Combined application of galactose oxidase and β-N-acetylhexosaminidase in the synthesis of complex immunoactive N-acetyl-D-galactosaminides

Article abstract of DOI:10.1002/adsc.200505041

A high-yield preparatory procedure for the synthesis of p-nitrophenyl 2-acetamido-2-deoxy-β-D-galacto-hexodialdo-1,5-pyranoside (2) using the galactose oxidase from Dactylium dendroides in a batch reactor was developed. Enzymatic recognition of this aldeh

Full text of DOI:10.1002/adsc.200505041

FULL PAPERS
Combined Application of Galactose Oxidase and
b-N-Acetylhexosaminidase in the Synthesis of Complex
Immunoactive N-Acetyl-d-galactosaminides
a
b
c
a
´
ˇ
ˇ
´
Pavla Fialova, Darius-Jean Namdjou, Rꢀdiger Ettrich, Vera Prikrylova,
a
a
a
b
´
ˇ
Jana Rauvolfova, Karel Krenek, Marek Kuzma, Lothar Elling,
Karel Bezouska, Vladimꢁr Kren *
a
a,
ˇ
ˇ
a
ˇ
´
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vꢁdenska 1083, 14220 Prague 4, Czech Republic
Fax: (þ420)-296-442-509, e-mail: kren@biomed.cas.cz
b
c
Laboratory for Biomaterials, Department of Biotechnology and Helmholtz Institute for Biomedical Engineering, Aachen
University, Worringerweg 1, 52074 Aachen, Germany
Fax: (þ49)-241-802-2387, e-mail: L.Elling@biotec.rwth-aachen.de
Laboratory of High Performance Computing, Institute of Physical Biology USB and Institute of Landscape Ecology,
´
´
Academy of Sciences of the Czech Republic, Zamek 136, 37333 Nove Hrady, Czech Republic
Fax: (þ420)-386-361-219, e-mail: ettrich@greentech.cz
Received: January 24, 2005; Accepted: March 18, 2005
Abstract: A high-yield preparatory procedure for the ceptor. The b-N-acetylhexosaminidase from Talaro-
synthesis of p-nitrophenyl 2-acetamido-2-deoxy-b-d- myces flavus gave the best yields (37%) of the trans-
galacto-hexodialdo-1,5-pyranoside (2) using the galac- glycosylation
product
2-acetamido-2-deoxy-b-d-
tose oxidase from Dactylium dendroides in a batch re- galacto-hexodialdo-1,5-pyranosyl-(1!4)-2-acetamido-
actor was developed. Enzymatic recognition of this al- 2-deoxy-d-glucopyranose, which was oxidised in situ
dehyde and the respective uronic acid 3 obtained by to yield the final product 2-acetamido-2-deoxy-b-d-
NaClO2 oxidation was studied using a set of 36 fungal galactopyranosyluronic
acid-(1!4)-2-acetamido-2-
b-N-acetylhexosaminidases from Acremonium, As- deoxy-d-glucopyranose (6). Compounds 3 and 6
pergillus, Penicillium and Talaromyces genera. The al- were shown to be high-affinity ligands for two natural
dehyde 2 was readily hydrolysed by all tested b-N- killer cell activation receptors, NKR-P1A and CD69.
acetylhexosaminidases but neither the uronic acid 3 For the latter receptor they turned out to be among
nor its methyl ester 4 were accepted. Molecular mod- the best ligands described so far. This increase was ob-
elling with docking into the active centre of the b-N- viously due to the presence of a carboxy moiety.
acetylhexosaminidase from Aspergillus oryzae re-
vealed that the aldehyde 2 is processed as a C-6 gemi- Keywords: b-N-acetylhexosaminidase; galactose oxi-
nal diol by the enzyme. The aldehyde 2 was tested for dase; glycosylation; modified substrate; molecular
transglycosylation reactions using GlcNAc as an ac- modelling; natural killer cell
Abbreviations: GalNAc: 2-acetamido-2-deoxy-d- acetamido-2-deoxy-d-glucopyranose; ManNAc: 2-
galactopyranose; GalNAcA: 2-acetamido-2-deoxy-d- acetamido-2-deoxy-d-mannopyranose; NK cell: natu-
galactopyranosuronic acid; b-d-GalNAcA-(1!4)-d- ral killer cell; pNP: p-nitrophenyl; pNP-b-GalNAc:
GlcNAc: 2-acetamido-2-deoxy-b-d-galactopyranosy- p-nitrophenyl 2-acetamido-2-deoxy-b-d-galactopyra-
luronic acid-(1!4)-2-acetamido-2-deoxy-d-glucopyr- noside; pNP-b-GalNAcA: p-nitrophenyl 2-acetami-
anose; GlcNAc: 2-acetamido-2-deoxy-d-glucopyra- do-2-deoxy-b-d-galactopyranosiduronic acid; UDP-
nose; LacdiNAc: b-d-GalNAc-(1!4)-d-GlcNAc or GalNAc: uridine diphosphate 2-acetamido-2-deoxy-
2-acetamido-2-deoxy-b-d-galactopyranosyl-(1!4)-2-
a-d-galactopyranose.
