Block synthesis of A tetrasaccharides (types 1, 3, and 4) related to the human ABO blood group system

Article abstract of DOI:10.1016/j.carres.2011.12.013

Blood group A tetrasaccharides of different types have the same terminal trisaccharide fragment that allows using a block scheme in their synthesis. 3-Aminopropyl glycosides of tetrasaccharides GalNAcα1-3(Fucα1-2) Galβ1-3GlcNAcβ (A type 1), GalNAcα1-3(Fuc

Full text of DOI:10.1016/j.carres.2011.12.013

Carbohydrate Research 351 (2012) 17–25
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Block synthesis of A tetrasaccharides (types 1, 3, and 4) related
to the human ABO blood group system
⇑
Ivan M. Ryzhov, Elena Yu Korchagina , Inna S. Popova, Nicolai V. Bovin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Received in revised form 13 December 2011
Accepted 15 December 2011
Available online 24 December 2011
Blood group A tetrasaccharides of different types have the same terminal trisaccharide fragment that
allows using a block scheme in their synthesis. 3-Aminopropyl glycosides of tetrasaccharides Gal-
NAc
GalNAc
2)Gal trichloroacetimidate as a glycosyl donor at the key stage.
a
1-3(Fuc
a
1-2)Galb1-3GlcNAcb (A type 1), GalNAc
a1-3(Fuc
a1-2)Galb1-3GalNAc
a
(A type 3), and
1-3(Fuc 1-
a
1-3(Fuc
a
1-2)Galb1-3GalNAcb (A type 4) were synthesised using acetylated Gal
a
a
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Block synthesis
ABO histo-blood group
Trichloroacetimidate
4,6-Di-tert-butyl-benzoxazol-2-yl
3-Aminopropyl glycoside
1. Introduction
expression of ABO antigens is often observed in the oncogenesis of
various organs and in vascular inflammatory processes.9–11
The main aim of our investigation is to develop a synthetic ap-
proach that will allow us to obtain complete library of blood
group ABO antigens. Such a library will provide a possibility for
wide-range fundamental biological studies and practical use in
the field of hematology and transplantology. To date, several
chemical5,12–19 and chemoenzymatic20–22 syntheses of different
types of histo-blood group antigens have been reported. The
methodology of stepwise elongation of the carbohydrate chain
starting from the reducing end is most often used in the synthesis
of blood group tetrasaccharides.5,12–18 In one paper the disaccha-
ride glycosyl donor has been used for the synthesis of blood
group tetrasaccharide A (type 3).19 We have recently reported a
block synthetic approach to obtaining blood group B tetrasaccha-
rides (types 1, 3, and 4) that allows us to minimize the number of
synthetic steps.23 Similar strategy has been chosen here for the
synthesis of blood group A tetrasaccharides 1–3. Blood group tet-
rasaccharides A of various types contain the same structural
trisaccharide fragment. This makes it possible to use the block
scheme ‘3+1’ in their synthesis, where ‘3’ is a glycosyl donor, in
particular, the acetylated glycosyl trichloroacetimidate 8 and ‘1’
is the corresponding glucosamine 9 or galactosamine 12 or 13
glycosyl acceptors.
ABO histo-blood group antigens are terminal oligosaccharides
in glycosphingolipids and glycoproteins expressed on human
erythrocytes and platelets that are widely distributed on vascular
endothelia, epithelia, and primary sensory neurons; soluble forms
of ABO antigens are found in plasma and body secretions, such as
saliva and urine.1–3 These antigens are major polymorphic alloan-
tigens and potent immunogens, which cause harmful immune
reactions in the cases of ABO incompatible transfusion, as well as
in bone marrow and organ transplantation. The ABO antigens can
be divided into six types in accordance with the structure of disac-
charide core (Table 1).4,5
Each type of ABO antigens is expressed tissue selectively, so type
1 antigens are expressed mainly in tissues of endodermal origin,
whereas type 2 antigens are expressed in various tissues of both
ecto- and endodermal origin and are known as key structures of hu-
man erythrocytes. Expression of type 3 and type 4 antigens is less
well characterized; biological significance of the structures was
shown, for example, by Henry and co-workers.6 The difference be-
tween two blood group A subgroups, A1 and A2, was first deter-
mined as a quantitative disparity of antigen presentation on the
erythrocyte surface,7,8 but later the difference in glycolipid struc-
tures was found. Thus A type 4 glycolipid was undetectable in the
A2 subgroup erythrocytes, but was present in the A1 ones.6 Aberrant
2. Results and discussion
The known aminopropyl glycoside 424 was used as the precur-
sor in trisaccharide glycosyl donor synthesis due to its availability
⇑
Corresponding author. Tel.: +7 495 330 0300; fax: +7 495 330 5592.
E-mail address: korchagina@carb.ibch.ru (E.Y. Korchagina).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.12.013

18
I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
Table 1
The structure of compound 5 was confirmed by mass spectrometry
(MS m/z 1123.4836 (M++H)) and 1H NMR spectroscopy data (two
singlets at d 1.37 (9H) and d 1.49 (9H) related to tert-butyl groups
and two doublets at d 7.28 (1H) and d 7.57 (1H) of aromatic pro-
Disaccharides cores of the ABO histo-blood group
antigens
Type
Disaccharide core
Type 1
Type 2
Type 3
Type 4
Type 5
Type 6
Galab1-3GlcNAcb
Galb1-4GlcNAcb
tons). Base-catalyzed acetolysis/acetylation of benzoxazole
5
yielded the peracetylated trisaccharide 6 (96%). Then selective 1-
O-deacetylation of derivative 6 with hydrazine acetate26 and treat-
ing with trichloroacetonitrile in the presence of 1,8-diazobicy-
clo[5.4.0]undec-7-ene (DBU) gave glycosyl trichloroacetimidate 8,
which was isolated by flash chromatography on silica gel in 90%
Galb1-3GalNAc
a
Galb1-3GalNAcb
Galb1-3Galb
Galb1-4Glcb
a
All monosaccharide residues are in pyranose form.
yield. We have to mention that only
a-trichloroacetimidate 8 (d
6.48, J1,2 3.7 Hz) of trisaccharide A was obtained. The synthesis of
peracetylated B trisaccharide trichloroacetimidate 8a reported ear-
in multi-gram quantity and its well-established scaled-upsynthe-
sis. The method of aminopropyl spacer elimination previously pub-
lished25 and optimized19 was used for obtaining of peracetylated
trisaccharide 6. Derivatization of aminopropyl glycoside 4 with
3,5-di-tert-butyl-1,2-benzoquinone, treatment of the resulting
azomethine with oxalic acid dihydrate, and acetylation afforded
the resulting benzoxazole derivative 5 in 92% yield (Scheme 1).
lier led to the mixture of a- and b-isomers under the same reaction
conditions.19 We cannot properly explain the differences in trichlo-
roacetimidate formation, as the only disparity in the trisaccharide
structures is the presence of an OAc-group (in the case of B trisac-
charide) and an NHAc-group (in the case of A trisaccharide) in po-
sition 2 of the terminal galactose unit.
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH OH
OH
OH OH
O
OH OH
O
O
O
O
HO
O
O
HO
HO
O
O
HO
O
O(CH2)3NH2
NHAc
O(CH2)3NH2
O
O
O
AcHN
O
O
AcHN
AcHN
AcHN
O
NHAc
O
O(CH2)3NH2
O
O
O
OH
O
OH
OH
HO
OH
HO
OH
HO
OH
3
1
2
A type 4
A type 1
A type 3
OAc
AcO
OH
OAc
OH
OAc
OH
O
O
OAc
O
OH
O
HO
O
O
O
NH2
AcHN
O
AcHN
O
(a)
N
O
O
5
4
O
OAc
O
OH
AcO
OAc
HO
OH
(b)
OAc
OAc
OAc
O
OAc
OAc
AcO
OAc
AcO
OAc
O
O
OAc
O
AcO
AcO
(d)
AcHN
O
AcHN
O
OR
O
O
O
OC(NH)CCl3
6: R = Ac
7: R = H
(c)
O
OAc
OAc
8
OAc
OAc
OAc
OAc
OAc
AcO
O
OAc
O
AcO
AcO
O
OC(NH)CCl3
OAc
O
O
8a
OAc
Scheme 1. Reagents and conditions: (a) (i) 3,5-di-tert-butyl-1,2-benzoquinone, MeOH; (ii) (COOH)2Á2H2O (pH 4); (iii) Ac2O/Py, 92% (total for three stages); (b) NaOAc, 1:1
Ac2O–AcOH, 100 °C, 10 days, 96%; (c) N2H4ÁHOAc, DMF, 50 °C, 95%; (d) Cl3CCN, DBU, CH2Cl2, 90%.

