Gram scale synthesis of A (type 2) and B (type 2) blood group tetrasaccharides through 1,6-anhydro-N-acetyl-β-D-glucosamine

Article abstract of DOI:10.1016/j.carres.2017.06.014

Gram scale synthesis of A (type 2) and B (type 2) tetrasaccharides in the form of 3-aminopropyl glycosides is proposed starting from 3-O-benzoyl-1,6-anhydro-N-acetylglucosamine. Its galactosylation followed by re-protection gave lactosamine derivative with single free 2′-OH group in total yield 75%. Standard fucosylation and next run of re-protection in total yield 88% gave a trisaccharide Fuc-Gal-anhydroGlcNAc with single free 3′-OH group. Its standard α-galactosylation gave protected B (type 2) tetrasaccharide. For synthesis of correspondent A tetrasaccharide seven different 2-azido-2-deoxygalactosyl (GalN₃) donors were tested: 6-O-acetyl-3,4-O-isopropylidene-GalN₃ thioglycoside was shown to provide the best yield (89%) and stereoselectivity (α/β = 24:1). Further 1,6-anhydro cycle opening, spacer-arming and complete deprotection resulted in the target 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides, yields 87 and 85% correspondingly.

Full text of DOI:10.1016/j.carres.2017.06.014

Accepted Manuscript
Gram scale synthesis of A (type 2) and B (type 2) blood group tetrasaccharides
through 1,6-anhydro-N-acetyl-β-D-glucosamine
Tatiana V. Tyrtysh, Elena Yu Korchagina, Ivan M. Ryzhov, Nicolai V. Bovin
PII:
S0008-6215(17)30293-8
10.1016/j.carres.2017.06.014
CAR 7411
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 17 April 2017
Revised Date: 21 June 2017
Accepted Date: 24 June 2017
Please cite this article as: T.V. Tyrtysh, E.Y. Korchagina, I.M. Ryzhov, N.V. Bovin, Gram scale
synthesis of A (type 2) and B (type 2) blood group tetrasaccharides through 1,6-anhydro-N-acetyl-β-D-
glucosamine, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.06.014.
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ACCEPTED MANUSCRIPT
Graphical abstract
1

ACCEPTED MANUSCRIPT
Gram scale synthesis of A (type 2) and B (type 2) blood group tetrasaccharides
through 1,6-anhydro-N-acetyl-β-D-glucosamine
Tatiana V. Tyrtysh , Elena Yu. Korchagina, Ivan M. Ryzhov, Nicolai V. Bovin*
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, Miklukho-Maklaya str. 16/10, Moscow, 117997, Russian Federation.
*
correspondence author: professorbovin@yandex.ru, phone +7(495)330-7138
Abstract
Gram scale synthesis of A (type 2) and B (type 2) tetrasaccharides in the form of 3-aminopropyl
glycosides is proposed starting from 3-O-benzoyl-1,6-anhydro-N-acetylglucosamine. Its
galactosylation followed by re-protection gave lactosamine derivative with single free 2'-OH
group in total yield 75%. Standard fucosylation and next run of re-protection in total yield 88%
gave a trisaccharide Fuc-Gal-anhydroGlcNAc with single free 3'-OH group. Its standard α-
galactosylation gave protected B (type 2) tetrasaccharide. For synthesis of correspondent A
tetrasaccharide seven different 2-azido-2-deoxygalactosyl (GalN ) donors were tested: 6-O-
3
acetyl-3,4-O-isopropylidene-GalN thioglycoside was shown to provide the best yield (89%) and
3
stereoselectivity (α/β=24:1). Further 1,6-anhydro cycle opening, spacer-arming and complete
deprotection resulted in the target 3-aminopropyl glycosides of A (type 2) and B (type 2)
tetrasaccharides, yields 87 and 85% correspondingly.
Keywords
ABO histo-blood group antigens; N-acetyl-1,6-anhydroglucosamine; A (type 2) tetrasaccharide;
B (type 2) tetrasaccharide.
1
. Introduction
Human histo-blood group oligosaccharides with -Galβ1-4GlcNAc inner core (know as type 2
oligosaccharides) dominate on the AB-serologically active glycoproteins and glycolipids of
human erythrocytes (RBCs). A (type 2), B(type 2) and H(type 2) oligosaccharides terminate N-
and O-chains in glycoproteins, as well as glycolipids in composition of practically all endothelial
2

ACCEPTED MANUSCRIPT
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, herein presented in the form of
aminopropyl glycosides) are minimal epitopes responsible for A and B antigenicity [3].
Chemical synthesis of these glycans was described in numerous works [4-10]. Nevertheless,
synthetic scheme leading to the tetrasaccharides in multigram quantities is now more necessary
than before, as a result of advances in modern 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 for ABO-typing; 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) during ABO-incompatible
organ transplantations [11].
Scaling the synthesis of carbohydrates requires high yields at each stage and
simplification of the synthetic protocol, namely, exclusion of rigorous reaction conditions and
minimization of the total amount of steps, particularly chromatographic isolations. Thus, new
approaches to the large-scale synthesis of oligosaccharides are in high demand. Recently we
have published the block synthesis of A (type 2) and B (type 2) tetrasaccharides, where the
GlcNAc unit was glycosylated with trichloroacetimidate of A or B trisaccharide [9] – approach
reasonable and convenient for obtaining a variety of blood group oligosaccharides, but
impractical for multi-gram synthesis of the tetrasaccharides. Herein we suggest a new synthetic
scheme that allows us scaling up the A (type 2) and B (type 2) tetrasaccharide synthesis. The use
of 1,6-anhydro of glucosamine is key feature of this approach.
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. H
(type 2) trisaccharide is obtained by consecutive elongation of the carbohydrate chain starting
from glucosamine: GlcNAc ⇒ Galβ1–4GlcNAc ⇒ Fucα1–2Galβ1–4GlcNAc. This approach
was applied in early versions of A (type 2) and B (type 2) tetrasaccharide synthesis [4,5] and is
3

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still in use [6-8]. Low reactivity of 4-OH group in glucosamine moiety hinders the formation of
β1–4 glycosidic bond, and, though the cases of efficient glycosylation of glucosamine
derivatives by orthoester [12], bromide [4], thioglycosides [7,13], or imidate [8] of galactose are
reported, the search of more effective synthetic route to lactosamine derivatives is being
continued. Particularly, obtaining of lactosamine from the derivatives with pre-existing 1–4
linkage (lactose [14-19] or lactulose [20,21]) has been reported. Another solution of the problem
found to be use of 1,6-anhydro-β-D-glucosamine derivatives, in which the reactivity of 4-OH is
1
increased due to the fixed С -conformation of GlcNAc moiety [22]. Earlier, we successfully
4
used 1,6-anhydro-N-acetyl-β-D-glucosamine in the synthesis of oligosaccharides containing
lactosamine block [23,24]. Herein we suggest convenient scheme for the synthesis of spacer-
armed A (type 2) 1 and B (type 2) 2 tetrasaccharides (Figure 1).
Figure 1. 3-Aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides.
2
. Results and Discussion
For further conjugation with lipids, affinity matrix, etc., the target A (type 2) and B (type
) tetrasaccharides were obtained in the form of 3-aminopropyl glycosides. The synthesis
2
includes obtaining of H (type 2) trisaccharide acceptor, its galactosaminylation or
galactosylation, spacer-arming followed by deprotection. We have started from 1,6-anhydro-3-
O-benzoyl-N-acetyl-β-D-glucosamine 4 [25] (Scheme 1); the 1,6-anhydro cycle was opened after
complete assembly of carbohydrate backbone.
2
.1 Synthesis of H (type 2) trisaccharide glycosyl acceptor
4

ACCEPTED MANUSCRIPT
1
,6-Anhydro-3-O-benzoyl-N-acetyl-β-D-glucosamine 4 was obtained from N-acetyl-D-
glucosamine by five step procedure as described in [25, 26]. First, 1,6-аnhydro-N-acetyl-β-D-
glucosamine was obtained by selective tosylation of 6-OH in GlcNAc followed by 1-6-
cyclization by the action of DBU as described by D. Lafont [26]. Next, 4-OH was selectively
tritylated, the obtained mono-O-Tr derivative was 3-O-benzoylated, and finally trityl group was
removed. Both 1,6-аnhydro-N-acetyl-β-D-glucosamine and its 3-O-Bz-derivative 4 are
crystalline compounds.
1
,6-Anhydro-3-O-benzoyl-N-acetyl-β-D-glucosamine 4 was glycosylated with galactosyl
bromide 3 [6] according to Helferich. The reaction mixture was subjected to flash
chromatography to give the target N-acetyllactosamine derivative 5 (Scheme 1) in 88% total
1
yield after crystallization. The minor α-isomer 6 was also isolated in 6% yield. H NMR data
II
confirmed the β-configuration of glycosidic bond for compound 5 (H-1 :
δ 4.63, J 7.9 Hz) and
1,2
II
the α-configuration for isomeric compound 6 (H-1 :
δ 5.42, J 3.7 Hz).
1,2
The selective acid-catalyzed methanolysis of O-acetyl groups in the presence of O-
benzoyl group [27] in disaccharide 5 gave triol 7. Along with O-deacetylation a side process of
N-deacetylation was observed resulting in approximately 10% of disaccharide 8 with free amino
1
group (according to TLC and H NMR data). It was re-N-acetylated with Ac O in NaHCO aq—
2
3
EtOH. Triol 7 was isolated in the total yield 92%.
5

