Enzymatic Discrimination of 2-Acetamido-2-deoxy-D-mannopyranose-Containing Disaccharides Using β-N-Acetylhexosaminidases

Article abstract of DOI:10.1002/adsc.200303002

β-N-Acetylhexosaminidase from Aspergillus oryzae selectively discriminates mixture of the disaccharides GlcNAcβ(1→4)GlcNAc (1) and GlcNAcβ(1→4)ManNAc (2). N,N′-Diacetylchitobiose (1) was selectively hydrolyzed by β-N-acetylhexosaminidase, whereas its C-2 epimer (2) was completely resistant to the enzyme hydrolysis. Analogous discrimination was observed also with GalNAcβ(1→4)GlcNAc (3) and GalNAcβ(1→ 4)ManNAc (4). β-N-Acetylhexosaminidases from A. terreus, A. flavus, bovine kidney and bovine epididymis displayed the same selectivity, whereas the enzymes from A. sojae, A. tamarii, Penicillium brasilianum, P. oxalicum, P. funiculosum, P. multicolor, Talaromyces flavus and jack beans hydrolyzed both types of disaccharides. Molecular modelling of β-N-acetylhexosaminidase from A. oryzae CCF 1066 and docking experiments with both types of disaccharides revealed that the ManNAc residue causes distortion of disaccharide molecule resulting in a steric conflict with a Trp⁴⁸² that causes diminished stabilization of the oxazolinium transition state by extending the distance of Asp³⁴⁵ in the active site. Both ManNAc-containing disaccharides 2 and 4 dock with similar steric energies into the active site but without cleaving and also without notable inhibition. This novel phenomenon enables also the preparative production of both disaccharides 2 and 4 starting from N, N′-diacetylchitobiose (1) or GalNAcβ(1→ 4)GlcNAc (3) followed by Lobry de Bruyn-Albreda van Ekenstein C-2 epimerization catalyzed by Ca(OH)₂. The resultant mixture of the respective epimers 1, 2 or 3, 4 that is hardly separable by, e.g., analytical HPLC can be treated with the β-N-acetylhexosaminidase from A. oryzae and the mixture of monosaccharides and target disaccharide can be easily separated using gel filtration.

Full text of DOI:10.1002/adsc.200303002

FULL PAPERS
Enzymatic Discrimination of 2-Acetamido-2-deoxy-d-
mannopyranose-Containing Disaccharides Using b-N-
Acetylhexosaminidases
a
a
a
œ ¬
¬
¬
¬
¬ œ
œ
Lucie Husakova, Eva Herkommerova-Rajnochova, Tomas Semenuk, Marek
a
a
a
b
a, c
¬
œ
œ
¬
œ
Kuzma, Jana Rauvolfova, Vera Prikrylova, R¸diger Ettrich, Ondrej PlÌhal,
a, c
a,
œ
œ
Karel Bezouska, VladimÌr Kren *
a
œ
¬
Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, VÌdenska 1083,
142 20 Prague 4, Czech Republic
Phone: ( ‡ 420)-296-442-510, Fax: ( ‡ 420)-296-442-509, e-mail: kren@biomed.cas.cz
b
c
¬
¬
Institute of Physical Biology, University of South Bohemia, Zamek 136, 373 33 Nove Hrady, Czech Republic
Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 40 Prague 2, Czech Republic
Received: January 8, 2003; Accepted: March 12, 2003
This paper is dedicated to Prof. David Herbert Gordon Crout on the occasion of his retirement.
Abstract: b-N-Acetylhexosaminidase from Aspergil- diminished stabilization of the oxazolinium transition
lus oryzae selectively discriminates mixture of the state by extending the distance of Asp345 in the active
disaccharides GlcNAcb(1!4)GlcNAc (1) and site. Both ManNAc-containing disaccharides 2 and 4
GlcNAcb(1!4)ManNAc (2). N,N'-Diacetylchito- dock with similar steric energies into the active site
biose (1) was selectively hydrolyzed by b-N-acetyl- but without cleaving and also without notable inhib-
hexosaminidase, whereas its C-2 epimer (2) was ition. This novel phenomenon enables also the
completely resistant to the enzyme hydrolysis. Anal- preparative production of both disaccharides 2 and
ogous discrimination was observed also with Gal- 4 starting from N, N×-diacetylchitobiose (1) or Gal-
NAcb(1!4)GlcNAc
(3)
and
Gal- NAcb(1!4)GlcNAc (3) followed by Lobry de
NAcb(1!4)ManNAc (4). b-N-Acetylhexosaminidas- Bruyn Albreda van Ekenstein C-2 epimerization
es from A. terreus, A. flavus, bovine kidney and catalyzed by Ca(OH)2. The resultant mixture of the
bovine epididymis displayed the same selectivity, respective epimers 1, 2 or 3, 4 that is hardly separable
whereas the enzymes from A. sojae, A. tamarii, Pen- by, e.g., analytical HPLC can be treated with the b-N-
icillium brasilianum, P. oxalicum, P. funiculosum, acetylhexosaminidase from A. oryzae and the mixture
P. multicolor, Talaromyces flavus and jack beans of monosaccharides and target disaccharide can be
hydrolyzed both types of disaccharides. Molecular easily separated using gel filtration.
