Detection of â-Galactosidase ActiVity by 19F NMR
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1997
20.54 (12s, 12 CH3CO), 19.76 (s, CH3-3). ESI-MS m/z 1178 [M+]
(28%), 1179 [M + 1] (12%). Anal. Calcd for C50H64NO30F: C,
50.96, H, 5.48, N, 1.19. Found: C, 50.92, H, 5.44, N, 1.16.
3-O-(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-r4,r5-di-O-
(2,3,4,6-tetra-O-acetyl-r-D-mannopyranosyl)-6-fluoro-
pyridoxol 11: 1.01 g, 78%, syrup, Rf 0.35 (1:3 cyclohexane/EtOAc).
NMR (CDCl3) δH 4.80 (1H, d, J1′,2′ ) 8.2 Hz, H-1′), 5.13 (1H, dd,
J2′,3′ ) 9.8 Hz, H-2′), 5.36 (1H, dd, J3′,4′ ) 4.2 Hz, H-3′), 5.30
(1H, dd, J4′,5′ ) 3.6 Hz, H-4′), 4.01 (1H, m, H-5′), 4.33 (1H, dd,
J5′,6a′ ) 3.2 Hz, J6a′,6b′ ) 10.0 Hz, H-6a′), 4.11 (1H, dd, J5′,6b′ ) 4.6
Hz, H-6b′), 4.71 (2H, d, J1′′,2′′ ) J1′′′,2′′′ ) 2.4 Hz, H-1′′ and H-1′′′),
4.74 (2H, dd, J2′′,3′′ ) J2′′′,3′′′ ) 6.2 Hz, H-2′′ and H-2′′′), 5.22 (2H,
dd, J3′′,4′′ ) J3′′′,4′′′ ) 3.8 Hz, H-3′′ and H-3′′′), 3.95 (2H, dd, J4′′,5′′
) J4′′′,5′′′ ) 2.0 Hz, H-4′′ and H-4′′′), 4.02 (2H, m, H-5′′ and H-5′′′),
4.11 (2H, dd, J5′′,6a′′ ) J5′′′,6a′′′ ) 2.0 Hz, J6a′′,6b′′ ) J6a′′,6b′′ ) 7.4 Hz,
H-6a′′ and H-6a′′′), 4.07 (2H, dd, J5′′,6b′′ ) J5′′′,6b′′′ ) 5.6 Hz, H-6b′′
and H-6b′′′), 4.87 (2H, d, JCH2-4a,CH2-4b ) JCH2-5a,CH2-5b ) 13.6
Hz, CH2-4a and CH2-5b), 4.67 (1H, d, JCH2-4a,CH2-4b ) JCH2-5a,CH2-5b
) 13.6 Hz, CH2-4b and CH2-5a), 2.33 (3H, s, CH3-2), 2.07, 2.04,
2.03, 2.00, 1.99, 1.98, 1.97, 1.96, 1.95, 1.94, 1.93, 1.92 (36H, 12s,
12 CH3CO). δC 171.27, 171.23, 171.15, 171.06, 170.87, 170.83,
170.76, 170.63, 170.58, 170.44, 170.29, 170.25 (12 CH3CO), 153.06
(1H, dd, J5′,6a′ ) 3.6 Hz, J6a′,6b′ ) 10.2 Hz, H-6a′), 4.31 (1H, dd,
J5′,6b′ ) 4.8 Hz, H-6b′), 4.84 (2H, d, J1′′,2′′ ) J1′′′,2′′′ ) 2.6 Hz, H-1′′
and H-1′′′), 4.68 (2H, dd, J2′′,3′′ ) J2′′′,3′′′ ) 6.0 Hz, H-2′′ and H-2′′′),
5.02 (2H, dd, J3′′,4′′ ) J3′′′,4′′′ ) 3.6 Hz, H-3′′ and H-3′′′), 4.05 (2H,
dd, J4′′,5′′ ) J4′′′,5′′′ ) 2.2 Hz, H-4′′ and H-4′′′), 3.94 (2H, m, H-5′′
and H-5′′′), 4.21 (2H, dd, J5′′,6a′′ ) J5′′′,6a′′′ ) 2.4 Hz, J6a′′,6b′′ ) J6a′′,6b′′
) 8.4 Hz, H-6a′′ and H-6a′′′), 4.17 (2H, dd, J5′′,6b′′ ) J5′′′,6b′′′ ) 6.5
Hz, H-6b′′ and H-6b′′′), 4.77 (2H, d, JCH2-4a,CH2-4b ) JCH2-5a,CH2-5b
) 11.6 Hz, CH2-4a and CH2-5b), 4.57 (1H, d, JCH2-4a,CH2-4b
)
JCH2-5a,CH2-5b ) 11.6 Hz, CH2-4b and CH2-5a), 2.45 (3H, s, CH3-
2), 4.30∼3.70 (12H, br, HO-2′, 3′, 4′, 6′, 2′′, 3′′, 4′′, 6′′, 2′′′, 3′′′,
4′′′, and 6′′′, exchangeable with D2O). δC 151.07 (d, 3JF-C ) 15.3
