Synthesis and NMR studies on the ABO histo-blood group antigens: Synthesis of type III and IV structures and NMR characterization of type I-VI antigens

Article abstract of DOI:10.1016/j.carres.2011.03.008

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also

Full text of DOI:10.1016/j.carres.2011.03.008

Carbohydrate Research 346 (2011) 1406–1426
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis
of type III and IV structures and NMR characterization of type I–VI antigens
Peter J. Meloncelli a, Lori J. West b, Todd L. Lowary a,
⇑
a Department of Chemistry and Alberta Ingenuity Centre for Carbohydrate Science, Gunning-Lemieux Chemistry Centre, University of Alberta, Edmonton, AB, Canada T6G 2G2
b Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2E1
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 11 March 2011
The ABO histo-blood group antigens are best known for their important roles in solid organ and bone
marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type
III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to
VI antigens, which were carried out to probe differences in overall conformation of the molecules. These
NMR investigations showed very little difference in the 1H chemical shifts, as well as 1H–1H coupling con-
stants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in
solution.
Dedicated to Professor András Lipták on the
occasion of his 75th birthday
Keywords:
ABO histo-blood group
ABO type III
Ó 2011 Elsevier Ltd. All rights reserved.
ABO type IV
Chemical synthesis
Trichloroacetimidate
7-Octen-1-yl
1. Introduction
(Table 1).4 Of these, type I–IV are considered to be the most impor-
tant.5 These antigens are differentially expressed on erythrocyte
Arguably one of the most important antigen families, the ABO
histo-blood group system has been the focus of much interest since
its discovery by Landsteiner in 1900.1 Despite the plethora of liter-
ature on the ABO antigens, many important questions remain
unanswered. For example, it is still unclear what role the ABO sub-
types I–VI (see below) play, and whether these structural differ-
ences are biologically relevant. One challenge to addressing this
question systematically is that, to the best of our knowledge, no
complete set of all 18 ABO antigen subtypes with a consistent
aglycone is available.
and tissue surfaces.6 For example, type II antigens are expressed
on the human lung vascular endothelial cells while type I, III and
IV moieties are not.7 In bronchial epithelial cells, secretors, individ-
uals who excrete their blood group structures in their bodily fluids
and secretions, express type I, III/IV, but not type II, antigens.7 In
the case of erythrocytes, type I–IV structures are expressed on
the cell surface.8 The expression of different subtypes of ABO epi-
topes is believed to have important biological implications. For
example, individuals who are of the A blood group can be broken
down into two subgroups, A1 and A2. In a study of the differences
between these blood group subtypes, it was shown by Enholm and
co-workers that A1 individuals have a higher antigen density than
those with the A2 blood type.9 Subsequently, Schachter and
co-workers demonstrated that A1 and A2 individuals have two
different N-acetyl-galactosaminyl transferases, termed GTA1 and
GTA2, respectively.10 Later, in a qualitative study, Clausen and co-
workers showed that GTA2 was unable to affect transfer on the H
type III precursor, suggesting that the differential glycosylation of
the H type I–IV antigens by GTA1 and GTA2 results in a greater A
antigen density in A1 individuals.11
The underlying carbohydrate epitopes (1–3, Fig. 1) responsible
for the ABO histo-blood group system were determined by
Watkins and Morgan in 1957.2 This work demonstrated that the
H(O) antigen is a disaccharide consisting of
L-fucose a-(1?2)
linked to -galactose (3, Fig. 1). The A and B blood group structures
D
(2, 3) result from further elaboration of the H antigen. Individuals
with the A and/or B histo-blood group genotype each produce one
additional enzyme, an N-acetyl-galactosaminyltransferase (GTA)
or a galactosyltransferase (GTB);3 these enzymes transfer either
an N-acetyl
D-galactosamine or D-galactose residue to the H
antigen.
Differences between the biological roles of the type I–VI sub-
groups could be the result of conformational changes induced by
the variable monosaccharide residue at the reducing end of the
antigen. Although conformational studies of these antigens have
been carried out, a systematic investigation of all of them has not
been reported; rather the focus has been on a relatively few
The ABO antigens can be further subdivided into six subtypes
(IꢀVI), based on the carbohydrate moiety at the reducing end
⇑
Corresponding author. Tel.: +1 780 492 1861; fax: +1 780 492 7705.
E-mail addresses: tlowary@ualberta.ca, carbres@chem.ualberta.ca (T.L. Lowary).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.008

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1407
HO
OH
HO
OH
O
HO
OH
O
HO
O
HO
O
OH
O
OH
HO
O
HO
O
OR
HO
HO
AcHN
OR
O
OR
O
O
O
OH
O
O
OH
OH
OH
HO
OH
OH
HO
HO
H(O) Antigen
A Antigen
B Antigen
3
1
2
Figure 1. Structures of the A, B and H(O) antigens. R = glycoprotein, glycolipid or free oligosaccharide.
the subtle conformational differences between the H type I trisac-
charide antigen (5) and the H disaccharide structure, 8. An impor-
tant point to note is that in all of the molecules, the fucose ring is
spatially orientated so that H3, H5, and H6 are in close proximity to
the reducing end moiety.
Table 1
Carbohydrate moieties responsible for the six subtypes (I–VI) of
the ABO histo-blood group antigens
Type
Carbohydrate
Type I
ABO-b-(1?3)-b-
ABO-b-(1?4)-b-
ABO-b-(1?3)-a-D-GalpNAc
ABO-b-(1?3)-b-
ABO-b-(1?3)-b-
ABO-b-(1?4)-b-
D
-GlcpNAc
-GlcpNAc
Systematically probing the effect of the non-reducing residue in
these tetrasaccharides on the overall conformation of the antigen
requires access to all 18 structures, ideally attached to the same
aglycone to minimize ambiguities arising from the presence of dif-
ferent groups at the reducing end. Although the synthesis of the
ABO histo-blood group antigens by either chemical and chemoen-
zymatic approaches has been the subject of a number of investiga-
tions, the vast majority of attention has focused on the type I and II
subtypes.15–19 In contrast, much less work has focussed on the
important type III and IV structures. In 1990, Bovin and Khorlin re-
ported the synthesis of the A type III antigen.20 Almost 20 years la-
ter, the same laboratory reported the synthesis of the B type III and
IV antigens.17 Both of these convergent syntheses used a [3+1]
block approach to enable ready access to these structures. In addi-
tion to these chemical synthetic approaches, a chemoenzymatic
synthesis of the A type IV tetrasaccharide was reported by Mazid
and co-workers.19 In this study, a synthetically prepared H type
IV trisaccharide was used as a substrate for a porcine GTA, afford-
ing the target antigen. We report here the synthesis of the ABO
type III and IV histo-blood group antigens (9–14, Fig. 4) attached
to an octen-1-yl aglycone. This work complements earlier work
from our group on the synthesis of the Type I, II, V, and VI
structures.21,22
Type II
Type III
Type IV
Type V
Type VI
D
D
D
D
-GalpNAc
-Galp
-Glcp
antigens. In 1982, Lemieux and co-workers examined the three-
dimensional structure of the blood group B trisaccharide methyl
glycoside (4) using molecular dynamics and NMR spectroscopy.12
Capitalizing on this work, in 1999, Otter and co-workers refined
the structural study, taking advantage of the significant develop-
ments in NMR spectroscopy that had occurred over the nearly
20 year period (Fig. 2).13 In addition, this latter report provided a
structure obtained by X-ray crystallography, which closely
matched the structure obtained from the NMR investigations.
Conformational studies have also been conducted on other
blood group epitopes. Of particular note is a study by Duus and
co-workers on the H type I antigen 5.14 These studies examined
the conformation and NOE interactions of the H type I antigen 5,
compared to the structural variants 6–8 (Fig. 3). In 5, the substitu-
ent at the 2 position of the reducing end is an N-acetyl group, in 6 it
is a hydroxyl group and 7 is the 2-deoxy analogue. In 8, the reduc-
ing end moiety was removed completely providing a disaccharide.
This work showed that in the case of 5, a strong NOE interaction
existed between the H3 of the fucose residue and the reducing
end N-acetyl group. This was supported by a 0.16 ppm upfield shift
of the fucose H3, compared to the H disaccharide 8. In the case of 6
and 7, only a small deviation was observed. This study highlights
2. Results and discussion
To prepare 9–14, we decided to employ a linear chemical syn-
thesis, similar to that used in our previous syntheses of type I, II,
(b)
(c)
(a)
HO
HO
HO
OH
OH
O
O
OH
OH
H
OH
H
H
HO
OH
O
O13Me
H HO
O
H
O
HO
O13Me
O
H
O
HO
H
O
H
H
O
O
OH
OH
H
CH3
OH
HO
OH
4
Figure 2. Crystal structure (b) and important NOE interactions (c) of B trisaccharide histo-blood group antigen 4.13

