4-deoxy-substrates for β-N-acetylhexosaminidases: How to make use of their loose specificity

Article abstract of DOI:10.1093/glycob/cwq058

β-N-Acetylhexosaminidases feature so-called wobbling specificity, which means that they cleave substrates both in gluco-and galacto-configurations, with the activity ratio depending on the enzyme source. Here we present the new finding that fungal β-N-acetylhexosaminidases are able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides with high yields. This clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose ring is not essential for substrate binding to the enzyme active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of β-Nacetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was synthesized and screened against a panel of β-N-acetylhexosaminidases (extracellular and intracellular) from various sources (fungal, human, animal, plant and bacterial) for hydrolysis. The results of this screening are reported here, as well as the structures of three novel 4′-deoxy-disaccharides prepared by transglycosylation reaction with high yields (52% total disaccharide fraction) using β-N-acetylhexosaminidase from Talaromyces flavus. The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Full text of DOI:10.1093/glycob/cwq058

Glycobiology vol. 20 no. 8 pp. 1002–1009, 2010
doi:10.1093/glycob/cwq058
Advance Access publication on April 14, 2010
4-Deoxy-substrates for β-N-acetylhexosaminidases: How to
make use of their loose specificity
Kristýna Slámová2,3, Radek Gažák2, Pavla Bojarová2,
Natallia Kulik4,5, Rudiger Ettrich4,5, Helena Pelantová6,
Petr Sedmera6,*, and Vladimír Křen1,2
human hexosaminidases result in serious lysosomal storage
disorders (Sandhoff and Tay-Sachs diseases), which draws at-
tention to the structure and catalytic mechanism of these
enzymes (Mark and James 2002; Maier et al. 2003; Mark et
al. 2003; Lemieux et al. 2006). In the past decade, we have
concentrated on the study of substrate specificities and structur-
al aspects of fungal β-N-acetylhexosaminidases, with respect
to their high synthetic potential for the preparation of bioactive
oligosaccharides (Fialová et al. 2004, 2005; Loft et al. 2009).
In this paper, we focused on the 4-hydroxyl group of the sub-
strate pyranosyl ring, which can be either axial (GalNAc) or
equatorial (GlcNAc). The substrates of both configurations
are cleaved by β-N-acetylhexosaminidases, with the
GlcNAc-ase/GalNAc-ase activity ratio depending on the en-
zyme source (Weignerová et al. 2003). This phenomenon is
called the “wobbling” specificity. In our previous work (Křen
et al. 1998), we have shown that, e.g., galactosyltransferase
from bovine milk is also able to wobble at the C-4 position
and catalyze the transfer of glucose.
4-Deoxy acetamido sugars and their derivatives have been
synthesized and studied for various biological activities by
several groups. Peracetylated 2-acetamido-2,4-dideoxy-4-
fluoro-D-glucosamine (4-F-GlcNAc) was used for the down-
regulation of the expression and ligand activity of T-cell
receptors (P- and E-selectin), lowering skin inflammatory re-
sponses and preventing the dermal dissemination of
cutaneous lymphomas (Descheny et al. 2006; Gainers et al.
2007). It was also shown that the treatment of ovarian tumor
cells with 4-F-GlcNAc resulted in an inhibition of cellular at-
tachment to galaptin, which plays a vital role in the adhesion
of tumor cells (Woynarowska et al. 1994). The structural re-
quirements of mammalian Gal/GalNAc-specific lectins were
studied using allyl 3- and 4-deoxy-GalNAc (Ichikawa et al.
1990). Other authors observed the biological role of 4-deoxy
hexosaminide derivatives as chain terminators in glycoconju-
gate (heparan sulfate) or chitin biosynthesis, which makes
these compounds potential anti-amyloid and antifungal thera-
peutic agents (Berkin et al. 2000, 2002, 2005; Kisilevsky et
al. 2004; Danac et al. 2007). Recently, Aerts' group presented
a novel substrate 4-methylumbelliferyl 4-deoxychitobiose for
an accurate and convenient chitotriosidase assay in patients
with Gaucher disease (Aguilera et al. 2003; Schoonhoven et
al. 2007). Finally, a number of deoxygenated donor/acceptor
substrates have been used in structure–function relationship
studies of various glycosyltransferases (Srivastava et al.
1990; Brockhausen et al. 1992; Kanie et al. 1993; Du and
Hindsgaul 1996; Kajihara et al. 1998).