Hz, Py-C2), 148.61 (d, 4JF-C ) 4.8 Hz, Py-C3), 144.27 (d, 3JF-C
)
2
3.6 Hz, Py-C4), 116.29 (d, JF-C ) 32.0 Hz, Py-C5), 157.25 (d,
1JF-C ) 233.5 Hz, Py-C6), 103.68 (s, C-1′), 98.56 (s, C-1′′), 98.68
(s, C-1′′′), 71.76 (s, C-2′), 70.66 (s, C-2′′), 70.86 (s, C-2′′′), 72.38
(s, C-3′), 71.46 (s, C-3′′), 71.32 (s, C-3′′′), 70.48 (s, C-4′), 66.87
(s, C-4′′), 67.90 (s, C-4′′′), 73.64 (s, C-5′), 72.20 (s, C-5′′), 72.65
(s, C-5′′′), 62.77 (s, C-6′), 63.56 (s, C-6′′), 64.83 (s, C-6′′′), 57.54
(s, CH2-4), 58.41 (s, CH2-5), 20.12 (s, CH3-3). ESI-MS m/z 673
[M+] (5%), 674 [M + 1] (9%). Anal. Calcd for C26H40NO18F: C,
46.34, H, 5.99, N, 2.08. Found: C, 46.31, H, 5.97, N, 2.05.
3
4
(d, JF-C ) 16.0 Hz, Py-C2), 149.41 (d, JF-C ) 4.6 Hz, Py-C3),
3-O-Benzyl-6-fluoropyridoxol 14. To a well-stirred CH2Cl2 (10
mL)/H2O (10 mL) biphasic mixture (pH 10∼11) of 1 (0.50 g, 2.67
mmol) and TBAB (0.10 g, 0.31 mmol), a solution of benzyl bromide
(0.51 g, 2.94 mmol, 1.1 equiv) in CH2Cl2 (10 mL) was added
dropwise over a period of 4∼5 h, while the reaction temperature
was maintained at 50 °C, and the stirring continued for an additional
hour. Products were extracted (CH2Cl2, 4 × 20 mL), washed free
of alkali, dried (Na2SO4), and concentrated, and the residue was
purified by column chromatography on silica gel with 1:2 cyclo-
hexane/EtOAc to afford major product 14 (0.56 g, 76%), white
crystalline, Rf 0.38 (1:2 cyclohexane/EtOAc). NMR (CDCl3) δH
3
2
145.38 (d, JF-C ) 4.6 Hz, Py-C4), 117.21 (d, JF-C ) 31.3 Hz,
Py-C5), 159.55 (d, 1JF-C ) 235.0 Hz, Py-C6), 103.62 (s, C-1′), 98.32
(s, C-1′′), 98.61 (s, C-1′′′), 70.75 (s, C-2′), 70.22 (s, C-2′′), 70.26
(s, C-2′′′), 71.83 (s, C-3′), 70.36 (s, C-3′′), 70.39 (s, C-3′′′), 69.38
(s, C-4′), 67.04 (s, C-4′′), 68.60 (s, C-4′′′), 72.54 (s, C-5′), 71.90
(s, C-5′′), 72.45 (s, C-5′′′), 61.47 (s, C-6′), 62.46 (s, C-6′′), 63.53
(s, C-6′′′), 56.44 (s, CH2-4), 56.46 (s, CH2-5), 21.29, 21.21, 21.19,
21.17, 21.13, 21.10, 21.08, 21.05, 21.00, 20.95, 20.90, 20.88 (12s,
12 CH3CO), 20.35 (s, CH3-3). ESI-MS m/z 1178 [M+] (20%), 1179
[M + 1] (17%). Anal. Calcd for C50H64NO30F: C, 50.96, H, 5.48,
N, 1.19. Found: C, 50.94, H, 5.45, N, 1.16.
7.39 (5H, m, Ar-H), 4.90 (2H, s, PhCH2), 4.75 (2H, d, JH-5,HO-5
)
3-O-(â-D-Galactopyranosyl)-r4,r5-di-O-(â-D-glucopyranosyl)-
6-fluoropyridoxol 12 and 3-O-(â-D-Galactopyranosyl)-r4,r5-di-
O-(r-D-mannopyranosyl)-6-fluoropyridoxol 13. Compounds 10
and 11 (1.00 g, 0.85 mmol) were deacetylated as described above
for 4 to yield 12 and 13 in quantitative yields.
