Function-spacer-lipid constructs of Lewis and chimeric Lewis/ABH glycans. Synthesis and use in serological studies

Article abstract of DOI:10.1016/j.carres.2016.09.016

Seven lipophilic constructs containing Lewis (Le^a, Le^b, Le^y) or chimeric Lewis/ABH (ALe^b, BLe^b, ALe^y, BLe^y) glycans were obtained starting from corresponding oligosaccharides in

Full text of DOI:10.1016/j.carres.2016.09.016

Accepted Manuscript
Function-spacer-lipid constructs of Lewis and chimeric Lewis/ABH glycans. Synthesis
and use in serological studies
Ivan M. Ryzhov, Elena Yu Korchagina, Alexander B. Tuzikov, Inna S. Popova,
Tatiana V. Tyrtysh, Galina V. Pazynina, Stephen M. Henry, Nicolai V. Bovin
PII:
S0008-6215(16)30290-7
10.1016/j.carres.2016.09.016
CAR 7266
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 4 August 2016
Revised Date: 26 September 2016
Accepted Date: 27 September 2016
Please cite this article as: I.M. Ryzhov, E.Y. Korchagina, A.B. Tuzikov, I.S. Popova, T.V. Tyrtysh,
G.V. Pazynina, S.M. Henry, N.V. Bovin, Function-spacer-lipid constructs of Lewis and chimeric Lewis/
ABH glycans. Synthesis and use in serological studies, Carbohydrate Research (2016), doi: 10.1016/
j.carres.2016.09.016.
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ACCEPTED MANUSCRIPT
Graphical abstract
HO OH
OH
O
OH
OH OH
O
O
O
b
O(CH ) NH
ALe : R=NHAc
2
3
2
O
O
b
NHAc
BLe : R=OH
O
O
R
O
HO
HO
OH
HO
OH
HO
OH
N
OH
O
O
O
O
H
O
H
O
O O H
O
O
H O
N
H
H
N
N
N
N
N
N
O
N
N
N
N
H
N
y
N
N
O
Sug
N
O
H O O
O
H O
O
O
O
H
O
H
O
H O
O
O
P
OH
OH
O
OH
a
b
y
b
b
y
Sug=Le , Le , Le , ALe , BLe , ALe , BLe

ACCEPTED MANUSCRIPT
Function-Spacer-Lipid Constructs of Lewis and Chimeric Lewis/ABH Glycans.
Synthesis and Use in Serological Studies
a
a
a
a
Ivan M. Ryzhov , Elena Yu. Korchagina *, Alexander B. Tuzikov , Inna S. Popova ,
a
a
b
a,b
Tatiana V. Tyrtysh , Galina V. Pazynina , Stephen M. Henry , Nicolai V. Bovin *
a
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation.
b
Centre for Kode Technology Innovation, School of Engineering, Auckland University of
Technology, Auckland, New Zealand.
*
ellyu@yandex.ru; korchagina@carb.ibch.ru (E. Yu. Korchagina),
professorbovin@yandex.ru (N.V. Bovin), phone +7 495 330 0300, fax +7 495 330 5592.
Abstract
a
b
y
Seven lipophilic constructs containing Lewis (Le , Le , Le ) or chimeric Lewis/ABH
b
b
y
y
(
ALe , BLe , ALe , BLe ) glycans were obtained starting from corresponding oligosaccharides
b
b
in form of 3-aminopropyl glycosides. ALe and BLe pentasaccharides were synthesized via
[
3+1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte
membrane, and obtained “kodecytes” were used to map the immunochemical specificity of
historical and contemporary monoclonal and polyclonal blood group system Lewis reagents.
Keywords
Neoglycolipid; Lewis antigens; ABO antigens; Lewis antigens; Block synthesis; FSL
b
b
constructs; kodecytes; ALe ; BLe
. Introduction
1
1
Lewis antigens are a family of structurally related fucosylated oligosaccharides,
a
represented by trisaccharide Galβ1→3(Fucα1→4)GlcNAcβ (Le ) and tetrasaccharide
b
Fucα1→2Galβ1→3(Fucα1→4)GlcNAcβ (Le ). Lewis glycans are found on cell surface in
composition of both glycoproteins and glycolipids. Glycans Galβ1→4(Fucα1→3)GlcNAcβ
x
y
(
Le ) and Fucα1→2Galβ1→4(Fucα1→3)GlcNAcβ (Le ) in spite of “Lewis” names and tightly

ACCEPTED MANUSCRIPT
related (isomeric) chemical structure in reality are not conventional antigens of the Lewis blood
group system, which leads to confusion, especially in chemical literature. As R.U. Lemieux
2
a
x
demonstrated in 1970s, stereochemically Le and Le are very similar because in both molecules
the Fuc and Gal residues form the same spatial pair (the same is true for Fuc/Fuc-Gal pair in case
b
y
of Le vs. Le ), and that due to similar conformations a number of serological anti-Lewis
x
y 3
reagents display cross-reactions with Le or Le . Of note, naturally occurring human antibodies
a
x
(
allo-antibodies) against Le never cross-react with constitutively expressed Le antigen on
human blood and epithelium cells, otherwise we could have auto-immune reactions.
b
y
b
The story is complicated by existence of “chimeric” structures, ALe , АLe , BLe , and
y
BLe possessing structural features of both Lewis antigens (the presence of α-Fuc residue at С-3
or С-4 of GlcNAc) and A or B motifs (the presence of α-GalNAc or α-Gal units at С-3 of β-
x
a
galactose). Also, Le and Le exist in form of sialylated and or sulfated glycans, known to be
4
ligands for selectins. The structural/conformational similarity in combination with occurrence of
resembling glycans makes immunochemical (with antibodies) identification and quantitation of
particular Lewis antigens a non-trivial task, requiring very precise tools for selection and
characterization of monoclonal reagents and polyclonal anti-Lewis sera. Here, we describe
synthesis of most complicated representatives of Lewis glycan superfamily, chimeric Le/ABH
glycans, and lipid-conjugated forms of the chimeric and their more simple “partial” glycans.
5
,6
Recently a new class of glycolipid-like constructs has been developed. These
compounds designed as conjugates with both variable glycans (having biological functions),
spacer-arms and lipids, are known as FSLs (Function-Spacer-Lipid, Figure 1).

ACCEPTED MANUSCRIPT
Figure 1. Schematic presentation of (a) FSL molecule design, (b) micelle formed by FSL in
aqueous solutions, (c) erythrocyte modified by FSL (kodecyte), (d) monolayer of FSL on the
solid support, its interaction with IgG and IgM antibodies followed by color reaction mediated
by secondary antibody (in green), actual result image (in grey) is shown as well, (e)
agglutination of erythrocytes due to bridging of cells with IgM antibody against glyco-part of
FSL.
Unlike natural glycolipids, FSLs are easily dispersible in water, saline, and biological
5
,6
media. They can incorporate into cell membrane without affecting of functionality or vitality.
Attachment of FSLs to live cells can be achieved in precise and controlled ways, and modified
5
,6
cells, “kodecytes”, possess known glycan density. Due to the properties described function-
spacer-lipid constructs are powerful tools for a wide range research into biological functions of
glycans on cell surfaces. The synthesized FSLs described in this paper were used for
7
glycomapping of monoclonal and polyclonal serological Lewis reagents and clinical samples.

ACCEPTED MANUSCRIPT
a
b
These FSL constructs were used to create both Lewis kodecytes expressing single Le , Le ,
b
b
x
y
y
y
ALe , BLe , Le , Le , ALe , or BLe glycans, and the same FSLs were inkjet printed in a
paper-based microplate and analyzed by enzyme immunoassay – for comparison with the cell
assay.
2
. Results and discussion
Function-spacer-lipid constructs 1–7 were obtained from 3-aminopropyl glycosides of
a 8
b 8
y 9,10
oligosaccharides 8–14 (Scheme 1). Synthesis of derivatives 8 (Le ) , 9 (Le ) , 10 (Le ) , 13
y 11
y 11
b
(
ALe ) , and 14 (BLe ) were described earlier. Aminopropyl glycosides 11 (ALe ) and 12
b
(
BLe ) were obtained via [3+1] block scheme. Their synthesis is described below.
OH
OH
O
O
O
H
O
H
O O
N
H
O
H
O
H
O
O
H
O
H
O
O
N
N
N
N
N
O
N
N
N
N
N
N
N
N
N
Sug
N
O
H
O O
O
H
O
O
O
H
O
H
O
H O
O
O
P
OH
OH
O
OH
1-7
Sug
NH
2
8-14
HO OH
HO OH
HO OH
OH
O
OH
O
OH
O
O
OH
O
OH
OH OH
OH OH
OH OH
OH
O
2, 9: Sug =
O
3, 10: Sug =
1, 8: Sug =
O
O
O
O
O
O
HO
O
O
HO
O
HO
O
NHAc
OH
NHAc
O
O
O
NHAc
Lea
O
Leb
O
OH
OH
Ley
HO
OH
HO
OH
HO OH
HO OH
OH
OH
O
O
OH
OH
4
, 11: Sug =
OH OH
5,12: Sug =
OH OH
O
O
O
O
O
O
NHAc
O
O
O
O
O
O
NHAc
O
O
O
NHAc
O
OH
HO
HO
O
OH
HO
HO
O
OH
ALeb
BLeb
HO
OH
HO
OH
HO
HO
HO OH
HO OH
OH
O
OH
O
O
O
OH OH
OH
OH OH
OH
O
6
, 13: Sug =
7, 14: Sug =
O
O
O
O
NHAc
O
O
NHAc
O
O
O
O
O
O
NHAc
O
OH
O
OH
HO
HO
O
OH
HO
HO
HO
OH
ALey
HO
OH
BLey
HO
HO
Scheme 1.
.1 Synthesis of pentasaccharides ALe and BLe
b
b
2

ACCEPTED MANUSCRIPT
Recently we proposed new approach to the synthesis of blood group A and B
1
2–14
tetrasaccharides of various types, namely block scheme [3+1].
The main advantage of this
strategy is employment of common A or B trisaccharide glycosyl donors – peracetylated
1
2
13
trichloracetimidetes of A or B trisaccharides 19 and 20 (Scheme 2). For the synthesis of each
type of target tetrasaccharides suitably protected monohydroxyl monosaccharide glycosyl
acceptor was used.
The use of glucosamine diol 18 as glycosyl acceptor in block scheme [3+1] could
potentially provide both tetrasaccharides of type 1 and type 2 simultaneously. Moreover, free
hydroxyl group at C-3 or C-4 of GlcNAc of obtained tetrasaccharides could give an opportunity
b
b
y
y
to synthesize fucosylated pentasaccharides ALe (BLe ) or ALe (BLe ), respectively.
8
Glucosamine diol 18 was easily obtained from known benzylidene derivative 15 in three
1
5
stages: acetylation of 3-OH, regioselective reductive ring-opening of benzylidene cycle, and
deacetylation (Scheme 2).

