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C. Podlipnik et al. / Carbohydrate Research 342 (2007) 1651–1660
1657
120 H+, filtered and the filtrate evaporated under re-
duced pressure to afford 8 (237 mg, 94%); amorphous
white solid; 1H NMR (400 MHz, CD3OD): d 4.04–
3.98 (m, 1H, H-1), 3.85–3.80 (m, 2H, H-2, 4), 3.68–
3.52 (m, 4H, H-3, 5, 6a, 6b), 3.05–2.95 (m, 2H,
CH2N), 2.01–1.85 (m, 2H, CH2CH2N); 13C NMR
(100 MHz, CD3OD): d 76.14 (CH), 74.99 (CH), 72.31
(CH), 71.53 (CH), 70.46 (CH), 66.36 (CH2), 63.86
(CH2), 33.9 (CH2).
(CH2), 28.61 (CH2); HRMS calcd for [M+H]+:
405.1274, [M+Na]+: 360.1423. Found: 383.1613,
405.1408.
Compound 13. Yield 72%; 1H NMR (400 MHz,
D2O): d 7.42 (d, J = 15.7 Hz, 1H, CH@), 7.17 (d,
J = 1.7 Hz, 1H, HAr), 7.11 (dd, J = 1.7, J = 8.0 Hz,
1H, HAr), 6.92 (d, J = 8.0 Hz, 1H, HAr), 6.45 (d,
J = 15.7 Hz, 1H, CH@), 6.02 (s, 2H, OCH2O), 4.38–
4.21 (m, 1H, H-1), 4.06–3.92 (m, 2H, H-2, 4), 3.84–
3.70 (m, 4H, H-3, 5, 6a, 6b), 3.43 (br t, 2H, CH2N),
2.04–1.90 (m, 1H, CHCH2N), 1.84–1.65 (m, 1H,
CHCH2N); 13C NMR (100 MHz, CD3OD): d 178.5
(C), 150.4 (2C), 143.4 (CH), 131.9 (C), 126.4 (CH),
124.8 (CH), 111.3 (CH), 109.1 (CH), 104.2 (CH2), 76.8
(CH), 74.7 (CH), 72.4 (CH), 71.6 (CH), 70.6 (CH),
64.0 (CH2), 39.2 (CH2), 26.2 (CH2); HRMS calcd for
[M+Na]+: 404.1321. Found 404.1257.
4.1.6. General procedure for amides 10–13. To a solu-
tion of the corresponding cinnamic acid derivative
(0.5 mmol) in THF (1 mL), at rt, oxalyl chloride
(0.2 mL/mmol) was added. The reaction was stirred
for 2.5 h, then the volatiles were evaporated under re-
duced pressure. The residue was dissolved in toluene
and added to a mixture of amine and Na2CO3 and
heated at reflux for 2.5 h. The solvent was evaporated
and the residue purified by flash column chromato-
graphy on silica gel, using a gradient EtOAc–MeOH,
10:0!8:2.
Compound 14. A solution of galactose amine 8
(16 mg, 0.078 mmol), diphenyl maleic anhydride
(24 mg, 0.097 mmol), Na2CO3 (33 mg, 0.31 mmol) and
H2O (100 lL) in MeOH and THF (1 mL, 1:1, vol:vol)
was stirred at rt for 1.5 h. The mixture was filtered,
the solvents were evaporated under reduced pressure
and the residue was purified using a solvent system gra-
dient EtOAc–MeOH, 10:0!6:4 to yield 10 mg of com-
1
Compound 10. Yield 86%; [a]D +0.183 (c 0.5); H
NMR (400 MHz, D2O): d 7.59–7.52 (m, 2H, HAr),
7.42–7.35 (m, 4H, HAr, CH@), 6.55 (d, J = 15.4 Hz,
1H, CH@), 4.15–4.08 (m, 1H, H-1), 3.91–3.87 (m, 2H,
H-2, 4), 3.70–3.60 (m, 4H, H-3, 5, 6a, 6b), 3.33–3.26
(m, 2H, CH2N), 1.91–1.85 (m, 1H, CHCH2N), 1.81–
1.72 (m, 1H, CHCH2N); 13C NMR (100 MHz, D2O):
d 171.4 (C), 143.7 (CH), 137.1 (C), 132.9 (CH), 131.7
(CH), 130.6 (CH), 122.8 (CH), 76.35 (CH), 74.63
(CH), 72.34 (CH), 71.70 (CH), 70.69 (CH), 63.87
(CH2), 39.26 (CH2), 25.93 (CH2); HRMS calcd for
[M+H]+: 338.1604, [M+Na]+: 360.1423. Found:
338.1765, 360.1302.
