2
A. Iqbal et al. / Carbohydrate Research 377 (2013) 1–3
Table 1
O
OH
Substrate and inhibitor properties of 5–8 with GlcH and GalH
O
HO
HO
OH
Compound Enzyme (mM) (mM) V (l )
K
m
K
i
max
mol min 1 mg
ꢀ
ꢀ1
1
a
1
5
7
6
8
GlcH
GlcH
GalH
GalH
4.77 (±0.53)
—
—
0.17 (±0.01)
—
e
a
c
a
3.00 (±0.57)
—
R
O
OMe
1.37 (±0.08)
0.74 (±0.02)
N
H
e
b
2.60 (±0.29)d
b
—
—
2
a
b
c
ꢀ1
No hydrolysis of 7 (4 mM) by GlcH (0.23 units mL ).
No hydrolysis of 8 (4 mM) by GalH (11.5 units mL ).
Competitive inhibition w.r.t. 5.
Competitive inhibition w.r.t. 6.
GalH is not inhibited by compound 7 and GalH is not inhibited by compound 8.
ꢀ1
H
O
R
N
d
e
R
N+
HO
HO
OMe
3
a
OMe
3
β-N(OMe)-glycoside
ensure that any absorbance increase at 340 nm could not be attrib-
uted to enzymatic hydrolysis of the N(OMe) linkage, solutions of 7
and 8 were freshly made on the day of use and were first pre-incu-
Scheme 1. Mechanism for aminooxy glycoside formation via reaction between
reducing sugars and O,N-disubstituted hydroxylamines.
+
bated with GDH and NADP (30 min) prior to the subsequent addi-
tion of the glycosidase enzyme. Under these conditions neither 7
nor 8 showed any evidence of enzymatic hydrolysis Table 1. Fur-
ther analysis of 7 and 8 showed them to be competitive inhibitors
X
OH
X
OH
OMe
4
Bn
O
O
Y
+
HN
Y
OBn
HO
HO
i m
of GalH and GlcH, respectively, with K values similar to the K
OH
OH
OH
values observed for their respective competing substrates, 5 and
6 (Table 1). This demonstrates that the N(OMe) group could serve
as a useful bioisostere for the exo-cyclic anomeric oxygen in the
design of glycosidase resistant glycoside analogues. It is notewor-
thy that previous studies of hydrolytic stability of aminooxy glyco-
conjugates under acidic conditions have demonstrated how they
become increasingly stable as the pH approaches neutrality.18 Col-
lectively, these results suggest that degradation of aminooxy glyco-
conjugates in vivo is more likely to arise from acid-catalysed,
rather than glycosidase-catalysed hydrolysis.
5
6
X=H, Y=OH
X=OH, Y=H
(
i)
X
OH
OMe
O
7
8
X = H, Y = OH (45%)
X = OH, Y = H (63%)
Y
N
HO
Bn
OH
1
1
. Experimental
Scheme 2. Reagents and conditions: (i) DMF/acetic acid (3:1), 40 °C, 6 days.
.1. General
X
OH
X
OH
GlcH
Chemical reagents and enzymes were purchased from Sigma–
5
pH 6.2
O
O
Aldrich. Sonication-mediated reactions were carried out using a
Branson model 2510 sonicator bath operating at a frequency of
Y
GDH
Y
GalH
pH 6.2
HO
OH
HO
6
O
OH
NADP+ NADPH
OH
1
13
40 kHz. H and C NMR spectra were measured on Varian Unity
Plus 400 NMR spectrometer and were referenced to the deuterated
D-Glc
(
X=H, Y=OH)
D
NMR solvent. Optical rotations ([a] ) were obtained with a Perkin-
D-Gal
X=OH, Y=H)
Elmer Model 241 Polarimeter, using the specified solvent and
(
ꢀ1
2
ꢀ1
concentration, and are quoted in units of 10 deg cm g . High-
resolution electrospray mass spectra were conducted at the EPSRC
National Mass Spectrometry Service Centre at Swansea University
using a Finnigan MAT 900 XLT with polyethyleneimine as the ref-
erence compound. T. acidophilum glucose dehydrogenase (GDH),
E. coli b-galactosidase (GalH) and almond b-glucosidase (GlcH)
were purchased from Sigma. One unit of GalH activity is defined
Scheme 3. Coupled assay for substrate/inhibitor analyses of 5–8.
Initial incubations of N(OMe)-galactoside 8 (600 lM) with GalH
under the coupled assay procedures described above (Scheme 3)
exhibited slow NADP+ consumption suggesting enzymatic libera-
+
tion of galactose from 8. However, the same rate of NADP con-
as the amount of GalH required to hydrolyse 1
phenyl-b- -galactoside per minute under saturating substrate con-
ditions at 37 °C and pH 7.3. One unit of GlcH activity is defined as
the amount of GlcH required to liberate 1 mol of glucose from sal-
lmol of O-nitro-
sumption was also observed when the experiment was repeated
in the absence of GalH suggesting either the presence of galactose
contamination in the assay mixture or that GDH was able to oxi-
D
l
+
dise 8 in an NADP -dependent manner. The latter was ruled out be-
icin per minute under saturating substrate conditions at 37 °C and
cause the increase in absorbance at 340 nm reached a plateau long
before NADPH levels were depleted. Based on the net increase in
absorbance at 340 nm the assay concentration of galactose was
pH 5.0.
1.2. Benzyl methoxyamine b-
D-glucopyranoside (7)
determined as 24 lM by enzymatic titration. These initial experi-
ments were performed using a stock solution of 8 (pH 6.2), which
had been prepared and stored at room temperature for 20 days.
When the assay was repeated using a freshly prepared stock solu-
tion of 8 no NADP+ consumption was observed indicating that 8
was subject to slow chemical hydrolysis upon storage in mildly
acidic solution. For subsequent enzymatic stability assays, to
A
solution of -glucose (360 mg, 2 mmol) and N-benzyl-
D
O-methyl hydroxylamine 4 (137 mg, 1 mmol) dissolved in a 3:1
solvent mixture of DMF/AcOH (16 mL) was stirred at 40 °C for
6 days. The reaction mixture was then concentrated on a rotary
evaporator and purified by chromatography eluting with a step-
2 2
wise solvent gradient of CH Cl /MeOH (9:1 to 8:2) to give 7
Iqbal, Amjid