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Scheme 1. Synthesis of protected H (type 2) trisaccharide. Reagents and conditions: (a)
Hg(CN) , HgBr , C H Cl ; (b) HCl/MeOH, CH Cl ; (c) Ac O, EtOH-H O (5:1), NaHCO ; (d)
2
2
2
4
2
2
2
2
2
3
Me C(OMe) , CH Cl , CSA; (e) Et NBr, MS 4Å, C H Cl ; (f) 95% aqueous TFA, CH Cl ; (g)
2
2
2
2
4
2
4
2
2
2
(i) MeC(OEt) , CH Cl , TsOH, (ii) 90% aqueous AcOH
3 2 2
Protection of 3'- and 4'-OH groups in triol 7 with 1,1’-dimethoxypropane catalyzed by
(1S)-(+)-camphor-10-sulfonic acid (CSA) provided disaccharide 9 in 93% yield. Its fucosylation
under Lemieux conditions [28] led to trisaccharide 11 (94%), deprotected with 95% aqueous
TFA to give diol 13 in 88% yield. Alternative deprotection protocol without isolation of 11 from
6

ACCEPTED MANUSCRIPT
reaction mixture gave trisaccharide 13 in 83% yield for two stages (Scheme 1). The main by-
product, β-fucosylated derivative 12 (4%) (or its diol 43), was easily separated by low pressure
chromatography. α-Configuration of Fuc1→2Gal linkage in trisaccharides 11 and 13 was proved
1
by H NMR spectroscopy data: anomeric proton of fucose appeared as a doublet at
δ
5.49 with
J 3.8 Hz (11) and at
δ 5.03 with J 3.6 Hz (13). Anomeric proton of fucose in trisaccharides
1
,2
1,2
1
2 and 43 with β-configuration of Fuc1→2Gal linkage appeared as a doublet at
.0 Hz (12) and at 4.53 with J 7.8 Hz (43).
δ 4.69 with J1,2
8
δ
1
,2
Glycosyl acceptor 14 was obtained from diol 13 (Scheme 1) via intermediate formation of
',4'-orthoacetate [29] and used in the reaction of α-galactosaminylation or α-galactosylation (see
3
below) without further purification.
.2 Synthesis of B (type 2) tetrasaccharide
2
Galactosyl donors with nonparticipating protecting groups at O-2, namely, benzylated α-
bromide [30], benzylated β-imidates [5,8], chlorobenzylated β-thioglycosyde [31] are typically
used for α-galactosylation at the final stage in the synthesis of B (type 2) tetrasaccharide. We
chose tetrabenzylgalactosyl bromide 15 as the simplest practical glycosyl donor of this type.
Condensation of 15 with trisaccharide acceptor 14 promoted by AgOTf led to B (type 2)
tetrasaccharide 16 (82%) and the by-product of β-galactosylation 17 (6%), which were easily
separated by low pressure chromatography (Scheme 2). Configuration of newly formed
1
glycosidic bond was determined by H NMR (see Table 1). Zemplen deacetylation,
hydrogenolysis, followed by acetylation (Ac O/Py, DMAP) and crystallization provided
2
acetylated derivative of B (type 2) tetrasaccharide 18 in 83% total yield for the three stages.
7

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of protected B (type 2) tetrasaccharide. Reagents and conditions: (a)
AgOTf, 1,1,3,3-tetramethylurea (TMU), C H Cl , MS 4Å, rt; (b) (i) MeONa/MeOH, (ii) H ,
2
4
2
2
Pd/C, (iii) Ac O/Py/DMAP
2
Table 1 Сonfiguration of glycosidic bonds in tetrasaccharides 16, 17, 29–34
IV
IV
Compound
Configuration H-1 : δ (ppm)
H-1 : J (Hz)
1,2
16
17
29
30
31
32
33
34
α
β
α
α
β
α
β
β
5.35
5.05
5.39
5.25
5.06
5.30
4.93
5.06
3.4
7.6
3.7
3.6
7.9
3.6
7.9
7.8
2
.3 Synthesis of A (type 2) tetrasaccharide
Our previous experience in glycosylation of constrained OH groups by 2-azido-2-
deoxydonors taught us that for high stereoselectivity donor should exactly match to reactivity of
particular glycosyl-acceptor. This is why we performed wide search of suitable glycosyl donor
8

ACCEPTED MANUSCRIPT
for reaction of trisaccharide acceptor 14; seven 2-azidogalactose derivatives (19–25) (Figure 2)
were compared.
Figure 2. 2-Azidogalactosyl donors
Syntheses of compounds 19 [32], 20 [33], 21 [34], 22 [35] and 24 [36] were described
earlier. Trichloracetimidates 23 and 24 were synthesized from thioglycoside 22 by selective
acetolysis (I /Ac O) [37], deacetylation of anomeric OH group (N H ⋅HOAc/DMF), followed by
2
2
2
4
treatment with Cl CCN in the presence of K CO [32] (Scheme 3). The obtained mixture of β-
3
2
3
and α-anomers 23 and 24 was separated by low pressure chromatography. The thioglycoside 25
was synthesized from corresponding acetylated thioglycoside 28 [35] (Scheme 3) in three stages
with the total yield 91%: Zemplen deacetylation of thioglycoside 28, 3,4-O-isopropylidene
acetalyzation according to Catelani procedure [38], and subsequent acetylation. The formation of
side 4,6-O-isopropylidene derivative was insignificant. The results of galactosaminylation with
1
various glycosyl donors are summarized in Table 2, H NMR data required to confirm
configurations of formed glycosidic linkages are presented in Table 1.
9

ACCEPTED MANUSCRIPT
Scheme 3.Glycosyl-donors used for α-galactosaminylation. Reagents and conditions: (a)
º
I /Ac O; (b) N H ⋅HOAc, DMF, 50 C; (c) Cl CCN, K CO , CH Cl ; (d) (i) MeONa/MeOH, (ii)
2
2
2
4
3
2
3
2
2
Me C(OMe) , CSA; (iii) MeOH-H O (10:1), boiling under reflux; (iv) Ac O/Py
2
2
2
2
Table 2.Glycosylation of trisaccharide 14 with derivatives of 2-azido-2-deoxygalactose 19–25
Yield, %
α / β ratio)
Donor
Reaction conditions
Products*
(
AcO OAc
AcO OAc
O
N3
1
O
AcO
O
CCl
NH
3
TMSOTf, C
r.t.
2
H
4
Cl
2
,
45
(α only)
AcO
N3
O
R
R
2
9
9
AcO OAc
AcO OAc
O
O
Hg(CN) , HgBr ,
40
(α only)
2
2
AcO
AcO
N3
C H Cl , r.t.
N3
Br
2
4
2
O
2
0
29
BnO OBn
BnO OBn
BnO
BnO
OBn
O
O
O
O
O
O
O
R
R
R
R
7
(3/2)
5
BnO
BnO
HgBr , C H Cl , r.t.
2
2
4
2
N3
N3
N
3O
Br
R
2
1
30
31
BnO OBn
BnO OBn
BnO OBn
O
O
O
NIS, TfOH, ether,
+5 C
80
(2/1)
SEt
BnO
BnO
BnO
o
N3
N
3O
N3
R
2
2
30
3
1
BnO OAc
BnO OAc
BnO OAc
O
O
O
BnO
O
CCl
NH
3
TMSOTf, C
r.t.
2
H
4
Cl
2
,
BnO
75
(3/2)
BnO
N3
N3
N3
O
R
R
3
2
2
3
3
33
BnO OAc
BnO OAc
BnO OAc
O
O
O
BnO
O
CCl
NH
3
TMSOTf, ether,
BnO
93
(3/1)
BnO
N3
o
N3
+
5 C
N3
O
3
2
2
33
1
0

ACCEPTED MANUSCRIPT
BnO OAc
BnO OAc
BnO OAc
O
O
O
BnO
O
R
TMSOTf, C H Cl ,
BnO
80
(3/2)
2
4
2
BnO
N3
CCl3
NH
O
N3
r.t.
N
3
O
R
3
2
3
3
2
4
1
. NIS, TfOH,
ether, r.t.
AcO OAc
AcO OAc
O
OAc
Me
Me
O
O
O
OR
2. 95% aqueous
TFA in CH Cl
AcO
89
O
SEt
AcO
N3
N3
N
3O
(24/1)
2
2
R
2
5
3. Ac
DMAP
O
2
O–Py (1:1),
2
9
34
OBz
O
OAc OBn
O
*
R = O
O
NHAc
O
O
OBn
OBn
BnO
Glycosylation of acceptor 14 with generally used peracetylated azidogalactose donors, β-
trichloracetimidate 19 and α-bromide 20, proceeded stereoselectively, but with low yields (40–
5%). The use of more active perbenzylated donors, bromide 21 and thioglycoside 22, resulted in
4
the considerable increase of glycosylation yields, but stereospecificity of reaction dramatically
dropped down. Galactosaminylation of 14 with bromide 21 promoted by HgBr in C H Cl led to
2
2
4
2
a mixture of α- and β-anomers 30 and 31 with total yield 75% (α/β 3:2); no reaction observed at
o
temperatures below 0 C. Glycosylation of 14 with thioglycoside 22 promoted by NIS/TfOH in
Et O gave a mixture of α- and β-anomers 2:1, yield 80%.
2
The use of anomeric 6-OAc-3,4-di-OBn-protected trichloroacetimidates 23 and 24 could
increase the yield of galactosaminylation comparing to peracetylated trichloroacetimidate 19
without the loss of its stereospecificity. However, in reality reaction in C H Cl promoted by
2
4
2
TMSOTf proceeded with low stereospecificity (α/β ≈ 3:2), independently on donor anomeric
configuration. This conformed to the S -1 mechanism of substitution and confirmed weak
N
influence of 6-OAc group in glycosyl donor on the reaction stereospecificity [36]. Carrying out
the condensation of compound 14 with β-imidate 23 in Et O improved α-stereoselectivity (α/β =
2
3
:1).
1
1

