130
K. Ajisaka et al.
HRMS m/z calcd. for C16H26NO13 [M-H+] 440.1399,
found 440.1390.
lization was conducted by rotation of the mixture at
40 °C for 48 h, and then, the suspension was cen-
trifuged at 2500 × g for 30 s. The beads were washed
with water (300 µL × 3 times) and methanol
(500 µL × 3 times) and finally dried in vacuo to obtain
D-mannobiose-linked BlotGlyco™ beads (5) with keep-
ing its cyclic conformation of D-mannose residue at the
reducing end (Fig. 1).
Analytical data of Manα1→3Man succinylamide
derivative.
Rf: 0.27 (CH3CN/H2O = 3:1). 1H-NMR
(600 MHz, D2O): δH 5.28 (0.09H, d, J = 1.95 Hz, H-
1α), 5.13 (0.91H, s, H-1β), 5.10 (0.09H, s, H-1α′), 4.97
(0.91H, d, J = 1.38 Hz, H-1β′), 3.95 (1H bd, J =
3.15 Hz, H-2′), 3.93 (1H, dd, J = 1.48, 3.06 Hz, H-2),
3.77–3.72 (3H, m, H-6, H-6, H-3), 3.70 (1H, dd, J =
3.32, 9.47 Hz, H-3), 3.67–3.56 (4H, m, H-6′, H-6′, H-
5′, H-4), 3.51 (1H, t, J = 9.66 Hz, H4), 3.36 (1H, ddd,
J = 2.00, 5.53, 9.56 Hz, H-5), 2.48 (4H, bs, –CH2–
CH2–), 13C-NMR (150 MHz, D2O): δC 178.78, 176.14
(C=O), 102.56 (C-1′), 80.86, 78.19, 78.23, 78.23,
74.44, 73.92, 70.91, 70.57, 70.36, 67,36, 66.52, 61.55
(C-6), 61.30 (C-6′), 31.12, 30.43 (–CH2–CH2–). HRMS
m/z calcd. for C16H26NO13 [M-H+] 440.1399, found
440.1393.
Determination of amounts of D-mannose linked to
beads.
The amount of D-mannobiose linked to
BlotGlyco™ beads was determined as follows. The
disaccharide-linked beads (5, 0.9–2.3 mg) were placed
into 4 M CF3COOH (300 µL) in an Eppendorf tube.
After incubating the tube at 100 oC for 3 h, N2 gas was
flashed into the tube for evaporation of CF3COOH and
water. D-Mannose residues released were dissolved in
distilled water (200 µL), and the sample was filtered
through a Millex filter (0.45 μm) (Merk Millipore
Corp., Billerica, MA) at 700 × g for 30 s. The amount
of D-mannose was determined by high-performance
liquid chromatography (HPLC) equipped with an evap-
orative light scattering detector using an Asahi-Pak
NH2-P50™ column (Showa Denko K.K., Tokyo).
Analytical data of Manα1→4Man succinylamide
derivative.
Rf: 0.25 (CH3CN/H2O = 3:1). 1H-NMR
(600 MHz, D2O): δH 5.31 (0.12H, d, J = 1.70 Hz,
H-1α), 5.14 (0.88H, d, J = 1.46 Hz, H-1β), 5.10 (1H, d,
J = 0.85 Hz, H-1β′), 3.94 (1H, dd, J = 1.70, 2.97 Hz,
H-2), 3.79 (1H, dd, J = 0.81, 3.09 Hz, H-2′), 3.78–3.74
(2H, m, H-6, H-6), 3.71–3.60 (4H, m), 3.59–3.52 (3H,
m), 3.43 (1H, ddd, J = 1.95, 5.10, 9.40 Hz, H-5), 2.59–
2.50 (4H, m, CH2–CH2–), 13C-NMR (150 MHz, D2O)
δC 177.17, 175.42 (C=O), 101.57 (C-1′), 80.86, 78.37,
76.83, 74.41, 74.27, 71.23, 70.87, 67,12, 61.54 (C-6),
61.48 (C-6′), 30.04, 28.91 (–CH2–CH2–). HRMS m/z
calcd. for C16H26NO13 [M-H+] 440.1399, found
440.1400.
