Synthesis of blood group A and B (type 2) tetrasaccharides. A strategy with fucosylation at the last stage

Article abstract of DOI:10.1016/j.carres.2020.108192

The traditionally used strategy for the synthesis of blood group A and B tetrasaccharides includes 2′-O-fucosylation of lactosamine followed by insertion of an α1-3 linked N-acetylgalactosamine or a galactose moiety. Here, we report the synthesis of 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides via an alternative sequence, i.e. α-galactosaminylation (or α-galactosylation) followed by α-fucosylation. This strategy allows us to synthesize fucose-free trisaccharides GalNAcα1-3Galβ1-4GlcNAc and Galα1-3Galβ1-4GlcNAc, which are promising targets for immunotherapy utilising human natural antibodies against the trisaccharides. The key stage in this scheme was the selective chloroacetylation of the 2′-OH group of βGal in the intermediate trisaccharides having the second (3-OH) unprotected group.The protocol is suitable for multigram syntheses and its further scaling up.

Full text of DOI:10.1016/j.carres.2020.108192

Journal Pre-proof
Synthesis of blood group A and B (type 2) tetrasaccharides. A strategy with
fucosylation at the last stage
Roman A. Kunetskiy, Galina V. Pazynina, Igor A. Ivanov, Nicolai V. Bovin
PII:
S0008-6215(20)30563-2
DOI:
https://doi.org/10.1016/j.carres.2020.108192
CAR 108192
Reference:
To appear in: Carbohydrate Research
Received Date: 20 September 2020
Revised Date: 31 October 2020
Accepted Date: 2 November 2020
Please cite this article as: R.A. Kunetskiy, G.V. Pazynina, I.A. Ivanov, N.V Bovin, Synthesis of blood
group A and B (type 2) tetrasaccharides. A strategy with fucosylation at the last stage, Carbohydrate
Research, https://doi.org/10.1016/j.carres.2020.108192.
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2020 Published by Elsevier Ltd.

Synthesis of blood group A and B (type 2) tetrasaccharides. A
strategy with fucosylation at the last stage
Roman A. Kunetskiy*, Galina V. Pazynina, Igor A. Ivanov, Nicolai V.Bovin*
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Miklukho-Maklaya str. 16/10, Moscow 117997, Russian Federation
Keywords: ABO histo-blood group antigens, A (type 2) tetrasaccharide, B (type 2)
tetrasaccharide, Galili trisaccharide
---------------------
*
Corresponding authors. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997,
Russian Federation. Tel.: +7 495 330 0300; fax: +7 495 330 5592.
E-mail addresses: roman.kunetskiy@gmail.com (R.A. Kunetskiy).
E-mail addresses: professorbovin@yandex.ru (N.V. Bovin).

Abstract
The traditionally used strategy for the synthesis of blood group A and B tetrasaccharides
includes 2’-O-fucosylation of lactosamine followed by insertion of an α1-3 linked N-
acetylgalactosamine or a galactose moiety. Here, we report the synthesis of 3-aminopropyl
glycosides of A (type 2) and B (type 2) tetrasaccharides via an alternative sequence, i.e. α-
galactosaminylation (or α-galactosylation) followed by α-fucosylation. This strategy allows us
to synthesize fucose-free trisaccharides GalNAcα1-3Galβ1-4GlcNAc and Galα1-3Galβ1-
4
GlcNAc, which are promising targets for immunotherapy utilising human natural antibodies
against the trisaccharides. The key stage in this scheme was the selective chloroacetylation of the
`-OH group of βGal in the intermediate trisaccharides having the second (3-OH) unprotected
group.The protocol is suitable for multigram syntheses and its further scaling up.
2

1
. Introduction
Human histo-blood group oligosaccharides with -Galβ1-4GlcNAc inner core (known as
type 2 glycans) dominate on the AB-serologically active glycoproteins and glycolipids of human
erythrocytes (RBC). A (type 2) and B (type 2) tetrasaccharides, and H (type 2) trisaccharide
terminate N- and O-chains in glycoproteins, as well as glycolipids in composition of practically
all endothelial and epithelial tissues, they are also found as free oligosaccharides in body
secretions [1],[2]. A (type 2) and B (type 2) tetrasaccharide terminations (Fig.1) are minimal
epitopes responsible for A and B antigens recognition by natural antibodies (known as allo-
antibodies, or isoagglutinins) [3]. Chemical synthesis of these glycans was described in
numerous works [4-14]. Nevertheless, synthetic scheme leading to the tetrasaccharides now are
still in demand because of recent advances in diagnostics and therapy. One example of medical
application is design of so called kodecytes (blood group O erythrocytes modified with lipophilic
derivatives of tetrasaccharides A and B), routinely used in the ABO-typing [15-17]; second
example is removal of anti-A and anti-B isoagglutinins from recipient's blood by means of
affinity adsorbents (A and B glycans immobilized on Sepharose) in ABO-incompatible organ
transplantation [18].
We recently published block synthesis of tetrasaccharides A (type 2) and B (type 2),
where the unit GlcNAc was glycosylated by the trichloroacetimidate form of protected
trisaccharides A and B [10], an approach that is acceptable and convenient for obtaining various
oligosaccharides containing these trisaccharide motifs, but inappropriate for multigram
tetrasaccharide synthesis; the same is true for a synthetic scheme based on the use of 1,6-
anhydroglucosamine as a key synthetic feature [19].

Fig. 1. Aminopropyl glycosides of A (type 2) 1a and B (type 2) 1b tetrasaccharides.
Traditionally, A (type 2) and B (type 2) tetrasaccharides are synthesized via
galactosaminylation or galactosylation of 3-OH in Gal residue of H (type 2) trisaccharide. This
approach was applied in early versions of A (type 2) and B (type 2) tetrasaccharide synthesis
[
5],[6] and is still in use [7-9],[20],[21]. Alternative scheme, GlcNAc → Galβ1–4GlcNAc →
Gal(NAc)α1–3Galβ1–4GlcNAc → Gal(NAc)α1–3(Fucα1–2)Galβ1–4GlcNAc, is at first glance
less rational since it requires two fucosylation reactions instead of one – i.e. in the synthesis of
each of the tetrasaccharides. However, the alternative scheme has undoubted advantage when
large-scale synthesis requires a fucose-free trisaccharide 2b and this is indeed the case, since
glycan 2b is used to deplete antibodies during xenotransplantation [22] and to improve the fight
against nosocomial infections [23].
Fig. 2. Trisaccharides 2a and 2b.

Since anti-GalNAcα1–3Galβ1–4GlcNAc (2a) immunoglobulin titers are even higher in
humans than against Galα1–3Galβ1–4GlcNAc (2b) [24], there is no doubt that the trisaccharide
(Fig. 2) will also soon be used in medicine. In this paper, we have demonstrated that the second
scheme is not just realizable, but very promising for practical scaling-up.