2Centre of Biocatalysis and Biotransformation, Institute of Microbiology,
Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ 14220 Praha
4, Czech Republic, 3Department of Biochemistry and Microbiology, Institute
of Chemical Technology, Prague, Technická 5, CZ 16628 Praha 4, Czech
Republic, 4Centre of Biocatalysis and Biotransformation, Institute of Systems
Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek
5
136, CZ 37333 Nové Hrady, Czech Republic, Institute of Physical Biology,
University of South Bohemia, Zámek 136, CZ 37333 Nové Hrady, Czech
Republic, and 6Laboratory of Characterization of Molecular Structure, Institute
of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083,
CZ 14220 Praha 4, Czech Republic
Received on January 8, 2010; revised on April 8, 2010; accepted on
April 8, 2010
β-N-Acetylhexosaminidases feature so-called wobbling
specificity, which means that they cleave substrates both
in gluco- and galacto- configurations, with the activity ra-
tio depending on the enzyme source. Here we present the
new finding that fungal β-N-acetylhexosaminidases are
able to hydrolyze and transfer 4-deoxy-N-acetylhexosami-
nides with high yields. This clearly demonstrates that the
4-hydroxy moiety at the substrate pyranose ring is not es-
sential for substrate binding to the enzyme active site,
which was also confirmed by molecular docking of the test-
ed compounds into the model of the active site of β-N-
acetylhexosaminidase from Aspergillus oryzae. A set of four
4-deoxy-N-acetylhexosaminides was synthesized and
screened against a panel of β-N-acetylhexosaminidases
(extracellular and intracellular) from various sources
(fungal, human, animal, plant and bacterial) for hydroly-
sis. The results of this screening are reported here, as well
as the structures of three novel 4′-deoxy-disaccharides
prepared by transglycosylation reaction with high yields
(52% total disaccharide fraction) using β-N-acetylhexosa-
minidase from Talaromyces flavus.
Keywords: β-N-acetylhexosaminidase/4-deoxy-glycoside/
enzymatic synthesis/modified substrate/transglycosylation
Introduction
In recent years, β-N-acetylhexosaminidases (EC 3.2.1.52,
CAZy GH20) have been intensively studied, especially their
structure, function and substrate specificity. Dysfunctions in
1To whom correspondence should be addressed: Tel.: +420-296442510; Fax:
+420-296442509; e-mail: kren@biomed.cas.cz
The first studies of the active site architecture and struc-
ture–function relationship in β-N-acetylglucosaminidases
*Dr. Petr Sedmera passed away during the preparation of this manuscript.
1002 © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

4-Deoxy-substrates for β-N-acetylhexosaminidases
HO
OH
OH
O
O
O
O
HO
HO
NHAc
O
NHAc
O
2
1
NO2
NO2
OH
OH
HO
O
O
HO
HO
NHAc
NHAc
3
4
Fig. 1. β-N-Acetylhexosaminidase substrates 1–4.
using deoxygenated substrates were performed in the 1970s.
Mega et al. (1973) determined the contribution of the binding
energies of the substrate hydroxyl moieties into the active site
of β-N-acetylglucosaminidase from Aspergillus oryzae by de-
termining the enzyme kinetic parameters for these phenyl
deoxy-hexosaminides (incl. compound 1 presented here)
and calculating the respective binding energy differences.
Later, a similar experiment with β-N-acetylglucosaminidase
from pig epididymis was described by Kolesnikov et al.
(1976), using p-nitrophenyl deoxy-hexosaminides as sub-
strates. The results of these projects suggested that the
pyranosyl 4-hydroxy group is not crucial to the activity of
this enzyme.
The aim of this work is to study the influence of the 4-
hydroxyl group (and its absence) in the reaction catalyzed
by β-N-acetylhexosaminidases from diverse sources and to
develop a method for the enzymatic synthesis of complex
4′-deoxy-disaccharides. Here we report on the synthesis of
4-deoxy-glycopyranosides 1 – 4 (Figure 1) and on their
use as substrates for a panel of one prokaryotic and 27 eu-
karyotic β-N-acetylhexosaminidases. The results of the
activity assays were compared with computational modeling
experiments. Furthermore, a high-yielding transglycosylation
reaction is reported, using 4-deoxy-glycoside 1 as a glycosyl
donor catalyzed by a fungal β-N-acetylhexosaminidase.
of unsaturated aldehyde 14 yielded 4-deoxy derivative 2. Com-
pound 4 was obtained by deacetylation of 12b.
Screening for hydrolysis of 4-deoxy-glycosides by β-N-
acetylhexosaminidases
Four 2-acetamido-2,4-dideoxy-β-D-hexopyranosides 1–4 were
tested as substrates with a large series of β-N-acetylhexosamini-
dases (24 fungal extracellular enzymes produced in the
Laboratory of Biotransformation and four commercial enzymes
from non-fungal sources, including a human one). The 4,5-unsat-
urated glycosides 3 and 4 were not cleaved by any of the enzymes
tested. 4-Deoxy-hexosaminides 1 and 2 proved to be good β-N-
acetylhexosaminidase substrates (Table I). Compound 1 in partic-
ular appeared to be a very good substrate for fungal β-N-
acetylhexosaminidases (up to 85% hydrolysis rate relative to phe-
nyl 2-acetamido-2-deoxy-β-D-glucopyranoside [Ph-GlcNAc]),
especially for the enzymes from the Penicillium and Talaromyces
genera (Table I). These results were supported by an in silico ex-
periment, in which glycosides 1–4 were docked into the
active site of a model of fungal β-N-acetylhexosaminidase
(A. oryzae). Generally, fungal enzymes were able to catalyze
the hydrolysis of 4-deoxy-glycosides more efficiently than
those of plant, bacterial or mammalian origin.