I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
19
OAc
OAc
OAc
OAc
O
O
Ph
OAc
O
OAc
AcO
O
OAc
O
OAc
O
O
O
O(CH2)3NHC(O)CF3
NHAc
AcO
AcO
AcHN
O
AcHN
O
O
O
OC(NH)CCl3
O
O
(a)
8
OAc
10
OAc
AcO
OAc
OAc
+
O
Ph
(b)
O
O
O(CH2)3NHC(O)CF3
HO
NHAc
OAc
OAc
9
OAc
OAc
O
OAc
O
O
AcO
AcO
O(CH2)3NHC(O)CF3
NHAc
O
AcHN
O
O
O
(c)
1
OAc
11
AcO
OAc
Scheme 2. Reagents and conditions: (a) TMSOTf, 3 Å MS, 1:1 CH3CN–CH2Cl2; (b) (i) AcOH (80%), 80 °C; (ii) Ac2O/Py, 56% (total for three stages); (c) (i) MeONa/MeOH; (ii) H2O,
97%.
The key stage of the tetrasaccharides synthesis was the glyco-
sylation of the known glycosyl acceptors 9,27 12,28 and 1328 with
trisaccharide trichloroacetimidate 8.
signal of an anomeric proton at d 4.69 as a doublet with
J = 4.1 Hz, which is consistent with the b-configuration of the gly-
cosidic linkage. 1H NMR of 22 showed a signal of an anomeric
proton at 5.28 as a doublet with a coupling constant of 4.1 Hz,
2.1. Synthesis of 3-aminopropyl glycoside of the A (type 1)
tetrasaccharide
consistent with the a-configuration of the glycosidic bond. For
the separation of the type 4 tetrasaccharides, the anomeric tetra-
saccharide mixture 15 was treated with AcOH and acetylated;
subsequent column chromatography on silica gel afforded the re-
The glycosylation of acceptor 9 with trichloroacetimidate 8
(Scheme 2) was carried out in acetonitrile–dichloromethane mix-
ture at room temperature using a twofold excess of the glycosyl
acceptor and trimethylsilyl triflate as the reaction promoter to
yield tetrasaccharide 10.
quired A (type 4) tetrasaccharide 21 (45%) and its
a-anomer 23
(22%). 1H NMR data confirmed the configuration of the formed
glycosidic bond: an anomeric proton appeared as a doublet at d
4.74 with J = 7.3 Hz (b-configuration) for tetrasaccharide 21, and
To facilitate purification, the product 10 was treated with AcOH
to remove the benzylidene acetal, acetylated (Ac2O/Py) followed by
column chromatography on silica gel afforded the peracetylated
derivative 11 in 56% yield (total for three stages). The stereochem-
istry of the glycosidic bond thus formed, was confirmed by NMR
spectroscopy; 11 showed a signal of an anomeric proton at d
4.37 as a doublet with J = 7.5 Hz, which is consistent with the b-
as a doublet at d 5.45 with a coupling constant of 3.4 Hz (
figuration) for tetrasaccharide 23.
a-con-
Though the b anomer is the major product of glycosylation un-
der the reaction conditions described above, the anomer is also
generated, with the b/ ratio about 2.3:1 in the case of the A (type
a
a
3) and 2:1 in the case of the A (type 4) tetrasaccharides.
To check the possibility of improving the b-stereoselectivity, the
coupling was performed at reduced temperature. The reaction was
carried out in acetonitrile in the presence of TMSOTf.
It is proposed that if low temperatures are employed in the gen-
eration of the glycosyl cation, this may be attacked by acetonitrile
configuration of glycosidic linkage. The corresponding
a-isomer
was not isolated. Complete deacetylation and removal of the triflu-
oroacetyl group in derivative 11 by aqueous alkali followed by
cation-exchange chromatography afforded the 3-aminopropygly-
coside of the A (type 1) tetrasaccharide 1, in a yield of 97%.
along the kinetically favored axial direction to form an a-nitrillium
ion.29 As a result, b glycosides can predominate in the reaction
products. In our case, performing the reaction at À20 °C did not re-
sult in the formation of any glycosylation product during 24 h.
Only traces of trichloroacetimidate-derived by-products were reg-
istered. After the reaction temperature was increased to 4 °C, the
2.2. Synthesis of 3-aminopropyl glycosides of the A (type 3) and
A (type 4) tetrasaccharides
In the synthesis of the A tetrasaccharides (type 3 and type 4),
coupling of the trichloroacetimidate 8 with glycosyl acceptors 12
and 13 (Scheme 3) in acetonitrile in the presence of TMSOTf at
room temperature gave corresponding mixtures 14 and 15 of
the anomeric tetrasaccharides. To facilitate the separation of the
type 3 tetrasaccharide, mixture 14 was treated with AcOH to re-
move the benzylidene acetal; subsequent column chromatogra-
glycosylation was completed in 1.5 h; the b/a-ratio was slightly
improved and reached 3.3:1, as compared to that of 2.1:1, when
the reaction proceeded at room temperature (Table 2). At the ini-
tial reaction temperature of À5 °C, only a trace amount of reaction
products was registered after 3 h. The increase of the reaction tem-
perature to 0 °C also had no effect on the glycosylation yield,
resulting in accumulation of by-products. After the reaction tem-
perature was raised to 4 °C, the glycosylation completed in 2 h,
phy on silica gel afforded the required
A
(type 3)
tetrasaccharide 16 (42%) and its anomer 18 (18%). Both com-
a
pounds were acetylated, and the structures of resulting deriva-
tives 20 and 22 were confirmed by 1H, 2D-COSY, and 13C NMR
and MS data. At this stage, the stereochemistry of the newly
formed glycosylic bond was confirmed: 1H NMR of 20 showed a
and the b/a-ratio was 3.0:1. The decrease of the yields of glycosyl-
ation products in the model experiments are likely caused by in-
creased time without reaction progress at minus temperatures
accompanied by the accumulation of by-products.

20
I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
Ph
OAc
OAc
OAc
OAc
Ph
OAc
AcO
O
O
OAc
AcO
OAc
O
O
OAc
O
O
O
AcO
(a)
O
AcO
O
AcHN
O
X
+
O
AcHN
O
O
O
O
X
HO
O
O
N
OC(NH)CCl3
3Y
14 or 15
N3
OAc
OAc
8
12 or 1Y3
OAc
OAc
(b)
OAc
OAc
OAc
OAc
OAc
O
OAc
OAc
O
O
OAc
O
OH OH
AcO
OH OH
AcO
O
+
AcHN
O
X
O
AcHN
O
O
O
O
X
O
O
O
N
3Y
N
3Y
OAc
16 or 17
18 or 19
OAc
AcO
OAc
AcO
OAc
(c)
(c)
OAc
OAc
OAc
OAc
OAc
OAc
OAc
O
OAc
OAc
O
O
OAc
O
OAc
OAc
AcO
O
AcO
X
O
AcHN
O
AcHN
O
O
O
N
3Y
O
O
X
O
O
OAc
N
22 or 23
3Y
20 or 21
OAc
AcO
OAc
AcO
OAc
12, 14, 16, 18, 20, 22: X = H, Y = O(CH2)3NHC(O)CF3
13, 15, 17, 19, 21, 23: X = O(CH2)3NHC(O)CF3, Y = H
Scheme 3. Reagents and conditions: (a) TMSOTf, 3 Å MS, CH3CN; (b) AcOH (80%), 80 °C; (c) Ac2O/Py.
Table 2
reduction of the azido group resulted in only 25% total yield of tet-
rasaccharide 29. Therefore, dithiothreitol (DTT) was used for the
Influence of temperature and solvent on the stereochemistry of the glycosylation
reaction
reduction of the azido group in the case of the
a anomer of the A
Glycosyl acceptor
Solvent
Temperature (°C)
b/aa
Yield (%)
(type 3) tetrasaccharide. Application of DTT for reduction of the su-
gar azides in organic solvents, dichloromethane,30 and acetoni-
trile31 is known. The use of DTT aqueous media for reduction of
azido nucleosides has also been reported.32 Using DTT in 0.2 M
aq NaHCO3 (pH 8.2) for reduction of the azide in 26 allowed us
to get tetrasaccharide 28 in 75% total yield.
12
12
13
13
13
CH3CN
CH3CN
CH3CN
CH3CN
CH2Cl2
rt
2.3:1
3.0:1
2.1:1
3.3:1
1:1.6
79
70
81
69
77
À5?4
rt
À20?4
rt
a
The values of b/
meric mixtures.
a
ratio were estimated from the 1H NMR spectra of the ano-
3. Conclusions
Glycosylation of monosaccharide acceptors (9, 12, and 13) with
A trisaccharide trichloroacetimidate 8 preferentially leads to the
formation of b glycosides in spite of the absence of 2-O-acyl partic-
ipation. We suppose that the glycosyl donor structure is the main
reason for b-stereoselectivity: the bulky substituent at C-2, acety-
lated fucose, and conformational rigidity of the molecule due to
the presence of two monosaccharide residues in adjacent positions
[Fuc at C-2 and GalNAc at C-3] hinder the nucleophilic attack from
The experiment using CH2Cl2 as a solvent was carried out for
additional confirmation of the co-participation of acetonitrile in
the coupling. In this case, the glycosylation proceeded with reverse
stereoselectivity; the b/a-ratio was 1:1.6.