2
. Results and discussion
Our synthesis for aminopropyl glycosides of A (type 2) 1a and B (type 2) 1b
tetrasaccharides is presented on Scheme 1 and 2. In conformity with the schemes, 3`-OH
lactosamine 3 (prepared according to literature procedure [25],[26]) was glycosylated with ethyl
thioglycosides 4a and 4b, with bromine activation and AgOTf as a promoter [25] at r.t. In both
cases α-selectivity was high; resulting trisaccharides 5a and 5b after partial purification were
deacetylated under the classical Zemplen conditions to trisacchrides 6a and 6b respectively; after
complete conversion, the basic components were removed using cation exchange resin in the
+
PyH form. This form was taken since in some cases it was previously found cation exchangers
+
in the form of H led to the replacement of the spacer by OMe, the latter coming from methanol.
Isolated trisaccharides 6a and 6b with four OH groups required selective protection before final
glycosylation. Therefore, in the next step, the 4`-OH and 6`-OH groups were protected with a
benzylidene group leading to diols 7a and 7b, where 3-OH must be protected in the presence of
2
`-OH. An attempt for di-O-acetylation and subsequent selective deacylation of the product
under controlled Zemplen conditions was unsuccessful - both 3-OAc and 2'-OAc showed the
same deacetylation rates. Selective protection of one of the hydroxyls with bulky silyl groups
was also unsuccessful. TBDMS chloride/imidazole in DMF [27] showed low selectivity, while
the more bulky TIPS chloride/imidazole in DMF [28] was almost inert to both hydroxyl groups
even at 60 °C. Effective and selective protection of 3-OH in the presence of 2`-OH in
compounds 7a and 7b was achieved using the following path: chloroacetylation-acetylation-
dechloroacetylation. Chloroacetic anhydride in the presence of collidine quickly reacted with 2`-
OH under selected conditions, in which the second hydroxyl was almost inert. This remaining
free 3-OH was then acetylated with acetyl chloride/collidine. Further gentle removal of the
chloroacetyl group with a water-pyridine mixture at 60 °C gave the desired glycosyl-acceptor
trisaccharides 8a and 8b, with similar selectivity and yields (Scheme 1).

OBn
O
BnO
BnO
OBn
O
OAc
OAc
OBn
O
OBn
O
BnO
BnO
AcO
HO
AcO
Br , AgOTf, TMU
2
O
O
SEt
O
AcO
R
O
AcO
Osp3
O
Osp3
CH Cl2
2
R
OAc
OAc
NHAc
NHAc
4
4
a: R = N3
b: R = OBn
3
5a (no isolation): R = N3
5b (no isolation): R = OBn
NaOMe
MeOH
Ph
OBn
O
OBn
O
BnO
BnO
BnO
BnO
O
OH
OBn
PhCH(OMe) , TsOH
2
OBn
O
HO
O
MeCN
O
O
O
R
O
HO
Osp3
NHAc
R
O
O
HO
Osp3
NHAc
O
OH
OH
7
7
a (90%): R = N3
b (89%): R = OBn
6a (44% for 2 steps): R = N3
6b (55% for 2 steps): R = OBn
i. (ClAc) O/Me Py
2
3
ii. AcCl/Me Py
3
o
iii. Py/H O, 60 C
2
Ph
OBn
O
BnO
BnO
O
OBn
O
O
O
O
R
Osp3
O
AcO
OH
NHAc
8
8
a (79% for 3 steps): R = N3
b (73% for 3 steps): R = OBn
Scheme 1. Syntheses of 8a and 8b trisaccharide glycosyl acceptors; sp3 = (CH ) NHCOCF .
2
3
3
The obtained trisaccharides 8a and 8b were fucosylated (Scheme 2) with ethyl thioglycoside 9
with bromine activation and AgOTf as a promoter [25] at r.t. In both cases α-selectivity was
high; after remove of benzylidene group, the tetrasaccharides 10a and 10b were partially purified
by column chromatography. Then, benzyl groups were removed with hydrogenolysis followed
by peracetylation to 11a and 11b respectively, the products were purified with chromatography
and completely deprotected by alkaline hydrolysis. Target tetrasaccharides with aminopropyl
+
linker, 1a and 1b, were isolated by ion exchange chromatography on H -cationite.

Ph
OBn
O
OBn
O
BnO
BnO
BnO
BnO
O
OH
OBn
O
OBn
O
O
HO
i. Br , AgOTf, TMU
ii. AcOH/H2O, 80°C
2
O
SEt
OBn
O
O
R
O
AcO
Osp3
R
O
O
AcO
Osp3
O
OBn
OH
BnO
O
NHAc
NHAc
O
OBn
8
a: R = N3
9
OBn
BnO
8b: R = OBn
1
1
0a (no isolation): R = N3
0b (no isolation): R = OBn
i. H2 (1 atm), Pd/C
ii. Ac O/Py
2
OH
OAc
HO
HO
AcO
AcO
OH
OAc
O
OH
O
AcO
OAc
HO
i. NaOMe, MeOH
ii. H2O
O
O
O
O
O
O
R
O
HO
O
NH2
R
O
AcO
Osp3
NHAc
O
O
NHAc
O
O
OH
OAc
OH
OAc
HO
AcO
1
1
a (90% for 2 steps): R = NHAc
b (90% for 2 steps): R = OH
11a (32% for 4 steps): R = NHAc
11b (35% for 4 steps): R = OAc
Scheme 2. Syntheses of A (type 2) 1a and B (type 2) 1b tetrasaccharides; sp3 =
CH ) NHCOCF .
(
2
3
3
The products 5a-8a and 5b-8b are protected trisaccharides 12a and 12b respectively (Scheme 3)
that are of independent value (in addition to the corresponding fucosylated forms). In particular,
1
2b is the well-known Galili xeno-antigen, its lipophilic form AGI-134 is being studied as a
potential anti-cancer therapeutic [29], while the polymeric form is a potential anti-bacterial
therapeutic [23]. The level of natural antibodies against trisaccharide 12a is even more than that
against the Galili trisaccharide [30], so, GalNAcα1-3Galβ1-4GlcNAc is also a potential
molecule for design of immunotherapeutics. For preparation of 12a and 12b (Scheme 3), 3`-OH
lactosamine 3 was glycosylated with ethyl thioglycosides 4a and 4b, resulting intermediate
trisaccharides 5a and 5b were partially purified as mentioned earlier (Scheme 1), but instead of
deacetylation, benzyl groups were removed by hydrogenolysis followed by peracetylation to 13a

and 13b respectively, the products were purified with chromatography and finally deacylated by
alkaline hydrolysis. Trisaccarides with aminopropyl linker, 12a and 12b, were isolated by ion
+
exchange chromatography on H -cationite.
Scheme 3. Syntheses of trisaccharides 12a and 12b; sp3 = (CH ) NHCOCF .
2
3
3
Complete prove of the structure of the target and intermediate oligosaccharides was performed
using 1H, 1H-2D-COSY and/or 1H, 13C-2D-HSQC experiments (available in Supporting
information).

3
. Conclusions
The proposed method for the synthesis of blood groups A and B tetrasaccharides through
fucosylation at the last stage uses a minimum of chromatographic procedures. This is
fundamentally important, since it opens up a real prospect for scaling this synthesis up for
medical purposes not only of tetra-, but also trisaccharides GalNAcβ1-3Gallβ1-4GlcNAc and
Galα1-3Gallβ1-4GlcNAc.

