Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases

Article abstract of DOI:10.1016/j.tet.2003.10.111

The hydrolytic and transglycosylation capabilities of 35 fungal β-N-acetylhexosaminidases with p-nitrophenyl 2-amino-2-deoxy-β-D- glucopyranoside and its four N-acyl derivatives (CH=O, COCH₂OH, COCH₂CH₃, COCF₃) as substrates were tested. The preparation of four novel p-nitrophenyl disaccharides from these unnatural substrates catalysed by enzymes from Aspergillus oryzae, Penicillium oxalicum and Talaromyces flavus represents a considerable extension of the synthetic potential of glycosidases.

Full text of DOI:10.1016/j.tet.2003.10.111

Tetrahedron 60 (2004) 693–701
Hydrolytic and transglycosylation reactions of N-acyl modified
substrates catalysed by b-N-acetylhexosaminidases
a
a
a
a
a
´
´
´
ˇ
ˇ
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ˇ
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Pavla Fialova, Lenka Weignerova, Jana Rauvolfova, Vera Prikrylova, Andrea Pisvejcova,
b
Rudiger Ettrich, Marek Kuzma, Petr Sedmera and Vladimır Kren
a
a
a
,
*
´
ˇ
¨
a
´
ˇ
´
Institute of Microbiology AS CR, Vıdenska 1083, CZ-14220 Prague, Czech Republic
Laboratory of High Performance Computing, Institute of Physical Biology USB and Institute of Landscape Ecology AS CR, Zamek 136,
´
CZ-37333 Nove Hrady, Czech Republic
b
´
Received 8 August 2003; revised 18 September 2003; accepted 17 October 2003
Abstract—The hydrolytic and transglycosylation capabilities of 35 fungal b-N-acetylhexosaminidases with p-nitrophenyl 2-amino-2-
deoxy-b-D-glucopyranoside and its four N-acyl derivatives (CHvO, COCH2OH, COCH2CH3, COCF3) as substrates were tested. The
preparation of four novel p-nitrophenyl disaccharides from these unnatural substrates catalysed by enzymes from Aspergillus oryzae,
Penicillium oxalicum and Talaromyces flavus represents a considerable extension of the synthetic potential of glycosidases.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
3,4-di-O-methyl and 3,4,6-tri-O-methyl groups,11 as well as
a range of N-acyl modified substrates12,13 were studied.
The recent dynamic development of glycosciences has
brought about the increasing need for new glycostructures.
Novel carbohydrate compounds can effectively be syn-
thesized using enzymatic methods.1 Glycosidases (EC 3.2),
although they often exhibit a poor regioselectivity and
sometimes low yields, are readily available from different
sources. They tolerate environmental stress and thanks to
their broad substrate specificity they are able to accept a
wide range of donors with different aglycon moieties and
acceptors.2–6
The affinity of various b-N-acetylhexosaminidases from
mushrooms and marine vertebrates to substrates modified
at C-2 amino group was thoroughly studied by
Molodtsov and Vafina.14–16 They isolated and characterised
the ‘b-N-glycylhexosaminidase’ from the marine helminth
Chaetopterus variopedatus17 and the ‘b-N-benzoylhexo-
saminidase’ from the scallop Mizuhopecten yessoensis.18
Besides, they detected a high tolerance to N-acyl modifi-
cations with the b-N-acetylhexosaminidase from the
fungus Hohenbuehelia serotina.19 A similar study was
carried out by Leaback and Walker with the commercial pig
epididymal b-N-acetylhexosaminidase.20
It has been revealed only recently that besides their natural
substrates (glycons), glycosidases are capable of accepting
substrates bearing various structural modifications. These
modified substrates, often exhibiting important biological
properties,7 find applications in many areas, such as
structure-activity relationship studies8–10 or treatment of
glycosidase-induced pathogenic states.9
Probably the first use of modified substrates in a
transglycosylation reaction was described by Wong and
Takayama with p-nitrophenyl 6-oxo-b-D-galactopyrano-
side.21 C-6 modifications were also studied by MacManus
et al.22 with a set of 10 p-nitrophenyl galactopyranosides
bearing different functionalities at C-6, such as methyl,
alkene, alkyne or fluorine. A b-N-acetylhexosaminidase
The first papers dealing with the enzymatic recognition of
substrates modified at the glycon moiety were published in
the early 70s, mostly investigating the substrate specificity
of the b-N-acetylhexosaminidase from Aspergillus
oryzae isolated from a digestive amylase preparation
Takadiastasew. Substrates bearing 3-O-methyl, 6-O-methyl,
ˇ´
´
was first used by Husakova et al. in a reaction with a 6-O-
acetylated glycosyl donor.23 To our best knowledge, no case
of transglycosylation with N-acyl modified substrates
catalysed by glycosidases has yet been published.
In the present paper, we report the affinity of a library of 35
fungal b-N-acetylhexosaminidases to various N-acyl modi-
fied substrates from the viewpoint of their hydrolytic and
transglycosylation potentials. Four novel p-nitrophenyl
Keywords: b-N-Acetylhexosaminidase; Transglycosylation; p-Nitrophenyl
2-acylamido-2-deoxy-b-D-glucopyranoside; N-Acyl modified substrate.
*
Corresponding author. Tel.: þ420-296442510; fax: þ420-296442509;
e-mail address: kren@biomed.cas.cz
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.111

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
694
Scheme 1.
disaccharides were prepared in transglycosylation reactions
with these new substrates. The experimental results are
discussed regarding molecular models of modified sub-
strates docked in the active centre of the b-N-acetylhexo-
saminidase from A. oryzae Culture Collection of Fungi 1066.
acetylhexosaminidases tested. On the other hand, substrates
6, 3 and 5 (from the best to the worst, respectively) were
cleaved by the majority of tested enzymes. The b-N-
acetylhexosaminidase from A. oryzae Culture Collection of
Fungi (CCF) 1066 did not substantially hydrolyse any of the
modified substrates 2–6 at a comparable level to the
standard substrate 1 (Table 1). These experimental results
were evaluated with regard to the interaction energies
calculated from the molecular model of this enzyme with
substrates 1–6 (Table 2). The decrease of the interaction
energy of an enzyme–substrate complex generally reflects
better binding. Neither of the respective modified substrates
exhibited any substantial steric conflict with the enzyme
active centre, as could be seen from minor differences in the
steric interaction energies. Surprisingly, the steric inter-
action energy slightly decreased with the size of the
substituent (formyl!acetyl!propionyl, 271, 284 and
2114 kJ/mol, respectively). More obvious differences
were observed in the electrostatic interaction energies.