Adv. Synth. Catal. 2005, 347, 997–1006
DOI: 10.1002/adsc.200505041
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FULL PAPERS
Introduction
oxy-b-d-galactopyranosyluronic acid moiety together
with other N-acetyl-d-hexosamines.
Enzymatic methods have already become a viable alter-
In this paper we demonstrate a high-yield preparative-
native to organic chemistry in the synthesis of complex scale procedure for p-nitrophenyl 2-acetamido-2-de-
carbohydrate structures,[1] especially due to their selec- oxy-b-d-galacto-hexodialdo-1,5-pyranoside (2) and its
tivity and simplicity. These methods are, however, often selective oxidation into the respective uronic acid 3.
limited by the strict specificity of the enzymes towards We studied the recognition of these glycosides by a set
substrates and, especially in case of glycosidases, by un- of 36 b-N-acetylhexosaminidases from our enzyme li-
satisfactory yields.[2] Ingenious combinations of various brary[12] and the results were evaluated using molecular
enzymes in a sequence or multi-enzyme one-pot meth- models of the modified substrates docked into the active
ods bring additional advantages and open new meth- centre of the b-N-acetylhexosaminidase from Aspergil-
odological ways.[3]
lus oryzae CCF 1066. A noveldisaccharide was prepared
Glycosidases (EC 3.2) are readily available in a large by a transglycosylation reaction and a subsequent chem-
repertoire from different sources. They tolerate envi- ical oxidation. Finally, all prepared hexosamine struc-
ronmental stress and have a broad substrate specificity tures were tested for their immunomodulatory activity
towards a variety of acceptors. Their application is using NK cell activation receptors NKR-P1A (rat) and
somehow limited in respect of glycosyl donors contrary CD 69 (human). Compounds 3 and 6 proved to be
to glycosyl transferases where a large number of modi- high-affinity ligands for these receptors, particularly
fied substrates – nucleotide sugars – were employed for CD69.
for synthetic purposes.[1b,4]
The first papers presenting the use of modified glyco-
syl donors with glycosidases were published in the late Results and Discussion
1990 s,[5,6] the first use of a b-N-acetylhexosaminidase
[7]
ˇ ´
´
was demonstrated by Husakova et al. in a reaction The C-6 oxidation of pNP-b-GalNAc (1) leading to the
with 6-O-acetylated glycosyl donors. A series of b-N- aldehyde 2 could theoretically be accomplished by vari-
acetylhexosaminidases with a broad substrate specifici- ous means, e.g., by laccase-TEMPO oxidation,[14] how-
ty was identified in a recent study dealing with p-nitro- ever, it is rather difficult to stop this reaction at the stage
phenyl 2-acylamido-2-deoxy-b-d-glucopyranosides.[8]
of the aldehyde and a mixture of the aldehyde and the
It is quite obvious that glycosidases do not tolerate uronic acid is usually formed. Therefore, we decided to
major changes in the structure of their substrates (gly- use galactose oxidase, which is known to oxidise galacto-
cone part). However, a large group of well-accepted pyranosyl[5,9] and 2-amino-2-deoxy-d-galactopyranosyl
modifications in the pyranose ring are those at the C-6 structures selectively and quantitatively.[15] Galactose
position.[5,7,9] The introduction of a highly reactive func- oxidase is highly specific for the carbohydrate C-6 posi-
tionality such as the aldehyde group brings about the tion[16] and its preparative application to GalNAc struc-
possibility of further modifications, e.g., reduction, oxi- tures has not been deeply exploited yet.[4,16] The problem
dation or conjugation (e.g., with amines or hydrazides). of using galactose oxidase is the oxygen supply. Al-
The latter has recently been accomplished by the use of though a simple diffusive gas transport through the sol-
biotinylated nucleotide sugars as substrates for glycosyl ution surface is mostly used in the literature,[9,17] the
transferases.[4c] It was already demonstrated that some yields turned to be unsatisfactory in our hands. Bubbling
galactosidases can tolerate the aldehyde group[5,9] and by air or oxygen quickly deactivates the enzyme, proba-
are able to use such modified substrates for transglyco- bly due to denaturation in the gas/liquid interphase area.
sylation reactions.
Therefore, we optimised a batch reactor with bubble-
Our research is oriented to biologically active glyco- free oxygen supply as described by Bꢀlter et al.[4c] by
sides and glycoconjugates,[10] which are able to stimulate doubling the reaction volume and reducing the concen-
the immune response of an organism by activation of tration of galactose oxidase twelve times compared to
NK cells through NKR-P1A protein and other cell sur- that used for the oxidation of UDP-GalNAc.[4c] This
face activation receptors. These immunoactive struc- method enabled a quantitative oxidation on a 100-mg
tures are mostly composed of N-acetyl-d-hexosamines scale with a considerably lower consumption of enzymes
(GalNAc, GlcNAc, ManNAc) and they can be prepared and time compared to literature reports.[4c,9,17] An enor-
in good yields using b-N-acetylhexosaminidases.[11,12] mous problem consisted in the purification of the alde-
GalNAc itself and glycosides containing b-GalNAc moi- hyde as the compound is relatively unstable due to its
ety possess a high immunomodulatory activity. More- high reactivity and a considerable decomposition occurs
over, the incorporation of groups able to carry a nega- in the presence of organic solvents, under even slightly
tive charge (COOH, SO3H) into such structures strongly acidic or basic conditions or under heating.[18] A good
improves the binding affinity towards the activation re- solution was Dowex 50W-X8 in the Ca2þ cycle, which,
ceptor of NK cells.[10a,13] Therefore, we were interested in however, required 100% conversion of the oxidation re-
the synthesis of glycosides bearing 2-acetamido-2-de- action because no starting material could be separated
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Synthesis of Complex Immunoactive N-Acetyl-d-galactosaminides
FULL PAPERS
by this method. p-Nitrophenyl 2-acetamido-2-deoxy-b- This corroborates our hypothesis that substrate 2 is rec-
d-galactopyranosiduronic acid (3) was prepared chemi- ognised by the enzyme as a geminal diol. In the cases of
cally[17] as well as its methyl ester 4.
