣), 4.48 (dd, J = 3.4, 11.2 Hz, 1H, H-6B),
4.47 (d, J = 7.7 Hz, H-1ꢀ), 4.22–4.00 (m, 4H, H-5, H-6A, H-6ꢀA, H-
6ꢀB), 3.86 (dt, J = 6.3, 1H, 5ꢀ-H), 3.75 (dd, J = 9.3 Hz, H-4␣), 2.15–1.96
(s, 21H, 7CH3).
over anhydrous Na2SO4, which was then removed by filtration and
concentrated under vacuum. Then, diethyl ether was added and
removed under vacuum to afford 6 as a white glassy solid (557 mg,
90%). TLC of 6: hexane:AcOEt 5:5 v/v, Rf = 0.33, HPLC (NH4H2PO4
10 mM buffer:ACN 7:3 v/v, pH 4) Rt = 5.2 min. 1H NMR (500 MHz,
CDCl3) ı: 6.42 (dd, J = 6.1 Hz, 1H, H-1), 4.95 (dd, J = 6.2, 2.0 Hz, 1H, H-
4), 4.84 (dd, J = 6.2, 3.2, 2.7 Hz, 1H, H-2), 4.43 (ddd, J = 6.7, 5.3 Hz, 1H,
H-5), 4.22–4.38 (m, 2H, H-6A, H-6B), 4.20–4.11 (dd, J = 6.2, 2.2 Hz,
1H, H-3), 2.55 (bs, 1H, OH), 2.16 (s, 3H, CH3), 2.11 (s, 3H, CH3).
2.3.3.
2-Acetamido-2-deoxy-1,3,4-tri-O-acetyl-˛-d-glucopyranose (3)
2-Acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-␣-d-
glucopyranose (720 mg, 1.85 mmol) was hydrolyzed in 100 mL
solution of sodium acetate (50 mM) (80%) and acetonitrile (20%)
at pH 5, using 3 g of the octyl-CRL immobilized preparation. When
the substrate disappeared, the aqueous solution was filtrated
and then extracted with ethyl acetate (3× 50 mL). The organic
layer was dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. The crude product was purified by flash
column chromatography (dichloromethane/methanol, 9.5:0.5) to
provide 3 (white solid, 530 mg, 82% yield). TLC of 3: CH2Cl2:CH3OH
(9:1), Rf = 0.52. HPLC (NH4H2PO4 10 mM buffer:ACN 8:2 v/v, pH
4) Rt = 5.70 min (␣-anomer), 9.33 ( anomer). 1H NMR (500 MHz,
CDCl3) ı: 6.19 (d, J = 3.3 Hz, 1H, H-1), 5.61 (d, J = 9 Hz, 1H, NH), 5.30
(t, J = 9.9 Hz, 1H, H-3), 5.16 (t, J = 9.7 Hz, 1H, H-4), 4.46 (m, J = 10.4,
8.4, 3.7 Hz, 1H, H-2), 3.81 (m, 1H, H-5), 3.59–3.71 (2dd, J = 8.5,
3.9, 12.4 Hz, 2H, H-6A, H-6B), 2.20 (s, 3H, CH3), 2.05–2.11 (2s, 6H,
2CH3), 1.96 (s, 3H, CH3).
