E Benesˇova´ et al.
transglycosylation products were calculated according to TLC
spot densities determined by program TotalLab (Nonlinear Dy-
namics, UK) using the data for the added donor molecule, ob-
tained transglycosylation products, and fucose released during
the reaction. The sum of all these data represents 100% of fu-
cosyl moieties in reaction mixtures.
p-Nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-mannopyrano-
side: 1H NMR: δ 8.21 (d, 2H, J = 9.2 Hz, p-NO2Ph), δ 7.25 (d,
2H, J = 9.2 Hz, p-NO2Ph), δ 5.76 (d, 1H, J = 1.5 Hz, H-1),
δ 4.52 (d, 1H, J = 7.8 Hz, H-1ꢁ), δ 4.33 (dd, 1H, J = 3.2 Hz,
H-2), δ 4.15 (dd, 1H, J = 3.2 Hz, 9.6 Hz, H-3), δ 3.82 (dd, 1H,
J = 9.7 Hz, H-4), δ 3.79 (m, 1H, H-5ꢁ), δ 3.75–3.66 (m, 3H, H-4ꢁ,
H-6, overlapped), δ 3.65 (dd, 1H, J = 3.6 Hz, 9.9 Hz, H-3ꢁ), δ
3.63 (m, 1H, H-5), δ 3.53 (dd, 1H, J = 7.9 Hz, 9.8 Hz, H-2ꢁ), δ
1.23 (d, 3H, J = 6.6 Hz, 6ꢁ-CH3).
Thin-layer chromatography
The samples of the reaction mixtures were spotted on the Silica
gel TLC plate (Fluka, USA) and developed by a solvent sys-
tem ethylacetate:acetic acid:water (7:2:2, v/v/v) (ethylacetate
purchased from Lach-Ner, s.r.o, CZ and acetic acid from Penta,
CZ). The spots of reaction products after separation were visual-
ized by UV detection (in the case of molecules containing the p-
nitrophenyl group) and by 0.1 M 2-methylresorcinol (Alfa Aesar
GmbH & Co. KG, Germany) dissolved in the 5% (v/v) solution
of sulfuric acid (Lach-Ner, s.r.o, CZ) in ethanol, detecting the
saccharidic parts of molecules (after heating of the TLC plate).
13C NMR: δ 160.8 (p-NO2Ph-1ꢁꢁ), 142.3 (p-NO2Ph-4ꢁꢁ),
126.1 (p-NO2Ph-3ꢁꢁ), 116.7 (p-NO2Ph-2ꢁꢁ), 101.2 (C-1ꢁ), 97.5
(C-1), 78.4 (C-3), 73.5 (C-5), 72.8 (C-3ꢁ), 71.3 (C-4ꢁ), 71.1
(C-5ꢁ), 70.6 (C-2ꢁ), 67.5 (C-2), 65.0 (C-4), 60.6 (6-CH2), 15.4
(6ꢁ-CH3).
p-Nitrophenyl β-D-fucopyranosyl-(1,3)-β-D-fucopyranoside:
1H NMR: δ 8.21 (d, 2H, J = 9.3 Hz, p-NO2Ph), δ 7.25 (d,
2H, J = 9.3 Hz, p-NO2Ph), δ 5.19 (d, 1H, J = 7.7 Hz, H-1), δ
4.58 (d, 1H, J = 7.8 Hz, H-1ꢁ), δ 4.04 (d, 1H, J = 3.1 Hz, H-4),
δ 4.00 (q, 1H, J = 6.6 Hz, H-5), δ 3.92 (dd, 1H, J = 7.7 Hz,
9.8 Hz, H-2), δ 3.87 (dd, 1H, J = 3.2 Hz, 9.8 Hz, H-3), δ 3.73
(q, 1H, J = 6.5, H-5ꢁ), δ 3.69 (d, 1H, J = 3.4 Hz, H-4ꢁ), δ 3.61
(dd, 1H, J = 3.4 Hz, 9.8 Hz, H-3ꢁ), δ 3.53 (dd, 1H, J = 7.8 Hz,
9.8 Hz, H-2ꢁ), δ 1.26 (d, 3H, J = 6.6 Hz, 6-CH3), δ 1.24 (d, 3H,
J = 6.5 Hz, 6ꢁ-CH3).
Identification and characterization of transglycosylation
products by mass spectrometry and NMR spectroscopy analysis
All transglycosylation products, obtained from transfucosyla-
tion reactions using p-nitrophenyl glycopyranosides and alco-
hols as acceptors, were confirmed by mass spectrometry analysis
in mode ESI+ (Q-Tof micro mass spectrometer, Waters Micro-
mass, USA) with direct inlet. In the case of alcohol acceptors
the whole reaction mixture (enzyme removed by filtration on
Vecta Spin Micro filters) was analyzed. Inasmuch as the re-
action mechanism allows only the formation of one type of
glycosidic bond (between the alcohol hydroxyl group and C1
of the fucosyl moiety), it was not necessary to analyze these
products by NMR spectroscopy. Products of transglycosyla-
tion reactions with p-nitrophenyl glycopyranosides as acceptors
were prepared by extraction into methanol directly from TLC
plates after separation. Methanol was evaporated for decreasing
the volume and samples were filtered using syringe filters with
the polytetrafluoroethylene membrane (0.45 μm) (Whatmann
International Ltd). Remaining methanol was evaporated and the
samples were dissolved in D2O, so that they were suitable not
only for mass spectrometry analysis, but also for NMR spec-
13C NMR: δ 161.8 (p-NO2Ph-1ꢁꢁ), 142.5 (p-NO2Ph-4ꢁꢁ),
126.1 (p-NO2Ph-3ꢁꢁ), 116.3 (p-NO2Ph-2ꢁꢁ), 104.1 (C-1ꢁ), 99.4
(C-1), 81.8 (C-3), 72.7 (C-3ꢁ), 71.3 (C-4ꢁ), 71.2 (C-2ꢁ), 70.9
(C-5), 70.8 (C-4, C-5ꢁ), 69.3 (C-2), 15.5, 15.3 (6-CH3, 6ꢁ-CH3).