ACCEPTED MANUSCRIPT
3
Ph
OR
OBn
O
O
(a)
O
O
R O
2
O
HO
HO
sp
sp
sp
1
HO
R O
NHAc
NHAc
3
NHAc
(c)
15
1
2
18
1
1
6: R = Ac, R , R = Bd
7: R = Ac, R = H, R = Bn
(b)
1
2
3
OBn
O
HO
O
sp
OAc
OAc
O
Ac A
9
tri
NHAc
O
NHAc
21
O
O
OC(NH)CCl3
+
+
O
Ac A
AcO
AcO
OAc
9
tri
OBn
OAc
OAc
18
d)
AcO
HO
O
sp
NHAc
O
(
Ac A
9
tri
22
OC(NH)CCl3
OBn
19
O
O
sp
NHAc
Ac A
9
tri
HO
2
3
OAc
OAc
O
O
OAc
O
O
OC(NH)CCl3
OAc
OBn
O
AcO
AcO
18
d)
O
HO
O
OAc
OAc
sp
NHAc
AcO
Ac B
9
tri
(
Ac B
24
9
tri
OC(NH)CCl3
20
sp = O(CH ) NHC(O)CF
3
2
3
Scheme 2. Reagents and conditions: (a) Ac O/Py, 97%, (b) NaBH CN, MsOH, THF, 80%, (c)
2
3
MeONa/MeOH, 96%, (d) TMSOTf, CH CN–CH Cl , 1:1, 33% (21), 10% (22), 5% (23), 43%
3
2
2
(24).
The glycosylation reactions of the acceptor 18 with the peracetylated trichloracetimidates
of A trisaccharide 19 and B trisaccharide 20 were carried out in CH CN–CH Cl (1:1) mixture to
3
2
2
1
6
enhance β-stereospecificity via nitrile effect. TMSOTf was used as catalyst. Coupling of the
diol 18 and the A trisaccharide imidate 19 provided the mixture of products (Scheme 2), from
which three tetrasaccharides: β-1→3-glycoside 21 (33%), α-1→3-glycoside 22 (10%), and β-

ACCEPTED MANUSCRIPT
1
→4-glycoside 23 (5%), were isolated. Condensation of the B trisaccharide trichloracetimidate
2
0 with the glycosyl acceptor 18 also resulted in complex multicomponent mixture. Only one
product, β-1→3-glycoside 24 was isolated as individual compound in 43% yield (Scheme 2).
1
Acetylated analogues 21Ac–24Ac were obtained in analytical amounts. All the signals in H and
1
3
C NMR spectra of the tetrasaccharides 21–24 and 21Ac–24Ac were identified with the use 2D-
1
1
1
13
H, H-COSY and 2D- H, C-HSQC experiments. Stereochemistry of newly formed glycosidic
linkages in 21–24 was confirmed using characteristic values of chemical shift and coupling
II
1
constant for H-1 signal in H NMR spectra (Table 1). To identify the position of the formed
1
glycosidic linkages H NMR spectra of the tetrasaccharides 21–24 were compared with the
spectra of acetylated analogues 21Ac–24Ac. Acetylation of free OH-group at C-3 or C-4 in
GlcNAc unit led to substantial (1–1.5 ppm) shift of signal of H-3 or H-4, respectively, in lower
field (Table 1). Thus, the position of the glycosidic linkages and the hydroxyls in 21–24 was
determined.
Table 1. Determination of configuration and position of newly formed glycosidic bonds in
tetrasaccharides 21–24.
Configuration and position of
formed glycosidic linkage
II
I
I
Compound
H-1 ,
δ
(ppm), J1,2 (Hz) H-3 ,
δ
(ppm)
H-4 ,
δ
(ppm)
2
2
2
2
2
2
2
2
1
β-1→3
4.50, J1,2 7.6
5.35, J1,2 3.6
4.08, J1,2 7.7
4.56–4.62
4.67–4.75
4.03
3.43
4.81
1Ас
2
α-1→3
β-1→4
β-1→3
3.72–3.78
5.03
2Ас
3
4.39
3.85–3.89
4.98
3.78
3Ас
4
3.90–4.09
3.59–3.64
4.92
4.55–4.65
3.98–4.04
4.23–4.35
4Ас
4.42, J1,2 7.6

ACCEPTED MANUSCRIPT
The yields of the tetrasaccharides A (type 1) 21 and B (type 1) 24 were lower comparing
1
2,13
with the glycosylation of monohydroxyl derivative 15.
The A (type 2) tetrasaccharide 23 was
isolated in minor quantities (5%), the B (type 2) tetrasaccharide was not isolated at all.
Nevertheless, the main products of glycosylation – the tetrasaccharides 21 and 24 with the free
OH-group at C-4 of the glucosamine unit – were convenient intermediates for the synthesis of
b
b
y
y
the fucosylated pentasaccharides ALe 11 and BLe 12. ALe and BLe pentasaccharides were
1
1
synthesized according to an alternative strategy.
Fucosylation of the acceptors 21 and 24 was carried out with the use of the known
1
7
fucosyl imidate 25 in Et O–CH Cl (3:1) mixture in the presence of TMSOTf. The
2
2
2
glycosylation of the A tetrasaccharide derivative 21 with the imidate 25 gave the mixture of two
1
products: required pentasaccharide 27 (60%) and its β-anomer 26 (13%) (Scheme 3). In H NMR
spectra the H-1 signal of 1→4-linked fucose appeared at δ 5.09 with J 3.7 Hz (consistent with
1
,2
α-glycosidic linkage) for 27 and at δ 4.52 with J 9.0 Hz (consistent with β-glycosidic linkage)
1
,2
for 26.

ACCEPTED MANUSCRIPT
BnO
BnO
O
BnO
OAc
OBn
O
OAc
O
O
sp
O
O
NHAc
NHAc
O
O
O
AcO
AcO
OAc
OAc
OBn
OAc
AcO
OAc
OAc
O
OC(NH)CCl3
OBn
O
O
HO
O
sp
NHAc
OBn
2
6
O
OBn
O
+
O
NHAc
25
a)
O
AcO
AcO
OAc
OAc
OAc
RO OR
(
AcO
OR
O
21
OAc
OR
OAc
O
O
O
sp
O
O
NHAc
O
O
NHAc
O
AcO
AcO
OAc
OAc
AcO
OAc
(c)
27: R = Bn
28: R = Ac
(b)
11
OBn
RO OR
OAc
OAc
O
OR
HO
O
O
O
sp
NHAc
O
OC(NH)CCl3
OBn
O
OAc
OR
OAc
OAc
O
O
OBn
O
O
O
sp
NHAc
OBn
O
AcO
AcO
OAc
O
25
O
OAc
OAc
OAc
O
O
AcO
(
a)
O
OAc
OAc
OAc
AcO
AcO
24
AcO
(c)
29: R = Bn
30: R = Ac
(
b)
1
2
sp = O(CH ) NHC(O)CF
3
2
3
Scheme 3. Reagents and conditions: (a) TMSOTf, Et O–CH Cl , 3:1, 13% (26), 60% (27), 75%
2
2
2
(
29), (b) (i) MeONa/MeOH, (ii) H , Pd/C, MeOH, (iii) Ac O/Py, 85% (28), 88% (30), (c) (i)
2 2
MeONa/MeOH, (ii) NaOH/H O, 94% (11), 94% (12).
2
Condensation of the derivative 25 with the B tetrasaccharide 24 provided only α-
1
fucosylated pentasaccharide 29 in 75% yield (Scheme 3). In H NMR spectrum of 29 H-1 of
1
→4-linked fucose appeared as the doubled at δ 5.14 with J 3.7 Hz, confirming formation of
1,2
α-glycosidic linkage.

ACCEPTED MANUSCRIPT
Removal of protecting groups by conventional methods (Scheme 3) provided
b
b
aminopropyl glycosides ALe 11 and BLe 12.
.2 Synthesis of function spacer lipids (FSLs)
2
Carboxymethylglycine peptide (CMG) spacer was chosen for FSLs 1–7. It was earlier
shown, that CMG possessed optimal length (~7 nm), enough for spacing glycan away from
membrane, does not bind any serum proteins and inducing maximal specific serological
5
,6
reactivity.
The synthesis of FSLs 1–7 was conducted in two steps: the aminopropyl glycosides 8–14
were reacted with Ad(ONSu) 31 to give activated ethers 32–38 (75–97%), which were then
2
condensed with the CMG-derivative 39 (Scheme 4). The compounds 1–7 were isolated from
reaction mixtures by gel filtration on Sephadex LH-20 in 71–90% yields. Structures of the
1
derivatives 1–7 were confirmed by H NMR spectroscopy and mass-spectrometry.
O
O
O
O
O
(
a)
Sug
NH2
H
N O
O N
O
Sug
+
N
O N
O
O
O
O
8-14
31
32–38
OH
OH
O
O
O
O
O
O
O
N
H
N
O
O
H
H
O
H
O
H
N
O
N
N
N
N
O
N
O
N
N
H
N
N
H
(b)
H N
2
N
N
O
O
O
H
H
O
O O
H
O
O
O
O
P
O
OH
OH
OH
39
OH
OH
O
O
O
O
O
H O
N
H
O
O
H
O H
N
O
H
N
O
H
N
Sug
N
N
N
N
O
N
N
N
O
N
N
N
N
H
N
O
H O O
O
H O
O
O
O
H
O
H
O
H O
P
O
O
OH
OH
O
OH
1-7
a
b
y
b
1
5
, 8, 32: Sug = Le ; 2, 9, 33: Sug = Le ; 3, 10, 34: Sug = Le ; 4, 11, 35: Sug = ALe ;
b
y
y
, 12, 36: Sug = BLe ; 6, 13, 37: Sug = ALe ; 7, 14, 38: Sug = BLe