5.4 Hz, CH2-5), 4.72 (2H, d, JH-4,HO-4 ) 6.0 Hz, CH2-4), 3.57
(1H, t, JH-5,HO-5 ) 5.4 Hz, R5-OH, exchangeable with D2O), 3.49
(1H, t, JH-4,HO-4 ) 6.0 Hz, R4-OH, exchangeable with D2O), 2.44
3
(3H, s, CH3-2). δC 151.34 (d, JF-C ) 9.6 Hz, Py-C2), 146.97 (d,
4JF-C ) 2.9 Hz, Py-C3), 149.55 (d, 3JF-C ) 3.1 Hz, Py-C4), 119.09
(d, 2JF-C ) 20.8 Hz, Py-C5), 156.30 (d, 1JF-C ) 216.2 Hz, Py-C6),
136.33, 128.96, 128.88, 128.57 (Ph-C), 55.99 (s, PhCH2, CH2-4),
56.76 (s, CH2-5), 19.31 (s, CH3-2). Anal. Calcd for C15H16NO3F:
C, 64.96, H, 5.82, N, 5.05. Found: C, 64.95, H, 5.79, N, 5.04.
3-O-(â-D-Galactopyranosyl)-r4,r5-di-O-(â-D-glucopyranosyl)-
6-fluoropyridoxol 12: 0.57 g, foam solid, Rf 0.20 (1:4 MeOH/
EtOAc). NMR (DMSO-d6) δH 5.01 (1H, d, J1′,2′ ) 8.2 Hz, H-1′),
5.22 (1H, dd, J2′,3′ ) 9.0 Hz, H-2′), 4.92 (1H, dd, J3′,4′ ) 4.6 Hz,
H-3′), 4.70 (1H, dd, J4′,5′ ) 2.6 Hz, H-4′), 3.91 (1H, m, H-5′), 4.12
(1H, dd, J5′,6a′ ) 3.2 Hz, J6a′,6b′ ) 10.2 Hz, H-6a′), 4.00 (1H, dd,
J5′,6b′ ) 5.6 Hz, H-6b′), 5.14 (2H, d, J1′′,2′′ ) 10.0 Hz, H-1′′ and
H-1′′′), 4.82 (2H, dd, J2′′,3′′ ) J2′′′,3′′′ ) 8.2 Hz, H-2′′ and H-2′′′),
4.69 (2H, dd, J3′′,4′′ ) J3′′′,4′′′ ) 3.4 Hz, H-3′′ and H-3′′′), 4.93 (2H,
dd, J4′′,5′′ ) J4′′′,5′′′ ) 3.2 Hz, H-4′′ and H-4′′′), 3.65 (2H, m, H-5′′
and H-5′′′), 3.55 (2H, dd, J5′′,6a′′ ) J5′′′,6a′′′ ) 4.8 Hz, J6a′′,6b′′ ) J6a′′′,6b′′′
3-O-Benzyl-r4,r5-di-O-(2,3,4,6-tetra-O-acetyl-â-D-glucopyra-
nosyl)-6-fluoropyridoxol 15 and 3-O-Benzyl-r4,r5-di-O-(2,3,4,6-
tetra-O-acetyl-r-D-mannopyranosyl)-6-fluoropyridoxol 16. Gly-
cosylation of 14 (0.46 g, 2.0 mmol) with 8 or 9 (1.83 g, 4.45 mmol,
1.1 equiv) was carried out as for 3, 10, and 11 to give 15 and 16,
respectively.