Thus, we can conclude that acetonitrile as a solvent and 4 °C are
the most favorable conditions for preferential formation of b glyco-
sides in glycosylation of galactosamine derivatives 12 and 13 with
the trisaccharide trichloroacetimidate 8.
the
a-side, thus resulting in the formation of b-glycosides. Addi-
The target A tetrasaccharides 2 (type 3) and 3 (type 4) were ob-
tained from the derivatives 20 and 21 by the following deprotec-
tion procedures: the Zemplén deacetylation, catalytic reduction
of azide over 10% Pd/C followed by N-acetylation, and alkali treat-
ment to remove the trifluoroacetic group (Scheme 4). Purification
of the tetrasaccharides by cation-exchange chromatography com-
pleted the synthesis and afforded the tetrasaccharides 2 and 3 in
77% and 64% yields, respectively.
tionally, the use of acetonitrile as a solvent upon glycosylation also
assists b-stereoselectivity.29
Synthesized here the A tetrasaccharides together with B tetra-
saccharides described earlier19 allowed us to perform two investi-
gations of carbohydrate-binding proteins. First, with the help of
Glyc-PAA-fluorescein probes [where Glyc are the blood group tet-
rasaccharides A and B; PAA is a poly(acrylamide) matrix] we have
found that tandem type galectins (-4, -8, and -9) are anchored on
the cell surface with the N-domain, whereas the C-carbohydrate
binding domain is exposed for external binding and displays high
Deprotection procedures for the
a anomer of the A (type 4) tet-
rasaccharide 23 were the same as those described for tetrasaccha-
ride 21. Unfortunately, the low yield at the step of catalytic

I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
21
OAc
OAc
OAc
OAc
OAc
OAc
AcO
O
OAc
O
OAc
O
OAc
O
OAc
O
OAc
OAc
AcO
AcO
X
AcHN
O
O
AcHN
O
O
O
O
O
O
X
N
O
3Y
N
3Y
OAc
OAc
AcO
OAc
OAc
22 or 23
20 or 21
(a)
(a)
OH
OH
OH
OH
OH
OH
OH
OH
O
O
OH
OH
OH
OH
HO
O
HO
O
O
X
O
AcHN
AcHN
O
O
O
O
N3
O
O
X
Y
N3
O
O
OH
OH
Y
HO
OH
HO
OH
26 or 27
(b) or (c)
24 or 25
(b)
OH
OH
O
OH
2 or 3
OH
O
OH
HO
OH
AcHN
O
O
O
20, 22, 24, 26: X = H, Y = O(CH2)3NHC(O)CF3
21, 23, 25, 27: X = O(CH2)3NHC(O)CF3, Y = H
28: X = H, Y = O(CH2)3NH2, 29: X = O(CH2)3NH2, Y= H
X
O
O
AcHN
OH
Y
HO
OH
28 or 29
Scheme 4. Reagents: (a) MeONa/MeOH; (b) (i) H2, Pd/C, MeOH/Ac2O; (ii) NaOH; (c) (i) DTT, aq NaHCO3 (pH 8.2), Ac2O; (ii) NaOH.
affinity for the blood group A and B tetrasaccharides.33 Second,
using the corresponding PAA-probes, and a printed glycan array
with the tetrasaccharides and Sepharose affinity adsorbents, we
have demonstrated that blood of the A blood group donors con-
tains formally autologous antibodies against the A antigen. These
antibodies incapable of binding A (type 2) tetrasaccharide are spe-
stants (J) were measured in Hertz. Signals of 1H NMR spectra were
assigned to the corresponding protons using 2D spectroscopy
(COSY). 13C NMR spectra were recorded at 150 MHz. Symbols of
monosaccharide residues in NMR spectra for trisaccharides: I—
b-Gal (reducing end), II— -Fuc, III— -GalNAc; for tetrasaccharides:
I— /b-GalNAc or b-GlcNAc (reducing end), II— /b-Gal, III— -Fuc,
IV— -GalNAc. ESIMS spectra were recorded on an Exactive Orbi-
a/
a
a
a
a
a
cific to the small GalNAc
a1-3Gal motif, and thus are not auto-
a
antibodies.34
trap (Thermo Fisher Scientific, Germany) spectrometer; HRESIMS
spectra were recorded on an Agilent 6224 TOF LC/MS instrument
(USA).
4. Experimental
4.1. 3-Aminopropyl 2-acetamido-2-deoxy-
anosyl-(1?3)-[ -fucopyranosyl-(1?2)]-b-
(1?3)-2-acetamido-2-deoxy-b- -glucopyranoside (1)
a
-
D
-galactopyr-
The reactions were performed with the use of commercial re-
agents (Acros, Aldrich, and Fluka); anhydrous solvents were puri-
a-
L
D-galactopyranosyl-
D
fied according to
the standard procedures. Column
chromatography was performed on Silica Gel 60 0.040–0.063 mm
(E. Merck), gel filtration was carried out on Sephadex LH-20 (Phar-
macia) columns (elution with 1:1 CHCl3–MeOH, unless otherwise
specified). Solvents were removed in vacuum at 30–40 °C. Thin-
layer chromatography (TLC) was performed on Silica Gel 60 F254
aluminium-backed plates (E. Merck). Spots of compounds were
visualized by dipping a TLC plate into an aqueous solution of
H3PO4 (8%) and subsequent heating (>150 °C). Deacetylation was
carried out in absolute MeOH by the addition of catalytic amount
of 2 M MeONa in MeOH (according to Zemplén). Na+ ions were
then removed by Dowex 50X4-400 (Acros) (H+) cation exchanger,
and the solution was evaporated. Hydrogenolysis was carried out
on 10% Pd/C (E. Merck) in the atmosphere of H2.
Tetrasaccharide 11 (85 mg, 0.065 mmol) was deacetylated
using Zemplén procedures. Water (2 mL) was then added to the
solution, MeOH was evaporated, and the reaction mixture was kept
for 3 h. Ion-exchange chromatography on Dowex 50X4-400 (H+)
(elution with 5% aq ammonia) afforded 50 mg (97%) of product 1
as a white foam: [
a]
+18 (c 0.50, 1:1 H2O–CH3CN), Rf 0.10
D
(100:10:10:10:2 EtOH–BuOH–Py–H2O–AcOH), 1H NMR (D2O):
(characteristic signals) d 1.25 (d, 3H, J5,6 6.6, H-6III), 1.90–1.99 (m,
2H, CCH2C), 2.04–2.09 (2s, 6H, 2 C(O)CH3), 3.05–3.09 (m, 2H,
NHCH2), 4.34 (q, 1H, J5,6 6.6, H-5III), 4.43 (d, 1H, J1,2 8.5, H-1I),
4.71 (d, 1H, J1,2 7.5, H-1II), 5.19 (d, 1H, J1,2 3.8, H-1IV), 5.27 (d, 1H,
J1,2 4.2, H-1III); 13C NMR (D2O): d 15.2 (C-6III), 22.0 (CH3C(O)),
22.3 (CH3C(O)), 26.8 (CCH2C), 37.5 (CCH2N), 49.7 (C-2IV), 54.8 (C-
2I), 60.7 (CCH2O), 61.2, 61.4, 63.0 (C-6I, C-6II, C-6IV), 66.7, 67.69,
67.70, 67.9, 68.6, 68.8, 69.8, 71.0, 71.9, 73.8, 74.9, 75.49, 75.53,
77.3 (C-2II, C-2III, C-3I, C-3II, C-3IV, C-3III, C-4I, C-4II, C-4IV, C-4III, C-
5I, C-5II, C-5III, C-5IV), 91.3 (C-1IV), 99.1, 100.0, 101.9 (C-1I, C-1II,
The values of optical rotations were measured on a Perkin–El-
mer 341 polarimeter at 21 2 °C. 1H NMR spectra were recorded
on a Bruker BioSpin GmbH (700 MHz) spectrometer at 30 °C;
chemical shifts (d-units) were referred to the peak of internal
D2O (d 4.750), CDCl3 (d 7.270), or CD3OD (d 3.500); coupling con-

22
I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
C-1III), 174.0 (CH3C(O)), 174.9 (CH3C(O)); HRESIMS, m/z: Calcd
[C31H55N3O20]H+: m/z 790.3452. Found: m/z 790.3453.