ACCEPTED MANUSCRIPT
Scheme 4. Reagents and conditions: (a) DMSO, 97% (32), 81% (33), 77% (34), 75% (35), 79%
(
36), 79% (37), 77% (38), (b) 50 mM NaHCO aq–i-PrOH, 2:1, DMSO, 78% (1), 74% (2), 78%
3
(3), 82% (4), 71% (5), 85% (6), 90% (7).
2
.3 Kodecyte and printed FSL-EIA biological assays
The extended biological analysis of monoclonal and polyclonal Lewis reagents, together
with clinical samples with Lewis antibodies using the FSL constructs described in this paper are
7
reported in detail elsewhere . Using red cells modified with FSL constructs (kodecytes) and the
same FSL constructs printed in solid phase on paper, a large series of monoclonal anti-Lewis
serological reagents were mapped. A compiled summary of the different observable reaction
patterns using natural cells, kodecytes and solid phase FSL constructs is shown in Figure 2. The
a
monoclonal reagents tested originally only mapped against natural red cells as either anti-Le or
b
anti-Le . However, when mapped against kodecytes prepared to carry a specific Lewis antigen,
these reagents could be seen to have significant serological cross-reactivity. The kodecytes were
prepared by mixing Lewis negative red cells with a Lewis FSL construct, which spontaneously
and stably inserts into the cell membrane, and creates a red cell with controlled levels of a
specific Lewis antigen.
When results from the kodecytes are compared with the same FSLs printed in solid phase
and visualized by EIA (Figure 2) they strongly correlated with each other, and revealed a
x
continuum of cross-reactivity from Le through to H. The FSL printed assay uses the same
constructs as used to prepare the kodecytes except they are applied to paper using an inkjet
printer (and printed in the name of the specificity they represent).
Together or alone the FSLs in the kodecytes and in the solid phase EIA assays were both
a
able to reveal fine specificity of the monoclonal reagents tested. Most monoclonal anti-Le
x
b
b
reagents could be shown to either crossreact with Le or Le , while most anti-Le reagents were
a
y
shown to crossreact with Le and Le . Significant differences were also observed in the
7
specificity of historical polyclonal reagents with modern monoclonal reagents . Obtained results

ACCEPTED MANUSCRIPT
clearly demonstrate that kodecytes, i.e. red cells inserted with neoglycolipids (FSLs), are an
indispensable analytical tool capable of discriminating the fine specificity of serological
reagents.
Figure 2. Mapping of monoclonal Lewis reagents with red cells modified with FSL constructs
(
Lewis kodecytes) and the same FSL constructs printed in solid phase on paper revealed almost
a
a
identical results. Monoclonal anti-Le reagents that showed only Le specificity against natural
a+x
a
a+b
b
red cells were subdivided into three related activities, Le , Le , and Le . The anti-Le reagents
a+b
a+b+y
b+y
b
were subdivided into four related specificities, Le , Le
, Le and Le . These fine
specificities of the reagents including their cross-reactivity with related structures were not
evident when tested against natural red cells. These results highlight the risk of defining reagents
purely on the basis of reactivity against natural cells and then using these serologically defined
Lewis reagents in assays other red cell serology.
3
. Conclusion

ACCEPTED MANUSCRIPT
A blockwise approach employing A and B blood group trisaccharides as glycosyl donors
b
b
was developed for the synthesis of ALe and BLe pentasaccharides. This methodology was
shown to be reasonable for obtaining of large library of structurally similar blood group antigens
b
b
(
ALe , BLe , and also A and B (types 1, 2, 3, and 4) tetrasaccharides, synthesis of which was
described earlier).
Conversion of C3 spacer-armed glycans into neoglycolipids (so called Functional Spacer
Lipids, FSLs) according to a standardized protocol proceeds with yields ~80%. Seven lipophilic
a
b
y
b
b
y
y
constructs (Le , Le , Le , ALe , BLe , ALe , BLe ) were obtained.
FSLs where L is phosphatidyl ethanolamine residue spontaneously insert into
erythrocytes in a controllable fashion giving rise to monospecific-antigen modified erythrocytes
(kodecytes). Naturally impossible kodecytes bearing only a single antigen of interest can be
rapidly created and used in routine serological methods to map the fine immunochemical
specificity of monoclonal and polyclonal blood group reagents.
4
4
. Experimental
.1 General procedures
Reactions were performed with the use of commercial reagents (Acros, Aldrich, and
Fluka); anhydrous solvents were purified according to the standard procedures. Column
chromatography was performed on Silica gel 60 0.040–0.063 mm (Merck), gel filtration was
carried out on Sephadex LH-20 (Pharmacia) columns. Solvents were removed in vacuum at 30–
о
4
0 С. Thin layer chromatography (TLC) was performed on Silica gel 60 F aluminum-backed
2
54
plates (Merck). Spots of compounds were visualized by dipping a TLC plate into aqueous
о
solution of H PO (8%) and subsequent heating (>150 С). Deacetylation was carried out in
3
4
absolute MeOH by the addition of catalytic amount of 2M MeONa in MeOH (according to
+
+
Zemplen). Na ions were then removed by Dowex 50X4-400 (Acros) (H ) cation exchanger and
solution was evaporated. Hydrogenolysis was carried out on 10% Pd/C (Merck) in the
atmosphere of H2.

ACCEPTED MANUSCRIPT
HO OH
HO OH
OH
O
III
OH
O
OH
OH OH
III
OH
I
O
O
O
O
OH OH
O
O
O
HO
O
O
NHAc
O
O
I
HO
II
NHAc
II
OH
O
OH
HO
OH
IV
a
b
y
Le (1)
Le , Le (2, 3)
HO OH
OH
O
O
III
OH
OBn
OAc
II
OAc
O
OH OH
O
O
HO
O
O
O
sp
NHAc
O
O
O
NHAc
IV
I
NHAc
II
O
O
I
O
NHAc
O
HO
HO
V
O
OH
AcO
AcO
O
OAc
OAc
OAc
HO
OH
HO
AcO
IV
III
b
b
y
y
Tetrasaccharides 21-24, 21Ac-24Ac
ALe , BLe , ALe , BLe
(
4, 5, 6, 7, 11, 12, 26, 27, 28, 29, 30)
Scheme 5. Symbols of monosaccharide units in NMR spectra.
The values of optical rotations were measured on a Perkin Elmer 341 polarimeter at 21±2
o
o
1
С. Н NMR spectra were recorded on a Bruker BioSpin GmbH (700 МHz) spectrometer at 30
С; chemical shifts (δ, ppm) were referred to the peak of internal D O (
δ
4.750), CDCl3 (δ
2
1
7
.270), or CD OD (
δ
3.500); coupling constants (J) were measured in Hz. Signals of Н NMR
3
1
3
spectra were assigned to the corresponding protons using 2D spectroscopy (COSY). C NMR
spectra were recorded at 150 MHz. Symbols of monosaccharide residues in NMR spectra are
shown on Scheme 5. ESIMS spectra were recorded on an Exactive Orbitrap (Thermo Fisher
Scientific, Germany) spectrometer; MALDI MS spectra were recorded on Brucker Daltonics
Ultraflex MALDI TOF/TOF Mass Spectrometer (Germany), HRESIMS spectra were recorded
on an Agilent 6224 TOF LC/MS instrument (USA).
b
b
4
.2 Synthesis of pentasaccharides ALe and BLe

ACCEPTED MANUSCRIPT
4
.2.1 3-Trifluoroacetamidopropyl 2-acetamido-6-O-benzyl-2-deoxy-β-
D-
glucopyranoside (18)
4
.2.1.1 3-Trifluoroacetamidopropyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-
-glucopyranoside (17)
Compound 15 (1.47 g, 3.18 mmol) was acetylated (Ac O/Py, 1:1, 50 ml, 20 h), the
D
2
reaction mixture was poured into ice and extracted with CHCl (3×250 ml). Organic fraction was
3
washed with H O (3×300 ml), water fraction was extracted with CHCl (2×50 ml). Combined
2
3
CHCl fractions were dried by filtration through cotton wool and concentrated. Column
3
chromatography on silica gel (CHCl –i-PrOH, 10:1) afforded 1.56 g (97%) of compound 16,
3
1
white foam; R 0.25 (CHCl –i-PrOH, 10:1); [α] –56° (C 1, CHCl –MeOH, 1:1); H NMR
f
3
D
3
(
DMSO-d ): δ 1.69–1.74 (m, 2H, CCH C), 1.78 and 1.96 (2s, 6H, OC(O)CH , NHC(O)CH ),
6 2 3 3
3
4
9
9
5
.16–3.24 (m, 2H, NCH ), 3.46–3.53 (m, 2H, OCH ), 3.72–3.80 (m, 4H, H-2, H-4, H-5, H-6a),
2 2
.24 (dd, 1H, J5,6b 5.0, J6a,6b 10.2, H-6b), 4.65 (d, 1H, J 8.4, H-1), 5.12 (dd, 1H, J 9.9, J
3,4
1,2
2,3
.9, H-3), 5.63 (s, 1H, PhCH), 7.35–7.39 (m, 5H, ArH), 7.91 (d, 1H, J2,NH 9.3, NHAc), 9.32–
+
.36 (m, 1H, NHC(O)CF ); MALDI MS: m/z calculated for [C H N O F ]H 505.18, found
3
22 27
2
8 3
05.14.
Compound 16 (1.63 g, 3.23 mmol) and THF (70 ml) were stirred with molecular sieves 4
Å (13 g) for 30 min, NaBH CN (1.83 g, 29.1 mmol) was added, and the mixture was stirred for
3
3
0 min. MsOH (14 ml of 2M solution in THF, 28 mmol) was then added to pH 3, and the
mixture was stirred for 3 h. The mixture was neutralized with Et N (50 µl), filtered, and
3
concentrated. The residue obtained was dissolved in CHCl (200 ml), washed with saturated
3
NaHCO aq (100 ml) and NaCl aq (0.9%, 6×100 ml). Aqueous fraction was extracted with
3
CHCl (2×100 ml). Combined CHCl fractions were dried by filtration through cotton wool and
3
3
concentrated. Column chromatography on silica gel (CHCl –i-PrOH, 10:1) and crystallization
3
from EtOAc–n-C H provided 1.31 g (80%) of compound 17, white crystals (Т 131 °C); R
f
6
14
m
1
0
.23 (CHCl –i-PrOH, 6:1); [α] –68° (C 1, CHCl ); H NMR (CDCl ): δ 1.81–1.88 (m, 2H,
3 D 3 3