3-O-Benzyl-r4,r5-di-O-(2,3,4,6-tetra-O-acetyl-â-D-glucopyra-
nosyl)-6-fluoropyridoxol 15: 0.32 g, 95%) syrup, Rf 0.35 (3:2
cyclohexane/EtOAc). NMR (CDCl3) δH 7.41 (5H, m, Ar-H), 5.36
(1H, d, J1′,2′ ) 8.2 Hz, H-1′), 5.41 (1H, d, J1′′,2′′ ) 8.2 Hz, H-1′′),
5.14 (2H, dd, J2′,3′ ) J2′′,3′′ ) 7.4 Hz, H-2′ and H-2′′), 4.45 (2H,
) 12.0 Hz, H-6a′′ and H-6a′′′), 3.31 (2H, dd, J5′′,6b′′ ) J5′′′,6b′′′
)
5.6 Hz, H-6b′′ and H-6b′′′), 4.29 (1H, d, JCH2-4a,CH2-4b ) 7.6 Hz,
CH2-4a), 4.36 (1H, d, JCH2-4a,CH2-4b ) 7.6 Hz, CH2-5a), 4.20 (2H,
d, JCH2-4a,CH2-4b ) JCH2-5a,CH2-5b ) 7.6 Hz, CH2-4b and CH2-5b),
4.18∼3.65 (12H, br, HO-2′, 3′, 4′, 6′, 2′′, 3′′, 4′′, 6′′, 2′′′, 3′′′, 4′′′,
and 6′′′, exchangeable with D2O), 2.42 (3H, s, CH3-2). δC 144.43
dd, J3′,4′ ) J3′′,4′′ ) 3.3 Hz, H-3′ and H-3′′), 4.84 (2H, dd, J4′,5′
)
3
4
J4′′,5′′ ) 3.8 Hz, H-4′ and H-4′′), 3.96 (2H, m, H-5′ and H-5′′),
4.80 (2H, dd, J5′,6a′ ) J5′′,6a′′ ) 2.6 Hz, J6a′,6b′ ) J6a′′,6b′′ ) 10.1 Hz,
H-6a′ and H-6a′′), 4.10 (2H, dd, J5′,6b′ ) J5′′,6b′′ ) 3.0 Hz, H-6b′
(d, JF-C ) 15.0 Hz, Py-C2), 136.26 (d, JF-C ) 3.8 Hz, Py-C3),
3
2
124.40 (d, JF-C ) 3.8 Hz, Py-C4), 120.39 (d, JF-C ) 32.8 Hz,
Py-C5), 148.98 (d, JF-C ) 259.7 Hz, Py-C6), 103.65 (s, C-1′),
1
and H-6b′′), 4.94 (2H, s, PhCH2), 4.55 (1H, d, JCH2-4a,CH2-4b
)
101.76 (s, C-1′′ and C-1′′′), 72.34 (s, C-2′), 71.28 (s, C-2′′ and
C-2′′′), 74.55 (s, C-3′), 73.88 (s, C-3′′ and C-3′′′), 69.82 (s, C-4′),
68.89 (s, C-4′′ and C-4′′′), 76.62 (s, C-5′), 77.29 (s, C-5′′ and C-5′′′),
61.36 (s, C-6′), 60.95 (s, C-6′′ and C-6′′′), 60.54 (s, CH2-4), 60.78
(s, CH2-5), 19.88 (s, CH3-3). ESI-MS m/z 673 [M+] (8%), 674 [M
+ 1] (14%). Anal. Calcd for C26H40NO18F: C, 46.34, H, 5.99, N,
2.08. Found: C, 46.32, H, 5.97, N, 2.07.
3-O-(â-D-Galactopyranosyl)-r4,r5-di-O-(r-D-mannopyranosyl)-
6-fluoropyridoxol 13: 0.57 g, foam solid, Rf 0.26 (1:4 MeOH/
EtOAc). NMR (DMSO-d6) δH 5.00 (1H, d, J1′,2′ ) 8.0 Hz, H-1′),
5.23 (1H, dd, J2′,3′ ) 10.0 Hz, H-2′), 5.16 (1H, dd, J3′,4′ ) 3.8 Hz,
H-3′), 5.08 (1H, dd, J4′,5′ ) 3.2 Hz, H-4′), 4.21 (1H, m, H-5′), 4.51
10.4 Hz, CH2-4a), 4.48 (1H, d, JCH2-4a,CH2-4b ) 10.4 Hz, CH2-4b),
4.60 (1H, d, JCH2-5a,CH2-5b ) 11.0 Hz, CH2-5a), 4.52 (1H, d,
JCH2-5a,CH2-5b ) 11.0 Hz, CH2-5b), 2.37 (3H, s, CH3-2), 2.00, 1.99,
1.98, 1.97, 1.96, 1.95, 1.94, 1.93 (24H, 8s, 8 CH3CO). δC 170.84,
170.76, 170.31, 170.29, 170.26, 169.95, 169.92, 169.84 (8 CH3CO),
3
4
152.18 (d, JF-C ) 14.5 Hz, Py-C2), 142.64 (d, JF-C ) 4.6 Hz,
Py-C3), 150.12 (d, 3JF-C ) 3.8 Hz, Py-C4), 116.20 (d, 2JF-C ) 32.0
Hz, Py-C5), 157.40 (d, 1JF-C ) 234.3 Hz, Py-C6), 136.37, 129.00,
128.94, 128.87, 128.16, 127.77 (Ph-C), 100.23 (s, C-1′), 100.41
(s, C-1′′), 71.41 (s, C-2′ and C-2′′), 72.08 (s, C-3′), 72.19 (s, C-3′′),
68.34 (s, C-4′), 68.51 (s, C-4′′), 72.86 (s, C-5′), 72.93 (s, C-5′′),