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M. Perez-Sanchez et al. / Tetrahedron 68 (2012) 2141e2145
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activity of the
adding a sample of enzyme solution (20
b
-galactosidases was assayed spectrophotometrically
L) with 80 L of sodium
citrate/phosphate buffer 50 mM, pH 5.0 containing p-nitrophenyl-
-galactopyranoside (pNP- -Gal) in order to obtain a final 5 mM
amino-2-deoxy-galactopyranosyl chloride (8.2 mmol) was dis-
m
m
solved in a 1:1 mixture of toluene and nitromethane (12 mL) and
mercury cyanide (8.2 mmol), MgSO4 and tetraethylene glycol
(321 mmol) were added. The mixture was stirred at room tem-
perature overnight. After addition of 20 mL CH2Cl2 the reaction
mixture was filtered over Celite and the solvent was evaporated
under reduced pressure. To the resulting syrup, 8.2 mmol NaOMe in
30 mL MeOH was added and the mixture was allowed to react for
3 h at room temperature. The reaction was neutralized with
Amberlite, the mixture was filtered and the solvent evaporated in
vacuo. The final product 6 (29%) was purified by flash chromatog-
raphy (CH2Cl2/MeOH 3:1). TLC 0.32 (CH2Cl2/MeOH 3:1). 1H NMR
(250 MHz, D2O) 5.32 (d, 1H, J¼5.3 Hz, H-4); 5.15 (dd, J1¼10.5 Hz,
J2¼7.8 Hz, H-2); 4.95 (dd, 1H, J1¼10.3 Hz, J2¼3.3 Hz, H-3); 4.52 (d,
1H, J¼7.8 Hz, H-1); 4.14 (m, 2H, H-6a, H-6b); 3.86 (m, 2H, H-5,
e(CH2)2e), 3.50e3.80 (m, 15H, e(CH2)2e); 2.17, 2.08, 2.07, 2.00 (s,
12H, CH3). 13C NMR (63 MHz, D2O): 174.3 (eCOe); 101.3 (H-1); 76.2,
74.2 (eCHe); 71.9, 70.1, 70.1, 69.9, 69.8, 68.7, 69.7 (eCH2e), 69.6
(eCHe), 60.9 (eCH2e), 60.6 (eCHe), 55.7 (eCH2e); 22.4 (eCH3).
b-
D
b
concentration (substrate). The reaction mixture was incubated for
3 min at 37 ꢁC. Absorbance was measured at 410 nm and the p-
nitrophenol (pNP) released was quantified against a standard curve
performed under the same experimental conditions. One unit of
enzyme activity was defined as the quantity of enzyme hydrolyzing
1 mmol of pNP-b-Gal per minute, under the conditions stated
above.
4.3. Semi-purification of enzymes
All operations were carried out at 4 ꢁC. A 5% m/v solution of
commercial
b-galactosidase from B. circulans in sodium citrate/
phosphate buffer 50 mM, pH 5.0 was precipitated by addition of
ammonium sulfate, (55% saturation). Then the enzyme was
centrifuged at 10,000 rpm, 20 min, 4 ꢁC with the aim to separate the
solid. The pellet was re-suspended in the same buffer and dialyzed
during 36 h (three changes of buffers) with 10 mM buffer solution.
Enzyme was lyophilized for 24 h. Proteic composition of final
sample was observed by sodium docedyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) according to Laemmli’s method,24 non
denaturing PAGE was performed under the same method without
4.5. General procedure for transglycosylation reactions using
glycoconjugates
A solution of 51.2 mg (0.17 M) p-nitrophenyl-b-D-galactopyr-
anoside (donor) and 188 mg (0.85 M) of N-acetylglucosamine (ac-
SDS, b-mercaptoethanol and pre-heating the sample, in both cases,
gels were running using 7.5% of acrylamide.
ceptor) in 1 mL of green solvent (2 M)-buffer mixture was pre-
equilibrated to 30 ꢁC. Afterwards, 155
mmol/min (U) of b-galacto-
sidase were added to the reaction mixture. Reaction was monitored
by HPLC UVevis and final products analysed by HPLC with an
evaporative light scattering detector (ELSD). The reaction was
stopped by heating the sample at 100 ꢁC for 5 min.
