J. Seibel et al. / Tetrahedron 61 (2005) 7081–7086
7085
3-H), 5.18–5.14 (dd, J2,3Z11.0 Hz, J2,1Z3.7 Hz, 1H, 2-H),
4.51–4.48 (t, JZ6.60 Hz, 1H, 50-H), 4.35–4.05 (m, 7H, 10-H2,
4-H, 60-H2, 6-H2). 13C NMR (100 MHz, CDCl3) d 170.46,
170.34, 170.10, 169.93, 169.87, 169.72 (7!COCH3), 103.58
(C-20), 90.41 (C-1), 78.72 (C-50), 75.46 (C-30), 74.63 (C-40),
68.00 (C-5), 67.47, 67.41, 67.12 (C-2, C-3, C-4), 63.90
(C-10), 63.08 (C-60), 61.69 (C-6), 20.67, 20.62, 20.57, 20.54
(COCH3).
membrane filter (Millipore, Germany) and analyzed, after
appropriate dilution. Analysis of the samples was carried
out using several chromatographic systems.
3.3.2. Preparative chromatography. Prior to preparative
chromatographic separation, the sucrose analogue solution
was subjected to an enzymatic treatment with a wild type
glycosyltransferase (Gtf) from Streptococcus oralis cloned
in Escherichia coli, kindly provided by Dr. Hofer (GBF
mbH, Germany). By this step, sucrose was converted into
dextran and fructose, which can be separated easily by
chromatography. The pH of the crude product solution was
adjusted to 5.4 and the reaction was started by adding
1 U Gtf mlK1 solution at 30 8C. After 2 h, the reaction was
stopped by heat denaturation.
3.2.4. b-D-Fructofuranosyl-a-D-galactopyranoside (Gal-
Fru) 6. To a stirred solution of 5 (100 mg, 147 mmol) in
MeOH (5 ml) was added NaOMe (200 ml of a 5 M solution
in MeOH, 1 mmol) dropwise at rt. After 10 min the solution
was neutralized to pH 7 with amberlite IR-120 HC, filtered
and concentrated. Purification by column chromatography
(6:1 CH3CN/H2O) gave the title compound (50.0 mg, 99%)
as a white solid.
Separation of sucrose analogues from the reaction mixture
was carried out with the PCR 6 in NaC form (300–330 mm,
Purolite, France), packed in a 2 m glass column (:Z
3.9 cm) (Borosilicat 3.3, QVF, Germany) and thermostated
at 70 8C.
White solid, mp: 160 8C, lit18 mp: 174–177 8C; [a]D C81.2
(c 1.0, H2O), lit.19 [a]D C79.0 (c 1.0, H2O); Rf 0.42 (6:3:1
EtOAc/isopropanol/H2O, 3 ascends); IR (cmK1): 3428,
1132, 1087, 1049, 1017; 1H NMR (400 MHz, D2O) d 5.40–
0
0
5.39 (d, J1,2Z3.9 Hz, 1H, 1-H), 4.18–4.15 (d, J3 ,4
Z
Z
Fifteen millilitre of Gtf (from S. oralis) reaction mixture
with a total sugar concentration of maximal 400 g lK1 was
subjected on the column and eluted with a flow rate of
4 ml minK1 distilled water. Equal fractions of 12 ml were
collected after measurement by differential refractometry.
8.7 Hz, 1H, 30-H), 4.11–4.07 (dt, J5,4Z0.9 Hz, J5,6
0
0
0
0
6.4 Hz, 1H, 5-H), 4.04–4.00 (t, 1H, J3 ,4 ZJ4 ,3 Z8.7 Hz,
40-H), 3.99–3.98 (dd, J4,5Z0.9 Hz, J4,3Z3.20 Hz, 1H, 4-H),
3.89–3.86 (dd, J3,2Z10.5 Hz, J4,3Z3.20 Hz, 1H, 3-H),
3.85–3.76 (m, 3H, 20-H, 50-H, 60-H2), 3.70–3.68 (t, JZ
6.4 Hz, 2H, 6-H2), 3.64 (s, 2H, 10-H2). 13C NMR (100 MHz,
D2O) d 106.2 (C-10), 94.87 (C-1), 83.86 (C-50), 79.12 (C-30),
76.72 (C-40), 73.99 (C-5), 71.700 (C-3), 71.67 (C-4), 70.55
(C-2), 64.93 (C-60), 64.08 (C-1 ), 63.44 (C-6). ESIMS: m/z
365.0 100% [MCNaC].