4
. Experimental
4
.1. General procedures
The reactions were performed with the use of commercial reagents (Acros, Aldrich, and Fluka);
anhydrous solvents were purified according to the standard procedures.
3
-Trifluoroacetamidopropyl O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-2-acetamido-3-
O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside 3 was prepared accordingly to lit. procedure
25]. Ethyl-2-azido-3,4,6-tri-O-benzyl-2-deoxy-1-thio-β-D-galactopyranoside 4a was prepared
[
accordingly to lit. procedure [31]. Ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside 4b
was prepared accordingly to lit. procedure [32]. Ethyl 2,3,4-tri-O-benzyl-1-thio-β-L-
fucopyranoside 9 was prepared accordingly to lit. procedure [33].
Column chromatography was performed on silica gel 60 0.040–0.063 mm (Merck). Solvents
were removed in vacuum at 30-40 °С. Thin layer chromatography (TLC) was performed on
silica gel 60 F254 aluminum-backed plates (Merck). The spots of compounds were visualized by
dipping a TLC plate into aqueous solution of H PO (8%) and subsequent heating (>150 °С).
3
4
The values of optical rotations were measured on a Perkin Elmer 341 and JASCO P-2000
polarimeters at 25°C.
1
H NMR spectra were recorded on a Bruker BioSpin GmbH (700 MHz) spectrometer at 30 °С;
chemical shifts (δ, ppm) were referred to solvent residual peak of internal D O (δ 4.79), CD OD
2
3
(
δ 3.31), CDCl (δ 7.27) and (CD ) SO (δ 2.50); coupling constants (J) were measured in Hz.
3 3 2
1
3
C NMR spectra were recorded at 150 MHz. Chemical shifts (δ, ppm) were referred to the peak
1
of internal CD OD (δ 49.0), CDCl (δ 77.16) and (CD ) SO (δ 39.62). Signals of H NMR and
3
3
3 2
1
3
C NMR spectra were assigned to the corresponding protons using 2D spectroscopy (COSY,
HSQC). 1D and 2D NMR spectra pictures are available in Supporting information.

4
.2.1 Synthesis of aminopropyl glycoside of A (type 2) tetrasaccharide 1a
3
-Trifluoroacetamidopropyl O-(2-azido-3,4,6-tri-O-benzyl-2-deoxy-
3)-O-( -D-galactopyranosyl)-(1 4)-2-acetamido-6-O-benzyl-2-deoxy-
glucopyranoside (6a)
α
-D-galactopyranosyl)-
(1
→
β
→
β
-D-
To ethyl thioglycoside 4a (11.8 g, 22.7 mmol) solution in abs. dichloromethane (120 mL) was
added bromine (1.27 mL, 25.0 mmol) at 0°С. The reaction was left at 0°C for 1 h. The solvent
and bromine was evaporated, residue was co-evaporated with toluene (3×100 mL) to remove
traces of bromine, almost colourless oil of glycosyl bromide was dried in vacuum at r.t.
To disaccharide 3 (12.0 g, 15.1 mmol) solution in abs. dichloromethane (140 mL) were added
freshly dried MS-4Å (25 mL) and tetramethylurea (3.40 mL, 27.8 mmol), the mixture stirred for
3
0 min, then AgOTf (6.40 g, 24.5 mmol) was added and the mixture was stirred for additional 15
min. To reaction mixture glycosyl bromide (see above) solution in abs. dichloromethane (70 mL)
was added drop-wise under intensive stirring. The reaction was left for 24 h with stirring (TLC
control in chloroform/methanol 10:1, Rf 0.47). Reaction mixture was neutralized with pyridine
(12 mL), filtered through Celite on glass filter, solid material was washed with portion of
dichloromethane and mixture of dichloromethane/methanol 10:1. The filtrate was poured in 10%
aq. solution of Na S O (170 mL), washed with cold sat. NaHCO (150 mL), dried with
2
2
3
3
Na SO /SiO .
2
4
2
After filtration, the solution was concentrated on rotary evaporator, co-evaporated with toluene,
dried in vacuum. The product 5a was partially purified with column chromatography on silica
gel (from toluene/acetone 5:1 to toluene/acetone 3:1), the fractions containing major product 5a
were collected, concentrated on rotary evaporator, co-evaporated with toluene, dried in vacuum
and used in next stage without additional purification (contains admixture of tetramethylurea).

The partially purified product 5a was dissolved in abs. methanol (500 mL), treated with 2M
NaOMe (50 mL, 100 mmol) and reaction mixture was left for 2 h. Reaction mixture was
+
neutralized with Dowex 50WX4 (100 g, PyH -form) and stirred for 15 min and filtered.
Obtained solution was concentrated on rotary evaporator, co-evaporated with ethyl acetate, dried
in vacuum. Crude material was diluted with ethyl acetate (360 mL) and product was precipitated
by slow addition of n-hexane (360 mL) under intensive stirring, left overnight in fridge for
complete crystallization. The crystalline product was collected on glass filter, washed with
mixture of n-hexane/ethyl acetate 3:1 and n-hexane subsequently, dried under fume-hood
overnight further by vacuum drying.
Yield of 6a for two steps: 44% (7.20 g, 6.62 mmol). White crystalline material; Rf 0.40
(
chloroform/ethyl acetate/methanol, 9:3:2); [α] could not be measured precisely owing to low
D
solubility of the compound in polar as well nonpolar solvents;
1
H NMR (CD OD, characteristic signals): δ 1.75-1.87 (m, 2H, OCH CH CH N), 1.98 (s, 3H,
3
2
2
2
II
I
NHC(O)CH ), 4.32 (d, 1H, J 7.7, H-1 ), 4.40 (d, 1H, J 8.4, H-1 ), 5.11 (d, 1H, J 3.6, H-
3
1,2
1,2
1,2
III 13
1
9
1
(
); C NMR (CD OD, characteristic signals): 22.6 (NHC(O)CH ), 29.7 (OCH CH CH N),
3 3 2 2 2
III
I
II
+
6.2 (C-1 ), 102.7 (C-1 ), 104.9 (C-1 ); ESI MS: m/z Calcd [C H F N O ]H : 1084.4. Found:
53
64
3
5
16
084.4
1
13
H, C, COSY and HSQC NMR spectra pictures can be found in Supporting information)
3
-Trifluoroacetamidopropyl O-(2-azido-3,4,6-tri-O-benzyl-2-deoxy-
3)-O-(4,6-O-benzylidene- -D-galactopyranosyl)-(1 4)-2-acetamido-6-O-benzyl-2-deoxy-
-D-glucopyranoside (7a)
α-D-galactopyranosyl)-
(1
→
β
→
β
Trisaccharide 6a (7.20 g, 6.62 mmol) from previous experiment was suspended in dry
acetonitrile (120 mL), then PhCH(OMe) (3.60 mL, 24.0 mol) and solution of TsOH·H O (150
2
2
mg, 0.80 mmol) in dry acetonitrile (5 mL) were added. Reaction mixture was stirred overnight at