Here, the standard substrate 1 displayed the strongest
electrostatic interaction (2216 kJ/mol), whereas the elec-
trostatic interaction energy of substrate 6 was significantly
higher (283 kJ/mol). Thus, the low cleavage of substrate 6
appears to be a result of a lower binding affinity. With
substrates 2–5 the situation is different: it may be supposed
from the interaction energy values that all these substrates
bind well to the active site of the enzyme and that their low
2. Results and discussion
Compounds 3, 5 and 6 were prepared from the correspond-
ing 2-acylamido-2-deoxy-D-glucopyranoses24 – 26 using
modified method27 (Scheme 1). The selective formylation
with p-nitrophenyl formiate28 as well as those using
HCOOH with Ac2O29,30 did not lead to the desired product,
and so the formylation with a mixed acetic formic anhydride
was performed.24 Substrate 2 was synthesized using
modified method31 (Scheme 2). In the synthesis of substrate
4 (Scheme 2), 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-
glucopyranose hydrochloride32 was trifluoroacetylated33 to
give 4a.
Substrates 2–6 were subjected to a screening for hydrolytic
cleavage comprising 35 fungal b-N-acetylhexosaminidases
from the genera of Acremonium, Aspergillus, Penicillium
and Talaromyces (Table 1).
Compounds 2 and 4 were poor substrates for all b-N-
Scheme 2.

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
695
Table 1. Screening on the hydrolysis of substrates 2–6 with fungal b-N-
acetylhexosaminidases
brasilianum CCF 2155 (6), P. brasilianum CCF 2171 (3), P.
oxalicum CCF 2315 (6), P. pittii CCF 2277 (3) and T. flavus
CCF 2686 (3, 5). The b-N-acetylhexosaminidase from A.
oryzae CCF 1066 displayed the lowest hydrolytic potential
of all tested enzymes with substrate 6 and, therefore, it was
selected for the catalysis of a transglycosylation reaction
with substrate 6 as a glycosyl acceptor.
Source of enzyme
2a
3a
4a
5a
6b
c
Acremonium persicinum CCF 1850
Aspergillus awamori CCF 763
A. caelatus CCF 3087
A. flavipes CCF 869
A. flavipes CCF 1895
A. flavipes CCF 3067
A. flavofurcatis CCF 107
A. flavus CCF 642
A. flavus CCF 3056
A. niger CCIM K2
A. niveus CCF 3057
A. nomius CCF 3086
A. oryzae CCF 147
A. oryzae CCF 1066
A. parasiticus CCF 141
A. parasiticus CCF 1298
A. phoenicis CCF 61
A. sojae CCF 3060
A. tamarii CCF 1665
A. terreus CCF 2539
A. terreus CCF 3059
þ
2
2
2
þ
2
2
2
2
2
2
2
2
2
2
2
2
þ
2
þ
þþþ
þ
2
þ
2
2
2
2
2
2
2
nd
2
2
2
2
2
2
2
2
2
2
þþþ
***c
*
*
*
þ
þ
þ
þ
þ
þ
þ
nd
nd
*
nd
*
nd
**
nd
**
*
nd
**
*
Substrates 3, 5 and 6 suffered from poor water solubility
(saturated solutions at 35 8C: substrate 3: 3.3 mM, substrate
5: 10.0 mM, substrate 6: 1.4 mM). To reach the optimum
concentration of substrates for transglycosylation reactions
(approx. 50 mM),1 MeCN was used as a cosolvent in the
reaction medium.1,34,35 It was shown before that MeCN is
well tolerated by respective b-N-acetylhexosaminidases.36
The activities of the selected enzymes in MeCN
(0–45% v/v) were tested (Table 3).
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þþ
þ
þ
þþ
þ
þ
þ
þ
þþ
þ
þ
*
*
***
*
þ
2
c
þþþ
þþþ
Table 3. Activity (%) of b-N-acetylhexosaminidases in MeCN
nd nd
nd nd
A. versicolor CCF 2491
Hamigera avellanea CCF 2923
Penicillium brasilianum CCF 2155
P. brasilianum CCF 2171
P. chrysogenum CCF 1269
P. funiculosum CCF 1994
P. multicolor CCF 2244
P. oxalicum CCF 1959
P. oxalicum CCF 2315
P. oxalicum CCF 2430
P. oxalicum CCF 3009
P. pittii CCF 2277
þ
þ
2
2
2
þ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
þ
nd
***
***c
nd
nd
**
**
**
***c
**
**
nd
nd
**
b-N-Acetylhexosaminidase
5% v/v MeCNa
45% v/v MeCNa
þþþ
þþþ
þþþ
þþ
þþ
þþþ
þþþ
þþ
A. persicinum CCF 1850
A. terreus CCF 2539
A. oryzae CCF 1066
P. brasilianum CCF 2155
P. brasilianum CCF 2171
P. oxalicum CCF 2315
P. pittii CCF 2277
84
77
107
125
81
93
87
107
0
33
7
c
nd nd
nd
2
2
2
2
2
þþ
þþ
29
22
43
55
þþþ
þþ
þþ
þþ
þþþ
þþþ
T. flavus CCF 2686
nd nd
nd
c
a
2
2
þ
þþþ
þþþ
þþþ
þþþ
Relative activities correlated to the values obtained in respective
reactions without MeCN.
P. spinulosum CCF 2159
Talaromyces flavus CCF 2686
þþþ
þþþ
c
c
All b-N-acetylhexosaminidases were inhibited by 45% v/v
MeCN, those from A. persicinum CCF 1850 and
P. brasilianum CCF 2155 were almost totally inactivated.
However, at 5% v/v, the cosolvent had a certain activation
effect on the b-N-acetylhexosaminidases from A. oryzae
CCF 1066, P. brasilianum CCF 2155 and T. flavus CCF
2686, the activity of which increased up to 125%.
a
Hydrolysis of substrates 2–5 was spectrophotometrically determined as
the liberated p-nitrophenol: the ratio of hydrolysis rates of the respective
modified substrate and substrate 1 was 24–12% (þþþ), 11–5% (þþ),
4–1% (þ) or lower than 1% (2).
b
Hydrolysis of substrate 6 was analysed by TLC: the ratio of hydrolysed
substrates 6 and 1 was 100–50% (***), 50–25% (**) or lower than 25%
(*). Both substrates were assayed at the starting concentration of 1 mM
due to poor water solubility of substrate 6 (sat. solution 1.4 mM) and
enzymatic activities were estimated according to TLC.
c
Three best hydrolysing enzymes for substrates 3, 5 and 6.
To test the synthetic ability of b-N-acetylhexosaminidases
with modified glycosyl donors and to optimise reaction
conditions, reactions with substrates 3, 5 and 6 were first
tested on an analytical scale and monitored by TLC and
HPLC. For each substrate, three best hydrolysing
enzymes (Table 1) were tested and classified regarding the
following criteria: (i) the most effective product formation;
(ii) the lowest impairment of the enzymatic activity by
MeCN. As a result, the following transglycosylation
reaction schemes were proposed: (i) substrates 3 or 5 with
the b-N-acetylhexosaminidase from T. flavus CCF 2686 in
45 and 5% v/v MeCN, respectively; (ii) substrate 6 with the
conversion into the hydrolytic product is caused by the
destabilisation of the oxazolinium reaction intermediate in
the proposed reaction mechanism,9 or at another level of the
‘dynamic’ part of the hydrolytic process.