ACCEPTED MANUSCRIPT
Conformationally rigid glycosyl donors with 4,6-di-tert-butylsilylidene or 3,4-
isopropylidene protection [10,39] are known to enhance α-stereospecificity of glycosylation.
Oxonium intermediates derived from such glycosyl donors have a bulky and rigid protecting
group which hinders nucleophilic attack from β-side [39]. 3,4-Isopropylidenated derivatives of 2-
deoxy-D-fucopyranose [39], galactopyranose [40], and 2-azido-2-deoxy-galactopyranose [41]
were successfully used for α-glycosylation. These data inspired us to test thioglycoside 25 with
3
,4-isopropylidene cycle as a glycosyl donor. The coupling of glycosyl acceptor 14 with
thioglycoside 25 was carried out in Et O at room temperature using NIS/ TfOH as the reaction
2
promoter to yield the required A (type 2) tetrasaccharide 35 (Scheme 4). To simplify the
isolation of the products, the reaction mixture was treated with 95% aqueous TFA in CH Cl to
2
2
remove isopropylidene acetal and acetylated. Subsequent low pressure chromatography afforded
the required A(type 2) tetrasaccharides 29 and its β-anomer 34 with 85% and 3.5% yields,
respectively, for three stages. Thus, the stereoselectivity of glycosylation with thioglycoside 25
was the highest in the series of glycosyl donors tested, the ratio of α/β-anomers being 24:1.
Peracetylated compound 36 was obtained from derivative 29 by the following
deprotection-reprotection procedures: Zemplen deacetylation, catalytic reduction of azido group
and hydrogenolysis of benzyl groups over 10% Pd/C and then reacetylation, chromatography and
crystallization with total yield 76% (Scheme 4).
1
2

ACCEPTED MANUSCRIPT
Scheme 4. Synthesis of protected A (type 2) tetrasaccharide. Reagents and conditions: (a) NIS,
TfOH, ether, r.t.; (b) (i) 95% aqueous TFA, CH Cl , (ii) Ac O/Py; (c) (i) MeONa/MeOH, (ii) H ,
2
2
2
2
Pd/C, Ac O/MeOH, (iii) Ac O/Py/DMAP
2
2
2
.4 Synthesis of spacer-armed A (type 2) and B (type 2) tetrasaccharides
Mild acetolysis of derivatives 36 and 18 (H SO /AcOH–Ac O) provided mixtures
2
4
2
containing approximately 80% (according to TLC) of oxazolines and 20% of 1-OAc derivatives
Scheme 5) for both tetrasaccharides. We found it unreasonable to use such mixtures for spacer-
(
arming; we also failed to obtain pure 1-OAc derivatives of A tetrasaccharide 37 and B
tetrasaccharide 38 without oxazoline admixtures applying different acetolysis conditions [42,43].
So, after the acetolysis the excess of NaOAc was added [22] to convert formed oxazolines into
peracetates. Treatment of the peracetates 37 and 38 with TMSOTf in C H Cl according to [44]
2
4
gave corresponding oxazolines 39 and 40, which were coupled with 3-
trifluoroacetamidopropanol in the presence of CSA to result in peracetylated spacer-armed
tetrasaccharides 41 and 42. After chromatography and crystallization, tetrasaccharides 41 and 42
were obtained with 87% and 85% yields, respectively, for two stages. β-Configuration of formed
1
I
glycosidic linkages in 41 and 42 was confirmed by H NMR spectroscopy (H-1 : δ 4.41, J 7.2
1,2
I
Hz (41) and H-1 :
δ
4.41, J 7.7 Hz (42)). Zemplen deacetylation and removal of trifluoroacetyl
1
,2
1
3

ACCEPTED MANUSCRIPT
protecting group with aqueous alkali yielded the target 3-aminopropyl glycosides of A (type 2)
and B (type 2) tetrasaccharides 1 and 2.
Scheme 5. Deprotection and spacer-arming of protected A and B tetrasaccharides. Reagents and
conditions: (a) (i) Ac O/AcOH/H SO , 1.5:1:0.018 (v/v/v), (ii) AcONa; (b) TMSOTf, C H Cl ,
2
2
4
2
4
2
+
4
0ºC; (c) HO(CH ) NHC(O)CF , C H Cl , H , 70ºC; (d) (i) MeONa/MeOH, (ii) NaOH/H O
2 3 3 2 4 2 2
3
. Conclusions
Thus, the proposed synthetic scheme allowed us to obtain spacer-armed A (type 2) and B
type 2) tetrasaccharides in gram amounts. The advantages of this scheme are: 1) high yields and
(
stereospecificity at the key stages, 2) technical simplicity of procedures (exclusion of low
temperature and inert atmosphere conditions for glycosylation, 3) purification of anhydro-
GlcNAc derivatives at several stages by crystallization. Besides improvement of 4-OH group
reactivity in GlcNAc due to its cyclic form, this approach gives an opportunity to introduce
1
4

ACCEPTED MANUSCRIPT
different spacer-arms at the final stage, as well as to use the tetrasaccharides as glycosyl donors
for the block synthesis of higher blood group oligosaccharides.
4
4
. Experimental
.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. Column
chromatography was performed on silica gel 60 0.040–0.063 mm (Merck), gel filtration was
carried out on Sephadex LH-20 (GE Healthcare Bio-Sciences AB) columns (elution with
CHCl –MeOH, 1:1). Solvents were removed in vacuo at 30–40 ºC. Thin layer chromatography
3
(TLC) was performed on silica gel 60 F254 aluminium-backed plates (Merck). The spots of
carbohydrates were visualized by dipping a TLC plate into aqueous solution of H PO (8%) and
3
4
subsequent heating (>150 ºC); carbohydrates with the free amino group were visualized by
dipping a TLC plate in a ninhydrin solution and subsequent heating (60-70 ºC). Molecular sieves
4
Å (Fluka) were dried at 300 ºC for 3 h. Deacetylation was carried out in 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 the solution was
evaporated. Hydrogenolysis was carried out on 10% Pd/C (Merck) in the atmosphere of H2.
The values of optical rotations were measured on a Perkin Elmer 341 polarimeter at 21±2
1
ºС. Н NMR spectra were recorded on a Bruker BioSpin GmbH (700 МHz) spectrometer at 30
o
С; chemical shifts (δ, ppm) were referred to the peak of internal D O (
δ
4.750), CDCl3 (
.270), CD OD (δ 3.500) or DMSO-d6 (δ 2.5); coupling constants (J) were measured in Hz and
3
δ
2
7
multiplicities are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
1
(
m), and broadened (br.). Signals of Н NMR spectra were assigned to the corresponding protons
1
3
using 2D spectroscopy (COSY,and DEPT-ed-HSQC). C NMR spectra were recorded at 176
1
3
MHz. Signals of C NMR spectra were assigned to the corresponding carbons using 2D
spectroscopy (HSQC, DEPT and DEPT-ed-HSQC). Symbols of monosaccharide residues in
1
5

ACCEPTED MANUSCRIPT
oligosaccharide NMR spectra: I – β-GlcNAc (reducing end), II – α/β-Gal, III – α/β-Fuc, IV –
α/β-GalN or α/β-Gal. MALDI TOF MS spectra were recorded on a Bruker Daltonics Ultraflex
MALDI TOF/TOF mass spectrometer (Germany).
2
,3,4-Tri-O-benzyl-α-
thioglycoside as described by H. Lönn [46]. 2,3,4-Tri-O-acetyl-6-O-benzyl-α-
galactopyranosyl bromide 3 [6], 2,3,4,6-tetra-O-benzyl-α- -galactopyranosyl bromide 15 [30]
and 2-azido-3,4,6-tri-O-benzyl-2-deoxy-α- -galactopyranosyl bromide 21 were prepared from
the corresponding thioglycosides [47,48,35] by the same procedure. Tri-O-acetyl-2-azido-2-
deoxy-α- -galactopyranosyl bromide 20 was obtained as described by H. Paulsen [33].
Oxazoline derivatives were prepared in accordance with S. Nakabayashi procedure [44].
L-fucosyl bromide 10 [45] was obtained from the corresponding
D-
D
D
D
1
3
Table 2. C NMR spectroscopy data for the derivatives of GalN
3
Сarbon
23
25
83.4
26
91.1
1
2
3
4
5
6
97.2
62.7
80.9
71.8
73.5
62.6
20.8
64.8
59.0
78.2
77.9
GalN
72.8
72.7
74.4
70.9
63.6
62.8
OC(O)CH3
C(CH3)2
20.8
20.8; 20.9
26.3; 28.2
CH C H
5
73.2; 74.7
72.7; 74.7
2
6
C H
6
128.0-128.7
128.0-128.7
5
CH (Et)
15.0
24.8
3
CH (Et)
2
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
1
3
Table 3. C NMR spectroscopy data for the derivatives of N-acetyl-lactosamine
Сarbon
5
6
7
8*
9
1
2
3
4
5
6
101.1
48.7
70.9
73.2
72.4
64.7
100.5
49.2
71.0
76.0
74.9
65.3
100.0
49.8
70.4
75.3
73.8
64.8
100.8
51.00
71.5
75.0
74.1
64.8
100.2
49.5
69.7
75.9
74.1
65.0
GlcN
1
6