1408
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
OH
OH
OH
H
H
HO
O
H
OH
OH
O
O
HO
HO
Gal
O
O
HO
OMe
OMe
HN
O
H
HO
O
HO
H
H
H
H
AcHN
O
GlcNAc
Ac
O
O
OH
H
OH
CH3
OH
HO
Fuc
OH
5
5
HO
OH
OH
O
O
HO
OMe
O
HO
OH
O
O
OH
OH
HO
6
HO
HO
OH
OH
O
OH
O
O
HO
OMe
O
HO
HO
OMe
O
O
O
O
OH
OH
OH
OH
HO
HO
7
8
Figure 3. Schematic representation of NOE interactions of 5 and structures 6, 7 and 8.14
V, and VI antigens.21,22 We also selected a 7-octen-1-yl aglycone,
identical that used in our type I/II22 and type V/VI21 syntheses. This
aglycone can be readily converted, by catalytic hydrogenation into
the corresponding octyl glycoside, which is a useful substrate for
enzyme kinetic studies.23 The alkene can also be used to introduce
a reactive linker via a thiol-ene ‘click reaction’.24
Introduction of the
plished using trichloroacetimidate 24 yielding the trisaccharide
25.26 This technique for preparing
-fucopyranosides was crucial
to the synthesis of all the type I–VI antigens. The ability to prepare
these linkages with excellent stereoselectivity and high yields out-
a-L-fucopyranoside residue was accom-
a
-L
weighed the limited disadvantages of the approach. The b-L-fuco-
pyranosyl trichloroacetimidate 24 can be straightforwardly
synthesized in large (10 g) quantitites; the only difficult step is its
isolation in pure form. With due care, chromatographic purification
of a highly activated fucopyranosyl trichloroacetimidate is possible
2.1. Synthesis of type III antigens
The synthesis of the ABO type III antigens (9–11) commenced
from the known trichloroacetimidate 15 (Scheme 1).25 Preparation
of the 7-octen-1-yl glycoside was achieved using standard condi-
as reported.26 The stereochemistry of the
a-L-fucopyranoside resi-
due was confirmed from the 1H NMR spectrum, which displayed
an anomeric signal for this moiety at 5.39 ppm as a doublet with
J = 3.4 Hz.
Removal of the pivaloate ester was achieved under forcing con-
ditions, using lithium methoxide in methanol at reflux. The main
disadvantage of this reaction was that prolonged reaction times
(7 days) were required to ensure complete conversion. This was
similar to what was observed in the syntheses of both the type I
and II22 and type V and VI21 antigens. Despite the length of this
deacylation reaction, the process gave an excellent yield (78%) of
the product 26 and thus alternatives were not explored.
tions to afford 16 as an inseparable mixture of anomers (a:b,
52:48). Deacetylation under Zemplén conditions afforded the triol
17 (87% yield from 15), which was immediately converted into the
benzylidene acetals 18 and 19 in an excellent 98% combined yield.
At this stage it was possible to separate the a- and b-anomers using
silica gel chromatography. The poor stereoselectivity observed in
the glycosylation reaction was fortuitous in some respects as it en-
abled preparation of the reducing end moiety for both the type III
and IV antigens, which differ in the anomeric stereochemistry of
this residue.
Glycosylation of acceptor 18 with trichloroacetimidate 2022
yielded disaccharide 21 (Scheme 2). To facilitate purification, the
product 21 was subjected to Zemplén deactylation to afford the
diol 22 in good yield (88%) and with complete stereoselectivity.
The stereochemistry of the newly formed glycosidic linkage was
confirmed using NMR spectroscopy; the 1H NMR spectrum of 22
showed an anomeric signal at 4.59 ppm as a doublet with a cou-
pling constant of 7.6 Hz, consistent with the b-stereochemistry.
Selective protection of the 30-hydroxyl group of 22 was achieved
by reaction with trimethylacetyl chloride in pyridine exclusively
providing 23 in 94% yield.
Access to the H type III antigen was achieved firstly by conver-
sion of the azide group in 26 into an N-acetyl group upon treat-
ment with thiolacetic acid in pyridine (Scheme 3). Unfortunately,
some acetylation of the alcohol in 27 was also observed; this side
reaction necessitated a second deacetylation step to convert the
O-acetylated product into 27 in 78% yield over the two steps.
Global deprotection of the resulting trisaccharide alcohol was
achieved using Birch reduction, affording 11 in excellent yield
(92%). For our conjugation studies,27 the 7-octen-1-yl aglycone
was desired. However, as discussed above, for studies such as the
examination of GTA and GTB kinetics, the octyl glycoside was

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1409
HO
OH
HO
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
OH
HO
HO
O
O
O
HO
AcHN
O
O
O
O
AcHN
O
AcHN
O
O
O
O
O
OH
OH
OH
OH
HO
HO
10
B Type III
9
A Type III
HO
HO
OH
HO
HO
OH
OH
OH
O
O
O
O
O
HO
O
O
HO
AcHN
O
AcHN
O
O
O
O
OH
OH
OH
OH
HO
HO
12
11
H Type IV
H Type III
HO
HO
OH
OH
O
O
OH
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
HO
HO
O
AcHN
HO
O
O
O
O
O
AcHN
O
AcHN
O
O
O
OH
OH
OH
OH
HO
HO
14
13
A Type IV
B Type IV
Figure 4. Structure of the ABO type III and IV histo-blood group antigens targeted for synthesis.
HO
AcO
AcO
OH
O
AcO
OAc
O
OAc
O
(a)
(b)
HO
O
CCl3
AcO
N3
17
N3
16
O
O
N3
NH
15
Ph
Ph
(c)
O
O
O
O
O
O
HO
O
HO
N3
N3
O
19
18
Scheme 1. Reagents: (a) HO(CH2)6CH@CH2, 4 Å MS, TMSOTf, CH2Cl2, (a:b, 52:48); (b) NaOCH3, CH3OH, 87% (two steps); (c) PhCH(OCH3)2, p-TsOH, DMF, 98%.
desired. Reduction of the alkene was therefore achieved using cat-
alytic hydrogenation to afford a 93% yield of octyl glycoside 28.
The A type III antigen was accessed from the trisaccharide inter-
mediate 26. Glycosylation using the trichloroacetimidate 15 under
standard conditions afforded the near pure tetrasaccharide 29 in
moderate (71%) yield (Scheme 4). To facilitate purification and re-
moval of trichloroacetimidate related impurities (<5% by 1H NMR
spectroscopy) produced during the glycosylation, the two azido

1410
Ph
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
O
O
O
Ph
Ph
AcO
Ph
Ph
AcO
O
O
CCl3
O
O
O
O
O
20
O
O
NH
(a)
O
O
Ph
O
O
O
O
PivO
O
RO
O
N3
HO
N3
RO
O
O
23
21 R = Ac
22 R = H
(b)
O
(c)
HO
N3
NH
O
O
OBn
CCl3
18
O
(d)
OBn
BnO
24
Ph
Ph
Ph
Ph
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
PivO
(e)
N3
N3
O
O
O
O
O
O
OBn
OBn
OBn
OBn
25
26
BnO
BnO
Scheme 2. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 88%; (c) (CH3)3CCOCl, pyridine, 94%; (d) TMSOTf, 4 Å MS, Et2O, 72%; (e) LiOCH3, CH3OH, 78%.
Ph
Ph
Ph
Ph
O
O
O
O
O
O
O
O
O
O
(a)
O
O
O
HO
O
HO
AcHN
O
N3
O
O
O
O
O
OBn
OBn
OBn
OBn
BnO
27
26
BnO
(b)
HO
O
HO
HO
OH
HO
O
HO
OH
OH
OH
O
O
O
O
HO
(c)
AcHN
O
AcHN
O
O
O(CH2)7CH3
O
O
OH
OH
OH
OH
28
11
HO
HO
Scheme 3. Reagents: (a) (i) AcSH, pyridine; (ii) NaOCH3, CH3OH, 78%; (b) Na, NH3, CH3OH, THF, 92%; (c) 10% Pd–C, H2, CH3OH, 93%.

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1411
Ph
Ph
O
O
O
O
O
O
Ph
Ph
O
HO
O
O
AcO
AcO
N3
O
OAc
O
O
O
O
O
OBn
O
O
N3
OBn
O
O
BnO
26
(a)
N3
O
O
O
AcO
AcO
OBn
OAc
O
29
OBn
BnO
O
CCl3
N3
NH
15
(b)
Ph
Ph
O
O
HO
O
RO
OH
O
OR
O
O
OH
OH
OH
OH
HO
RO
O
O
O
O
O
AcHN
AcHN
O
O
O
O
(d)
AcHN
AcHN
O
O
O
O
O
OH
OBn
OH
OBn
HO
9
BnO
30 R = Ac
31 R = H
(c)
Scheme 4. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 71%; (b) AcSH, pyridine, 76%; (c) NaOCH3, CH3OH, 99%; (d) Na, NH3, CH3OH, THF, 96%.
groups in 29 were converted, in 76% yield, into the corresponding
acetamides using thiolacetic acid in pyridine. Confirmation of the
glycosidic linkage stereochemistry was possible at this stage; the
3J1,2 for the terminal GalNAc residue was 3.7 Hz, indicative of the
end acceptor. Not unsurprisingly, therefore, the preparation of the
type IV structures mirrored the approach described above for the
Type III antigens, using a different starting monosaccharide. In
the latter case the 7-octenyl-a-glycoside 18 was used, while in
a
-stereochemistry. Deacetylation of 30 was achieved using stan-
the former case the corresponding b-glycoside (19) was used.
As detailed in Scheme 6, the synthesis of the type IV antigens
commenced from the acceptor 19. Glycosylation using the trichlo-
roacetimidate 2022 afforded the disaccharide 35. To facilitate puri-
fication and isolation, Zemplén deacetylation was conducted to
provide 36 in 76% yield over the two steps. Protection of the
3-OH group of the galactose ring at the non-reducing end of the
molecule using trimethylacetyl chloride provided 37 in excellent
dard Zemplén conditions to afford 31. Final global deprotection
was achieved using a dissolving metal reduction to provide the A
type III antigen 9 in 95% overall yield from 30.
Access to the B type III antigen commenced from the trisaccha-
ride intermediate 26. Treatment of 26 with the galactosyl trichlo-
roacetimidate 3228 using TMSOTf as the promoter afforded the
tetrasaccharide 33 (Scheme 5). As was the case with the prepara-
tion of 29, trace amounts of trichloroacetimidate related byprod-
ucts (<5% by 1H NMR spectroscopy) hampered obtaining 33 in
pure form. A small amount of the acceptor was isolated as the
TMS ether, which explains the modest yield (68%) of this glycosyl-
ation. To facilitate the purification, the azide moiety was converted
into an N-acetyl group, enabling the isolation of 34 in high purity,
but in moderate yield over the two steps (52%). Complete debenzy-
lation and removal of the benzylidene acetals in 34 using Birch
reduction afforded 10 in excellent yield (92%).
yield (98%). Installation of the
a-L-fucopyranoside moiety was
achieved in 75% yield using activation of the glycosyl donor 24
with TMSOTf, described initially by Gerhard and Schmidt.26
Removal of the pivaloate ester from 38 was achieved, as it was in
the case of the Type III structures, by heating in lithium methoxide
in methanol at reflux to provide 39 in 80% yield.
Access to both 7-octen-1-yl and octyl glycoside analogues of the
H type IV antigen first required conversion of the azide in 39 to an
N-acetyl group (Scheme 7). Thiolacetic acid in pyridine was again
used to affect this conversion. As was the case in the H type III anti-
gen synthesis, some acetylation of the galactopyranose 3-OH group
was observed, but this problem could be remedied using a
Zemplén deacetylation step to afford 40 in 60% yield over the
two steps. The lower yield was due to difficulties in forcing the
2.2. Synthesis of type IV antigens
The type IV antigens are very similar to the type III structures,
the only difference is in the anomeric configuration of the reducing