Transglycosylation reactions using 4-deoxy-N-
acetylhexosaminide 1 as a glycosyl donor
Results
The conditions of the transglycosylation reaction using com-
pound 1 as a glycosyl donor, GlcNAc (5) as an acceptor, and
β-N-acetylhexosaminidase from Talaromyces flavus CCF 2686
were first optimized at analytical scale by varying the concen-
trations and ratios of the reaction components. The reactions
were monitored by thin-layer chromatography (TLC), and
the following reaction conditions were identified as the most
efficient: 75 mM donor 1, 300 mM acceptor 5, incubation
for 5—6 h at 35°C. The preparative transglycosylation reaction
was performed using the optimum conditions described above,
yielding a mixture of three 4-deoxy-disaccharides (Figure 3)
with an outstanding yield of 52% of total disaccharide fraction.
The reaction mixture was separated by size exclusion chroma-
tography, the disaccharide fraction eluted from the column as a
single peak. For the separation of individual products, a new
Synthesis of 4-deoxy-glycopyranosides 1–4
Starting compounds phenyl and p-nitrophenyl 2-acetamido-2-
deoxy-β-D-glucopyranosides (7, 8) were prepared from chlo-
ride 6 (Horton 1966). Following the procedure described by
Berkin et al. (2002), providing straightforward access to all
four desired compounds 1–4 (Figure 1), we prepared phenyl
and p-nitrophenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-
glucopyranosides 11a and 11b (instead of the originally used
methyl glycosides), which were oxidized by SO3-pyridine in
DMSO/TEA to yield α,β-unsaturated aldehydes 12a and 12b
(Figure 2). Reduction of the carbonyl group in 12a and 12b by
NaBH4 in MeOH yielded α,β-unsaturated derivatives 3 and
13, respectively. Catalytic hydrogenation of the double bond
of 13 on Pd/C yielded 4-deoxy-glycoside 1; hydrogenation
1003

K Slámová et al.
OH
OTr
a
O
R
O
O
R
O
HO
HO
HO
HO
NHAc
NHAc
9a: R = H
9b: R = NO2
7: R = H
8: R = NO2
b
OH
O
OTr
O
c
O
R
O
R
AcO
AcO
AcO
AcO
NHAc
NHAc
O
11a: R = H
10a: R = H
10b: R = NO2
11b: R = NO2
d
R
O
R
OH
O
e
O
O
13: R = H
3: R = NO2
12a: R = H
12b: R = NO2
HO
AcO
NHAc
NHAc
g
f
O
R
OH
OH
HO
HO
O
O
O
HO
14: R = H
4: R = NO2
NHAc
NHAc
h
1
OH
HO
HO
O
O
NHAc
2
Fig. 2. Synthesis of compounds 1–4. Reagents and conditions: (a) TrCl, Py, r.t., 2 d, 85%; (b) Ac2O, Py, r.t., 12 h, 95%; (c) HCOOH/Et2O (1:1), 1.5 h, r.t., 90%; (d)
SO3·Py, DMSO/Et3N, 1.5 h, r.t., 93%; (e) NaBH4, MeOH, 20 min, r.t., 64%; (f) K2CO3, MeOH/H2O (10:1), 1 h, r.t., 90%.; (g) H2-Pd/C, MeOH, 3 d, r.t., 60%; (h)
H2-Pd/C, MeOH, 4 h, r.t., 90%.
semipreparative ion-exchange high-performance liquid chro-
matography (HPLC) method was developed, capable of
separating even the 1-4 and 1-6 regioisomers (Figure 4, Supple-
mentary material). The disaccharides were eluted in a mobile
phase containing 9 mM sulfuric acid, which required neutraliza-
tion. Final purification and desalting was accomplished on a
microcrystalline cellulose column (Avicel® PH-101). Finally,
after lyophilization, three novel 4′-deoxy-disaccharides 4-deoxy-
β-GlcNAc-(1→4)-GlcNAc (15) 7%, 4-deoxy-β-GlcNAc-(1→6)-
GlcNAc (16) 6% and 4-deoxy-β-GlcNAc-(1→6)-4-deoxy-β-
GlcNAc-O-Ph (17) 14% were obtained in sufficient purity for
mass spectrometry (MS) and nuclear magnetic resonance
(NMR) characterization.
In an analogous preparative transglycosylation reaction,
containing only substrate 1 both as glycosyl donor and accep-
tor (200 mM) and catalyzed by β-N-acetylhexosaminidase
from T. flavus CCF 2686, the phenyl disaccharide 17 was ob-
tained in a similar yield (14%). The reaction was stopped after
2 h since the longer incubation time leads to the undesired
cleavage of the product. The reaction mixture was separated
on a Biogel P2 column, and the product 17 was eluted as a pure
compound, lyophilized and characterized.