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
99.3
69.2
70.4
67.4
72.9
67.6
98.5
68.0
67.4
68.8
69.0
68.6
23.3
102.9
71.2
73.5
69.1
73.9
69.3
21.3
102.9
71.2
73.5
69.1
73.9
69.4
102.0
73.5
79.1
73.6
72.5
69.1
23.1
Gal
NC(O)CH3
OC(O)CH3
22.8
2
0.5; 20.6;
20.6 (3Ac)
2
1.1
C(CH3)2
CH C H
5
26.3; 28.1
73.3
73.8
127.9-133.6
73.6
72.9
72.9
2
6
C H
127.8–134.4
127.0–133.0
127.4–133.3
127.6–133.8
6
5
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
1
3
Table 4. C NMR spectroscopy data for the derivatives of H (type 2) trisaccharide
Сarbon
11
100.3
49.6
69.7
76.6
74.5
65.0
101.8
76.9
79.6
73.6
72.0
69.1
96.6
76.3
79.0
77.8
66.8
16.9
23.2
12
99.6
49.8
68.7
76.6
74.2
64.4
103.3
78.5
77.6
73.8
70.5
69.0
103.6
79.2
83.2
76.2
71.8
16.9
23.0
13
100.5
49.1
43*
99.5
49.3
68.8
76.0
74.2
64.0
102.7
80.2
71.6
67.5
72.7
68.8
103.7
79.0
82.71
76.1
70.8
16.4
22.9
14
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
100.5
49.2
70.2
74.2
72.6
64.9
100.5
82.8
71.7
68.6
72.7
68.2
101.6
77.1
79.3
77.4
68.4
16.7
22.9
20.8
70.3
GlcN
74.3 or 73.5
72.8
64.9
100.6
82.4
72.9
Gal
67.8
74.3 or 73.5
69.0
101.4
77.5
79.5
Fuc
77.4
68.4
16.7
NC(O)CH3
OC(O)CH3
C(CH3)2
23.0
26.4; 27.8
26.5; 27.9
7
7
3.1; 73.1;
3.3; 74.9
72.9; 72.9;
74.8; 75.2
72.8; 73.5;
75.0; 75.0
73.1; 73.1;
74.9; 74.9
73.0; 73.5;
74.6; 74.9
CH C H
5
2
6
1
27.4–133.6
127.3–133.8
(5 Ph)
127.5–133.6
(5 Ph)
126.6–133.7
(5 Ph)
127.5–133.7
(5 Ph)
C H
6
5
(
5 Ph)
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
1
7

ACCEPTED MANUSCRIPT
1
3
Table 5. C NMR spectroscopy data for the derivatives of A (type 2) tetrasaccharide (part 1)
Сarbon
29
100.6
49.0
70.2
75.0
72.5
65.0
101.9
78.5
73.1
64.0
71.7
67.7
99.2
75.7
79.5
78.2
67.4
16.5
93.1
57.7
68.4
68.0
66.8
62.5
22.9
0.6–20.8
33*
100.4
49.2
69.8
76.0
73.5
64.9
102.1
78.0
75.3
69.9
72.3
68.1
98.6
75.5
79.5
77.7
67.2
16.6
100.3
61.6
70.7
66.3
70.4
61.0
22.8
30
100.7
49.1
70.3
74.9
72.2
65.0
101.8
78.7
72.2
64.3
72.0
67.9
98.4
76.8
78.9
77.3
67.5
16.6
93.3
59.8
77.5
72.9
69.6
70.0
22.9
31*
100.3
49.1
69.7
75.7
73.4
64.8
102.1
79.0
74.6
69.7
72.6
68.6
98.0
76.0
80.5
77.6
66.9
16.3
100.0
63.8
79.2
72.5
74.0
69.5
22.7
35
100.5
49.1
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
70.0
GlcN
75.4
72.9
65.0
102.3
79.5 or 79.4
72.8
Gal
64.1
71.7
67.7
99.3
76.3
79.5 or 79.4
77.2
Fuc
67.4
16.7
92.8
60.9
73.0
Gal(N)
73.1
66.3
64.1
NC(O)CH3
OC(O)CH3
22.9
2
20.6–20.8
(4 Ac)
20.7
20.4
21.0; 20.6
(
4 Ac)
7
1.5; 72.3;
72.3; 72.4;
73.1; 73.2;
73.4; 74.3;
74.9
7
7
3.4; 73.6;
3.6; 75.3
72.8; 73.3;
73.6; 75.0
73.1; 73.5;
74.0; 74.4;
72.2; 73.6;
73.8; 75.3
CH C H
5
2
6
7
5.2
1
26.9–133.7
5 Ph)
127.2–133.7
(5 Ph)
125.0–130.0
(8 Ph)
125.0–130.0
(8 Ph)
126.9–133.7
(5 Ph)
C H
6
5
(
C(CH3)2
26.2; 28.3
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
1
3
Table 5. C NMR spectroscopy data for the derivatives of A (type 2) tetrasaccharide (part 2)
3
7
37
Сarbon
GlcN
32*
33*
36
41
(
α anomer)
(β anomer)
1
100.4
100.3
100.6
90.6
92.7
101.4
1
8

ACCEPTED MANUSCRIPT
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
48.9
69.9
75.0
72.4
64.8
101.9
78.4
72.3
64.1
71.8
67.8
98.6
76.7
79.2
77.3
67.3
16.2
93.1
59.6
77.4
72.6
69.0
64.0
22.6
49.1
68.7
75.7
73.4
64.8
102.1
78.1
75.3
69.8
72.6
68.5
98.1
76.0
79.4
77.7
67.0
16.4
100.4
63.6
80.3
71.9
72.1
62.6
22.7
50.0
70.6
76.2
73.0
65.1
101.1
76.6
73.6
67.5
70.7
62.1
96.5
67.8
67.8
71.1
65.6
15.8
93.4
47.2
67.7
65.6
67.0
62.3
22.8; 22.9
50.8
70.5
73.4
70.5
61.9
101.0
74.0
77.0
66.2
70.8
61.0
96.3
67.5
67.8
71.1
65.4
15.8
96.9
48.0
67.6
67.4
68.4
62.6
23.1; 23.1
52.5
71.9
73.5
73.8
62.2
101.0
75.0
77.5
66.2
70.8
61.2
96.6
67.5
67.7
71.2
65.6
15.8
96.8
48.0
67.7
67.4
68.3
62.5
23.1; 23.2
53.9
72.1
74.0
73.1
62.0
100.9
74.4
77.2
66.5
70.7
61.0
96.4
67.2
67.8
71.1
65.4
15.7
97.5
48.1
67.5
67.4
68.4
62.6
23.0; 23.3
Gal
Fuc
Gal(N)
NC(O)CH3
OC(O)CH3
2
0.6–21.3
9 Ac)
20.6–21.0
(11 Ac)
20.6–21.0
(11 Ac)
20.5–20.9
(10Ac)
20.27; 20.57 20.52; 20.53
(
OCH CH CH N
67.1
28.6
37.3
2
2
2
OCH CH CH N
2
2
2
OCH CH CH N
2
2
2
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
1
3
Table 6. C NMR spectroscopy data for the derivatives of B (type 2) tetrasaccharide
3
8
38
Сarbon
16*
17*
18
42
(
α anomer)
(β anomer)
1
2
3
4
5
6
1
2
100.6
49.0
70.1
74.9
72.3
64.8
102.1
78.3
100.3
49.4
69.8
76.1
73.8
64.9
102.5
77.0
100.4
48.8
69.7
76.8
72.7
64.9
102.8
78.0
90.6
92.6
51.4
71.6
72.3
73.19
62.5
101.9
77.5
101.3
53.3
50.9
70.5
72.1
GlcN
72.5
73.1
70.5
72.84
62.4
61.9
101.8
77.1
101.9
77.1
Gal-1
1
9

ACCEPTED MANUSCRIPT
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
72.0
64.6
72.0
68.1
98.4
77.4
78.9
77.6
67.3
16.4
93.0
76.7
78.7
74.7
69.2
69.8
22.7
76.1
70.5
73.8
69.7
97.4
75.8
78.5
78.2
76.7
16.6
101.8
79.8
82.1
73.9
72.8
68.8
22.9
71.7
64.1
72.7
64.4
70.7
61.2
97.7
68.1
67.6
71.1
65.9
16.1
92.5
67.1
67.0
68.2
67.0
61.7
23.1
72.3
64.4
70.8
61.2
97.8
68.2
67.5
71.1
66.1
16.2
92.4
67.1
67.0
68.2
66.8
61.6
23.2
72.6
64.4
70.7
61.1
97.7
68.0
67.5
71.0
65.9
16.1
92.5
67.1
66.9
68.1
66.8
61.5
23.3
71.0
61.4
97.9
67.0 or 68.5
67.0 or 68.5
71.0
Fuc
66.1
16.0
91.8
67.0
67.1
Gal-2
68.0
66.2
61.4
NC(O)CH3
OC(O)CH3
23.0
2
(
0.3–21.1
10 Ac)
20.4–21.1
(12 Ac)
20.4–21.1
(12 Ac)
20.3–21.5
(11 Ac)
20.3
20.7
7
7
7
2.3; 72.6;
3.3; 73.3;
3.6; 73.8;
72.1; 72.6;
73.3; 72.8;
73.5; 74.2;
75.0; 75.4
CH C H
5
2
6
7
4.4; 75.2
1
26.9–133.6
127.0–133.6
(9 Ph)
C H
6
5
(
9 Ph)
OCH CH CH N
66.9
28.5
37.3
2
2
2
OCH CH CH N
2
2
2
OCH CH CH N
2
2
2
1
3
*
C NMR data was taken from DEPT-ed-HSQC spectrum.
.2 2,3,4-Tri-O-acetyl-6-O-benzyl-β- -galactopyranosyl-(1→4)-2-acetamido-1,6-
anhydro-3-О-benzoyl-2-deoxy-β- -glucopyranose (5).
4
D
D
A mixture of compound 4 [25] (4.70 g, 15.3 mmol), Hg(CN) (6.58 g, 26.0 mmol), HgBr2
2
(
0.94 g, 2.6 mmol), anhydrous C H Cl (130 ml) and molecular sieves 4Å (24 g) was stirred for
2
4
2
1
.5 h at rt. The mixture was heated to 50 °C and the solution of bromide 3 [6] (obtained from the
-galactopyranoside [47] (11.46 g, 26.02 mmol)
ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-1-thio-β-
D
2
0

