836
T. Sato et al. / Carbohydrate Research 343 (2008) 831–838
2
2
efficiency would be determined by varying the hydro-
carbon chain length.
(88.1%). Mp 160–162 °C, lit. mp 161 °C, [a]D ꢀ18.8
1
(c 0.12, CH OH). H NMR (CD OD): d 4.38 (d, 1H,
3
3
In conclusion, saccharide primers such as GlcNAc-
C12 and LacNAc-C12 were developed to synthesize
neolacto-series oligosaccharides using mammalian cells.
The glycosylated products were separated by HPLC,
and the sequences were determined by enzymatic diges-
tion and mass spectrometry. The saccharide primers
employed in this study are expected to be useful for syn-
thesizing oligosaccharides expressed in mammalian cells.
J1,2 6.1 Hz, H-1), 3.91–3.83 (m, 2H, H-6a, NH), 3.70–
3.58 (m, 2H, H-2, H-5), 3.48–3.40 (m, 2H, H-3, H-6b),
3.34–3.27 (m, 3H, H-4, OCH ), 1.97 (s, 3H, Ac), 1.53–
2
1.51 (m, 2H, OCH CH (CH ) CH ), 1.34–1.22 (m,
2
2
2 9
3
18H, OCH CH (CH ) CH ), 0.89 (t, 3H, OCH CH -
2
2
2 9
3
2
2
(CH ) CH ). MALDI-TOFMS: calcd for C H NO :
2
9
3
20 39
6
+
+
(M+Na) , 412.3, Found: (M+Na) , 412.3. Anal. Calcd
for C H NO ꢂ0.3H O (398.68): C, 60.82; H, 10.11; N,
2
0
39
6
2
3
.55. Found: C, 60.81; H, 10.04; N, 3.54.
4. Experimental
4.2. Synthesis of dodecyl b-D-galactopyranosyl-(1?4)-2-
acetamido-2-deoxy-b-D-glucopyranoside (LacNAc-C12)
4.1. Synthesis of dodecyl 2-acetamido-2-deoxy-b-D-
glucopyranoside (GlcNAc-C12)
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1?4)-2-
acetamido-1,3,6-tri-O-acetyl-2-deoxy-b-D-glucopyranos-
ide (Ac-LacNAc) was prepared by mixing N-acetyl-
lactosamine (982 mg, 2.56 nmol, LacNAc, Yaizu
2
-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopy-
ranoside (Ac -GlcNAc) was prepared by reacting
4
N-acetylglucosamine (2.5 g, 11.3 nmol, GlcNAc, Sigma)
Suisankagaku Industry Co. Ltd, Japan) with Ac O
2
with Ac O (15 mL, 159 mmol, Wako Pure Chemicals) in
(5 mL, 52.9 mmol) in 10 mL of pyridine. Ac-LacNAc
(0.799 g, 1.88 mmol) was mixed with TMS-OTf
(0.24 mL, 1.33 mmol) in CH Cl under nitrogen. The
solution was refluxed at 50 °C with stirring for 12 h.
After evaporation, followed by neutralization with
2
1
9
3
0 mL of pyridine according to the literature. Ac -Glc-
4
NAc (3 g, 7.71 mmol) was mixed with TMS-OTf
4.0 mL, 21.9 mmol, E. Merck) in CH Cl under nitro-
2
2
(
2
2
2
0
gen. The solution was refluxed at 50 °C with stirring
for 7 h. After evaporation followed by neutralization
Et N, the product was chromatographed on silica gel
3
with Et N, the product was chromatographed on silica
gel to examine the progress of the reaction. The reaction
mixture was mixed with 1-dodecanol (3.6 mL,
to examine the progress of reaction. After evaporation,
the product was collected by column chromatography
(Silica Gel 60, 2 ꢁ 23 cm, 1:2:0.01 toluene–EtOAc–
3
1
7
4
5.5 mmol, Wako Pure Chemicals), BF ꢂOEt (21 mL,
Et N). The collected products were mixed with 1-dodec-