ACCEPTED MANUSCRIPT
CCH C), 1.95, 2.11 (2s, 6H, C(O)CH ), 3.09–3.21 (m, 1H, OH), 3.23–3.32 (m, 1H, NHCHH),
2
3
3
2
8
.53–3.64 (m, 3H, H-5, NHCHH, OCHH), 3.71 (dd, 1H, J 9.2, J 9.4, H-4), 3.75–3.79 (m,
3,4 4,5
H, H-6a, H-6b), 3.90 (m, 1H, OCHH), 3.95 (dd, 1H, J 8,4, J 10.6, H-2), 4.49 (d, 1H, J
1,2
1
,2
2,3
.4, H-1), 4.57 (dd, 2H, J 11.9, CH Ph), 4.91 (dd, 1H, J 10.6, J 9.2, H-3), 6.04 (d, 1H, J 9.0,
2
2,3
3,4
1
3
NHAc), 7.28–7.40 (m, 5H, ArH), 7.50–7.57 (m, 1H, NHC(O)CF ); C NMR (DMSO-d ): δ
3
6
2
6
1
0.7 (OC(O)CH ), 22.6 (NC(O)CH ), 28.3 (CCH C), 36.5 (NCH ), 53.1 (C-2), 66.1 (OCH ),
3 3 2 2 2
8.0 (C-5), 69.3 (C-6), 72.3 (PhCH ), 75.3 (C-3), 76.0 (C-4), 100.4 (C-1), 115.1 (C(O)CF ),
2
3
27.3 (C-Ar), 128.1 (C-Ar), 138.6 (C-Ar), 156.2 (C(O)CF ), 168.9 (C(O)CH ), 169.8
3
3
+
(
C(O)CH ); MALDI MS: m/z calculated for [C H N O F ]H 507.20, found 507.22.
3 22 29 2 8 3
4
.2.1.2 3-Trifluoroacetamidopropyl 2-acetamido-6-O-benzyl-2-deoxy-β-
D-
glucopyranoside (18)
The derivative 17 (600 mg, 1.19 mmol) was deacetylated under Zemplen conditions. In 3
h the mixture was neutralized with AcOH (10 µl) and concentrated. Crystallization from
acetone–n-C H provided 530 mg (96%) of diol 18, white crystals (Т 135 °C); [
α
]D –30° (c 1,
6
14
m
1
CHCl –MeOH, 1:1); H NMR (CDCl –CD OD): δ 1.93–2.05 (m, 2H, CCH C), 2.23 (s, 3H,
3
3
3
2
NHC(O)CH ), 3.40–3.46 (m, 1H, NHCHH), 3.55 (d, 1H, J 9.5, H-4), 3.58–3.72 (m, 4H, H-3,
3
3,4
H-5, NHCHH, OCHH), 3.81 (dd, 1H, J 10.1, H-2), 3.85 (dd, 1H, J 6.0, J6a,6b 10.9, H-6a),
2
,3
5,6a
4
.03 (dd, 1H, J5,6b 7.8, H-6b), 4.05–4.09 (m, 1H, OCHH), 4.55 (d, 1H, J 8.3, H-1), 4.76 (d, 2H,
1,2
1
3
CH Ph), 7.43–7.61 (m, 5H, ArH); C NMR (CDCl ): δ 22.2 (NC(O)CH ), 28.5 (CCH C), 36.7
2
3
3
2
(
NCH C), 56.2 (C-2), 66.6 (OCH ), 69.7 (C-6), 71.1 (C-4), 73.4 (PhCH ), 75.0 (C-5), 75.4 (C-3),
2 2 2
1
1
01.2 (C-1), 114.4 (NHC(O)CF ), 127.58, 127.63, 128.2, 137.9 (C-Ar), 157.9 (NHC(O)CF )
3 3
+
72.9 (NC(O)CH ); MALDI MS: m/z calculated for [C H N O F ]K 503.14, found 503.50.
3
20 27
2
7 3
4
.2.2 3-Aminopropyl 2-acetamido-2-deoxy-α-
D
-galactopyranosyl-(1→3)-[α-
L-
fucopyranosyl-(1→2)]-β-
acetamido-2-deoxy-β- -glucopyranoside (11)
D-galactopyranosyl-(1→3)-[α-L-fucopyranosyl-(1→4)]-2-
D

ACCEPTED MANUSCRIPT
4
.2.2.1 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α- -fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-
galactopyranosyl-(1→3)-2-acetamido-6-O-benzyl-2-deoxy-β- -glucopyranoside (21)
A mixture of trichloracetimidate 19 (279 mg, 0.276 mmol), diol 18 (256 mg, 0.552
mmol), anhydrous CH CN (7 ml), and anhydrous CH Cl (7 ml) was stirred with molecular
D-
L
D-
D
3
2
2
sieves 3Å (1 g) for 30 min. A solution of TMSOTf (5 µl, 0.03 mmol) in CH Cl (1 ml) was then
2
2
added. The mixture was stirred for 48 h, neutralized with Et N (10 µl), filtered, and concentrated.
3
Gel filtration and subsequent column chromatography on silica gel (n-C H –CHCl –i-PrOH,
6
14
3
4
:3:1→3:3:1) gave products 21 (119 mg, 33%), 22 (36 mg, 10%), and 23 (18 mg, 5%).
Compound 21: white foam; R 0.18 (n-C H –CHCl –i-PrOH, 3:3:1); [α] +23° (c 1,
f
6
14
3
D
1
13
CHCl ); H NMR (CDCl ): see Table 2; C NMR (DEPT, CDCl ): see Table 3; MALDI MS:
3
3
3
+
m/z calculated for [C H N O F ]Na 1334.44, found 1334.46.
5
6
76
3
29 3
1
Compound 22: white foam; R 0.27 (n-C H –CHCl –i-PrOH, 3:3:1); H NMR (CDCl ):
f
6
14
3
3
1
3
see Table 2; C NMR (DEPT, CDCl ): see Table 3; MALDI MS: m/z calculated for
3
+
[
C H N O F ]Na 1334.44, found 1334.40.
5
6
76
3
29 3
1
Compound 23: white foam; R 0.13 (n-C H –CHCl –i-PrOH, 3:3:1); H NMR (CDCl ):
f
6
14
3
3
1
3
see Table 2; C NMR (DEPT, CDCl ): see Table 3; MALDI MS: m/z calculated for
3
+
[
C H N O F ]Na 1334.44, found 1334.43.
5
6
76
3
29 3
Tetrasaccharides 21 (15 mg, 0.011 mmol), 22 (10 mg, 0.008 mmol), and 23 (10 mg,
.008 mmol) were acetylated (Ac O/Py, 1:1, 1 ml, 40 h). The reaction mixtures were
0
2
coevaporated with PhCH . Column chromatography on silica gel (n-C H –CHCl –i-PrOH,
3
6
14
3
4
:3:1) provided products 21Ac (14 mg, 94%), 22Ac (9 mg, 83%), and 23Ac (9 mg, 83%),
respectively.
Compound 21Ac: white foam; R 0.13 (n-C H –CHCl –i-PrOH, 4:3:1); [α] +17° (c 1,
f
6
14
3
D
1
13
CHCl ); H NMR (CDCl ): see Table 2,; C NMR (DEPT, CDCl ): see Table 3; MALDI MS:
3
3
3
+
m/z calculated for [C H N O F ]Na 1376.45, found 1376.47.
5
8
78
3
30 3

ACCEPTED MANUSCRIPT
Compound 22Ac: white foam; R 0.39 (n-C H –CHCl –i-PrOH, 3:3:1); [α] +32° (c
f
6
14
3
D
1
13
0
.82, CHCl ); H NMR (CDCl ): see Table 2,; C NMR (DEPT, CDCl ): see Table 3; MALDI
3 3 3
+
MS: m/z calculated for [C H N O F ]Na 1376.45, found 1376.41.
58
78
3
30 3
Compound 23Ac: white foam; R 0.25 (n-C H –CHCl –i-PrOH, 3:3:1); [α] –20° (c
f
6
14
3
D
1
13
0
.60, CHCl ); H NMR (CDCl ): see Table 2,; C NMR (DEPT, CDCl ): see Table 3; MALDI
3 3 3
+
MS: m/z calculated for [C H N O F ]Na 1376.45, found 1376.50.
58
78
3
30 3
4
.2.2.2 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α- -fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-
galactopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-α- -fucopyranosyl-(1→4)]-2-acetamido-6-O-
benzyl-2-deoxy-β- -glucopyranoside (27)
D-
L
D-
L
D
A mixture of tetrasaccharide 21 (95 mg, 0.071 mmol), trichloracetimidate 25 (82 mg,
0
.14 mmol), anhydrous Et O (3 ml), and anhydrous CH Cl (1 ml) was stirred with molecular
2
2
2
sieves 3Å (300 mg) in the atmosphere of Ar for 30 min. A solution of TMSOTf (6.5 µl, 0.036
mmol) in CH Cl (100 µl) was added. The mixture was stirred for 4 h, neutralized with Et N (10
2
2
3
µl), filtered, and concentrated. Gel filtration and subsequent column chromatography on silica
gel (n-C H –CHCl –i-PrOH, 3:3:1) gave products 26 (16 mg, 13%), 27 (74 mg, 60%), and
6
14
3
starting tetrasaccharide 21 (14 mg, 15%).
Compound 26: white foam; R 0.36 (n-C H –CHCl –i-PrOH, 3:3:1); [α] –10° (c 1.3,
f
6
14
3
Hg
1
13
CHCl ); H NMR (CDCl ): see Table 4; C NMR (CDCl , DEPT): see Table 5; MALDI MS:
3
3
3
+
m/z calculated for [C H N O F ]K 1767.62, found 1767.93.
8
3
104
3
33 3
Compound 27: white foam; R 0.21 (n-C H –CHCl –i-PrOH, 3:3:1); [α] –17° (c 1.0,
f
6
14
3
Hg
1
13
CHCl ); H NMR (CDCl ): see Table 4; C NMR (CDCl , DEPT): see Table 5; MALDI MS:
3
3
3
+
m/z calculated for [C H N O F ]H 1728.66, found 1767.85.
8
3
104
3
33 3
4
.2.2.3 3-Trifluoroacetamidopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
D-
galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α- -fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-D-
L