its color from dark brown to green in 5 min. The solution was
stirred for 3 h, then treated with (COOH)2Á2H2O (to pH 4), and
the reaction mixture was left overnight at À20 °C. The solution
was concentrated in vacuo; the residue was washed with 2:1
EtOAc–C6H6 (100 mL) and acetylated (Ac2O/Py, 48 h). The reaction
mixture was poured into ice and extracted with CHCl3
(3 Â 70 mL). The organic fraction was washed with H2O
(3 Â 50 mL), the aq fraction was then extracted with CHCl3
(2 Â 20 mL). The combined CHCl3 fractions were dried by filtra-
tion through cotton wool and concentrated. Column chromatog-
raphy on silica gel (6:3:1 n-C6H14–CHCl3–2-PrOH) yielded
4.2. 3-Aminopropyl 2-acetamido-2-deoxy-
osyl-(1?3)-[ -fucopyranosyl-(1?2)]-b- -galactopyranosyl-
(1?3)-2-acetamido-2-deoxy- -galactopyranoside (2)
a-D-galactopyran-
a
-L
D
a-D
Tetrasaccharide 20 (62 mg, 0.048 mmol) was deacetylated un-
der Zemplén conditions, purified by gel filtration (1:1 CH3CN–
H2O), and subjected to hydrogenolysis (3 h) in a mixture of MeOH
(5 mL) and Ac2O (200
and concentrated. The residue was dissolved in H2O (3 mL), an aq
solution of NaOH (2 M, 5 L) was added, and the reaction mixture
lL). The reaction mixture was then filtered
benzoxazole 5 (1.04 g, 92%) as white foam, [
a]
À2 (c 1, CHCl3),
D
l
Rf 0.50 (4:3:1 n-C6H14–CHCl3–2-PrOH),1H NMR (CDCl3): d 1.08
(d, 3H, J5,6 6.5, H-6II), 1.37 (s, 9H, C(CH3)3), 1.49 (s, 9H, –
C(CH3)3), 1.96–2.17 (9s, 27 H, 8 OC(O)CH3, NHC(O)CH3), 3.31–
3.35 (m, 2H, CCH2), 3.79–3.83 (m, 2H, H-2I, OCHH), 3.88 (dd,
1H, J2,3 9.3, J3,4 3.5, H-3I), 4.02–4.16 (m, 5H, H-6aI, H-6bI, H-6aIII,
H-6bIII, OCHH), 4.27 (dd, 1H, J5,6a 6.1, J5,6b 6.1, H-5III), 4.41–4.51
(m, 3H, H-5I, H-2III, H-5II), 4.52 (d, 1H, J1,2 7.4, H-1I), 5.02 (dd,
1H, J2,3 11.4, J3,4 3.2, H-3III), 5.23–5.26 (m, 3H, H-1III, H-3II, H-
4II), 5.32 (dd, 1H, J1,2 3.7, J2,3 11.2, H-2II), 5.38 (d, 1H, J3,4 3.2, H-
4III), 5.47 (d, 1H, J3,4 3.5, H-4I), 5.51 (d, 1H, J1,2 3.7, H-1II), 6.13
(d, 1H, J2,NH 8.4, NHAcIII), 7.28 (d, 1H, J 1.7, ArH), 7.57 (d, 1H, J
1.7, ArH); 13C NMR (CDCl3): d 15.9 (C-6II), 20.6–20.6 (8OC(O)CH3),
23.2 (NC(O)CH3), 29.3 (CCH2), 29.9 (C(CH3)3), 31.8 (C(CH3)3), 34.5
(C(CH3)3), 35.2 (C(CH3)3), 47.9 (C-2III), 61.1 (C-6I), 62.8 (C-6III),
65.2 (C-5II), 66.5 (C-4III), 66.8 (OCH2), 67.2 (C-4I), 67.39 (C-2II),
67.43 (C-3II), 67.7 (C-3III), 68.1 (C-5III), 70.3 (C-5I), 71.2 (C-4II),
75.5 (C-2I), 77.8 (C-3I), 96.7 (C-1II), 97.1 (C-1III), 102.6 (C-1I),
113.3, 119.9, 134.0, 139.1, 146.8, 148.4, 163.5 (C-Ar), 169.8–
171.0 (9C(O)CH3); HRESIMS: Calcd [C53H74N2O24]H+: m/z
1123.4704. Found: m/z 1123.4836.
was kept for 3 h at rt. Ion-exchange chromatography on Dowex
50X4-400 (H+) (elution with 5% aq ammonia) gave 29 mg (77%)
of product 2 as white foam: [a] +99 (c 0.4, 1:1 H2O–CH3CN), Rf
D
0.17 (100:10:10:10:2 EtOH–BuOH–Py–H2O–AcOH), 1H NMR
(D2O): (characteristic signals) d 1.21 (d, 3H, J5,6 6.6, H-6III), 1.95–
2.00 (m, 2H, CCH2C), 2.03–2.08 (2s, 6H, 2 C(O)CH3), 3.09–3.13 (m,
2H, NHCH2), 4.30 (q, 1H, J5,6 6.6, H-5III), 4.66 (d, 1H, J1,2 7.5, H-
1II), 4.88 (d, 1H, J1,2 3.5, H-1I), 5.16 (d, 1H, J1,2 3.8, H-1IV), 5.27 (d,
1H, J1,2 4.2, H-1III); 13C NMR (D2O): d 15.3 (C-6III), 21.9 (CH3C(O)),
22.0 (CH3C(O)), 26.8 (CCH2C), 37.1 (CCH2N), 49.4, 49.6 (C-2I, C-
2IV), 61.1, 61.3, 61.5 (C-6I, C-6II, C-6IV), 64.9 (CCH2O), 63.1, 66.9,
67.7, 67.8, 68.6, 69.2, 70.0, 70.7, 71.0, 71.8, 72.8, 74.4, 74.9, 75.5
(C-2II, C-2III, C-3I, C-3II, C-3III, C-3IV, C-4I, C-4II, C-4III, C-4IV, C-5I, C-
5II, C-5III, C-5IV), 91.3 (C-1IV), 96.7 (C-1I), 98.8 (C-1III), 102.2 (C-
1II), 173.6 (CH3C(O)), 174.9 (CH3C(O)); HRESIMS, Calcd
[C31H55N3O20]H+: m/z 790.3452. Found: m/z 790.3451.
4.3. 3-Aminopropyl 2-acetamido-2-deoxy-
a-D-
galactopyranosyl-(1?3)-[ -fucopyranosyl-(1?2)]-b-D-
a-
L
galactopyranosyl-(1?3)-2-acetamido-2-deoxy-b-D-
galactopyranoside (3)
4.5. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-D-
galactopyranosyl-(1?3)-[2,3,4–tri-O-acetyl-a-L-fucopyranosyl-
Tetrasaccharide 21 (102 mg, 0.079 mmol) was deacetylated un-
der Zemplén conditions. The product obtained was purified by gel
filtration (1:1 CH3CN–H2O) and subjected to hydrogenolysis (5 h)
(1?2)]-4,6-di-O-acetyl-b-D-galactopyranoside
trichloroacetimidate (8)
in a mixture of MeOH (5 mL) and Ac2O (300
was then filtered and concentrated. The residue was dissolved in
H2O (4 mL), an aq solution of NaOH (2 M, 5 L) was added, and
l
L). Reaction mixture
A solution of benzoxazole 5 (1.04 g, 0.930 mmol) and NaOAc
(2.5 g, 31 mmol) in a mixture of 1:1 AcOH–Ac2O (37 mL) was
sealed in glass ampoules and kept at 100 °C for 10 days. The am-
poules were then opened, and reaction mixture was poured onto
ice and extracted with CHCl3 (3 Â 75 mL).The organic fraction
was washed with cold satd aq NaHCO3 (2 Â 150 mL) and H2O
(2 Â 150 mL), and the aqueous fraction was extracted with CHCl3
(2 Â 100 mL). The combined CHCl3 fractions were dried by filtra-
tion through cotton wool and concentrated. Column chromatogra-
phy on silica gel (6:3:1 n-C6H14–CHCl3–2-PrOH) yielded the
mixture of anomeric acetates 6 (810 mg, 96%) as white foam, Rf
0.42 (4:3:1 n-C6H14–CHCl3–2-PrOH), ESIMS: Calcd [C38H53NO24]H+
m/z 908.30. Found: m/z 908.34.
A solution of anomeric acetates 6 (300 mg, 0.330 mmol) in 3 mL
of DMF was heated to 50 °C with stirring, and N2H4ÁHOAc (43 mg,
0.46 mmol) was then added. The reaction was stirred for 20 min,
diluted with CHCl3 (10 mL), and washed with H2O (3 Â 20 mL).
The aq fraction was extracted with CHCl3 (2 Â 5 mL). The com-
bined CHCl3 fractions were dried by filtration through cotton wool
and concentrated. Column chromatography on silica gel (5:3:1 n-
C6H14–CHCl3–2-PrOH) afforded 270 mg (95%) of hemiacetal 7 as
white foam, Rf 0.26 (4:3:1 n-C6H14–CHCl3–2-PrOH).