pNP-b-GalNAcA (3) and its methyl ester 4, both sub-
Compounds 2, 3 and 4 were subjected to a screening strates obviously bind weakly into the active site due
for hydrolytic cleavage comprising 36 fungal b-N-acetyl- to a loss in hydrogen bonding and, correspondingly, no
hexosaminidases from the genera of Acremonium, As- hydrolysis was observed. Both substrates 3 and 4 have
pergillus, Penicillium and Talaromyces. Compounds 3 similarly low interaction energies, which demonstrates
and 4 proved to be poor substrates for all b-N-acetylhex- that the carboxylic group is not accepted due to its struc-
osaminidases tested, with the cleavage rate lower than tural features and not because of the charge and possible
1% compared to the standard substrate 1. On the other anion solvation.
hand, all tested enzymes cleaved substrate 2, e.g., the b-
b-GalNAc structures bearing a carboxy group show
N-acetylhexosaminidase from Aspergillus oryzae CCF strong immunoactivation properties.[10a,13] Therefore,
1066 hydrolysed it at a rate of 10% compared to 1 (Ta- we aimed to synthesise a disaccharide of b-d-GalNAcA-
ble 1).
(1!4)-d-GlcNAc (6) – a LacdiNAc analogue. This
Regarding the hydrolysis of the aldehyde 2, we should could be accomplished either by enzymatic transfer
consider the somehow neglected[5,9] question of whether of a 2-acetamido-2-deoxy-b-d-galactopyranosyluronic
b-N-acetylhexosaminidases recognise the aldehyde as acid moiety onto GlcNAc or by transfer of a 2-acetami-
such or in its hydrated form as a geminal diol (see do-2-deoxy-b-d-galacto-hexodialdo-1,5-pyranosyl moi-
Scheme 1), which is the prevailing form in the water sol- ety followed by subsequent oxidation. The former meth-
ution.[18,19] The answer to this question as well as the con- od was impossible because pNP-b-GalNAcA (3) was
firmation of the other experimental results could be not accepted by b-N-acetylhexosaminidases, so we opt-
reached with the help of molecular modelling. The ed for the aldehyde 2 as a donor with GlcNAc (5) as an
modified substrates 2, 3 and 4 were docked into the ac- acceptor under the catalysis by the b-N-acetylhexosami-
tive centre of the b-N-acetylhexosaminidase from As- nidase from Talaromyces flavus CCF 2686. The desired
pergillus oryzae CCF 1066. The stability of the en- structure 6 bearing a carboxy group was obtained by in
zyme-substrate complex reflects the drop of interaction situ oxidation of the reaction mixture with NaClO2. In-
energy (steric and electrostatic contributions, Table 2).
terestingly, under the given conditions no oxidation at
The steric energy contribution is in the same range for the free anomeric position occurred and the disacchar-
all examined compounds and is considerably lower than ide 6 was isolated in 37% yield (Scheme 2).
for the standard pNP-b-GalNAc (1, Figure 1). This is
The affinity of compounds 1–4 towards two represen-
well understandable as glycosidases are known to be tative NK cell activation receptors, rat NKR-P1A, and
sensitive to any structural modifications of their sub- human CD69 proteins, was tested. Due to their good
strates. On the other hand, major changes between the availability and stability, monomeric soluble forms of
modified substrates are observed in the electrostatic these proteins, i.e., NKR-391 and CD69CWTY, which
part, in our case in the hydrogen bonding between sub- encompass the ligand-binding domains, were used for
strate and enzyme (Figures 1 and 2). In case of the sub- the tests.[20,21] To verify the identity and homogeneity
strate 2, its hydrated form exhibits stabilisation by hy- of the protein preparations, the technique of Fourier
drogen bonding with six amino acids whereas the alde- transform-ion cyclotron resonance mass spectrom-
hyde form lacks hydrogen bonding with two of them. etry[22] was employed in addition to the standard analyt-
Scheme 1. Chemoenzymatic synthesis of substrates 2 – 4.
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Table 1. Hydrolysis of substrate 2 by fungal b-N-acetylhexo-
saminidases.
Source of enzyme
2[a]
Acremonium persicinum CCF 1850
Aspergillus (A.) awamori CCF 763
A. caelatus CCF 3087
A. flavipes CCF 1895
A. flavipes CCF 3067
A. flavofurcatis CCF 3061
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 1298
A. sojae CCF 3060
A. tamarii CCF 3085
þ þ
þ þ
þ
þ
þ þ
þ
þ þ
þ þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
A. terreus CCF 2539
A. terreus USA
Figure 1. pNP-b-GalNAc (1) docked into the active site of
the b-N-acetylhexosaminidase from A. oryzae. The catalytic
glutamic acid 346 is shown in yellow, substrate 1 in violet.
