Synthesis of β-(1→6)-linked N-acetyl-d-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B

Article abstract of DOI:10.1016/j.carres.2011.03.029

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-d-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were sy

Full text of DOI:10.1016/j.carres.2011.03.029

Carbohydrate Research 346 (2011) 1445–1453
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of b-(1?6)-linked N-acetyl-D-glucosamine oligosaccharide
substrates and their hydrolysis by Dispersin B
Anikó Fekete a, Anikó Borbás a, Gyöngyi Gyémánt b, , Lili Kandra b, Erika Fazekas b, Narayanan Ramasubbu c,
⇑
Sándor Antus a,d
a Research Group for Carbohydrates of the Hungarian Academy of Sciences, PO Box 94, H-4010 Debrecen, Hungary
b Department of Inorganic and Analytical Chemistry, University of Debrecen, PO Box 21, H-4010 Debrecen, Hungary
c Department of Oral Biology, University of Medicine and Dentistry of New Jersey, 185 South Orange Ave, Newark, NJ 07103, United States
d Department of Organic Chemistry, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a b-hexosaminidase exhibiting biofilm
Received 21 January 2011
Received in revised form 6 March 2011
Accepted 16 March 2011
Available online 23 March 2011
detachment activity. A series of b-(1?6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with
degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was
studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was
applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been
ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-
hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed
that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB
on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the
dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB.
The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also
examined using mutant enzymes.
Keywords:
Biofilm
Dispersin B
b-(1?6)-Oligoglucosamine
Synthesis
Mutation
Substrate specificity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
glucosamines consisting of 5, 7, 9 and 11 monomers, using a con-
vergent blockwise approach.5 Homologous cyclic oligo-(1?6)-b-
D-
Glucosamines are common components of many biologically
important oligosaccharides and frequently encountered in glyco-
proteins and glycolipides.1 Isolation of glucosamine oligosaccha-
rides from natural sources is a difficult task, therefore, the
chemical synthesis would be important to provide substrates for
biochemical studies. Oligo-b-(1?6)-glucosamine derivatives at-
tract growing interest being fragments of the bacterial surface
polysaccharides. However, only a few papers dealing with the
preparation of b-(1?6)-linked glucosamine oligosaccharides have
been published to date.2–8 Yang and coworkers reported the first
glucosamines consisting of two to seven residues have been ob-
tained by intramolecular glycosylation of the linear oligosaccha-
rides.6 Unprotected N-acetyl-
D-glucosamine (GlcNAc) could be
also utilized for regio- and stereoselective synthesis of b-(1?6)-
linked oligomers by means of an acid reversion reaction in hydro-
gen fluoride.7,8
These synthetic oligosaccharides represent fragments of the
bacterial surface polysaccharide produced by numerous bacterial
pathogens such as Staphylococci. The most notable staphylococci,
Staphylococcus aureus and Staphylococcus epidermidis bring about
infections related to colonization of medical implants and forma-
tion of biofilms. Biofilms formed on different medical devices are
one of the most serious problems of present-day clinical practice.9
According to the National Institute of Health (US) 80% of all infec-
tions are biofilm related.10 The treatment of biomaterial-related
infections has not been fully developed, but enzymatic ap-
proaches are promising for both erasing a developed biofilm and
prevention its formation.11,12 A major component of the biofilm
synthesis of (1?6)-b-D
-glucosamine hexa- and nonasaccharides2
using isopropyl thioglycosides as suitable donors in all coupling
steps. Efficient one-pot syntheses of tri- and tetraglucosamines
were carried out by Fridman et al. exploiting the different
reactivity of N-trichloroethoxycarbonyl- and N-phthaloyl-
protected glucosamine donors.3 Melean et al. applied glucosamine
trichloroacetimidates and phosphates for the construction of
b-(1?6)-oligoglucosamines in solution and solid-phase.4 Gening
et al. synthesized a series of 3-b-acetamidopropyl oligo-b-(1?6)-
matrix is a poly-b-D-(1?6)-N-acetyl-glucosamine (PNAG) that
mediates intercellular adhesion.13,14
Kaplan et al. have reported15 that Dispersin B (DspB, EC
3.2.1.52) a soluble b-N-acetylglucosaminidase originated from
⇑
Corresponding author. Tel.: +36 52 512900; fax: +36 52 518660.
E-mail address: gyemant@science.unideb.hu (G. Gyémánt).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.029

1446
A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
periodontal pathogen Aggregatibacter actinomycetemcomitans16
was able to disperse and detach biofilms produced by S. epidermi-
dis. On the basis of biofilm-releasing activity of DspB the authors
suggested that this enzyme could be used as an antibiofilm agent
to remove S. epidermidis biofilms from medical devices. Combina-
tion of Dispersin B and antiseptic triclosan showed synergistic
broad-spectrum antibiofilm and antimicrobial activity against S.
aureus, S epidermidis and Escherichia coli.17
DspB belongs to family 20 glycoside hydrolases. In general,
these hydrolases exhibit broad substrate specificity, but DspB,
according to our earlier ligand docking and hydrolysis experi-
ments, appears to be specific to a linear polymer of b-(1?6)-linked
group by acidic hydrolysis (2?3?4) afforded acceptor 4 in an
overall 65% yield. Probably, acyl migration from O-4 to O-6 under
the used acidic conditions for detritylation could cause this moder-
ate yield.21 Then chloroacetyl group, as a temporary protecting
group, was introduced with chloroacetyl chloride in dichlorometh-
ane in the presence of pyridine at ꢀ10 °C to give derivative 5 in
good yield (75%) (Scheme 1).
After bromination of 5 with bromine at room temperature the
obtained bromo sugar without purification was coupled with
acceptor 4 using AgOTf promotion to give the disaccharide 6 in
77% yield. Treatment of 6 with thiourea gave disaccharide 7 which
was used as an acceptor for preparation of trisaccharide 9 by its
N-acetyl-
D
-glucosamine (GlcNAc) and removes the terminal
coupling with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-glu-
GlcNAc moiety from the non-reducing end.18 For these studies
b-(1?6)-linked GlcNAc oligomers with DP 2, 4 and 6 were synthe-
sized as p-methoxyphenyl glycosides. The results suggest that b-
(1?6)-linked GlcNAc oligomer bound to the active site better than
b-(1?4)-linked GlcNAc oligosaccharide and DspB is an exo-acting
enzyme. Unfortunately, the beta-linked p-methoxyphenyl agly-
cone of the hydrolysis products can be further hydrolysed by DspB
disturbing the precise HPLC measurements. In addition, due to the
applied blockwise approach only the even numbered oligomers
were available.
copyranosyl bromide (8)22 (Scheme 2).
Disaccharide 6 was also converted to glycosyl bromide whose
coupling with disaccharide acceptor 7 afforded tetrasaccharide
10 in 75% yield.
Since the yield of the formation of the tetrasaccharide 10 was
higher than in the case of trisaccharide 9, we planned to use 1+4
block synthesis for the preparation of pentasaccharide 12 instead
of 2+3. Thus, synthesis of pentasaccharide 12 was achieved starting
from compound 10 in similar manner (10?11?12) as the prepa-
ration of trisaccharide 9 (Scheme 3).
Here we present the synthesis of a novel thiophenyl glycoside
series of b-(1?6)-linked oligosaccharides and its application to
further delineate the substrate specificity of DspB. Since the thio-
glycosidic bond is stable to enzymatic hydrolysis the reducing
end products bear always a chromophore group allowing the
detection of the substrate and the reducing end hydrolysis prod-
ucts with UV detection after HPLC separation.
The importance of aromatic amino acids at the active centre of
DspB has been studied by Manuel and coworkers using mutant en-
zymes.19 Mutation of W237 completely abolished the hydrolytic
Deprotection of oligosaccharides 6, 9, 10 and 12 by ethylenedi-
amine in ethanol, followed by acetylation afforded 13, 14, 15 and
16 in overall 83%, 81%, 73%, and 71%, yields, respectively (Scheme
4). We performed peracetylation after the removal of the phthaloyl
groups since dephthaloylation resulted in a rather complex reac-
tion mixture and the isolation of the fully acetylated derivatives
was easier from it by silica gel column chromatography than the
selectively N-acetylated derivatives.