1412
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
Ph
Ph
O
O
O
O
Ph
Ph
O
O
O
BnO
OBn
O
O
HO
O
O
O
N3
O
O
BnO
O
O
O
BnO
OBn
O
O
(a)
OBn
N3
O
26
BnO
O
O
OBn
OBn
BnO
OBn
33
OBn
O
O
CCl3
BnO
OBn
NH
(b)
32
Ph
Ph
HO
O
BnO
BnO
OH
O
O
OBn
O
O
O
OH
OH
OH
OH
O
HO
O
O
O
HO
BnO
O
O
O
O
(c)
AcHN
O
AcHN
O
O
O
O
O
OH
OBn
OH
OBn
HO
BnO
10
34
Scheme 5. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 68%; (b) AcSH, pyridine, 76%; (c) Na, NH3, CH3OH, THF, 92%.
reaction to completion; unreacted starting material (28%) was
recovered and the structure confirmed by 1H NMR spectroscopy.
Complete deprotection using a dissolving metal reduction pro-
vided (87% yield) the H type IV antigen with the 7-octen-1-yl agly-
cone required for conjugation to a linker. Catalytic reduction of the
alkene in 12 resulted in the successful preparation of the octyl gly-
coside 41.
Using intermediate 39, glycosylation with trichloroacetimidate
15 provided the near pure tetrasaccharide 42 (Scheme 8) in 79%
yield. Again, as was the case in the A type III synthesis, contamina-
tion with trichloroacetimidate related impurities (<5% by 1H NMR
spectroscopy) was observed. Consequently, treatment of 42 with
thiolacetic acid in pyridine resulted in conversion of the two azide
groups into the expected acetamides and the preparation, in 83%
yield, of pure 43. Exhaustive deprotection, firstly by Zemplén
deacetylation and secondly by Birch reduction provided the A type
IV antigen 13 in 84% yield. At this stage the stereochemistry of the
newly formed glycosidic linkage could be confirmed using 1H NMR
spectroscopy; the anomeric signal appeared as a doublet at
5.15 ppm with a coupling constant of 3.7 Hz.
2.3. NMR analysis of ABO type I–VI antigens
With a collection of all 18 ABO type I–VI antigens in hand, pro-
vided as described above, or through our previous work,21,22 our
attention shifted to NMR studies aimed at assessing any conforma-
tional differences between the subtypes. As an initial gauge of dif-
ferences or similarities, 1H chemical shifts and 1H–1H coupling
constants were measured on all 18 compounds under identical
conditions. One of the main complications in carrying out these
analyses was the significant spectral overlap in the 1H NMR spec-
tra. To overcome this problem a gradient-enhanced chemical shift
selective filtering (ge-CSSF) technique was used. In previous stud-
ies, 1D-ge-CSSF-TOCSY experiments have been shown29,30 to be
effective in assigning the coupling constants and chemical shifts
for the type I and II antigens,22 as well as other oligosaccha-
rides.31–33 A sample 1D-ge-CSSF-TOCSY is included in Supplemen-
tary data. This technique is essentially a 1D version of the standard
2D-TOCSY, with each carbohydrate ring presented as a single 1D
1H NMR spectrum. However, significantly better resolution can
be obtained. The main limitation of this technique is the same as
Access to the B type IV antigen was achieved from the trisaccha-
ride 39. Glycosylation with the trichloroacetimidate 32 afforded
the tetrasaccharide 45 (Scheme 9), which was contaminated with
trichloroacetimidate related impurities (<5% by 1H NMR spectros-
copy). Instead, conversion of the azide in the partially purified
product into an N-acetyl group was required to enable isolation
of the pure tetrasaccharide in 78% yield from 39. The anomeric sig-
that of a traditional TOCSY experiment: the signal transfer
throughout each ring is dependent upon the size of the coupling
constants. In systems such as glucose, this is typically not a prob-
lem. Galactose and fucose, on the other hand, when irradiated at
3
H1, suffer poor signal transfer from H3 to H4 (due to JH3,H4
<3 Hz). In most cases this problem could be partially circumvented
by irradiating at other proton signals (H4, H5 or H6).
nal of the newly formed
blet at 5.33 ppm with
deprotection of the benzyl ethers and benzylidene acetals using a
Birch reduction afforded 14 in excellent (94%) yield.
a
-
a
D
-galactopyranoside appeared as a dou-
coupling constant of 3.6 Hz. Final
The chemical shift and coupling constant data are presented in
three sets of two tables (Tables 2–7). The first table of each set (Ta-
bles, 2, 4 and 6) details the 1H chemical shift and multiplicity for
each of the antigens; the second table of each set (Tables 3, 5

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1413
Ph
O
O
O
Ph
AcO
Ph
Ph
Ph
AcO
O
CCl3
O
O
O
O
O
O
O
O
NH
20
(a)
O
O
O
O
Ph
O
O
O
PivO
O
RO
O
N3
HO
N3
RO
(b)
37
O
35 R = Ac
36 R = H
O
NH
(c)
O
HO
N3
O
OBn
CCl3
O
(d)
19
OBn
BnO
Ph
24
Ph
Ph
Ph
O
O
O
O
O
O
O
O
O
O
O
O
O
(e)
O
O
O
PivO
O
HO
N3
O
N3
O
O
OBn
OBn
OBn
OBn
38
39
BnO
BnO
Scheme 6. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 76%; (c) (CH3)3CCOCl, pyridine, 98%; (d) TMSOTf, 4 Å MS, Et2O, 75%; (e) LiOCH3, CH3OH, 80%.
Ph
Ph
Ph
Ph
O
O
O
O
O
O
O
O
O
O
O
(a)
O
O
O
HO
O
O
HO
AcHN
O
N3
O
O
O
OBn
OBn
OBn
OBn
39
40
BnO
BnO
(b)
HO
HO
HO
HO
HO
OH
OH
OH
OH
O
(c)
O
O
O
O
O(CH2)7CH3
O
O
HO
AcHN
AcHN
O
O
O
O
OH
OH
41
Scheme 7. Reagents: (a) (i) AcSH, pyridine; (ii) NaOCH3, CH3OH, 60%; (b) Na, NH3, CH3OH, THF, 87%; (c) Pd–C, H2, CH3OH, 73%.
OH
OH
HO
HO
12
and 7) details the 1H–1H coupling constants. Figure 6 details the
nomenclature system used for the NMR spectroscopic data, using
tetrasaccharide 14 as an example. The work by Duus and
co-workers, described above, showed that the H3, H5 and H6
signals of the fucose on the H type I antigen were the most prone
to variation, albeit small.14 In the case of the A and B antigens

1414
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
Ph
Ph
O
O
Ph
Ph
O
O
O
O
AcO
OAc
O
O
O
O
O
O
O
HO
AcO
N3
O
O
O
N3
O
(a)
O
O
O
N3
O
OBn
OBn
O
BnO
39
OBn
42
OBn
BnO
AcO
AcO
OAc
O
O
CCl3
N3
NH
(b)
15
Ph
Ph
O
HO
O
RO
RO
OH
O
OR
O
O
O
OH
OH
O
OH
OH
HO
O
O
O
(d)
O
AcHN
AcHN
O
O
O
O
O
AcHN
O
AcHN
O
O
O
OH
13
OBn
OH
OBn
HO
BnO
43 R = Ac
44 R = H
(c)
Scheme 8. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 79%; (b) AcSH, pyridine, 83%; (c) NaOCH3, CH3OH, 92%; (d) Na, NH3, CH3OH, THF, 91%.
studied here (Tables 2–4), there is very little variation in both the
chemical shifts and coupling constants. The only significant varia-
tion observed was in the chemical shifts of the A and B type V anti-
gens. The chemical shift of the H5 proton of the fucose residue of A
type V resonated at 4.65 ppm, a deviation of 0.33 ppm from the
4.32 ppm average (Table 2). A similar observation was made of
the fucose H5 in the B type antigen series (Table 4). The H3 and
H6 protons of the fucose residue also showed some minor devia-
tion in the case of the A and B type V antigens, although this was
far less pronounced than was the case with the H5 proton. In the
H antigen series, very little deviation across the subtypes was
observed, even in the fucose H3, H5 and H6 signals. As shown in
Tables 3, 5 and 7, the 1H–1H coupling constants across all antigen
subtypes were also nearly identical.
In addition to the 1D-ge-CSSF-TOCSY experiments, we also con-
ducted 2D TROESY experiments on both the A type IV 13 and A
type V 47 antigens. The A type V antigen was chosen as it showed
the most significant deviation in 1H NMR chemical shifts in the
previous experiments. The A type IV antigen was chosen as a point
of comparison. Not surprisingly, the vast majority of the interac-
tions were identical in both the A type IV and the A type V antigens.
The only difference is that in the case of the A type IV antigen a
strong ROSEY interaction between H10 and H5 was observed. A
summary of the main interactions is shown in Figure 5, more de-
tailed ROSEY data are included in Supplementary data.
limited number of exceptions, the reducing end moiety in these
antigen subtypes does not appear to influence significantly the
conformation of the canonical A, B and O(H) antigens. Therefore,
it does not appear that differential recognition of these structures
by various proteins or receptors, such as the different catalytic
activities of GTA1 and GTA2 observed by Clausen and co-workers,11
arises from conformational differences.
2.4. Conclusions
In summary, we report the linear synthesis of the ABO type III
and IV histo-blood group antigens with a 7-octen-1-yl linker. The
syntheses described here complete the goal of preparing all 18
known ABO antigens attached to a common aglycone.21,22 The high
yields and stereoselectivity observed with the trichloroacetimidate
glycosylations ensured each antigen was prepared in an efficient
manner. Of particular note was the glycosylation using the
b-
by Gerhard and Schmidt,26 which was used to stereoselectively
introduce the -fucopyranosyl moiety in 25 and 38. The use of
L-fucopyranosyl trichloroacetimidate 24, originally developed
a
-L
an azido precursor for the amino group enabled the later conver-
sion to an N-acetyl group using mild conditions. In addition, Birch
reduction was effectively used on several occasions to remove all
of the benzyl ethers and benzylidene acetals without any reduction
of the 7-octen-1-yl aglycone. Finally, NMR analysis was conducted
on all 18 ABO antigens. The 1H chemical shift and 1H–1H coupling
constant data obtained indicate that residue at the reducing end of
The conclusion that can be drawn from the chemical shift and
coupling constant data reported in Tables 2–7 is that, with a very