ACCEPTED MANUSCRIPT
and Br (1.477 ml, 28.62 mmol)) in C H Cl (40 ml) was then added dropwise. The mixture was
2
2
4
2
vigorously stirred for 2 h at 50 °C and 16 h at rt, then filtered, and concentrated. The residue was
dissolved in CHCl (300 ml), washed with H O (2×75 ml), 1M KI (150 ml), H O (100 ml); dried
3
2
2
by filtration through cotton wool; and concentrated. Column chromatography on silica gel (210
g) (PhCH –acetone, 4:1) followed by crystallization from acetone–Et O afforded disaccharide 5
3
2
(9.23 g, 88%) and its α-isomer 6 (0.58 g, 6%).
Compound 5: colorless crystals, mp 98–100 °C; [α]546 –88 (с 1, CHCl ); R 0.29 (PhCH –
3
f
3
1
acetone, 3:1); H NMR (CDCl ):
δ
2.02, 2.11, 2.11 and 2.14 (4s, 4×3H, 3×OC(O)CH3,
3
II
II
NHC(O)СН ), 3.55 (dd, 1H, J 6.4, J6a,6b 9.8, H-6a ), 3.62 (dd, 1H, J 6.3, J6b,6a 9.8, H-6b ),
3
6a,5
6b,5
I
I
3
.87 (dd, 1H, J6a,5 5.6, J6a,6b 7.6, H-6a ), 3.88 (br. s, 1H, H-4 ), 3.91 (br. t, 1H, J 6.4, J5,6b 6.4,
5,6a
II
I
I
H-5 ), 4.18 (d, 1H, J6 7.6, H-6b ), 4.28 (br. d, 1H, J
9.9, H-2 ), 4.46 and 4.57 (2d, 2×1H,
b,6a
2,NH
I
II
Jgem 12.0, CH Ph), 4.53 (br. d, 1H, J 5.6, H-5 ), 4.63 (d, 1H, J 7.9, H-1 ), 5.12 (dd, 1H, J
2
5,6a
1,2
3,2
II
I
II
1
0.6, J 3.5, H-3 ), 5.15 (br. s, 1H, H-3 ), 5.28 (dd, 1H, J 7.9, J 10.6, H-2 ), 5.40 (br. s, 1H,
3
,4
2,1
2,3
I
II
I
H-1 ), 5.51 (br. d, 1H, J 3.5, H-4 ), 6.41 (d, 1H, J
9.9, NHAc ), 7.32–7.23 (m, 5H,
4
,3
NH,2
C H CH ), 7.47 (2t, 2H, J 7.8, m-ArH ), 7.60 (t, 1H, J 7.4, p-ArH), 8.04 (2d, 2H, J 8.0, o-ArH).
6
5
2
+
MALDI MS: m/z Calcd [C H NO ]Na : 708.2. Found: 708.1.
34
39
14
1
Compound 6: colorless glass, [α]546 +39 (с 1, CHCl ); R 0.25 (PhCH –acetone, 3:1); H
3
f
3
NMR (CDCl3): δ 2.03, 2.08, 2.11 and 2.13 (4s, 4×3H, 3×OC(O)CH , 1×NHC(O)СН ), 3.53 (dd,
3 3
II
II
I
1
3
H, J6a,5 5.3, J6a,6b 10.1, H-6a ), 3.55 (dd, 1H, J 7.0, J6b,6a 10.1, H-6b ), 3.66 (br. s, 1H, H-4 ),
6b,5
I
I
.69 (dd, 1H, J6a,5 5.7, J6a,6b 7.7, H-6a ), 4.02 (d, 1H, J6b,6a 7.7, H-6b ), 4.27 (br. d, 1H, J
9.7,
2,NH
I
II
H-2 ), 4.46 and 4.52 (2d, 2×1H, Jgem 11.8, CH Ph), 4.49 (dd, 1H, J 5.3, J5,6b 7.0, H-5 ), 4.67
2
5,6a
I
I
II
(
br. d, 1H, J5,6a 5.7, H-5 ), 4.82 (br. s, 1H, H-3 ), 5.27 (dd, 1H, J 3.7, J 10.9, H-2 ), 5.42 (d,
2,1
2,3
II
II
I
1
H, J 3.7, H-1 ), 5.47 (dd, 1H, J 11.0, J 3.5, H-3 ), 5.48 (br. s, 1H, H-1 ), 5.53 (br. d, 1H,
1
,2
3,2
3,4
II
I
J 3.5, H-4 ), 6.14 (d, 1H, J
9.7, NHAc ), 7.24–7.34 (m, 5H, C H CH ), 7.47 (2t, J 7.5, 2H,
4
,3
NH,2
6
5
2
m-ArH), 7.61 (t, 1H, J 7.5, p-ArH), 8.04 (2d, J 8.0, 2H, o-ArH). MALDI MS: m/z Calcd
+
[
C H NO ]Na : 708.2. Found: 708.1.
3
4
39
14
2
1

ACCEPTED MANUSCRIPT
4
.3 6-O-Benzyl-β-
D-galactopyranosyl-(1→4)-2-acetamido-1,6-anhydro-3-О-benzoyl-
2
-deoxy-β- -glucopyranose (7).
D
A solution of compound 5 (9.20 g, 13.4 mmol) in C H Cl (50 ml) was treated with
2
4
2
methanolic HCl prepared by addition of acetyl chloride (8.10 ml, 114 mmol) to dry MeOH (200
ml) at 0 ºC. The mixture was kept for 1 day at rt, neutralized with excess of aqueous KHCO3
(
19.0 g, 190 mmol), diluted with CHCl (600 ml), washed with H O (100 ml), dried, and
3 2
concentrated. The residue (containing triol 7 and GlcNH -derivative 8 (~10%)) was dissolved in
2
EtOH–H O (5:1, 84 ml), NaHCO (353 mg, 4.20 mmol) and Ac O (132 µl, 1.40 mmol) were
2
3
2
added, and the mixture was vigorously stirred for 0.5 h. The mixture was concentrated, dissolved
in CHCl (100 ml), washed with H O (70 ml), the chloroform fraction was dried by filtration
3
2
through cotton wool and concentrated. Column chromatography on silica gel (300 g, CHCl3–
MeOH, 3→12%) provided 6.9 g (92%) of triol 7 as colorless foam; R 0.33 (CHCl –MeOH, 9:1),
f
3
1
[
α]546 –39 (с 1, CHCl ); H NMR (CD OD):
δ
2.18 (s, 3H, NHC(O)СН ), 3.73 (dd, 1H, J 9.6,
3
3
3
3,2
II
II
II
J 3.4, H-3 ), 3.80 (dd, 1H, J 7.8, J 9.6, H-2 ), 3.92 (dd, 1H, J 5.8, J6a,6b 10.4, H-6a ),
3
,4
2,1
2,3
6a,5
II
I
3
.94 (dd, 1H, J6b,5 6.3, J6b,6a 10.4, H-6b ), 3.99 (dd, 1H, J 5.9, J6a,6b 7.7, H-6a ), 4.01 (br. t, 1H,
6a,5
II
II
I
J5,6a 6.2, J5,6b 6.2, H-5 ), 4.05 (br. d, 1H, J 3.4, H-4 ), 4.07 (br. s, 1H, H-4 ), 4.14 (br. s, 1H, H-
4,3
I
I
2
7
), 4.36 (d, 1H, J6b,6a 7.7, H-6b ), 4.71 and 4.74 (2d, 2×1H, J 11.7, CH Ph), 4.76 (d, 1H, J
gem
2
1,2
II
I
I
I
.8, H-1 ), 4.9 (br. d, 1H, J 5.5, H-5 ), 5.43 (br. s, 1H, H-3 ), 5.61 (br. s, 1H, H-1 ), 7.34–7.43
5
,6a
(
m, 5H, C H CH ), 7.62 (2t, J 7.8, 2H, m-ArH), 7.76 (t, 1H, J 7.4, p-ArH), 8.16 (2d, J 8.0, 2H,
6 5 2
+
o-ArH). MALDI MS: m/z Calcd [C H NO ]Na : 582.2. Found: 582.1.
28
33
11
1
GlcNH -derivative 8: colorless glass; R 0.11 (MeOH); H NMR (CD OD): δ 3.11 (br. s,
2
f
3
I
II
II
1
H, H-2 ), 3.73 (dd, 1H, J 9.7, J 3.4, H-3 ), 3.79 (dd, 1H, J 7.7, J 9.7, H-2 ), 3.92 (dd,
3
,2
3,4
2,1
2,3
II
II
1
H, J6a,5 4.2, J6a,6b 10.1, H-6a ), 3.98 (dd, 1H, J 7.2, J6b,6a 10.2, H-6b ), 4.01 (dd, 1H, J 4.2,
6b,5
5,6a
2
2