Finally, de-O-acetylation of 13–16 by Zemplén transesterifica-
tion gave the intended b-(1?6)-linked GlcNAc oligosaccharides
17, 18, 19 and 20 (Scheme 4) which were tested as substrates for
DspB as follows.
activity on 4-nitrophenyl N-acetyl-b-D-glucosaminide (PNP-b-Glc-
NAc) substrate but showed a low biofilm detachment activity.
Mutation of Y187 and Y278 amino acids decreased the hydrolytic
activity measured on both PNP-b-GlcNAc and PNAG substrate.
Activity of mutant enzymes on the new oligomer substrate series
was also studied.
2.2. Hydrolysis of thioglycosides catalysed by Dispersin B
Substrate specificity of DspB was studied separately on each
thiophenyl glycoside 17– 20. Product analysis was carried out by
HPLC resulted in base-line separation of 17–20 and phenyl 2-
2. Results and discussion
deoxy-2-acetamido-1-thio-b-D
-glucopyranoside (21)23 as shown
2.1. Synthesis of substrate series
Fig. 4 in Section 3.
Figure 1 shows the change of product distribution of thiophenyl
glycoside substrates during 4 h incubation with DspB. These dia-
grams demonstrate that hydrolysis occurs step-by-step from the
non-reducing end of the substrates leading to the formation of
shorter chromophore-containing reducing end products. Dimer
substrate gives monomer product, dimer and monomer formed
from trimer substrate, while tetramer hydrolysis results in trimer,
dimer and monomer thioglycoside products. Product distribution
of pentamer exhibits the most interesting profile; while the
amount of the tetrasaccharide 19, after a short increasing, is prac-
tically constant, the quantity of trimer 18 and dimer 17 increase
linearly and the monomer appears after a longer incubation time.
Effect of the DP on hydrolysis rate shows that b-(1?6)-linked
GlcNAc thioglycosides longer than the dimer 17 are good
Fully protected 2-phthalimido-containing thioglucosides (5, 6
and 10) equipped with monochloroacetate ester as temporary pro-
tecting group and acetate esters as permanent protecting groups
were utilized for the synthesis of the desired b-(1?6)-linked Glc-
NAc oligosaccharides. These key-intermediates were transformed
either to a donor by bromination or to an acceptor by selective lib-
eration of the terminal primary hydroxyl group. The 6-O-chloro-
acetate proved to be stable to both bromination and the
glycosylation reaction but could be readily removed with thiourea
in an assisted cleavage.
The known phenyl 2-deoxy-2-phthalimido-1-thio-b-D-glucopy-
ranoside (1)20 was resorted as starting compound. Tritylation of 1
gave compound 2, whose acetylation followed by removal of trityl
RO
AcO
AcO
RO
HO
HO
CAO
O
O
O
AcO
ii
iv
SPh
SPh
SPh
AcO
NPhth
NPhth
NPhth
120 R=H
2 R=Tr
3 R=Tr
4 R=H
5
iii
i
Scheme 1. Reagents and conditions: (i) triphenylmethyl chloride, DMAP, Py, rt, 24 h; (ii) Ac2O, Py, rt, 24 h; (iii) 80% aq acetic acid, 50 °C, 3 h, 65% for 4 for three steps; (iv)
chloroacetyl chloride, Py, DCM, ꢀ20 °C?rt, 75%.

A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
1447
AcO
O
AcO
Br
AcO
RO
AcO
AcO
NPhth
O
i
O
AcO
AcO
O
822
iii
O
Ac
5
O
4
AcO
SPh
SPh
NPhth
AcO
NPhth
NPhth
3
6 R=CA
7 R=H
9
ii
Scheme 2. Reagents and conditions: (i) (a) Br2, DCM, rt, 1 h; (b) AgOTf in toluene; collidine, DCM, ꢀ74 °C?rt, 24 h, 77%; (ii) thiourea, DCM–MeOH, 50 °C, 24 h, 76%; (iii) AgOTf
in toluene; collidine, DCM, ꢀ74 °C?rt, 24 h, 53%.
The conversions determined on substrates with different DP are
summarised in Table 1.
O
O
AcO
AcO
R
Ac
i
822
O
O
AcO
6
SPh
AcO
SPh
iii
7
Results show that Y187A mutation decreased hydrolytic activ-
ity and changed substrate specificity. Highest conversion was mea-
sured on pentamer substrate, hydrolysis rate of tetramer was
lower (ꢁ80% of 20), followed by trimer (46% of 20) and dimer
showed the lowest conversion (<10% of 20) in case of wild type en-
zyme. In contrast, the conversions were similar on trimer and di-
mer in Y187A catalysed reaction, 85% and 80% of 20, respectively.
Our results verify the presence of multiple subsites, since mutation
of Tyr187 (an amino acid farther away form the cleavage site)
influenced rather the hydrolysis of tetramer and pentamer than
that of trimer or dimer.
In conclusion, our measurements confirm the applicability of
new synthetic compounds 17–20 for investigation of Dispersin B
and its mutants. Substrate series are suitable for activity measure-
ment, studying the effect of mutation and exploring the active site
composition/structure. These experiments will be extended to
study new mutants for detailed understanding of enzyme function.
NPhth
NPhth
4
5
10 R=CA
11 R=H
12
ii
Scheme 3. Reagents and conditions: (i) (a) Br2, DCM, rt, 1 h; (b) AgOTf in toluene;
collidine, DCM, ꢀ74 °C?rt, 24 h, 72%; (ii) thiourea, DCM–MeOH, 50 °C, 24 h, 76%;
(iii) AgOTf in toluene; collidine, DCM, ꢀ74 °C?rt, 24 h, 72%.
substrates and linear decrease in peak area% of substrate versus
time can be observed using HPLC product distribution measure-
ments for the oligomers (Fig. 2). After 4 h incubation time the con-
version of dimer 17 was very low (3.6%), while hydrolysis of trimer
18, tetramer 19 and pentamer 20 resulted in higher conversion
19.3%, 33.2%, and 39.6%, respectively. However, it is interesting
that the reaction rates (slopes of the curves) for the hydrolysis of
tetramer 19 and pentamer 20 were similar. Reaction rate increased
with degree of polymerisation until tetramer, but the fifth GlcNAc
unit in case of pentamer did not result in further increase of veloc-
ity. These results indicate that effective binding requires at least
four glucosamine residues in substrate molecule. The points of
pentamer hydrolysis deviate from the straight line above 30% con-
version due to the decrease of substrate concentration.
3. Experimental
3.1. Chemistry
Optical rotations were measured at room temperature with a
Perkin–Elmer 241 automatic polarimeter in CHCl3. TLC was per-
formed on Kieselgel 60 F254 (Merck) with detection by charring
with 50% aqueous sulfuric acid. Column chromatography was per-
formed on Silica Gel 60 (Merck 63–200 mesh). The 1H (360 and
400 MHz) and 13C NMR (90.54 and 128 MHz) spectra were re-
corded with Bruker AM-360 and Bruker DRX-400 spectrometers.
Internal references: TMS (0.000 ppm for 1H), CDCl3 (77.00 ppm
for 13C for organic solutions). The 1H and 13C NMR assignments
have been established from 1D NMR spectra and the proton-signal
assignments were supported by analysis of two-dimensional
1H–1H correlation spectra (COSY), as well as the carbon-signal
assignments by two-dimensional 13C-1H correlation maps (HET-
COR). MALDI TOF-MS analyses of compounds were carried out in
the positive reflectron mode using a BIFLEX III mass spectrometer.
Elemental analyses were performed at the analytical laboratories
in Debrecen.
2.3. Effect of mutation of aromatic amino acids at the active
centre of DspB
Hydrolytic activity of Y187A, Y278A and W237A mutants was
also studied on thiophenyl glycosides 17–20 as substrates and
compared to hydrolytic activity of wild type enzyme. To compare
the reaction rates of enzymes on pentasaccharide substrate 20
the conversions were plotted versus reaction time (Fig. 3).
We have found that wild type enzyme is a much more active on
pentamer substrate 20 than Y187A mutant and Y278A mutant
shows a low activity. W237A mutant was completely inactive on
pentamer substrate, therefore, it was not represented in the graph.
