102
C. Albermann et al. / Carbohydrate Research 334 (2001) 97–103
2%-Fucosyllactose (h-
2)-i- -galactopyranosyl-(14)-
A solution containing GDP- -fucose (35 mg,
L
-fucopyranosyl-(1
course of each reaction to GDP-b- -fucose
L
D
D
-glucose).—
was monitored by HPLC analysis with UV-
detection (Beckman, Munich, Germany) at
260 nm. As the mobile phase, a phosphate-
buffer (30 mM potassium phosphate pH 6.0; 5
mM tetrabutylammonium hydrogensulfate,
4% MeCN) and MeCN were used in combina-
tion with a reversed-phase column Eurospher
ODS18, 5 mm, 250×4.6 mm (Knauer, Berlin,
Germany).27
L
57 mmol sodium salt), lactose (30 mg, 80
mmol), the GST-FucT2 protein (10 nkat) was
incubated at 37 °C for 8 h in reaction buffer
(10 mM MnCl2, 100 mM NaCl in 50 mM
Tris–HCl pH 7.5). Boiling and centrifugation
removed the proteins. The mixture was
purified by an anion-exchange column chro-
matography (Dowex 1×8 resin, mesh 200–
400; Cl-form, Serva, 1×10 cm, water). The
eluate was concentrated under diminished
pressure and then the product was further
purified by gel filtration on a column of Sep-
hadex G-10 (2.5×100 cm, water; Pharmacia,
Germany), equipped with a RI-detector
(Knauer, Germany), to give 2%-fucosyllactose
(18 mg, 65%). 2%-Fucosyllactose and other sac-
charides were analysed by means of HPAEC–
PAD using a Dionex DX-500 system equipped
with a PA10 column (Dionex, Sunnyvale,
GDP-b-
-fucose was purified from NADP+
L
and NADPH by preparative HPLC. The
chromatography was performed with an Eu-
rospher ODS18 (20×250 mm) column
(Knauer, Berlin, Germany) using the same
conditions described as above for the analyti-
cal chromatography. GDP- -fucose-contain-
L
ing fractions were pooled and evaporated to a
volume of 12 mL under diminished pressure
(20 mbar) at 20–25 °C. The preparation was
desalted by gel filtration on a Sephadex G-10
column (SR 25/100; Pharmacia, Freiburg,
Germany), in a total volume of 398 mL and
eluted at a flow rate of 1 mL/min. The GDP-
USA). Spectrometric data for a-
L
-fucopyran-
osyl-(12)-b- -galactopyranosyl-(14)- -
D
D
1
glucose: H NMR (400 MHz, D2O): l 5.27 (d,
H-1%%), 5.18 (d, J1,2 3,3 Hz, H-1a), 4.60 (d, J1,2
7.7 Hz, H-1b), 4.48 (d, J1,2 7.6 Hz, H-1%), 4.21
(m, J5,6 6.5 Hz, H-5%%), 3.79 (d, J2,3 9.8 Hz,
H-2%%), 3.25 (dd, J1,2 7.7 Hz, H-2b), 1.19 (d, J5,6
6.5 Hz, H-6%%); 13C NMR (100 MHz, D2O): l
102.51 (C-1%), 101.59 (C-1%%), 98.16 (C-1a),
94.08 (C-1b), 78.56 (C-4a), 78.36 (C-4b), 78.15
(C-3%%), 77.48 (C-5%%), 76.64 (C-3b), 76.56 (C-
5b), 76.22 (C-3%), 75.87 (C-2%%), 74.82 (C-4%%),
73.94 (C-3b), 73.56 (C-2b), 71.93 (C-2a), 71.42
(C-5%), 71.40 (C-5a), 70.46 (C-2%%), 69.16 (C-4%),
62.45 (C-6b), 62.35 (C-6a), 61.91 (C-6%), 17,49
(C-6%%); ESIMS: m/z [M+Na]+ 511.04.
b- -fucose-containing fractions were pooled
L
and applied to a membrane anion exchanger
Q15 (Sartorius, Go¨ttingen, Germany) and the
GDP-b- -fucose preparation was equilibrated
L
against 150 mM NaCl to yield the Na+ form.
After another volume reduction by evapora-
tion to a volume of about 12 mL and a further
gel filtration (Sephadex G-10 column), the
sodium GDP-b- -fucose was lyophilised
L
(Cryograph LCD-1, Christ, Osterrode, Ger-
many) (78 mg, 78%).
1H NMR (400 MHz, D2O): l 1.1 (d, H-6%%,
3
3J5%%6%% 6.4 Hz); 3.55 (dd H-2%%, J2%%3%% 11 Hz,
3
3J1%%2%% 8.1 Hz); 3.65 (dd, H-3%%, J3%%4%% 3.5 Hz,
3
3J2%%3%% 11 Hz); 3.71 (m, H-4%%, J3%%4%% 3.5 Hz);
3.75 (m, H-5%%); 4.2 (m, 2 H, H-5a%H-5b%); 4.52
(m, H-4%); 4.59 (dd, H-3%); 4.8 (dd, H-2%,
Acknowledgements
3
3
3J1%2%6.1 Hz, J2%3%3.7 Hz); 4,91 (dd, H-1%%, J1%%2%%
3
3
8.1 Hz, JP-2H1%% 8.3 Hz); 5.9 (d, H-1%, J1%2%6.1
We thank Roche Diagnostics, Penzberg,
Germany, for financial support and for the
preparation of the H. pylori DNA. We are
grateful to Professor Dr J. Thiem and his
coworkers for the synthesis of acceptor
oligosaccharides used in this study. We also
thank Y. Rockser and E. Sauerbier for techni-
cal assistance.
13
Hz); 8,1 (s, H-1); C NMR (100 MHz, D2O):
l 17.5 C%-6%%; 33.2 C-2%; 64.6 C-5%; 73.1 73.2
72.5 C-3%% C-4%% C-5%; 74.5 C-3%%; 75.8 C-2%%;
100.4 C-1%%; 118.3 C-5; 139.6 C-1; 153.9 C-4;
156.2 C-2; 161.2 C-6; 31P NMR (160 MHz,
2
D2O): l −9.7 (P-1, JPP 19.9 Hz); −11.5
2
3
(P-2, Jpp 19.9 Hz, JP-2H-1%%8.3 Hz).