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1415
Ph
Ph
O
O
O
O
O
O
Ph
Ph
O
O
HO
O
BnO
BnO
O
N3
O
OBn
O
O
O
O
OBn
O
O
(a)
BnO
OBn
O
O
O
39
BnO
N3
O
O
OBn
45
BnO
BnO
OBn
O
OBn
BnO
O
CCl3
OBn
NH
(b)
32
Ph
Ph
HO
HO
O
BnO
BnO
O
OH
O
OBn
O
O
O
OH
OH
OH
OH
O
O
O
O
HO
BnO
O
O
O
O
O
O
(c)
AcHN
O
AcHN
O
O
O
OH
OBn
OH
OBn
HO
BnO
46
14
Scheme 9. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 86%; (b) AcSH, pyridine, 91%; (c) Na, NH3, CH3OH, THF, 94%.
the molecule influence the global conformation of the molecule
very little. One exception is the chemical shift for H5 of the fucose
residue of the A type V tetrasaccharide, which resonated around
4.65 ppm, a deviation of 0.33 ppm from the 4.32 ppm average.
The significance of this observation is currently unclear.
identify individual proton resonances. Samples for 1D-ge-CSSF-
TOCSY experiments were prepared in CD3OD at a concentration
of 21 mg/mL. Spectra were obtained using CD3OD as the solvent
as spectra obtained using D2O exhibited significant line broadening
that interfered with the selective irradiation of the desired proton.
On average, each of the 18 antigens required approximately 8 h of
instrument time on a Varian Direct Drive three channel 600 MHz
spectrometer. 13C NMR (APT) spectra were recorded at 125 or
100 MHz, and 13C chemical shifts were referenced to the peak for
internal CDCl3 (77.1 ppm, CDCl3) or CD3OD (49.0, CD3OD). All spec-
tra were recorded in CDCl3 unless specified otherwise. Melting
points were measured using a PerkinElmer Thermal Analysis. Elec-
trospray mass spectra were recorded on samples suspended in
mixtures of THF with CH3OH and added NaCl.
3. Experimental
All reagents were purchased from commercial sources and were
used without further purification, unless otherwise stated. Reac-
tion solvents were purchased and were used without purification;
dry solvents were purified by successive passage through columns
of alumina and copper under nitrogen. All reactions were carried
out at room temperature under a positive pressure of argon, unless
otherwise stated. Thin layer chromatography (TLC) was performed
on Merck Silica Gel 60 F254 aluminum-backed plates that were
stained by heating (>200°) with either p-anisaldehyde in 5% sulfu-
ric acid in EtOH or 10% ammonium molybdate in 10% sulfuric acid.
Unless otherwise indicated, all column chromatography was per-
3.1. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-deoxy-
a-
D-galactopyranosyl)-2-O-(
a-L-fucopyranosyl)-b-D-galactopyr-
anosyl)-2-deoxy- -galactopyranoside (9)
a
-D
A solution of the tetrasaccharide 30 (400 mg, 0.283 mmol) in
CH3OH (20 mL) was treated with a catalytic amount of NaOCH3
in CH3OH and the solution was stirred for 2 h. The solution was
then neutralized with Amberlite IR 120 (H+), filtered and the resi-
due subjected to flash chromatography (CH2Cl2–CH3OH 10:1) to af-
ford 31 (360 mg, 99%) as a colorless oil. Redistilled liquid ammonia
(20 mL) was collected in a flask cooled to (ꢀ78 °C) and treated with
sodium until the blue color persisted. A solution of the tetrasaccha-
formed on Silica Gel 60 (40–60 lM). C18 silica gel (35–70 lM)
was manufactured by Toronto Research Chemicals. Optical rota-
tions were measured at 589 nm, at 22 2 °C and are in units of
deg dmꢀ1 cm3 gꢀ1, in all cases the concentrations are in the units
g/100 mL.
1H NMR spectra were recorded at 400 and 500 MHz, chemical
shifts were referenced to the peak for TMS (0.0 ppm, CDCl3) or
CD3OD (3.30 ppm, CD3OD). For final compounds, NMR analysis
was conducted using 1D-ge-CSSF-TOCSY29,30 at 600 MHz, to
ride 31 (271 mg, 0.210 mmol) in THF (4 mL) and CH3OH (42
lL,
1.05 mmol) was added dropwise and the mixture was stirred