Our results confirm the conclusion of Manuel and coworkers,19
who find the mutant to be inactive on monomer and polymer sub-
strate, and suggested that W237 is an important participant of
hydrophobic substrate binding pocket of DspB.
O
O
O
H
Ac
R
O
O
O
i
ii
AcO
AcO
AcO
AcO
HO
SPh
SPh
SPh
HO
NPhth
NHAc
NHAc
n
n
n
17 n=2
13 n=2
14 n=3
15 n=4
16 n=5
6 R=CA, n=2
9 R=Ac, n=3
18 n=3
19 n=4
20 n=5
10 R=CA, n=4
12 R=Ac, n=5
Scheme 4. Reagents and conditions: (i) (a) ethylenediamine, EtOH, 70 °C, 24 h; (b) Ac2O, Py, rt, 24 h, 83% for 13 for, 81% for 14, 73% for 15, 71% for 16; (ii) NaOMe, MeOH, 86%
for 17, 86% for 18, 88% for 19, 85% for 20.

1448
A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
Area
1000
Area
600
800
600
400
200
0
400
200
0
0
50
100
150
200
250
0
50
100
150
200
250
Time (min)
Time (min)
Area
Area
400
250
200
150
100
50
300
200
100
0
0
0
50
100
150
200
250
0
50
100
150
200
250
Time (min)
Time (min)
Figure 1. HPLC product distribution of DspB catalysed hydrolysis of thiophenyl glycosides. Measured area for: x monomer 21, s dimer 17, j trimer18,
pentamer 20.
D
tetramer 19, d
Area %
100
40
80
60
20
40
20
0
0
200
400
600
0
Time (min)
0
50
100
150
200
250
Time (min)
Figure 3. Conversion of pentamer substrate 20 hydrolysis catalysed by: . DspB, +
Y187A and Y278A mutants. Data of fitted curves: DspB m = 0.17 b = 13.76
r = 0.997; Y187A m = 0.073 b = 10.5 r = 0.999; Y278A m = 0.013 b = 10.3 r = 0.973.
h
Figure 2. Time course of hydrolysis reaction on thiophenyl glycosides 17, 18, 19
and 20 catalysed by DspB. [S] = 0.4 mM; [E] = 2
19, d 20.
lM; pH 5.9; T = 25 °C; s 17, j 18, 4
Table 1
Conversion% after 6 h incubation of substrates in hydrolysis reaction catalysed by
DspB and mutants
3.1.1. Typical procedure A for glycosylation reaction I
To a solution of starting material (2.66 mmol) in dry CH2Cl2
Substrate
Enzyme
Y187A
(10 mL) bromine (150 lL, 3.07 mmol) was added at room temper-
ature. After stirring for 1 h the reaction mixture was concentrated
Wild type
Y278A
and then was co-evaporated with toluene. To a solution of crude
Dimer 17
Trimer 18
Tetramer 19
Pentamer 20
4.3
23.1
39.8
49.8
2.8
14.4
15.4
18.0
0
bromo sugar (2.66 mmol) and acceptor (1.77 mmol) in dry CH2Cl2
2.6
4.36
7.7
0
(10 mL) were added collidine (500
lL, 2.79 mmol) and 4 ÅA molec-
ular sieves and cooled to ꢀ74 °C. After stirring for 30 min at

A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
1449
3.1.6. Phenyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-
D
-glucopyranoside (4)
To solution of phenyl 2-deoxy-2-phthalimido-1-thio-
-glucopyranoside 120 (3 g, 7.48 mmol) in pyridine (50 mL)
a
b-
D
triphenylmethyl chloride (5.22 g, 18.76 mmol) and 4-dimethyl-
aminopyridine (62 mg) were added. After stirring for 1 day at room
temperature the mixture was concentrated and then was co-evap-
orated with toluene. To a solution of crude product 2 (7.48 mmol)
in pyridine (30 mL) was added Ac2O (15 mL). After stirring for
1 day at room temperature the mixture was concentrated. The res-
idue was diluted with CH2Cl2, extracted with aq 1 M HCl and satu-
rated NaHCO3 solution, dried and concentrated. Compound 3 thus
obtained was used in the next step without further purification. A
solution of compound 3 (7.48 mmol) in 80% aq acetic acid (100 mL)
was stirred at 50 °C for 3 h. Then the mixture was concentrated and
then was co-evaporated with toluene. The crude product was puri-
fied by silica column chromatography (CH2Cl2–acetone 95:5) to
Figure 4. HPLC separation of 1 mM standard mixture of GlcNAc oligomers 17–20
and monomer 21.
yield 4 (2.4 g, 65%) as a colourless syrup. [
a]
D
+65.8 (c 0.12 in
CHCl3); 1H NMR (CDCl3): d 7.92–7.73 (m, 4H, Ar), 7.45–7.24 (m,
5H, Ar), 5.85 (dd, 1H, J3,4 9.5 Hz, H-3), 5.77 (d, 1H, J1,2 10.5 Hz, H-
1), 5.11 (t, 1H, J4,5 9.5 Hz, H-4), 4.36 (t, 1H, J2,3 10.5 Hz, H-2),
3.84–3.61 (m, 3H, H-5, H-6, H-60), 2.41 (s, 1H, OH), 2.05 and 1.87
(2s, 6H, 2COCH3); 13C NMR (CDCl3): d 170.06 (2COCH3), 82.90
(C-1), 78.22, 71.41 and 68.94 (C-3, C-4, C-5), 61.49 (C-6), 53.64
(C-2), 20.59 and 20.35 (2COCH3). Anal. Calcd for C24H23NO8S:
C, 59.37; H, 4.77. Found: C, 59.42; H, 4.69.
ꢀ74 °C, silver triflate (927 mg, 3.48 mmol) dissolved in toluene
was added. Then the reaction mixture was left to attain room tem-
perature over night. The mixture was diluted with CH2Cl2 and fil-
tered through Celite. The filtrate was washed with 10% aq
Na2S2O3 and water, dried and concentrated. The crude product
was purified by silica column chromatography.
3.1.2. Typical procedure B for dechloroacetylation
3.1.7. Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-
To a solution of starting material (1.03 mmol) in dry CH2Cl2
phthalimido-1-thio-b-
To a solution of compound 4 (500 mg, 1.03 mmol) in dry CH2Cl2
(10 mL) pyridine (100 L, 1.24 mmol) and chloroacetyl chloride
(99
D-glucopyranoside (5)
(10 mL) pyridine (100
(99
30 min the reaction was quenched with methanol (0.5 mL) diluted
with CH2Cl2, extracted with aq 1 M HCl and saturated NaHCO3
solution, dried and concentrated. The crude product was purified
by silica column chromatography.
lL, 1.24 mmol) and chloroacetyl chloride
L, 1.24 mmol) were added at ꢀ10 °C. After stirring for
l
l
l
L, 1.24 mmol) were added at ꢀ10 °C. After stirring for
30 min the reaction was quenched with methanol (0.5 mL) diluted
with CH2Cl2, extracted with aq 1 M HCl and saturated NaHCO3
solution, dried and concentrated. The crude product was purified
by silica column chromatography (hexane–acetone 3:2) to yield
3.1.3. Typical procedure C for glycosylation reaction II
5 (435 mg, 75%) as a colourless syrup. [a]D +52.23 (c 0.43 in CHCl3);
To a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl bromide (8)22 (174 mg, 0.35 mmol) and acceptor
(0.23 mmol) in dry CH2Cl2 (5 mL) collidine (66 L, 0.35 mmol)
D-
1H NMR (CDCl3): d 7.91–7.71 (m, 4H, Ar), 7.43–7.25 (m, 5H, Ar),
5.81 (dd, 1H, J3,4 9.5 Hz, H-3), 5.75 (d, 1H, J1,2 10.5 Hz, H-1), 5.13
(t, 1H, J4,5 9.5 Hz, H-4), 4.42–4.30 (m, 3H, H-2, H-6, H-60), 4.10 (s,
2H, COCH2Cl), 3.97–3.90 (m, 1H, H-5), 2.03 and 1.85 (2s, 6H,
2COCH3); 13C NMR (CDCl3): d 169.95, 169.41 and 166.93 (CO),
82.95 (C-1), 75.54, 71.38 and 68.47 (C-3, C-4, C-5), 63.59 (C-6),
53.42 (C-2), 40.58 (COCH2Cl), 20.50 and 20.28 (2COCH3). Anal.