ACCEPTED MANUSCRIPT
galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-(1→4)]-2-acetamido-6-O-
acetyl-2-deoxy-β- -glucopyranoside (28)
D
Pentasaccharide 27 (72 mg, 0.042 mmol) was deacetylated under Zemplen conditions and
subjected to (45 mg Pd/C, 4 ml MeOH, 2 h). The mixture was filtered and concentrated. The
residue obtained was acetylated (Ac O/Py, 1:1, 3 ml, 20 h) and coevaporated with PhCH .
2
3
Column chromatography on silica gel (n-C H –CHCl –i-PrOH, 3:2:1) afforded pentasaccharide
6
14
3
2
8 (55 mg, 85%), white foam; R 0.18 (n-C H –CHCl –i-PrOH, 2:3:1); [α] –42° (c 1.0,
f 6 14 3 Hg
1
13
CHCl ); H NMR (CDCl ): see Table 4; C NMR (CDCl , DEPT): see Table 5; MALDI MS:
3
3
3
+
m/z calculated for [C H N O F ]K 1575.48, found 1575.69.
6
3
89
3
37 3
4
.2.2.4 3-Aminopropyl 2-acetamido-2-deoxy-α-
fucopyranosyl-(1→2)]-β- -galactopyranosyl-(1→3)-[α-
acetamido-2-deoxy-β- -glucopyranoside (11)
D
-galactopyranosyl-(1→3)-[α-
L-
D
L-fucopyranosyl-(1→4)]-2-
D
Pentasaccharide 28 (55 mg, 0.036 mmol) was deacetylated under Zemplen conditions.
MeOH was then partially evaporated, H O (1 ml) added, and the mixture was kept for 4 h at rt.
2
+
Ion exchange chromatography on Dowex 50XW-400 (H ) (elution with 5% NH aq) provided 32
3
mg (94%) of product 11, white foam; R 0.13 (EtOH–BuOH–Py–H O–AcOH, 100:10:10:10:3);
f
2
1
[
α] –18° (c 1.2, H O–CH CN, 1:1); H NMR (D O, characteristic signals): δ 1.27–1.33 (m, 6H,
Hg
2
3
2
III
IV
H-6 , H-6 ), 1.89–2.00 (m, 2H, CCH C), 2.04, 2.09 (2s, 6H, 2 NHC(O)CH ), 3.05–3.10 (m,
2
3
IV
I
II
2
4
H, NCH ), 4.39 (q, 1H, J 6.8, H-5 ), 4.42 (d, 1H, J 8.5, H-1 ), 4.72 (d, 1H, J 7.6, H-1 ),
2
5,6
1,2
1,2
III
III
IV
V
13
.86 (q, 1H, J 6.7, H-5 ), 5.04 (d, 1H, J1,2 3.9, H-1 ), 5.22–5.24 (m, 2H, H-1 , H-1 ); C
5
,6
III
IV
V
NMR (D O, DEPT): δ 15.4, 15.5 (C-6 , C-6 ), 26.8 (CCH C), 37.5 (NCH ), 49.9 (C-2 ), 55.6
2
2
2
I
I
II
V
(
C-2 ), 59.5 , 61.5 , 61.6 (C-6 , C-6 , C-6 ), 62.3, 66.5, 67.2, 67.6, 67.8, 67.9, 68.0, 68.6, 69.2,
I
I
I
II
II
6
4
9
9.8, 71.0, 72.0, 72.1, 72.4, 74.2, 74.5, 74.5, 75.5, 76.0 (OCH , C-3 , C-4 , C-5 , C-2 , C-3 , C-
2
II
II
III
III
III
III
IV
IV
IV
IV
V
V
V
V
, C-5 , C-2 , C-3 , C-4 , C-5 , C-2 , C-3 , C-4 , C-5 , C-3 , C-4 , C-5 ), 91.0 (C-1 ),
III
IV
I
II
8.0 (C-1 ), 99.3 (C-1 ), 100.4 (C-1 ), 101.9 (C-1 ); MALDI MS: m/z calculated for
+
[
C H N O ]H 959.39, found 959.29.
3
7
65
3
24

ACCEPTED MANUSCRIPT
4
.2.3. 3-Aminopropyl α-
D
-galactopyranosyl-(1→3)-[α-
L-fucopyranosyl-(1→2)]-β-D-
galactopyranosyl-(1→3)-[α-
L
-fucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-
glucopyranoside (12)
4
.2.3.1 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-acetyl-α-
1→3)-[2,3,4-tri-O-acetyl-α- -fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-
1→3)-2-acetamido-6-O-benzyl-2-deoxy-β- -glucopyranoside (24)
A mixture of trichloracetimidate 20 (200 mg, 0.198 mmol), diol 18 (184 mg, 0.397
mmol), anhydrous CH CN (5 ml), and hydrous CH Cl (5 ml) was stirred with molecular sieves
D
-galactopyranosyl-
(
(
L
D-galactopyranosyl-
D
3
2
2
3
Å (500 mg) under the atmosphere of N for 30 min. A solution of TMSOTf (4 µl, 0.02 mmol) in
2
CH Cl (100 µl) was added. The mixture was stirred for 20 h, neutralized with Et N (10 µl),
2
2
3
filtered, and concentrated. Gel filtration and subsequent column chromatography on silica gel (n-
C H –CHCl –i-PrOH, 6:3:1) and repeated column chromatography on silica gel (PhCH –
6
14
3
3
EtOAc, 1:5) gave product 24 (122 mg, 43%), white foam; R 0.20 (PhCH –EtOAc, 1:5); [α]
Hg
f
3
1
13
+
8° (c 0.70, CHCl ); H NMR (CDCl ): see Table 2; C NMR (CDCl , DEPT): see Table 3;
3 3 3
+
MALDI MS: m/z calculated for [C H N O F ]K 1352.40, found 1352.43.
56
76
2
30 3
Tetrasaccharide 24 (10 mg, 0.0076 mmol) was acetylated (Ac O/Py, 1:1, 1 ml, 20 h), the
2
mixture was coevaporated with PhCH . Column chromatography on silica gel (n-C H –CHCl –
3
6
14
3
i-PrOH, 5:3:1) provided compound 24Ac (9 mg, 87%), white foam; R 0.17 (n-C H –CHCl –i-
f
6
14
3
1
13
PrOH, 5:3:1); [α] +18° (c 0.79, CHCl ); H NMR (CDCl ): see Table 2; C NMR (CDCl ,
Hg
3
3
3
+
DEPT): see Table 3; MALDI MS: m/z calculated for [C H N O F ]Na 1378.44, found
58
78
2
31 3
1
378.43.
4
.2.3.2 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-acetyl-α-
D
-galactopyranosyl-
(
1→3)-[2,3,4-tri-O-acetyl-α-
L
-fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-
D-galactopyranosyl-
(
1→3)-[2,3,4-tri-O-benzyl-α-
L
-fucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-D-
glucopyranoside (29)

ACCEPTED MANUSCRIPT
A mixture of tetrasaccharide 24 (75 mg, 0.057 mmol), trichloracetimidate 25 (65 mg,
0
.11 mmol), anhydrous Et O (3 ml), and anhydrous CH Cl (1 ml) was stirred with molecular
2
2
2
sieves 3Å (300 mg) under the atmosphere of Ar for 30 min. A solution of TMSOTf (5 µl, 0.03
mmol) in CH Cl (100 µl) was added. The mixture was stirred for 3 h, neutralized with Et N (5
2
2
3
µl), filtered, and concentrated. Gel filtration and subsequent column chromatography on silica
gel (n-C H –CHCl –i-PrOH, 7:3:1) provided product 29 (75 mg, 76%), white foam; R 0.33 (n-
6
14
3
f
1
13
C H –CHCl –i-PrOH, 5:3:1); [α] –10° (c 1.0, CHCl ); H NMR (CDCl ): see Table 4; C
6
14
3
Hg
3
3
+
NMR (CDCl , DEPT): see Table 5; MALDI MS: m/z calculated for [C H N O F ]K 1576.46,
3
63 88
2
38 3
found 1576.45.
4
.2.3.3 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-acetyl-α-
D
-galactopyranosyl-
-fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β- -galactopyranosyl-
-fucopyranosyl-(1→4)]-2-acetamido-6-O-acetyl-2-deoxy-β-
(
1→3)-[2,3,4-tri-O-acetyl-α-
L
L
D
(
1→3)-[2,3,4-tri-O-acetyl-α-
D-
glucopyranoside (30)
Pentasaccharide 29 (75 mg, 0.043 mmol) was deacetylated under Zemplen conditions,
subjected to hydrogenolysis (70 mg Pd/C, 5 ml MeOH, 3 h), filtered, and concentrated. The
residue obtained was acetylated (Ac O/Py, 1:1, 3 ml, 24 h) and coevaporated with PhCH .
2
3
Column chromatography on silica gel (n-C H –acetone, 1:1) provided pentasaccharide 30 (59
6
14
1
mg, 88%), white foam; R 0.23 (n-C H –acetone, 1:1); [α] –29° (c 1.0, CHCl ); H NMR
f
6
14
Hg
3
1
3
(
CDCl ): see Table 4; C NMR (CDCl , DEPT): see Table 5; MALDI MS: m/z calculated for
3 3
+
[
C H N O F ]K 1576.46, found 1576.45.
6
3
88
2
38 3
4
.2.3.4 3-Aminopropyl α-D-galactopyranosyl-(1→3)-[α-L-fucopyranosyl-(1→2)]-β-D-
galactopyranosyl-(1→3)-[α-
L
-fucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-
glucopyranoside (12)
Pentasaccharide 30 (59 mg, 0.038 mmol) was deacetylated under Zemplen conditions.
MeOH was then partly evaporated, H O (1 ml) was added, and the mixture was kept for 4 h at rt.
2