1416
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
Table 2
1H Assignments (ppm) of the A type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
Proton
A Type I
A Type II
A Type III
A Type IV
A Type V
A Type VI
H1000
H2000
H3000
H4000
H5000
H6000
H6000
5.15 (d)
5.15 (d)
5.13 (d)
4.31 (dd)
3.87 (dd)
3.90 (d)
4.27–4.24 (m)
3.74–3.67 (m)
3.74–3.67 (m)
5.15 (d)
4.30 (dd)
3.84 (dd)
3.88 (d)
4.21–4.17 (m)
3.68 (dd)
3.79–3.72 (m)
5.15 (d)
5.15 (d)
4.29 (dd)
3.85 (dd)
3.87 (d)
4.20 (dd)
3.75 (dd)
3.68 (dd)
4.32 (dd)
3.82 (dd)
3.89 (d)
4.16 (dd)
3.76 (dd)
3.68 (dd)
4.32 (dd)
3.83 (dd)
3.90 (d)
4.17 (dd)
3.75 (dd)
3.69 (dd)
4.32 (dd)
3.82 (dd)
3.89 (d)
4.16 (dd)
3.76 (dd)
3.69 (dd)
H100
H200
H300
H400
H500
H600
5.23 (s)
3.69 (dd)
3.65 (dd)
3.72 (d)
4.36 (q)
1.19 (d)
5.33 (d)
3.72 (dd)
3.68 (dd)
3.69 (d)
4.32 (q)
1.21 (d)
5.20 (d)
3.73 (dd)
3.63 (dd)
3.63 (d)
4.31 (q)
1.23 (d)
5.26 (d)
3.70 (dd)
3.59 (dd)
3.73 (d)
4.32 (q)
1.22 (d)
5.29 (d)
3.71 (dd)
3.81 (dd)
3.71 (d)
4.65 (q)
1.13 (d)
5.34 (d)
3.73 (dd)
3.67 (dd)
3.69 (d)
4.31 (q)
1.21 (d)
H10
H20
H30
H40
H50
H60
H60
4.57 (d)
3.91 (dd)
3.86 (dd)
4.09 (d)
3.49 (dd)
3.77–3.73 (m)
3.66–3.65 (m)
4.52 (d)
3.99 (dd)
3.90 (dd)
4.09 (d)
3.29–3.25 (m)
3.81–3.77 (m)
3.72–3.66 (m)
4.64 (d)
3.91 (dd)
3.87 (dd)
4.09 (d)
3.48 (dd)
3.73 (dd)
3.69 (d)
4.56 (d)
3.98 (dd)
3.87 (dd)
4.10 (d)
3.51 (dd)
3.78–3.71 (m)
3.78–3.71 (m)
4.66 (d)
4.51 (d)
4.01 (dd)
3.90 (dd)
4.11 (d)
3.48 (dd)
3.73 (dd)
3.69 (dd)
4.00 (dd)
3.91 (dd)
4.11 (d)
3.53 (dd)
3.76 (dd)
3.67 (dd)
H1
H2
H3
H4
H5
H6
H6
4.25–4.20 (m)
3.86–3.79 (m)
3.86–3.79 (m)
3.41–3.37 (m)
3.32–3.28 (m)
3.78–3.74 (m)
3.69–3.66 (m)
4.39 (d)
3.71 (dd)
3.69 (dd)
3.59 (dd)
3.28 (dd)
3.90 (d)
3.78 (d)
4.89 (d)
4.25 (dd)
3.97 (dd)
4.19 (d)
3.84 (dd)
3.74–3.67 (m)
3.74–3.67 (m)
4.19 (d)
4.23–4.20 (m)
3.61–3.54 (m)
3.61–3.54 (m)
4.11 (s)
3.54–3.50 (m)
3.75–3.71 (m)
3.75–3.71 (m)
4.26 (d)
4.07 (dd)
3.79–3.72 (m)
4.10 (d)
3.46 (dd)
3.73 (dd)
3.69 (dd)
3.25 (dd)
3.45 (dd)
3.65 (dd)
3.28 (ddd)
3.89 (dd)
3.75 (dd)
CH2O
CH2O
3.91–3.87 (m)
3.44–3.40 (m)
4.99–4.95 (m)
4.91–4.89 (m)
5.84–5.76 (dddd)
2.08–2.01 (m)
1.57–1.47 (m)
1.42–1.26 (m)
1.99 (s)
3.86–3.83 (m)
3.45–3.41 (dt)
5.00–4.94 (m)
4.92–4.88 (m)
5.84–5.76 (dddd)
2.07–2.01 (m)
1.58–1.48 (m)
1.42–1.27 (m)
2.00 (s)
3.74–3.66 (m)
3.37–3.32 (dt)
5.00–4.95 (m)
4.92–4.88 (m)
5.84–5.76 (dddd)
2.07–2.02 (m)
1.60–1.53 (m)
1.44–1.29 (m)
1.99 (s)
3.90–3.83 (m)
3.45–3.38 (m)
4.99–4.94 (m)
4.92–4.88 (m)
5.80 (dddd)
2.06–2.01 (m)
1.58–1.47 (m)
1.42–1.27 (m)
2.00 (s)
3.89–3.86 (m)
3.57–3.53 (m)
5.00–4.95 (m)
4.93–4.88 (m)
5.85–5.76 (dddd)
2.07–2.01 (m)
1.65–1.58 (m)
1.43–1.29 (m)
2.00 (s)
3.56–3.51 (m)
3.91–3.85 (m)
5.00–4.95 (m)
4.92–4.88 (m)
5.85–5.76 (dddd)
2.08–2.01 (m)
1.65–1.57 (m)
1.43–1.29 (m)
2.00 (s)
CH@CH2
CH@CH2
CH@CH2
(CH2)5
(CH2)5
(CH2)5
CH3CO
CH3CO
1.99 (s)
1.96 (s)
1.98 (s)
1.98 (s)
Table 3
2JH,H 3JH,H (Hz) of the A type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
,
Coupling
A Type I
A Type II
A Type III
A Type IV
A Type V
A Type VI
3JH-1
3.8
10.8
3.4
0.0
7.6
3.7
11.0
3.0
0.0
7.2
3.7
10.8
3.2
0.0
m
3.7
10.9
3.2
0.0
4.2
m
3.7
11.0
3.1
0.0
7.4
3.6
11.0
3.0
0.0
7.4
000 ,H-2000
3JH-2
000 ,H-3000
3JH-3
000 ,H-4000
3JH-4
000 ,H-5000
3JH-5
000 ,H-6a000
3JH-5
4.1
11.7
4.5
11.5
m
m
4.5
11.6
4.3
11.5
000 ,H-6b000
2JH-6a
11.6
000 ,H-6b000
3JH-1
3.9
10.5
3.1
3.9
9.5
3.3
6.6
4.0
9.8
3.5
6.6
4.1
10.2
3.2
3.9
10.2
3.2
3.9
9.9
3.1
6.6
00 ,H-200
3JH-2
00 ,H-300
3JH-3
00 ,H-400
3JH-5
6.5
6.6
6.4
00 ,H-600
3
0
0
0
0
JH-1 ,H-2
7.3
9.9
3.1
7.9
4.3
m
7.7
9.8
3.0
m
m
m
7.2
9.6
2.9
6.8
5.6
11.2
7.5
9.9
3.1
6.0
6.0
m
7.7
9.8
3.0
7.0
5.1
11.4
7.7
9.8
2.9
7.1
4.9
11.3
3
3
3
3
2
0
JH-2 ,H-3
JH-3 ,H-4
JH-5 ,H-6a
JH-5 ,H-6b
0
0
0
0
0
0
0
JH-6a ,H-6b
3JH-1,H-2
3JH-2,H-3
3JH-3,H-4
3JH-4,H-5
3JH-5,H-6a
3JH-5,H-6b
2JH-6a,H-6b
m
m
m
m
m
m
m
8.4
9.3
8.9
9.8
4.9
0.0
11.2
3.6
11.2
3.1
0.0
6.1
6.1
m
8.4
10.8
3.1
0.0
6.0
6.0
m
m
m
m
m
m
m
m
7.8
8.5
9.1
9.4
4.9
1.1
11.9
CH2O
m
9.7, 6.6
10.0, 6.4
m
m
m
CH@CH2
17.1, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1417
Table 4
1H Assignments (ppm) of the B type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
Proton
B Type I
B Type II
B Type III
B Type IV
B Type V
B Type VI
H1000
H2000
H3000
H4000
H5000
H6000
H6000
5.14 (d)
5.15 (d)
3.84 (dd)
3.78 (dd)
3.9 (d)
4.14–4.10 (m)
3.76–3.71 (m)
3.70–3.66 (m)
5.14 (d)
5.14 (d)
3.84 (dd)
3.80 (dd)
3.89 (d)
4.16 (dd)
3.75–3.71 (m)
3.70–3.66 (m)
5.15 (d)
5.15 (d)
3.85 (dd)
3.81 (dd)
3.90 (d)
4.14–4.10 (m)
3.75–3.72 (m)
3.70–3.67 (m)
3.83 (dd)
3.81 (dd)
3.89 (dd)
4.16 (dd)
3.73 (dd)
3.68 (dd)
3.86–3.80 (m)
3.86–3.80 (m)
3.91 (d)
4.23 (dd)
3.76–3.71 (m)
3.71–3.67 (m)
3.85 (dd)
3.80 (dd)
3.91 (d)
4.16 (dd)
3.73 (dd)
3.69 (dd)
H100
H200
H300
H400
H500
H600
5.21 (d)
3.7 (dd)
3.65 (dd)
3.71 (d)
4.45 (q)
1.18 (d)
5.32 (m)
3.73 (m)
3.69–3.66 (m)
3.69–3.66 (m)
4.29 (q)
5.20 (d)
3.71 (dd)
3.63 (dd)
3.61 (d)
4.29 (q)
1.22 (d)
5.25 (d)
3.71 (dd)
3.58 (dd)
3.72 (d)
4.31 (q)
1.21 (d)
5.29 (d)
3.72 (dd)
3.80 (dd)
3.71 (d)
4.61 (q)
1.20 (d)
5.33 (d)
3.73 (dd)
3.69–3.66 (m)
3.69–3.66 (m)
4.29 (q)
1.20 (d)
1.19 (d)
H10
H20
H30
H40
H50
H60
H60
4.60–4.56 (m)
3.93–3.87 (m)
3.93–3.87 (m)
4.10 (s)
3.54 (dd)
3.79 (dd)
4.53 (d)
4.63 (d)
4.57 (d)
3.98 (dd)
3.91 (dd)
4.11 (d)
3.53–3.48 (m)
3.77–3.71 (m)
3.77–3.71 (m)
4.66 (d)
4.01 (dd)
3.93 (dd)
4.13 (d)
3.53 (m)
3.74–3.65 (m)
3.74–3.65 (m)
4.52 (d)
3.98 (dd)
3.94 (dd)
4.13 (d)
3.58–3.54 (m)
3.72–3.65 (m)
3.84–3.75 (m)
3.98 (dd)
3.94 (dd)
4.12 (d)
3.60–3.54 (m)
3.79 (dd)
3.67 (dd)
3.93–3.86 (m)
3.93–3.86 (m)
4.10 (d)
3.53 (dd)
3.75–3.53 (m)
3.75–3.53 (m)
3.68 (dd)
H1
H2
H3
H4
H5
H6
H6
4.25–4.19 (m)
3.86–3.77 (m)
3.86–3.77 (m)
3.41–3.37 (m)
3.31–3.28 (m)
3.93–3.83 (m)
3.76–3.64 (m)
4.36 (d)
4.87 (d)
4.24 (dd)
3.95 (dd)
4.18 (d)
3.84 (dd)
3.72–3.69 (m)
3.72–3.69 (m)
4.19 (d)
4.07 (dd)
3.77 (dd)
4.08 (d)
3.53–3.48 (m)
3.77–3.71 (m)
3.77–3.71 (m)
4.23–4.20 (m)
3.61–3.55 (m)
3.61–3.55 (m)
4.12 (s)
3.54–3.51 (m)
3.74–3.65 (m)
3.74–3.65 (m)
4.26 (d)
3.70 (dd)
3.59 (dd)
3.66 (dd)
3.30–3.26 (m)
3.90 (dd)
3.77 (dd)
3.25 (dd)
3.46 (dd)
3.65 (dd)
3.28 (ddd)
3.89 (dd)
3.76 (dd)
CH2O
CH2O
3.92–3.83 (m)
3.46–3.37 (m)
4.99–4.95 (m)
4.92–4.89 (m)
5.84–5.75 (dddd)
2.07–2.01 (m)
1.57–1.46 (m)
1.42–1.27 (m)
1.99 (s)
3.91–3.83 (m)
3.45 (dt)
3.74–3.66 (m)
3.35 (dt)
3.90–3.83 (m)
3.42 (dt)
3.91–3.85 (m)
3.59–3.51 (m)
5.00–4.95 (m)
4.93–4.88 (m)
5.84–5.76 (dddd)
2.07–2.01 (m)
1.65–1.59 (m)
1.43–1.31 (m)
2.00 (s)
3.92–3.85 (m)
3.57–3.51 (m)
4.99–4.94 (m)
4.92–4.87 (m)
5.84–5.76 (dddd)
2.08–1.99 (m)
1.66–1.57 (m)
1.43–1.24 (m)
CH@CH2
CH@CH2
CH@CH2
(CH2)5
(CH2)5
(CH2)5
CH3CO
CH3CO
5.00–4.95 (m)
4.93–4.88 (m)
5.84–5.76 (dddd)
2.08–2.01 (m)
1.58–1.49 (m)
1.43–1.25 (m)
1.95 (s)
4.99–4.95 (m)
4.92–4.89 (m)
5.80 (dddd)
2.07–2.01 (m)
1.60–1.54 (m)
1.45–1.27 (m)
1.99 (s)
5.00–4.94 (m)
4.92–4.88 (m)
5.80 (dddd)
2.07–2.01 (m)
1.59–1.46 (m)
1.43–1.27 (m)
1.99 (s)
Table 5
2JH,H 3JH,H (Hz) of the B type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
,
Coupling
B Type I
B Type II
B Type III
B Type IV
B Type V
B Type VI
3JH-1
3.5
10.2
3.1
0.8
7.3
3.8
10.1
2.9
0.0
m
2.8
m
3.6
10.1
3.0
0.0
6.7
4.6
m
3.7
10.1
3.1
0.0
7.1
3.7
10.1
3.1
0.0
m
000 ,H-2000
3JH-2
000 ,H-3000
3JH-3
1.3
0.0
6.7
5.7
m
000 ,H-4000
3JH-4
000 ,H-5000
3JH-5
000 ,H-6a000
3JH-5
4.3
11.4
m
m
4.6
11.4
m
m
000 ,H-6b000
2JH-6a
000 ,H-6b000
3JH-1
4.1
9.3
3.3
6.6
3.9
9.9
m
4.0
9.4
3.2
6.5
4.1
10.2
3.0
3.9
10.1
3.2
4.0
10.7
m
00 ,H-200
3JH-2
00 ,H-300
3JH-3
00 ,H-400
3JH-5
6.6
6.5
6.6
6.6
00 ,H-600
3
0
0
0
0
JH-1 ,H-2
m
m
0.0
7.8
4.4
11.4
7.4
9.7
2.7
7.3
3.9
11.6
6.7
m
2.0
6.5
5.3
m
7.6
9.6
2.8
m
m
m
7.6
9.6
2.9
m
m
m
7.7
9.7
2.9
m
m
m
3
3
3
3
2
0
JH-2 ,H-3
JH-3 ,H-4
JH-5 ,H-6a
JH-5 ,H-6b
0
0
0
0
0
0
0
JH-6a ,H-6b
3JH-1,H-2
3JH-2,H-3
3JH-3,H-4
3JH-4,H-5
3JH-5,H-6a
3JH-5,H-6b
2JH-6a,H-6b
m
m
m
m
m
m
m
8.4
10.4
8.8
9.2
5.0
3.7
11.1
3.0
0.0
5.5
5.5
m
8.4
10.8
3.0
0.0
m
m
m
m
m
m
m
m
7.8
9.2
9.3
9.3
4.8
1.7
11.9
1.8
11.9
m
m
CH2O
m
9.7, 6.6
10.0, 6.3
9.6, 6.7
m
m
CH@CH2
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7