Calcd for C26H24ClNO9S: C, 55.57; H, 4.30. Found: C, 55.44; H, 4.42.
l
and 4 Å molecular sieves were added and cooled to ꢀ74 °C. After
stirring for 30 min at ꢀ74 °C, silver triflate (120 mg, 0.46 mmol)
dissolved in toluene was added. Then the reaction mixture was left
to attain room temperature over night. The mixture was diluted
with CH2Cl2 and filtered through Celite. The filtrate was washed
with 10% aq Na2S2O3 and water, dried and concentrated. The crude
product was purified by silica column chromatography.
3.1.8. Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-
phthalimido-b-
deoxy-2-phthalimido-1-thio-b-
D
-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-
3.1.4. Typical procedure D for dephthaloylation and
peracetylation of the obtained amine
D-glucopyranoside (6)
Compound 5 (1.5 g, 2.66 mmol) was reacted with bromine and
then the obtained bromo sugar was coupled with acceptor 4
according to typical procedure A. The crude product was purified
by silica column chromatography (hexane–acetone 1:1) to yield 6
To a solution of starting material (0.18 mmol) in ethanol
(15 mL) was added ethylene diamine (5 equiv per phthaloyl group
of starting material). After stirring for 1 day at reflux temperature
the mixture was concentrated. The residue was dissolved in pyri-
dine (10 mL) and acetic anhydride (5 mL) was added. After stirring
for 1 day at room temperature the mixture was concentrated and
then was co-evaporated with toluene. The residue was diluted
with CH2Cl2, washed with water, dried and concentrated. The
crude product was purified by silica column chromatography.
(1.28 g, 77%) as a colourless syrup. [
a] +46.76 (c 0.20 in CHCl3);
D
1H NMR (CDCl3): d 7.86–7.61 (m, 8H, Ar), 7.32–7.23 (m, 5H, Ar),
5.75 (dd, 1H, J2 ,3 10.5 Hz, H-30), 5.70 (dd, 1H, J3,4 9.5 Hz, H-3),
0
0
5.58 (d, 1H, J1,2 10.5 Hz, H-1), 5.54 (d, 1H, J1 ,2 8.5 Hz, H-10), 5.15
0
0
(t, 1H, J4 ,5 9.5 Hz, H-40), 4.86 (t, 1H, J4,5 9.5 Hz, H-4), 4.41–4.28
(m, 3H, H-20, H-6a0, H-6b0), 4.20 (t, 1H, J2,3 10.5 Hz, H-2), 4.20 (s,
2H, COCH2Cl), 3.92–3.80 (m, 3H, H-5, H-50, H-6a), 3.69 (dd, 1H,
J5,6b 6.5 Hz, J6a,6b 10.5 Hz, H-6b), 2.06, 1.94, 1.87 and 1.77 (4s,
12H, 4COCH3); 13C NMR (CDCl3): d 170.01, 169.94, 169.45, 169.39,
167.63, 167.14 and 166.75 (CO), 98.24 (C-10), 82.23 (C-1), 76.84
(C-5), 71.66 (C-50), 71.34 (C-3), 70.59 (C-30), 69.25 (C-4), 68.58 (C-
40), 69.13 (C-6), 63.32 (C-60), 54.27 (C-20), 53.32 (C-2), 40.70
0
0
3.1.5. Typical procedure E for de-O-acetylation
To a solution of starting material (0.13 mmol) in methanol
(15 mL) catalytic amount of NaOMe (pH ꢁ8) was added. After stir-
ring for 1 day at room temperature the mixture was diluted with
water, neutralized with Amberlite IR-120 H+ ion-exchange resin,
filtered and concentrated. The residue was lyophilized.

1450
A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
(COCH2Cl), 20.57, 20.49, 20.36 and 20.29 (4COCH3). Anal. Calcd for
44H41ClN2O17S: C, 56.38; H, 4.41. Found: C, 56.47; H, 4.50.
8.5 Hz, H-100), 5.18 (t, 1H, J3
9.5 Hz, H-4000), 4.92 (t, 1H, J3 ,4
0
0
000,4000
00 00
C
9.5 Hz, H-40), 4.81 (dd, 1H, J3 ,4 8 Hz, H-400), 4.79 (t, 1H, J4,5 10 Hz,
000,6b000
H-4), 4.45 (dd, 1H, J6a
12 Hz, J5
5 Hz, H-6a000), 4.39–4.33
000,6a000
3.1.9. Phenyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-
1-thio-b- -glucopyranoside (7)
Compound 6 (0.9 g, 0.96 mmol) was treated with thiourea
according to typical procedure B. The crude product was purified
by silica column chromatography (CH2Cl2–acetone 95:5) to yield
D
-
(m, 2H, H-2000, H-6b000), 4.26–4.16 (m, 2H, H-20, H-200), 4.22 (s, 2H,
COCH2Cl), 4.13 (t, 1H, J2,3 10 Hz, H-2), 3.99–3.65 (m, 6H, H-5000, H-
6a0, H-6a, H-50, H-6a00, H-6b0), 3.62–3.56 (m, 1H, H-5), 3.52–3.39
(m, 3H, H-500, H-6b00, H-6b), 2.05, 1.95, 1.93, 1.86, 1.79, 1.75 and
1.60 (8s, 24H, 8COCH3); 13C NMR (CDCl3): d 169.82, 169.39,
169.22, 167.52, 167.04 and 166.62 (CO), 97.80 (C-1000), 97.69 (C-
100), 97.37 (C-10), 82.23 (C-1), 76.75 (C-5), 72.73 (C-50, C-500), 71.60
(C-5000), 71.35 (C-3), 70.47 (C-3000), 70.39 (C-30, C-300), 69.33 (C-40,
C-400), 68.78 (C-4), 68.60 (C-4000), 68.22 (C-6), 67.98 (C-60), 67.39
(C-600), 63.29 (C-6000), 54.26 (C-2000), 54.15 (C-20), 54.11 (C-200),
53.17 (C-2), 40.62 (COCH2Cl), 20.46, 20.34 and 20.21 (8COCH3).
MALDI TOF-MS calcd for C80H75ClN4O33S: 1686.37 [M]. Found:
1709.11 [M+Na]+.
D
7 (625 mg, 76%) as a colourless syrup. [
a]D +42.0 (c 0.37 in CHCl3);
1H NMR (CDCl3): d 7.93–7.56 (m, 8H, Ar), 7.44–7.16 (m, 5H, Ar),
5.78 (t, 1H, J3 ,4 10 Hz, H-30), 5.68 (t, 1H, J3,4 10 Hz, H-3), 5.58 (d,
0
0
1H, J1,2 10 Hz, H-1), 5.53 (d, 1H, J1 ,2 8.5 Hz, H-10), 5.12 (t, 1H, J4 ,5
0
0
0
0
10 Hz, H-40), 4.94 (t, 1H, J4,5 10 Hz, H-4), 4,30 (dd, 1H, J2,3 9 Hz, H-
20), 4.19 (t, 1H, J 10 Hz, H-2), 3.95 (dd, 1H, J6a,6b 10 Hz, J5,6a 3 Hz
H-6a), 3.86–3.76 (m, 2H, H-5, H-6a0), 3.86–3.76 (m, 3H, H-50, H-
6b, H-6b0), 2.78 (br s, 1H, OH), 2.07, 1.97, 1.87 and 1.78 (4s, 12H,
4COCH3); 13C NMR (CDCl3): d 170.14, 169.91, 167.70 and 166.81
(CO), 97.83 (C-10), 82.38 (C-1), 76.52 (H-5), 74.16 (H-50), 71.39 (C-
3), 70.68 (C-30), 69.80 (C-4), 69.17 (C-40), 69.01 (C-6), 61.21 (C-60),
54.39 (C-20), 53.39 (C-2), 20.70 and 20.36 (4COCH3). Anal. Calcd
for C42H40N2O16S: C, 58.60; H, 4.68. Found: C, 58.71; H, 4.76.