ACCEPTED MANUSCRIPT
+
Ion exchange chromatography on Dowex 50XW-400 (H ) (elution with 5% NH aq) provided 32
3
mg (94%) of product 12, white foam; R 0.16 (EtOH–BuOH–Py–H O–AcOH, 100:10:10:10:3);
f
2
1
[
α] –155° (c 0.27, H O–CH CN, 1:1); H NMR (D O, characteristic signals): δ 1.28–1.31 (m,
Hg 2 3 2
III
IV
6
H, H-6 , H-6 ), 1.89–2.02 (m, 2H, CCH C), 2.11 (s, 3H, NHC(O)CH ), 3.06–3.10 (m, 2H,
2 3
IV
I
II
NCH ), 4.40 (q, 1H, J 6.9, H-5 ), 4.42 (d, 1H, J 8.5, H-1 ), 4.73–4.74 (m, 1H, H-1 ), 4.84
2
5,6
1,2
III
III
V
(
q, 1H, J 6.6, H-5 ), 5.05 (d, 1H, J 3.9, H-1 ), 5.19 (d, 1H, J 4.2, H-1 ), 5.25 (d, 1H, J
5
,6
1,2
1,2
1,2
IV 13
III
IV
3
5
6
.8, H-1 ); C NMR (D O, DEPT): δ 15.4, 15.5 (C-6 , C-6 ), 26.8 (CCH C), 37.5 (NCH ),
2
2
2
I
I
II
V
5.7 (C-2 ), 59.6, 61.3, 61.6 (C-6 , C-6 , C-6 ), 63.5, 66.5, 67.2, 67.8, 68.1, 68.6, 69.1, 69.3,
I
I
I
II
II
9.4, 69.7, 71.3, 72.1, 72.1, 72.2, 74.0, 74.4, 74.8, 75.5, 76.5, 77.0 (C-3 , C-4 , C-5 , C-2 , C-3 ,
II
II
III
III
III
III
IV
IV
IV
IV
V
V
V
V
C-4 , C-5 , C-2 , C-3 , C-4 , C-5 , C-2 , C-3 , C-4 , C-5 , C-2 , C-3 , C-4 , C-5 ), 93.5
V
III
IV
I
II
(
C-1 ), 97.9 (C-1 ), 99.4 (C-1 ), 100.7 (C-1 ), 102.0 (C-1 ); MALDI MS: m/z calculated for
+
[
C H N O ]H 896.38, found 896.10.
3
5
63
2
24
4
.3 Synthesis of FSL (Sug-Ad-CMG-Ad-DOPE) 1–7
4
.3.1 Synthesis of N-succinimide derivatives (general procedure)
a
b
A solution of aminopropyl glycoside (0.010 mmol, 5.9 mg for 8 (Le ), 7.3 mg for 9 (Le ) and 10
y
b
y
b
y
(
Le ), 9.4 mg for 11 (ALe ) and 13 (ALe ), 9.0 mg for 12 (BLe ) and 14 (BLe )) in 0.5 ml of
DMSO was added by 5 equal portions (100 µl in every 10 min) to a solution of Ad(ONSu) (34
2
mg, 0.10 mmol) in 0.5 ml of DMSO. After the last portion was added, the mixture was kept for
2
0 min at rt and subjected to gel filtration (CH CN–H O, 0.3% AcOH) to give N-succinimide
3 2
derivatives 32–38.
a
Le AdONSu (32): 7.9 mg (97%), R 0.42 (CHCl –EtOH–H O, 4:9:2, 0.1% AcOH),
f
3
2
b
y
Le AdONSu (33): 7.6 mg (81%), R 0.38 (CHCl –EtOH–H O, 4:9:2, 0.1% AcOH), Le AdONSu
f
3
2
b
(
34): 7.4 mg (77%), R 0.45 (CHCl –EtOH–H O, 4:9:2, 0.1% AcOH), ALe AdONSu (35): 8.7
f 3 2
b
mg (75%), R 0.37 (CHCl –EtOH–H O, 4:9:2, 0.1% AcOH), BLe AdONSu (36): 8.9 mg (79%),
f
3
2
y
R 0.40 (CHCl –EtOH–H O, 4:9:2, 0.1% AcOH), ALe AdONSu (37): 9.2 mg (79%), R 0.41
f
3
2
f

ACCEPTED MANUSCRIPT
y
(
CHCl –EtOH–H O, 4:9:2, 0.1% AcOH), BLe AdONSu (38): 8.6 mg (77%), R 0.37 (CHCl –
3
2
f
3
EtOH–H O, 4:9:2, 0.1% AcOH).
2
4
.3.2 Synthesis of FSLs (general procedure)
A solution of N-succinimide derivative (0.0043 mmol, 3.5 mg for 32, 4.1 mg for 33 and
4, 5.0 mg for 35 and 37, 4.8 mg for 36 and 38) in 300 µl of DMF was added by 3 equal portions
3
(100 µl every 1.5 h) to the solution of CMG-Ad-DOPE 39 (5.3 mg, 0.0029 mmol) in the mixture
of NaHCO aq (50 mM, 0.5 ml) and i-PrOH (0.25 ml) (pH 8.5). The mixture was left overnight,
3
then neutralized with AcOH (2 µl), and subjected to gel filtration (i-PrOH–H O, 1:2, 0.25%
2
AcOH, 0.5% Py) to give neoglycolipids 1–7.
a
Le -Ad-CMG-Ad-DOPE (1): 6.0 mg (78%), white foam; R 0.39 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.57 (m, 4H,
3
2
2
3
III
2
-CH=CH-), 5.43–5.47 (m, 1H, OCH CHOCH O-), 5.20 (d, 1H, J 3.9, H-1 ), 5.04 (q, 1H,
2
2
1,2
III
II
I
J 6.6, H-5 ), 4.68 (d, 1H, J 8.4, H-1 ), 4.66 (d, 1H, J 7.7, H-1 ), 4.63 (dd, 1H, J 2.4, J
5
,6
1,2
1,2
1
2.3, -C(O)OCHHCHOCH O-), 2.41–2.57 (m, 12H, 6 -CH CO), 2.17–2.22 (m, 11H, 2 -CH -
2 2 2
CH=CH-CH - and NHC(O)CH ), 1.90–1.98 (m, 2H, OCH CH CH NH), 1.74–1.84 (m, 12H, 2
2
3
2
2
2
COCH CH CH CH CO and 2 COCH CH -), 1.41–1.55 (m, 40H, 20 CH ), 1.37 (d, 3H, J 6.6,
2
2
2
2
2
2
2
5,6
III
+
+
H-6 ), 1.06 (t, J 7.1, 6H; 2 CH ); MALDI MS: m/z 2549 (M+Na+H) , 2565 (M+K+H) ; 2571
3
+
+
+
+
(
(
(
M+2Na+H) , 2587 (M+Na+K+H) , 2593 (M+3Na+H) , 2609 (M+2Na+K+H) , 2615
+
+
+
+
M+4Na+H) , 2631 (M+3Na+K+H) , 2647 (M+2Na+2K+H) , 2653 (M+4Na+K+H) , 2669
+
M+3Na+2K+H) .
b
Le -Ad-CMG-Ad-DOPE (2): 5.7 mg (74%), white foam; R 0.41 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.57 (m, 4H,
3
2
2
3
IV
2
3
-CH=CH-), 5.43–5.47 (m, 1H, -OCH CHOCH O-,), 5.33 (br.s, 1H, H-1 ), 5.19 (d, 1H, J
2 2 1,2
III
III
II
.8, H-1 ), 5.03 (q, 1H, J 6.8, H-5 ), 4.83 (d, 1H, J 7.3, H-1 ), 4.63 (dd, 1H, J 2.4, J 12.3, -
5
,6
1,2
IV
I
C(O)OCHHCHOCH O-), 4.45 (q, 1H, J 6.7, H-5 ), 4.53 (d, 1H, J 8.5, H-1 ), 2.49–2.56 (m,
2
5,6
1,2
1
2H, 6 CH CO), 2.24 (s, 3H, NHC(O)CH ), 2.17–2.22 (m, 8H, 2 -CH CH=CHCH -), 1.88–1.96
2 3 2 2

ACCEPTED MANUSCRIPT
(
)
(
m, 2H, OCH CH CH NH), 1.74–1.84 (m, 12H, 2 (COCH CH CH CH CO) and 2 (COCH CH -
2 2 2 2 2 2 2 2 2
III
IV
, 1.41–1.56 (m, 46H, H-6 , H-6 , 20 CH ), 1.06 (t, J 7.0, 6H; 2 CH ); MALDI MS: m/z 2698
2
3
+
+
+
M+Na+H) , 2714 (M+K+H) ; 2720 (M+2Na) .
y
Le -Ad-CMG-Ad-DOPE (3): 6.0 mg (78%), white foam; R 0.45 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.56 (m, 4H,
3
2
2
3
IV
2
-CH=CH-), 5.43–5.46 (m, 1H, OCH CHOCH O-), 5.43 (d, 1H, J 3.3, H-1 ), 5.26 (d, 1H,
2
2
1,2
III
III
I
II
J 3.9, H-1 ), 5.05 (q, 1H, J 6.5, H-5 ), 4.66–4.70 (m, 2H, H-1 , H-1 ), 4.63 (dd, 1H, J 2.3, J
1
,2
5,6
1
2.0, -C(O)OCHHCHOCH O-), 2.40–2.56 (m, 12H, 6 -CH CO), 2.17–2.22 (m, 11H, 2 -CH -
2 2 2
CH=CH-CH -, NHC(O)CH ), 1.90–1.97 (m, 2H, OCH CH CH NH), 1.74–1.84 (m, 12H, 2
2
3
2
2
2
III
IV
COCH CH CH CH CO and 2 COCH CH -), 1.40–1.55 (m, 46H, H-6 , H-6 , 20 CH ), 1.06 (t,
2
2
2
2
2
2
2
+
+
+
J 7.1, 6H; 2 CH ); MALDI MS: m/z 2697 (M+Na) , 2719 (M+2Na) ; 2742 (M+3Na) .
3
b
ALe -Ad-CMG-Ad-DOPE (4): 6.8 mg (82%), white foam; R 0.42 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.57 (m, 4H,
3
2
2
3
2
1
4
-CH=CH-), 5.44–5.47 (m, 1H, -OCH CHOCH O), 5.41, 5.39 (d, 1H, J 3.8 and br.s, 1H, H-
2 2 1,2
IV
V
III
III
II
, H-1 ), 5.20 (d, 1H, J 3.8, H-1 ), 5.01 (q, 1H, J 6.9, H-5 ), 4.89 (d, 1H, J 7.4, H-1 ),
1
,2
5,6
1,2
IV
.63 (dd, 1H, J 2.2, J 12.1, -C(O)OCHHCHOCH O-), 4.61 (q, 1H, J 6.6, H-5 ), 4.53 (d, 1H,
2
5,6
I
J 8.3, H-1 ), 2.41–2.56 (m, 12H, 6 -CH CO), 2.24 (s, 3H, NHC(O)CH ), 2.22 (m, 11H,
1
,2
2
3
NHC(O)CH , 2 CH CH=CHCH -), 1.86–1.96 (m, 2H, O-CH CH CH -NH), 1.72–1.84 (m, 12H,
3
2
2
2
2
2
III
IV
2
-COCH CH CH CH CO and 2 -COCH CH -), 1.40–1.56 (m, 46H, H-6 , H-6 , 20 CH ), 1.06
2 2 2 2 2 2 2
+
+
+
(
t, J 7.1, 6H; 2 CH ); MALDI MS: m/z 2923 (M+2Na) , 2945 (M+3Na) , 2962 (M+2Na+K) .
3
b
BLe -Ad-CMG-Ad-DOPE (5): 5.8 mg (71%), white foam; R 0.52 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.52–5.58 (m, 4H,
3
2
2
3
IV
2
1
4
-CH=CH-), 5.43–5.47 (m, 1H, -OCH CHOCH O-), 5.39 (d, 1H, J 3.8, H-1 ), 5.34 (br. s,
2 2 1,2
V
III
III
II
H, H-1 ), 5.20 (d, 1H, J 3.5, H-1 ), 5.00 (q, 1H, J 6.8, H-5 ), 4.90 (d, 1H, J 7.2, H-1 ),
1
,2
5,6
1,2
IV
.63 (dd, 1H, J 2.1, J 12.0, -C(O)OCHHCHOCH O-), 4.61 (q, 1H, J 6.7, H-5 ), 4.53 (d, 1H,
2
5,6
I
J 8.7, H-1 ), 2.40–2.60 (m, 12H, 6 CH -CO), 2.25 (s, 3H, NHC(O)CH ), 2.17–2.22 (m, 8H, 2 -
1
,2
2
3