The ligand is fixed by hydrogen bonding and aromatic stack-
ing of the phenol ring with Trp 482.
Fusarium oxysporum CCF 377
Hamigera avellanea CCF 2923
Chaetomium globosum CCF 430
Penicillium (P.) brasilianum CCF 2155
P. brasilianum CCF 2171
P. chrysogenum CCF 1269
P. funiculosum CCF 2985
P. melinii CCF 2440
P. multicolor CCF 2244
P. oxalicum CCF 1959
P. oxalicum CCF 2315
P. oxalicum CCF 2430
P. pittii CCF 2277
þ þ þ
þ
þ
þ þ
þ þ
þ
þ þ þ
þ
11630.1 for the native (oxidised) forms of NKR-P1A
and CD69, respectively. The biological activity of the
soluble receptors after 125I iodination was verified using
simple sugars. d-Mannose served as a negative (non-
inhibitory) control carbohydrate. GlcNAc (5), used as
þ þ þ
þ þ
þ þ þ
þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ
þ þ þ
þ þ
a reference compound, provided IC50 values of 10ꢀ5
M
(NKR-P1A) and 10ꢀ3 M (CD69), which are in accord-
ance with the previously reported values.[20,21]
P. spinulosum CCF 2159
Talaromyces (T.) flavus CCF 2573
T. flavus CCF 2686
T. ohiensis CCF 2229
T. striatus CCF 2232
All inhibition assays were performed three times in
duplicates and the results for NKR-P1 and CD69 are
given in Figures 3 and 4, respectively, as ꢀlog IC50
with standard deviations. With the monomeric NKR-
P1A, pNP-b-GalNAc (1; IC50 ¼1.3ꢁ10ꢀ6 M) had a
ten-fold higher inhibitory activity compared to the refer-
ence substance GlcNAc (5).[10a] While the oxidation of 1
into the aldehyde 2 caused only a negligible increase in
IC50, further oxidation to the uronic acid 3 resulted in
an additional ten-fold increase in the inhibitory activity
(IC50 ¼1.6ꢁ10ꢀ7 M). A similar trend was observed in
the disaccharide series: whilst a disaccharide of b-d-
GalNAc-(1!4)-d-GlcNAc had an IC50 of 1.6ꢁ10ꢀ7
M, the analogous structure 6 bearing a GalNAcA moi-
ety gave rise to a very potent disaccharide inhibitor
with an IC50 of 10ꢀ8 M.
Trichoderma harzianum CCF 2687
[a]
Hydrolysis of both the standard substrate 1 and the alde-
hyde 2 was spectrophotometrically determined as the lib-
erated p-nitrophenol: the ratio of hydrolysis rates of 2 to
1 was 21–12% (þ þ þ ), 11–7% (þ þ ), 6–1% (þ).
Table 2. Interaction energies of substrates 1 – 4 with the b-N-
acetylhexosaminidase from A. oryzae.
Substrate
Interaction energy [kJ/mol]
Total
Steric
Electrostatic
1
ꢀ242
ꢀ180
ꢀ241
ꢀ136
ꢀ131
ꢀ121
ꢀ89
ꢀ86
ꢀ94
ꢀ79
ꢀ121
ꢀ91
2 (aldehyde)
2 (diol)
3
4
With the monomeric CD69 protein, the increase in the
inhibitory potency of the uronic acid derivatives was
even more profound. Here, a gradual increase in the
IC50 values was observed by pNP-b-GalNAc (1) and
ꢀ155
ꢀ42
ꢀ52
the aldehyde 2, reaching IC50 values of 1.6ꢁ10ꢀ4
M
and 10ꢀ5 M, respectively, compared to GlcNAc (5; Fig-
ical techniques described.[20,21] This method showed the ure 4). However, the uronic acid 3 exhibited a significant
proteins to be homogenous with m/z¼11965.4 and increase with IC50 ¼10ꢀ7 M. Even more interestingly,
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Synthesis of Complex Immunoactive N-Acetyl-d-galactosaminides
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Figure 2. Substrates 2 – 4 docked into the active site of the b-N-acetylhexosaminidase from A. oryzae. A: aldehydic form of
substrate 2; B: geminal diol form of substrate 2; C: pNP-b-GalNAcA (3); D: methyl ester 4.