3.1.12. Phenyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-
b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-
phthalimido-b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-
deoxy-2-phthalimido-1-thio-b- -glucopyranoside (11)
D-
D
D
D
Compound 10 (240 mg, 0.14 mmol) was treated with thiourea
according to typical procedure B. The crude product was purified
by silica column chromatography (CH2Cl2–acetone 9:1) to yield
3.1.10. Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-
b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-
phthalimido-1-thio-b-
D-
D
11 (173 mg, 76%) as a colourless syrup. [
a
]
+46.45 (c 0.16 in
D
D-glucopyranoside (9)
CHCl3); 1H NMR (CDCl3): d 8.03–7.54 (m, 16H, Ar), 7.33–7.18 (m,
Bromo sugar 822 (174 mg, 0.35 mmol) was coupled with accep-
tor 7 according to typical procedure C. The crude product was puri-
fied by silica column chromatography (CH2Cl2–acetone 95:5) to
5H, Ar), 5.83 (dd, 1H, J3
9.5 Hz, H-3000), 5.68 (dd, 1H, J3 ,4 9.5 Hz,
000,4000
0
0
H-30), 5.64 (t, 1H, J3,4 10 Hz, H-3), 5.58 (dd, 1H, J3 ,4 9.5 Hz, H-300),
00 00
5.52 (d, 1H, J1
8.5 Hz, H-1000), 5.51 (d, 1H, J1,2 10.5 Hz, H-1), 5.39
000,2000
0
0
00 00
yield 9 (155 mg, 53%) as a colourless syrup. [
a
]
+40.07 (c 0.28 in
(d, 1H, J1 ,2 8.5 Hz, H-10), 5.32 (d, 1H, J1 ,2 8.5 Hz, H-100), 5.16 (t,
D
CHCl3); 1H NMR (CDCl3): d 7.95–7.55 (m, 12H, Ar), 7.32–7.19 (m,
1H, J4
9.5 Hz, H-4000), 5.04 (t, 1H, J4 ,5 9.5 Hz, H-40), 4.87 (t, 1H,
0
0
000,5000
5H, Ar), 5.79 (dd, 1H, J2 ,3 10.5 Hz, H-300), 5.63 (t, 1H, J3,4 10 Hz,
J4 ,5 9.5 Hz, H-400), 4.81 (t, 1H, J4,5 10 Hz, H-4), 4.33 (dd, 1H, J2
00 00
00 00
000,3000
H-3), 5.59 (t, 1H, J2 ,3 10.5 Hz, H-30), 5.56 (d, 1H, J1 ,2 10 Hz, H-
10.5 Hz, H-2000), 4.24 (dd, 1H, J2 ,3 10.5 Hz, H-20), 4.21 (dd, 1H, J2 ,3
10.5 Hz, H-200), 4.15 (t, 1H, J2,3 10 Hz, H-2), 4.01–3.95 (m, 1H, H-
6a000), 3.90–3.51 (m, 10H, H-5, H-50, H-500, H-5000, H-6a, H-6b, H-6a0,
0
0
00 00
0
0
00 00
100), 5.51 (d, 1H, J1,2 10 Hz, H-1), 5.33 (d, 1H, J1 ,2 8.5 Hz, H-10),
0
0
5.19 (t, 1H, J3 ,4 9.5 Hz, H-400), 4.92 (t, 1H, J3 ,4 9.5 Hz, H-40), 4.80
(t, 1H, J4,5 9.5 Hz, H-4), 4.41–4.32 (m, 2H, H-6a00, H-200), 4.25–4.17
(m, 2H, H-20, H-6b00), 4.14 (t, 1H, J2,3 10 Hz, H-2), 3.97–3.84 (m,
3H, H-500, H-6a, H-6a0), 3.77–3.55 (m, 3H, H-6b0, H-50, H-5), 3.45
(dd, 1H, J5,6b 6 Hz, J6a,6b 10 Hz, H-6b), 2.14, 2.04, 1.95, 1.92, 1.86,
1.79 and 1.75 (7s, 21H, 7COCH3); 13C NMR (CDCl3): d 170.68,
170.02, 169.98, 169.44, 169.31, 167.65 and 166.75 (CO), 97.81 (C-
10), 97.78 (C-100), 82.29 (C-1), 77.42 (C-5), 73.21 (C-50), 71.87 (C-
500), 71.45 (C-3), 70.64 (C-300, C-30), 69.42 (C-40), 69.01 (C-4), 68.78
(C-400), 68.52 (C-6), 67.63 (C-60), 61.83 (C-600), 54.37 (C-200), 54.26
(C-20), 53.28 (C-2), 20.74, 20.58, 20.44, 20.37 and 20.31 (7COCH3).
MALDI TOF-MS calcd for C62H59N3O25S: 1277.31 [M]. Found:
1300.15 [M+Na]+.
00 00
0
0
H-6b0, H-6a00, H-6b000), 3.47 (dd, 1H, J5 ,6b 6 Hz, J6a ,6b 11 Hz H-
6b00),, 3.05 (br s, 1H), 2.08, 1.97, 1.94, 1.88, 1.80 and 1.77 (8s, 24H,
8COCH3); 13C NMR (CDCl3): d 170.05, 169.95, 169.66, 169.56,
169.36, 167.62, 167.54 and 166.74 (CO), 97.82 (C-100), 97.56 (C-10),
97.40 (C-1000), 82.37 (C-1), 76.96 (C-5), 74.18 (C-5000), 72.82 (C-500),
72.39 (C-50), 71.46 (C-3), 70.72 (C-3000), 70.57 (C-300, C-30), 69.89 (C-
40), 69.69 (C-400), 69.13 (C-4000), 68.96 (C-4), 68.44 (C-600), 67.99 (C-
60), 67.76 (C-6000), 61.11 (C-6), 54.38 (C-2000), 54.25 (C-20, C-200),
53.30 (C-2), 20.62, 20.60, 20.51, 20.45, 20.37 and 20.32 (8COCH3).
MALDI TOF-MS calcd for C78H74N4O32S: 1610.40 [M]. Found:
1633.24 [M+Na]+.
00
00
00
00
3.1.13. Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-
b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-
phthalimido-b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-
deoxy-2-phthalimido-b- -glucopyranosyl-(1?6)-3,4-di-O-
-glucopyranoside (12)
D-
3.1.11. Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-
phthalimido-b-
deoxy-2-phthalimido-b-
acetyl-2-deoxy-2-phthalimido-b-
D
-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-
-glucopyranosyl-(1?6)-3,4-di-O-
-glucopyranosyl-(1?6)-3,4-
D
D
D
D
D
di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-
D-glucopyranoside
acetyl-2-deoxy-2-phthalimido-1-thio-b-D
(10)
Bromo sugar 822 (75 mg, 0.15 mmol) was coupled with acceptor
11 according to typical procedure C. The crude product was puri-
fied by silica column chromatography (CH2Cl2–acetone 95:5) to
Compound 6 (340 mg, 0.36 mmol) was reacted with bromine
and then the obtained bromo sugar was coupled with acceptor 7
according to typical procedure A. The crude product was purified
by silica column chromatography (hexane–acetone 1:1) to yield
yield 12 (146 mg, 72%) as a colourless syrup. [a] +38.44 (c 0.16
D
in CHCl3); 1H NMR (CDCl3): d 7.96–7.55 (m, 20H, Ar), 7.32–7.17
(m, 5H, Ar), 5.80 (dd, 1H, J 9 Hz, J 10.5 Hz, H-30000), 5.72–5.44 (m,
6H, H-3, H-30, H-300, H-3000, H-10000, H-1), 5.34–5.26 (m, 3H, H-10, H-
100, H-1000), 5.21 (t, 1H, J 9.5 Hz, H-40000), 4.93 (t, 1H, J 9.5 Hz, H-4000),
4.88–4.74 (m, 3H, H-4, H-40, H-400), 4.43–4.34 (m, 2H, H-20000, H-
6a0000), 4.28–4.08 (m, 5H, H-6b0000, H-2, H-20, H-200, H-2000), 3.99–3.34
(m, 13H, H-50000, H-6a, H-6a0, H-6a00, H-6a000, H-5, H-50, H-500, H-5000,
10 (280 mg, 72%) as a colourless syrup. [
a
]
+44.04 (c 0.13 in
D
CHCl3); 1H NMR (CDCl3): d 7.95–7.54 (m, 16H, Ar), 7.30–7.19 (m,
10.5 Hz, H-3000), 5.67 (dd, 1H, J2 ,3
0
0
000,3000
5H, Ar), 5.80 (dd, 1H, J2
10.5 Hz, H-30), 5.63 (t, 1H, J3,4 10 Hz, H-3), 5.57 (dd, 1H, J2 ,3
00 00
10.5 Hz, H-300), 5.51 (d, 1H, J1
8.5 Hz, H-1000), 5.50 (d, 1H, J1,2
000,2000
10 Hz, H-1), 5.34 (d, 1H, J1 ,2 8.5 Hz, H-10), 5.29 (d, 1H, J1 ,2
0
0
00 00

A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
1451
H-6b, H-6b0, H-6b00, H-6b000), 2.26, 2.17, 2.05, 1.97, 1.94, 1.87, 1.79
and 1.75 (11s, 33H, 11COCH3); 13C NMR (CDCl3): d 170.60,
169.86, 169.39, 169.29, 167.58 and 166.68 (CO), 97.89 (C-10000),
97.79, 97.54 and 97.40 (C-10, C-100, C-1000), 82.29 (C-1), 77.61 (C-5),
72.86, 72.76 and 72.51 (C-50, C-500, C-5000), 71.88 (C-50000), 71.44,
70.61 and 70.43 (C-3, C-30, C-300, C-3000, C-30000), 69.45, 69.33 and
68.86 (C-4, C-40, C-400, C-4000), 68.79 (C-40000), 68.29, 67.99, 67.66
and 67.15 (C-6, C-60, C-600, C-6000), 61.84 (C-60000), 54.29 and 54.20
(C-20, C-200, C-2000, C-20000), 53.24 (C-2), 20.69, 20.53, 20.40 and
20.27 (11COCH3). MALDI TOF-MS calcd for C98H93N5O41S:
2027.51 [M]. Found: 2050.66 [M+Na]+.