The three best hydrolysing enzymes for each of substrates 3,
5 and 6 (Table 1) were selected as potential candidates for
catalysing transglycosylation reactions, namely b-N-acetyl-
hexosaminidases from Acremonium persicinum CCF 1850
(substrates 5 and 6), A. terreus CCF 2539 (5), Penicillium
Table 2. Interaction energies of substrates with the b-N-acetylhexosaminidase from A. oryzae CCF 1066
Substrate
Total interaction energy (kJ/mol)
Steric contribution (kJ/mol)
Electrostatic contribution (kJ/mol)
1
2
3
4
5
6
2299
2251
2227
2226
2274
2194
284
287
271
2123
2111
2114
2216
2164
2156
2103
2163
283

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
696
Scheme 3.
b-N-acetylhexosaminidase from P. oxalicum CCF 2315 in
45% v/v MeCN; (iii) substrates 1 and 6 with the b-N-
acetylhexosaminidase from A. oryzae CCF 1066 in 45% v/v
MeCN.
modified substrate 6 than to the standard substrate 1. This
concept widely extends the synthetic potential of b-N-
acetylhexosaminidases.
The above reactions were performed on a semi-preparative
scale (Scheme 3) under conditions optimised by HPLC
analyses. Products 7, 8 and 9 were obtained in reasonable
yields (78% for product 8) and with a high selectivity
(exclusive formation of b(1!4) bonds). The formation of
product 10 was unexpected. Due to the molar ratio of
substrates 1: 6¼1:2 and the low hydrolysis rate of substrate
6 by the b-N-acetylhexosaminidase from A. oryzae CCF
1066 we assumed the formation of p-nitrophenyl
2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-deoxy-
2-propionamido-b-D-glucopyranoside as a major product.
However, the enzyme preferred to transfer the modified
glycosyl moiety to p-nitrophenyl 2-acetamido-2-deoxy-b-
D-glucopyranoside and only products 9 and 10 were isolated
from the reaction mixture. This demonstrates the high
affinity of the b-N-acetylhexosaminidase from A. oryzae
CCF 1066 to the modified substrate 6.
4. Experimental
4.1. Materials
Citrate/phosphate buffer (Mc Ilvaine) pH 5.0 was prepared
by mixing 0.1 M citric acid (24.3 mL) and 0.2 M Na2HPO4
(25.7 mL), by diluting with water to 100 mL and adjusting
pH to 5.0. The fungal strains producing b-N-acetylhexo-
saminidases (EC 3.2.1.52) originated from the CCF,
Department of Botany, Charles University, Prague, or
from the Culture Collection of the Institute of Microbiology
(CCIM), Prague. The strains were cultivated as described
previously.37,38 Flasks (500 mL) with medium (100 mL)
were inoculated with the suspension of spores in 0.1%
Tween 80 and cultivated on a rotary shaker at 28 8C. Three
media were used. Mineral medium (for A. oryzae CCF
1066), [g/l]: yeast extract (0.5), KH2PO4 (3), NH4H2PO4
(5), (NH4)2SO4 (2), GlcNAc (5), NaCl (15), pH 6.0. Peptone
medium (for Aspergillus awamori CCF 763, A. niger CCIM
K2, A. phoenicis CCF 61 and P. chrysogenum CCF 1269),
[g/l]: yeast extract (0.5), mycological peptone (5), KH2PO4
(3), NH4H2PO4 (5), casein acid hydrolysate (7.5), pH 6.0. In
inductor supplemented medium (for all other strains in
Table 1), the casamino acids were replaced by crude chitin
hydrolysate (2 g/l). After sterilisation each flask was
supplemented with 0.5 mL 10% MgSO4·7H2O. Enzymes
were isolated by fractional (NH4)2SO4 precipitation (20–
80% sat.) and the precipitates were directly used for
respective reactions.
3. Conclusion
The acetamido group is a crucial structural feature of
substrates for b-N-acetylhexosaminidases, which are quite
sensitive to its modification. This study clearly demonstrates
for the first time that besides cleavage, fungal b-N-
acetylhexosaminidases are able to catalyse synthetic
transglycosylation reactions with N-acyl modified sub-
strates. They tolerate certain sterical changes at C-2 (shorter
or longer acyls, a hydroxyl instead of a hydrogen).
Nevertheless, they do not accept highly electronegative
acyls, for example, trifluoroacetyl, nor the free amino group.
Transglycosylation products were obtained selectively and
in good yields. Moreover, the b-N-acetylhexosaminidase
from A. oryzae CCF 1066 exhibited a higher affinity to the
4.2. Methods
4.2.1. Analytical methods. TLC was performed on pre-
coated Merck silica gel DC-Alufolien Kieselgel 60 F254

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
697
plates. The spots were visualised by charring with 5%
H2SO4 in EtOH. Optical rotation was measured on a
Perkin–Elmer 241 polarimeter at 589 nm. CD spectra were
measured on Jobin–Yvon/Spex CD 6 spectrophotometer in
the spectral region of 184–400 nm in H2O (compounds 7, 8)
ligands described.39 The non-binding interaction energy
between the model and the ligands within the optimised
complex was calculated using the TRIPOS force field. This
estimation of a real interaction energy neglects solvation
and desolvation effects.
1
or MeOH (compounds 9, 10). H- and 13C NMR spectra
were recorded on a Varian INOVA-400 spectrometer
(399.89 MHz and 100.55 MHz, respectively) at 30 8C in
the indicated solvents. Chemical shifts are expressed in the
d-scale and were measured with digital resolution justifying
the reported values to three or two decimal places,
respectively. HMQC and HMBC readouts are accurate to
one decimal place only. Residual solvent signals were used
for referencing (CDCl3: dH 7.265, dC 77.00; CD3OD: dH
3.330, dC 49.30), internal acetone (dH 2.030, dC 30.50) was
used for D2O solutions. The reported assignment is based on
gCOSY, HMQC, HMBC, and 1D-TOCSY experiments.