modelling of b-N-acetylhexosaminidase from A. ory-
zae CCF 1066 and docking experiments with both
types of disaccharides revealed that the ManNAc Keywords: acetamido sugars; alkali-catalyzed epime-
residue causes distortion of disaccharide molecule rization; Aspergillus oryzae; N,N'-diacetylchitobiose;
resulting in a steric conflict with a Trp482 that causes molecular modelling
Abbreviations: CCF: Culture Collection of Fungi,
Department of Botany, Charles University, Prague;
GlcNAc: 2-acetamido-2-deoxy-d-glucopyranose; Man-
NAc: 2-acetamido-2-deoxy-d-mannopyranose; pNP-
bGlcNAc: p-nitrophenyl 2-acetamido-2-deoxy-b-d-
glucopyranoside; pNP-bGalNAc: p-nitrophenyl 2-
acetamido-2-deoxy-b-d-galactopyranoside; GlcNAcb-
(1!4)GlcNAc: 4-O-(2-acetamido-2-deoxy-b-d-gluco-
pyranosyl)-2-acetamido-2-deoxy-d-glucopyranose; Glc-
NAcb(1!4)ManNAc: 4-O-(2-acetamido-2-deoxy-b-
d-glucopyranosyl)-2-acetamido-2-deoxy-d-mannopyr-
anose; GalNAcb(1!4)GlcNAc: 4-O-(2-acetamido-2-
deoxy-b-d-galactopyranosyl)-2-acetamido-2-deoxy-d-
glucopyranose; GalNAcb(1!4)ManNAc: 4-O-(2-
acetamido-2-deoxy-b-d-galactopyranosyl)-2-acetami-
do-2-deoxy-d-mannopyranose; GlcNAcb(1!6)Glc-
NAc:
6-O-(2-acetamido-2-deoxy-b-d-glucopyrano-
syl)-2-acetamido-2-deoxy-d-glucopyranose; GlcNAcb
(1!6)ManNAc: 6-O-(2-acetamido-2-deoxy-b-d-glu-
copyranosyl)-2-acetamido-2-deoxy-d-mannopyranose.
Enzyme: b-N-acetylhexosaminidase (EC 3.2.1.52)
Adv. Synth. Catal. 2003, 345, 735 742
DOI: 10.1002/adsc.200303002
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Introduction
2-Acetamido-2-deoxy-d-mannose (ManNAc) is a fre-
quently occurring glycosyl residue in many bacterial
capsular polysaccharides and lipopolysaccharides (e.g.,
from Haemophillus influenzae and Streptococcus pneu-
moniae).[1,2] In Gram-positive bacteria, such as Staph-
ylococcus aureus H, and Bacillus subtilis, the b-ManNAc
residue is a component of the ™linkage unit∫ attaching
teichoic acids to the peptidoglycan.[2b]
Despite an extensive literature search no disacchar-
ides containing ManNAc at the reducing end have been
described
except for GlcNAcb(1!4)ManNAc (2)
published in our previous work.[3,4]
Recently, ManNAc was identified as the strongest
monosaccharidic ligand for the natural killer cell
activating protein NKR-P1.[3] Nevertheless, the stron-
gest holosaccharidic ligand (besides glycoconjugates
and glycodendrimers) is probably the disaccharide
GlcNAcb(1!4)ManNAc (2) or GalNAcb(1!4)
ManNAc (4).[3]
Scheme 1. Selective removal of GlcNAcb(1!4)GlcNAc (1)
and GalNAcb(1!4)GlcNAc (3) from the epimerization
mixtures by b-N-acetylhexosaminidase from Aspergillus
oryzae.
The interesting biological activities of this saccharide
stimulated a high demand for this substance for in vivo distinguish disaccharides bearing ManNAc at their
tests and for the preparation of its derivatives. This reducing end. This unique reactivity has been evaluated
compound was prepared for the first time in our using mixtures of GlcNAcb(1!4)ManNAc (2) and
laboratory by Lobry de Bruyn Alberda van Ekenstein N,N×-diacetylchitobiose (1) as well as GalNAcb
[catalyzed by aqueous Ca(OH)2] epimerization of N, N×- (1!4)ManNAc (4) and GalNAcb(1!4)GlcNAc (3).
diacetylchitobiose.[4] The resulting mixture of both 2- This reaction can be employed for a large-scale prepa-
epimers (containing only 25% of the desired product 2) ration of the ManNAc-containing disaccharides that are
was, however, separable only by multiple preparative important components of microbial cell walls, and can
HPLC runs. This enabled us to obtain only milligram be utilized as an immunoactive compounds.
amount of the compound needed for its spectral
characterization and preliminary biological tests.
