J. Li et al. / Bioorg. Med. Chem. 7 (1999) 1549±1558
1557
and lyophilized to aord the white powder 15A in
quantitative yield. 15A: Selected 13C NMR (D2O) 103.5,
102.7, 99.1 (mannose anomeric carbon) 96.0 (a-galac-
tose anomeric carbon), 79.2 (C-4 of glucose), 77.8 (C-3
of b-galactose).
1.38±1.28 (m, 2H). 13C d174.7, 171.1, 171.0, 170.8, 170.7,
170.5, 170.3, 169.8, 101.7, 101.2, 77.0, 73.5, 73.3, 72.4,
71.7, 71.3, 70.5, 69.8, 67.3, 62.7, 61.5, 34.6, 29.7, 26.0,
25.2, 21.54, 21.49, 21.4(b), 21.3(b), 21.2(b). HRFABMS
calcd for C33H48O20Na+ 787.2637, found 787.2618.
Polymer (15B). Acceptor glycopolymer 14B (120 mg,
93 mmol of lactose), UDP-Gal (80 mg) and ra1-3GalT
(15 unit) were incubated in a solution of Tris±HCl buf-
fer (50 mM, pH 7) containing Mn2+ (10 mM) for 48 h at
ambient temperature. The mixture was directly applied
to Bio-Gel P-2 with water as the eluent. The fractions
containing the glycopolymer were collected and lyophi-
lized to aord the white powder 15B in quantitative
yield. 15B: Selected 13C NMR (D2O) d 103.4, 102.7,
100.4 (mannose anomeric carbon) 95.9 (a-galactose
anomeric carbon), 79.1 (C-4 of glucose), 77.7 (C-3 of b-
galactose).
Compound 17. To a solution of 16 (3 g) in anhydrous
methanol (200 mL) at 0ꢀC was added NaOMe until pH
10. The solution was stirred for 3 h. Dowex cation
exchange resin (H form) was added to adjust the pH to
6±7 and ®ltered. The ®ltrate was evaporated to give a
residue which was dissolved in MeOH:H2O (1:1). To the
above solution was added LiOH until pH 11. The solu-
tion was stirred for 1 h. Dowex cation exchange resin (H
form) was used to neutralize the above solution which
was concentrated to aord a syrup 17 in quantitative
1
yield. 17: H NMR (D2O) d 4.45 (d, J=8.0 Hz, 1H),
4.41 (d, J=7.6 Hz, 1H), 3.96±3.49 (m, 13 H), 3.26 (t,
J=8.0 Hz, 1H), 2.22 (t, J=7.2 Hz, 2H), 1.64±1.53 (m,
4H), 1.38±1.32 (m, 2H) 13C d183.3, 103.5, 102.6, 79.0,
76.0, 75.4, 75.1, 73.5, 73.1, 71.6, 71.1, 69.2, 61.6, 60.7,
37.1, 29.1, 25.8, 25.5. HRFABMS calcd for C18H32O13
Na+ 479.1741, found 479.1762.
Poly {acrylamide-co-2-[2-(2-N-acrylamidoethoxy)ethoxy]-
ethoxyl-ꢀ-d-mannopyranoside} (15C). Copolymer 15C
was synthesized using compound 8 following the proce-
dure described for terpolymer 14A. The average incor-
poration molar ratio is 3:1 (acrylamide:mannose).
Compound 18. To a solution of 17 (612 mg) and p-ami-
nophenyl-a-d-mannopyranoside (400 mg) in DMF
(10 mL) at 0ꢀC was added DPPA (328 mL) and TEA
(400 mL). The solution was stirred vigorously overnight.
The solution was concentrated in vacuo, followed by gel
®ltration through Bio-Gel P-2 column eluted by water.
The fractions containing the glycoconjugate were col-
lected and lyophilized to aord 18 628 mg (66% yield).
Selected 1H NMR (D2O) d5.39 (s, 1H, anomeric proton
of mannose), 4.26 (d, J=7.5 Hz, 2H, two anomeric
protons of lactose). 18: Selected 13C (103.4 (C-1 of glu-
cose), 102.5 (C-1 of galactose), 99.0 (C-1 of mannose).