Positive-ion electrospray (ESI) mass spectra were recorded
on an LCQDECA ion trap mass spectrometer (Finnigan, San
Jose, USA) equipped with an ESI ion source. Spray voltage
was set at 5.0 kV, tube lens voltage was 210 V. Samples
dissolved in 40% MeCN were continuously infused into the
ion source via a linear syringe pump at a flow rate of 3 mL/
min. Full scan spectra were acquired over the m/z range of
150–1000 Da. A 0.1% solution of Ultramark 1621 (PCR,
Inc., Gainesville, FL) in MeCN was used to calibrate the m/z
scale of the instrument. Analytical HPLC was carried out on
a Spectra Physics modular analytical system (San Jose,
USA) comprised of an SP 8800 ternary gradient pump, an
SP 8880 autosampler and a Spectra Focus scanning UV/VIS
detector. Preparative HPLC was performed on a Spectra
Physics modular preparative system (San Jose, USA)
comprised of an SP 8810 Ti pump, a Rheodyne injection
port with a 100 mL sample load, a Spectra 100 variable
wavelength UV/VIS detector and a ChromJet SP 4400
integrator. A Lichrospher 100-5 NH2 column (Watrex, CR,
250£4 mm—analytical and 250£8 mm—preparatory) with
a mobile phase MeCN–H2O 79:21 was used at ambient
temperature. Compounds were detected at 200 nm.
4.3. Synthesis of substrates
4.3.1. General procedure. Compound 3a24 (5a25 or 6a;26
22.0 mmol) was dissolved in acetyl chloride (15 mL) and
refluxed under argon. After 12 h, the reaction mixture was
diluted with CH2Cl2 (80 mL) and extracted with ice cold
water (100 mL) and ice cold sat. NaHCO3 (2 £ 100 mL), all
within 10–15 min. The organic phase was dried with
Na2SO4 and evaporated in vacuo. The crude compound 3b
(5b or 6b), tetrabutyl ammonium hydrogen sulphate
(22.0 mmol) and p-nitrophenol (44.0 mmol), were dissolved
in a mixture of CH2Cl2 (80 mL) and 1 M NaOH (80 mL)
and stirred vigorously for 4 h. The reaction was monitored
by TLC (AcOEt–hexane 4:1). The mixture was extracted
with CH2Cl2 (400 mL), the organic phase was separated,
washed with water (2£400 mL), dried with Na2SO4 and
evaporated in vacuo, yielding the crude acetate 3c (5c or
6c), which was further purified.
4.3.2. p-Nitrophenyl 2-deoxy-2-formamido-b-D-gluco-
pyranoside (3). Following Section 4.3.1, compound 3c
(2.45 g, 5.39 mmol; 25%) was prepared from the starting
material 3a (4.56 g, 22.0 mmol) and purified by column
chromatography on Merck silica gel 60 (40–63 mm)
(AcOEt–hexane 3:1). The deacetylation according to
41
´
Zemplen
yielded the title compound
3
(1.69 g,
5.15 mmol; 96%). White crystals; [a]2D3¼227.7 (c 0.141,
MeOH) (Lit.12 [a]1D8¼224.2 (DMF)); according to NMR it
1
is a mixture of 73% trans- and 27% cis-amide. H NMR
(CD3OD) trans-amide: d 3.468 (dd, 1H, J¼9.8, 8.7 Hz,
H-4), 3.564 (ddd, 1H, J¼9.8, 5.7, 2.3 Hz, H-5), 3.661 (dd,
1H, J¼10.3, 8.7 Hz, H-3), 3.572 (dd, 1H, J¼12.1, 5.7 Hz,
H-6u), 3.954 (dd, 1H, J¼12.1, 2.3 Hz, H-6d), 4.023 (ddd,
1H, J¼10.3, 8.4, 1.1 Hz, H-2), 5.290 (d, 1H, J¼8.4 Hz,
H-1), 7.206 and 8.225 (4H, AA0BB0, SJ¼9.3 Hz, p-NP),
8.213 (d, 1H, J¼1.1 Hz, CHvO); cis-amide: d 3.472 (dd,
1H, J¼10.4, 8.5 Hz, H-4), 3.473 (dd, 1H, J¼10.3, 7.9 Hz,
H-2), 3.551 (ddd, 1H, J¼10.4, 5.5, 2.2 Hz, H-5), 3.552 (dd,
1H, J¼10.3, 8.5 Hz, H-3), 3.757 (dd, 1H, J¼12.1, 5.5 Hz,
H-6u), 3.953 (dd, 1H, J¼12.1, 2.2 Hz, H-6d),05.182 (d, 1H,
J¼7.9 Hz, H-1), 7.237 and 8.242 (4H, AA0BB , SJ¼9.2 Hz,
p-NP), 8.138 (s, 1H, CHvO); 13C NMR (CD3OD, HMQC
readouts) trans-amide: d 55.6 (C-2), 61.9 (C-6), 71.1 (C-4),
74.7 (C-3), 77.9 (C-5), 99.2 (C-1), 117.0 (2£C-ortho), 125.9
(2£C-meta), 163.7 (CHvO); cis-amide: d 59.6 (C-2), 62.9
(C-6), 71.1 (C-4), 74.7 (C-3), 77.9 (C-5), 99.1 (C-1), 117.0
(2£C-ortho), 126.0 (2£C-meta), 167.2 (CHvO); ESI-MS:
m/z calcd for C13H16N2O8Na [MþNa]þ 351.1, found 351.3.
4.2.2. Molecular modelling. The primary sequence of the
b-N-acetylhexosaminidase from A. oryzae CCF 1066 was
aligned with the known X-ray structures of the b-N-
acetylhexosaminidases from Serratia marcescens and
Streptomyces plicatus, extracted from the Brookhaven
Protein Database (PDB entry: 1QBA and 1HP4,
respectively). The sequence data are available in
DDBJ/EMBL/GeneBank databases (http://www.ncbi.nlm.
nih.gov/) under the accession AY091636. 3D models were
generated by Modeller6 package.40 For model refinement
and minimisation, the SYBYL package with the TRIPOS
force field (TRIPOS Associates Inc.) were used. The
complete modelling including the alignment and energy
minimisation was done exactly as described previously.39
The docking of ligands was performed as described
earlier.39 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 on
electrostatic contributions from any atomic charges present
in the ligand. Exact positioning of the ligand was done by a
two step procedure, that is, energy minimisation followed
by a molecular dynamics, in exactly the same manner for all
4.3.3. p-Nitrophenyl 2-deoxy-2-glycoloylamido-b-D-glu-
copyranoside (5). Following Section 4.3.1, compound 5c
(3.20 g, 6.07 mmol; 28%) was prepared from the starting
material 5a (6.14 g, 22.0 mmol) and purified by column
chromatography on silica gel (AcOEt–PE 2:1). White
powder; 1H NMR (CDCl3): d 2.070 (6H, 2£Ac), 2.086 (3H,
Ac), 2.155 (3H, Ac), 3.937 (ddd, 1H, J¼9.8, 5.6, 2.6 Hz,
H-5), 4.192 (dd, 1H, J¼12.3, 2.6 Hz, H-6u), 4.270 (ddd, 1H,

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
698
J¼10.7, 8.6, 8.1 Hz, H-2), 4.290 (dd, 1H, J¼12.3, 5.6 Hz,
H-6d), 4.432 and 4.611 (2H, AB, J¼15.5 Hz, CH2O-), 5.168
(dd, 1H, J¼9.8, 9.3 Hz, H-4), 5.370 (d, 1H, J¼8.1 Hz, H-1),
5.397 (dd, 1H, J¼10.7, 9.3 Hz, H-3), 6.275 (d, 0 1H,
J¼8.6 Hz, NH), 7.076 and 8.194 (4H, AA BB0,
SJ¼9.3 Hz, p-NP); 13C NMR (CDCl3, HMQC and
HMBC readouts): d 20.5 (4£Ac), 54.2 (C-2), 61.9 (C-6),
62.7 (CH2O2), 68.4 (C-4), 71.2 (C-3), 72.6 (C-5), 98.3
(C-1), 116.7 (2£C-ortho), 125.7 (2£C-meta), 143.2
(C-para), 161.3 (C-ipso), 167.6 (2-CO), 169.2 (4-CO),
169.5 (CH2OCO), 170.3 (6-CO), 171.1 (3-CO); ESI-MS:
m/z calcd for C12H16N2O7Na [MþNa]þ 323.1, found
323.2.