1418
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
Table 6
1H Assignments (ppm) of the O type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
Proton
H Type I
H Type II
H Type III
H Type IV
H Type V
H Type VI
H100
H200
H300
H400
H500
H600
5.20 (d)
5.21 (d)
5.12 (d)
3.76 (dd)
3.70 (dd)
3.65 (d)
4.22 (q)
1.28 (d)
5.21 (d)
3.74 (dd)
3.66 (dd)
3.69 (d)
4.25 (q)
1.23 (d)
5.18 (d)
3.76 (dd)
3.79 (dd)
3.67 (d)
4.35 (q)
1.23 (d)
5.23 (d)
3.76 (dd)
3.73 (dd)
3.66 (d)
4.17 (q)
1.20 (d)
3.73 (dd)
3.68 (dd)
3.71–3.69 (m)
4.29 (q)
3.77–3.71 (m)
3.77–3.71 (m)
3.66 (d)
4.17 (q)
1.20 (d)
1.19 (d)
H10
H20
H30
H40
H50
H60
H60
4.54–4.51 (m)
3.71–3.65 (m)
3.71–3.65 (m)
3.80–3.78 (m)
3.52 (dd)
4.50–4.44 (m)
3.75–3.68 (m)
3.75–3.68 (m)
3.78 (d)
3.54 (dd)
3.65–3.61 (m)
3.75–3.73 (m)
4.59 (d)
4.53 (d)
3.71 (dd)
3.67 (dd)
3.80 (d)
3.53–3.48 (m)
3.82–3.68 (m)
3.82–3.68 (m)
4.63 (d)
3.72 (dd)
3.69 (dd)
3.83 (d)
3.52 (dd)
3.78–3.71 (m)
3.78–3.71 (m)
4.47 (d)
3.73 (dd)
3.70 (dd)
3.78 (d)
3.55–3.52 (m)
3.69–3.62 (m)
3.69–3.62 (m)
3.69–3.64 (m)
3.69–3.64 (m)
3.82 (d)
3.54 (dd)
3.75–3.66 (m)
3.75–3.66 (m)
3.67–3.64 (m)
3.77–3.74 (m)
H1
H2
H3
H4
H5
H6
4.28–4.22 (m)
3.84–3.78 (m)
3.84–3.78 (m)
3.42–3.38 (m)
3.32–3.28 (m)
3.74–3.65 (m)
4.37 (d)
4.96 (d)
4.23 (d)
4.26 (d)
3.64 (dd)
3.60 (dd)
4.10 (d)
3.54–3.52 (m)
3.78–3.71 (m)
4.26 (d)
3.72 (dd)
3.59 (dd)
3.69 (dd)
3.33–3.29 (m)
3.89 (dd)
4.25 (dd)
3.98 (dd)
4.17 (d)
3.83 (dd)
3.74–3.68 (m)
4.10 (dd)
3.82 (dd)
4.06 (d)
3.51 (dd)
3.82–3.68 (m)
3.24 (dd)
3.48 (dd)
3.65 (dd)
3.31 (ddd)
3.70–3.62 (m)
H6
CH2O
CH2O
CH@CH2
CH@CH2
CH@CH2
(CH2)5
(CH2)5
(CH2)5
CH3CO
3.92–3.84 (m)
3.45–3.37 (m)
3.91–3.83 (m)
4.99–4.93 (m)
4.92–4.88 (m)
5.84–5.75 (dddd)
2.07–2.00 (m)
1.58–1.46 (m)
1.41–1.26 (m)
1.97 (s)
3.83 (dd)
3.74–3.68 (m)
3.36 (dt)
3.82–3.68 (m)
3.43 (dt)
3.87 (m)
4.99–4.94 (m)
4.93–4.89 (m)
5.80 (dddd)
2.08–2.01 (m)
1.59–1.47 (m)
1.43–1.27 (m)
1.96 (s)
3.78–3.71 (m)
3.59–3.51 (m)
3.91–3.86 (m)
5.00–4.95 (m)
4.92–4.88 (m)
5.80 (dddd)
2.07–2.01 (m)
1.66–1.57 (m)
1.43–1.26 (m)
3.90–3.85 (m)
3.55–3.52 (m)
3.91–3.82 (m)
5.00–4.95 (m)
4.92–4.88 (m)
5.80 (dddd)
2.07–2.00 (m)
1.65–1.57 (m)
1.43–1.26 (m)
3.47–3.42 (m)
3.91–3.82 (dt)
4.99–4.94 (m)
4.92–4.88 (m)
5.83–5.75 (dddd)
2.07–2.01 (m)
1.58–1.48 (m)
1.41–1.28 (m)
1.96 (s)
3.74–3.65 (m)
5.00–4.94 (m)
4.93–4.89 (m)
5.80 (dddd)
2.08–2.02 (m)
1.59–1.51 (m)
1.42–1.30 (m)
1.98 (s)
Table 7
2JH,H 3JH,H (Hz) of the O type I–VI antigens, recorded at 600 MHz with CD3OD as solvent
,
Coupling
H Type I
H Type II
H Type III
H Type IV
H Type V
H Type VI
3JH-1
3.8
10.0
3.1
6.6
3.3
m
2.8
6.6
3.8
10.0
3.3
6.6
3.9
10.0
3.5
6.5
3.3
10.2
2.9
3.4
10.2
2.8
00 ,H-200
3JH-2
00 ,H-300
3JH-3
00 ,H-400
3JH-5
6.5
6.5
00 ,H-600
3
0
0
0
0
JH-1 ,H-2
m
m
m
7.7
4.3
m
m
m
4.0
7.6
4.0
m
6.5
m
2.7
6.6
5.6
m
7.3
9.6
3.1
m
m
m
7.2
9.6
3.0
6.3
4.7
m
6.9
9.4
3.0
m
m
m
3
3
3
3
2
0
JH-2 ,H-3
JH-3 ,H-4
JH-5 ,H-6a
JH-5 ,H-6b
0
0
0
0
0
0
0
JH-6a ,H-6b
3JH-1,H-2
3JH-2,H-3
3JH-3,H-4
3JH-4,H-5
3JH-5,H-6a
3JH-5,H-6b
2JH-6a,H-6b
m
m
m
m
m
m
m
8.4
10.5
8.8
10.4
4.4
1.9
3.6
11.2
3.0
0.0
6.5
6.5
m
8.4
10.8
3.0
0.0
6.0
6.0
m
7.6
9.6
3.0
0.0
m
7.8
9.2
9.4
9.4
4.2
1.9
m
m
m
12.0
CH2O
m
9.6, 6.6
10.0, 6.3
9.7, 6.6
m
m
CH@CH2
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
17.0, 10.2, 6.7, 6.7
(ꢀ78 °C, 1 h). The reaction was then quenched by the addition of
CH3OH (4 mL) and the ammonia evaporated. The solution was ta-
ken up in CH3OH (100 mL), neutralized with Amberlite IR 120
(H+), filtered and the residue subjected to C18 chromatography
(CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide
(C2, C2000), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O),
30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.2
(CH@CH2(CH2)5CH2O), 22.85 (CH3CO), 22.84 (CH3CO), 16.8 (C600).
ESIMS: m/z Calcd [C36H62N2O20]Na+: 865.3788. Found: 865.3785.
9 (170 mg, 96%) as a colorless oil. [
a
] +253.8 (c 0.4, CH3OH); 1H
3.2. 7-Octen-1-yl 2-acetamido-3-O-(2-O-(
O-( -galactopyranosyl)-b- -galactopyranosyl)-2-deoxy-
galactopyranoside (10)
a-L-fucopyranosyl)-3-
NMR data see Tables 2 and 3. 13C NMR (CD3OD, 125.7 MHz): dC
174.4 (C@O), 173.3 (C@O), 140.0 (CH2@CH), 114.8 (CH2@CH),
104.2 (C10), 100.5 (C100), 98.1 (C1), 93.9 (C1000), 77.6, 77.4, 76.2,
75.8, 73.4, 72.7, 71.8, 71.6, 70.6, 70.1, 70.0, 69.9, 68.4, 64.9 (C20,
C2000, C3, C30, C300, C3000, C4, C40, C400, C4000, C5, C50, C500, C5000), 68.9
(CH@CH2(CH2)5CH2O), 63.4, 62.9, 62.5 (C6, C60, C6000), 51.4, 50.9
a
-D
D
a-D-
Redistilled liquid ammonia (25 mL) was collected in a flask
cooled to ꢀ78 °C and treated with sodium until the blue color per-
sisted. A solution of the tetrasaccharide 34 (281 mg, 0.0.175 mmol)