l
the reaction mixture was kept for 2 h. Cation-exchange chromatog-
raphy on Dowex 50X4-400 (H+) (elution with 5% aq ammonia) gave
40 mg (64%) of product 3 as white foam: [a] +3 (c 0.4, 1:1 H2O–
D
CH3CN), Rf 0.14 (100:10:10:10:2 EtOH–BuOH–Py–H2O–AcOH), 1H
NMR (D2O): (characteristic signals) d 1.21 (d, 3H, J5,6 6.6, H-6III),
1.88–1.98 (m, 2H, CCH2C), 2.03–2.07 (2s, 6H, 2C(O)CH3), 3.06–
3.09 (m, 2H, NHCH2), 4.33 (d, 1H, J1,2 8.2, H-1I), 4.66 (d, 1H, J1,2
7.8, H-1II), 5.17 (d, 1H, J1,2 3.7, H-1IV), 5.28 (d, 1H, J1,2 4.0, H-1III);
13C NMR (D2O): d 15.3 (C-6III), 22.0 (CH3C(O)), 22.3 (CH3C(O)),
26.7 (CCH2C), 37.7 (CCH2N), 49.6, 51.3 (C-2I, C-2IV), 61.0, 61.1,
61.4 (C-6I, C-6II, C-6IV), 63.1 (CCH2O), 66.9, 67.7, 67.8, 68.0, 68.54,
68.56, 68.57, 69.9, 71.0, 71.8, 72.8, 74.9, 75.6, 77.1 (C-2II, C-2III, C-
3I, C-3II, C-3III, C-3IV, C-4I, C-4II, C-4III, C-4IV, C-5I, C-5II, C-5III, C-
5IV), 91.3 (C-1IV), 98.8 (C-1III), 102.2 (C-1II), 102.7 (C-1I) 174.0
(CH3C(O)), 174.9 (CH3C(O)), HRESIMS, Calcd [C31H55N3O20]H+: m/z
790.3452. Found: m/z 790.3453.
4.4. 2-(4,6-Di-tert-butylbenzoxazol-2-yl)ethyl 2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-
tri-O-acetyl- -fucopyranosyl-(1?2)]-4,6-di-O-acetyl-b-
galactopyranoside (5)
a-D-galactopyranosyl-(1?3)-[2,3,4–
a-
L
D
-
A stirred mixture of derivative 7 (270 mg, 0.312 mmol), Cl3CCN
(334
l
L, 3.37 mmol), and CH2Cl2 (5 mL) was cooled to À20 °C at
stirring, and then DBU (5
l
L, 0.03 mmol) was added. The reaction
3,5-Di-tert-butyl-o-benzoquinone (448 mg, 2.00 mmol) was
added with stirring to a solution of trisaccharide 4 (590 mg,
1.01 mmol) in 30 mL of MeOH. The reaction mixture changed
mixture was stirred for 3.5 h and concentrated. Column chroma-
tography on silica gel (1:3 PhCH3–EtOAc, 1% Et3N) gave trichloro-
acetimidate 8 (284 mg, 90%) as white foam: Rf 0.25 (1:1 n-C6H14
–

I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
23
acetone, 1% Et3N), 1H NMR (CDCl3): d 1.09 (d, 3H, J5,6 6.5, H-6II),
1.94–2.22 (9s, 27 H, 8 OC(O)CH3, NHC(O)CH3), 4.04–4.09 (m, 3H,
H-6aI, H-6aIII, H-6bIII), 4.11 (dd, 1H, J1,2 3.7, J2,3 9.8, H-2I), 4.18–
4.23 (m, 2H, H-6bI, H-5II), 4.28 (dd, 1H, J5,6a 6.6, J5,6b 6.6, H-5III),
4.30 (dd, 1H, J2,3 9.8, J3,4 3.7, H-3I), 4.37 (dd, 1H, J5,6a 6.6, J5,6b 6.6,
H-5I), 4.55 (ddd, 1H, J1,2 3.6, J2,3 11.3, J2,NH 8.8, H-2III), 5.01 (dd,
1H, J2,3 11.3, J3,4 3.1,H-3III), 5.22–5.24 (m, 2H, H-3II, H-4II), 5.31 (d,
1H, J1,2 3.6, H-1III), 5.32–5.35 (m, 1H, H-2II), 5.39 (d, 1H, J1,2 3.6,
H-1II), 5.44 (d, 1H, J3,4 3.1, H-4III), 5.54 (d, 1H, J3,4 3.7, H-4I), 5.90
(d, 1H, J2,NH 8.8, NHAcIII), 6.48 (d, 1H, J1,2 3.7, H-1I), 8.62 (s, 1H,
4.7. 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy- -galactopyranosyl-(1?3)-[2,3,4-tri-O-acetyl-
fucopyranosyl-(1?2)]-4,6-di-O-acetyl-b- -galactopyranosyl-
(1?3)-4,6-di-O-acetyl-2-azido-2-deoxy- -galactopyranoside
(20) and 3-trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy- -galactopyranosyl-(1?3)-[2,3,4-tri-O-acetyl-
-fucopyranosyl-(1?2)]-4,6-di-O-acetyl- -galactopyran-
-galactopyran-
a
-
D
a-L-
D
a-D
a-D
a-
L
a-D
osyl-(1?3)-4,6-di-O-acetyl-2-azido-2-deoxy-a-D
oside (22)
C(NH)CCl3); 13C NMR (CDCl3):
d
16.0 (C-6II), 20.5–20.8 (8
A solution of trichloroacetimidate 8 (411 mg, 0.407 mmol) and
glycosyl acceptor 12 (366 mg, 0.821 mmol) in anhyd CH3CN
(18 mL) was stirred with 3 Å molecular sieves (700 mg) for
30 min at rt under an atmosphere of N2. A solution of TMSOTf
OC(O)CH3), 23.2 (NC(O)CH3), 47.9 (C-2III), 61.4 (C-6I), 62.1 (C-6III),
65.8 (C-5II), 66.9 (C-2II), 67.1 (C-4I), 67.28 (C-4III), 67.34 (C-3II),
67.76 (C-5III), 68.8 (C-5I), 71.1 (C-4II), 73.5 (C-3I), 74.6 (C-2I), 90.7
(C(NH)CCl3), 94.7 (C-1I), 96.8 (C-1III), 98.3 (C-1II), 160.8
(C(NH)CCl3), 169.8–170.6 (9C(O)CH3), ESIMS: Calcd [C38H51N2
O23Cl3]H+: m/z 1011.20. Found: m/z 1011.24.
(7
ture was stirred for 3 h, and it was then neutralized by Et3N
(10 L), filtered, and concentrated. Gel filtration followed by col-
lL, 0.04 mmol) in anhyd CH3CN (100 lL) was added, the mix-
l
umn chromatography on silica gel (6:3:1?4:3:1 n-C6H14–CHCl3–
2-PrOH) yielded a mixture of anomers 14 (416 mg, 79%) with a
4.6. 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-
b/
a
ratio of 2.5:1.
2-deoxy-
a
-D
-galactopyranosyl-(1?3)-[2,3,4-tri-O-acetyl-
a-L-
A mixture of anomeric glycosides 14 (416 mg, 0.322) was dis-
fucopyranosyl-(1?2)]-4,6-di-O-acetyl-b-
(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-b-D-
glucopyranoside (11)
D
-galactopyranosyl-
solved in 20 mL of AcOH (80%), kept for 1 h at 80 °C, concentrated,
and co-evaporated with PhCH3 (2 Â 15 mL). Column chromatogra-
phy on silica gel (3:3:1 n-C6H14–CHCl3–2-PrOH) resulted in a frac-
tion (212 mg) containing tetrasaccharide 16, Rf 0.12 (3:3:1 n-
C6H14–CHCl3–2-PrOH), and pure tetrasaccharide 18 (91 mg, 18%)
A mixture of trichloroacetimidate 8 (269 mg, 0.266 mmol), gly-
cosyl acceptor 9 (246 mg, 0.532 mmol), anhyd CH3CN (7 mL), and
anhyd CH2Cl2 (7 mL) was stirred with 3 Å molecular sieves
(500 mg) for 30 min under an atmosphere of N2. A solution of
as white foam, [
CHCl3–2-PrOH).
a]
+90 (c 1, CHCl3), Rf 0.25 (3:3:1 n-C6H14–
D
The ‘mix fraction’ was acetylated (Ac2O/Py, 12 h) and the solu-
tion was evaporated. Column chromatography on silica gel (6:3:1
n-C6H14–CHCl3–2-PrOH) afforded tetrasaccharide 20 (220 mg,
TMSOTf (16
added, and the reaction mixture was stirred for 5 h. The mixture
was neutralized with Et3N (50 L) and filtered. The filtrate was
lL, 0.090 mmol) in anhyd CH3CN (0.3 mL) was then
l
42%) as white foam, [a] +64 (c 1, CHCl3), Rf 0.50 (3:3:1 n-C6H14–
D
CHCl3–2-PrOH). 1H NMR (CDCl3): d 1.11 (d, 3H, J5,6 6.6, H-6III),
1.94–2.00 (m, 14 H, CCH2C, 4 OC(O)CH3), 2.06–2.20 (7s, 21 H, 6
OC(O)CH3, NHC(O)CH3), 3.52 (dd, 1H, J1,2 3.6, J2,3 10.9, H-2I),
3.53–3.58 (m, 2H, NCH2), 3.59–3.64 (m, 1H, OCHH), 3.78 (dd, 1H,
J1,2 7.4, J2,3 9.3, H-2II), 3.82–3.86 (m, 2H, H-5II, OCHH), 3.87 (dd,
1H, J2,3 9.3, J3,4 3.4, H-3II), 4.00–4.19 (m, 7H, H-5I, H-6aI, H-6bI, H-
6aII, H-6bII, H-6aIV, H-6bIV), 4.19–4.23 (m, 2H, H-3I, H-5IV), 4.47
(ddd, 1H, J1,2 3.4, J2,3 11.2, J2,NH 8.2, H-2IV), 4.67 (q, 1H, J5,6 6.6, H-
5III), 4.69 (d, 1H, J1,2 7.4, H-1II), 5.01 (dd, 1H, J2,3 11.2, J3,4 3.1, H-
3IV), 5.06 (d, 1H, J1,2 3.6, H-1I), 5.26 (d, 1H, J1,2 3.4, H-1IV), 5.28–
5.35 (m, 3H, H-2III, H-3III, H-4III), 5.38 (d, 1H, J3,4 3.4, H-4II), 5.47
(br s, 1H, H-4IV), 5.52–5.55 (m, 2H, H-4I, H-1III), 6.31 (d, 1H, J2,NH
8.2, NHAcIV), 6.83–6.90 (m, 1H, NHC(O)CF3); 13C NMR (CDCl3): d
15.8 (C-6III), 20.5–20.8 (10 OC(O)CH3), 23.0 (NC(O)CH3), 28.7
(CCH2C), 37.7 (CCH2N), 48.2 (C-2IV), 58.6 (C-2I), 61.0 (C-6I), 62.4
(C-6II), 62.8 (H-6IV), 64.9 (H-5III), 66.5 (CCH2O), 66.8 (C-4II), 67.2
(C-2III), 67.4 (C-3IV), 67.5 (C-4IV), 67.6 (C-5IV), 67.8 (C-3III), 68.8
(C-5I), 70.0 (C-4I), 70.6 (C-5II), 71.6 (C-4III), 73.0 (C-3I), 74.1 (C-
2II), 78.9 (C-3II), 96.2 (C-1III), 98.1 (C-1IV), 98.8 (C-1I), 102.5 (C-
1II), 115.9 (C(O)CF3), 157.2 (C(O)CF3), 169.3–171.2 (11C(O)CH3);
HRESIMS: Calcd [C50H70N5O30F3]H+: m/z 1290.4130. Found: m/z
1290.4047.