Isolation of disaccharides was done by carbon/Celite (50% m/m),
chromatography, the column was eluted with milliQ water and
ethanol gradient (from 5% to 15% v/v).21,26 The structures of the
4.4. Synthesis of the functionalized monosaccharides
4.4.1. Synthesis of 1-pentenyl-2-acetamido-2-deoxy-b-D-glucopyr-
anoside (1). This compound was synthesized as previously de-
scribed by de Paz et al.25 To a solution of 2-acetamido-1,3,4,6-tetra-
O-acetyl-2-deoxy-D-glucopyranoside (1 g, 2.5 mmol) in dry CH2Cl2
(15 mL), TMSOTf (3.63 mmol) was added and the mixture stirred at
30 ꢁC for 72 h. Then, 4-pentenol (7.5 mmol) was added and the
reaction stirred at that temperature for an additional 3 h. The
suspension was neutralized with Et3N, filtered and the solvent was
evaporated to dryness. The residue was purified by flash chroma-
tography (CH2Cl2/acetone 9:3) to yield 60%. TLC 0.75 (CH2Cl2/ace-
tone 3:1). 1H NMR:25 (250 MHz, CDCl3) 5.75 (m, 1H, eCH]CH2);
5.61 (d, 1H, J¼8.6 Hz, eNHe); 5.27 (t, 1H, J¼9.9 Hz, H-3); 5.03 (t, 1H,
J¼9.6 Hz, H-4); 4.98e4.92 (m, 2H, eCH]CH2); 4.64 (d,1H, J¼8.3 Hz,
H-1); 4.22 (dd,1H, J¼4.8,12.2 Hz, H-6a); 4.09 (dd,1H, J¼2.3,12.2 Hz,
H-6b); 3.86e3.77 (m, 2H, H-2, eCH2eOe); 3.67 (m, 1H, eCH2eOe);
2.17 (m, 4H, J¼7.5 Hz, e(CH2)2e); 2.04, 1.99, 1.98, 1.91 (4s, 12H,
eCOCH3); 2.05e1.59 (m, 4H, e(CH2)2). 13C NMR (63 MHz, CDCl3):
171.3, 171.2, 170.6, 169.9 (eCOe); 138.7 (eCHe); 115.4 (eCH2e);
101.1 (H-1); 72.7, 72.1 (eCHe); 69.5, 69.0, 62.6 (eCH2e); 55.3
(eCHe); 30.3, 28.9 (eCH2e); 23.8, 21.2, 21.1, 21.0 (eCH3e).
disaccharides (Gal-b(1/4)GlcNAc and Gal-b(1/6)GlcNAc) were
assigned by 1H and 13C NMR (D2O, 700 MHz), spectra were identical
to previous references.21,26,27
In order to determine the effect of non-proteic substances
present in Biolacta preparation in the reaction yield, trans-
glycosylation reactions were carried out using semipurified (ly-
ophilized) enzyme. The best performing solvents were chosen in
view of the previous results, using crude extracts and setting the
reactions with semipurified enzymes. The transglycosylation con-
ditions were the same mentioned above and the reaction was
monitored using HPLC-ELSD.
For functionalized disaccharides the same protocol previously
described was used, and different glycoconjugates as acceptors
were added. A solution of 51.2 mg (0.17 M) pNP-b-Gal (donor) and
the different monosaccharides functionalized 1 and 2 (0.51 M).
This compound (2 g) was dissolved in dry MeOH (2 mL) and
NaOMe (2 g) was added. After stirring over the night at room
temperature the reaction was neutralized with Amberlite IR-
120Hþ. The mixture was filtered and the solvent was evaporated in
vacuo to obtain compound 5. TLC 0.62 (isopropanol/nitromethane/
water 10:9:2) 1H NMR:25 (250 MHz, D2O) 84e5.68 (m, 1H, eCH]
CH2); 5.00e4.80 (m, 2H, eCH]CH2); 4.43 (d, 1H, J¼8.4 Hz, H-1);
3.67e3.32 (m, 2H, H6a, eCH2eOe); 3.63e3.25 (m, 6H, H-4,
eCH2eOe, H-3, H-5, H6b); 2.01e1.95 (m, 4H, e(CH2)2e); 1.58e1.50
(m, 3H, eCOCH3e). 13C NMR (63 MHz, D2O): 174.8 (eCOe); 131.9
(eCH2e); 115.2 (eCH¼); 101.4 (H-1); 76.1, 74.1, 70.2 (eCH2e); 69.9,
61.0, 55.9, 29.5 (eCHe); 28.7 (eCH3e); 22.5 (eCH2e).
Acknowledgements
This work has been supported by the MICINN (‘Ministerio de
ꢀ
Ciencia e Innovacion’), CTQ2009-11801. M.S. is grateful for a Doc-
toral fellowship granted by Universidad Nacional de Costa Rica. We
also thank the kind donation of Bio-solvents from Cognis IP Man-
agement GmbH, now part of BASF.
References and notes
1. Caines, M. E. C.; Zhu, H.; Vuckovic, M.; Willis, L. M.; Withers, S. G.; Wakarchuk,
W. W.; Strynadka, N. C. J. J. Biol. Chem. 2008, 283, 31279e31283.
2. Shirato, H.; Ogawa, S.; Ito, H.; Sato, T.; Kameyama, A.; Narimatsu, H.; Xiaofan, Z.;
Miyamura, T.; Wakita, T.; Ishii, K.; Takeda, N. J. Virol. 2008, 82, 10756e10767.
3. Springer, G. F. Science 1984, 224, 1198e1206.
4.4.2. Synthesis of 1-tetraethylenglycol-2-acetamido-2-deoxy-b-D-
glucopyranoside (2). This compound was synthesized as previously
4. Springer, G. F.; Desai, P. R.; Wise, W.; Carlstedt, S. C.; Tegtmeyer, H.; Stein, R.;
Scanlon, E. F. Immunol. Ser. 1990, 53, 587e612.
22
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described by Perez et al. Starting with N-acetyl-tri-O-acetyl-2-