N. Nagahori and S.-I. Nishimura
Compound 1: N-Iodosuccinimide (NIS; 50 mg, 0.222 mmol, 1.2 equiv)
was added to a solution of 7 (100 mg, 0.185 mmol, 1 equiv) and 8
Transmission electron microscopy (TEM): The samples dissolved in de-
ionized water were put on the carbon-grid substrate. The TEM image of
the nanoparticles was obtained by using the JEOL JEM-100S instrument,
operating at 75 kV.
(
140 mg, 0.277 mmol, 1.5 equiv) in dry dichloromethane (5 mL) at 08C.
The mixture was stirred for 30 min, then triflic acid (TfOH; 19.6 mL,
.222 mmol, 1.2 equiv) was added. When the reaction was complete
within 1 h), the solution was neutralized with triethylamine and washed
with 5% Na and brine, and dried over MgSO . The residue was con-
0
(
Enzymatic glycosylation on GCNPs bearing 1: The enzymatic glycosyla-
tion of the GCNPs bearing GlcNAc derivative 1 by recombinant human
b-1,4-galactosyltransfease (b-1,4-GalT) and a-1,3-fucosyltransferase VI
2
S
2
O
3
4
centrated and purified by using silica gel chromatography (eluent gradi-
(
a-1,3-FucT) was carried out as follows. b-1,4-GalT(4 mU) was added to
ent of 0–50% EtOAc in hexane) to give 9 (140 mg, 0.152 mmol, 82%).
a solution of GCNPs (50 mm, calculated as the concentration of GlcNAc
1
[
22]
3
H NMR (CDCl , 600 MHz): d=7.85, 7.74 (2m, 4H; NPh); 5.81 (t, 1H;
residues conjugated according to the method reported previously ) and
UDP-Gal (400 mm) in HEPES buffer (10 mm; pH 7.4, containing 10 mm
MnCl2 and 150 mm NaCl), and the mixture was incubated at 258C. The
reaction was monitored directly by LDI-TOF MS analysis using 0.5 mL of
a sample taken at 5, 10, 15, 20, 30, 40, 50 and 60 min. The sample
(0.5 mL) was directly placed on the MALDI plate without any purifica-
tion and was employed for further mass analysis without a matrix. When
the GalTreaction was complete, the mixture was subjected to purifica-
tion by means of ultrafiltration using Microcon YM-50 (Millipore) and
the GCNPs were washed with water three times. Next, the GCNPs were
dissolved in HEPES buffer solution (50 mL) containing GDP-Fuc
(400 mm), then a-1,3-FucT(5 mU) was added. Th e reaction was moni-
tored directly by LDI-TOF MS analysis as described above.
H-3); 5.43 (d, J=8.46 Hz, 1H; H-1); 5.17 (t, 1H; H-4); 4.32 (m, 2H; H-2,
H-6); 4.18 (d, 1H; H-6); 3.87 (m, 1H; H-5); 3.65–3.32 (m, 28H; -CH -O-,
CH -Br); 2.12, 2.03, 1.87 (3s, Ac); 1.86 (m, 2H; -CH -CH -Br); 1.58–
.28 ppm (m, 14H; -CH -); MALDI-TOF MS: m/z calcd for
64BrNO16: 917.3408; found: 940.076 [M+Na] .