r.t. The reaction was neutralised with pyridine (12 mL). Diethyl ether (360 mL) was added under
stirring, and formed crystalline product was collected on glass filter, washed with diethyl ether,
n-hexane, dried in vacuum.
Yield of 7a: 90% (6.95 g, 5.95 mmol). White crystalline material; Rf 0.65 (chloroform/ethyl
acetate/methanol, 9:3:2); [α] +36° (c 0.7, CHCl );
D
3
1
H NMR (CDCl , characteristic signals): δ 1.74-1.89 (m, 2H, OCH CH CH N), 2.01 (s, 3H,
3
2
2
2
I
III
NHC(O)CH ), 4.64 (d, 1H, J 8.4, H-1 ), 5.11 (d, 1H, J 3.5, H-1 ), 5.52 (s, 1H, PhCH), 6.34
3
1,2
1,2
1
3
(
d, 1H, J2,NH 6.8, NHCOCH ); C NMR (CDCl , characteristic signals): δ 23.5 (NHC(O)CH ),
3
3
3
III
I
II
2
8.5 (OCH CH CH N), 97.1 (C-1 ), 100.9 (PhCH), 101.1 (C-1 ), 103.7 (C-1 ); ESI HRMS: m/z
2 2 2
+
Calcd [C H F N O ]H : 1172.4686. Found: 1172.4623.
60
68
3
5
16
1
13
(
H, C and HSQC spectra pictures can be found in Supporting information)
3
-Trifluoroacetamidopropyl O-(2-azido-3,4,6-tri-O-benzyl-2-deoxy-
3)-O-(4,6-O-benzylidene- -D-galactopyranosyl)-(1 4)-2-acetamido-3-O-acetyl-6-O-
benzyl-2-deoxy- -D-glucopyranoside (8a)
α-D-galactopyranosyl)-
(1
→
β
→
β
To powder of trisaccharide 7a (6.95 g, 5.95 mmol) from previous experiment was added solution
of chloro acetic anhydride (3.10 g, 18.1 mmol) and sym-Collidine (16 mL, 120 mmol) in abs.
dichloromethane (40 mL) at 0°C under intensive stirring. The mixture was stirred for 1.5 h. The
reaction was quenched with methanol (15 mL), concentrated on rotary evaporator, co-evaporated
with toluene, dried in vacuum.
Resulting oil was dissolved in abs. dichloromethane (90 mL) followed by addition of sym-
Collidine (18.0 mL, 137 mmol) and acetyl chloride (9.0 mL, 126 mmol). Reaction mixture was
left for 24 h at r.t. Reaction was quenched with methanol (40 mL), concentrated on rotary
evaporator, co-evaporated with toluene. The residue was diluted with toluene (200 mL), stirred

for 15 min, collidinium salt was collected on glass filter, washed few times with toluene. The
filtered solution was concentrated on rotary evaporator, dried in vacuum.
The residue was dissolved in pyridine (200 mL), water (100 mL) was added and the mixture was
warmed at 60 °C for 24 h. The mixture was concentrated on rotary evaporator, co-evaporated
with toluene, dried in vacuum.
The product 8a was purified with column chromatography on silica gel (chloroform/acetone,
from 5:1 to 3:1, 0.5% pyridine) , the fractions containing major product were collected,
concentrated on rotary evaporator (not to dryness). Concentrated solution was diluted with ethyl
acetate (100 mL), n-hexane (300 mL) was added drop-wise under stirring to precipitate pure
trisaccharide 8a and the mixture was left overnight in fridge for complete crystallization.
Solid product was collected on glass filter, washed with little portion of n-hexane/ethyl acetate
3
:1, pure n-hexane, dried in vacuum.
Yield of 8a for three steps: 79% (5.70 g, 4.69 mmol). White crystalline material; Rf 0.50
(
chloroform/methanol, 10:1); [α] +25° (c 0.3, CHCl );
D 3
1
H NMR (CDCl , characteristic signals): δ 1.48-1.56 (m, 1H, OCH CHHCH N), 1.59-1.68 (m,
3
2
2
1
H, OCH CHHCH N), 1.99, 2.15 (2s, 6H, OC(O)CH and NHC(O)CH ), 3.07-3.14 (m, 1H,
2 2 3 3
OCH CH CHHN), 3.34-3.40 (m, 1H, OCHHCH CH N), 4.10 (ddd, 1H, J ≈J ≈J
9.0, H-
2
2
2
2
1,2 2,3
2,NH
I
II
I
III
2
1
), 4.18 (d, 1H, J 7.6, H-1 ), 4.39 (d, 1H, J 8.4, H-1 ), 5.09 (d, 1H, J 3.5, H-1 ), 5.22 (dd,
1
,2
1,2
1,2
I
13
H, J 9.0, J 10.5, H-3 ), 5.51 (s, 1H, PhCH), 6.44 (d, 1H, J
8.9, NHCOCH ); C NMR
3
2
,NH
(
CDCl , characteristic signals): δ 21.0, 23.4 (OC(O)CH and NHC(O)CH ), 28.4
3
3
3
I
III
(
OCH CH CH N), 37.9 (OCH CH CH N), 53.8 (C-2 ), 97.1 (C-1 ), 101.1 (PhCH), 101.9 (C-
2
2
2
2
2
2
I
II
+
1
), 103.4 (C-1 ); ESI HRMS: m/z Calcd [C H F N O ]H : 1214.4792. Found: 1214.4781.
62
70
3
5
17
1
13
(
H, C, COSY and HSQC NMR spectra pictures can be found in Supporting information)
3
-Trifluoroacetamidopropyl O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-
α
galactopyranosyl)-(1
→
3)-O-[2,3,4-tri-O-acetyl- -L-fucopyranosyl-(1→2)]-O-(4,6-di-O-acetyl-
α

β
-D-galactopyranosyl)-(1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranoside
(11a)
To ethyl thioglycoside 9 (4.34 g, 9.08 mmol) solution in abs. dichloromethane (50 mL) was
added bromine (0.52 mL, 10.0 mmol) at 0°С. The reaction was left at 0°C for 1 h. The solvent
and bromine was evaporated, residue was co-evaporated with toluene (3×50 mL) to remove
traces of bromine, almost colourless oil of glycosyl bromide was dried in vacuum at r.t.
To trisaccharide 8a (5.50 g, 4.53 mmol) solution in abs. dichloromethane (100 mL) were added
freshly dried MS-4Å (40 mL) and tetramethylurea (1.2 mL, 10.0 mmol), the mixture stirred for
3
0 min, then AgOTf (2.32 g, 9.0 mmol) was added and the mixture was stirred for additional 15
min. To reaction mixture glycosyl bromide (see above) solution in abs. dichloromethane (50 mL)
was added drop-wise under intensive stirring. The reaction was left for 24 h with stirring (TLC
control in ethyl acetate/toluene 3:1, Rf 0.50). Reaction mixture was neutralized with pyridine (7
mL), filtered through Celite on glass filter, solid material was washed with portion of
dichloromethane and mixture of dichloromethane/methanol 10:1.
Filtered solution was concentrated on rotary evaporator, co-evaporated with toluene, dried in
vacuum. The residue was dissolved in acetic acid (200 mL), water (50 mL) was added under
stirring and the mixture was warmed at 80 °C for 3 h (TLC control in chloroform/propan-2-ol
1
0:1, Rf 0.37). The mixture was concentrated on rotary evaporator, co-evaporated with toluene,
dried in vacuum.
Crude product 10a was partially purified with column chromatography on silica gel (from
chloroform/propan-2-ol 20:1 to 15:1, 0.5% pyridine) , the fractions containing major product
were collected (TLC control in chloroform/propan-2-ol 10:1+0.5% pyridine), concentrated on
rotary evaporator, co-evaporated with toluene, dried in vacuum and used in next stage without
additional purification (contains admixture of tetramethylurea).