1004

4-Deoxy-substrates for β-N-acetylhexosaminidases
Table I. Hydrolysis of phenyl 4-deoxy-N-acetylhexosaminides 1, 2 by β-N-
OH
O
OH
NHAc
acetylhexosaminidasesa
OH
O
HO
Ph-glycoside 1b
Ph-glycoside 2b
O
Enzyme source
HO
+
HO
NHAc
Acremonium persicinum CCF 1850
Aspergillus awamori CCF 763
Aspergillus flavofurcatis CCF 3061
Aspergillus flavus CCF 642
Aspergillus niger CCIM K2
Aspergillus niveus CCF 3057
Aspergillus nomius CCF 3086
Aspergillus oryzae CCF 147
A. oryzae CCF 1066
Aspergillus parasiticus CCF 1298
Aspergillus tamarii CCF 3085
Aspergillus versicolor CCF 2491
Fusarium oxysporum CCF 377
Penicillium brasilianum CCF 2171
Penicillium chrysogenum CCF 1269
Penicillium oxalicum CCF 1959
P. oxalicum CCF 2315
++
+++
+
++
++
+
++
++
++
++
++
+
+++
++
+++
++++
++++
+++
++++
++++
++++
++++
+++
+++
++
+
+
1
5
β-N-acetylhexosaminidase
(Talaromyces flavus)
35 °C, pH 5.0
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
++
++
++
+
OH
O
OH
O
O
HO
HO
OH
NHAc
NHAc
15
+
OH
O
O
HO
O
HO
O
OH
NHAc
NHAc
+
O
P. oxalicum CCF 2430
17
O
HO
Penicillium pittii CCF 2277
Talaromyces flavus CCF 2573
T. flavus CCF 2686
Talaromyces ohiensis CCF 2229
Talaromyces striatus CCF 2232
Trichoderma harzianum CCF 2687
Canavalia ensiformis (Jack Bean)
Streptococcus pneumoniae
O
HO
NHAc
HO
OH
16
NHAc
Fig. 3. Transglycosylation reaction catalyzed by β-N-acetylhexosaminidase
from T. flavus using substrate 1 as glycosyl donor and GlcNAc (5) as acceptor.
−
+
−
−
−
+
+
+
Bovine kidney
Human placenta (HEX A)
result, substrate 1 was the most promising candidate for hydro-
lysis by β-N-acetylhexosaminidase from A. oryzae. Substrates
2–4 show considerably lower binding energies, approaching
the range of non-specific binding (cf. 2, 3). This is caused by
diminished hydrogen bonding and by the loss of stacking with
Trp 517 and Trp 482 as a result of an improper spacial orienta-
tion of the substrates. Consequently, these substrates are not
hydrolyzed by β-N-acetylhexosaminidases, though they appar-
ently bind to the active site as indicated by their relatively high
binding energies.
ap-Nitrophenyl glycosides 3 and 4 were not cleaved by any of the enzymes.
b(−) <3%, (+) 4–9%, (++) 10–24%, (+++) 25–50%, (++++) >50% hydro-
lysis rate related to hydrolysis of standard substrate phenyl N-acetyl-β-D-
glucosaminide 7.
Docking and simulations of molecular dynamics
Binding energies and overall changes in the active site of β-N-
acetylhexosaminidase from A. oryzae were analyzed during the
simulation (10 ns) of molecular dynamics (MD). Table II pre-
sents binding parameters, binding energies and active-site
interactions calculated after 10 ns of MD. Notable changes
are observed in the interactions with the C-4 and C-3 of the
substrate pyranose ring. In contrast to p-nitrophenyl 2-acetami-
do-2-deoxy-β-D-glucopyranoside (pNP-GlcNAc) and similar
to p-nitrophenyl 2-acetamido-2-deoxy-β-D-galactopyranoside
(pNP-GalNAc), the lack of C-4 hydroxyl in 4-deoxy-substrates
causes the absence of hydrogen bonding with active sites Arg
193 and Glu 418. This causes an altered position of the sub-
strate pyranose ring in the active site.
Hydrogen bonding involving the hydroxyl at position C-3 is
altered even more profoundly. For substrates 2 and 4, no hy-
drogen bonding with Arg 193 is observed as the amino acid is
significantly shifted in the direction of the substrate diol group.
In comparison, pNP-GalNAc also lacks this interaction, but
there the C-3 hydroxyl is stabilized by an extra hydrogen bond
with Arg 308. Supposedly, these changes in the enzyme active
site influence the charge distribution during the formation of
the oxazolinium reaction intermediate.
Discussion
For synthesizing 4-deoxy-β-N-acetylhexosaminides, we fol-
lowed the procedure by Berkin et al. (2002) based on
oxidation-β-elimination. This method provided an easy access
to compounds 1–4 (Figure 2) with similar yields to the re-
ported methyl glycosides (Berkin et al. 2002). Due to the
prolonged reaction time, over-reduction of 14 directly led to
alcohol 1. Glycosides 2, 3 and 4 have been prepared in this
way for the first time. This reaction pathway impedes the pres-
ence of p-nitrophenyl leaving group in substrates 1 and 2.
Instead, these compounds were prepared as phenyl glycosides,
which, fortunately enough, have a similar reaction kinetics to
the standardly used p-nitrophenyl glycosides.
Phenyl glycoside 1 was a very good substrate for fungal β-
N-acetylhexosaminidases, (Table I), whereas aldehyde 2 was
hydrolyzed much more slowly, and unsaturated compounds 3
and 4 were not cleaved at all. β-N-Acetylhexosaminidase from
T. flavus, used in the transglycosylation reaction with 1, exhi-
bits extremely broad substrate specificity, as shown in previous
studies (Fialová et al. 2004, 2005; Loft et al. 2009). The higher
tolerance of β-N-acetylhexosaminidases of Penicillium sp. to
the configuration of C-4 hydroxyl is presumably related to
Substrate 1 exhibits the highest binding energy of all 4-deoxy
compounds tested—its value lies between that of pNP-GalNAc
and pNP-GlcNAc. Correspondingly, the position of 1 at the ac-
tive site after 10 ns of MD was similar to pNP-GlcNAc. As a
1005

K Slámová et al.