HRFABMS calcd for C30H47NO18Na+ 732.2691,
found 732.2665.
Poly {acrylamide-co-N-acryloyl-aminophenyl ꢀ-d-manno-
pyranoside} (15D). Copolymer 15D was synthesized
using compound 13 following the procedure described
before.8 The average incorporation molar ratio is 6:1
(acrylamide:mannose).
Poly {acrylamide-co-2-[2-(2-N-acrylamidoethoxy)ethoxy]-
ethoxyl-ꢁ-lactoside} (15E). Copolymer 15E was synthe-
sized using compound 5 following the procedure descri-
bed for terpolymer 14A. The average incorporation
molar ratio is 1:12 (lactose:acrylamide).
Polymer (15F). Copolymer 15F was synthesized follow-
ing the procedure described for polymer 15A. The
enzymatic reaction aorded white powder 15F in quan-
titative yield.
Compound 19. Acceptor glycoconjugate 18 (250 mg),
UDP-Gal (256 mg), 0.1% BSA and ra1-3GalT (15 unit)
were incubated in a solution of Tris±HCl buer
(50 mM, pH 7) containing Mn2+ (10 mM) for 48 h at
ambient temperature. The mixture was directly applied
to Bio-Gel P-2 with water as the eluent. The fractions
containing the product were collected and lyophilized to
aord the white powder 19 187 mg (61% yield). 19:
Selected 1H NMR (D2O) d 5.52 (s, 1H, anomeric proton
of mannose), 5.09, (d, J=3.2 Hz, 1H, anomeric proton
of a-galactose), 4.45 (d, J=7.6 Hz, 1H, anomeric pro-
ton), 4.40 (d, J=8.0 Hz, 1H, anomeric proton). Selected
13C d 103.9 (C-1 of glucose), 103.1 (C-1 of b-galactose),
99.5 (C-1 of mannose), 96.5 (C-1 of a-galactose).
HRFABMS calcd for C36H57NO23Na+ 894.3219,
found 894.3223.
Compound 16. 2,3,4,6-Hepta-O-acetyl-a-lactosyl bro-
mide (5 g, 7.1 mmol) and methyl 6-hydroxyhexanoate
(1.3 g, 8.9 mmol) were added to a previously ¯ame-dried
¯ask containing 7 g MS 4 A and 30 mL anhydrous
CHCl3. The resulting suspension was stirred for half an
hour. HgO (1.5 g) and HgBr2 (257 mg) were added to
the suspension. The mixture was stirred in the dark at
ambient temperature for 48 h. The resulting mixture was
passed through a Celite packed glass funnel, and
washed with saturated NaHCO3 and water. The organic
phase was dried over anhydrous Na2SO4 and con-
centrated in vacuo. The resulting residue was chroma-
tographed (hexane:EtOAc, 1:1) to aord product 16
(4.0 g, 74%). 16: 1H NMR (CDCl3) d 5.32 (d, J=
2.5 Hz, 1H), 5.17 (t, J=9.0 Hz, 1H), 5.08 (dd, J=8.0,
10.5, 1H), 4.93 (dd, J=3.5, 10.5, 1H), 4.85 (dd, J=8.0,
10.5, 1H), 4.47±4.44 (m, 2H), 4.42 (d, J=8.0 Hz, 1H),
4.11±4.04 (m, 3H), 3.87±3.80 (m, 2H), 3.77 (t, J=
9.5 Hz, 1H), 3.64 (s, 3H), 3.59±3.55 (m, 1H), 3.46±3.41
(m, 1H), 2.27 (t, J=7.5 Hz, 2H), 2.13, 2.10, 2.04, 2.021,
2.020, 2.01, 1.94 (7s, 7 acetyl groups), 1.64±1.50 (m, 4H),
Agglutination assay. E. coli K12 HB101 was grown
overnight at 37ꢀC in static LB media, washed, and
suspended with PBS to a ®nal concentration of 1Â108
cells/mL. Yeast (S. cerevisiae, wild type) was incubated
on YPD media at 25ꢀC for 48 h, washed, and sus-
pended with PBS to a ®nal dilution of 4Â107 cells/mL.
Agglutination assays were carried out on a 9-well spot