ACCEPTED MANUSCRIPT
II
I
II
J5,6b 7.2, H-5 ), 4.02 (dd, 1H, J 5.7, J
7.8, H-6a ), 4.04 (br. d, 1H, J 3.4, H-4 ), 4.13 (br.
6a,5
6a,6b
4,3
I
I
I
s, 1H, H-4 ), 4.41 (d, 1H, J
7.8, H-6b ), 4.99 (br. d, 1H, J 5.7, H-5 ), 4.71 and 4.75 (2d,
6
b,6a
5,6a
II
I
2
×1H, Jgem 11.6, CH Ph), 4.78 (d, 1H, J 7.7, H-1 ), 5.56 (br. s, 1H, H-3 ), 5.64 (br. s, 1H, H-
2 1,2
I
1
), 7.34–7.44 (m, 5H, C H CH ), 7.63 (2t, J 8.1, 2H, m-ArH), 7.77 (t, 1H, J 7.5, p-ArH), 8.15
6
5
2
(
2d, J 8.3, 2H, o-ArH).
.4 6-O-Benzyl-3,4-O-isopropylidene-β-
anhydro-3-О-benzoyl-2-deoxy-β- -glucopyranose (9).
4
D-galactopyranosyl-(1→4)-2-acetamido-1,6-
D
A mixture of disaccharide 7 (5.0 g, 8.9 mmol), 2,2-dimethoxypropane (3.28 ml, 26.7
mmol), CSA (104 mg, 0.449 mmol), and CH Cl (50 ml) was stirred for 1 h at rt, diluted with
2
2
CHCl (100 ml), washed with saturated aqueous NaHCO (70 ml), H O (50 ml), the chloroform
3
3
2
fraction was dried by filtration through cotton wool and concentrated. Column chromatography
on silica gel (80 g, PhCH –acetone, 20→40%) afforded 4.96 g (93%) of disaccharide 9 as white
3
powder: R 0.53 (PhCH –acetone, 1:1); R 0.34 (CHCl –MeOH, 93:7); [α] –25 (с 1, CHCl3);
f
3
f
3
546
1
H NMR (CDCl3): δ 1.38, 1.55 (2s, 2×3H, C(CH ) ), 1.99 (s, 3H, NHC(O)СН ), 2.49 (d, 1H, J
3 2 3
2-
II
II
3
.2, 2-OH ), 3.72 (ddd, 1H, J 8.3, J 7.3, J
3.2, H-2 ), 3.78 (dd, 1H, J
6a,5
5.5, J6a,6b
OH,2
2,1
2,3
2,2-OH
II
I
II
1
0.1, H-6a ), 3.79 (br. s, 1H, H-4 ), 3.82 (dd, 1H, J 7.0, J6b,6a 10.1, H-6b ), 3.85 (dd, 1H, J
6
b,5
6a,5
I
I
II
5
.8, J6a,6b 7.7, H-6a ), 4.12 (d, 1H, J6b,6a 7.7, H-6b ), 4.15 (dd, 1H, J 7.1, J 5.5, H-3 ), 4.15
3,2
3,4
I
II
(
br. d, 1H, J2,NH 8.5, H-2 ), 4.21 (ddd, 1H, J 2.1, J 5.5, J5,6b 7.0, H-5 ), 4.23 (dd, 1H, J 5.5,
5,4 5,6a 4,3
II
I
J 2.1, H-4 ), 4.54 and 4.59 (2d, 2×1H, J =11.9, CH Ph), 4.66 (br. d, 1H, J 5.8, H-5 ), 4.70
4
,5
gem
2
5,6a
II
I
I
(
d, 1H, J 8.3, H-1 ), 5.42 (br. s, 1H, H-3 ), 5.55 (br. s, 1H, H-1 ), 6.38 (d, 1H, J
8.5,
1
,2
NH,2
I
NHAc ), 7.19–7.28 (m, 5H, C H CH ), 7.48 (2t, J 7.8, 2H, m-ArH), 7.629 (t, 1H, J 7.8, p-ArH),
6
5
2
+
8
.04 (2d, J 8.2, 2H, o-ArH). MALDI MS: m/z Calcd [C H NO ]H : 600.2. Found: 600.2.
58 65 15
2
3

ACCEPTED MANUSCRIPT
4
.5 2,3,4-Tri-O-benzyl-α-
-galactopyranosyl-(1→4)-2-acetamido-1,6-anhydro-3-О-benzoyl-2-deoxy-β-
glucopyranose (11)
L-fucopyranosyl-(1→2)-6-O-benzyl-3,4-O-isopropylidene-β-
D
D-
A suspension of disaccharide 9 (4.71 g, 7.86 mmol), Et NBr (6.87 g, 23.8 mmol) and 4Å
4
molecular sieves (45 g) in mixture of anhydrous CH Cl (230 ml) and DMF (9 ml) was stirred at
2
2
rt for 2 h. A solution of tribenzylfucosyl bromide 10 (obtained from the ethyl 2,3,4-tri-O-benzyl-
1-thio-α- -fucopyranoside (11.4 g, 23.8 mmol) and Br (1.351 ml, 26.18 mmol) in CH Cl (25
-
L
2
2
2
ml)) was then added. The reaction mixture was stirred at rt for 24 h. Methanol (20 ml) was then
added, and the mixture was stirred at rt for 2 h, filtered, concentrated, and coevaporated with
PhCH (3×50 ml). The residue was dissolved in CHCl (100 ml), washed with saturated aqueous
3
3
NaHCO (50 ml) and H O (50 ml); the chloroform fraction was dried by filtration through cotton
3
2
wool and concentrated. Column chromatography on silica gel (350 g, PhCH –acetone, 5→20%)
3
afforded 7.51 g (94%) of trisaccharide 11 and 320 mg (4%) of its β-isomer 12.
Compound 11: colorless foam; [α]546 –60 (с 1, CHCl ); R 0.2 (PhCH –acetone, 9:1) and
3
f
3
1
III
R 0.30 (CHCl –MeOH, 98:2); H NMR (CDCl ):
δ
1.16 (d, J 6.5, 3H, H-6 ), 1.37 and 1.51
f
3
3
6,5
I
(
2s, 2×3H, C(CH ) ), 1.96 (s, 3H, NHC(O)CH ), 3.63 (br. s, 1H, H-4 ), 3.67 (br. d, 1H, J 2.4,
3
2
3
4,3
III
I
II
H-4 ), 3.75 (dd, 1H, J6 5.7, J6a,6b 7.7, H-6a ), 3.77 (dd, 1H, J 5.8, J6a,6b 10.1, H-6a ), 3.80
a,5
6a,5
II
II
(
dd, 1H, J 7.9, J 6.5, H-2 ), 3.81 (dd, 1H, J 6.7, J6b,6a 10.1, H-6b ), 3.88 (dd, 1H, J 10.1,
2,1 2,3 6b,5 3,2
III
I
III
J 2.4, H-3 ), 4.05 (d, 1H, J
7.7, H-6b ), 4.09 (dd, 1H, J 3.8, J 10.1, H-2 ), 4.10–4.13
2,1 2,3
3
,4
6b,6a
I
II
III
II
(
m, 2H, H-2 , H-5 ), 4.16 (q, 1H, 1H, J 6.5, H-5 ), 4,20 (dd, 1H, J 5.7, J 1.3, H-4 ), 4.325
5,6 4,3 4,5
II
I
(
br. t, 1H, J 6.1, J 6.1, H-3 ), 4.38 (br. d), 1H, J 5.6, H-5 ), 4.53 and 4.58 (2d, 2×1H, J
3
,2
3,4
5,6a
gem
a
b
1
1.8, CH Ph ), 4.66 and 4.99 (2d, 2×1H, J 11.6, CH Ph ), 4.74 and 4.86 (2d, 2×1H, J 11.9,
2
gem
2
gem
c
II
d
I
CH Ph ), 4.77 (d, 1H, J 7.9, H-1 ), 4.79 (s, 2H, CH Ph ), 5.44 (br. s, 1H, H-3 ), 5.48 (br. s, 1H,
2
1,2
2
2
4

ACCEPTED MANUSCRIPT
I
III
H-1 ), 5.49 (d, 1H, J 3.8, H-1 ), 6.39 (d, 1H, J
8.5, NHAc), 7.16–8.10 (m, 25H, 5×C H ).
6 5
1
,2
NH,2
+
MALDI MS: m/z Calcd [C H NO ]Na : 1038.4. Found: 1038.3.
58
65
15
Compound 12: colorless foam; [α]546 +11 (с 1, CHCl ); R 0.19 (PhCH –acetone, 9:1)
3
f
3
1
III
and R 0.26 (CHCl –MeOH, 98:2); H NMR (CDCl ):
δ
1.21 (d, J 6.4, 3H, H-6 ), 1.36 and
f
3
3
6,5
I
1
1
1
3
7
4
1
1
.53 (2s, 2×3H, C(CH ) ), 1.91 (s, 3H, NNC(O)CH ), 2.98 (d, 1H, J
8.0, H-6a ), 3.11 (br. s,
3
2
3
6a,6b
I
I
III
H, H-4 ), 3.28 (dd, 1H, J6b,5 5.8, J6b,6a 8.0, H-6b ), 3.49 (dd, 1H, J 9.7, J 2.9, H-3 ), 3.51 (q,
3,2
3,4
III
III
II
H, J 6.4, H-5 ), 3.60 (br. d, 1H, J 2.9, H-4 ), 3.73 (dd, 1H, J 5.5, J6a,6b 10.4, H-6a ),
5
,6
4,3
6a,5
II
III
.75 (dd, 1H, J 8.0, J 7.4, H-2 ), 3.79 (dd, 1H, J 8.0, J 9.7, H-2 ), 3.80 (dd, 1H, J
2
,1
2,3
2,1
2,3
6b,5
II
I
II
.5, J6b,6a 10.4, H-6b ), 3.91 (br. d, 1H, J2,NH 7.6, H-2 ), 4.20 (dd, 1H, J 5.4, J 2.2, H-4 ),
4,3
4,5
II
II
.25 (ddd, 1H, J 2.2, J 5.5, J5,6b 7.5, H-5 ), 4.27 (dd, 1H, J 7.4, J 5.4, H-3 ), 4.37 (br. d),
5
,4
5,6a
3,2
3,4
I
a
H, J5,6a 5.4, H-5 ), 4.51 and 4.57 (2d, 2×1H, Jgem 11.9, CH Ph ), 4.66 and 4.93 (2d, 2×1H, J
2
gem
b
III
c
1.8, CH Ph ), 4.69 (d, 1H, J 8.0, H-1 ), 4.71 and 4.78 (2d, 2×1H, J 10.7, CH Ph ), 4.75
2
1,2
gem
2
d
II
I
and 4.78 (2d, 2×1H, Jgem 10.8, CH Ph ), 4.76 (d, 1H, J 8.0, H-1 ), 5.25 (br. s, 1H, H-3 ), 5.46
2
1,2
I
(
br. s, 1H, H-1 ), 6.39 (d, 1H, J
7.6, NHAc), 7.16–8.10 (m, 25H, 5×C H ). MALDI MS: m/z
NH,2
6
5
+
Calcd [C H NO ]Na : 1038.4. Found: 1038.3.
58
65
15
4
.6 2,3,4-Tri-O-benzyl-α-
L
-fucopyranosyl-(1→2)-6-O-benzyl-β-D-galactopyranosyl-
(1→4)-2-acetamido-1,6-anhydro-3-О-benzoyl-2-deoxy-β-D-glucopyranose (13)
А. A solution of compound 11 (7.50 g, 7.38 mmol) in CH Cl (100 ml) was treated with
2
2
9
5% aqueous CF COOH (10 ml) at rt. After 10 min the mixture was diluted with PhCH (100
3 3
ml), evaporated to 30% of volume, diluted with CHCl (100 ml), washed with saturated aqueous
3
NaHCO (70 ml) and H O (30 ml); the chloroform fraction was dried by filtration through cotton
3
2
wool and concentrated. Column chromatography on silica gel (380 g, PhCH –acetone, 5→15%)
3
afforded 6.34 g (88%) of diol 13 as colorless foam; R 0.50 (PhCH –acetone, 2:1); [α] –67 (с
f
3
546
2
5