Scheme 2. Chemoenzymatic synthesis of disaccharide 6.
the introduction of a carboxyl moiety into a disaccharide Conclusion
of b-d-GalNAc-(1!4)-d-GlcNAc led to a remarkable
increase in IC50 of over four orders of magnitude, i.e., In this paper we have demonstrated an optimised proce-
from 1.3ꢁ10ꢀ4 M (neutral disaccharide) to 1.5ꢁ10ꢀ8 dure for the preparation of p-nitrophenyl 2-acetamido-
M (the oxidised disaccharide 6). Although the high af- 2-deoxy-b-d-galacto-hexodialdo-1,5-pyranoside
(2).
finities of certain negatively charged oligosaccharides We found that this glycoside, contrary to its derivatives
for NKR-P1A have been reported,[13] this is to the best (pNP-b-GalNAcA, 3), its methyl ester 4, was readily
of our knowledge the first systematic study of the struc- accepted by fungal b-N-acetylhexosaminidases and we
ture-activity relationship using a complete series of supported the experimental data by molecular model-
these compounds. Moreover, the very high inhibitory ling. A novel, highly immunoactive disaccharide 6 was
potency of the oxidised disaccharide 6 for CD69 (mak- prepared by a transglycosylation reaction and a subse-
ing it one of the best ligands known so far) represents quent chemical oxidation. This concept demonstrates
an entirely novel observation, which could be effectively the synthetic potential of the multi-enzyme approach
applied in the development of therapeutically useful and offers further possibilities for a selective and high
glycomimetics.[10e]
yielding preparation of oligosaccharidic structures.
Adv. Synth. Catal. 2005, 347, 997–1006
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Figure 3. Affinity of new carbohydrate ligands towards rat NK cell activation receptor, NKR-P1A, expressed in the logarith-
mic scale (ꢀlog IC50). Data are average values from three independent experiments with standard deviations indicated by
error bars.
Figure 4. Affinity of new carbohydrate ligands towards human NK cell activation receptor, CD69, expressed in the logarithmic
scale (ꢀlog IC50). Data are average values from three independent experiments with standard deviations indicated by error
bars.
chemical Corporation (Canada) and used in the form of ali-
quots (100 mL, 0.5 U/mL) in 0.02 M Na2HPO4/NaH2PO4 buffer,
Experimental Section
pH 6.0. The catalase from bovine liver (EC 1.11.1.6) was pur-
chased from Sigma. The fungal strains producing b-N-acetyl-
Enzymes
Citrate/phosphate buffer (McIlvaine) pH 5.0 was prepared by
mixing 0.1 M citric acid (24.3 mL) and 0.2 M Na2HPO4
(25.7 mL), then diluting with water to 100 mL and adjusting
the pH to 5.0. The galactose oxidase from Dactylium den-
droides (EC 1.1.3.9) was purchased from Worthington Bio-
hexosaminidases (EC 3.2.1.52) originated from the Culture
Collection of Fungi (CCF), Department of Botany, Charles
University Prague, or from the Culture Collection of the Insti-
tute of Microbiology (CCIM), Prague,[12] and were cultivated
as described previously.[8]
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Synthesis of Complex Immunoactive N-Acetyl-d-galactosaminides
FULL PAPERS
ic screening, 36 b-N-acetylhexosaminidases were tested. They
were classified according to the ratio of hydrolysis rates of
the respective modified substrate and substrate 1 assayed un-
der the same conditions and extrapolated to the same amount
of enzyme.
Analytical Methods
TLC was performed on precoated Merck silica gel DC-Alufo-
lien Kieselgel 60 F254 plates. The spots were visualised by char-
ring with 5% H2SO4 in EtOH. Analytical HPLC was carried
out on a Spectra Physics modular analytical system (San Jose,
U. S.A) consisting of an SP 8800 ternary gradient pump, an
SP 8880 autosampler and a Spectra Focus scanning UV/VIS de-
tector. A Luna C8 (2) column, 250ꢁ4.6 mm with a guard col-
umn 4ꢁ3 mm (Phenomenex, USA) was used at ambient tem-
perature with a mobile phase of MeCN: H2O, 20: 80, and a flow
rate of 0.5 mL/min. Retention times were: 14.28 min (1),
11.45 min (2) and 5.89 min (3). Compounds were detected at
Molecular Modelling
The primary sequence of the b-N-acetylhexosaminidase from
A. oryzae CCF 1066 was aligned with the known X-ray struc-
tures of the b-N-acetylhexosaminidases from Serratia marces-
cens and Streptomyces plicatus, extractedfrom the Brookhaven
Protein Database (PDB entries: 1QBA and 1HP4, respective-
ly). The sequence data are available in DDBJ/EMBL/Gene-
Bank databases (http://www.ncbi.nlm.nih.gov/) under the ac-
cession number AY091636. 3D models were generated by
Modeller6 package.[24] For model refinement and minimisa-
tion, the SYBYL package with the TRIPOS force field (TRI-
POS Associates Inc.) were used. The complete modelling in-
cluding the alignment and energy minimisation was done ex-
actly as described previously.[8] The docking of ligands was per-
formed as described earlier.[8] The non-binding interaction en-
ergy between the model and the ligands within the optimised
complex was calculated using the TRIPOS force field. This es-
timationof areal interaction energy neglects solvation and des-
olvation effects.
