Scheme 1. Preparation of Coumarin-Conjugated Glycosidesa
a Abbreviations: EDC ) 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide hydrochloride; TBAHS ) tetrabutylammonium hydrogen sulfate;
BTA-Cl ) benzyltriethylammonium chloride; HMPA ) hexamethylphosphoramide.
centration; 7.5 µM) for 1 h at 25 °C or 37 °C. When two or
more glycosides were hydrolyzed by an enzyme, the amount
of the enzyme (0.001-1 U) was varied to obtain the relative
rates of hydrolysis. p-Nitrophenol (14) was used as an
internal standard to quantitate the amount of products
obtained from the enzymatic reactions.
Initially, catalytic activities of â-galactosidases (0.01-
0.1 U) from Aspergillus oryzae, KluyVeromyces lactis,
Bacillus circulans, and Escherichia coli were examined. All
of these â-galactosidases catalyze the hydrolysis of nonre-
ducing terminal â-galactose-containing substrates (1 and 6,
Figure 1b).10 Coumarin â-galactoside (1) is a better substrate
than coumarin â-lactoside (6) for all the glycosidases (Table
2). Also, â-galactosidase from B. circulans was found to
enzymatic reaction was performed in the presence of the
â-GlcNAcase inhibitor (500 µM) [O-(2-acetamido-2-deoxy-
D-glucopyranosylidene)amino-N-phenylcarbamate, PUG-
NAc].12 In this case, glycoside 5 was not hydrolyzed by
â-galactosidase from B. circulans, indicating that this
compound is cleaved by the contaminated â-GlcNAcase and
not by â-galactosidase. It was also found that glycosidases
with low catalytic activities (up to 0.1 mU) were readily
detected by this method.
To extend this strategy to profiling of the glycosidase
activities in cell lysates, the catalytic activity of E. coli
â-galactosidase (LacZ) in cultures of E. coli BL21, induced
with IPTG, was determined. Cell lysates, containing 6 µg
of proteins, were incubated with the glycoside cocktail, and
the reaction mixture was analyzed by HPLC. As shown in
Figure 1c, substrates 1 and 6, each containing a â-galactose
moiety, were hydrolyzed by this enzyme in cell lysates in a
manner analogous to that observed with the free â-galac-
tosidases.
Table 2. Catalytic Activities of Glycosidases Determined by
the Use of Glycoside Cocktails
glycosidases
catalytic activity
To further demonstrate the utility of this method, additional
enzymatic reactions were performed with â-N-acetylglu-
cosaminidase, R-mannosidase, R-fucosidase, â-glucuronidase,
and R- and â-glucosidases. The results show that â-GlcNA-
â-galactosidase from A. oryzae, Gal-â-R1 (1) > Lac-â-R4 (6)
K. lactis, B. circulans, E. coli
â-N-acetylglucosaminidase
from C. ensiformis
R-mannosidase
GlcNAc-â-R3 (5) ∼
GalNAc-â-R5 (9)
Man-R-R5 (11)
from C. ensiformis
R-fucosidase from bovine kidney Fuc-R-R5 (13)
â-glucuronidase from E. coli
R-glucosidase from S. cerevisiae Glc-R-R5 (8) > Mal-â-R2 (2)
(9) (a) Carrie`re, D.; Meunier, S. J.; Tropper, F. D.; Cao, S.; Roy, R. J.
Mol. Catal. A: Chem. 2000, 154, 9-22. (b) Brubaker, A. N.; DeRuiter, J.;
Whitmer, W. L. J. Med. Chem. 1986, 29, 1094-1099. (c) Gerber, S. A.;
Turecˇek, F.; Gelb, M. H. Bioconjugate Chem. 2001, 12, 6030-615. (d)
Wang, L.-X.; Pavlova, N. V.; Li, S.-C.; Li, Y.-T.; Lee, Y. C. Glycoconjugate
J. 1996, 13, 359-365. (e) Rothermael, J.; Faillard, H. Carbohydr. Res. 1990,
196, 29-40. (f) Christine, M.; Duchateau, C.; Veyrie`res, A. Carbohydr.
Res. 1978, 65, 23-33.
GlcA-â-R5 (7) ∼ GalA-â-R5 (10)
â-glucosidase from almond
Cell-â-R3 (4) > Gal-â-R1 (1) >
Xyl-â-R1 (3) ∼ Lac-â-R4 (6)
(10) Wallenfels, K.; Weil, R. The Enzymes, 3rd ed.; Boyer, P. D., Ed.;
Academic Press: New York, 1972; Vol 7, pp 617-663.
(11) Usui, T.; Kubota, S.; Ohi, H. Carbohydr. Res. 1993, 244, 315-
323.
(12) Horsch, M.; Hoesch, L.; Vasella, A.; Rast, D. M. Eur. J. Biochem.
1991, 197, 815-818.
cleave coumarin â-N-acetylglucosaminide (5). To determine
if this unexpected finding is a result of the action of a â-N-
acetylglucosaminidase (â-GlcNAcase) contaminant,11 the
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