Culture and labeling with a fluorescent dye of
bacteria.
E. coli (strain W3110)14) donated by Dr.
M. Fukuda of Nagaoka University of Technology was
grown in a Luria–Bertani medium at 37 °C for 14–20 h
by shaking. After centrifugation, the bacteria were sus-
pended in Diluent C (1 mL) from the cell membrane
labeling kit, mixed with 10 µL of PKH67 solution
(4 µL of PKH67 mixed with 996 µL of Diluent C),
and incubated at 37 °C for 10 min to label the bacteria
with the fluorescent dye according to the manufac-
turer’s protocol. In this study, more than 90% of
E. coli was labeled with the dye and alive during the
period of experiments, which was confirmed by deter-
mining the growth rate of the fluorescence-labeled E
coli in culture. Higher percentages of cells labeled with
this dye together with a longer survival time of the
dye-labeled cells were reported.15)
Analytical data of Manα1→6Man succinylamide
derivative.
Rf: 0.25 (CH3CN/H2O = 3:1). 1H-NMR
(600 MHz, D2O): δH 5.31 (0.11H, d, J = 1.70 Hz, H-
1α), 5.11 (0.89H, d, J = 0.97 Hz, H-1β), 4.77 (0.88H,
d, J = 1.59 Hz, H-1β′), 4.75 (012H, d, J = 1.64 Hz, H-
1α′), 3.86 (1H, dd, J = 1.62, 3.98 Hz, H-2), 3.83 (1H,
dd, J = 1.02, 2.98 Hz, H-2′), 3.81 (1H, dd, J = 5.13,
11.46 Hz, H-6), 3.75 (1H, dd, J = 2.04, J = 12.17 Hz,
H-6), 3.71 (1H, dd, J = 3.18, 8.93 Hz, H-3′), 3.63 (1H,
dd, J = 5.56, 12.04, H-6′), 3.59 (1H, dd, J = 5.47,
11.64, H-6′), 3.58 (1H, dd, J = 3.92, 9.58, H-3), 3.58–
3.51 (3H, m, H-4, H-4′, H-5′), 3.47 (1H, ddd, J = 1.95,
5.10, 9.40 Hz, H-5), 2.59–2.52 (4H, m, –CH2–
CH2–),13C-NMR (150 MHz, D2O) δC 177.66, 175.79
(C=O), 100.10 (C-1′), 78.27, 76.34, 74.01, 73.27,
71.04, 74.01, 70.04, 67,23, 66.89, 66.28 (C-6), 61.44
(C-6′), 30.55, 29.40 (–CH2–CH2–). HRMS m/z calcd.
for C16H26NO13 [M-H+] 440.1399, found 440.1394.
Assay for binding of bacteria to D-mannobiose-linked
beads.
Fluorescence-labeled E. coli suspended in a
Luria-Bertani medium (OD600 = 0.1, 200 µL) was
mixed with 1 mg of D-mannobiose-linked beads sus-
pended in 40 µL of 10 mM phosphate-buffered saline
(pH 7.4) (PBS) in a glass-bottom dish and incubated at
37 °C for 1–2 h. Then, beads were washed with PBS
(200 µL) three times and suspended in PBS (200 µL)
for observation under a confocal laser microscope. In
some experiments, the bacteria and the beads were
incubated in the presence of 0.1 M methyl
α-D-mannopyranoside.
Immobilization of D-mannobiose isomers on beads.
To D-mannobiosyl succinylamide derivative (4, 2 mg,
4.5 µmol) dissolved in distilled water (300 μL), 8 mg
of BlotGlyco™ beads (Sumitomo Bakelite Co. Ltd.,
Tokyo)13) and water-soluble carbodiimide (WSC)
(86.3 mg, 450 µmol) was added (Fig. 1). The immobi-
Results and discussion
Preparation of D-mannobiose isomer-linked beads
In order to determine the binding preference of
E. coli with type 1 fimbria among D-mannobiose