Numerous attempts for its chemical synthesis based Results and Discussion
on GlcNAc substitution of the ManNAc derivatives
failed. We have also tried the b-N-acetylhexosaminidase
Attempted Enzymatic Synthesis of
(EC 3.2.1.52)-catalyzed transfer of a b-GlcNAc residue
GlcNAcb(1!4)ManNAc (2)
onto ManNAc that proved to be effective in many
previous cases.[5,6] This approach failed as well, despite
many enzymes tested. Only the enzymes from Asper- The reaction mixture after the attempted transglycosy-
gillus flavofurcatis CCF 3061 and A. tamarii CCF 1665 lation of ManNAc with b-N-acetylhexosaminidase us-
gave traces of 2 together with the other products as ing pNP-bGlcNAc as a donor was pre-separated by gel
identified by HPLC.
filtration to obtain the disaccharide fraction. This
It is generally accepted that glycosidases unable to fraction was analyzed by TLC and HPLC for the
cleave certain glycosidic linkages are often unable to occurrence of 2. The authentic sample of this compound
synthesize the same type of the bond (often regioisom- prepared previously[4] was used as a standard.
ers). Adopting an ™inverse approach∫ led us to the
The formation of a minor amount of 2 was observed in
presumption that the enzyme(s) unable to synthesize two cases only (Aspergillus flavofurcatis CCF 3061 and
the respective glycosidic linkage would not be able to A. tamarii CCF 1665) in the HPLC pattern and this was
hydrolyze it. This proved to be true in our case when the further proved by co-chromatography with the authen-
b-N-acetylhexosaminidase from A. oryzae selectively tic compound. In some cases the formation of the other
hydrolyzed N,N×-diacetylchitobiose (1), whereas the (unidentified) regioisomers was observed. These data
epimer (2) remained intact. The resulting mixture of should only demonstrate the difficulty of the enzymatic
mono- and disaccharides is then easily separable by gel reaction leading to the desired product.
filtration (Scheme 1).
We report here also a possible molecular mechanism
by which microbial b-N-acetylhexosaminidases can
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Adv. Synth. Catal. 2003, 345, 735 742

Enzymatic Discrimination of 2-Acetamido-2-deoxy-d-mannopyranose-Containing Disaccharides
Table 1. Cleavage of GlcNAcb(1!4)ManNAc (2) by the b-N-acetylhexosaminidases.
FULL PAPERS
Source of b-N-acetylhexosaminidase
Hydrolytic activity[a]
10 min
30 min
60 min
180 min
Aspergillus oryzae CCF 1066
A. flavipes CCF 1895
A. sojae CCF 3060
A. tamarii CCF 1665
A. terreus CCF 2539
‡
‡
‡
‡
‡
‡‡
‡‡
‡‡
‡‡‡
‡‡‡
‡‡‡
A. flavus CCF 3056
Mucor dimorphosporus CCF 2609
Penicillium brasilianum CCF 2155
P. oxalicum CCF 2430
P. funiculosum CCF 2984
P. chrysogenum CCF 1269
P. multicolor CCF 2244
Talaromyces flavus CCF 2686
Bovine kidney (Sigma)
Bovine epididymis (Sigma)
Jack beans (Sigma)
‡‡
‡‡
‡
‡‡
‡
‡‡‡
‡‡
‡
‡
‡‡
‡
‡‡
‡
‡‡
‡
‡‡‡
‡‡‡
‡‡
‡‡‡
‡‡‡
‡‡‡
Lysozyme (Sigma)
Rat plasma
[a]
‡ Monosaccharides formation (GlcNAc and ManNAc) from 2 ( ‡< 10%, ‡‡> 10%, ‡‡‡> 25%); no hydrolysis.
Selectivity of the Hydrolysis of
GlcNAcb(1!4)ManNAc (2) with Various b-N-
Acetylhexosaminidases
ination of 4 the same enzyme from A. oryzae as in the
previous case. The disaccharide 4 has not been described
so far.
A large panel of various b-N-acetylhexosaminidases
was tested for their ability to hydrolyze the disaccharide Inhibition of b-N-Acetylhexosaminidases by
2 (Table 1). Only those enzymes that were found to be GlcNAcb(1!4)ManNAc (2)
completely unable to cleave this disaccharide would be
suitable for its discrimination in the presence of N,N×- The compound 2 was tested as a potential inhibitor of
diacetylchitobiose. The selected enzymes, e.g., from fungal b-N-acetylhexosaminidases from A. oryzae CCF
A. oryzae, A. terreus, and A. flavus, were incubated with 1066, P. oxalicum CCF 2430, Mucor dimorphosporus
the substrate 2 for a prolonged time (24 hours) without CCF 2609 and Talaromyces flavus CCF 2686. Virtually
any detectable hydrolysis (TLC, HPLC). All tested b-N- no inhibition was observed (Table 2).
acetylhexosaminidases cleaved N,N×-diacetylchito-
biose.
The animal b-N-acetylhexosaminidases were tested Mechanism of the Discrimination of ManNAc-
for the same reaction mainly to prove the applicability of Containing Disaccharides by b-N-
this immunoactive saccharide in vivo. None of the Acetylhexosaminidase from A. oryzae
animal b-N-acetylhexosaminidases including samples of
fresh rat plasma (containing b-N-acetylhexosaminidase The b-N-acetylhexosaminidases, similar to most other
and lysozyme) hydrolyzed this disaccharide. This glycosidases, are rather promiscuous towards an agly-
proved the potential stability of the substance in body cone (or second saccharide). The phenomenon ob-
fluids and its possible in vivo applicability.
served by us, e.g., the discrimination according to the
reducing saccharide was, therefore, unusual, and we
have attempted to explain this effect.