concentrated and subjected to gel filtration followed by column
chromatography on silica gel (1:1?1:2 n-C6H14–acetone) to af-
ford compound 10 (282 mg) as a colorless oil containing about
10% of impurities, which was immediately used for the next step.
The colorless oil was dissolved in AcOH (80%), kept for 1 h at
80 °C, concentrated, and co-evaporated with PhCH3 (2 Â 15 mL).
The residue was acetylated (Ac2O/Py, 10 h), concentrated, co-
evaporated with PhCH3 (4 Â 10 mL), and subjected to column
chromatography on silica gel (2:3 n-C6H14–acetone) to give tetra-
saccharide 11 (194 mg, 56%) as white foam: [a] +13 (c 1, CHCl3),
D
Rf 0.22 (2:3 n-C6H14–acetone), 1H NMR (CDCl3): d 1.21 (d, 3H, J5,6
6.5, H-6III), 1.85–1.94 (m, 2H, CCH2C), 1.98–2.18 (12s, 36 H, 10
OC(O)CH3, 2 NHC(O)CH3), 2.91–2.98 (m, 1H, H-2I), 3.48–3.54 (m,
2H, NCH2), 3.68–3.78 (m, 4H, H-5I, H-3II, H-5II, OCHH), 3.81 (dd,
1H, J1,2 7.5, J2,3 9.6, H-2II), 3.90–3.95 (m, 1H, OCHH), 3.98–4.04
(m, 1H, H-6aIV), 4.05–4.10 (m, 3H, H-6aI, H-6bI, H-6bIV), 4.11–
4.15 (m, 1H, H-5IV), 4.18 (dd, 1H, J5,6a 2.4, J6a,6b 12.3, H-6aII),
4.24 (dd, 1H, J5,6b 4.9, J6a,6b 12.3, H-6bII), 4.37 (d, 1H, J1,2 7.5, H-
1II), 4.48 (ddd, 1H, J1,2 3.5, J2,3 11.5, J2,NH 9.1, H-2IV), 4.68–4.75
(m, 2H, H-3I, H-5III), 4.84 (dd, 1H, J3,4 9.4, J4,5 9.6, H-4I), 4.98
(dd, 1H, J2,3 11.5, J3,4 3.1, H-3IV), 5.02 (d, 1H, J2,1 8.0, H-1I), 5.23
(d, 1H, J1,2 3.5, H-1IV), 5.27 (br s, 1H, H-4III), 5.31–5.35 (m, 2H,
H-2III, H-3III), 5.36 (d, 1H, J3,4, H-4II), 5.41 (br s, 1H, H-4IV), 5.63
(d, 1H, J2,1 2.6, H-1III), 6.30–6.45 (m, 1H, NHAcIV), 6.92 (d, 1H,
J2,NH 6.1, NHAcI), 7.14–7.21 (m, 1H, NHC(O)CF3); 13C NMR (CDCl3):
d 15.4 (C-6III), 20.6–20.8 (10 OC(O)CH3), 23.0 (NC(O)CH3), 23.1
(NC(O)CH3), 28.3 (CCH2C), 37.4 (CCH2N), 48.1 (C-2IV), 58.8 (C-2I),
60.6 (C-6I), 62.2 (C-6II), 62.8 (H-6IV), 65.5 (H-5III), 66.9 (C-4II),
67.2 (C-3IV), 67.29 (C-2III), 67.35 (C-4IV), 67.7 (CCH2O), 68.1 (C-
3III), 68.4 (C-5IV), 68.9 (C-4I), 70.5 (C-5II), 71.4 (C-4III), 72.0 (C-
5I), 72.6 (C-2II), 75.3 (C-3I), 79.6 (C-3II), 95.7 (C-1III), 98.3 (C-1IV),
99.3 (C-1I), 101.5 (C-1II), 116.0 (C(O)CF3), 157.3 (C(O)CF3),
169.3–172.3 (12C(O)CH3); HRESIMS: Calcd [C53H74N3O31F3]H+:
m/z 1306.4331. Found: m/z 1306.4353.
Tetrasaccharide 18 was acetylated (Ac2O/Py, 12 h), and the
solution was evaporated. Column chromatography on silica gel
(n-C6H14–CHCl3–2-PrOH, 6:3:1) gave tetrasaccharide 22 (90 mg,
92%) as white foam: [a] +68 (c 1, CHCl3), Rf 0.53 (3:3:1 n-C6H14–
D
CHCl3–2-PrOH), 1H NMR (CDCl3): d 1.17 (d, 3H, J5,6 6.5, H-6III),
1.90–1.96 (m, 5H, CCH2C, OC(O)CH3), 1.97–2.21 (10s, 30 H, 9
OC(O)CH3, NHC(O)CH3), 3.33–3.41 (m, 1H, NCHH), 3.52–3.59 (m,
1H, OCHH), 3.63–3.70 (m, 1H, NCHH), 3.76–3.84 (m, 1H, H-2I),
3.87–3.83 (m, 1H, OCHH), 3.97–4.06 (m, 4H, H-2II, H-6aII, H-6bII,
H-6aIV), 4.10–4.18 (m, 5H, H-3I, H-5I, H-6aI, H-6bI, H-6bIV), 4.18–
4.22 (m, 1H, H-5IV), 4.24 (dd, 1H, J2,3 8.5, J3,4 3.3, H-3II), 4.25–4.29
(m, 2H, H-5II, H-5III), 4.51 (ddd, 1H, J1,2 3.5, J2,3 11.8, J2,NH 8.6, H-

24
I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
2IV), 5.00 (d, 1H, J1,2 3.6, H-1I), 5.07–5.13 (m, 1H, H-3IV), 5.16 (dd,
1H, J2,3 10.8, J3,4 3.6, H-3III), 5.25 (d, 1H, J1,2 3.5, H-1IV), 5.27 (d,
1H, J3,4 3.6, H-4III), 5.28 (d, 1H, J1,2 4.1, H-1II), 5.32 (dd, 1H, J1,2
2.4, J2,3 10.8, H-2III), 5.37–5.40 (m, 1H, H-1III), 5.42 (d, 1H, J3,4 2.7,
H-4IV), 5.47 (dd, 1H, J3,4 3.3, J4,5 3.1, H-4II), 5.69 (d, 1H, J3,4 2.65,
H-4I), 6.26 (d, 1H, J2,NH 8.6, NHAcIV), 7.38–7.42 (m, 1H, NHC(O)CF3);
13C NMR (CDCl3): d 15.9 (C-6III), 20.5–20.8 (10 OC(O)CH3), 23.0
(NC(O)CH3), 28.3 (CCH2C), 38.1 (CCH2N), 48.3 (C-2IV), 59.8 (C-2I),
60.7 (C-6II), 62.2 (C-6I), 62.5 (H-6IV), 65.0 (C-5III), 66.8 (C-3III),
66.9 (C-4I), 67.3 (CCH2O), 67.4 (C-4IV), 67.47 (C-3IV), 67.53 (C-3I),
67.6 (C-4II), 68.21 (C-2III), 68.23 (C-5IV), 68.6 (C-5II), 70.9 (C-4III),
72.37 (C-5I), 72.39 (C-2II), 74.2 (C-3II), 94.8 (C-1II), 96.3 (C-1III),
97.9 (C-1I), 98.3 (C-1IV), 116.0 (C(O)CF3), 157.3 (C(O)CF3), 169.8–
170.7 (11C(O)CH3); HRESIMS: Calcd [C50H70N5O30F3]H+: m/z
1290.4130. Found: m/z 1290.4024.
jected to column chromatography on silica gel (4:3:1 n-C6H14–
CHCl3–2-PrOH, for tetrasaccharide 17, and 5:3:1 n-C6H14–CHCl3–
2-PrOH, for tetrasaccharide 19 correspondingly) to afford tetrasac-
charide 21 (232 mg, 45%), and tetrasaccharide 23 (114 mg, 22%).