4.3.6. p-Nitrophenyl 2-deoxy-2-trifluoroacetamido-b-D-
glucopyranoside (4). Crude syrupy 1,3,4,6-tetra-O-acetyl-
2-deoxy-2-trifluoroacetamido-/-D-glucopyranosyl bromide
(4a)33 (318 mg, 0.685 mmol) was dried with 4A molecular
sieve (250 mg) in vacuo for 2 h. Dry p-nitrophenol
(191.6 mg, 1.4 mmol) was mixed with molecular sieve A4
(290 mg), the mixture was suspended in dry MeCN (4 mL)
under argon and stirred for 10 min. 2,6-Dimethylpyridine
(160 mL) was added dropwise and after 10 min of stirring,
m/z calcd for C20H24N2O12Na [MþNa]þ 549.1, found
41
´
´
549.2. The deacetylation according to Zemplen and
Fetison reagent (50% Ag2CO3/celite; 221 mg, 0.368 mmol)
crystallisation from hot water yielded the title compound
5 (1.59 g, 4.43 mmol; 73%). White crystals; [a]2D3¼228.9
(c 0.135, H2O).
was added. After 10 more minutes the solution of 4a in
MeCN (3£2 mL) was added via canula. The reaction was
monitored by TLC (AcOEt–hexane 1:1). After 2 h the
reaction mixture was diluted with CH2Cl2 (50 mL),
extracted with water (50 mL), 0.02 M H2SO4 (50 mL) and
with water (3£50 mL) again. The organic phase was dried
with Na2SO4, concentrated in vacuo and recrystallised twice
from hot EtOH yielding p-nitrophenyl 3,4,6-tri-O-acetyl-2-
4.3.4. p-Nitrophenyl 2-deoxy-2-propionamido-b-D-glu-
copyranoside (6). Following Section 4.3.1, compound 6c
(3.6 g, 7.5 mmol; 34%) was prepared from the starting
material 6a (5.18 g, 22.0 mmol) and purified by crystal-
lisation from hot EtOH. The deacetylation according to
41
deoxy-2-trifluoroacetamido-b-D-glucopyranoside
(4b)
(142 mg, 0.272 mmol; 40%). White crystals; 1H NMR
(CDCl3): d 2.075 (s, 3H, 4-Ac), 2.086 (s, 3H, 3-Ac), 2.097
(s, 3H, 6-Ac), 3.975 (ddd, 1H, J¼9.8, 5.6, 2.6 Hz, H-5),
4.177 (dd, 1H, J¼12.4, 2.6 Hz, H-6u), 4.304 (dd, 1H,
J¼12.4, 5.6 Hz, H-6d), 4.313 (ddd, 1H, J¼10.6, 8.8, 8.1 Hz,
H-2), 5.182 (dd, 1H, J¼9.8, 9.2 Hz, H-4), 5.336 (d, 1H,
J¼8.1 Hz, H-1), 5.405 (dd, 1H, J¼10.6, 9.2 Hz, H-3), 6.706
(d, 1H, J¼8.8 Hz, 2-NH), 7.076 and 8.204 (4H, AA0BB0,
SJ¼9.3, p-NP); 13C NMR (CDCl3): d 20.37 (4-Ac), 20.50
(3-Ac), 20.64 (6-Ac), 54.63 (C-2), 61.87 (C-6), 68.14 (C-4),
70.95 (C-3), 72.44 (C-5), 97.58 (C-1), 114.42 (CF3,
JC,F¼287.9 Hz), 116.63 (2£C-ortho), 125.78 (2£C-meta),
143.29 (C-para), 157.48 (2-CvO, JC,F¼37.9), 161.09
(C-ipso), 169.30 (4-CvO), 170.51 (6-CvO), 170.99
(3-CvO); ESI-MS: m/z calcd for C20H21F3N2O11Na
[MþNa]þ 545.1, found 545.2. Compound 4b (142 mg,
0.272 mmol) was mixed with sat. NH4OH–MeOH 1:10
(5 mL) and left for 6 h at ambient temperature (TLC
MeOH–CH2Cl2 1:2).43 The evaporation in vacuo afforded 4
(107 mg, 0.270 mmol; 99%). Yellowish crystals;
[a]2D3¼29.2 (c 0.229, H2O) (Lit.13 [a]2D5¼210.8 (c 0.157,
DMF)).
´
Zemplen and crystallisation from hot water yielded the
title compound 6 (1.67 g, 4.7 mmol; 62%). White crystals;
[a]2D3¼217.3 (c 0.196, MeOH) (Lit.12 [a]1D8¼219.7
1
(DMF)); H NMR (CD3OD): d 1.141 (t, 3H, J¼7.7 Hz,
Et), 2.227 (dq, 1H, J¼16.7, 7.7 Hz), 2.278 (dq, 1H, J¼16.7,
7.7 Hz), 3.453 (dd, 1H, J¼9.8, 8.6 Hz, H-4), 3.537 (ddd, 1H,
J¼9.8, 5.7, 2.3 Hz, H-5), 3.626 (dd, 1H, J¼10.4, 8.6 Hz,
H-3), 3.744 (dd, 1H, J¼12.1, 5.7 Hz, H-6u), 3.952 (dd, 1H,
J¼12.1, 2.3 Hz, H-6d), 3.982 (dd, 1H, J¼10.4, 8.5 Hz,
H-2), 5.229 (d, 1H, J¼8.5 Hz, H-1), 7.195 and 8.226 (4H,
AA0BB0, SJ¼9.3 Hz, p-NP); 13C NMR (CD3OD): d 11.4
(CH3CH2), 31.5 (CH3CH2), 58.2 (C-2), 63.4 (C-6), 72.5
(C-4), 76.0 (C-3), 79.2 (C-5), 100.8 (C-1), 118.0
(2£C-ortho), 127.1 (2£C-meta), 144.9 (C-para), 164.5
(C-ipso), 178.3 (CvO); ESI-MS: m/z calcd for
C15H20N2O8Na [MþNa]þ 379.1, found 379.2.