Selectivity of the Hydrolysis of
GalNAcb(1!4)ManNAc (4) by Fungal b-N-
Acetylhexosaminidases
Recently, the b-N-acetylhexosaminidase from A. ory-
zaewassequencedanditsmodelbasedonhighhomology
with the known sequences and the crystal structure[7] was
constructed. We carried out a simulation of the docking
Screening for hydrolysis of 4 was performed analogously of the respective disaccharides into this model of the
as in the case of 2 and quite similar results were obtained active center of the b-N-acetylhexosaminidase from
(data not shown). Therefore, we used for the discrim- A. oryzae and the following data were obtained.
Adv. Synth. Catal. 2003, 345, 735 742
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Lucie Husakova et al.
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Table 2. Inhibition of b-N-acetylhexosaminidases by GlcNAcb(1!4)ManNAc (2).
Residual activity [%]
Substrate
pNP-GlcNAc
GlcNAcb(1!4)GlcNAc (1)
Inhibitor concentration[a]
0.04 mM
0.4 mM
0.04 mM
0.4 mM
b-N-Acetylhexosaminidase
Aspergillus oryzae CCF 1066
Penicillium oxalicum CCF 2430
Mucor dimorphosporus CCF 2609
Talaromyces flavus CCF 2686
90
97
95
96
98
82
85
99
86
102
95
102
109
71
79
78
[a]
The concentration of both substrates was 0.4 mM.
Scheme 2. Proposed catalytic mechanism of b-N-acetylhexosaminidase from A. oryzae.[8]
N,N'-Diacetylchitobiose (1) (non-reducing GlcNAc considerably higher ( À 53.1 kJ ¥ molÀ1) (Table 3) than
moiety) participates in the hydrogen bonding between that in the case of N, N×-diacetylchitobiose. This may
Asp345, Tyr445 and the acetamido group, which may also explain the facts that the enzyme does not attack the
eventually stabilize the oxazolinium transition state substrate (2) but it shows virtually no inhibition
(Scheme 2). The non-reducing end of the disaccharide is (Table 2).
locked into the active site owing to the hydrogen bonds
The situation of the analogous b(1!6) linked di-
of Arg193, Trp482 and Glu519with the C-4 and C-5 OH saccharides is completely different. We were unable to
groups (Figure 1). The calculated steric interaction observe the discrimination of 5 and 6 despite extensive
energy was À 101.6 kJ ¥ molÀ1 (Table 3).
screening of our b-N-acetylhexosaminidase library (40
On the other hand, the molecule of 2 is slightly enzymes tested, data not shown). Only minor differ-
distorted and, therefore, there is a steric conflict with ences in the hydrolysis rate were observed. In the case of
Trp482. The reorientation of the ligand due to this steric the b-N-acetylhexosaminidase from A. oryzae the hy-
conflict during the molecular mechanics simulation drolysis of 6 is even somehow faster than that of 5 as
leads to a 0.3 ä higher distance between Asp345 and observed by HPLC.
the acetamido group that obviously results in a dimin-
This situation might be well explained also by the
ished stabilization of the oxazolinium transition state. docking experiments into the active site model. We
The other interactions stabilizing the non-reducing could observe that the position of the non-reducing
carbohydrate moiety seem to remain intact. Due to GlcNAc of both disaccharides in the active site is
this steric conflict the steric interaction energy is virtually identical and the respective hydrogen bonds
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Enzymatic Discrimination of 2-Acetamido-2-deoxy-d-mannopyranose-Containing Disaccharides
FULL PAPERS
Figure 1. A simulation of the docking of the respective disaccharides into the model of the active center of b-N-
acetylhexosaminidase from A. oryzae. (A) GlcNAcb(1!4)GlcNAc (1), (B) GlcNAcb(1!4)ManNAc (2), (C)
GlcNAcb(1!6)GlcNAc (5), (D) GlcNAcb(1!6)ManNAc (6).
reflected also by substantial lowering of the steric
interaction energy for 6 ( À 103.7 kJ ¥ molÀ1) compared
to 5 ( À 43.1 kJ ¥ molÀ1).
The discrimination of ManNAc-containing disacchar-
ides by the b-N-acetylhexosaminidases is a rather
unexpected and unusual phenomenon. It can be effec-
tively utilized for the production of rare ManNAc-
containing saccharides.
Scheme 3. Action of b-N-acetylhexosaminidase from A. or-
yzae upon b(1!6) linked hexosamine disaccharides.
Conclusions
Table 3. Interaction energies of GlcNAcb(1!4)GlcNAc (1),
GlcNAcb(1!4)ManNAc (2), GlcNAcb(1!6)GlcNAc (5)
and GlcNAcb(1!6)ManNAc (6) with b-N-acetylhexosami-
nidase from Aspergillus oryzae (molecular modelling).