1
200 nm. H and 13C NMR spectra were recorded on a Varian
INOVA-400 spectrometer (399.89 MHz and 100.55 MHz, re-
spectively) at 308C in the indicated solvents. Chemical shifts
are expressed in the d scale and were measured with digital res-
olution justifying the reported values to three or two decimal
places, respectively. HMQC and HMBC readouts are accurate
at one decimal place only. Residual solvent signals DMSO-d6:
dH ¼2.50, dC ¼39.60; CD3OD: dH ¼3.33, dC ¼49.30) or internal
acetone for D2O solutions (dH ¼2.030, dC ¼30.50) were used
for referencing. The reported assignments are based on gCO-
SY, HMQC, HMBC, and 1D-TOCSY experiments. Positive-
ion MALDI mass spectra were measured on a Bruker BIFLEX
II reflectron time-of-flight mass spectrometer (Bruker Dalton-
ics, USA) equipped with a nitrogen laser (337 nm) and a grid-
less delayed extraction ion source. Ion acceleration voltage was
19 kVand the reflectron voltage was set to 20 kV. Spectra were
externally calibrated using the monoisotopic [MþH]þ ion of
angiotensin I (Sigma). A saturated solution of a-cyano-4-hy-
droxycinnamic acid (CCA) in 50% ACN/0.3% acetic acid
was used as MALDI matrix. The matrix solution (1 mL) was
premixed with a sample and loaded on the target and a droplet
(1 mL) was allowed to dry at ambient temperature. The MAL-
DI-TOF spectra were collected in reflectron mode. Positive ion
electrospray (ESI) mass spectra were recorded on an LCQDECA
ion trap mass spectrometer (Finnigan, San Jose, U. S.A) equip-
ped with a nano-ESI ion source. Spray voltage was set at 2.0 kV.
Samples dissolved in 50% MeOH were continuously infused
into the ion source via a coated microcapillary. Full scan spec-
tra were acquired over the m/z range of 50–500 Da. A 0.1%
solution of Ultramark 1621 (PCR, Inc., Gainesville, FL,
U. S. A.) in MeCN was used to calibrate the m/z scale of the in-
strument. Optical rotation was measured on a Perkin-Elmer
241 polarimeter at 589 nm.
Tests of Affinity to NK Cell Activation Receptors,
NKR-P1A and CD69[10a]
Compounds 1–6 were tested for their affinity towards two rep-
resentative NK cell activation receptors, NKR-P1A and CD69
proteins. NKR-391 protein, the major activation receptor of rat
NK cells, was expressed and purified as described previous-
ly.[10a,20] CD69CWTY protein that contained the soluble li-
gand-binding domain of the earliest activation receptor of lym-
phocytes and NK cells, CD69 antigen, was prepared as describ-
ed earlier.[21] The proteins were purified from inclusion bodies
that were dissolved in 6 M guanidine-HCl and 100 mM DTT,
and refolded by dropwise addition into a hundred times larger
volume of 50 mM Tris-HCl buffer pH 8.5 containing 0.4 M ar-
ginine, redox buffer (10 mM cysteamine and 1 mM cystamine),
1 mM PMSF, 1 mM leupeptin and 1 mM pepstatin. The folded
protein was dialysed against low salt buffer, and purified by a
combination of ion-exchange chromatography, reversed phase
separation, and gel filtration as described previously.[21] The
identity and homogeneity of the proteins were verified using
SDS-PAGE under both reducing and non-reducing conditions,
N-terminal sequencing (10 cycles of automated Edman degra-
dation), and MALDI mass spectrometry, as described previ-
ously.[21] Moreover, Fourier transform-ion cyclotron resonance
mass spectrometry was employed (APEX-Q, Bruker Dalton-
ics, Bremen, Germany).[22] The proteins were radiolabelled
with Na125I using Iodogen (Pierce, Rockville, IL, USA). Bind-
ing and inhibition assays were performed as described pre-
viously.[10a] Briefly, 96-well polyvinyl chloride microplates (Ti-
tertek Immuno Assay-Plate, ICN Flow, Irvine, Scotland) were
coated overnight at 48C with GlcNAc23BSA (50 mL, Sigma) in
TBSþC buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2
and 1 mM NaN3). Plates were blocked with 1% BSA in TBSþ
Activity Assay for b-N-Acetylhexosaminidases[23]
The reaction mixture containing substrate 1 (2 mM, starting
concentration) and b-N-acetylhexosaminidase (0.02–0.03 U/
mL) in buffer (assay volume 50 mL) was incubated in micro-
plates at 358C for 10 min. The assay was stopped by adding
0.1 M Na2CO3 (150 mL). Liberated p-nitrophenol was deter-
mined spectrophotometrically (414 nm) on a microplate read-
er Titertek Multiscanꢃ MCC/ 340 (Flow Laboratories,
McLean, USA). One unit of enzymatic activity is defined as
the amount of enzyme that releases 1 mmol of p-nitrophenol
per minute under the above conditions. The activity towards
modified substrates 2–4 was determined analogously, with
the amount of enzyme being 0.25–0.40 U/mL (for substrate
2) and 0.45–0.70 U/mL (for substrates 3 and 4). In the enzymat-
Adv. Synth. Catal. 2005, 347, 997–1006
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Pavla Fialova et al.
FULL PAPERS
C for 2 h at 48C, incubated with the concentration of the radio-
labelled protein corresponding to half of the saturating amount
and various dilutions of the inhibitors (total reaction volume
100 mL), washed three times with TBSþC and drained. Scintil-
lation solution (100 mL) was added, and the radioactivity in the
individual wells was counted by b-counting (Microbeta, Wal-
lac, Turku, Finland). All experiments were performed in dupli-
cates and the inhibition degree was calculated with regard to
the wells containing no inhibitor.
