Kwok-Kong Tony Mong, Yu-Chie Chen et al.
water was used as the eluent). Hexavalent glycodendritic molecule 3a
was obtained as yellowish amorphous powder after lyophilization
(yield: 650 mg, 84%). For 3a: Rf =0.3 (IPA/EtOH/H2O/AcOH 5:1:1:1);
complete conversion of Cl!N3 was confirmed by using 13C NMR spec-
troscopy. The DMF solvent was removed by distillation under reduced
pressure and the reaction residue was dissolved in H2O (5 mL) and puri-
fied by using size-exclusion chromatography (70ꢁ1.5 cm TOSPEAR HW
40-F column with deionized water as an eluent). Upon lyophilization, de-
sired nonavalent glycodendritic molecule 4a was obtained as white amor-
phous powder (yield: 1.24 g, 87%). For 4a: Rf =0.35 (IPA/EtOH/H2O/
AcOH 4:1:2:1); GPC retention time 9.660 min; PDI 1.029 (Jordi Gel
DVB 500A column (ꢁ2), DMF elution at 0.8 mLminꢃ1); 1H NMR
(300 MHz, D2O): d=7.97–7.91 (brm, 12H; triazole-Hꢁ12), 4.48–3.26
(brm, 199H), 1.87 (s, 3H; CH3C=Oꢁ1), 1.73 (brs, 18H; C6-linker-CH2 ꢁ
9), 1.40 (brs, 18H; C6-linker-CH2 ꢁ9), 1.18 (brs, 18H; C6-linker-CH2 ꢁ9),
1.11 ppm (brs, 18H; C6-linker-CH2 ꢁ9); MALDI-TOF: m/z calcd for
C194H326N39O87: 4594.2; found: 4594.1 [M+H]+.
a
GPC retention time 9.947 min; PDI 1.112 (Jordi Gel DVB 500A column
1
(ꢁ2), DMF elution at 0.8 mLminꢃ1); H NMR (300 MHz, D2O): d=8.07–
8.03 (brm, 8H; triazole-H), 5.24 (s, 1H; Gal H-4), 4.79–3.35 (m, 138H),
2.13, (s, 3H; CH3C=O), 1.92–1.86 (brs, 15H; acetyl-CH3 ꢁ1 and C6-
linker-CH2 ꢁ6), 1.51 (brs, 12H; C6-linker-CH2 ꢁ6), 1.29–1.24 ppm (brs,
24H; C6-linker-CH2 ꢁ12); MALDI-TOF: m/z calcd for C136H227N27NaO62
:
3253.5; found: 3253.4 [M+Na]+.
Synthesis of hexavalent glycodendritic molecule 3b
A solution of hexavalent glycodendritic molecule 3a (520 mg, 0.16 mmol)
in MeOH (5 mL) was treated with freshly cut Na (ꢂ10 mg) and stirred
at RT. Upon completion of deacetylation (ꢂ8 h), as determined by using
1H NMR spectroscopy, the reaction mixture was neutralized with resin
IR-120 H+, filtered, concentrated, and then dialyzed in deionized water
for 5 h (2ꢁ2 L). Upon lyophilization, hexavalent glycodendritic molecule
3b was obtained as a yellow amorphous powder (yield: 440 mg, 86%).