We have shown that the b-N-acetylhexosaminidase
from Aspergillus oryzae and some other b-N-acetylhex-
osaminidases from a filamentous fungi selectively dis-
criminate between disaccharides containing ManNAc
and GlcNAc at the reducing end. Disaccharides con-
taining the ManNAc moiety (at reducing end) are
resistant to the cleavage. This unusual effect was
explained by docking experiments of the respective
disaccharide into the active site model of the b-N-
acetylhexosaminidase from A. oryzae. This method
opens a possibility for the preparative-scale synthesis
of these disaccharides that are interesting for their
Interaction energy [kJ ¥ molÀ1]
Substrate
Total
Steric
Electrostatic
1
2
5
6
À 268.8
À 246.2
À 165.1
À 227.8
À 101.6
À 53.1
À 43.1
À 103.7
À 166.8
À 193.1
À 122.1
À 126.2
remain conserved. There is, however, a different sit- immunoactive properties.
uation at the reducing carbohydrate moiety, where, in
the case of 6, a new hydrophobic interaction of CH3 and
of NHAc group with Trp482 can be observed. This is
Adv. Synth. Catal. 2003, 345, 735 742
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FULL PAPERS
mixture was evaporated and dissolved in citrate-phosphate
buffer (50 mM, pH 5.3, 25 mL) and the b-N-acetylhexosami-
nidase from A. oryzae (20 U) was added and the mixture was
incubated at 378C (typically 2 hours) until all N,N×-diacetyl-
chitobiose had disappeared (TLC). The enzyme was then
deactivated by 5 min boiling and the mixture was evaporated
to 1 2 mL. The sample was loaded onto Bio Gel P2 (BioRad,
USA) column (2.6 Â 80 cm, flow rate 12 mL ¥ hÀ1, dead volume
190 mL) eluted with H2O. The isolated yield of 2 was 18% and
its structure was confirmed by NMR, MS and optical rotation
as described previously.[4]
Experimental Section
b-N-Acetylhexosaminidase
All b-N-acetylhexosaminidases (EC 3.2.1.52) used in this
work, besides the commercial ones, originated from the library
of fungal glycosidases of the Laboratory of Biotransformation
in Prague and were prepared by cultivation of the respective
fungi as described previously.[9] The producing strains are
deposited with the Czech Collection of Fungi (CCF) at the
Department of Botany of the Charles University, Prague.
TLC
4-O-(2-Acetamido-2-deoxy-b-d-galactopyranosyl)-2-
acetamido-2-deoxy-d-glucopyranose (3)
Thin layer chromatography was carried out using silica Gel 60
GF254 (Merck) with the solvent system 2-propanol/water/28%
ammonia (7/2/1, v/v), twice developed. The spots were
visualized by charring with 5% H2SO4 in ethanol.
The compound 3 was prepared using a significantly modified
procedure[11] for a transglycosylation reaction catalyzed by
b-N-acetylhexosaminidase from A. oryzae CCF 1066.
p-Nitrophenyl 2-acetamido-2-deoxy-b-d-galactopyranoside
(12.5 mg, 73 mmol) and 2-acetamido-2-deoxy-b-d-glucopyra-
noside (31.5 mg, 287 mmol) were dissolved in citrate-phos-
phate buffer (50 mM, pH 5.0, 1.25 mL) and the enzyme (2 U)
was added. The mixture was incubated at 378C for 1 h and the
reaction was stopped by 5 min boiling. The reaction mixture
was extracted with diethyl ether (3 Â 250 mL) to remove the
bulk of the liberated p-nitrophenol and after partial evapo-
ration loaded onto Toyopearl (HW-40F, TOSOH Corp., Japan)
column (2.6 Â 80 cm, flow rate 12 mL ¥ hÀ1, dead volume
220 mL) eluted with H2O. The isolated yield of 3 was 84.5%
(13.1 mg) referred to pNP-bGalNAc.
HPLC
The analysis were carried out using an HPLC system consisting
of a solvent-delivery system 600 (Waters), a photo-diode array
detector 996 (Waters) and a Polymer IEX H -form column
‡
(8 mm, 250 Â 8 mm, Waters). As mobile phase 9 mM H2SO4
was employed at a flow rate 0.5 mL ¥ minÀ1 and 358C.
Compounds were detected at 210 nm. Retention times were
9.56 min for N,N×-diacetylchitobiose (1), 10.04 min for
GlcNAcb(1!4)ManNAc (2), 10.59 min for GalNAcb
(1!4)GlcNAc (3), 11.21 min for GalNAcb(1!4)ManNAc
(4), 10.49 min for GlcNAcb(1!6)GlcNAc (5) and 9.58 min for
GlcNAcb(1!6)ManNAc (6).
4-O-(2-Acetamido-2-deoxy-b-d-galactopyranosyl)-2-
acetamido-2-deoxy-d-mannopyranose (4)
NMR
The compound 3 (51 mg, 0.12 mmol) was epimerized under the
same conditions as above. The enzymatic separation of 3 and 4
was done analogously as in the case of 1 and 2. The isolated
yield of 4 was 21.6% (11 mg). The structure was determined by
NMR. [a]58920: ‡ 3.98 (c 0.73 in water).