1H, H-4), 4.419 (dd, J¼0.7, 1.4 Hz, 1H, H-5), 5.040 (d, J¼
6.1 Hz, 1H, 3-OH), 5.147 (dd, J¼0.7, 4.4 Hz, 1H, 4-OH),
5.318 (d, J¼8.5 Hz, 1H, H-1), 7.244 (m, 2H, H-ortho), 7.825
(d, J¼8.9 Hz, 1H, 2-NH), 8.208 (m, 2H, H-meta), 9.500 (s,
1H, H-6); 13C NMR (DMSO-d6): d¼23.0 (2-Ac), 51.5 (C-2),
68.1 (C-4), 70.1 (C-3), 79.5 (C-5), 98.2 (C-1), 116.7 (C-ortho),
125.6 (C-meta), 141.9 (C-para), 162.1 (C-ipso), 169.7 (2-CO),
199.7 (C-6).
1
Geminal diol 2: H NMR (DMSO-d6): d¼1.802 (s, 3H, 2-
Ac), 3.274 (dd, J¼0.2, 6.6 Hz, 1H, H-5), 3.568 (ddd, J¼3.1,
6.1, 10.4 Hz, 1H, H-3), 3.912 (ddd, J¼0.2, 3.1, 4.2 Hz, 1H, H-
4), 4.033 (ddd, J¼8.5, 9.0, 10.4 Hz, 1H, H-2), 4.705 (d, J¼
4.2 Hz, 1H, 4-OH), 4.801 (d, J¼6.1 Hz, 1H, 3-OH), 4.899
(ddd, J¼6.3, 6.6, 7.3 Hz, 1H, H-6), 5.073 (d, J¼8.5 Hz, 1H,
H-1), 5.841 (d, J¼7.3 Hz, 1H, 6-OH), 5.962 (d, J¼6.3 Hz,
1H, 6-OH), 7.200 (m, 2H, H-ortho), 7.655 (d, J¼9.0 Hz, 1H,
2-NH), 8.178 (m, 2H, H-meta); 13C NMR (DMSO-d6): d¼
23.0 (2-Ac), 51.7 (C-2), 67.4 (C-4), 71.1 (C-3), 77.8 (C-5), 88.2
(C-6), 99.1(C-1), 116.7 (C-ortho), 125.6 (C-meta), 141.7 (C-
para), 162.5 (C-ipso), 169.6 (2-CO); MS (MALDI-TOF): m/
z¼363 [MþNa]þ, 341 [MþH]þ, 323.
Oxidation by the Galactose Oxidase from Dactylium
dendroides in a Batch Reactor with Bubble-Free
Aeration[4c]
Oxidation of p-nitrophenyl 2-acetamido-2-deoxy-b-d-galacto-
pyranoside (1) was performed in a batch reactor with bubble-
free aeration.[4c] The mixture of glycoside
1 (140 mg,
0.409 mmol), the galactose oxidase from Dactylium dendroides
(100 U) and the catalase from bovine liver (40,500 U) in 0.02 M
Na2HPO4/NaH2PO4 buffer pH 6.0 (100 mL) was pumped
through a thin-walled silicon tube [Tygonꢃ 3350 (P-lab, CZ),
wall thickness 0.8 mm, i.d. 4 mm, length 600 mm] placed in
an oxygen-storage flask with an oxygen pressure of 50 kPa.
Both the inlet and the outlet of the tube were air-tightly con-
nected to a batch reactor (200 mL) where the reaction mixture
was slowly stirred. The reaction was monitored by TLC
(propan-1-ol:H2O:NH3 aq., 7:2:1) and HPLC and stopped af-
ter 5 h by freezing. Lyophilisation gave the crude product 2,
which was further purified.
p-Nitrophenyl 2-acetamido-2-deoxy-b-D-galactopyranosi-
duronic acid (3): The starting material
2
(112 mg,
0.329 mmol) was suspended in water (41 mL) under stirring
at 08C and NaClO2 (82 mg, 0.907 mmol) was added in several
portions during 1 h. The reaction was monitored by TLC
(AcOEt:MeOH:H2O, 7:3:0.5) and HPLC. It was stopped after
7 h by evaporation under vacuum to dryness. The crude com-
pound 3 was purified by column chromatography on Merck
silica gel 60 (40–63 mm) (AcOEt:MeOH:H2O, 7:3:1) afford-
ing compound
3 as a yellowish honey; yield: 97 mg
1
(0.272 mmol, 83%); [a]2D0: ꢀ11.98 (c 0.30, water). H NMR
(D2O): d¼1.788 (s, 3H, 2-Ac), 3.700 (dd, J¼3.4, 10.9 Hz, 1H,
H-3), 4.009 (dd, J¼8.4, 10.9 Hz, 1H, H-2), 4.042 (d, J¼
1.3 Hz, 1H, H-5), 4.096 (dd, J¼1.3, 3.4 Hz, 1H, H-4), 4.996
(d, J¼8.4 Hz, 1H, H-1), 6.976 (m, 2H, H-ortho), 8.008 (m,
2H, H-meta); 13C NMR (D2O): d¼22.3 (2-Ac), 52.1 (C-2),
69.5 (C-4), 70.9 (C-3), 75.7 (C-5), 99.2 (C-1), 116.9 (2ꢁC-or-
tho), 126.3 (2ꢁC-meta), 142.8 (C-para), 162.4 (C-ipso), 174.6
(C-6), 175.4 (2-CO); MS (ESI): m/z¼401 [Mþ2 Na]þ, 379
[MþNa]þ, 357 [MþH]þ.