3.1.16. Phenyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-
-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-
b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-
deoxy-1-thio-b- -glucopyranoside (15)
Compound 10 (180 mg, 0.107 mmol) was treated with ethylene
diamine and then the obtained amine was peracetylated according
to typical procedure D. The crude product was purified by silica
column chromatography (CH2Cl2–methanol 95:5) to yield 15
D-
D
D
D
(102 mg, 73%) as a colourless syrup. [
a
]
ꢀ1.90 (c 0.16 in CHCl3);
D
1H NMR (CDCl3): d 7.91 (d, 1H, J 9 Hz, NH), 7.58–7.51 (m, 2H, Ar),
7.46 (d, 1H, J 9 Hz, NH), 7.40–7.26 (m, 4H, Ar, NH), 7.18 (d, 1H, J
9 Hz, NH), 5.38 (t, 1H, J2 ,3 10 Hz, H-30), (t, 1H, J2,3 10 Hz, H-3),
0
0
3.1.14. Phenyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-1-
thio-b- -glucopyranoside (13)
D-
5.08 (t, 1H, J 10 Hz, H-3000), 5.03 (t, 1H, J3 ,4 10 Hz, H-400), 4.94 (t,
00 00
000,4000
1H, J3
9 Hz, H-4000), 4.91 (d, 1H, J1,2 10.5 Hz, H-1), 4.89 (t, 1H,
D
J3,4 9.5 Hz, H-4), 4.74 (t, 1H, J3 ,4 9.5 Hz, H-40), 4.73 (d, 1H, J1 ,2
0
0
00 00
Compound 6 (170 mg, 0.18 mmol) was treated with ethylene
diamine and the obtained amine was peracetylated according to
typical procedure D. The crude product was purified by silica
column chromatography (CH2Cl2–acetone 1:1) to yield 13
9 Hz, H-100), 4.70 (d, 1H, J1 ,2 10 Hz, H-10), 4.44 (dd, 1H, J6a
0
0
000,6b000
12 Hz, J5
6 Hz, H-6a000), 4.38–4.18 (m, 4H, H-1000, H-5, H-2000, H-
000,6a000
2), 4.11–4.00 (m, 3H, H-200, H-50, H-6b000), 3.92–3.65 (m, 6H, H-5000,
H-20, H-500, H-6a00, H-6b00, H-6a), 3.60–3.44 (m, 2H, H-6b, H-6a0),
(110 mg, 83%) as a colourless syrup. [
a
]
ꢀ32.36 (c 0.15 in
D
3.32 (dd, 1H, J6a ,6b 12, J5 ,6b 8 Hz, H-6b0), 2.16, 2.10, 2.08, 2.06,
2.05, 2.03, 2.01, 2.00 1.97, 1.94 and 1.88 (10s, 39H, 13COCH3);
0
0
0
0
CHCl3); 1H NMR (CDCl3): d 7.53–7.30 (m, 5H, Ar), 5.59 (d, 1H,
J 9 Hz, NH), 5.48 (d, 1H, J 9 Hz, NH), 5.17 (t, 1H, J3,4 10 Hz, H-
13C NMR (CDCl3):
d 171.50, 170.92, 170.73, 170.07, 170.02,
3), 5.16 (d, 1H, J2 ,3 10 Hz, H-30), 5.03 (t, 1H, J3 ,4 9.5 Hz, H-40),
4.98 (t, 1H, J4,5 10 Hz, H-4), 4.86 (d, 1H, J1,2 10 Hz, H-1), 4.51
0
0
0
0
169.93 and 169.56 (CO), 103.56 (C-1000), 102.70 (C-100), 101.26 (C-
10), 86.75 (C-1), 75.39 (C-5), 74.20 (C-3), 73.48 (C-5000), 72.59 (C-
30), 72.39 (C-500, C-3000), 71.96 (C-300), 71.63 (C-50), 70.92 (C-60),
70.77 (C-6, C-600), 70.41 (C-4), 70.36 (C-40), 69.59 (C-4000), 69.08
(C-400), 62.69 (C-6000), 55.27 (C-20), 54.67 (C-200), 54.12 (C-2), 52.92
(C-2000), 23.25, 23.17, 23.05, 20.91, 20.68 and 20.61 (COCH3). MALDI
TOF-MS calcd for C56H76N4O29S: 1300.43 [M]. Found: 1323.44
[M+Na]+.
(d, 1H, J1 ,2 8.5 Hz, H-10), 4.25 (dd, 1H, J6a ,6b 12, J5,6a 4.5 Hz,
0
0
0
0
0
H-6a0), 4.11 (dd, 1H, J5,6b 10.5 Hz, H-6b0), 3.96 (t, 1H, J2,3
0
10 Hz, H-2), 3.93–3.86 (m, 2H, H-6a, H-20), 3.71–3.59 (m, 2H,
H-5, H-50), 3.53 (dd, 1H, J6a,6b 12, J5,6b 6 Hz, H-6b), 2.07, 2.02,
2.00, 1.96, 1.80 and 1.63 (7s, 21H, 7COCH3); 13C NMR (CDCl3):
d
170.98, 170.82, 170.75, 170.54, 169.78 and 169.43 (CO),
100.74 (C-10), 86.14 (C-1), 76.92 (C-5), 73.64 (C-3), 72.66 (C-30),
71.66 (C-50), 68.73 (C-4), 68.41 (C-40), 68.10 (C-6), 61.80 (C-60),
54.00 (C-20), 52.69 (C-2), 22.67, 22.61, 20.48 and 20.37 (COCH3).
3.1.17. Phenyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-
-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-
b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-
deoxy-b- -glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-
2-deoxy-1-thio-b- -glucopyranoside (16)
D-
MALDI TOF-MS calcd for
C32H42N2O15S: 726.23 [M]. Found:
749.01 [M+Na]+.