NMR spectra were recorded on a Varian UNITY Inova-
400 MHz spectrometer (399.90 MHz for H, 100.55 MHz for
1
13C) in D2O (99.95% D, Chemtrade) at 303 K. The assignments
are based on COSY, HMQC, HMBC and 1D TOCSY experi-
ments. All 2D experiments were done using the manufacturer×s
software. Acetone signal [dH and dC (30.50)] was used as a
reference. Carbon chemical shifts and some proton chemical
shifts were determined from 2D spectra with lower accuracy.
4 a-anomer: 1H NMR: d ˆ 1.810, 1.837(2  3H, s, CH3CO-2,
CH3CO-2'), 3.49 (1H, m, H-5'), 3.497 (1H, dd, J ˆ 4.5, 12.1 Hz,
H-6a), 3.552 (1H, dd, J ˆ 9.1, 9.9 Hz, H-4), 3.54 (1H, m, H-3'),
3.56 (2H, m, H-6'), 3.577 (1H, dd, J ˆ 2.1, 12.1 Hz, H-6b), 3.663
(1H, ddd, J ˆ 2.1, 4.5, 9.9, H-5), 3.693 (1H, dd, J ˆ 8.5, 10.8 Hz,
H-2'), 3.709 (1H, m, H-4'), 3.927 (1H, dd, J ˆ 4.8, 9.1 Hz, H-3),
4.096 (1H, dd, J ˆ 1.8, 4.8 Hz, H-2), 4.291 (1H, d, J ˆ 8.5 Hz, H-
1'), 4.893 (1H, d, J ˆ 1.8 Hz, H-1); 13C NMR: d ˆ 22.1, 22.4 (2 Â
q, CH3CO-2, CH3CO-2'), 52.8 (C-2, C-2'), 61.2 (C-6'), 62.3 (C-
6), 67.8 (C-3), 67.9 (C-4'), 70.6 (C-5), 70.7 (C-3'), 75.5 (C-5'),
77.0 (C-4), 93.0 (C-1), 101.9 (C-1').
Optical Rotation
Optical rotation was measured on a Perkin-Elmer 241 polar-
imeter.
4 b-anomer: 1H NMR: d ˆ 1.834, 1.851 (2  3H, s, CH3CO-2,
CH3CO-2'), 3.251 (1H, ddd, J ˆ 2.1, 4.7, 9.9 Hz, H-5), 3.451
(1H, dd, J ˆ 9.4, 9.9 Hz, H-4), 3.470 (1H, dd, J ˆ 4.7, 12.0 Hz, H-
6a), 3.49 (1H, m, H-5'), 3.52 (1H, m, H-3'), 3.56 (2H, m, H-6'),
3.622 (1H, dd, J ˆ 2.1, 12.0 Hz, H-6b), 3.684 (1H, dd, J ˆ 8.3,
10.8 Hz, H-2'), 3.709 (1H, m, H-4'), 3.725 (1H, dd, J ˆ 4.6,
9.4 Hz, H-3), 4.255 (1H, dd, J ˆ 1.7, 4.6 Hz, H-2), 4.268 (1H, d,
J ˆ 8.3 Hz, H-1'), 4.786 (1H, d, J ˆ 1.7 Hz, H-1); 13C NMR: d ˆ
22.3, 22.4 (CH3CO-2, CH3CO-2'), 52.9 (C-2'), 53.5 (C-2), 61.2
4-O-(2-Acetamido-2-deoxy-b-d-glucopyranosyl)-2-
acetamido-2-deoxy-d-mannopyranose (2)
N,N'-Diacetylchitobiose (1, 500 mg, 1.18 mmol) obtained by
acidic cleavage of chitin[10] was dissolved in a saturated aqueous
Ca(OH)2 solution (25 mL) and left overnight at a laboratory
‡
temperature.[4] Dowex 50WX2 in H form (Supelco, USA) was
used for the neutralization and the removal of Ca2‡ ions. The
740
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Adv. Synth. Catal. 2003, 345, 735 742

Enzymatic Discrimination of 2-Acetamido-2-deoxy-d-mannopyranose-Containing Disaccharides
FULL PAPERS
(C-6'), 62.2 (C-6), 67.9 (C-4'),70.9 (C-3'), 71.0 (C-3), 75.3 (C-5),
75.5 (C-5−), 76.6 (C-4), 93.2 (C-1), 102.0 (C-1').
P. funiculosum CCF 1994, P. funiculosum CCF 1995, P. funi-
culosum CCF 2984, Talaromyces flavus CCF 2686) was added.
The reaction mixture was incubated at 378C for 8 hours and the
reaction was monitored by HPLC.