Partially purified 10a was dissolved in methanol (300 mL), then 10% Pd/C (4.0 g) was added
and reaction mixture was degassed in vacuum further by filling with H (1 atm) three times. The
2
reaction was left under H (1 atm) with stirring for 12 h. Acetic anhydride (15 mL) was added to
2
reaction mixture under intensive stirring and the mixture was additionally stirred for 15 min. The
solution was degassed in vacuum further by filling with H (1 atm) three times. The reaction was
2
left under H (1 atm) with stirring for 24 h. Conversion was controlled by TLC (propan-2-
2
ol/ethyl acetate/water 2:3:1, product Rf 0.34). Reaction mixture was filtered through Celite on
glass filter, solid material was washed with methanol, portion of 50% aq. methanol and resulting
solution was concentrated on rotary evaporator, co-evaporated with toluene, dried in vacuum.
The residue was dissolved in pyridine (100 mL) and Ac O (50 mL), reaction mixture was left at
2
4
0 °C for 48 h. Reaction mixture was concentrated on rotary evaporator, co-evaporated with
toluene.
Crude product 11a was purified with column chromatography on silica gel (from pure ethyl
acetate to ethyl acetate/ propan-2-ol 20:1), the fractions containing major product were collected
(TLC control in ethyl acetate/ propan-2-ol 50:1 and chloroform/ propan-2-ol 9:1), concentrated
on rotary evaporator, co-evaporated with toluene, co-evaporated with chloroform, dried in
vacuum.
Yield of 11a for four steps: 32% (1.91 g, 1.46 mmol). White solid material; Rf 0.24 (n-
hexane/acetone, 1:2); [α]546 -22° (c 1, CHCl ), lit. [19] -22°;
3
1
1
H spectrum was identical to early described in lit. [10], [19]; H NMR (CDCl ): δ 1.17 (d, 3H,
3
III
J 6.5, H-6 ), 1.77–1.84 (m, 1H, OCH CHHCH N), 1.86–1.94 (m, 1H, OCH CHHCH N),
5
,6
2
2
2
2
1
.95, 1.98, 1.98, 2.00, 2.07, 2.09, 2.09, 2.12, 2.12, 2.14, 2.14, 2.16 (12s, 36H, 10 OC(O)CH , 2
3
NHC(O)CH ), 3.24–3.31 (m, 1H, OCH CH CHHN), 3.58–3.67 (m, 2H, OCH CH CHHN,
3
2
2
2
2
I
OCHHCH CH N), 3.69 (ddd, 1H, J 9.6, J 4.6, J5,6b 1.9, H-5 ), 3.75 (dd, 1H, J 7.2, J 9.1,
2
2
4,5
5,6a
1,2
2,3
II
II
I
H-2 ), 3.82 (dd, 1H, J5 6.8, J5,6b 6.8, H-5 ), 3.86 (dd, 1H, J 9.2, J 9.6, H-4 ), 3.87 (dd, 1H,
,6a
3,4
4,5
II
J 9.1, J 3.2, H-3 ), 3.91–3.95 (m, 1H, OCHHCH CH N), 3.98 (ddd, 1H, J 8.1, J 10.1,
2
,3
3,4
2
2
1,2
2,3

I
II
II
IV
IV
J2
,NH
8.9, H-2 ), 4.05–4.15 (m, 4H, H-6a , H-6b , H-6a , H-6b ), 4.18 (dd, 1H, J
5,6a
6.0, J5,6b
IV
I
II
6
6
1
.0, H-5 ), 4.32 (dd, 1H, J 4.6, J6a,6b 12.2, H-6a ), 4.42 (d, 1H, J 7.2, H-1 ), 4.44 (q, 1H, J
5,6a 1,2 5,6
III
IV
I
.5, H-5 ), 4.46–4.49 (m, 1H, H-2 ), 4.50 (d, 1H, J 8.1, H-1 ), 4.55 (dd, 1H, J 1.9, J6a,6b
1
,2
5,6b
I
IV
I
2.2, H-6b ), 5.02 (dd, 1H, J 11.3, J 3.0, H-3 ), 5.09 (dd, 1H, J 10.1, J 9.2, H-3 ), 5.12
2
,3
3,4
2,3
3,4
III
IV
(
dd, 1H, J 11.1, J 3.3, H-3 ), 5.22 (d, 1H, J 3.5, H-1 ), 5.28 (dd, 1H, J 3.7, J 11.1, H-
2
,3
3,4
1,2
1,2
2,3
III
III
II
IV
2
), 5.30 (br. d, 1H, J 3.3 H-4 ), 5.38 (br. d, 1H, J 3.2, H-4 ), 5.45 (br. s, 1H, H-4 ), 5.51
3
,4
3,4
III
I
IV
(
d, 1H, J 3.7, H-1 ), 5.76 (d, 1H, J
8.9, NHAc ), 6.17 (d, 1H, J
7.9, NHAc ), 7.30–
1
,2
2,NH
2,NH
7
.36 (m, 1H, NHC(O)CF3).
1
(
H NMR spectrum picture can be found in Supporting information)
3
-Aminopropyl O-(2-acetamido-2-deoxy-
2)]-O-( -D-galactopyranosyl)-(1
glucopyranoside (1a)
α
-D-galactopyranosyl)-(1
→3)-O-[α-L-
fucopyranosyl-(1
→
β
→
4)-2-acetamido-2-deoxy-β-D-
Tetrasaccharide 11a (1.91 g, 1.46 mmol) was dissolved in abs. methanol (115 mL), treated with
M NaOMe (5.62 mL, 11.3 mmol) and reaction mixture was left for 2 h. The mixture was
2
concentrated in vacuum (not to dryness) and diluted with water (115 mL), reaction mixture was
left overnight.
Target deprotected tetrasaccharide was isolated with cation-exchange chromatography on Dowex
5
0X4-400 (H+) by subsequent elution with water, 1M pyridine water solution and 1M aqueous
ammonia, fractions containing the carbohydrate were collected, concentrated in vacuum and
freeze dried twice.
Yield of 1a for two steps: 90% (1.03 g, 1.31 mmol). White solid material; [α] +26° (c 1, H O)
D
2
lit. [19] +26°;
1
1
H spectrum was identical to early described in lit. [7], [19]; H NMR (D O+TFA, characteristic
2
signals): δ 1.29 (d, 3H, J 6.6, FucMe), 1.94-2.03 (m, 2H, OCH CH CH N), 2.08, 2.09 (2s, 6H,
5
,6
2
2
2

I
2
NHC(O)CH ), 3.12 (t, 2H, J 6.7, OCH CH CH N), 4.53, 4.63 (2d, 2H, J 8.5, J 7.7, H-1
3 2 2 2 1,2 1,2
II
III
IV
and H-1 ), 5.22, 5.39 (2d, 2H, J 3.8, J 4.1, H-1 and H-1 ).
1
,2
1,2
1
(
H NMR spectrum picture can be found in Supporting information)
4
.2.2 Synthesis of aminopropyl glycoside of B (type 2) tetrasaccharide 1b
3
-Trifluoroacetamidopropyl O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→3)-O-(β-D-
galactopyranosyl)-(1
→
4)-2-acetamido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (6b)
To ethyl thioglycoside 4b (15.8 g, 27.0 mmol) solution in abs. dichloromethane (150 mL) was
added bromine (1.53 mL, 29.7 mmol) at 0°С. The reaction was left at 0°C for 1 h. The solvent
and bromine was evaporated, residue was co-evaporated with toluene (3×100 mL) to remove
traces of bromine, almost colourless oil of glycosyl bromide was dried in vacuum at r.t.
To disaccharide 3 (14.3 g, 18.0 mmol) solution in abs. dichloromethane (180 mL) were added
freshly dried MS-4Å (45 mL) and tetramethylurea (3.9 mL, 32.4 mmol), the mixture stirred for
3
0 min, then AgOTf (7.63 g, 29.7 mmol) was added and the mixture was stirred for additional 15
min. To reaction mixture glycosyl bromide (see above) solution in abs. dichloromethane (90 mL)
was added drop-wise under intensive stirring. The reaction was left for 24 h with stirring (TLC
control in chloroform/methanol 10:1, Rf 0.36). Reaction mixture was neutralized with pyridine
(15 mL), filtered through Celite on glass filter, solid material was washed with portion of
dichloromethane and mixture of dichloromethane/methanol 10:1.
The filtrate was poured in 10% aq. solution of Na S O (180 mL), washed with cold sat.
2
2
3
NaHCO (170 mL), dried with Na SO /SiO . After filtration, the solution was concentrated on
3
2
4
2
rotary evaporator, co-evaporated with toluene, dried in vacuum. The product 5b was partially
purified with column chromatography on silica gel (from ethyl acetate/n-hexane 1:1 to ethyl
acetate/n-hexane 7:1), the fractions containing major product 5b were collected, concentrated on