D
D
D
3.1.15. Phenyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-
-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-
1-thio-b- -glucopyranoside (14)
D-
D
Compound 12 (136 mg, 0.067 mmol) was treated with ethyl-
ene diamine and then the obtained amine was peracetylated
according to typical procedure D. The crude product was purified
by silica column chromatography (CH2Cl2–methanol 95:5) to
D
D
Compound 9 (81 mg, 0.06 mmol) was treated with ethylene
diamine and then the obtained amine was peracetylated accord-
ing to typical procedure D. The crude product was purified by
silica column chromatography (CH2Cl2–methanol 95:5) to yield
yield 16 (76 mg, 71%) as a colourless syrup. [
a
]
ꢀ8.24 (c 0.12
D
in CHCl3); 1H NMR (CDCl3): d 8.18 (d, 1H, J 9.5 Hz, NH), 7.72 (d,
2H, J 9 Hz, NH), 7.69–7.61 (m, 2H, Ar), 7.40–7.33 (m, 3H, Ar),
7.13 (d, 1H, J 9 Hz, NH), 6.93 (br s, 1H, NH), 5.35–5.23 (m, 2H,
H-3000, H-30000), 5.21–4.81 (m, 8H, H-3, H-30, H-300, H-40, H-400,
H-4000, H-40000, H-1), 4.66 (t, 1H, J 9.5 Hz, H-4), 4.63–4.46 (m, 5H,
H-10, H-100, H-1000, H-10000, H-6a0000), 4.41–4.13 (m, 6H, H-2, H-20,
H-200, H-2000, H-20000, H-5), 4.10–3.06 (m, 13H, H-6b0000, H-6a, H-6b,
14 (52 mg, 81%) as a colourless syrup. [
a
]
ꢀ16.98 (c 0.17 in
D
CHCl3); 1H NMR (CDCl3): d 7.58–7.29 (m, 5H, Ar), 6.45 (d, 1H,
J 9 Hz, NH), 6.40 (d, 1H, J 9 Hz, NH), 5.98 (d, 1H, J 8.5 Hz, NH),
5.24 (t, 1H, J3,4 10 Hz, H-3), 5.18 (t, 2H, J 9.5 Hz, H-30, H-300),
5.04 (t, 1H, J3 ,4 9.5 Hz, H-400), 4.93 (t, 2H, J 10 Hz, H-4, H-40),
00 00
4.91 (d, 1H, J1,2 10 Hz, H-1), 4.58 (d, 1H, J1 ,2 8 Hz, H-10), 4.48
0
0
H-50, H-500, H-5000, H-50000
,
H-6a0, H-6b0, H-6a00, H-6b00, H-6a000,
(d, 1H, J1 ,2 8.5 Hz, H-100), 4.26 (dd, 1H, J6a ,6b 12 Hz, J5,6a
00 00
00
00
00
H-6b000), 2.23, 2.15, 2.11, 2.08, 2.07, 2.05, 2.03, 2.02, 2.00 1.99
and 1.95 (16s, 48H, 16COCH3); 13C NMR (CDCl3): d 171.74,
171.29, 171.11, 171.01, 170.96, 170.38, 170.27, 170.12, 170.05,
169.86, 169.66 and 169.49 (CO), 104.43, 104.16, 103.35 and
101.81 (C-10000, C-1000, C-100, C-10), 86.96 (C-1), 74.64, 74.41, 73.66,
73.52, 72.78, 72.51 and 72.22 (C-3, C-30, C-300, C-3000, C-30000, C-5,
C-50, C-500, C-5000, C-50000), 71.71, 71.53 and 71.44 (C-6, C-60,
C-600, C-6000), 70.91, 70.86, 70.60, 69.65 and 69.06 (C-4, C-40, C-400,
C-4000, C-40000), 63.02 (C-60000), 54.38, 54.32, 54.19 and 53.01
(C-2, C-20, C-200, C-2000, C-20000), 23.33, 23.30, 23.19, 23.04,
21.13, 20.83, 20.70 and 20.63 (COCH3). MALDI TOF-MS
5 Hz, H-6a00), 4.12 (dd, 1H, J6a ,6b 12 Hz, J5,6b 2 Hz, H-6b00), 4.01
00
00
00
(t, 1H, J2 ,3 8.5 Hz, H-200), 3.98 (t, 1H, J2,3 10 Hz, H-2), 3.92–
00 00
3.77 (m, 4H, H-20, H-6a0, H-6b0, H-5), 3.74–3.57 (m, 3H, H-500,
H-50, H-6a), 3.45 (dd, 1H, J6a,6b 11.5 Hz, J5,6b 6 Hz, H-6b), 2.12,
2.08, 2.05, 2.04, 2.02, 1.97, 1.95 and 1.83 (10s, 30H, 10COCH3);
13C NMR (CDCl3):
d 171.14, 171.09, 170.94, 170.89, 170.83,
170.69, 170.66, 170.20, 169.97 and 169.54 (CO), 101.50 (C-100),
100.85 (C-10), 86.10 (C-1), 76.48 (C-5), 73.72 (C-3), 72.87 (C-
300), 72.71 (C-30), 72.58 (C-50), 71.74 (C-500), 69.21 (C-4), 69.06
(C-40), 68.65 (C-400), 68.58 (C-6), 68.48 (C-60), 62.11 (C-600),
54.01 (C-2), 53.80 (C-200), 52.78 (C-20), 20.74, 20.58, 20.44,
20.37 and 20.31 (7COCH3). MALDI TOF-MS calcd for
calcd for
C68H93N5O36S: 1587.53 [M]. Found: 1610.29
[M+Na]+.
C
44H59N3O22S: 1013.33 [M]. Found: 1036.36 [M+Na]+.

1452
A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
3.1.18. Phenyl 2-acetamido-2-deoxy-b-
D
-glucopyranosyl-(1?6)-
in H2O); 1H NMR (DMSO): d 7.59–7.27 (m, 5H, Ar), 4.87 (d, 1H, J
2-acetamido-2-deoxy-1-thio-b-
D
-glucopyranoside (17)
10.5 Hz, H-1), 4.50–4.39 (m, 4H, H-10, H-100, H-1000, H-10000), 4.16–
3.97 (m, 4H, H-6a, H-6a0, H-6a00, H-6a000), 3.83–3.14 (m, 26H,
H-6a0000, H-2, H-5, H-6b, H-6b0, H-6b00, H-6b000, H-6b0000, H-20, H-200,
Compound 13 (95 mg, 0.13 mmol) was treated with NaOMe
according to typical procedure E. The crude product was lyophi-
lized to give 17 (58 mg, 86%) as a white solid. [
a
]
D
ꢀ53.23 (c
H-2000, H-20000, H-3, H-30, H-300, H-3000, H-30000, H-50, H-500, H-5000, H-50000
,
0.14 in H2O); 1H NMR (D2O): d 7.58–7.40 (m, 5H, Ar), 4.94 (d,
H-4, H-40, H-400, H-4000, H-40000), 2.09, 2.06, 2.01 and 1.98 (5s, 15H,
5COCH3); 13C NMR (DMSO): d 170.34, 170.31, 170.28, 170.19 and
170.02 (CO), 102.25, 102.16 and 102.03 (C-10, C-100, C-1000, C-10000),
86.13 (C-1), 78.77 (C-5), 76.96, 75.66, 75.40, 75.28, 75.15, 74.79,
74.28, 74.19, 74.11, 71.25, 70.70 and 70.63 (C-50, C-500, C-5000, C-
50000, C-3, C-30, C-300, C-3000, C-30000, C-4, C-40, C-400, C-4000, C-40000), 70.01,
69.94, 69.84 and 69.52 (C-6, C-60, C-600, C-6000), 61.18 (C-60000), 55.68
(C-20, C-200, C-2000, C-20000), 54.37 (C-2), 23.22, 23.17 and 23.13
(5COCH3). MALDI TOF-MS calcd for C46H71N5O25S: 1125.42 [M].
Found: 1148.44 [M+Na]+.
1H, J1,2 10 Hz, H-1), 4.58 (d, 1H, J1 ,2 8.5 Hz, H-10), 4.22 (dd, 1H,
0
0
0
0
0
J5,6a 11 Hz, J6a,6b 1 Hz, H-6a), 3.97 (dd, 1H, J5,6a 11 Hz, J6a ,6b
1 Hz, H-6a0), 3.83 (t, 1H, J2,3 10 Hz, H-2), 3.85–3.72 (m, 3H, H-
6b, H-6b0, H-20), 3.70–3.43 (m, 6H, H-5, H-3, H-30, H-40, H-4, H-
5), 2.04 and 1.96 (2s, 6H, 2COCH3); 13C NMR (D2O): d 175.38,
175.17 (CO), 102.40 (C-10), 87.56 (C-1), 79.78 (C-5), 76.92 (C-50),
76.22 (C-3), 75.00 (C-30), 70.97 (C-4), 70.75 (C-40), 69.59 (C-6),
61.76 (C-60), 56.50 (C-2), 55.47 (C-20), 23.18 and 23.14 (2COCH3).