ACCEPTED MANUSCRIPT
1
III
1
, CHCl ); H NMR (CDCl ): δ 1.04 (d, 3H, J 6.5, H-6 ), 1.92 (s, 3H, NHC(O)CH ), 2.45 (br.
3 3 6,5 3
II
III
II
s, 1H, OH ), 3.66 (br. d, 1H, J 2.7, H-4 ), 3.66 (dd, 1H, J 7.0, J 9.5, H-2 ), 3.86 (dd, 1H,
6
,5
2,1
2,3
II
I
II
II
II
J 9.5, J 3.0, H-3 ), 3.72–3.77 (m, 4H, H-4 , H-5 , H-6a , H-6b ), 3.78 (dd, 1H, J 5.3,
3
,2
3,4
6a,5
I
II
III
J6a,6b 7.7, H-6a ), 3.93 (br. d, 1H, J 3.0, H-4 ), 3.94 (dd, 1H, J 10.0, J 2.7, H-3 ), 4.02 (q,
4,3
3,2
3,4
III
I
III
1
H, J 6.5, H-5 ), 4.03 (d, 1H, J
7.7, H-6b ), 4.09 (dd, 1H, J 3.6, J 10.0, H-2 ), 4.21
5
,6
6b,6a 2,1 2,3
I
a
(
br. d, 1H, J2,NH 9.0, H-2 ), 4.54 and 4.56 (2d, 2×1H, J 12.1, CH Ph ), 4.61 and 4.98 (2d,
gem 2
b
II
II
2
×1H, Jgem 11.5, CH Ph ), 4.68 (br. s, 1H, OH ), 4.70 (d, 1H, J 7.0, H-1 ), 4.72 and 4.92 (2d,
2 1,2
c
I
2
×1H, Jgem 11.0, CH Ph ), 4.38 (br. d, 1H, J 5.3, H-5 ), 4.79 and 4.81 (2d, 2×1H, J 12.0,
2
5,6a
gem
d
III
I
I
CH Ph ), 5.03 (d, 1H, J 3.6, H-1 ), 5.18 (br. s, 1H, H-3 ), 5.46 (br. s, 1H, H-1 ), 6.78 (d, 1H,
2
1,2
JNH,2 9.0, NHAc), 7.15–7.44 (m, 20H, 4×C H CH ), 7.47 (2t, 2H, J 7.8, m-ArH, 7.61 (t, 1H, J
6
5
2
+
7
.4, p-ArH), 8.04 (2d, 2H, J 7.2, o-ArH). MALDI MS: m/z Calcd [C H NO ]Na : 998.4.
55 61 15
Found: 998.2.
B. Deprotection of trisaccharide 11 without isolation from the reaction mixture
The fucosylation of disaccharide 9 (6.06 g, 10.1 mmol) followed by washing the reaction
mixture was performed as described above. The crude product containing trisaccharide 11 was
disolved in CHCl (150 ml) without purification and treated with 95% aqueous CF COOH (15
3
3
ml) at rt. Standart washing procedures as described above and column chromatography on silica
gel (500 g, PhCH –acetone, 5→15%) gave 8.19 g (83%) of diol 13 and 390 mg (4%) of β-
3
fucoside 43.
Compound 43: colorless crystals, mp 176–178 °С (crystallization from EtOAc–Et O); R
2
f
1
0
.40 (CHCl –MeOH, 95:5) and R 0.18 (PhCH –acetone, 2:1); [α] –4 (с 1, CHCl ); H NMR
3
f
3
546
3
III
I
(CDCl3):
δ
1.24 (d, 3H, J 6.2, H-6 ), 1.91 (s, 3H, NHC(O)CH ), 2.86 (d, 1H, J
8.1, H-6a ),
6,5
3
6a,6b
I
I
3
3
3
.12 (dd, 1H, J6b,5 5.5, J6b,6a 8.2, H-6b ), 3.18 (br. s, 1H, H-4 ), 3.57 (dd, 1H, J 10.1, J 2.8, H-
3,2 3,4
III
III
III
II
III
), 3.58 (q, J 6.2, H-5 ), 3.63 (br. d, 1H, J 2.8, H-4 ), 3.69–3.83 (m, 5Н, H-2 , H-2 , H-
5
,6
4,3
II
II
II
II
I
, H-6a , H-6b ), 3.91 (br. t., 1H, J 6.0, J5,6b 6.0, H-5 ), 3.99 (br. d, 1H, J
8.0, H-2 ), 4.09
5
,6a
2,NH
II
I
III
(
br. s, 1H, H-4 ), 4.24 (br. d, 1H, J 5.2, H-5 ), 4.53 (d, 1H, J 7.8, H-1 ), 4.54 (s, 2H,
5,6a 1,2
2
6

ACCEPTED MANUSCRIPT
a
b
CH Ph ), 4.67 and 4.97 (2d, 2×1H, J 11.7, CH Ph ), 4.75 and 4.81 (2d, 2×1H, J 11.3,
2
gem
2
gem
c
d
II
CH Ph ), 4.78 and 4.81 (2d, 2×1H, J 11.75, CH Ph ), 4.87 (d, 1H, J 6.6, H-1 ), 5.25 (br. s,
2
gem
2
1,2
I
I
1
H, H-3 ), 5.39 (br. s, 1H, H-1 ), 6.40 (d, 1H, J
8.0, NHAc), 6.75–8.05 (м, 25H, 5×C H ).
NH,2 6 5
+
MALDI MS: m/z Calcd [C H NO ]H : 976.4. Found: 976.2.
55
61
15
4
.7 2,3,4-Tri-O-benzyl-α-
L
-fucopyranosyl-(1→2)-4-O-acetyl-6-O-benzyl-β-D-
galactopyranosyl-(1→4)-2-acetamido-1,6-anhydro-3-О-benzoyl-2-deoxy-β-D-glucopyranose
(14)
A mixture of diol 13 (4.16 g, 4.26 mmol), CH C(OEt) (1.60 ml, 8.52 mmol), TsOH⋅H O
3
3
2
(
84 mg, 0.44 mmol), and CH Cl (55 ml) was stirred at rt for 2 h. The mixture containing
2 2
orthoester (R 0.53, PhCH –acetone, 3:1) was treated with 90% aqueous AcOH (20 ml) and after
f
3
1
5 min neutralized with Py (25 ml) and coevaporated with PhCH (4×100 ml). The residue was
3
dissolved in CHCl (100 ml), washed with saturated aqueous NaHCO (50 ml) and H O (50 ml);
3
3
2
the chloroform fraction was dried by filtration through cotton wool and concentrated.
Trisaccharide 14 (4.29 g, 99%) was obtained as yellowish foam; R 0.47 (PhCH –acetone, 3:1);
f
3
1
III
[
α]546 –58 (с 1, CHCl ); H NMR (CDCl ):
δ
1.03 (d, 3H, J 6.5, H-6 ), 1.87, 2.08 (2s, 2×3H,
3
3
6,5
II
OC(O)CH , NHC(O)СН ), 3.51 (dd, 1H, J 6.1, J6a,6b 9.7, H-6a ), 3.55 (dd, 1H, J 6.7, J6b,6a
3
3
6a,5
6b,5
II
II
III
9
.7, H-6b ), 3.63 (dd, 1H, J 7.4, J 9.7, H-2 ), 3.64 (br. d, 1H, J 2.5, H-4 ), 3.76 (br. s, 1H,
2,1 2,3 4,3
I
I
II
H-4 ), 3.79 (dd, 1H, J6 5.8, J6a,6b 7.5, H-6a ), 3.86 (dd, 1H, J 9.7, J 3.1, H-3 ), 3.91 (br. t,
a,5
3,2
3,4
II
III
III
1
4
H, J5,6a 6.5, J5,6b 6.5, H-5 ), 3.93 (dd, 1H, J 10.0, J 2.5, H-3 ), 4.01 (q, 1H, J 6.5, H-5 ),
3,2 3,4 5,6
I
III
.03 (d, 1H, J6b,6a 7.5, H-6b ), 4.06 (dd, 1H, J 3.6, J 10.0, H-2 ), 4.20 (br. d, 1H, J 9.0,
2,NH
2,1
2,3
I
a
II
II
H-2 ), 4.46 and 4.51 (2d, 2×1H, Jgem 1.8, CH Ph ), 4.47 (br. s, 1H, OH ), 4.55 (br. s, 1H, OH ),
2
b
c
4
.61 and 4.98 (2d, 2×1H, Jgem 11.5, CH Ph ), 4.72 and 4.91 (2d, 2×1H, J 12.0, CH Ph ), 4.38
2 gem 2
2
7