4.3.5. p-Nitrophenyl 2-amino-2-deoxy-b-D-glucopyrano-
side (2). 3,4,6-Tri-O-acetyl-2-amino-2-deoxy-a-D-gluco-
pyranosyl bromide hydrobromide (2a)42 (8.98 g,
20.0 mmol) was dissolved in dry MeCN (150 mL), silver
p-nitrophenolate (5 g, 20.3 mmol) was added and the
mixture was stirred at ambient temperature overnight. The
reaction was monitored by TLC (AcOEt–hexane 4:1).
The insoluble materials were removed by filtration through
Celite and the filtrate was evaporated in vacuo yielding the
crude acetate 2b, which was purified by column chroma-
tography on silica gel (AcOEt–hexane 4:1). Acetate 2b
4.4. Solubility of the N-acyl modified substrates 3, 5 and
6
The solubility of substrates 3, 5 and 6 in MeCN/buffer
mixture was measured by a gradual addition of MeCN to a
suspension of substrates (1–2 mg) in citrate/phosphate
buffer (pH 5.0) at 37 8C, so that the final substrate
concentration would be about 50 mM and the MeCN
concentration would not exceed 50% v/v, which is generally
the highest possible concentration tolerated by most fungal
b-N-acetylhexosaminidases. Substrates 3 (50 mM) and 6
(46 mM) were soluble in 45% v/v MeCN, whereas for
substrate 5 (50 mM), 5% v/v MeCN was sufficient.
(3.2 g, 6.9 mmol; 35%) was deprotected according to
41
Zemplen and crystallised from hot MeOH affording the
´
title compound 2 (991 mg, 3.3 mmol; 48%). Yellowish
crystals; [a]D23¼291.1 (c 0.214, H2O) (Lit.42 [a]2D0¼242 (c
1
0.4, MeOH) for hydrochloride); H NMR (D2O): d 2.737
(dd, 1H, J¼9.9, 8.2 Hz, H-2), 3.278 (m, 1H, H-3), 3.296 (m,
1H, H-4), 3.471 (ddd, 1H, J¼9.6, 5.7, 2.3 Hz, H-5), 3.556
(dd, 1H, J¼12.4, 5.7 Hz, H-6u), 3.730 (dd, 1H, J¼12.4,
2.3 Hz, H-6d),0 4.960 (d, 1H, J¼8.2 Hz, H-1), 7.026 and
8.039 (4H, AA BB0, SJ¼9.4 Hz, p-NP); 13C NMR (D2O): d
56.49 (C-2), 60.72 (C-6), 69.67 (C-4), 75.72 (C-3), 76.54
(C-5), 100.52 (C-1), 116.61 (2£C-ortho), 126.25
(2£C-meta), 142.72 (C-para), 161.92 (C-ipso); ESI-MS:
4.5. Enzymatic methods
4.5.1. Enzyme activity assay.2 The reaction mixture
containing substrate 1 (2 mM, starting concentration) and

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
699
b-N-acetylhexosaminidase (0.02–0.03 U) in buffer was
incubated with shaking at 35 8C for 10 min. Liberated
p-nitrophenol was determined spectrophotometrically
(420 nm) under alkaline conditions (0.1 M Na2CO3). One
unit of enzymatic activity was defined as the amount of
enzyme that releases 1 mmol of p-nitrophenol per minute
under the above conditions. The activity towards substrates
2–5 was determined analogously, with the amount of
enzyme being 0.07–0.15 U. In the case of substrate 6,
reaction mixtures containing 1 mM substrate 6 and b-N-
acetylhexosaminidase (0.07 U) in buffer at 35 8C were
analysed by TLC after 10 min (AcOEt–MeOH–sat.
NH4OH 7:3:1). In the enzymatic screening, the enzymes
were classified according to the ratio of hydrolysis rates of
the respective modified substrate and substrate 1 assayed
under the same conditions.
AA0BB0, SJ¼9.3 Hz, p-NP), 7.946 (s, 2H, 2£CHvO);
cis-/trans-amide and trans-/cis-amide: d 3.920 (0dd, 1H,
J¼10.4, 8.3 Hz, H-2), 4.479 (d, 1H, J¼8.3 Hz, H-1 ), 5.151
(d, 1H, J¼8.5 Hz, H-1), 8.027 (4H, AA0BB0, SJ¼8.6 Hz,
p-NP), 7.818 (s, 2H, 2£CHvO); 13C NMR (D2O) trans-/
trans-amide: d 53.64 (C-2), 54.54 (C-20), 60.04 (C-6), 60.72
(C-60), 69.88 (C-30), 71.98 (C-5), 73.38 (C-40), 75.03 (C-3),
76.09 (C-50), 78.79 (C-4), 98.30 (C-1), 101.20 (C-10), 116.71
(2£C-ortho), 126.22 (2£C-meta), 142.89 (C-para), 161.74
(C-ipso), 165.02 (CHvO), 165.08 (CHvO); cis-/cis-
amide: d 58.32 (C-2), 59.24 (C-20), 59.67 (C-6), 60.68
(C-60), 69.74 (C-30), 71.83 (C-3), 73.11 (C-40), 75.03 (C-5),
75.09 (C-50), 78.46 (C-4), 97.85 (C-1), 100.65 (C-10), 116.71
(2£C-ortho), 126.22 (2£C-meta), 142.92 (C-para), 161.48
(C-ipso), 168.43 (CHvO), 168.52 (CHvO); long-range
coupling between aldehyde protons and the respective H-2
protons was observed in the trans-series. CHvO also
showed coupling to C-2 and H-2 to the aldehydic carbonyl
(HMBC); ESI-MS: m/z calcd for C20H27N3O13Na
[MþNa]þ 540.1, found 540.3.
4.5.2. Analytical transglycosylation reactions. The reac-
tion mixture contained 11–13 mg of substrate 3, 5 (starting
concentration 50 mM) or 6 (starting concentration 46 mM)
in MeCN/buffer (45% v/v for substrates 3, 6; 5% v/v for
substrate 5). The reaction was started by the addition of
b-N-acetylhexosaminidase (0.8–8.2 U) and was incubated
at 37 8C with shaking for 24 h. Aliquots were analysed by
TLC (AcOEt–MeOH–sat. NH4OH 7:3:1) and by HPLC
(reactions with substrates 3 and 5).