Analytical Transglycosylation Reactions Catalysed by
b-N-Acetylhexosaminidases
Substrate 2 (4 mg, 0.012 mmol) and 2-acetamido-2-deoxy-d-
glucopyranose (5; 26 mg, 0.118 mmol) were dissolved in cit-
rate/phosphate buffer pH 5.0 (392 mL). The reaction was start-
ed by the addition of b-N-acetylhexosaminidase (0.75 U) and
was incubated at 378C with shaking for 24 h. Aliquots were an-
alysed by TLC (propan-1-ol:H2O:NH3 aq., 7:2:1).
Methyl (p-nitrophenyl 2-acetamido-2-deoxy-b-d-galacto-
pyranosid)uronate (4): Compound 3 (45 mg, 0.126 mmol)
was dissolved in dry MeOH (25 mL) and stirred with Dowex
50W-X2 in Hþ cycle (0.5 mL of the suspension) for 15 min.
Dowex was then filtered off and the reaction mixture was con-
centrated to a volume of 10 mL. 1 M CH2N2 in dry MeOH (ap-
proximately 2 mL) was added dropwise until the reaction
reached equilibrium (did not colour off any more). TLC
(AcOEt:MeOH:H2O, 7: 3: 1) confirmed complete conversion.
Immediately, the reaction mixture was evaporated under vac-
uum to dryness and the crude compound 4 was purified by col-
umn chromatography on silica gel (gradient of CH2Cl2:MeOH
12:1 to 6:1). Pure compound 4 was obtained as a white solid;
yield: 19 mg (0.051 mmol, 41%); [a]2D0: ꢀ36.58 (c 0.20, water).
1H NMR (CD3OD): d¼1.993 (s, 3H, Ac), 3.808 (s, 3H,
OMe), 3.905 (dd, J¼10.8, 3.3 Hz, 1H, H-3), 4.248 (dd, J¼
10.8, 8.5 Hz, 1H, H-2), 4.269 (dd, J¼3.3, 1.4 Hz, 1H, H-4),
4.574 (d, J¼1.4 Hz, 1H, H-5), 5.312 (d, J¼8.5 Hz, 1H, H-1),
7.225 and 8.222 (AA’BB’, SJ¼9.3 Hz, 4H, pNP); 13C NMR
(CD3OD), HMQC and HMBC readouts: d¼23.1 (Ac), 52.8
(OMe), 53.7 (C-2), 70.7 (C-4), 71.9 (C-3), 75.8 (C-5), 99.9 (C-
Synthesis of Substrates
p-Nitrophenyl 2-acetamido-2-deoxy-b-d-galacto-hexodialdo-
1,5-pyranoside (2): Compound 2 was prepared by oxidation
catalysed by the galactose oxidase from Dactylium dendroides
in a batch reactor with bubble-free aeration from the starting
material 1. After lyophilisation, the reaction mixture was dis-
solved in water (30 mL), centrifuged (Beckman Model JS-21
centrifuge, Beckman Coulter, Inc., U. S. A.; 15,000 rpm,
15 min) and the supernatant was purified by column chroma-
tography on Dowex 50W-X8 (Sigma) in Ca2þ cycle with water
as a mobile phase and a flow rate of 60 mL/h. The pure com-
pound 2 was obtained as a white solid; yield: 124 mg
(0.364 mmol; 89%); [a]2D0: ꢀ138.78 (c 0.21, water). According
to NMR, compound 2 is a mixture of the geminal diol and
the free aldehyde (diol/aldehyde¼4.26).
Free aldehyde 2: 1H NMR (DMSO-d6): d¼1.808 (s, 3H, 2-
Ac), 3.722 (ddd, J¼3.4, 6.1, 10.4 Hz, 1H, H-3), 4.064 (ddd,
J¼8.5, 8.9, 10.4 Hz, 1H, H-2), 4.163 (ddd, J¼1.4, 3.4, 4.4 Hz,
1004
ꢂ 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Adv. Synth. Catal. 2005, 347, 997–1006

Synthesis of Complex Immunoactive N-Acetyl-d-galactosaminides
FULL PAPERS
1), 117.8 (2ꢁC-ortho), 126.7 (2ꢁC-meta), 144.2 (C-para),
163.7 (C-ipso), 170.2 (C-6), 174.3 (2-CO); MS (ESI): m/z¼
393 [MþNa]þ.
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2-Acetamido-2-deoxy-b-d-galactopyranosyluronic
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(1!4)-2-acetamido-2-deoxy-d-glucopyranose (6): Substrate
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1.845 (s, 3H, 2’-Ac), 3.366 (dd, J¼8.3, 10.0 Hz, 1H, H-4),
3.437 (dd, J¼4.9, 12.1 Hz, 1H, H-6u), 3.557 (dd, J¼2.2,
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(C-6), 69.33 (C-4’), 69.53 (C-3), 70.11 (C-5), 71.06 (C-3’),
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(C-3’), 72.86 (C-3), 74.79 (C-5), 75.47 (C-5’), 80.18 (C-4),
95.13 (C-1), 101.72 (C-1’), 174.32 (C-6’), 174.99 (2-CO, 2’-
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