rotary evaporator, co-evaporated with toluene, dried in vacuum and used in next stage without
additional purification (contains admixture of tetramethylurea).
Partially purified product 5b was dissolved in abs. methanol (900 mL), treated with 2M NaOMe
(90 mL, 180 mmol) and reaction mixture was left for 4 h at r.t. Reaction mixture was neutralized
+
with Dowex 50WX4 (180 g, PyH -form) and stirred for 15 min and filtered. Obtained solution
was concentrated on rotary evaporator, co-evaporated with ethyl acetate, dried in vacuum. Crude
material was diluted with ethyl acetate (400 mL), suspension was warmed up to boiling,
acetonitrile (~250 mL) was added under reflux to complete dissolving of the crystals. Clear
solution was cooled down and left overnight in fridge for complete crystallization. The
crystalline product was collected on glass filter, washed with cold ethyl acetate and n-hexane
subsequently, dried under fume-hood overnight further by vacuum drying.
Yield of 6b for two steps: 55% (11.3 g, 9.90 mmol). White crystalline material; Rf 0.18
(
chloroform/methanol, 10:1); [α] could not be measured precisely owing to low solubility of the
D
compound in polar as well nonpolar solvents;
1
H NMR ((CD ) SO, characteristic signals): δ 1.67-1.74 (m, 2H, OCH CH CH N), 1.81 (s, 3H,
3
2
2
2
2
I
II
NHC(O)CH ), 3.15-3.27 (m, 2H, OCH CH CH N), 4.24 (d, 1H, J 7.5, H-1 or H-1 ), 5.19 (d,
3
2
2
2
1,2
III
13
1
H, J 3.5, H-1 ), 7.74 (d, 1H, J
8.3, NHCOCH ), 9.34 (br. t, 1H, NHCOCF ); C NMR
1
,2
2,NH 3 3
(
(
[
(
(CD ) SO, characteristic signals): δ 22.9 (NHC(O)CH ), 28.4 (OCH CH CH N), 36.5
3 2 3 2 2 2
III
I
II
OCH CH CH N), 92.5 (C-1 ), 100.8, 103.9 (C-1 and C-1 ); ESI HRMS: m/z Calcd
2
2
2
+
C H F N O ]H : 1149.4778. Found: 1149.4742.
6
0
71
3
2
17
1
13
H, C and HSQC NMR spectra pictures can be found in Supporting information)
3
-Trifluoroacetamidopropyl O-(2,3,4,6-tetra-O-benzyl-
O-benzylidene- -D-galactopyranosyl)-(1 4)-2-acetamido-6-O-benzyl-2-deoxy-
glucopyranoside (7b)
α
-D-galactopyranosyl)-(1
→
3)-O-(4,6-
β
→
β-D-

Trisaccharide 6b (11.2 g, 9.78 mmol) was suspended in dry acetonitrile (150 mL), then
PhCH(OMe) (4.5 mL, 30.0 mol) and solution of TsOH·H O (180 mg, 0.98 mmol) in dry
2
2
acetonitrile (3 mL) were added. Reaction mixture was stirred overnight at r.t. The reaction was
neutralised with pyridine (5 mL). Diethyl ether (300 mL) was added under stirring, and formed
crystalline product was collected on glass filter, washed with diethyl ether, n-hexane, dried in
vacuum.
Yield of 7b: 89% (10.8 g, 8.70 mmol). White crystalline material; Rf 0.30 (chloroform/methanol,
1
0:1); [α] +24° (c 0.9, CHCl );
D 3
1
H NMR ((CD ) SO, characteristic signals): δ 1.66-1.76 (m, 2H, OCH CH CH N), 1.81 (s, 3H,
3
2
2
2
2
III
NHC(O)CH ), 3.15-3.27 (m, 2H, OCH CH CH N), 5.33 (d, 1H, J 3.5, H-1 ), 5.64 (s, 1H,
3
2
2
2
1,2
1
3
PhCH), 7.75 (d, 1H, J2,NH 8.3, NHCOCH ), 9.34 (br. t, 1H, NHCOCF ); C NMR ((CD ) SO,
3
3
3 2
characteristic signals): δ 22.9 (NHC(O)CH ), 28.4 (OCH CH CH N), 36.5 (OCH CH CH N),
3
2
2
2
2
2
2
III
I
II
9
1.8 (C-1 ), 99.7 (PhCH), 100.9, 103.4 (C-1 and C-1 ); ESI HRMS: m/z Calcd
+
[
(
C H F N O ]H : 1237.5091. Found: 1237.5033.
6
7
75
3
2
17
1
13
H, C, COSY and HSQC NMR spectra pictures can be found in Supporting information)
3
-Trifluoroacetamidopropyl O-(2,3,4,6-tetra-O-benzyl-
O-benzylidene- -D-galactopyranosyl)-(1 4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-
D-glucopyranoside (8b)
α-D-galactopyranosyl)-(1→3)-O-(4,6-
β
→
β-
To powder of trisaccharide 7b (10.2 g, 8.24 mmol) was added solution of chloro acetic
anhydride (4.23 g, 24.7 mmol) and sym-Collidine (22.0 mL, 165 mmol) in abs. dichloromethane
55 mL) under intensive stirring. The mixture was stirred for 3 h at 0°С. The reaction was
(
quenched with methanol (20 mL), concentrated on rotary evaporator, co-evaporated with
toluene, dried in vacuum.