Table II. Binding parameters of compounds docked into the model of β-N-acetylhexos-aminidase from A. oryzae during molecular dynamics experiments
Estimated
binding
energy
π–π of
aglycon
with Trp
482
π–π stacking
of pyranosyl
ring with Trp
517
Number of
hydrogen
bonds
Distance from
catalytic Glu
(nm)
Substrate
(kcal/mol)
pNP-GlcNAc
pNP-GalNAc
1
2
3
4
−8.06
−6.69
−7.31
−5.74
−4.16
−6.98
8
6
5
6
6
4
0.41
0.54
0.40
0.61
0.42
0.60
+
+
+
−
−
−
+
+
−
−
−
−
their exceptionally high GalNAc-ase/GlcNAc-ase activity ratio
(>1) (Weignerová et al. 2003). The ability of bacterial, plant
and mammalian β-N-acetylhexosaminidases to cleave sub-
strates 1 and 2 was much lower than that observed in the
fungal enzymes. Presumably, fungi produce extracellular gly-
cosidases to hydrolyze polymeric substrates in order to access
low-molecular-weight sugars from the environment, and a
high flexibility in the substrate specificity of these enzymes
is of advantage. In contrast, (intracellular) enzymes of other
origins play a more specific role in cell metabolism, e.g., gly-
coprotein processing, and therefore their specificity needs to
be more focused.
Molecular modeling using the model of β-N-acetylhexosa-
minidase from A. oryzae (Ettrich et al. 2007) correlates well
with “wet” experiments, suggesting that substrate 1 is the only
substrate expected to be hydrolyzed, and showing the distortion
of the active site caused by the docking of compounds 3 and 4,
which are not able to reach the conformation necessary to form
the oxazoline intermediate (Figures 5 and 6, Supplementary
material). Moreover, molecular dynamics simulations revealed
that a successful cleavage of substrates by hexosamininidase
from A. oryzae requires a fixed position of the C-3 hydroxyl
of the substrate pyranose ring in the active site. This is mainly
ensured by Arg 193 and Asp 345, and this condition is not met
in substrates 2 and 4.
erably cheaper than the modified nucleoside required for the
transferase-catalyzed reaction.
Materials and methods
All NMR and MS data are in the Supplementary material avail-
able online.
Synthesis of 4-deoxy-hexosaminides 1–4
Phenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (7) was
prepared from 5 according to published procedure (Roy and
Tropper 1990).
p-Nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (8)
was prepared following the method by Begbie (1969).
p-Nitrophenyl 2-acetamido-2-deoxy-6-O-triphenylmethyl-β-D-
glucopyranoside (9b) was prepared according to published
procedure (Ekborg et al. 1982).
p-Nitrophenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-
glucopyranoside (11b) was prepared according to Matta and
Barlow (1975).
Phenyl 2-acetamido-2-deoxy-6-O-triphenylmethyl-β-D-
glucopyranoside (9a). Ph3CCl (0.488 g, 1.750 mmol) was
added to a solution of phenyl 2-acetamido-2-deoxy-β-D-
glucopyranoside (7) (0.4 g, 1.347 mmol) in anhydrous pyridine
(8 mL). The mixture was stirred at room temperature for 48 h.
The resulting mixture was concentrated under reduced pressure,
the residue co-evaporated with toluene and the crude product
purified by flash chromatography (CH2Cl2/MeOH 10:1)
yielding 9a (0.615 g, 85%) as a white solid.
Phenyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-
glucopyranoside (11a). Compound 9a (0.590 g, 1.095
mmol) was dissolved in CH2Cl2 (20 mL), treated with Ac2O
(1 mL) and pyridine (2 mL) and the reaction mixture stirred
overnight at room temperature. The mixture was washed
sequentially with an ice-cooled saturated solution of NaHCO3
(2 × 25 mL) and H2O, dried (Na2SO4) and concentrated under
reduced pressure to yield crude 10a (0.670 g, 98%) as a white
solid, which was used in the next reaction step without further
purification. A solution of 1:1 HCOOH–Et2O (50 mL) was
added to compound 10a (0.670 g, 1.075 mmol) and the mixture
stirred for 1.5 h at room temperature. The mixture was concen-
trated under reduced pressure and the residue co-evaporated
with toluene to yield a white solid. Flash chromatography
(CH2Cl2/MeOH 10:1) on silica gel yielded 11a (0.390 g,
95%) as a white, amorphous solid.
Glycosylation of GlcNAc (5) with 4-deoxy-glycoside 1 as a
glycosyl donor catalyzed by β-N-acetylhexosaminidase from
T. flavus yielded three novel 4′-deoxy-disaccharides (15, 7%;
16, 6%; 17, 14%). The isolated yield of the disaccharide frac-
tion was exceptionally high in this reaction, namely 52%.
Respective disaccharides were formed in ca 1:1:1 ratio, as
determined by the TLC and HPLC. The transglycosylation
mode was strongly preferred over simple cleavage since
the formation of the hydrolytic product, 2-acetamido-2,4-di-
deoxy-xylo-pyranose, was negligible. The relatively poor
reaction regioselectivity may be improved by a selective pro-
tection of acceptor hydroxyls (Weignerová et al. 1999). As
anticipated, using compound 1 both as a glycosyl donor
and acceptor, the transglycosylation reaction catalyzed by
β-N-acetylhexosaminidase from T. flavus yielded the disac-
charide 17 (14%). An analogous transfer of a 4-deoxy-
glycoside using N-acetylglucosaminyltransferase I was de-
scribed by Srivastava et al. (1990); however, this reaction
was only performed at an analytical scale, with the yield
1
of 7% (220 μg) estimated from H NMR signal integration.