MALDI TOF-MS calcd for
C22H32N2O10S: 516.18 [M]. Found:
539.18 [M+Na]+.
3.2. Enzymatic studies
3.1.19. Phenyl 2-acetamido-2-deoxy-b-
2-acetamido-2-deoxy-b- -glucopyranosyl-(1?6)-2-acetamido-
2-deoxy-1-thio-b- -glucopyranoside (18)
Compound 14 (46 mg, 0.045 mmol) was treated with NaOMe
according to typical procedure E. The crude product was lyophi-
D-glucopyranosyl-(1?6)-
D
3.2.1. Expression and purification of DspB and mutants
The enzyme DspB was expressed and purified using the plas-
mid pRC3 that carried the dspB gene encoding amino acids 21–
381 fused directly to a hexahistidine metal-binding C-terminal
tail located downstream from an IPTG-inducible tac promoter as
previously described.18 Y187A, Y278A and W237A mutants of
DspB were generated using the primers and method published
earlier.19
D
lized to give 18 (28 mg, 86%) as a white solid. [
a
]
ꢀ70.37 (c 0.07
D
in H2O); 1H NMR (DMSO): d 7.58–7.31 (m, 5H, Ar), 4.87 (d, 1H,
J1,2 10.5 Hz, H-1), 4.45 (d, 2H, J 8.5 Hz, H-10, H-100), 4.09 (dd, 1H,
J6a ,6b 10 Hz, J5 ,6a 2 Hz, H-6a00), 3.82 (dd, 1H, J6a,6b 10 Hz, J5,6a
1 Hz, H-6a), 3.74 (t, 1H, J2,3 10 Hz, H-2), 3.70–3.55 (m, 7H, H-5,
H-6b, H-20, H-200, H-6b00, H-6a0, H-6b0), 3.51 (t, 1H, J3,4 10 Hz, H-3),
3.47–3.35 (m, 3H, H-30, H-300, H-500), 3.31–3.15 (m, 4H, H-4, H-50,
H-40, H-400), 1.98, 1.90 and 1.89 (3s, 9H, 3COCH3); 13C NMR (DMSO):
d 170.48, 170.40 and 170.24 (CO), 102.23 and 102.05 (C-10, C-100),
86.31 (C-1), 79.03 (C-5), 76.97 (C-50), 75.81 (C-500), 75.35 (C-3),
74.56 (C-300), 74.35 (C-30), 70.88 (C-4), 70.65 (C-40), 70.43 (C-400),
69.70 (C-6), 69.54 (C-60), 61.22 (C-600), 55.72 (C-20, C-200), 54.42
(C-2), 23.23 and 23.15 (3COCH3). MALDI TOF-MS calcd for
00
00
00
00
3.2.2. HPLC separation of substrates and products of the
enzymatic reaction
Compounds were separated on a reversed phase column (Zor-
bax Eclipse XDB-C18 150 ꢂ 4.6 mm, 5
lm) with acetonitrile/water
(10:90) as the mobile phase at a flow rate of 1 mL/min at 25 °C
using a Hewlett–Packard 1090 Series II liquid chromatograph
equipped with diode array detector and automatic sampler. Five
microlitres of the standard mixture or the reaction mixture was in-
jected into the chromatographic column. Effluent was monitored
for the products containing thiophenyl group at 254 nm. The HPLC
profile of the standard mixture (Fig. 4) shows that substrates (DP
2–5, 1 mM) and products (DP 1–4) are separated very well with
reasonable retention time. Retention order is reversed on C18 col-
umn, first eluted peak is the substrate, followed by the shorter
hydrolysis products. The products of the hydrolysis were identified
and measured by using relevant standards with application of
CHEMSTATION software. The quality of acetonitrile was HPLC gradient
grade. Purified water was obtained from a laboratory purification
system equipped with both ion exchange and carbon filters (Milli-
pore, Bedford, MA, USA).
C
30H45N3O15S: 719.26 [M]. Found: 742.36 [M+Na]+.
3.1.20. Phenyl 2-acetamido-2-deoxy-b-
2-acetamido-2-deoxy-b- -glucopyranosyl-(1?6)-2-acetamido-
2-deoxy-b- -glucopyranosyl-(1?6)-2-acetamido-2-deoxy-1-
thio-b- -glucopyranoside (19)
D-glucopyranosyl-(1?6)-
D
D
D
Compound 15 (92 mg, 0.07 mmol) was treated with NaOMe
according to typical procedure E. The crude product was lyophi-
lized to give 19 (58 mg, 88%) as a white solid. [
a]
ꢀ27.34 (c 0.08
D
in H2O); 1H NMR (DMSO): d 7.53–7.31 (m, 5H, Ar), 4.88 (d, 1H,
J1,2 10.5 Hz, H-1), 4.49–4.39 (m, 3H, H-10, H-100, H-1000), 4.11–4.01
(m, 3H, H-6a, H-6a0, H-6a00), 3.86–3.34 (m, 15H, H-6a000, H-2, H-6b,
H-6b0, H-6b00, H-6b000, H-20, H-200, H-2000, H-5, H-5000, H-3, H-30, H-300,
H-3000), 3.31–3.16 (m, 6H, H-50, H-500, H-4, H-40, H-400, H-4000), 1.99,
1.98, 1.91 and 1.90 (4s, 12H, 4COCH3); 13C NMR (DMSO): d
170.84, 170.73 and 170.51 (CO), 102.30, 102.24 and 102.08 (C-10,
C-100, C-1000), 86.31 (C-1), 78.98 (C-5), 77.00, 75.71, 75.50, 75.43,
74.74 and 74.33 (C-50, C-500, C-5000, C-3, C-30, C-300, C-3000), 71.10,
70.73 and 70.60 (C-4, C-40, C-400, C-4000), 69.82, 69.77 and 69.40 (C-
6, C-60, C-600), 61.30 (C-6000), 55.84 (C-20, C-200, C-2000), 54.52 (C-2),
23.25 and 23.18 (4COCH3). MALDI TOF-MS calcd for C38H58N4O20S:
922.34 [M]. Found: 945.44 [M+Na]+.
3.2.3. Hydrolysis on oligomer substrates catalysed by DspB
enzyme and its mutants
Hydrolytic reactions were carried out on b-(1?6)-linked Glc-
NAc oligosaccharides and the reaction products were analysed by
HPLC. Incubations of oligosaccharides DP 2–5 at substrate concen-
tration of 0.4–2 mM were carried out in 50 mM phosphate buffer
(pH 5.9) containing 100 mM NaCl at room temperature (25 °C).
The reactions were initiated by the addition of enzyme (2
To follow the reaction 5 L reaction mixture was injected into
lM).
l
the chromatographic column at different time intervals. Reaction
products were separated as described above. The hydrolysis con-
version is defined as the percentage of substrate converted to
shorter products. The conversions were calculated from HPLC peak
area of substrate and products. The sum of peak areas of products
was divided by the total area of substrate and products. This ratio
was multiplied with 100 to get conversion% of the enzyme
reactions.
3.1.21. Phenyl 2-acetamido-2-deoxy-b-
2-acetamido-2-deoxy-b- -glucopyranosyl-(1?6)-2-acetamido-
2-deoxy-b- -glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-2-acetamido-2-deoxy-1-thio-b-D-
glucopyranoside (20)
D-glucopyranosyl-(1?6)-
D
D
D-
Compound 16 (72 mg, 0.045 mmol) was treated with NaOMe
according to typical procedure E. The crude product was lyophi-
lized to give 20 (43 mg, 85%) as a white solid. [
a]
ꢀ37.23 (c 0.10
D

A. Fekete et al. / Carbohydrate Research 346 (2011) 1445–1453
1453
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This work was supported by the Hungarian Scientific
Research Fund (Project No. PD73064) and by the TÁMOP 4.2.1./
B-09/1/KONV-2010-0007 project. The project is implemented
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