Resulting oil was dissolved in abs. dichloromethane (107 mL) followed by addition of sym-
Collidine (25 mL, 190 mmol) and acetyl chloride (10.0 mL, 140 mmol). Reaction mixture was
left for 24 h at r.t. Reaction was quenched with methanol (40 mL), concentrated on rotary
evaporator, co-evaporated with toluene. The residue was diluted with toluene (200 mL), stirred
for 15 min, collidinium salt was collected on glass filter, washed few times with toluene. The
filtered solution was concentrated on rotary evaporator, dried in vacuum.
The residue was dissolved in pyridine (200 mL), water (100 mL) was added and mixture warmed
at 60 °C for 24 h. The mixture was concentrated on rotary evaporator, co-evaporated with
toluene, dried in vacuum.
Crude product 8b was purified with column chromatography on silica gel (chloroform/acetone,
from 5:1 to 3:1, 0.5% pyridine), the fractions containing major product were collected,
concentrated on rotary evaporator (not to dryness). Concentrated solution was diluted with ethyl
acetate (100 mL), n-hexane (100 mL) was added drop-wise under stirring to precipitate pure
trisaccharide 8b and the mixture was left overnight in fridge for complete precipitation.
Solid product was collected on glass filter, washed with little portion of n-hexane/ethyl acetate
1
:1, n-hexane/ethyl acetate 3:1, pure n-hexane, dried in vacuum.
Yield of 8b for three steps: 73% (7.74 g, 6.05 mmol). White crystalline material; Rf 0.45 (n-
hexane/chloroform/propan-2-ol, 4:2:1); [α] +16° (c 1.2, CHCl );
D
3
1
H NMR (CDCl , characteristic signals): δ 1.47-1.71 (m, 1H, OCH CHHCH N), 1.98, 2.09 (2s,
3
2
2
6
H, OC(O)CH and NHC(O)CH ), 2.91-3.024 (m, 1H, OCH CH CHHN), 5.14 (d, 1H, J 3.2,
3 3 2 2 1,2
III
I
H-1 ), 5.28 (dd, 1H, J ≈ J 9.8, H-3 ), 5.42 (s, 1H, PhCH), 5.72 (d, 1H, J 7.1,
2,NH
2
,3
3,4
13
NHCOCH ); C NMR (CDCl , characteristic signals): 21.1, 23.5 (OC(O)CH and
3
3
3
I
III
NHC(O)CH ), 28.3 (OCH CH CH N), 38.2 (OCH CH CH N), 73.4 (C-3 ), 94.6 (C-1 ), 101.5
3
2
2
2
2
2
2
+
(
PhCH); ESI HRMS: m/z Calcd [C H F N O ]H : 1279.5196. Found: 1279.5111.
69 77 3 2 18
1
13
(
H, C, COSY and HSQC NMR spectra pictures can be found in Supporting information)

3
-Trifluoroacetamidopropyl O-(2,3,4,6-tetra-O-acetyl-
tri-O-acetyl- -L-fucopyranosyl-(1 2)]-O-(4,6-di-O-acetyl-
acetamido-3,6-di-O-acetyl-2-deoxy- -D-glucopyranoside (11b)
α
-D-galactopyranosyl)-(1
→
3)-O-[2,3,4-
4)-2-
α
→
β
-D-galactopyranosyl)-(1→
β
To ethyl thioglycoside 9 (5.78 g, 12.1 mmol) solution in abs. dichloromethane (75 mL) was
added bromine (0.69 mL, 13.3 mmol) at 0°С. The reaction was left at 0°C for 1 h. The solvent
and bromine was evaporated, residue was co-evaporated with toluene (3×50 mL) to remove
traces of bromine, almost colourless oil of glycosyl bromide was dried in vacuum at r.t.
To 8b (7.74 g, 6.05 mmol) solution in abs. dichloromethane (150 mL) were added freshly dried
MS-4Å (75 mL) and tetramethylurea (1.6 mL, 13.3 mmol), the mixture stirred for 30 min, then
AgOTf (3.11 g, 12.1 mmol) was added and the mixture was stirred for additional 15 min. To
reaction mixture glycosyl bromide (see above) solution in abs. dichloromethane (75 mL) was
added drop-wise under intensive stirring. The reaction was left for 24 h with stirring (TLC
control in ethyl acetate/toluene 3:1, Rf 0.50). Reaction mixture was neutralized with pyridine (6
mL), filtered through Celite on glass filter, solid material was washed with portion of
dichloromethane and mixture of dichloromethane/methanol 10:1.
Filtered solution was concentrated on rotary evaporator, co-evaporated with toluene, dried in
vacuum. The residue was dissolved in acetic acid (200 mL), water (50 mL) was added under
stirring and the mixture was warmed at 80 °C for 3 h (TLC control in pure ethyl acetate, Rf
0
.45). The mixture was concentrated on rotary evaporator, co-evaporated with toluene, dried in
vacuum.
Crude product 10b was partially purified with column chromatography on silica gel (from
toluene/ethyl acetate 1:1 to pure ethyl acetate), the fractions containing major product were
collected (TLC control in pure ethyl acetate), concentrated on rotary evaporator, co-evaporated
with toluene, dried in vacuum and used in next stage without additional purification (contains
admixture of tetramethylurea).

The product 10b was dissolved in methanol (300 mL), then 10% Pd/C (3.0 g) was added and
reaction mixture was degassed in vacuum further by filling with H (1 atm) three times. The
2
reaction was left under H (1 atm) with stirring for 48 h at r.t. Conversion was controlled by TLC
2
(propan-2-ol/ethyl acetate/water 2:3:1, product Rf 0.35). Reaction mixture was filtered through
Celite on glass filter, solid material was washed with methanol, portion of 50% aq. methanol and
resulting solution was concentrated on rotary evaporator, co-evaporated with toluene, dried in
vacuum. The residue was dissolved in pyridine (100 mL) and Ac O (50 mL), reaction mixture
2
was left at 40 °C for 24 h. Reaction mixture was concentrated on rotary evaporator, co-
evaporated with toluene.
Crude product 11b was purified with column chromatography on silica gel (from pure ethyl
acetate to ethyl acetate/ propan-2-ol 50:1), the fractions containing major product were collected
(TLC control in ethyl acetate/ propan-2-ol 50:1 and chloroform/ propan-2-ol 9:1), concentrated
on rotary evaporator, co-evaporated with toluene, dried in vacuum.
Prepurified product 11b was dissolved in ethyl acetate (100 mL). To resulting solution was
added n-hexane drop-wise under stirring till light turbidity appeared, after crystallization started,
addition of n-hexane (to whole solution volume 400 mL) was continued, the mixture allowed to
crystallize for 12 h. Crystalline material was collected on glass filter, washed with n-
hexane/ethyl acetate 3:1, pure n-hexane. Recrystallization procedure was repeated with the same
amount of solvents, obtained crystalline material was dried in vacuum.
Yield of 11b for four steps: 35% (2.78 g, 2.12 mmol). White crystalline material; Rf 0.35
(
chloroform/propan-2-ol, 9:1); [α]546 -11° (c 1, CHCl ), lit. [19] -11°;
3
1
1
H spectrum was identical to early described in lit. [10], [19]; H NMR (CDCl ): δ 1.16 (d, 3H,
3
III
J 6.5, H-6 ), 1.78–1.84 (m, 1H, OCH CHHCH N), 1.86–1.90 (m, 1H, OCH CHHCH N),
5
,6
2
2
2
2
1
.91, 1.97, 1.99, 1.99, 2.07, 2.07, 2.07, 2.08, 2.10, 2.11, 2.15, 2.16 (12s, 36H, 11 OC(O)CH , 1
3
II
NHC(O)CH ), 3.24–3.31 (m, 1H, OCH CH CHHN), 3.56 (dd, 1H, J 7.6, J 10.0, H-2 ),
3
2
2
1,2
2,3
3
.57–3.61 (m, 1H, OCHHCH CH N), 3.61–3.66 (m, 1H, OCH CH CHHN), 3.78 (dd, 1H, J
2 2 2 2 5,6a