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1419
OH
OH
OH
OH
O
O
OH
HO
O
OH
HO
O
HO
HO
OH
OH
HO
HO
H
H
H
H
H
H
O
O
O
O
AcHN
AcHN
H
H
O
O
O
O
H
NHAc
HO
O
O
H
H
H
H
H
H
H
H
O
O
OH
H
OH
H
OH
OH
OH
OH
A Type V
A Type IV
47
13
Figure 5. Summary of main ROSEY interactions between 13 and 47.
HO
OH
O
51.1 (C2), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O),
30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.2
(CH@CH2(CH2)5CH2O), 22.8 (CH3CO), 16.9 (C600). ESIMS: m/z Calcd
[C28H49NO15]Na+: 662.2994. Found: 662.29867.
OH
OH
OH
OH
O
HO
O
HO
O
H1
O
O
H1′′′
O
NHAc
H1′
3.4. 7-Octen-1-yl 2-acetamido-3-O-(2-O-(
a-L-fucopyranosyl)-b-
H1′′
OH
O
D
-galactopyranosyl)-2-deoxy-b- -galactopyranoside (12)
D
14
OH
Redistilled liquid ammonia (10 mL) was collected in a flask
HO
B Type IV
cooled to ꢀ78 °C and treated with sodium until the blue color per-
Figure 6. Nomenclature system used for NMR spectroscopic data, using B type IV
antigen 14 for illustrative purposes.
sisted. A solution of the trisaccharide 40 (254 mg, 0.2341 mmol) in
THF (4 mL) and CH3OH (47 lL, 1.17 mmol) was added dropwise
and the solution was stirred (ꢀ78 °C, 2 h). The reaction was then
quenched by the addition of CH3OH (4 mL) and the ammonia was
evaporated to dryness. The solution was taken up in CH3OH
(100 mL), neutralized with Amberlite IR 120 (H+), filtered and the
residue subjected to C-18 chromatography (CH3OHꢀH2O, 1:1) to af-
ford the fully deprotected trisaccharide 12 as a colorless oil (130 mg,
in THF (4 mL) and CH3OH (63 lL, 1.57 mmol) was added dropwise
and the solution stirred (ꢀ78 °C, 1 h). The reaction was then
quenched by the addition of CH3OH (4 mL) and the ammonia was
evaporated. The solution was taken up in CH3OH (100 mL), neutral-
ized with Amberlite IR 120 (H+), filtered and the residue subjected to
C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotec-
87%). [
a
] ꢀ67.2 (c 0.4, CH3OH); 1H NMR data see Tables 6 and 7. 13
C
ted tetrasaccharide 10 (132 mg, 94%) as a colorless oil. [ ] +106.8
a
NMR (CD3OD, 125.7 MHz): dC 173.2 (C@O), 140.1 (CH2@CH), 114.8
(CH2@CH), 104.1, 103.7 (C1, C10), 101.2 (C100), 79.2, 78.5, 76.8, 76.4,
75.4, 73.5, 71.5, 70.6, 70.4, 69.7, 68.3 (C20, C200, C3, C30, C300, C4, C40,
C400, C5, C50, C500), 70.5 (CH@CH2(CH2)5CH2O), 62.6 (2C, C6, C60),
52.7 (C2), 34.9 (CH@CH2(CH2)5CH2O), 30.7 (CH@CH2(CH2)5CH2O),
30.2 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0
(CH@CH2(CH2)5CH2O), 23.4 (CH3CO), 16.7 (C600). ESIMS: m/z Calcd
[C28H49NO15]Na+: 662.2994. Found: 662.2995.
(c 0.7, CH3OH); 1H NMR data see Tables 4 and 5. 13C NMR (CD3OD,
125.7 MHz): dC 173.3 (C@O), 140.0 (CH2@CH), 114.8 (CH2@CH),
104.2 (C10), 100.5, 98.1 (C1, C100), 96.3 (C1000), 79.2, 77.6, 76.0, 75.7,
73.4, 73.0, 71.8, 71.6, 71.31, 71.26, 70.0 (3C), 68.3, 65.6 (C20, C200,
C2000, C3, C30, C300, C3000, C4, C40, C400, C4000, C5, C50, C500, C5000), 68.9
(CH@CH2(CH2)5CH2O), 63.3, 62.9, 62.5 (C6, C60, C6000), 50.9 (C2),
34.9 (CH@CH2(CH2)5CH2O), 30.5 (CH@CH2(CH2)5CH2O), 30.1
(CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.2 (CH@CH2-
(CH2)5CH2O), 22.9 (CH3CO), 16.8 (C600). ESIMS: m/z Calcd
[C97H109NO20]Na+: 824.3523. Found: 824.3517.
3.5. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-deoxy-
a-
D-galactopyranosyl)-2-O-(
a-
L-fucopyranosyl)-b-
D-
galactopyranosyl)-2-deoxy-b-
D-galactopyranoside (13)
3.3. 7-Octen-1-yl 2-acetamido-3-O-(2-O-(
a-L-fucopyranosyl)-b-
D
-galactopyranosyl)-2-deoxy- -galactopyranoside (11)
a
-
D
A solution of tetrasaccharide 43 (490 mg, 0.346 mmol) in
CH3OH (20 mL) was treated with a catalytic amount of NaOCH3
in CH3OH and the solution was stirred for 2 h. The solution was
then neutralized with Amberlite IR 120 (H+), filtered and the resi-
due subjected to flash chromatography (CH2Cl2–CH3OH 10:1) to af-
ford 44 (410 mg, 92%) as a colorless oil. Redistilled liquid ammonia
(20 mL) was collected in a flask cooled to (ꢀ78 °C) and treated with
sodium until the blue color persisted. A solution of the tetrasaccha-
Redistilled liquid ammonia (10 mL) was collected in a flask
cooled to ꢀ78 °C and treated with sodium until the blue color per-
sisted. A solution of the trisaccharide 27 (175 mg, 0.161 mmol) in
THF (4 mL) and CH3OH (33 lL, 0.806 mmol) was added dropwise
and the solution was stirred (ꢀ78 °C, 2 h). The reaction was then
quenched by the addition of CH3OH (4 mL) and the ammonia was
evaporated to dryness. The solution was taken up in CH3OH
(100 mL), neutralized with Amberlite IR 120 (H+), filtered and the
residue subjected to C-18 chromatography (CH3OH–H2O, 1:1) to af-
ford the fully deprotected trisaccharide 11 as a colorless oil (95 mg,
ride 44 (228 mg, 0.177 mmol) in THF (4 mL) and CH3OH (36 lL,
0.885 mmol) was added dropwise and the mixture was stirred
(ꢀ78 °C, 1 h). The reaction was then quenched by the addition of
CH3OH (4 mL) and the ammonia evaporated. The solution was ta-
ken up in CH3OH (100 mL), neutralized with Amberlite IR 120
(H+), filtered and the residue subjected to C18 chromatography
(CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide
92%). [a C
] +32.9 (c 0.8, CH3OH); 1H NMR data see Tables 6 and 7. 13
NMR (CD3OD, 125.7 MHz): dC 173.4 (C@O), 140.0 (CH2@CH), 114.8
(CH2@CH), 103.7 (C1), 101.9, 98.0 (C1, C100), 80.8, 77.8, 76.6, 74.9,
73.3, 71.8, 71.6, 70.6, 70.1, 69.1, 69.0 (C20, C200, C3, C30, C300, C4, C40,
C400, C5, C50, C500), 68.9 (CH@CH2(CH2)5CH2O), 62.8, 62.5 (C6, C60),
13 (135 mg, 91%) as a colorless oil. [
a
] +52.6 (c 0.9, CH3OH); 1H
NMR data see Tables 2 and 3. 13C NMR (CD3OD, 125.7 MHz): dC