p-Nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-glucopyrano-
side: 1H NMR: δ 8.22 (d, 2H, J = 9.3 Hz, p-NO2Ph), δ 7.25 (d,
2H, J = 9.3 Hz, p-NO2Ph), δ 5.80 (d, 1H, J = 3.5 Hz, H-1),
δ 4.60 (d, 1H, J = 7.8 Hz, H-1ꢁ), δ 4.05 (dd, 1H, J = 9.3 Hz,
H-3), δ 3.92 (dd, 1H, J = 3.5 Hz, 9.7 Hz, H-2), δ 3.80 (q, 1H,
J = 6.5 Hz, H-5ꢁ), δ 3.73 (d, 1H, J = 3.0 Hz, H-4ꢁ), δ 3.62–3.70
(m, 4H, H-6a, H-5, H-6b, H-3ꢁ, overlapped with impurity), δ
3.45–3.60 (m, 2H, H-2ꢁ, H-4, overlapped with impurity), δ 1.23
(d, 3H, J = 6.5 Hz, 6ꢁ-CH3).
13C NMR: δ 161.2 (p-NO2Ph-1ꢁꢁ), 142.4 (p-NO2Ph-4ꢁꢁ),
126.0 (p-NO2Ph-3ꢁꢁ), 116.7 (p-NO2Ph-2ꢁꢁ), 103.2 (C-1ꢁ), 96.5
(C-1), 82.2 (C-3), 72.7 (C-3ꢁ), 72.6 (C-5), 71.2 (C-2ꢁ), 71.0
(C-4ꢁ), 71.0 (C-5ꢁ), 70.2 (C-2), 67.8 (C-4), 60.2 (6-CH2), 15.4
(6-CH3, 6ꢁ-CH3).
1
troscopy analysis. H, 13C, COSY, HMQC and HMBC, NOE,
and TOCSY spectra were measured on a Bruker AdvanceIII
600 (Bruker Corporation, Germany) spectrometer operating at
600.13 MHz for 1H and 150.92 MHz for 13C. All spectra were
acquired at 298 K in D2O. Chemical shifts are given in δ-units
(ppm) and are referenced to TMS.
Funding
The Ministry of Education, Youth and Sports (MSM
6046137305).
p-Nitrophenyl β-D-fucopyranosyl-(1,6)-α-D-galactopyrano-
1
side: H NMR: δ 8.21 (d, 2H, J = 9.2 Hz, p-NO2Ph), δ 7.26
(d, 2H, J = 9.2 Hz, p-NO2Ph), δ 5.79 (d, 1H, J = 3.7 Hz, H-1),
δ 4.23 (d, 1H, J = 7.9 Hz, H-1ꢁ), δ 4.15 (m, 1H, H-5), δ 4.04–4.09
(m, 2H, H-3, H-4), δ 3.98 (dd, 1H, J = 3.7 Hz, 9.7 Hz, H-2),
δ 3.92 (dd, 1H, J = 4.6 Hz, 11.3 Hz, H-6a), δ 3.74 (dd, 1H,
J = 7.3 Hz, 11.3 Hz, H-6b), δ 3.61–3.65 (m, 2H, H-4ꢁ, H-5ꢁ,
overlapped with impurity), δ 3.46 (dd, 1H, J = 3.4 Hz, 9.9 Hz,
H-3ꢁ), δ 3.28 (dd, 1H, J = 7.9 Hz, 9.9 Hz, H-2ꢁ), δ 1.11 (d, 3H,
J = 6.5 Hz, 6ꢁ-CH3).
Abbreviations
CIAP, calf intestinal alkaline phosphatase; DMF, dimethylfor-
mamide; DNA, deoxyribonucleic acid; E. coli, Escherichia coli;
IPTG, isopropyl β-D-thiogalactopyranoside; LBA, ampicillin
containing Luria-Bertani medium; LB, Luria-Bertani medium;
NMR, nuclear magnetic resonance; OD, optical density; P. thi-
aminolyticus, Paenibacillus thiaminolyticus; PAGE, polyacry-
lamide gel; PCR, polymerase chain reaction; pNPβ-D-Fuc,
p-nitrophenyl β-D-fucopyranoside; pNPα-D-Gal, p-nitro-
phenyl α-D-galactopyranoside; pNPβ-D-Gal, p-nitrophenyl
13C NMR: δ 161.7 (p-NO2Ph-1ꢁꢁ), 142.4 (p-NO2Ph-4ꢁꢁ),
126.1 (p-NO2Ph-3ꢁꢁ), 117.0 (p-NO2Ph-2ꢁꢁ), 102.8 (C-1ꢁ), 97.0
(C-1), 72.9 (C-3ꢁ), 71.2 (C-5ꢁ), 70.9 (C-5), 70.8 (C-4ꢁ), 70.4 (C-
2ꢁ), 69.1 (C-3, C-4), 68.6 (6-CH2), 67.8 (C-2), 15.3 (6ꢁ-CH3).
450