3
2
3
.9 mmol, Wako Pure Chemicals) in the presence of
anol (1.3 mL, 5.89 mmol), (R,S)-camphor sulfonate
˚
A molecular sieves (2.5 g), and stirred at room temper-
(27 mg, 0.12 mmol, Wako Pure Chemicals) in the pre-
2
1
˚
ature for 22 h. BF ꢂOEt (20.1 mL, 0.79 mmol) was
sence of 4 A molecular sieves (350 mg), and refluxed
3
added at 18 h to complete the reaction. The mixture
for 6 h. The mixture was neutralized with Et N. After
3
was neutralized with Et N. After evaporation, the prod-
evaporation of the solvent, the product was purified by
3
uct was purified by column chromatography (Silica Gel
column chromatography (Silica Gel 60, 2 ꢁ 35 cm, 2:3
1
6
6
8
0, E. Merck, 7 ꢁ 30 cm, 1:1 n-hexane–EtOAc). Yield:
n-hexane–EtOAc). Yield: 50% (478 mg). H NMR
1
3.8% (2.53 g). H NMR(CDCl ): d 5.51 (d, 1H, J
(CDCl ): d: 5.63 (d, 1H, J
9.3 Hz, NH), 5.35 (d,
3
2,NH
3
NH,2
0
0
.8 Hz, NH), 5.31 (dd, 1H, J2,3 10.1 Hz, J3,4 9.5 Hz,
1H, J
0
0
2.9 Hz, H-4 ), 5.11 (dd, 1H, H-2 ), 5.06 (dd,
3 ;4
H-3), 5.06 (dd, 1H, J3,4 9.5 Hz, J4,5 9.9 Hz, H-4), 4.65
d, 1H, J1,2 8.4 Hz, H-1), 4.26 (dd, 1H, J5,6b 4.7 Hz,
J6,gem 12.3 Hz, H-6a), 4.12 (dd, 1H, J5,6a 2.4 Hz, J6,gem
1H, J
8.1 Hz, H-3), 4.97 (dd, 1H, J
0 0
10.3 Hz,
3,4
2 ;3
0
0
(
H-3 ), 4.51–4.46 (m, 2H, H-1 , H-6a), 4.43 (d, 1H, J
7.3, H-1), 4.15–4.09 (m, 3H, H-6b, H-6b , H-6a ), 4.03
(dd, 1H, J2,3 9.3 Hz, H-2), 3.87 (ddd, 1H, H-5 ), 3.78
(dd, 1H, H-4), 3.62 (ddd, 1H, J4,5 5.6 Hz, H-5), 3.41
(dd, 2H, OCH CH (CH ) CH ), 2.15–1.96 (m, 21H,
1,2
0
0
0
1
8
9
1
2.3 Hz, H-6b), 3.89–3.75 (m, 2H, J2,NH 8.8 Hz, J1,2
.4 Hz, H-2, OCH CH (CH ) CH ), 3.69 (ddd, J
4,5
2
2
2 9
3
.9 Hz, J5,6a 2,4 Hz, J5,6b 4.7 Hz, H-5), 3.50–3.42 (m,
H, OCH CH (CH ) CH ), 1.94, 2.02, 2.02, and 2.08
2
2
2 9
3
Ac), 1.60–1.46 (m, 2H, OCH CH (CH ) CH ), 1.30–
2
2
2 9
3
2
2
2 9
3
(
s, each 3H, Ac), 1.60–1.50 (m, 2H, OCH CH -
1.18 (m, 18H, OCH CH (CH ) CH ), 0.87 (t, 3H,
2 2 2 9 3
2
2
(
CH ) CH ), 1.35–1.14 (m, 18H, OCH CH (CH ) CH ),
OCH (CH ) CH ).
2 2 10 3
2
9
3
2
2
2 9
3
0
.87 (t, 3H,OCH CH (CH ) CH ).
Dodecyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
Dodecyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1?4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b-D-gluco-
pyranoside (478 g, 0.56 mmol) in 25 mL of MeOH was
deacetylated by the addition of NaOMe (160 mg,
2.97 mmol) with stirring for 3 h. The reactant was
concentrated after treating with Amberlite IR-120B
(Organo Co., Japan). LacNAc-C12 was purified by dis-
2
2
2 9
3
glucopyranoside (2.5 g, 4.85 mol) in 100 mL of MeOH
was deacetylated in the presence of NaOMe (270 mg,
5
.0 mmol, Wako Pure Chemicals). Deprotection was
carried out with stirring for 40 min. After decolorization
on charcoal in EtOH, the product GlcNAc-C12 was
obtained by recrystallization in ethanol. Yield: 1.70 g
tilling with EtOH, toluene, and CHCl . Yield: 326 mg
3