For 3b: Rf (IPA/EtOH/H2O/AcOH 5:1:1:1) 0.30. Because 3b was used as
an intermediate for the subsequent CuAAC reaction, and the NMR spec-
Synthesis of nonavalent glycodendritic molecule 4b
A solution of nonavalent glycodendritic molecule 4a (1.02 g, 0.22 mmol)
in MeOH (10 mL) was treated with freshly cut sodium (ꢂ20 mg) and
stirred at RT. Upon completion of deacetylation (ꢂ8 h), as judged by
using 1H NMR spectroscopy, the reaction mixture was neutralized with
resin IR-120 H+, filtered, concentrated, and then dialyzed with deionized
water for 5 h (2ꢁ2 L) to obtain nonavalent glycodendritic molecule 4b as
a yellowish amorphous powder (yield: 710 mg, 70%). For 4b: Rf =0.35
(IPA/EtOH/H2O/AcOH 4:1:2:1); 1H NMR (300 MHz, CD3OD): d=
8.13–7.97 (brm, 12H; triazole-Hꢁ12), 4.74 (s, 9H; Man H-1ꢁ9), 4.69–
4.55 (m, 20H), 4.44–4.36 (m, 18H), 4.31 (d, J=9.0 Hz, 4H; Gal H-1ꢁ4),
4.00–3.55 (m, 136H), 3.50–3.36 (m, 12H), 1.92–1.90 (m, 18H; C6-linker-
CH2 ꢁ9), 1.59–1.57 (m, 18H; C6-linker-CH2 ꢁ9), 1.42–1.31 ppm (m, 36H;
troscopy was skipped. MALDI-TOF: m/z calcd for C132H223N27NaO60
:
3169.5; found: 3169.7 [M+Na]+.
Synthesis of FITC-labeled hexavalent glycodendritic ligand 3c
A mixture of hexavalent glycodendritic molecule 3b (95 mg, 0.030 mmol)
and CuSO4·5H2O (3.8 mg, 0.015 mmol) in CH3CN/H2O (3 mL, 4:1 v/v)
was stirred for 15 min at RT, followed by addition of freshly prepared Na
ascorbate (5 wt%, 12 mg, 0.060 mmol) and propargyl fluorescein deriva-
tive 8 (17 mg, 0.045 mmol). The resulting mixture was stirred at 558C for
18 h and then the reaction temperature was brought to RT. The upper
CH3CN layer of reaction mixture was removed and the lower aqueous
phase was concentrated by rotary evaporator for size-exclusion chroma-
tography (elution over 70ꢁ1.5 cm TOSPEAR HW 40-F column with de-
ionized water as the eluting buffer at 0.3 mLminꢃ1). Upon removal of
water by lyophilization, hexavalent glycodendritic mannosyl ligand 3c
was obtained as a yellow amorphous powder (yield: 92 mg, 72%). For
3c: Rf =0.25 (IPA/EtOH/H2O/AcOH 4:1.5:1.5:0.6); 1H NMR (300 MHz,
CD3OD): d=8.13 (brs, 1H; triazole-H), 7.94–7.81 (m, 10H; triazole-Hꢁ
8 and Ar-Hꢁ2), 7.61 (m, 1H; Ar-H), 7.21–7.11 (brs, 4H; Ar-H), 6.93
(brs, 1H; Ar-H), 6.60 (brm, 1H; Ar-H), 6.51 (brs, 1H; Ar-H), 5.21 (brs,
2H; -OCH2C-triazole), 4.67–3.37 (brm, 86H; signals are partly over-
lapped with residual H2O and broaden; thus the observed H-integral is
less than theoretical 139H), 1.72 (brs, 12H; linker-CH2 ꢁ6), 1.38 (brs,
12H; linker-CH2 ꢁ6), 1.16 ppm (brs, 24H; linker-CH2 ꢁ12); MALDI-
TOF: m/z calcd for C155H237N27NaO65: 3539.6; found: 3539.7 [M+Na]+.
C6-linker-CH2 ꢁ18); MALDI-TOF: m/z calcd for C192H322N39NaO86
:
4574.2; found: 4574.6 [M+Na]+.
Procedure of ligand-binding-induced Con A precipitation (Figure 4 and
5a)
The examined ligands (1, 2c, and 3c; 1 mm) in Tris (pH 7.4, 75 mL) were
mixed individually with solutions of Con A (50 mL; 10, 20, or 40 mm) in
Tris (12.5 mm, pH 7.4) and allowed to stand at RT. Photographs of these
mixtures under UV illumination (lmax =365 nm) were taken at time
points of 0, 5, and 30 min (Figure 4). In the control experiments, Con A
(50 mL; 10, 20, or 40 mm) was mixed with a blank Tris buffer (75 mL,
12.5 mm, pH 7.4). After standing for 30 min at RT, the samples were cen-
trifuged at 2000 rpm for 10 min, then the supernatant was decanted. The
resulting ligand-bound precipitate pellets were washed with fresh Tris
buffer (125 mL, 12.5 mm, pH 7.4), followed by centrifugation at 8000 rpm.