In contrast, our method using β-N-acetylhexosaminidase is
robust, and the donor for the glycosidase reaction is consid-
1006

4-Deoxy-substrates for β-N-acetylhexosaminidases
Phenyl 2-acetamido-3-O-acetyl-2,4-dideoxy-α-L-threo-hex-4-
enodialdo-1,5-pyranoside (12a). Et3N (5 mL) at 0°C was
added to a solution of 11a (0.870 g, 2.283 mmol) in dimethyl
sulfoxide (DMSO) (11 mL). Then, SO3-pyridine (2.9 g,
18.221 mmol) in DMSO (22 mL) was added dropwise under
stirring over 1.5 h. The mixture was diluted with CH2Cl2
(150 mL) and washed sequentially with ice-cooled aqueous
tartaric acid (sat), aq NaHCO3 (sat) and H2O, dried (Na2SO4)
and concentrated under reduced pressure to yield a residue.
The residue was subjected to flash chromatography on silica
gel (CH2Cl2/MeOH 10:1) to yield 12a (0.680 g, 93%) as a
white amorphous solid.
p-Nitrophenyl 2-acetamido-3-O-acetyl-2,4-dideoxy-α-L-
threo-hex-4-enodialdo-1,5-pyranoside (12b). Compound 12b
(0.180 g, 88%) was prepared via the same procedure as 12a,
starting from 8b (0.240 g, 0.563 mmol).
Phenyl 2-acetamido-2,4-dideoxy-α-L-threo-hex-4-enopyrano-
side (13). NaBH4 (0.151 g, 3.992 mmol) was added to a
solution of 12a (0.680 g, 2.125 mmol) in MeOH (20 mL).
The mixture was stirred for 20 min at room temperature
and concentrated under reduced pressure; the residue was
co-evaporated with MeOH several times. The resulting resi-
due was subjected to flash chromatography on silica gel
(CH2Cl2/MeOH 10:1) to yield 13 (0.380 g, 64%) as a white
amorphous solid.
Hydrolysis of 4-deoxy-hexosaminides by β-N-
acetylhexosaminidases
General: pH 5.0 citrate/phosphate buffer (McIlvaine) was pre-
pared by mixing 0.1 M citric acid (24.3 mL) and 0.2 M
Na2HPO4 (25.7 mL), diluting with water to 100 mL and adjust-
ing the pH to 5.0. The fungal strains producing β-N-
acetylhexosaminidases (EC 3.2.1.52) originated from the Culture
Collection of Fungi (CCF), Department of Botany, Charles Uni-
versity in Prague or from the Culture Collection of the Institute
of Microbiology (CCIM), Prague. The strains were cultivated in
liquid media as described previously (Fialová et al. 2004). En-
zymes were obtained by (NH4)2SO4 precipitation (80% sat) of
the cultivation media, and the precipitates were used directly for
screening. β-N-Acetylhexosaminidases from non-fungal
sources (Canavalia ensiformis, Streptococcus pneumoniae, bo-
vine kidney and human placenta (HexA)) were purchased from
Sigma. HPLC was carried out on a modular system consisting
of DeltaChrom SDS 020 and 030 pumps (Watrex, CZ), a Spec-
tra 100 Variable UV/VIS CE detector (Thermo Separation
Products, USA), Basic Marathon Plus autosampler (Watrex,
CZ). The software Clarity AS (Chromservis, CZ) was used
for evaluation.
Enzyme activity assay—phenyl glycosides. The reaction mix-
tures (total volume 50 µL) containing Ph-GlcNAc 7 as a
standard substrate or 4-deoxy-glycosides 1, 2 (starting concen-
tration 2 mM) and β-N-acetylhexosaminidase (1.6–2.5 mU for
7, 6.4–10 mU for 1 and 2) in pH 5.0 McIlvaine buffer were
incubated in a Thermomixer (Eppendorf, DE) with shaking
at 35°C for 10 min. The reaction was stopped by heating to
100°C for 3 min and centrifuged (13,000 rpm, 10 min) to re-
move the denatured proteins. The supernatant (40 µL) was
mixed with HPLC mobile phase (40 µL; MeCN:NaOAc buff-
er, 50 mM, pH 5.0 = 35:65), and the amount of liberated
phenol was determined by HPLC. The HPLC analysis was per-
formed on a C18 (Phenomenex, USA) column, 250 × 4.6 mm,
at ambient temperature (mobile phase MeCN:NaOAc buffer,
50 mM pH 5.0 = 35:65, flow rate 0.8 mL/min, injection vol-
ume 20 µL), and phenol was detected at 275 nm (Rt (phenol) =
5.8 min, Rt (7, 1 and 2) = 2.7 min). In the enzymatic screening,
28 β-N-acetylhexosaminidases were tested (24 fungal, four
from other sources), the enzymes classified according to the ra-
tio of hydrolysis rates of the modified substrates 1 and 2) and
substrate 7 assayed under the same conditions and extrapolated
to the same amount of enzyme. One unit of enzymatic activity
was defined as the amount of enzyme that releases 1 µmol of
phenol per minute under the above conditions. Each reaction
was prepared in duplicate, and each was analyzed twice.
p-Nitrophenyl 2-acetamido-2,4-dideoxy-α-L-threo-hex-4-eno-
pyranoside (3). Compound 3 (0.090 g, 96%) was prepared
via the same procedure as 13, starting from 12b (0.105 g,
0.288 mmol).