II
I
II
6
1
.6, J5,6b 6.6, H-5 ), 3.79–3.81 (m, 1H, H-5 ), 3.88 (dd, 1H, J 10.0, J 2.8, H-3 ), 3.91 (dd,
2,3 3,4
I
H, J 8.8, J 8.2, H-4 ), 3.92–3.94 (m, 1H, OCHHCH CH N), 4.02 (ddd, 1H, J 7.7, J 9.3,
3
,4
4,5
2
2
1,2
2,3
I
II
II
IV
J2,NH 8.8, H-2 ), 4.06–4.13 (m, 3H, H-6a , H-6b , H-6a ), 4.24 (dd, 1H, J 6.9, J6a,6b 11.2, H-
5,6b
IV
II
I
III
I
6
5
8
2
b ), 4.39 (d, 1H, J 7.6, H-1 ), 4.39–4.43 (m, 2H, H-6a , H-5 ), 4.44–4.48 (m, 2H, H-6b , H-
1,2
IV
I
III
), 4.49 (d, 1H, J 7.7, H-1 ), 5.05 (dd, 1H, J 11.1, J 3.4, H-3 ), 5.07 (dd, 1H, J 9.3, J
1
,2
2,3
3,4
2,3
3,4
I
III
III
.8, H-3 ), 5.21 (dd, 1H, J 3.7, J 11.1, H-2 ), 5.25 (br. d, 1H, J 3.4, H-4 ), 5.32–5.36 (m,
1
,2
2,3
3,4
IV
IV
II
IV
III
H, H-1 , H-2 ), 5.39–5.44 (m, 2H, H-4 , H-3 ), 5.45 (d, 1H, J 3.7, H-1 ), 5.60 (br. d, 1H,
1
,2
IV
I
J 2.7, H-4 ), 5.86 (d, 1H, J
8.8, NHAc ), 7.39–7.44 (m, 1H, NHC(O)CF );
3
3
,4
2,NH
1
(
H NMR spectrum picture can be found in Supporting information)
3
-Aminopropyl O-(
α
-D-galactopyranosyl)-(1
→
3)-O-[α-L-fucopyranosyl-(1→2)]-O-(β-D-
galactopyranosyl)-(1
→
4)-2-acetamido-2-deoxy-
β-D-glucopyranoside (1b)
Tetrasaccharide 11b (2.50 g, 1.98 mmol) was dissolved in abs. methanol (150 mL), treated with
M NaOMe (7.50 mL, 15 mmol) and reaction mixture was left for 2 h. The mixture was
2
concentrated in vacuum (not to dryness) and diluted with water (150 mL), reaction mixture was
left overnight.
Target deprotected tetrasaccharide was isolated with cation-exchange chromatography on Dowex
5
0X4-400 (H+) by subsequent elution with water, 1M pyridine water solution and 1M aqueous
ammonia, fractions containing the carbohydrate were collected, concentrated in vacuum and
freeze dried twice.
1
Yield of 1b for two steps: 90% (1.33 g, 1.78 mmol). White solid material; H spectrum was
identical to early described in lit. [7], [19]; [α] -10° (c 1, H O), lit. [19] -10°;
D
2
1
H NMR (D O+TFA, characteristic signals): δ 1.29 (d, 3H, J 6.6, FucMe), 1.97-2.05 (m, 2H,
2
5,6
OCH CH CH N), 2.11 (s, 3H, NHC(O)CH ), 3.14 (t, 2H, J 7.0, OCH CH CH N), 4.55, 4.67
2
2
2
3
2
2
2
I
II
III
IV
(
2d, 2H, J 8.5, J 7.7, H-1 and H-1 ), 5.30, 5.38 (2d, 2H, J <2.0, J 4.2, H-1 and H-1 ).
1,2 1,2 1,2 1,2

1
(
H NMR spectrum picture can be found in Supporting information)

4
.2.3 Synthesis of trisaccharide 12a
3
-Aminopropyl O-(2-acetamido-2-deoxy-
α
-D-galactopyranosyl)-(1→3)-O-(β-D-
galactopyranosyl)-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (12a)
To ethyl thioglycoside 4a (3.76 g, 7.24 mmol) solution in abs. dichloromethane (40 mL) was
added bromine (0.41 mL, 8.0 mmol) at 0°С. The reaction was left at 0°C for 1 h. The solvent and
bromine was evaporated, residue was co-evaporated with toluene (3×30 mL) to remove traces of
bromine, almost colourless oil of glycosyl bromide was dried in vacuum at r.t.
To disaccharide 3 (3.82 g, 4.82 mmol) solution in abs. dichloromethane (50 mL) were added
freshly dried MS-4Å (8 mL) and tetramethylurea (1.08 mL, 8.87 mmol), the mixture stirred for
3
0 min, then AgOTf (2.04 g, 7.82 mmol) was added and the mixture was stirred for additional 15
min. To reaction mixture glycosyl bromide (see above) solution in abs. dichloromethane (20 mL)
was added drop-wise under intensive stirring. The reaction was left for 24 h with stirring (TLC
control in chloroform/methanol 10:1, Rf 0.47). Reaction mixture was neutralized with pyridine (4
mL), filtered through Celite on glass filter, solid material was washed with portion of
dichloromethane and mixture of dichloromethane/methanol 10:1. The filtrate was poured in 10%
aq. solution of Na S O (60 mL), washed with cold sat. NaHCO (50 mL), dried with
2
2
3
3
Na SO /SiO .
2
4
2
After filtration, the solution was concentrated on rotary evaporator, co-evaporated with toluene,
dried in vacuum. The product 5a was partially purified with column chromatography on silica
gel (from toluene/acetone 5:1 to toluene/acetone 3:1), the fractions containing major product 5a
were collected, concentrated on rotary evaporator, co-evaporated with toluene, dried in vacuum
and used in next stage without additional purification (contains admixture of tetramethylurea).
The partially purified product 5a was dissolved in methanol (300 mL), then 10% Pd/C (4.0 g)
was added and reaction mixture was degassed in vacuum further by filling with H (1 atm) three
2

times. The reaction was left under H (1 atm) with stirring for 12 h. Acetic anhydride (15 mL)
2
was added to reaction mixture under intensive stirring and the mixture was additionally stirred
for 15 min. The solution was degassed in vacuum further by filling with H (1 atm) three times.
2
The reaction was left under H (1 atm) with stirring for 24 h. Conversion was controlled by TLC
2
(propan-2-ol/ethyl acetate/water 2:10:1). Reaction mixture was filtered through Celite on glass
filter, solid material was washed with methanol, portion of 50% aq. methanol and resulting
solution was concentrated on rotary evaporator, co-evaporated with toluene, dried in vacuum.
The residue was dissolved in pyridine (100 mL) and Ac O (50 mL) and left at 40 °C for 48 h.
2
Reaction mixture was concentrated on rotary evaporator, co-evaporated with toluene.
Crude product 13a (chloroform/methanol 10:1, Rf 0.36 )was partially purified with column
chromatography on silica gel (from pure ethyl acetate to ethyl acetate/propan-2-ol 20:1), the
fractions containing major product were collected, concentrated on rotary evaporator, co-
evaporated with toluene, co-evaporated with chloroform, dried in vacuum. The product 13a
contains admixture of tetramethylurea, but being amorphous solid cannot be completely purified
with crystallization [34].
Partially purified trisaccharide 13a was dissolved in abs. methanol (230 mL), treated with 2M
NaOMe (11.2 mL, 22.6 mmol) and reaction mixture was left for 2 h. The mixture was
concentrated in vacuum (not to dryness) and diluted with water (230 mL), reaction mixture was
left overnight.
Target deprotected trisaccharide was isolated with cation-exchange chromatography on Dowex
5
0X4-400 (H+) by subsequent elution with water, 1M pyridine water solution and 1M aqueous
ammonia, fractions containing the carbohydrate were collected, concentrated in vacuum and
freeze dried twice.
Yield of 12a for five steps: 49% (1.50 g, 2.34 mmol). White solid material; [α]546 +128° (c 0.3,
MeCN-H O, 1:1), lit. [34] +128°;
2