After decanting the supernatant, the washed precipitate pellets (if any)
were examined under UV illumination (lmax =365 nm).
Procedure of competitive ligand binding experiment (Figure 5b)
Synthesis of nonavalent glycodendritic molecule 4a
A series of (ligand 1)-bound Con A precipitate pellets were obtained
from mixing a solution of Con A (0.1 mm, 10 mL) in Tris (12.5 mm,
Gal branching unit 7 (210 mg, 0.43 mmol) and CuSO4·5H2O (10 mg,
0.04 mmol) in THF/H2O (10 mL, 1:1) was stirred at RT for 15 min, fol-
lowed by addition of freshly prepared 5 wt% sodium ascorbate (85 mg,
0.43 mmol) and glycodendritic molecule 2b (2.08 g, 1.52 mmol). The reac-
tion was stirred at 608C for 40 h and monitored by using TLC. Upon
completion of click coupling, the solvent was removed by rotary evapora-
tor and filtered through a syringe filter. The resulting residue was dis-
solved in H2O (10 mL) for purification with size-exclusion chromatogra-
phy (70ꢁ1.5 cm TOSPEAR HW 40-F column with deionized water as an
eluent). Removal of the residual copper ions was achieved by dialysis
(0.25 wt% EDTA (2ꢁ2 L) and deionized water (2ꢁ2 L)) and followed
by lyophilization. The nonavalent glycodendritic molecule was obtained
as a yellowish amorphous powder (yield: 1.43 g, 62%). Rf =0.35 (IPA/
EtOH/H2O/AcOH 4:1:2:1); 1H NMR (300 MHz, D2O): d=7.97–7.91
(brm, 12H; triazole-Hꢁ12), 4.48–3.26 (brm, 199H), 1.87 (s, 3H; CH3C=
Oꢁ1), 1.73 (brs, 18H; C6-linker-CH2 ꢁ9), 1.40 (brs, 18H; C6-linker-CH2 ꢁ
9), 1.18–1.11 ppm (m, 36H; C6-linker-CH2 ꢁ18). The aforementioned
nonavalent glycodendritic molecule (1.43 g, 0.31 mmol) was dissolved in
DMF (5.0 mL), and followed by the addition of NaN3 (101 mg,
1.56 mmol). The mixture was stirred at 908C under N2 for ꢂ18 h. The
pH 7.4) with monovalent mannose ligand
1 (1.0 mm, 15 mL) in Tris
(12.5 mm, pH 7.4) for 1 h, followed by centrifugation at 4000 rpm (48C)
for 10 min. After decanting the supernatants, the ligand-bound precipi-
tate pellets were then mixed with Tris buffer, a solution of saccharide
(0.1m; mannose (Man), Glucose (Glc), sucrose (Suc), maltose (Mal),
xylose (Xyl), arabinose (Ara), fucose (Fuc), rhamnose (Rha), or galac-
tose (Gal)) or unmodified fluorescein (25 mL) for 30 min, followed by
centrifugation at 4000 rpm (48C) for 10 min. After decanting the super-
natants, the remaining precipitate pellets were examined under UV illu-
mination (lmax =365 nm).
Fluorescence polarization binding assay
The measurement of fluorescence polarization assays is based on the ro-
tation speed of a fluorophore. When a fluorophore-containing compound
is bound to the protein counterpart (e.g., Con A herein), the fluorophore
rotates at a slower rate than when it is unbound and the resulting fluores-
cence polarization is higher. Herein, we carried out all measurements ac-
cording to a reported procedure.[27] A fluorescent compound was added
to the final sample volume (70 mL) in each assay to achieve a final con-
Chem. Asian J. 2014, 9, 1786 – 1796
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