2.3.7. 1,2,3,4-Tetra-O-acetyl-ˇ-d-galactopyranose (7)
Penta-O-acetyl-galactopyranose (320 mg, 0.82 mmol) was
hydrolyzed in 50 mL solution of sodium acetate (50 mM) (80%) and
acetonitrile (20%) at pH 5, using 3 g of octyl-TLL preparation. After 4
days, the substrate completely disappeared. The aqueous solution
was filtrated and saturated with NaCl and then extracted with
ethyl acetate (5× 50 mL). The collected organic layers were dried
over anhydrous Na2SO4, which was then removed by filtration
and concentrated under vacuum. Then, diethyl ether was added
and removed under vacuum to afford 7 as a white glassy solid
(243 mg, 85%). TLC of 7: hexane:ethyl acetate (1:1), Rf = 0.23. HPLC
(NH4H2PO4 10 mM buffer:ACN 7:3 v/v, pH4) Rt = 10.6 min. 1H NMR
(500 MHz, CDCl3) ı: 5.73 (d, J = 8.2 Hz, 1H, H-1), 5.44 (d, J = 3.3 Hz,
1H, H-4), 5.32 (t, J = 8.3 Hz, 1H, H-3), 5.13 (dd, J = 3.4, 10.4 Hz, 1H,
H-2), 3.91 (dt, J = 6.4 Hz, 1H, H-5), 3.8–3.51 (m, 2H, H-6A, H-6B),
2.14–1.97 (s, 12H, 4CH3).
2.3.4. 2,3,4-Tri-O-acetyl-1-O-methyl-ˇ-d-glucopyranose (4)
Tetra-O-acetyl-1-O-methyl-glucopyranose
(727 mg,
2.01 mmol) was hydrolyzed in 100 mL solution of sodium acetate
(50 mM) (80%) and acetonitrile (20%) at pH 5, using 3 g of octyl-CRL
preparation. After 4 h, the substrate completely disappeared. The
aqueous solution was filtrated and saturated with NaCl and then
extracted with ethyl acetate (5× 50 mL). The collected organic lay-
ers were dried over anhydrous Na2SO4, which was then removed
by filtration and concentrated under vacuum. Then, diethyl ether
was added and removed under vacuum to afford 4 as a white glassy
solid (529 mg, 82%). TLC of 4: hexane:ethyl acetate (1:1), Rf = 0.23.
HPLC (NH4H2PO4 10 mM buffer:ACN 7:3 v/v, pH 4) Rt = 6.7 min.
1H NMR (500 MHz, CDCl3) ı: 5.04 (t, J = 9.5 Hz, 1H, H-3), 5.00 (t,
J = 9.8 Hz, 1H-4), 4.94 (dd, J = 9.7, 8 Hz, 1H, H-2), 4.46 (d, J = 7.9 Hz,
1H, H-1), 3.6–3.8 (m, J = 7.9, 2, 12.5 Hz, 2H, H-6A, H-6B), 3.53 (m,
1H-5), 3.52 (s, 3H, CH3), 1.92–2.01 (s, 9H, 3CH3).
2.3.8. 2,3,4-Tetra-O-acetyl-1-thioisopropyl-˛-d-glucopyranose
(8)
1-Thioisopropyl-per-O-acetyl
glucopyranose
(681 mg,
1.67 mmol) was hydrolyzed in 100 mL solution of sodium acetate
(50 mM) (80%) and acetonitrile (20%) at pH 5, using 3 g of the octyl-
CRL immobilized preparation. When the substrate disappeared
(checked by TLC and HPLC), the aqueous solution was filtrated and
saturated with NaCl and then extracted with ethyl acetate (5×
50 mL). The collected organic layers were dried over anhydrous
Na2SO4, which was then removed by filtration and concentrated
under vacuum to afford 8 as a white glassy solid (583 mg, 96%).
TLC of 8: hexane: ethyl acetate (1:1), Rf = 0.29. 1H NMR (500 MHz,
CDCl3) ı: 5.40 (d, J = 2.9 Hz, 1H, H-4), 5.20 (t, J = 9.9 Hz, 1H, H-2),
5.08 (dd, J = 6.6, 3.1 Hz, 1H, H-3), 4.58 (d, J = 10.0 Hz, 1H, H-1),
3.78–3.68 (m, 2H, H-5, H-6A), 3.52–3.46 (m, 1H, H-6B), 3.23–3.14
(m, J = 6.6 Hz, 1H, CH), 2.16 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.99 (s,
3H, CH3), 1.30 (m, 6H, 2CH3).