Preparation of GlcNAcb(1!6)GlcNAc (5) and
GlcNAcb(1!6)ManNAc (6)
Hydrolysis of the Mixture of GlcNAcb(1!6)ManNAc
(6) and GlcNAcb(1!6)GlcNAc (5) by b-N-
Acetylhexosaminidases
The compound 5 was prepared as described previously.[12]
The mixture of 5 and 6 was prepared by Ca(OH)2-catalyzed
epimerization of 5 (53.6 mg, 0.13 mmol) under the same
conditions as described above which gave a 20% yield
(13 mg) of 6 under the thermodynamic equilibrium.
The reaction mixture of 5 and 6 (2.4 mg) was dissolved in
citrate-phosphate buffer (50 mM, pH 5.0, 0.2 mL) and 1 U of
the respective b-N-acetylhexosaminidase was added. The
reaction was monitored by HPLC. All enzymes tested (as in
the Table 1) hydrolyzed both disaccharides at a similar rate and
no discrimination was observed.
Cleavage of GlcNAcb(1!4)ManNAc (2) by the b-N-
Acetylhexosaminidases
Compound 2 (1 mg, 2.3 mmol) was dissolved in citrate-phos-
phate buffer (50 mM, pH 5.0, 100 mL) and 0.5 U of the
respective b-N-acetylhexosaminidase was added. For use of
rat plasma substrate 2 (1 mg, 2.3 mmol) was dissolved in fresh
rat plasma (0.1 mL). The reactions were incubated at 378C and
monitored by TLC and HPLC. For use of lysozyme (Sigma,
from chicken egg white, EC 3.2.1.17): the substrate 2 (1 mg)
was dissolved in citrate-phosphate buffer (50 mM, pH 6.2,
200 mL) at 378C and 5 U (and after 20 hours another 40 U) of
lysozyme were added (Table 1). The reactions were monitored
by TLC and HPLC. The b-N-acetylhexosaminidase activity
was assayed using pNP-bGlcNAc.[13] One unit of b-N-acetyl-
hexosaminidase activity was defined as the amount releasing
1 mmol of p-nitrophenol per minute at pH 5.0 and 378C.
Molecular Modelling
The complete primary sequence of the b-N-acetylhexosamini-
dase from A. oryzae that was cloned by us[14] was aligned with
the known X-ray structure of the b-N-acetylhexosaminidase
from Seratia marcescens and Streptomyces plicatus, extracted
from the Brookhaven Protein Database (PDB entry: 1QBA
and 1HP4, respectively, http://www.rcsb.org/pdb/). These pro-
teins show a high degree of primary structure similarity: 44%.
3D Models constituted by all non-hydrogen atoms were
generated by Modeller6 package.[15] For model refinement
and minimization the SYBYL package with the TRIPOS force
field (TRIPOS Associates Inc.) was used. Finally, the tertiary
structure models were checked with Pro Check[16]. With the
complete modelling including the alignment and energy
minimization, we used exactly the parameters and methods
published previously.[7]
Inhibition of the b-N-Acetylhexosaminidases by
GlcNAcb(1!4)ManNAc (2)
For the docking experiments our model structure was fitted
onto the crystal structure of 1QBB, a bacterial chitobiase
complexed with N,N×-diacetylchitobiose (1). Compound 1 was
placed in an arbitrary position according to the ligand co-
ordinates in the bacterial chitobiase complex. The positioning
of the ligand in the arbitrary site was done with the DOCK
module included in SYBYL/MAXIMIN2 that calculates
energies of interaction based on steric contributions from the
TRIPOS force field and electrostatic contributions from any
atomic charges present in the ligand. Exact positioning of the
ligand was done by a two-step procedure, energy minimization
followed by a molecular dynamics. The ligand-protein system
was minimized by 1000 interactions with the Powell minimizer
and the TRIPOS force field including electrostatic interactions
based on H¸ckel[18] partial charge distributions using a
dielectric constant with a distance dependent function e ˆ 4r
and non-bonded interaction cut-off of 8 ä.[17,18] A molecular
dynamics simulation at 290 K followed the minimization with
the NTVensemble over 15 ps. The resulting structure was then
minimized with the same parameters as above to the con-
vergence of the energy gradient less than 0.04 kJ ¥ molÀ1. For
the other ligands described in this paper the procedure was
exactly the same. The non-bonded interaction energy between
the model and the ligands within optimized complex was
calculated using the TRIPOS force field. This estimation of
real interaction energy neglects solvation and desolvation
effects.
The disaccharide 2 was tested as a potential inhibitor of fungal
b-N-acetylhexosaminidases from A. oryzae CCF 1066, P. ox-
alicum CCF 2430, Mucor dimorphosporus CCF 2609 and
Talaromyces flavus CCF 2686 (phylogenetically distant spe-
cies) (Table 2).
The
mixtures
of
either
pNP-bGlcNAc
or
GlcNAcb(1!4)GlcNAc (1) (final concentration 0.4 mM)
and the disaccharide 2 (final concentration 0.04 or 0.4 mM)
in citrate-phosphate buffer (50 mM, pH 5.0, 50 mL) were
supplemented with 0.5 U of the respective b-N-actylhexosa-
minidase. The mixtures were incubated at 378C for 5 minutes.
Controls were identical without 2. The activity was determined
according to the released p-nitrophenol (detection at 420 nm)
[13] or by HPLC measuring the released GlcNAc.