Compound 21: white foam, [
a]
+4 (c 1, CHCl3), Rf 0.25 (n-
D
C6H14–CHCl3–i-PrOH, 4:3:1), 1H NMR (CDCl3): d 1.17 (d, 3H, J5,6
6.5, H-6III), 1.94–2.00 (m, 11 H, CCH2C, 3 OC(O)CH3), 2.05–2.19
(8s, 24 H, 7 OC(O)CH3, NHC(O)CH3), 3.50–3.54 (m, 1H, NCHH),
3.55–3.60 (m, 1H, NCHH), 3.64–3.70 (m, 2H, H-2I, H-3I), 3.75 (dd,
1H, J1,2 7.3, J2,3 9.1, H-2II), 3.76–3.81 (m, 2H, H-3II, H-5II), 3.83–
3.87 (m, 2H, H-5I, OCHH), 3.97–4.01 (m, 1H, OCHH), 4.05–4.19
(m, 6H, H-6aI, H-6bI, H-6aII, H-6bII, H-6aIV, H6bIV), 4.23 (dd, 1H,
J5,6a 5.8, J5,6b 5.8, H-5IV), 4.42 (d, 1H, J1,2 7.2, H-1I), 4.49 (ddd, 1H,
J1,2 3.3, J2,3 11.3, J2,NH 7.2, H-2IV), 4.56 (q, 1H, J5,6 6.5, H-5III), 4.74
(d, 1H, J1,2 7.3, H-1II), 5.03 (dd, 1H, J2,3 11.3, J3,4 2.9, H-3IV), 5.26
(d, 1H, J1,2 3.3, H-1IV), 5.30 (dd, 1H, J1,2 3.4, J2,3 10.2, H-2III), 5.31–
5.34 (m, 2H, H-3III, H-4III), 5.37–5.39 (m, 2H, H-4I, H-4II), 5.47 (d,
1H, H-4IV), 5.56 (d, 1H, J1,2 3.37, H-1III), 6.21 (d, 1H, J2,NH 7.2, NHA-
cIV), 6.97–7.02 (m, 1H, NHC(O)CF3); 13C NMR (CDCl3): d 16.1 (C-6III),
20.5–20.7 (10OC(O)CH3), 23.0 (NC(O)CH3), 28.5 (CCH2C), 38.0
(CCH2N), 48.1 (C-2IV), 61.1 (C-6I), 62.1 (C-6II), 62.3 (C-2I), 62.6 (C-
6IV), 65.3 (C-5III), 66.3 (C-4II), 67.4 (C-4IV), 67.5 (C-3IV), 67.67 (C-
3III), 67.72 (C-2III), 68.4 (C-5IV), 68.7 (C-4I), 68.8 (CCH2O), 70.6 (C-
3I), 71.4 (C-4III), 71.8 (C-5I), 74.8 (C-2II), 75.4 (C-3II), 77.6 (C-5II),
96.2 (C-1III), 97.2 (C-1IV), 102.1 (C-1I), 102.8 (C-1II), 115.9
(C(O)CF3), 157.2 (C(O)CF3), 169.5–170.9 (11C(O)CH3); HRESIMS:
Calcd [C50H70N5O30F3]H+: m/z 1290.4130. Found: m/z 1290.4047.
4.8. 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-
fucopyranosyl-(1?2)]-4,6-di-O-acetyl-b-
(1?3)-4,6-di-O-acetyl-2-azido-2-deoxy-b-
(21) and 3-trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy- -galactopyranosyl-(1?3)-[2,3,4-tri-O-acetyl-
-fucopyranosyl-(1?2)]-4,6-di-O-acetyl- -galactopyran-
-galactopyran-
a
-D
-galactopyranosyl-(1?3)-[2,3,4-tri-O-acetyl-
-galactopyranosyl-
-galactopyranoside
a-L-
D
D
a-D
a-
L
a-D
osyl-(1?3)-4,6-di-O-acetyl-2-azido-2-deoxy-b-
D
oside (23)
A solution of trichloroacetimidate 8 (400 mg, 0.396 mmol) and
glycosyl acceptor 13 (353 mg, 0.791 mmol) in anhyd CH3CN
(18 mL) was stirred with 3 Å molecular sieves (700 mg) for
30 min at rt under an atmosphere of N2. A solution of TMSOTf
Compound 23: white foam, [a] +39 (c 1, CHCl3), Rf 0.36 (4:3:1
D
n-C6H14–CHCl3–2-PrOH), 1H NMR (CDCl3–CD3OD, 1:1): d 1.37 (d,
3H, J5,6 6.5, H-6III), 2.08–2.12 (m, 2H, CCH2C), 2.13–2.39 (11s, 33
H, 10 OC(O)CH3, NHC(O)CH3), 3.57–3.68 (m, 2H, NCH2), 3.81 (dd,
1H, J1,2 7.8, J2,3 10.6, H-2I), 3.86–3.90 (m, 1H, OCHH), 3.92 (dd,
1H, J2,3 10.6, J3,4 3.0, H-3I), 4.05 (dd, 1H, J5,6a 6.3, J5,6b 6.3, H-5I),
4.15–4.19 (m, 1H, OCHH), 4.22 (dd, 1H, J5,6a 6.9, J6,6b 11.1, H-6aI),
4.24–4.30 (m, 3H, H-2II, H-6aI, H-6aII), 4.30–4.36 (m, 3H, H-6bI,
H-6bII, H6bIV), 4.36–4.39 (m, 1H, H-5IV), 4.40 (dd, J2,3 9.3, J3,4 2.9,
H-3II), 4.44–4.49 (m, 1H, H-5III), 4.57 (d, 1H, J1,2 7.8, H-1I), 4.58–
4.60 (m, 1H, H-5II), 4.62 (dd, 1H, J1,2 3.4, J2,3 11.6, H-2IV), 5.27 (d,
1H, J2,3 11.6, H-3IV), 5.41–5.44 (m, 2H, H-1IV, H-2III), 5.45 (d, 1H,
J1,2 3.4, H-1II), 5.47 (dd, 1H, J2,3 10.8, J3,4 3.3, H-3III), 5.50 (d, 1H,
J3,4 3.3, H-4III), 5.61 (d, 1H, J3,4 2.5, H-4IV), 5.62–5.64 (m, 1H, H-
1III), 5.66–5.70 (m, 2H, H-4I, H-4II); 13C NMR (CDCl3): d 16.0 (C-
6III), 20.5–20.8 (10 OC(O)CH3), 23.0 (NC(O)CH3), 28.4 (CCH2C),
38.2 (CCH2N), 48.4 (C-2IV), 61.2 (C-6I), 61.8 (C-6II), 62.4 (C-6IV),
62.7 (C-2I), 65.1 (C-5III), 65.4 (C-4II), 67.1 (C-2III), 67.4 (C-4IV),
67.5 (C-3IV), 67.6 (C-4I), 68.0 (C-3III), 68.1 (C-5IV), 68.9 (C-5II),
69.1 (CCH2O), 71.0 (C-4III), 71.3 (C-3I), 72.4 (C-5I), 73.6 (C-3II),
74.4 (C-2II), 94.5 (C-1II), 96.4 (C-1III), 97.6 (C-1IV), 102.5 (C-1I),
116.0 (C(O)CF3), 157.1 (C(O)CF3), 169.8–170.7 (11C(O)CH3); HRE-
SIMS: Calcd [C50H70N5O30F3]H+: m/z 1290.4130. Found: m/z
1290.4046.
(7
the reaction mixture was stirred for 3 h. The mixture was neutral-
ized with Et3N (10 L), filtered, and concentrated. Column chroma-
lL, 0.04 mmol) in anhyd CH3CN (100 lL) was then added, and
l
tography on silica gel (1:1?2:3 n-C6H14–acetone) afforded the
mixture of anomeric tetrasaccharides 15 (413 mg, 81%) with a b/
a
ratio of 2:1.