Potassium thioacetate (AcSK; 41 mg, 0.26 mmol) and Bu
2
-
2
2
2
1
2
+
42
C H
4
NI were added
to the solution of compound 9 (110 mg, 0.12 mol) in butanone (10 mL)
and the mixture was stirred for 3 h at 608C. The mixture was extracted
with EtOAc and washed with water. The organic layer was dried and
evaporated. The crude product was purified by using silica gel chroma-
tography (eluent gradient of 0–80% EtOAc in hexane) to give 10
1
(
110 mg, 0.112 mmol, 99%). H NMR (CDCl
3
, 600 MHz): d=7.85, 7.74
(
NPhta); 5.81 (t, 1H; H-3); 5.42 (d, J=8.52 Hz, 1H; H-1); 5.17 (t, 1H;
LDI-TOF MS: m/z calcd for LacNAc 4: 1197.726; found: 1221.810
+
+
x
H-4); 4.32 (m, 2H; H-2, H-6); 4.18 (d, 1H; H-6); 3.89 (m, 1H; H-5);
[M+Na] , 1234.539 [M+K] ; m/z calcd for Le 5: 1343.730; found:
+
3
2
1
.64–3.31 (m, 26H; -CH
.11, 2.03, 1.86 (3s, Ac); 1.56 (m, 4H; -CH
.26 ppm (m, 12H; -CH -); MALDI-TOF MS: m/z calcd for
67NO17S: 913.4130; found: 952.056 [M+K] .
Ethylenediamine (1 mL, 15 mmol) was added to a solution of compound
0 (100 mg, 0.11 mmol) in ethanol (10 mL), and the mixture was stirred
2
-O-); 2.85 (t, 2H; -CH
2
-SAc); 2.32 (s, 3H; SAc);
1367.609 [M+Na] .
2
-CH
2
-O-, -CH -CH -SAc);
2
2
During the reactions, the yield of the product (%) was estimated by
using the ratio of the peak intensities from the starting material and the
product. For example, the yield of product 4 was calculated as shown in
Equation (1):
2
+
44
C H
1
at 808C for 18 h. When the reaction was finished, the solvent was evapo-
rated. Methanol was added to the mixture, which was subsequently co-
evaporated with methanol three times. The mixture was dissolved in pyri-
½compound 4
Yield ð%Þ ¼
ꢁ 100 ð%Þ
ð1Þ
ð½compound 4 þ ½compound 3Þ
dine (Pyr; 4 mL) at 08C and acetic anhydride (Ac
The reaction mixture was kept at 238C for 21 h. The solvent was evapo-
rated and the crude product was purified by using silica gel chromatogra-
2
O; 1 mL) was added.
Inhibition assay for b-1,4-GalT: The inhibitory effect of UDP on the re-
combinant human b-1,4-GalTreaction was investigated under the same
conditions as described above for the galactosylation of GCNPs contain-
ing GlcNAc derivative 1. Reaction mixtures of b-1,4-GalTand UDP-Gal
in the presence of UDP (0, 20, 50, 80, 100, 200, 300, 400, and 500 mm)
were incubated for 60 min at 258C and the degree of inhibition by UDP
was determined by the intensities of the ion peaks of 3 and 4 observed in
the LDI-TOF MS analysis. The inhibition (%) was estimated by calculat-
ing the ratio of the ion peak intensities [Eq. (2)] due to product 4 and
hetero-disulfide ion 3, which is predominantly generated from the start-
ing material (compound 1 attached to the GCNPs):
phy (eluent of chloroform with a methanol gradient from 1 to 2%) to
1
afford 11 (155 mg, 90%). H NMR (CDCl
3
, 600 MHz): d=6.52 (d, 2H;
NHAc); 5.08 (2s, 4H; H-3, H-4); 4.78 (d, J=8.4 Hz, 2H; H-1); 4.25
dd, 2H; H-6); 4.13 (d, 2H; H-6); 4.13 (m, 2H; H-2); 3.87–3.57 (m, 50H;
H-5, -CH -O-); 3.43 (t, 2H; -O-CH -CH -CH -); 2.67 (t, 4H; -CH -S-);
.31 (NHAc); 2.08, 2.00, 1.96 (Ac); 1.65 (m, 4H; -CH -); 1.55 (m, 4H;
-); MALDI-TOF MS: m/z calcd for
(
2
2
2
2
2
2
2
-
C
CH
2
-); 1.52–1.27 ppm (m, 24H; -CH
: 1564.799; found: 1587.340 [M+Na] .
2
+
72
H
128
N
2
O
30
S
2
NaOMe (200 mL, 28% methanol) was added to a solution of compound
1 (100 mg, 64 mmol) in methanol (10 mL). The reaction mixture was stir-
red at 238C for 19 h, then neutralized by using Dowex 50W-X8 (H ).