3.3.3. b-D-Fructofuranosyl-a-D-mannopyranoside (Man-
Fru) 10. [a]D C18.2 (c 1.0, H2O), lit.11 [a]D C19.1 (c 1.2,
H2O); Rf 0.40 (6:3:1 EtOAc/isopropanol/H2O, 3 ascends);
1H NMR (400 MHz, D2O) d 5.30–5.29 (d, J1,2Z1.9 Hz, 1H,
0
0
0
1-H), 4.14–4.12 (d, J3 ,4 Z8.07 Hz, 1H, 3 -H), 4.02–3.99
0
0
0
0
(t, J4 ,3 ZJ4 ,5 Z8.7 Hz, 1H, 4 -H), 3.86–3.67 (m, 9H, 2-H,
3-H, 4-H, 5-H, 6-H2, 50-H, 60-H2), 3.61 (s, 10-H2). 13C NMR
(100 MHz, D2O) d 106.55 (C-20), 96.18 (C-1), 83.93 (C-50),
78.58 (C-30), 76.51 (C-40), 75.91, 73.68, 72.70 (C-2, C-3,
C-5), 69.02 (C-4), 64.98 (C-60), 63.55 (C-10), 63.21 (C-6).
ESIMS: m/z 365.0 100% [MCNaC].
3.3. Enzymatic synthesis of sucrose analogues
3.3.1. General description of the fructosylation reaction.
For the cultivation of B. subtilis NCIMB 11871 a liquid
mineral salt medium containing 2.5% sucrose (w/v) was
prepared. The mineral salt medium contained (in mg/100 ml):
KH2PO4-136; Na2HPO4$2H2O-267; (NH4)2SO4-60;
MgSO4$7H2O-20; CaCl2$2H2O-1; FeSO4$7H2O-0.5;
MnSO4$H2O-0.18 and Na2MoO4$2H2O-0.25. Shaken
culture was incubated at 30 8C and 150 rpm for 48 h.
3.3.4. b-D-Fructofuranosyl-a-D-xylopyranoside (Xyl-Fru)
12. White solid, mp 120 8C; [a]D C59.5 (c 1.1, H2O), lit.20
[a]D C62 (c 1.0, H2O); Rf 0.46 (6:3:1 EtOAc/isopropanol/
1
H2O, 2 ascends); IR (cmK1): 3412, 1121, 1046; H NMR
(400 MHz, D2O) d 5.30–5.29 (d,0 J1,2Z3.6 Hz, 1H, 1-H),
0
0
0
0
4.17–4.15 (d, J3 ,4 Z8.09 Hz, 1H, 3 -H), 4.07–4.02 (t, J4 ,3
Z
When reaching the stationary phase, the cells were
separated by centrifugation at 5000!g for 15 min at 4 8C
(SORVALw Centrifuge, USA) and then discarded. The
supernatant obtained was analyzed undiluted, as crude
enzyme solution for the characterisation but also as
concentrated solution (ultrafiltration).
0
0
0
0
J4 ,5 Z8.9 Hz, 1H, 4 -H), 3.85–3.81 (dt, J5 ,4 Z8.9 Hz,
0
0
0
0
0
0
0
J5 ,6 Z2.8 Hz,0 1H, 5 -H), 3.78–3.74 (2d, J6a ,5 ZJ6b ,5
Z
0
2.8 Hz, 2H, 6a -H, 6b -H), 3.68–3.60 (m, 2H, 3-H, 5-H), 3.60
(s, 2H, 10-H2), 3.56–3.54 (m, 1H, 4-H), 3.50–3.46 (dd,
J2,3Z9.9 Hz, J2,1Z3.6 Hz, 1H, 2-H). 13C NMR (1000 MHz,
D2O) d 106.35 (C-20), 94.97 (C-1), 84.01 (C-5 ), 78.83
(C-30), 76.26 (C-40), 75.40 (C-3), 73.67 (C-2), 71.82 (C-4),
64.45 (C-60), 64.38 (C-5), 63.48 (C-10). ESIMS: m/z 335.0
100%, [MC NaC].
To a reaction mixture containing 40% (w/v) sucrose as
substrate and 40% (w/v) glycopyranoside as acceptor in
5.0 ml phosphate buffer (pH 6) was added the equivalent
volume of FTF supernatant (25 mU FTF per 5.0 ml
supernatant). The sucrose analogue formation was investi-
gated by discontinuous analysis of aliquots from the
reaction mixture at suitable time intervals up to 48 h.
3.3.5. b-D-Fructofuranosyl-b-L-fucopyranoside (Fuc-
Fru) 14. White solid, mp 120 8C; [a]D K18.8 (c 0.6,
H2O); Rf 0.42 (6:3:1 EtOAc/isopropanol/H2O, 2 ascends);
1
IR (cmK1): 3440, 1117, 1046, 1012; H NMR (400 MHz,
The enzyme was inactivated by boiling the samples in a
water-bath for 10 min. After cooling, the inactivated
samples were filtered through a 0.22 mm nitrocellulose
D2O) d 4.74–4.71 (d, J1,2Z8.0 Hz, 1-H), 4.20–4.16 (m, 1H,
0
40-H), 4.18–4.16 (d, J3 ,4 Z7.8 Hz, 1H, 3 -H), 3.87–3.84 (m,
0
0
0
1H, 50-H), 3.82–3.77 (m, 2H, 6a -H, 5-H), 3.73–3.70 (m, 2H,