4.5.4. p-Nitrophenyl 2-deoxy-2-glycoloylamido-b-D-glu-
copyranosyl-(1!4)-2-deoxy-2-glycoloylamido-b-D-glu-
copyranoside (8). Substrate 5 (41 mg, 0.114 mmol) was
dissolved in 5% v/v MeCN/buffer (2.3 mL), the b-N-
acetylhexosaminidase from T. flavus CCF 2686 (3.16 U)
was added and the mixture was shaken at 37 8C. After
100 min the reaction was stopped by diluting with MeOH
and boiling for 5 min. The reaction mixture was evaporated
and chromatographed on a silica gel column (AcOEt–
MeOH–sat. NH4OH 7:3:1.5) yielding the title compound 8
(25.6 mg, 0.044 mmol; 78%). Colourless syrup; [Q]¼þ36.2
(194 nm), 24.52 (220 nm), þ1.11 (280 nm), 20.534
(340 nm); 1H NMR (D2O): d 3.276 (dd, 1H, J¼9.9,
8.4 Hz, H-40), 3.317 (m, 1H, H-50), 3.478 (dd, 1H, 0J¼12.3,
2.6 Hz, H-6u), 3.479 (dd, 1H, J¼10.4, 8.4 Hz, H-3 ), 3.548
(dd, 1H, J¼12.3, 5.6 Hz, H-60u), 3.553 (m, 1H, H-5), 3.567
(dd, 1H, J¼9.4, 8.3 Hz, H-4), 3.627 (dd, 1H, J¼10.4,
8.4 Hz, H-20), 3.682 (dd, 1H, J¼12.3, 10.9 Hz, H-6d), 3.728
(dd, 1H, J¼12.3, 2.1 Hz, H-60d), 3.734 (dd, 1H, J¼10.5,
8.3 Hz, H-3), 3.867 and 3.893 (2H, AB, J¼16.6 Hz, CH2O-
), 3.920 (dd, 1H, J¼10.5, 8.4 Hz, H-2), 3.936 and 3.961(2H,
AB, J¼16.6 Hz, CH2O–), 4.506 (d, 1H, J¼8.4 Hz, H-10),
5.170 (d, 1H, J¼8.4 Hz, H-1), 6.959 and 8.025 (4H,
AA0BB0, SJ¼9.3 Hz, p-NP); 13C NMR (D2O): d 54.71
(C-2), 55.63 (C-20), 60.20 (C-6), 60.89 (C-60), 61.28
(2£CH2O–), 70.08 (C-40), 72.19 (C-3), 73.58 (C-30),
75.20 (C-5), 76.23 (C-50), 79.21 (C-4), 98.51 (C-1),
101.42 (C-10), 116.85 (2£C-ortho), 126.36 (2£C-meta),
143.02 (C-para), 161.87 (C-ipso), 175.86 (CvO), 176.03
(CvO); ESI-MS: m/z[MþNa]þ 600.2, found 600.3.
4.5.3. p-Nitrophenyl 2-deoxy-2-formamido-b-D-gluco-
pyranosyl-(1!4)-2-deoxy-2-formamido-b-D-glucopyra-
noside (7). Substrate 3 (82 mg, 0.250 mmol) was dissolved
in 45% v/v MeCN/buffer (5 mL) and after the addition of
the b-N-acetylhexosaminidase from T. flavus CCF 2686
(13.6 U) the mixture was shaken at 37 8C. After 3 h the
reaction was stopped by diluting with MeOH and boiling for
5 min. The reaction mixture was evaporated and chromato-
graphed on a silica gel column (AcOEt–MeOH–sat.
NH4OH 7:3:1.5) yielding the title compound 7 (10.5 mg,
0.020 mmol; 16%). White crystals; [Q]¼þ35.0† (194 nm),
24.01 (220 nm),þ1.25 (280 nm), 20.662 (340 nm).
According to NMR the ratio of trans-/cis-amide isomers
was 74:26, however, four species containing all possible
combinations were distinguished, albeit not completely; 1H
NMR (D2O) trans-/trans-amide: d 3.271 (dd, 1H, J¼10.2,
10.1 Hz, H-30), 3.306 (ddd, 1H, J¼8.3, 5.3, 2.2 Hz, H-50),
3.415 (dd, 1H, J¼10.2, 8.3 Hz, H-40), 3.512 (dd, 1H,
J¼12.2, 4.5 Hz, H-6u), 3.545 (dd, 1H, J¼12.2, 5.3 Hz,
H-60u), 3.559 (ddd, 1H, J¼8.4, 4.5, 3.2 Hz, H-5), 3.606 (dd,
1H, J¼10.50, 10.2 Hz, H-3), 3.626 (ddd, 1H, J¼10.1, 8.4,
0.7 Hz, H-2 ), 3.679 (dd, 1H, J¼10.5, 8.4 Hz, H-4), 3.691
(dd, 1H, J¼0 12.2, 3.2 Hz, H-6d), 3.724 (dd, 1H, J¼12.2,
2.2 Hz, H-6 d), 3.915 (ddd, 1H, J¼10.2, 8.4, 0.6 Hz, H-2),
4.473 (d, 1H, J¼8.4 Hz, H-10), 5.114 (d, 1H, J¼8.4 Hz,
H-1), 6.965 and 8.022 (4H, AA0BB0, SJ¼9.3 Hz, p-NP),
8.004 (d, 1H, J¼0.6 Hz, 2-CHvO), 8.056 (d, 1H,
J¼0.7 Hz, 20-CHvO); cis-/cis-amide: d 3.120 (dd, 1H,
J¼10.2, 8.0 Hz, H-20), 3.267 (dd, 1H, J¼9.7, 8.6 Hz, H-40),
3.306 (ddd, 1H, J¼9.7, 5.3, 2.2 Hz, H-50), 3.391 (dd, 1H,
J¼10.2, 8.6 Hz, H-30), 3.444 (dd, 1H, J¼10.0, 8.2 Hz, H-2),
3.547 (dd, 1H, J¼12.2, 5.3 Hz, H-60u), 3.725 (dd, 1H,
J¼12.2, 2.2 Hz, H-60;d), 4.438 (d, 1H, J¼8.3 Hz, H-10),
5.126 (d, 1H, J¼8.5 Hz, H-1), 7.000 and 8.033 (4H,
4.5.5. p-Nitrophenyl 2-deoxy-2-propionamido-b-D-glu-
copyranosyl-(1!4)-2-deoxy-2-propionamido-b-D-gluco-
pyranoside (9). Substrate 6 (60.8 mg, 0.171 mmol) was
dissolved in 45% v/v MeCN/buffer (3.7 mL) and the b-N-
acetylhexosaminidase from P. oxalicum CCF 2315 (51 U)
was added. The mixture was shaken at 37 8C. After 16 h the
substrate was practically consumed and the reaction was
stopped by diluting with MeOH (10 mL) and boiling for
5 min. The solids were filtered off; the reaction mixture was
evaporated to dryness, dissolved in water and extracted into
a solid phase on Amberlite XAD-4 resin (BDH Chemicals,
†
Molar elipticity [Q] is reported in units of 103 deg cm2/dmol.