ACCEPTED MANUSCRIPT
CH CH=CHCH -), 1.88–1.96 (m, 2H, OCH CH CH NH), 1.71–1.84 (m, 12H, 2
2
2
2
2
2
III
IV
(
COCH CH CH CH CO) and 2 (COCH CH -), 1.41–1.55 (m, 46H, H-6 , H-6 , 20 CH ),
2 2 2 2 2 2 2
+
+
1
.04–1.08 (m, 6H; 2 CH ); MALDI MS: m/z 2860 (M+Na+H) , 2882 (M+2Na+H) , 2904
3
+
(
M+3Na+H) .
y
ALe -Ad-CMG-Ad-DOPE (6): 7.1 mg (85%), white foam; R 0.60 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.57 (m, 4H,
3
2
2
3
IV
2
-CH=CH-), 5.50 (d, 1H, J1,2 4.1, H-1 ), 5.43–5.47 (m, 1H, -OCH CHOCH O-) 5.38 (d, 1H,
2
2
V
III
III
J 3.7, H-1 ), 5.28 (d, 1H, J 4.0, H-1 ), 5.05 (q, 1H, J 6.8, H-5 ), 4.75 (d, 1H, J 7.3, H-
1
,2
1,2
5,6
1,2
II
I
1
1
), 4.68 (d, 1H, J 7.8, H-1 ), 4.64 (dd, 1H, J 2.4, J 12.2, -C(O)OCHHCHOCH O-), 4.52 (q,
1
,2
2
IV
H, J 6.8, H-5 ), 2.40–2.57 (m, 12H, 6 CH CO), 2.16–2.23 (m, 14H, 2 -CH CH=CHCH -, 2
5
,6
2
2
2
NHC(O)CH ), 1.90–1.96 (m, 2H, OCH CH CH NH), 1.74–1.84 (m, 12H, 2
3
2
2
2
III
IV
(
COCH CH CH CH CO) and 2 (COCH CH -), 1.41–1.55 (m, 46H, H-6 , H-6 , 20 CH ), 1.06
2 2 2 2 2 2 2
+
+
+
(
t, J 7.1, 6H; 2 CH ); MALDI MS: m/z 2901 (M+Na+H) , 2923 (M+2Na) ; 2939 (M+Na+K) ,
3
+
+
+
2
945 (M+3Na) ; 2962 (M+2Na+K) , 2967 (M+4Na) .
y
BLe -Ad-CMG-Ad-DOPE (7): 7.4 mg (90%), white foam; R 0.45 (i-PrOH–MeOH–
f
1
CH CN–H O, 4:3:6:4); H NMR (D O–CD OD, 1:1, characteristic signals): δ 5.51–5.57 (m, 4H,
3
2
2
3
IV
2
-CH=CH-), 5.47 (d, 1H, J 4.1, H-1 ), 5.43–5.46 (m, 1H, -OCH CHOCH O-), 5.40 (d, 1H,
1,2 2 2
V
III
III
J 3.1, H-1 ), 5.27 (d, 1H, J 3.7, H-1 ), 5.06 (q, 1H, J 6.5, H-5 ), 4.76 (d, 1H, J 7.6, H-
1
,2
1,2
5,6
1,2
II
I
1
), 4.66–4.70 (m, 1H, H-1 ), 4.63 (dd, 1H, J 2.3, J 12.3, -C(O)OCHHCHOCH O-), 4.51 (q, 1H,
2
IV
J 6.5, H-5 ), 2.40–2.56 (m, 12H, 6 CH -CO), 2.16–2.22 (m, 11H, 2 -CH CH=CHCH - and
5
,6
2
2
2
NHC(O)CH ), 1.90–1.96 (m, 2H, OCH CH CH NH), 1.74–1.84 (m, 12H, 2 -
3
2
2
2
III
IV
COCH CH CH CH CO- and 2 -COCH CH -), 1.40–1.55 (m, 46H, H-6 , H-6 , 20 CH ), 1.06
2
2
2
2
2
2
2
+
+
+
(
t, J 7.0, 6H; 2 CH ); MALDI MS: m/z 2859 (M+Na) , 2881 (M+2Na) ; 2903 (M+3Na) .
3

ACCEPTED MANUSCRIPT
1
Table 2. H NMR spectra (
δ
(ppm) and J (Hz)) of tetrasaccharides 21–24 and 21Ac–24Ac.
Proton
21
21Ac
22
22Ac
23
23Ac
24
24Ac
I
I
I
I
I
4.82, d
J1,2 8.1
4.77, d
J1,2 8.2
3.46, ddd, J1,2 8.2,
J2,3 9.6, J2,NH 7.9
4.03, dd
5.23, d
J1,2 7.7
3.09, ddd, J1,2 7.7,
J2,3 8.9, J2,NH 6.7
4.39, dd
J2,3 8.9, J3,4 8.9
5.03, dd
J3,4 8.9, J4,5 9.4
4.81, d
J1,2 8.2
4.47, d
J1,2 8.3
4.99, d
J1,2 7.8
H-1
H-2
H-3
H-4
H-5
4.96–5.03
4.55–4.65
2.97–3.08
4.56–4.62
2.92–2.99
3.37–3.43
3.85–3.89
3.90–4.09
3.28–3.41
4.23–4.35
4.98
J2,3 10.4, J3,4 9.6
4.67–4.75
4.81, dd
3.98–4.04
3.59–3.64
3.49–3.52
J2,3 9.6, J3,4 9.1
3.43, dd
J3,4 9.0, J4,5 9.4
3.54, ddd, J4,5 9.4,
J5,6a 6.0, J5,6b 1.8
3.66, dd
3.78, dd
J3,4 9.2, J4,5 9.2
4.92, dd
J3,4 9.1, J4,5 9.1
3.72–3.78
3.52–3.57
3.90–4.09
3.37–3.45
J3,4 9.5, J4,5 9.5
3.69–3.74
3.81–3.85
3.44–3.48
3.63–3.69
I
3.53, dd
3.55, dd
J5,6a 5.2, J6a,6b 10.8
3.59, dd
3.84, dd
J5,6a 1.0, J6a,6b 10.8
3.71, dd
J5,6a 5.8, J5,6b 10.7
3.55, dd
J5,6a 5.9, J6a,6b 10.6
3.59, dd
J5,6b 3.4, J6a,6b 10.6
4.42, d
H-6a
3.72–3.78
3.81, dd
3.85–3.89
3.93, dd
J5,6a 6.0, J6a,6b 10.8 J5,6a 5.8, J6a,6b 10.7
3.84, dd 3.56, dd
J5,6b 1.8, J6a,6b 10.8 J5,6b 2.6, J6a,6b 10.7 J5,6b 3.5, J6a,6b 10.4 J5,6b 2.8, J6a,6b 10.8 J5,6b 2.8, J6a,6b 10.9
I
H-6b
3.90–4.09
3.90–4.09
3.56–3.62
3.37–3.45
3.80–3.84
4.55–4.65
3.73–3.78
II
4.50, d
J1,2 7.6
3.94, dd
4.38, d
J1,2 7.5
3.81, dd
5.35, d
J1,2 3.6
3.99, dd
4.96, d
J1,2 3.0
3.90, dd
4.08, d
J1,2 7.7,
3.72, dd
H-1
J1,2 7.6
II
H-2
3.63–3.69
J1,2 7.6, J2,3 9.5
J1,2 7.5, J2,3 9.4
J1,2 3.6, J2,3 10.2
4.20, dd
J2,3 10.2, J3,4 3.4
J1,2 3.0, J2,3 9.5
4.08, dd
J2,3 9.5, J3,4 3.7
5.47, br. d
J3,4 1.6
J1,2 7.7, J2,3 9.1
II
3.87, dd
J2,3 9.8, J3,4 2.6
3.81, dd
J2,3 10.4, J3,4 2.7
H-3
3.81–3.84
3.74–3.77
5.32–5.37
3.52–3.59
5.24–5.29
II
5.41, br. d
J3,4 2.6
5.19, br. d
J3,4 3.3
H-4
5.44, br.s
5.38–5.42
5.37–5.41
II
4.28, dd
J5,6a 6.8, J5,6b 6.8
3.71, dd
J5,6a 6.9, J5,6b 6.9
H-5
3.87–3.91
4.05–4.15
4.05–4.15
3.74–3.77
4.05–4.09
4.05–4.09
4.34–4.37
3.52–3.59
4.05–4.08
3.37–3.45
3.90–4.09
3.90–4.09
3.80–3.84
3.98–4.04
II
H-6a
H-6b
4.07–4.12
4.13–4.16
4.01–4.06
II
4.18, dd
J5,6b 5.0, J6a,6b 11.5
5.31, d
4.17, dd
J5,6b 4.2, J6a,6b 11.5
4.07–4.12
4.05–4.08
5.50, d
J1,2 3.6, H-1
5.37, dd
J1,2 3.6, J2,3 10.9
4.01–4.06
III
5.67, d
J1,2 3.3
5.25, dd
5.62, d
J1,2 1.6
5.37, d
J1,2 3.3
5.31, dd
5.45, d
J1,2 3.6
H-1
H-2
H-3
H-4
III
5.55, br. s
5.12–5.22
5.12–5.22
5.24–5.29
4.55–4.65
5.55–5.58
J1,2 3.8
III
5.27, dd
J1,2 3.8, J2,3 11.0
5.32–5.37
5.32–5.37
5.25, br. s
4.67–4.75
5.38–5.41
5.37–5.41
J1,2 3.3, J2,3 11.0
5.33, dd
J2,3 11.0, J3,4 3.0
5.26, br. d
J3,4 3.0
J1,2 3.3, J2,3 10.8
III
III
III
5.27, dd
J2,3 11.1, J3,4 3.2
5.34, br. d
5.23, dd
J2,3 10.9, J3,4 3.3
5.25, br. d
J3,4 3.3,
5.35–5.37
5.24–5.29
5.24–5.29
4.42–4.48
5.08–5.14
5.27, br. d
J3,4 3.4,
4.38, q
J5,6 6.5
1.14, d
5.24, br. d
J3,4 2.3
4.63, q
J5,6 6.4
1.24, d
J6,5 6.4
J3,4 3.2
4.48, q
J5,6 6.5
1.14, d
J5,6 6.5
H-5
4.56–4.62
4.13–4.16
III
1.19, d
J5,6 6.5
1.20, d
J5,6 6.5
1.15, d
J5,6 6.5
1.11, d
J5,6 6.5
1.25, d
J5,6 6.4
H-6 (3H)
J5,6 6.5