ACCEPTED MANUSCRIPT
I
II
(
br. d, 1H, J5,6a 5.3, H-5 ), 4.75 (d, 1H, J 7.0, H-1 ), 4.76 and 4.80 (2d, 2×1H, J 11.7,
1,2 gem
d
III
I
II
CH Ph ), 4.89 (d, 1H, J 3.6, H-1 ), 5.16 (br. s, 1H, H-3 ), 5.45 (br. d, 1Н, J 3.1, H-4 ), 5.47
2
1,2
4,3
I
(
br. s 1H, H-1 ), 6.77 (d, 1H, J
9.0, NHAc), 7.15–7.44 (m, 20H, 4×C H CH ), 7.48 (2t, 2H, J
NH,2
6
5
2
7
.8, m-ArH), 7.62 (t, 1H, J 7.4, p-ArH), 8.05 (2d, 2H, J 7.5, o-ArH). MALDI MS: m/z Calcd
+
[
C H NO ]H : 1018.4. Found: 1018.3.
5
7
63
16
4
.8 2,3,4,6-Tetra-O-benzyl-α-
D
-galactopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-α-
-galactopyranosyl-(1→4)-2-acetamido-1,6-
-glucopyranose (16)
L-
fucopyranosyl-(1→2)]-4-O-acetyl-6-O-benzyl-β-
anhydro-3-О-benzoyl-2-deoxy-β-
D
D
A solution of trisaccharide 14 (2.82 g, 2.77 mmol) and TMU (1.01 ml, 8.37 mmol) in
PhCH –C H Cl (7:1, 135 ml) was stirred with MS 4Å (3g) for 1 h at rt. Then AgOTf (1.45 g,
3
2
4
2
5
.64 mmol) and MS 4Å (2.5 g) were added. The mixture was cooled to –15 °С and freshly
prepared solution of bromide 15 (obtained from the ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-
galactopyranoside [30] (2.75 g, 4.71 mmol) and Br (0.267 ml, 5.18 mmol)) in PhCH (40 ml))
D
-
2
3
was added dropwise with stirring. The reaction mixture was stirred in the darkness at –15 °С for
h and overnight at rt, neutralisated with Et N (5 ml), filtered, and concentrated. Column
1
3
chromatography on silica gel (PhCH –EtOAc, 4:1) gave tetrasaccharide 16 (3.50 g, 82%) R 0.30
3
f
(
PhCH –EtOAc, 3:1), R 0.25 (PhCH –acetone, 8:1); and its β-isomer 17 (256 mg, 6%) R 0.27
3
f
3
f
(
PhCH –EtOAc, 3:1), R 0.23 (PhCH –acetone, 8:1).
3
f
3
1
Compound 16: colorless foam; [α]546 –16 (с 1, CHCl ); H NMR (CDCl ):
δ
0.96 (d, 1H,
3
3
III
J 6.4, H-6 ), 1.79 and 2.80 (2s, 2 × 3H, OC(O)CH , NHC(O)СН ), 3.16 (br. d, 1H, J 2.4, H-
6
,5
3
3
3,4
III
II
II
4
1
), 3.45 (dd, 1H, J6a,5 6.0, J6a,6b 9.8, H-6a ), 3.52 (dd, 1H, J 6.7, J6b,6a 9.8, H-6b ), 3.59 (dd,
6b,5
IV
IV
H, J6a,5 5.2, J6a,6b 8.9, H-6a ), 3.65 (dd, 1H, J 7.5, J6b,6a 8.8, H-6b ), 3.76 (dd, 1H, J 5.8,
6b,5
6a,5
2
8

ACCEPTED MANUSCRIPT
I
I
III
J6a,6b 7.4, H-6a ), 3.80 (br. s, 1H, H-4 ), 3.81 (dd, 1H, J 10.19, J 2.4, H-3 ), 3.87 (br. t, 1H,
2,3
3,4
II
III
III
J5,6a 6.5, J5,6b 6.5, H-5 ), 3.90 (dd, 1H, J 3.8, J 10.1, H-2 ), 3.91 (q, 1Н, J 6.4, H-5 ), 3.93
2,1
2,3
5,6
II
I
IV
(
dd, 1H, J 7.6, J 9.6, H-2 ), 3.99 (d, 1H, J
7.4, H-6b ), 4.02 (br. d, 1H, J 2.7, H-4 ),
2
,1
2,3
6b,6a
3,4
II
IV
4
8
4
.03 (dd, 1H, J 9.6, J 3.2, H-3 ), 4.08 (dd, 1H, J 3.3, J 10.1, H-2 ), 4.14 (br. d, 1H, J
3
,2
3,4
2,1
2,3
2,NH
I
a
IV
.9, H-2 ), 4.20 and 4.24 (2d, 2×1H, Jgem 11.6, CH Ph ), 4.30 (dd, 1H, J 10.1, J 2.8, H-3 ),
2
3,2
3,4
b
c
.31 and 4.72 (2d, 2×1H, Jgem 11.0, CH Ph ), 4.32 and 4.40 (2d, 2×1H, J 10.9, CH Ph ), 4.43
2
gem
2
d
e
and 4.61 (2d, 2×1H, Jgem 11.3, CH Ph ), 4.46 and 4.48 (2d, 2×1H, J 11.8, CH Ph ), 4.51 (br. t,
2
gem
2
IV
f
1
2
H, J5,6a 6.4, J5,6b 6.4, H-5 ), 4.53 and 4.87 (2d, 2×1H, J 11.8, CH Ph ), 4.68 and 4.70 (2d,
gem 2
g
h
×1H, Jgem 11.6, CH Ph ), 4.70 and 4.76 (2d, 2×1H, J 12.1, CH Ph ), 4.71 (br. d, 1H, J 5.7,
2
gem
2
5,6a
I
II
III
I
H-5 ), 4.79 (d, 1H, J 7.5, H-1 ), 5.23 (d, 1H, J 3.8, H-1 ), 5.28 (br. s, 1H, H-3 ), 5.35 (d, 1H,
1
,2
1,2
IV
I
II
J 3.4, H-1 ), 5.44 (br. s, 1H, H-1 ), 5.63 (br. d, 1H, J 2.9, H-4 ), 6.84 (d, 1H, J 8.9,
NH,2
1
,2
3,4
+
NHAc), 7.10–8.10 (m, 45H, 8×C H CH , C H CO). MALDI MS: m/z Calcd [C H NO ]Na :
6
5
2
6
5
91 97
21
1
563.6. Found: 1563.6.
Compound 17: colorless foam; [α]546 –31 (с 1, CHCl ); H NMR (CDCl ):
1
δ
1.10 (d, 1H,
3
3
III
J 6.4, H-6 ), 1.90 and 1.99 (2s, 2 × 3H, OC(O)CH , NHC(O)СН ), 3.20 (dd, 1H, J 9.8, J
6
,5
3
3
3,2
3,4
IV
II
II
2
3
3
.9, H-3 ), 3.30 (br. t, 1H, J5 6.0, J5,6b 6.0, H-5 ), 3.41 (dd, 1H, J 5.6, J6a,6b 9.7, H-6a ),
,6a 6a,5
III
IV
IV
.43 (br. s, 1H, H-4 ), 3.51 (dd, 1H, J 7.5, J6a,6b 10.2, H-6a ), 3.54 (br. d, 1H, J 2.9, H-4 ),
6
a,5
3,4
IV
II
.55 (dd, 1H, J6b,5 4.8, J6b,6a 10.3, H-6b ), 3.59 (dd, 1H, J 6.7, J6b,6a 9.7, H-6b ), 3.68 (dd, 1H,
6b,5
IV
I
I
J 7.6, J 9.8, H-2 ), 3.71 (dd, 1H, J 5.8, J6a,6b 7.7, H-6a ), 3.71 (br. s, 1H, H-4 ), 3.79 (dd,
2
,1
2,3
6a,5
III
III
1
4
H, J 3.4, J 10.3, H-2 ), 3.82 (dd, 1H, J 10.4, J 2.5, H-3 ), 3.88 (dd, 1H, J 7.4, J5,6b
2,1 2,3 2,3 3,4 5,6a
IV
I
III
II
I
.8, H-5 ), 3.99 (d, 1H, J6 7.7, H-6b ), 4.05–4.10 (m, 3Н, H-5 , H-2 , H-2 ), 4.19 (dd, 1H,
b,6a
II
a
J 9.0, J 3.9, H-3 ), 4.39 and 4.49 (2d, 2×1H, J 11.6, CH Ph ), 4.42 and 4.44 (2d, 2×1H,
3
,2
3,4
gem
2
b
I
c
Jgem 12.0, CH Ph ), 4.45 (br. d, 1H, J 5.7, H-5 ), 4.51 and 4.56 (2d, 2×1H, J 12.1, CH Ph ),
2
5,6a
gem
2
d
e
4
.52 and 4.88 (2d, 2×1H, Jgem 11.2, CH Ph ), 4.54 and 4.58 (2d, 2×1H, J 11.9, CH Ph ), 4.55
2 gem 2
f
g
and 4.90 (2d, 2×1H, Jgem 11.4, CH Ph ), 4.63 and 4.68 (2d, 2×1H, J 12.4, CH Ph ), 4.71 and
2
gem
2
h
II
IV
4
.82 (2d, 2×1H, Jgem 10.4, CH Ph ), 4.79 (d, 1H, J 7.5, H-1 ), 5.05 (d, 1H, J 7.6, H-1 ),
2 1,2 1,2
2
9