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P. Fialova et al. / Tetrahedron 60 (2004) 693–701
700
Ltd). After washing with water, p-nitrophenyl glycosides
were eluted with MeOH, the eluent was evaporated and
separated on Sephadex LH-20 (25–100 mm, Pharmacia)
with a mobile phase of MeOH–H2O 4:1 and a flow rate of
22 mL/h, which afforded the title compound 9 (11.8 mg,
0.021 mmol; 24%). White crystals; [Q]¼þ56.6 (194 nm),
25.95 (220 nm),þ1.83 (280 nm), 20.669 (340 nm); 1H
NMR (CD3OD): d 1.136 (t, 3H, J¼7.6 Hz, 2-Et), 1.189 (t,
3H, J¼7.6 Hz, 20-Et), 2.232 (dq, 1H, J¼15.2, 7.6 Hz,
2-CH2-u), 2.246 (dq, 1H, J¼15.2, 7.6 Hz, 2-CH2-d), 2.305
(dq, 1H, J¼15.2, 7.6 Hz, 20-CH2-u), 2.330 (dq, 1H, J¼15.2,
7.6 Hz, 20-CH2-d), 3.335 (dd, 1H, J¼9.8, 8.3 Hz, H-40),
3.390 (ddd, 1H, J¼9.8, 6.4, 2.3 Hz, H-50), 3.479 (dd, 1H,
J¼10.3, 8.3 Hz, H-30), 3.601 (ddd, 1H, J0¼9.7, 4.6, 2.0 Hz,
H-5), 3.674 (dd, 1H, J¼11.9, 4.6 Hz, H-6 u), 3.674 (dd, 1H,
J¼9.7, 8.2 Hz, H-4), 3.695 (dd, 1H, J¼12.2, 4.6 Hz, H-6u),
3.777 (dd, 1H, J¼10.3, 8.5 Hz, H-20), 3.786 (dd, 1H,
J¼10.4, 8.2 Hz, H-3), 3.874 (dd, 1H, J¼0 12.2, 2.0 Hz,
H-6d), 3.943 (dd, 1H, J¼11.9, 2.2 Hz, H-6 d), 4.059 (d0d,
1H, J¼10.4, 8.4 Hz, H-2), 4.556 (d, 1H, J¼8.5 Hz, H-1 ),
5.220 (d, 1H, J¼8.4 Hz, H-1), 7.181 and 8.221 (4H,
AA0BB0, SJ¼0 9.3 Hz, p-NP); 13C NMR (CD3OD): d 10.2
(Et),0 10.5 (Et ), 30.5 (CH20)0, 30.6 (CH2), 56.4 (C-2), 57.3
(C-20), 61.8 (C-6), 62.7 (C-6 ),072.3 (C-40), 74.0 (C-3), 76.0
(C-3 ), 77.1 (C-5), 78.2 (C-5 ), 81.0 (C-4), 100.2 (C-1),
103.3 (C-10), 118.0 (2£C-ortho), 0126.9 (2£C-meta), 144.3
(C-para), 163.7 (C-ipso), 177.2 (2 -CvO), 177.8 (2-CvO);
ESI-MS: m/z calcd for C24H35N3O13Na [MþNa]þ 596.2,
found 596.5.
5.224 (d, 1H, J¼8.5 Hz, H-1). 7.186 and 8.223 (4H,
AA0BB0, SJ¼9.3 Hz, p-NP); 13C NMR (CD3OD, HMQC
and HMBC readouts): d 10.4 (CH3CH2), 23.0 (Ac), 30.6
(CH3CH2), 56.7 (C-2), 57.5 (C-20), 61.8 (C-6), 62.8 (C-60),
72.4 (C-40), 74.2 (C-3), 76.1 (C-30), 77.1 (C-5), 78.5 (C-50),
81.1 (C-4), 100.2 (C-1), 103.4 (C-10), 118.0 (2£C-ortho),
126.9 (2£C-meta), 144.7 (C-para), 164.2 (C-ipso), 174.2
(CH3CO), 177.8 (CH3CH2CO); ESI-MS: m/z calcd for
C23H33N3O13Na [MþNa]þ 582.2, found 582.4.
4.5.7. NMR characterisation of transglycosylation pro-
ducts. All prepared disaccharides showed the expected
pseudomolecular ions in the MS spectra. According to J1,2
values (1H NMR), 0the anomeric configuration was b in all
cases. H-1 and H-1 protons were differentiated on the basis
of the coupling of the former to C-ipso of p-NP. The (1!4)-
linkage was inferr0ed from heteronuclear couplins of H-10 to
C-4 or H-4 to C-1 observed in HMBC. The downfield shift
of C-4 with respect to the parent compound provided a
supporting argument. The attachment of a substituent to C-2
is apparent from the coupling of H-2 and side chain protons
to the same carbonyl.
Acknowledgements
This work was supported by Czech Science Foundation GA
CR No. 204/02/P096. The preparation of enzymes used in
this work was done under the support of GA CR grant No.
203/01/1018. Molecular modelling was financed by
Ministry of Education of the Czech Republic (MSMT
4.5.6. p-Nitrophenyl 2-deoxy-2-propionamido-b-D-glu-
copyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-gluco-
pyranoside (10). Substrates 1 (40.4 mg, 0.118 mmol) and 6
(80.6 mg, 0.226 mmol) were dissolved in 45% v/v MeCN/
buffer (4.9 mL) and the b-N-acetylhexosaminidase from
A. oryzae CCF 1066 (48 U) was added. The mixture was
shaken at 37 8C. The enzyme was added twice more (24 U
after 7 h and 12 U after 13 h). After 15.5 h substrate 1 was
consumed and the reaction was stopped by diluting with
MeOH (10 mL) and boiling for 5 min. The solids were
filtered off and the reaction mixture was evaporated to
dryness, dissolved in water and loaded onto Amberlite
XAD-4 resin. After washing with water, p-nitrophenyl
glycosides were eluted with MeOH, the eluent was
evaporated and separated on Sephadex LH-20 (mobile
phase MeOH–H2O 4:1, flow rate 24 mL/h) affording a
mixture of 9 and 10, which was further separated by
preparative HPLC. Besides product 9 (2.9 mg, 0.005 mmol;
yield 4.3% referred to substrate 1), the title compound 10
(1.2 mg, 0.002 mmol; yield 1.8%) was obtained. White
crystals; [Q]¼þ91.8 (194 nm), 29.25 (220 nm),þ2.75
ˇ
123100001). We thank Dr K. Bezouska (Fac. Sci., Charles
University, Prague) for providing computing facilities for
molecular modelling and Dr P. Halada (Inst. Microbiol.,
Prague) for the MS measurements.
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