ACCEPTED MANUSCRIPT
IV
IV
IV
5.21, d
J1,2 3.6
5.22, d
J1,2 3.5
5.23, d
J1,2 3.4
5.20, d
J1,2 3.4
5.20, d
J1,2 3.5
5.15, d
J1,2 3.4
5.35, d
J1,2 3.8
5.34, d
J1,2 3.7
H-1
H-2
H-3
4.51–4.54
4.44–4.48
4.50–4.55
4.46–4.50
4.42–4.48
4.43–4.47
5.24–5.29
5.29–5.33
5.05, dd
J2,3 11.3, J3,4 3.1
5.04, dd
J2,3 11.2, J3,4 3.1
5.00, dd
J2,3 11.4, J3,4 3.1
5.38, br. d
4.95, dd
J2,3 11.4, J3,4 3.1
4.90, dd
J2,3 11.7, J3,4 2.9
4.96–5.03
5.38–5.42
5.37–5.41
IV
IV
H-4
H-5
5.42, br. s
4.05–4.15
4.05–4.15
5.40 , br. s
4.11–4.15
3.97–4.03
5.46 , br. s
4.22–4.26
5.42, br. s
4.16–4.20
5.37–5.41
4.10–4.14
3.90–4.09
5.57, br. s
4.36–4.44
3.98–4.04
5.55–5.58
4.42–4.46
J3,4 3.1
4.13–4.16
IV
IV
I
3.99, dd
J5,6a 7.7, J6a,6b 11.1
H-6a
H-6b
4.07–4.12
4.13, dd
3.99–4.05
4.11, dd
3.97–4.04
4.10, dd
4.05–4.15
4.05–4.09
3.90–4.09
4.24–4.30
7.00–7.04
4.23–4.35
6.81–6.92
J5,6b 5.3, J6a,6b 12.1 J5,6b 6.0, J6a,6b 11.7 J5,6b 4.2, J6a,6b 11.7
6.99, d
J2,NH 5.0
6.86, d
J2,NH 6.3
6.14, d
J2,NH 8.0
5.98, d
6.44, d
J2,NH 6.7
6.03, d
5.90, d
J2,NH 7.1
5.71, d
J2,NH 8.7
NHAc
IV
NHAc
6.02–6.14
1.84–1.94
3.47–3.51
6.31–6.45
1.83–1.94
3.44–3.54
6.37–6.44
6.52–6.62
J2,NH 7.9
J2,NH 8.8
1
.75–1.82
.85–1.91
.34–3.40
.52–3.57
3.58–3.63
3.89–3.94
1.93–2.16
(10s, 30H)
1.80–1.86
1.87–1.93
3.37–3.43
3.52–3.59
3.59–3.64
3.97–4.04
2.00–2.16
(9s, 30H)
1.79–1.86
1.87–1.94
3.25–3.32
3.63–3.70
3.56–3.62
3.90–4.09
1.97–2.16
(8s, 33H)
1.80–1.85
1.86–1.91
3.33–3.40
3.53–3.58
3.65–3.69
3.92–3.96
1.92–2.17
(8s, 30H)
-
CH CH CH -
1.78–1.90
1.79–1.91
3.41–3.50
2
2
2
1
3
3
3.36–3.43
3.44–3.50
3.64–3.70
3.91–3.95
1.97–2.17
(9s, 33H)
-
CH CH N-
2 2
3
3
1
.72–3.77
.87–3.91
.97–2.18
3.69–3.74
3.88–3.93
1.96–2.17
(11s, 33H)
3.63–3.69
3.91–3.97
1.92–2.17
(10s, 33H)
-
OCH CH -
2
2
-
C(O)CH3
(
9s, 30H)
-NHC(O)CF3
7.42–7.49
7.24–7.28
7.31–7.34
7.27–7.38
7.35–7.42
7.30–7.35
7.69–7.87
7.45–7.56
4
4
.49, d, J 12.1
.55,d, J 12.1
4.57, d, J 11.8
4.62, d, J 11.8,
4.46, d, J 12.1
4.58, d, J 12.1
4.42–4.48
4.80, d, J 12.4
4.43, d, J 12.1
4.83, d, J 12.1
4.51, d, J 12.0
4.54, d, J 12.0
PhCH2-
4.56–4.62
4.55–4.65
7
7
.28–7.30,
.31–7.34
ArH (5H)
OH
7.27–7.36
3.81–3.84
7.28–7.36
7.27–7.38
7.35–7.42
7.35–7.42
7.26–7.36
7.25–7.37
1
3
Table 3. C NMR spectra (DEPT, δ (ppm)) of tetrasaccharides 21–24 and 21Ac–24Ac.
Carbon
21
99.8
57.6
81.2
21Ac
99.3
22
100.7
56.3
81.8
22Ac
99.2
23
100.6
57.9
71.6
23Ac
101.8
54.0
24
24Ac
100.0
57.3
I
C-1
101.1
I
C-2
59.1
58.6
I
C-3
75.7
77.0
72.6
71.0
71.7

ACCEPTED MANUSCRIPT
I
I
I
C-4
C-5
C-6
69.7
75.2
69.3
101.8
72.3
77.2
65.6
70.9
61.3
95.4
67.6
67.7
70.8
65.4
15.5
95.7
47.6
67.3
67.1
67.9
62.2
28.0
37.3
67.5
69.8
73.3
69.1
101.6
72.7
70.5
66.9
70.5
60.7
95.8
67.3
68.0
71.5
65.6
15.6
98.2
48.1
67.4
67.4
68.5
62.8
28.3
37.5
72.3
74.2
69.8
98.7
73.5
73.1
67.2
66.9
61.5
96.5
67.3
67.7
71.1
66.2
15.9
96.8
48.2
67.8
67.5
68.2
62.3
28.6
37.4
67.1
69.9
72.7
68.7
96.8
75.7
74.1
68.3
68.2
62.3
98.0
66.6
67.8
71.0
65.7
16.0
97.7
48.4
67.6
67.3
68.2
62.2
28.4
37.6
77.8
74.2
67.5
100.6
74.4
79.3
67.3
70.7
61.1
96.5
66.9
67.8
71.2
65.1
15.8
99.2
48.3
67.3
67.4
68.7
62.9
28.5
37.6
67.4
72.8
74.8
67.3
99.9
74.3
70.2
68.0
70.2
60.9
96.4
66.5
68.0
71.4
65.0
15.6
100.3
48.3
67.1
67.5
68.8
63.2
28.6
37.5
67.0
70.0
75.1
69.6
102.5
70.9
72.4
64.0
71.1
62.3
96.6
67.8
67.2
70.9
66.3
16.2
91.8
67.2
67.1
67.7
66.7
61.2
28.4
37.5
70.6
70.4
69.5
102.9
73.4
73.1
64.1
70.4
60.8
97.2
67.7
67.5
70.9
66.2
16.1
92.5
67.1
66.9
67.8
66.3
61.1
28.2
37.3
67.3
II
II
II
II
II
II
C-1
C-2
C-3
C-4
C-5
C-6
III
III
III
III
III
III
IV
IV
IV
IV
IV
IV
C-1
C-2
C-3
C-4
C-5
C-6
C-1
C-2
C-3
C-4
C-5
C-6
-
CH CH CH -
2 2 2
-
CH CH N-
2 2
-
OCH CH -
67.9
68.1
2
2
2
0.4–21.0
20.5–20.8
(8C)
20.5–20.9
(9C)
20.5–21.0
(9C)
-OC(O)CH3
20.0–20.6 (8C)
20.4–21.0 (8C)
20.1–21.1 (9C)
20.1–21.1 (10C)
(9C)
-
NC(O)CH3
PhCH2-
22.7, 22.9
73.3
23.1, 23.4
73.6
23.1, 23.5
73.6
23.2, 23.6
23.1, 23.6
73.5
23.1, 23.4
73.8
23.0
73.6
23.2
73.5
73.5