Pure tetrasaccharide 15-b was also isolated in analytical
amounts as white foam: [
a]
À18 (c 1, CHCl3), Rf 0.25 (4:3:1 n-
D
C6H14–CHCl3–2-PrOH), 1H NMR (CDCl3): d 0.70 (d, 3H, J5,6 6.4, H-
6III), 1.95–2.06 (m, 17 H, CCH2C, 5 OC(O)CH3), 2.11–2.18 (4s, 12H,
3 OC(O)CH3, NHC(O)CH3), 3.44 (br s, 1H, H-6aI), 3.51–3.58 (m,
1H, NCHH), 3.60 (dd, 1H, J2,3 10.7, J3,4 3.2, H-3I), 3.61–3.65 (m,
1H, NCHH), 3.74–3.78 (m, 1H, OCHH), 3.80 (dd, 1H, J5,6a 6.6, J5,6b
6.6, H-5II), 3.87 (dd, 1H, J2,3 9.4, J3,4 3.3, H-3II), 3.92 (dd, 1H, J1,2
7.4, J2,3 9.4, H-2II), 3.97–4.00 (m, 1H, H-6aIV), 4.02 (dd, 1H, J1,2
7.9, J2,3 10.7, H-2I), 4.06–4.14 (m, 5H, H-5I, H-6aII, H-5IV, H-6bIV,
OCHH), 4.19 (dd, 1H, J5,6b 6.6, J6,6b 11.2, H-6bII), 4.24 (dd, 1H, J3,4
3.2, H-4I), 4.30 (d, 1H, J5,6a 11.8, H-6bI) 4.45 (d, 1H, J1,2 7.9, H-1I),
4.47 (ddd, 1H, J1,2 3.4, J2,3 11.4, J2,NH 8.4, H-2IV), 4.53 (q, 1H, J5,6
6.4, H-5III), 4.87 (d, 1H, J1,2 7.4, H-1II), 4.98 (dd, 1H, J2,3 11.4, J3,4
3.0, H-3IV), 5.19 (d, 1H, J3,4 3.1, H-4III), 5.25 (d, 1H, J1,2 3.4, H-1IV),
5.28 (dd, 1H, J1,2 3.4, J2,3 11.0, H-2III), 5.31 (dd, J2,3 11.0, J3,4 3.1,
H-3III), 5.38 (d, 1H, J3,4 3.0 H-4IV), 5.40 (d, 1H, J3,4 3.3, H-4II), 5.56
(s, 1H, PhCH), 5.59 (d, 1H, J1,2 3.4, H-1III), 6.39 (d, 1H, J2,NH 8.4, NHA-
cIV), 7.12–7.16 (m, 1H, NHC(O)CF3), 7.32–7.38 (m, 3H, ArH), 7.47–
7.50 (m, 2H, ArH), ESIMS: Calcd [C54H70N5O28F3]Na+: m/z
1316.41. Found: m/z 1316.46.
4.9. 3-Aminopropyl 2-acetamido-2-deoxy-
a-D-
galactopyranosyl-(1?3)-[ -fucopyranosyl-(1?2)]-a-D-
a-
L
galactopyranosyl-(1?3)-2-acetamido-2-deoxy-a-D-
galactopyranoside (28)
A
mixture of anomeric tetrasaccharides 15 (413 mg,
Tetrasaccharide 22 (22 mg, 0.017 mmol) was deacetylated un-
der Zemplén conditions, and product 26 that was obtained was
purified by gel filtration (1:1 CH3CN–H2O). A mixture of tetrasac-
charide 26 (15 mg, 0.017 mmol), DTT (8 mg. 0.05 mmol), and aq
NaHCO3 (1.5 mL, 50 mM, pH 8.2) was stirred for 1.5 h under an
0.319 mmol) was dissolved in 20 mL of AcOH (80%), kept for 3 h
at 70 °C, concentrated, and co-evaporated with PhCH3
(4 Â 15 mL). Column chromatography on silica gel (3:3:1 n-
C6H14–CHCl3–2-PrOH) gave a fraction containing tetrasaccharide
17 (230 mg), Rf 0.11 (3:3:1 n-C6H14–CHCl3–2-PrOH), and a fraction
atmosphere of Ar, Ac2O (50 lL) was then added and the reaction
containing tetrasaccharide 19 (111 mg), Rf 0.22 (3:3:1 n-C6H14
CHCl3–2-PrOH). Both fractions also contained some impurities.
Both fractions were separately acetylated (Ac2O/Py, 15 h) and sub-
–
was stirred for 45 min. The mixture was subjected to gel filtration
(1:1 CH3CN–H2O) followed by column chromatography (elution
with 6:5:1 CH2Cl2–EtOH–H2O) and concentrated. The residue was

I. M. Ryzhov et al. / Carbohydrate Research 351 (2012) 17–25
25
dissolved in H2O (1 mL), aq NaOH (2 M, 2 lL) was added, and the
Supplementary data
reaction mixture was kept for 3 h. Cation-exchange chromatogra-
phy on Dowex 50X4-400 (H+) (elution with 5% aq ammonia) gave
10 mg (75%) of product 28 as white foam: [a] +129 (c 0.9, H2O–
D
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.12.013.
CH3CN, 1:1), Rf 0.19 (100:10:10:10:2 EtOH–BuOH–Py–H2O–AcOH),
1H NMR (D2O): (characteristic signals) d 1.27 (d, 3H, J5,6 6.6, H-6III),
1.98–2.05 (m, 2H, CCH2C), 2.05–2.08 (2s, 6H, 2 C(O)CH3), 3.11–3.16
(m, 2H, NHCH2), 4.93 (d, 1H, J1,2 3.8, H-1I), 5.18 (d, 1H, J1,2 3.7, H-
1IV), 5.19 (d, 1H, J1,2 3.8, H-1II), 5.28 (d, 1H, J1,2 4.0, H-1III); 13C
NMR (D2O): d 15.4 (C-6III), 22.0 (CH3C(O)), 22.1 (CH3C(O)), 26.8
(CCH2C), 37.1 (CCH2N), 48.0, 49.6 (C-2I, C-2IV), 61.11, 61.14, 61.2
(C-6I, C-6II, C-6IV), 65.0 (CCH2O), 63.8, 65.2, 67.4, 67.8, 67.9, 68.5,
69.4, 69.6, 70.7, 70.9, 71.3, 71.75, 71.76, 72.8 (C-2II, C-2III, C-3I, C-
3II, C-3III, C-3IV, C-4I, C-4II, C-4III, C-4IV, C-5I, C-5II, C-5III, C-5IV),
91.3 (C-1IV), 94.6 (C-1II), 97.2 (C-1I), 99.2 (C-1III), 174.2 (CH3C(O)),
174.8 (CH3C(O)), HRESIMS, Calcd [C31H55N3O20]H+: m/z 790.3452.
Found: m/z 790.3439.
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4.10. 3-Aminopropyl 2-acetamido-2-deoxy-
a-D-
galactopyranosyl-(1?3)-[ -fucopyranosyl-(1?2)]-a-D-
a
-L
galactopyranosyl-(1?3)-2-acetamido-2-deoxy-b-D-
galactopyranoside (29)
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(7 h) in a mixture of MeOH (4 mL) and Ac2O (200
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solved in H2O (3 mL), an aq solution of NaOH (2 M, 3 L) was
lL). The reaction
l
20. Blixt, O.; Razi, N. Methods Enzymol. 2006, 415, 137–153.
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added, and the reaction mixture was kept for 4 h. Cation-exchange
chromatography on Dowex 50X4-400 (H+) (elution with 5% aq
ammonia) gave 9 mg (25%) of product 29 as white foam: [a]
D
+91 (c 0.9, H2O–CH3CN, 1:1), Rf 0.18 (100:10:10:10:2 EtOH–
BuOH–Py–H2O–AcOH), 1H NMR (D2O): (characteristic signals) d
1.27 (d, 3H, J5,6 6.6, H-6III), 1.94–2.01 (m, 2H, CCH2C), 2.05–2.07
(2s, 6H, 2 C(O)CH3), 3.10–3.13 (m, 2H, NHCH2), 4.53 (d, 1H, J1,2
8.5, H-1I), 5.18 (m, 2H, H-1II H-1IV), 5.28 (d, 1H, J1,2 4.0, H-1III);
,
13C NMR (D2O): d 15.4 (C-6III), 22.0 (CH3C(O)), 22.3 (CH3C(O)),
26.7 (CCH2C), 37.7 (CCH2N), 49.6, 50.7 (C-2I, C-2IV), 60.9, 61.0,
61.2 (C-6I, C-6II, C-6IV), 63.7 (CCH2O), 64.5, 67.4, 67.87, 67.90,
68.0, 68.5, 69.3, 69.6, 70.9, 71.2, 71.7, 71.8, 74.7, 75.5 (C-2II, C-2III,
C-3I, C-3II, C-3III, C-3IV, C-4I, C-4II, C-4III, C-4IV, C-5I, C-5II, C-5III, C-
5IV), 91.3 (C-1IV), 94.9 (C-1II), 99.2 (C-1III), 101.6 (C-1I) 174.6
(CH3C(O)), 174.9 (CH3C(O)); HRESIMS: Calcd [C31H55N3O20]H+: m/
z 790.3452. Found: m/z 790.3459.
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Pazynina, G. V.; Severov, V. V.; Kaltner, H.; André, S.; Gabius, H.-J.; Bovin, N. V.
Biochemistry (Moscow) 2011, 76, 1185–1192.
Acknowledgments
34. Obukhova, P.; Korchagina, E.; Henry, S.; Bovin, N. Transfusion 2011. doi:
10.1111/j1537-2995.2011.03381.x.
This work was supported by the Russian Academy of Sciences
Presidium program ‘Molecular and Cell Biology’ and RFBR Grant
No. 10-04-01-693. Authors are grateful to Alexander Paramonov
for the NMR spectra and Dr. Konstantin Antonov for the HRESIMS
determinations.