Phenyl 2-acetamido-2,4-dideoxy-β-D-xylo-hexopyranoside
(1). A mixture of 13 (0.360 g, 1.290 mmol) and 10% Pd–C
(0.400 g) in MeOH (30 mL) was subjected to a hydrogen-
balloon pressure for 3 days. The mixture was filtered
through Celite 521 (Aldrich), the residue washed with
MeOH and the combined filtrate and washings concentrat-
ed under reduced pressure to give a residue which was
purified by flash chromatography on silica gel (CH2Cl2/
MeOH 10:1) to yield 1 (0.220 g, 60.6%) as a white amor-
phous solid.
Phenyl 2-acetamido-2,4-dideoxy-α-L-threo-hex-4-enodialdo-
1,5-pyranoside (14). K2CO3 (0.052 g, 0.376 mmol) was
added to a solution of 12a (0.120 g, 0.376 mmol) in
MeOH/H2O (20 mL, 9:1, v/v) and the mixture stirred for 1
h at room temperature. Amberlite IR-120 was added to the
mixture to neutral pH, the ion exchanger was then filtered
off and the filtrate evaporated to dryness by co-evaporation
with toluene, yielding pure 14 (0.108 g, 98%).
p-Nitrophenyl 2-acetamido-2,4-dideoxy-α-L-threo-hex-4-eno-
dialdo-1,5-pyranoside (4). Compound 4 (0.110 g, 98%) was
prepared via the same procedure as 14, starting from 12b
(0.120 g, 0.329 mmol).
Enzyme activity assay—p-nitrophenyl glycosides. The reaction
mixtures (total volume 55 µL) containing pNP-GlcNAc (8) as
a standard substrate (2 mM, starting concentration) and β-N-
acetylhexosaminidase (0.6–0.8 mU) in McIlvaine buffer pH
5.0 were incubated in 96-well microtitration plates with shak-
ing at 35°C. After 10 min, the reaction was stopped by adding
1 M Na2CO3 (150 µL). Liberated p-nitrophenol was detected
spectrophotometrically at 420 nm (Sunrise Absorbance Reader,
Tecan, A). Their activity towards modified substrate 3 or 4 was
determined analogously, with the amount of enzyme being 12–
16 mU. One unit of enzymatic activity was defined as the
amount of enzyme that releases 1 µmol of p-nitrophenol per
minute under the above conditions. In the enzymatic screening,
Phenyl 2-acetamido-2,4-dideoxy-β-D-xylo-1,5-dialdo-hexo-
pyranoside (2). Pd/C (0.150 g, 10% Pd w/w) was added to
a solution of 14 (0.150 g, 0.511 mmol) in MeOH (20 mL)
and the resulting mixture stirred under hydrogen for 4 h.
The mixture was filtered through Celite 521 (Aldrich), the res-
idue washed with MeOH, and the combined filtrate and
washings were concentrated under reduced pressure to obtain
a residue, which was purified by flash chromatography on sil-
ica gel (CH2Cl2/MeOH 10:1) to yield 2 (0.136 g, 90%) as a
white amorphous solid.
1007

K Slámová et al.
28 β-N-acetylhexosaminidases were tested (24 fungal, four from
other sources). They were classified according to the ratio of the
hydrolysis rates of modified substrate 3 or 4 and substrate 8 as-
sayed under the same conditions and extrapolated to the same
amount of enzyme. Each reaction was performed in triplicate.
β-N-acetylhexosaminidase from T. flavus CCF 2686 (1 U),
the mixture was shaken at 35°C. After 2 h, the reaction was
stopped by heating to 100°C for 3 min. The reaction mixture
was centrifuged (13,000 rpm, 10 min) and loaded onto a Bio-
gel P2 column (Bio-Rad, USA) (120 cm × 2.5 cm, water, flow
rate 10 mL/h). The disaccharide 17 was freeze-dried and ob-
tained as a white solid (5 mg, 14%).
Analytical transglycosylation reaction
The reaction mixtures (total volume 250 µL) contained 1.7–5.1
mg of donor 1 (25–75 mM) and 6.9–16.5 mg of acceptor 5
(125–300 mM) in McIlvaine buffer pH 5.0. The reaction
was started by the addition of β-N-acetylhexosaminidase from
T. flavus CCF 2686 (0.2–0.7 U) and was incubated at 35°C
with shaking for 6 h. The reaction progress was monitored
by TLC (propane-2-ol: water: ammonia = 7:2:1) on aluminum
sheets precoated with Silica Gel 60 (F254 Merck, G), and the
plates were visualized by UV light (254 nm) and charring with
a mixture of 5% sulfuric acid in ethanol.
Docking and simulations of molecular dynamics
Substrates were built in YASARA, force field parameters were
derived using the AutoSMILES approach (Krieger et al.