1
pound 14 (28%); H NMR (400 MHz, D2O): d 7.30–
7.21 (m, 6H, HAr), 7.18–7.04 (m, 4H, HAr), 4.18–4.05
(m, 1H, H-1), 4.00–3.92 (m, 2H, H-2, 4), 3.82–3.61 (m,
4H, H-3, 5, 6a, 6b), 3.44–3.31 (m, 2H, CH2N), 2.02–
1.90 (m, 1H, CHCH2N), 1.89–1.80 (m, 1H, CHCH2N);
13C NMR (100 MHz, CD3OD): d 174.7 (C), 146.8 (C),
139.4 (C), 138.6 (C), 136.5 (C), 133.0 (CH), 132.6
(CH), 132.3 (CH), 131.9 (CH), 131.6 (CH), 131.1
(CH), 130.9 (CH), 130.7 (CH), 130.6 (CH), 130.5
(CH), 74.8 (CH), 72.4 (2 · CH), 71.7 (CH), 70.9 (CH),
63.9 (CH2), 39.4 (CH2), 26.1 (CH2); HRMS calcd for
[M+NaꢀH2O]+: 462.1529. Found: 462.1719.
Compound 11. Yield 70%; 1H NMR (400 MHz,
D2O): d 7.31 (d, J = 15.7 Hz, 1 H, CH@), 6.82 (s, 2H,
HAr), 6.45 (d, J = 15.7 Hz, 1H, CH@), 4.13–4.07 (m,
1H, H-1), 3.98–3.90 (m, 2H, H-2, 4), 3.85–3.65 (m,
13H, H-3, 5, 6a, 6b, 3OCH3), 3.38 (br t, 2H, CH2N),
1.98–1.83 (m, 2H, CH2CH2N); 13C NMR (100 MHz,
D2O): d 180.4 (C), 155.2 (C), 143.3 (CH), 133.6 (C),
122.6 (CH), 108.0 (CH), 74.63 (CH), 72.33 (CH), 71.66
(CH), 70.67 (CH), 63.84 (CH2), 63.55 (CH), 58.63
(CH3), 39.20 (CH2), 23.63 (CH2); HRMS calcd for
[M+H]+: 428.1921. Found: 428.1967.
4.2. SPR measurements
The SPR measurements were performed on a double
channel BIOCORE instrument at 25 ꢁC, in HBS buffer.
The glycoprotein asialofetuin (ASF) was covalently
attached to a dextran functionalized gold SPR chip via
NHS active ester intermediacy following a protocol pre-
viously established.37 A series of measurements with
increasing CTB5 concentrations yielded a binding iso-
therm and a Kchip of 70 lM.38 After each binding exper-
iment the surface was regenerated with 10 mM NaOH
for 2.5 min. In a typical competition experiment,
CTB5 (70 lM in HBS buffer) was mixed with variable
concentrations of the ligands and allowed to reach equi-
librium for 5 min at rt. The equilibrated samples were
then introduced to the chip-immobilized protein. These
measurements generated a new series of binding iso-
therms, from which equilibrium constants (Req) were
Compound 12. Yield 75%; 1H NMR (400 MHz,
CD3OD): d 8.29–8.20 (m, 2H, HAr), 7.80–7.75 (m,
2H, HAr), 7.59 (d, J = 15.7 Hz, 1H, CH@), 6.81 (d,
J = 15.7 Hz, 1H, CH@), 4.12–4.04 (m, 1H, H-1), 3.96–
3.82 (m, 3H, H-2, 4, 6a), 3.80–3.76 (m, 1H, H-5),
3.72–3.65 (m, 2H, H-3, 6b), 3.59–3.50 (m, 1H, CHN),
3.48–3.362 (m, 1H, CHN), 1.97–1.80 (m, 2H,
CH2CH2N); 13C NMR (100 MHz, CD3OD): d 170.6
(C), 152.3 (C), 145.5 (C), 141.9 (CH), 132.6 (CH),
129.0 (CH), 127.9 (CH), 77.22 (CH), 76.91 (CH), 74.65
(CH), 72.93 (CH), 72.93 (CH), 65.07 (CH2), 40.92