ꢀ
ꢁ
1
½product 4
Inhibition ð%Þ ¼ 1ꢀ
ꢁ 100
ð2Þ
+
ð½product 4 þ ½compound 3Þ
The mixture was purified by using silica gel chromatography (eluent of
chloroform with a methanol gradient 0–50%) to afford compound 1
Sugar-elongation reaction by N. meningitidis b-1,4-GalT expressed in E.
1
(
74 mg, 88%). H NMR (CD
3
OD, 600 MHz): d=4.48 (d, J=8.5 Hz, 2H;
-O-), 3.57 (m,
-O-), 3.43 (t, 2H), 3.30 (m, 2H), 2.68 (t, 4H;
coli
ꢀ
1
H-1), 3.94 (m, 2H), 3.87 (dd, 2H), 3.71–3.63 (m, 48H; -CH
H), 3.47 (t, 4H; -CH
2
Step i (Figure 6a): To a cell-free extract of E. coli (8.5 mgmL as esti-
[
23]
4
2
mated by Bradford assay ), which produces N. meningitidis b-1,4-GalT,
GCNPs containing 1 and UDP-Gal were added to make a final volume
of 80 mL. The final concentrations of the GlcNAc residues chemisorbed
on the GCNPs and UDP-Gal were adjusted to be 120 mm and 500 mm in
HEPES buffer (pH 7.4 containing 0.1% Triton X-100), respectively. The
mixture was incubated at 258C for 20 h.
-
-
CH
CH
2
-S-), 1.99 (s, 6H; NHAc), 1.68 (m, 4H; -CH
-CH -CH -O-), 1.42–1.29 ppm (m, 24H; -CH
2
-CH
2
3
-S-), 1.56 (m, 4H;
1
2
2
2
2
-); C NMR (CD
3
OD,
1
5
50 MHz): d=171.3 (NHAc), 100.2 (C-1), 75.5, 73.8, 69.9, 69.1, 69.0, 68.7,
4.9 (C-2), 37.3 (-CH -S-), 28.2, 28.1, 28.0, 27.8, 27.7, 26.9, 24.7 ppm;
2
MALDI-TOF MS: m/z calcd for compound 1: 1312.736; found: 1335.461
+
+
[
M+Na] , 1351.361 [M+K] .
Step ii (Figure 6a): The GCNPs were simply purified by means of ultra-
filtration using the Microcon YM-50 instrument (Millipore) and washed
with water three times. The residual GCNPs were re-suspended in a
small amount of water and used for further LDI-TOF MS analyses. LDI-
Preparation of the GCNPs bearing GlcNAc derivatives 1 and 2a: NaBH
4
(
(
70 mg, 1.6 mmol) dissolved in water (5 mL) was added to a solution of 1
10 mg, 7.6 mmol), 2a (52 mg, 69 mmol), and HAuCl (25.5 mg, 75 mmol)
4
+
in methanol (35 mL). The clear yellow solution immediately became
dark brown. After the solution had been stirred at 238C for 12 h, the re-
action mixture was subjected to ultrafiltration (Microcon YM50, Milli-
pore) and the collected material was washed with water and methanol
and then lyophilized.
TOF MS: m/z calcd for LacNAc 4: 1197.726; found: 1221.656 [M+Na] .
Comparison of the ionization efficiency of GCNPs and the gold-coated
plate: GCNPs bearing 2a were prepared as described previously. The sol-
ution of GCNPs (20 pmol) was applied to the MALDI-TOF MS plate in
a spot with a diameter of 2 mm and the plate was dried before use. For
the preparation of the monolayer of 2a, the solution of 2a (25 pmol) in
MALDI-TOF MS: m/z calcd for compound 2a: 758.503; found 781.102
+
+
[
M+Na] ; m/z calcd for disulfide 3: 1035.619; found: 1058.297 [M+Na] .
methanol was placed in
a
2 mm-diameter spot on the gold-coated
6484
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6478 – 6485