2004). In the first step, YASARA calculated semi-empirical
AM1 Mulliken point charges that were corrected by the as-
signment of AM1BCC atom types and improved AM1BCC
charges by fragments of molecules with known RESP charges,
to more closely resemble the actual RESP charges. The
corresponding bond, angle and torsion potential parameters
were taken from the General AMBER force field. Docking
of substrates was done manually by overlaying with chitobiose
docked in the active site of β-N-acetylhexosaminidase from A.
oryzae (the model was reported previously) (Ettrich et al.
2007). Substrate–enzyme complexes were neutralized and
minimized in water (TIP3P) with YASARA. MD simulation
was started in YASARA with the following parameters: NPT
ensemble (287 K, 0.997 g/mL), at pH 7, intramolecular forces
were calculated every 1.5 fs, intermolecular—every 3 fs, force
field Yamber2, Particle Mesh Ewald algorithm was used for
long-range interaction and cutoff 0.78 nm for short-range, at
the periodic boundary conditions. Estimated binding energies
of the final enzyme–substrate complexes were calculated with
AutoDock4 (Morris et al. 1996).
Preparation of 4-deoxy-disaccharides: 2-acetamido-2,4-
dideoxy-β-D-xylo-hexopyranosyl-(1→4)-2-acetamido-2-deoxy-
D-glucopyranose (15), 2-acetamido-2,4-dideoxy-β-D-xylo-
hexopyranosyl-(1→6)-2-acetamido-2-deoxy-D-glucopyranose
(16) and phenyl 2-acetamido-2,4-dideoxy-β-D-xylo-
hexopyranosyl-(1→6)-2-acetamido-2,4-dideoxy-β-D-xylo-
hexopyranoside (17)
Phenyl glycoside 1 (41 mg, 0.15 mmol, 75 mM) and acceptor 5
(132 mg, 0.6 mmol, 300 mM) were dissolved in McIlvaine
buffer pH 5.0 (1952 µL). β-N-Acetylhexosaminidase from T.
flavus CCF 2686 (6 U) was added, and the mixture was shaken
at 35°C. After 5.5 h, the reaction was stopped by heating to
100°C for 3 min. The reaction mixture was centrifuged
(13,000 rpm, 10 min) and loaded onto a Biogel P2 column
(Bio-Rad, USA) (120 cm × 2.5 cm, water, flow rate 7 mL/
h). Fractions containing a mixture of disaccharides were col-
lected, lyophilized and further purified by cation exchange
semipreparative HPLC. The mixture of disaccharides was sep-
arated on a Polymer IEX H+ column (Watrex, CZ), 250 ×
8 mm, at ambient temperature (mobile phase 9 mM sulfuric
acid, injection 13 × 70 µL, UV detection 210 nm). The flow
rate was 0.5 mL/min during the first 20 min of the run, then it
was increased to 1.2 mL/min to elute the last compound (Rt
(15) = 12.9 min, Rt (16) = 14.6 min, Rt (17) = 60.9 min).
The fractions containing the separated disaccharides were col-
lected, immediately neutralized (100 mM sodium hydroxide),
dried under vacuum and desalted on a microcrystalline cellu-
lose (Avicel PH-101, Fluka) column (25 cm × 1 cm, mobile
phase MeCN: water = 7:3); this step was repeated twice to re-
move the salt completely. The presence of salt (Na2SO4) was
verified by the formation of a precipitate with BaCl2. After ly-
ophilization, disaccharides 15, 16 and 17 were obtained as
white solids in the following yields: 15, 7% (3.7 mg, 0.009
mmol); 16, 6% (3.4 mg, 0.008 mmol); and 17, 14% (4.3 mg,
0.009 mmol); relative to substrate 1.
Supplementary Data
Supplementary data for this article is available online at http://
glycob.oxfordjournals.org/.
Funding
The Czech Science Foundation (203/09/P024 to P.B., 305/
09/H008), the Ministry of Education of the Czech Republic
(LC06010) and the research concepts of the Institute of
Microbiology AV0Z50200510 and of the Institute of Sys-
tems Biology and Ecology AV0Z60870520 are thanked for
financial support of this work. Access to METACentrum
computing facilities is provided under the research concept
MSM6383917201.
Abbreviations
CCF, Culture Collection of Fungi; CCIM, Culture Collection
of the Institute of Microbiology; DMSO, dimethyl sulfoxide;
GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glu-
cosamine; 4-F-GlcNAc, 2-acetamido-2,4-dideoxy-4-fluoro-D-
glucosamine; MD, molecular dynamics; MS, mass spectrom-
etry; NMR, nuclear magnetic resonance; Ph-GlcNAc, phenyl
2-acetamido-2-deoxy-β-D-glucopyranoside; Ph-GalNAc, phe-
nyl 2-acetamido-2-deoxy-β-D-galactopyranoside; pNP-
Preparation of phenyl 2-acetamido-2,4-dideoxy-β-D-xylo-
hexopyranosyl-(1→6)-2-acetamido-2,4-dideoxy-β-D-xylo-
hexopyranoside (17)
Substrate 1 (43 mg, 0.16 mmol, 200 mM) was dissolved in
McIlvaine buffer pH 5.0 (764 µL), and after the addition of
1008

4-Deoxy-substrates for β-N-acetylhexosaminidases
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