2.3.5. 3,4-Di-O-acyl glucal (5)
Tri-O-acetyl-glucal (730 mg, 2.68 mmol) was hydrolyzed in
100 mL solution of sodium acetate (50 mM) (80%) and acetonitrile
(20%) at pH 5 using 3 g of octyl-CRL preparation. After the substrate
completely disappeared, the aqueous solution was filtrated and sat-
urated with NaCl and then extracted with ethyl acetate (5× 50 mL).
The collected organic layers were dried over anhydrous Na2SO4,
which was then removed by filtration and concentrated under vac-
uum. Then, after purification on a flash column, the product was
washed with diethyl ether and removed under vacuum to afford
5 as a white solid (430 mg, 70%). TLC of 5: hexane:ethyl acetate
(1:1), Rf = 0.29. HPLC (NH4H2PO4 10 mM buffer:ACN 7:3 v/v, pH 4)
Rt = 6.7 min. 1H NMR (500 MHz, CDCl3) ı: 6.49 (dd, J = 6.1 Hz, 1H,
H-1), 5.41–5.50 (m, 1H, H-3), 5.22 (dd, J = 9.0, 6.5 Hz, 1H, H-4), 4.81
(dd, J = 5.9, 2.8 Hz, 1H, H-2), 3.98–4.09 (m, 1H, H-5), 3.66–3.86 (m,
2H, H-6A, H-6B), 2.07–2.13 (2s, 6H, 2CH3).
2.3.9. 3,4-Di-O-acetyl-d-galactal (9)
Tri-O-acetyl-galactal (720 mg, 2.64 mmol) was hydrolyzed in
100 mL solution of sodium acetate (50 mM) (80%) and acetoni-
trile (20%) at pH 5, using 3 g of octyl-CRL immobilized preparation.
After 24 h, the substrate completely disappeared (checked by TLC
and HPLC). The aqueous solution was filtrated and saturated with
NaCl and then extracted with ethyl acetate (5× 50 mL). The col-
lected organic layers were dried over anhydrous Na2SO4, which was
then removed by filtration and concentrated under vacuum. Then,
diethyl ether was added and removed under vacuum to afford 9
as a white glassy solid (603 mg, 99%). TLC of 9: CH2Cl2:CH3OH 9:1
v/v, Rf = 0.67, HPLC (NH4H2PO4 10 mM buffer:ACN 7:3 v/v, pH 4)
Rt = 5.2 min. 1H NMR (500 MHz, CDCl3) ı: 6.50 (dd, J = 6.2, 1.8 Hz,
1H, H-1), 5.60–5.55 (m, 1H, H-3), 5.50–5.44 (dt, J = 7.4, 3.6 Hz, 1H,
H-4), 4.73 (dt, J = 5.9, 2.8 Hz, 1H, H-2), 4.22–4.16 (m, J = 7 Hz, 1H, H-
5), 3.83–3.61 (dd, J = 11.6, 5.8 Hz, 2H, H-6A, H-6B), 2.31 (bs, 1H, OH),
2.16 (s, 3H, CH3), 2.05 (s, 3H, CH3).
2.3.6. 4,6-Di-O-acetyl-d-glucal (6)
Tri-O-acetyl-glucal (727 mg, 2.68 mmol) was hydrolyzed in
100 mL solution of sodium acetate (50 mM) (80%) and acetonitrile
(20%) at pH 5 using 1 g of CNBr-CAL-B immobilized preparation.
After 18 h, the substrate completely disappeared. The aqueous solu-
tion was filtrated and saturated with NaCl and then extracted with
ethyl acetate (5× 50 mL). The collected organic layers were dried
2.3.10. 4,6-Di-O-acetyl-d-galactal (10)
Tri-O-acetyl-galactal (720 mg, 2.64 mmol) was hydrolyzed in
100 mL solution of sodium acetate (50 mM) (80%) and acetonitrile