Transglycosylation Activities of Fungal b-N-
Acetylhexosaminidases towards ManNAc
The reaction components pNP-bGlcNAc (2.6 mg, 7.6 mmol)
and ManNAc (7.4 mg, 33.7 mmol) were dissolved in citrate-
phosphate buffer (50 mM, pH 5.0, 130 mL) and 0.5 U of
respective enzyme (Aspergillus flavofurcatis CCF 3061, A. per-
sicinum CCF 1850, A. tamarii CCF 1665, A. oryzae CCF 1063,
A. oryzae CCF 1066, A. sojae CCF 3060, A. flavus CCF 3056,
Penicillium oxalicum CCF 2315, P. parasiticus CCF 1298,
Adv. Synth. Catal. 2003, 345, 735 742
asc.wiley-vch.de
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
741

œ ¬
¬
Lucie Husakova et al.
FULL PAPERS
œ
œ
[7] J. Sklenµr, O. PlÌhal, P. Novµk, R. Ettrich, V. Kren, K.
Acknowledgements
œ
Bezouska, Effect of glycosylation on structure and
The authors would like to acknowledge support from the Czech
National Science Foundation (No. 524/00/1275 and 203/01/
1018), MSMT projects OC D25.002 and MSM113100001,
function of fungal b-hexosaminidase. Manuscript in
preparation for Arch. Biochem. Biophys.
[8] M. Horsch, Ch. Mayer, U. Sennhauser, D. M. Rast,
œ
Institutional researchconcept (Inst. Microbiol., Prague)
AV0Z5020903, from the Volkswagen Foundation (I/74679),
and from the COST actions D13 and D25.
Pharmacol. Ther. 1997, 76, 187 218.
œ
œ
¬
œ
œ
¬
¬
[9] Z. Hunkova, V. Kren, M. Scigelova, L. Weignerova, O.
Scheel, J. Thiem, Biotechnol. Lett. 1996, 18, 725 730.
[10] J. A. Rupley, Biochim. Biophys. Acta 1964, 83, 245 255.
[11] D. H. G. Crout, S. Singh, B. E. P. Swoboda, P. Critchley,
W. T. Gibson, J. Chem. Soc. Chem. Commun. 1992, 704
705.
References
[1] J. J. Gridley, H. M. I. Osborn, J. Chem. Soc. Perkin Trans.
1 2000, 1471 1491.
¬
œ ¬
¬
œ
¬
œ
[12] E. Rajnochova, J. Dvorakova, Z. Hunkova, V. Kren,
Biotechnol. Lett. 1997, 19, 869 872.
[2] a) M. Nilsson, T. Norberg, J. Chem. Soc. Perkin Trans. 1
1998, 1699 1704; b) K. Yokoyama, H. Mizuguchi, Y.
Araki, S. Kaya, E. Ito, J. Bacteriol. 1989, 171, 940 946;
c) C. J. Lee, S. D. Banks, J. P. Li, Crit. Rev. Microbiol.
1991, 18, 89 114; d) C. J. Lee, B. A. Fraser, J. Biol.
Chem. 1980, 255, 6847 6853.
[13] T. Mega, T. Ikenaka, Y. Matsushima, J. Biochem. 1970,
68, 109 117.
¬ œ
¬
œ
[14] O. PlÌhal, J. Sklenar, P. Matousek, P. Man, V. HavlÌcek, L.
¬
œ
¬
œ
œ
Weignerova, A. P ivsejcova, V. Kren, K. Bezouska, A
large N-terminal domain of fungal b-hexosaminidase is
cleaved before the secretion. Manuscript submitted to
Biochem. Biophys. Res. Commun.
¬
¬
¬
[3] P. Krist, E. Herkommerova-Rajnochova, J. Rauvolfova,
œ
œ
¬
œ
œ
T. Semenuk, P. Vavruskova, J. PavlÌcek, K. Bezouska, L.
[15] A. Sali, T. L. Blundell, J. Mol. Biol. 1993, 234, 779 815.
[16] R. A. Laskowski, M. W. MacArthur, D. S. Moss, J. M.
Thornton, J. Appl. Crystallogr. 1993, 26, 283 291.
[17] M. Clark, R. D. Cramer, N. Van Opdenbosch, J. Comput.
Chem. 1989, 10, 982 1012.
œ
œ
Petrus, V. Kren, Biochem. Biophys. Res. Commun. 2001,
287, 11 20.
œ
¬
œ
¬
[4] P. Sedmera, V. Prikrylova, K. Bezouska, E. Rajnochova,
œ
J. Thiem, V. Kren, J. Carbohydr. Chem. 1998, 17, 1351
1357.
[18] J. Gasteiger, M. Marsili, Tetrahedron 1980, 36, 3219
3238.
[5] S. Singh, J. Packwood, Ch. J. Samuel, P. Critchley,
D. H. G. Crout, Carbohydr. Res. 1995, 279, 293 305.
[6] S. Singh, D. H. G. Crout, J. Packwood, Carbohydr. Res.
1995, 279, 321 325.
742
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2003, 345, 735 742