1420
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
174.4 (C@O), 173.3 (C@O), 140.1 (CH2@CH), 114.7 (CH2@CH),
104.3, 104.1 (C1, C10), 100.1 (C100), 93.5 (C1000), 79.6, 77.7, 76.5,
76.4, 74.0, 72.8, 71.6, 70.6, 70.03, 69.98, 69.9, 68.0, 64.8 (C20, C200,
C3, C30, C300, C3000, C4, C40, C400, C4000, C5, C50, C500, C5000), 70.6
(CH@CH2(CH2)5CH2O), 63.5, 62.6, 62.5 (C6, C60, C6000), 52.5, 51.4
(C2, C2000), 34.9 (CH@CH2(CH2)5CH2O), 30.7 (CH@CH2(CH2)5CH2O),
30.2 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0
(CH@CH2(CH2)5CH2O), 22.8 (CH3CO), 16.6 (C600). ESIMS: m/z Calcd
[C36H62N2O20]Na+: 865.3788. Found: 865.3784.
CH@CH2(CH2)5CH2O), 3.54 (1H, dd, J2,3 10.4 Hz, J1,2 3.4 Hz, H2),
3.53–3.49 (1H, m, CH@CH2(CH2)5CH2O), 2.53–2.39 (1H, br s, OH),
2.11–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.69–1.58 (2H, m,
CH@CH2(CH2)5CH2O), 1.43–1.30 (6H, m, CH@CH2(CH2)5CH2O);
13C NMR (125.7 MHz): dC 138.9 (CH@CH2), 137.3 (Ph), 129.3 (Ph),
128.3 (Ph), 126.2 (Ph), 114.3 (CH@CH2), 101.3 (PhCH), 98.6 (C1),
75.6 (C4), 69.3 (C6), 68.7 (CH@CH2(CH2)5CH2O), 67.3 (C3), 62.7
(C5), 60.7 (C2), 33.6 (CH@CH2(CH2)5CH2O), 29.3 (CH@CH2(CH2)5-
CH2O), 28.80 (CH@CH2(CH2)5CH2O), 28.76 (CH@CH2(CH2)5CH2O),
25.9 (CH@CH2(CH2)5CH2O), ESIMS: m/z Calcd [C21H29N3O5]Na+:
426.1999. Found: 426.1997. Further elution (EtOAc–hexanes, 1:2)
3.6. 7-Octen-1-yl 2-acetamido-3-O-(2-O-(
O-( -galactopyranosyl)-b- -galactopyranosyl)-2-deoxy-b-
galactopyranoside (14)
a-L-fucopyranosyl)-3-
a-D
D
D
-
afforded the b-glycoside 19 as a colorless oil (8.0 g, 47%). [
a]
+151.6 (c 0.9, CH2Cl2); Rf 0.63 (EtOAc–hexanes, 3:7). [
a] ꢀ4.5 (c
0.27, CH2Cl2); Rf 0.28 (EtOAc–hexanes, 3:7); 1H NMR (500 MHz):
dH 7.53–7.48 (2H, m, Ph), 7.40–7.36 (3H, m, Ph), 5.87–5.77 (1H,
m, CH@CH2), 5.54 (1H, s, PhCH), 5.03–4.98 (1H, m, CH@CH2),
4.95–4.92 (1H, m, CH@CH2), 4.33 (1H, dd, J6,6 12.5 Hz, J5,6 1.5 Hz,
H6), 4.28 (1H, d, J1,2 3.4 Hz, H1), 4.15 (1H, dd, J3,4 3.8 Hz, J4,5
1.1 Hz, H4), 4.07 (1H, dd, J6,6 12.5 Hz, J5,6 1.9 Hz, H6), 3.99 (1H,
ddd, J 9.3 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.54 (1H, dd,
J2,3 10.2 Hz, J1,2 7.8 Hz, H2), 3.56–3.50 (2H, m, H3, CH@CH2-
(CH2)5CH2O), 3.46–3.40 (1H, m, H5), 2.61 (1H, d, J 9.2 Hz, OH),
2.09–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.71–1.61 (2H, m,
CH@CH2(CH2)5CH2O), 1.47–1.31 (6H, m, CH@CH2(CH2)5CH2O);
13C NMR (125.7 MHz): dC 139.0 (CH@CH2), 137.3 (Ph), 129.3 (Ph),
128.2 (Ph), 126.3 (Ph), 114.2 (CH@CH2), 102.1 (C1), 101.4 (PhCH),
74.6 (C4), 71.4 (C3), 70.2 (CH@CH2(CH2)5CH2O), 69.0 (C6), 66.4 (C5),
64.1 (C2), 33.6 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O),
28.82 (CH@CH2(CH2)5CH2O), 28.77 (CH@CH2(CH2)5CH2O), 25.7
(CH@CH2(CH2)5CH2O), ESIMS: m/z Calcd [C21H29N3O5]Na+: 426.1999.
Found: 426.2000.
Redistilled liquid ammonia (20 mL) was collected in a flask
cooled to (ꢀ78 °C) and treated with sodium until the blue color per-
sisted. A solution of the tetrasaccharide 46 (281 mg, 0.175 mmol) in
THF (4 mL) and CH3OH (63 lL, 1.57 mmol) was added dropwise and
the mixture was stirred (ꢀ78 °C, 1 h). The reaction was then
quenched by the addition of CH3OH (4 mL) and the ammonia evap-
orated. The solution was taken up in CH3OH (100 mL), neutralized
with Amberlite IR 120 (H+), filtered, and the residue was subjected
to C18 chromatography (CH3OH–H2O 1:1) to afford the fully depro-
tected tetrasaccharide 14 (132 mg, 94%) as a colorless oil. [a] 12.8
(c 0.7, CH3OH); 1H NMR data see Tables 4 and 5. 13C NMR (CD3OD,
125.7 MHz): dC 173.3 (C@O), 140.1 (CH2@CH), 114.7 (CH2@CH),
104.3, 104.2 (C1, C10), 100.2 (C100), 96.1 (C1000), 79.7, 79.6, 76.4, 76.2,
74.1, 73.5, 73.1, 71.6, 71.3, 70.01, 69.98, 67.9, 65.7 (C20, C200, C2000,
C3, C30, C300, C3000, C4, C40, C400, C4000, C5, C50, C500, C5000), 70.6
(CH@CH2(CH2)5CH2O), 34.9 (CH@CH2(CH2)5CH2O), 30.7 (CH@CH2-
(CH2)5CH2O), 30.2 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5-
CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.4 (CH3CO) 16.6 (C600). ESIMS:
m/z Calcd [C34H59NO20]Na+: 824.3523. Found: 824.3515.
3.8. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-b-
D-
galactopyranosyl)-2-deoxy- -galactopyranoside (22)
a
-D
3.7. 7-Octen-1-yl 2-azido-4,6-O-benzylidene-2-deoxy-
galactopyranoside (18) and 7-Octen-1-yl 2-azido-4,6-O-
benzylidene-2-deoxy- -galactopyranoside (19)
a-D-
A solution of the acceptor 18 (949 mg, 2.35 mmol) in dry CH2Cl2
(20 mL) was stirred over 4 Å molecular sieves (1.5 g) and the mix-
ture was stirred (rt, 1 h). The solution was then cooled (ꢀ40 °C),
treated with TMSOTf (0.2 mL, 1.1 mmol) followed by dropwise
addition of the trichloroacetimidate 2034 (2.33 g, 4.71 mmol) and
then the mixture was allowed to warm to 0 °C (1 h). The mixture
was neutralized with Et3N (2 mL), concentrated and subjected to
flash chromatography (EtOAc–hexanes, 1:1) to afford compound
21 as a colorless oil, which was immediately used in the next step.
The colorless oil was taken up in CH3OH (100 mL), treated with a
solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution
was neutralized with Amberlite IR 120 (H+), filtered, and subjected
to flash chromatography (EtOAc–hexanes, 1:1) to afford diol 22 as
a-D
A stirred solution of the trichloroacetimidate25 15 (23.12 g,
48.8 mmol) and 7-octen-1-ol (8.88 mL, 59.0 mmol) in dry Et2O
(200 mL) was treated with 4 Å molecular sieves (5 g) and the mix-
ture was stirred (rt, 1 h). The mixture was cooled (ꢀ10 °C), treated
with TMSOTf (0.5 mL, 2.8 mmol) and allowed to slowly warm to
0 °C (1 h). The mixture was neutralized by the addition of Et3N
(1 mL), filtered, concentrated, and subjected to flash chromatogra-
phy (EtOAc–hexanes 1:3) to give an inseparable
a/b mixture
(52:48, ratio determined by 1H NMR spectroscopy) 16, which used
immediately in the subsequent step. The oil was taken up in
CH3OH (200 mL) and treated with a catalytic amount of NaOCH3
in CH3OH and the solution stirred (rt, 1 h). The solution was then
neutralized with Amberlite IR 120 (H+) and the mixture filtered;
concentration followed by flash chromatography (EtOAc–hexanes
a colorless oil (1.35 g, 88%). [a] +104.5 (c 0.4, CH2Cl2); Rf 0.19
(EtOAc–hexanes, 1:1); 1H NMR (500 MHz): dH 7.58–7.47 (4H, m,
Ph), 7.37–7.29 (6H, m, Ph), 5.87–5.76 (1H, m, CH2@CH), 5.58 (1H,
s, PhCH), 5.53 (1H, s, PhCH), 5.04 (1H, d, J1,2 3.6 Hz, H1), 5.03–
4.98 (1H, m, CH2@CH), 4.97–4.94 (1H, m, CH2@CH), 4.59 (1H, d,
5:1) yielded the triol 17 (13.3 g, 87%) as an inseparable
a/b mix-
J1 ,2 7.6 Hz, H10), 4.50 (1H, d, J3,4 3.2 Hz, H4), 4.28 (1H, dd, J6 ,6
0
0
0
0
ture. A solution of the triol 17 (13.3 g, 42.2 mmol) in dry DMF
(100 mL) was then treated with benzaldehyde dimethyl acetal
(7.6 mL, 50.0 mmol) and p-TsOH (300 mg) and the solution stirred
(50 °C, 18 h). The solution was treated with Et3N (1.5 mL), concen-
trated and the residue subjected to flash chromatography (EtOAc–
10.5 Hz, J5 ,6 1.5 Hz, H60), 4.25 (1H, d, J6,6 10.5 Hz, H6), 4.22 (1H,
0
0
0
0
0
0
dd, J2,3 10.8 Hz, J3,4 3.2 Hz, H3), 4.15 (1H, dd, J3 ,4 3.7 Hz, J4 ,5
0.7 Hz, H40), 4.06 (1H, dd, J6 ,6 10.5 Hz, J5 ,6 1.7 Hz, H60), 4.04 (1H,
d, J6,6 10.5 Hz, H6), 3.91 (1H, dd, J1,2 3.6 Hz, J2,3 10.8 Hz, H2), 3.79
0
0
0
0
(1H, dd, J2 ,3 8.3 Hz, J1 ,2 7.6 Hz, H20), 3.74–3.64 (3H, m, H5, H30,
0
0
0
0
hexanes 1:4) to firstly afford the
a-glycoside 18 as a colorless oil
(8.6 g, 51%). [ ] +151.6 (c 0.9, CH2Cl2); Rf 0.63 (EtOAc–hexanes,
a
CH@CH2(CH2)5CH2O), 3.52 (1H, ddd, J 9.8 Hz, 6.7 Hz, 6.7 Hz,
3:7); 1H NMR (500 MHz): dH 7.52–7.47 (2H, m, Ph), 7.42–7.36
(3H, m, Ph), 5.87–5.77 (1H, m, CH@CH2), 5.58 (1H, s, PhCH),
5.03–4.98 (1H, m, CH@CH2), 5.01 (1H, d, J1,2 3.4 Hz, H1), 4.96–
4.93 (1H, m, CH@CH2), 4.31–4.26 (2H, m, H4, H6), 3.70 (1H, dd,
J2,3 10.4 Hz, J3,4 3.3 Hz, H3), 4.09 (1H, dd, J6,6 12.6 Hz, J5,6 1.8 Hz,
H6), 3.75–3.74 (1H, m, H5), 3.71 (1H, ddd, J 9.7 Hz, 6.7 Hz, 6.7 Hz,
CH@CH2(CH2)5CH2O), 3.46–3.43 (1H, m, H50), 2.95 (1H, s, OH),
2.67 (1H, d, J 7.5 Hz, OH), 2.10–2.02 (2H, m, CH@CH2(CH2)5CH2O),
1.68–1.60 (2H, m, CH@CH2(CH2)5CH2O), 1.45–1.31 (6H, m,
CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz): dC 139.0 (CH2@CH),
137.8 (Ph), 137.7 (Ph), 129.2 (Ph), 128.9 (Ph), 128.3 (Ph), 128.2
(Ph), 126.4 (Ph), 126.4 (Ph), 114.3 (CH2@CH), 104.0 (C10), 101.3

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1421
(PhCH), 101.0 (PhCH), 98.5 (C1), 76.3 (C4), 75.1, 74.7 (C3, C40), 72.2,
71.5 (C20, C30), 69.21, 69.19, 68.7 (C6, C60, CH@CH2(CH2)5-
CH2O), 66.7 (C50), 63.1, 59.1 (C2, C5), 33.7, (CH@CH2(CH2)5CH2O),
29.4 (CH@CH2(CH2)5CH2O), 28.82 (CH@CH2(CH2)5CH2O), 28.78
(CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5CH2O). ESIMS: m/z
Calcd [C41H51N3O10]Na+: 676.2841. Found: 676.2839.
(125.7 MHz): dC 177.9 (C@O), 139.0 (2C, Ph), 138.9 (CH2@CH),
138.6 (Ph), 137.9 (Ph), 137.7 (Ph), 128.94 (Ph), 128.87 (Ph), 128.3
(Ph), 128.24 (Ph), 128.17 (Ph), 128.14 (2C, Ph), 128.09 (Ph), 127.5
(Ph), 127.42 (Ph), 127.41 (Ph), 127.34 (Ph), 127.29 (Ph), 126.3
(Ph), 126.0 (Ph), 114.3 (CH2@CH), 102.0 (C10), 100.9 (PhCH), 100.8
(PhCH), 99.0 (C1), 97.0 (C100), 79.5, 78.4, 76.5, 76.4, 76.1 (C200, C300,
C4, C40, C400), 74.9 (PhCH2), 73.1 (PhCH2), 72.8 (PhCH2), 72.5, 72.2,
70.2 (C20, C3, C30), 69.1, 69.0, 68.7 (C6, C60, CH@CH2(CH2)5CH2O),
66.5, 66.1 (C50, C500), 63.3 (C5), 58.8 (C2), 38.9 ((CH3)3C),
33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.82
(CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 27.0 ((CH3)3-
C), 26.0 (CH@CH2(CH2)5CH2O), 16.3 (C600). ESIMS: m/z Calcd
[C66H79N3O15]Na+: 1176.5403. Found: 1176.5396.
3.9. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-pivaloyl-
b-D-galactopyranosyl)-2-deoxy-a-D-galactopyranoside (23)
A solution of diol 22 (660 mg, 1.01 mmol) in dry pyridine (10 mL)
was treated with trimethylacetyl chloride (150 L, 1.2 mmol) and
l
the solution was stirred (rt, 2 h). The solution was concentrated
and the residue was subjected to flashchromatography(EtOAc–hex-
anes, 1:3) to afford 23 as a colorless oil (700 mg, 94%). [
a
] +127.7 (c
3.11. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-2-O-(2,3,4-
0.6, CH2Cl2); Rf 0.41 (EtOAc–hexanes, 3:7); 1H NMR (500 MHz): dH
7.55–7.54 (4H, m, Ph), 7.36–7.26 (6H, m, Ph), 5.87–5.77 (1H, m,
CH2@CH), 5.57 (1H, s, PhCH), 5.52 (1H, s, PhCH), 5.06 (1H, d, J1,2
3.4 Hz, H1), 5.04–4.98 (1H, m, CH2@CH), 4.97–4.94 (1H, m,
tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-
deoxy- -galactopyranoside (26)
a
-D
A solution of the pivaloyl ester 25 (667 mg, 0.58 mmol) in
CH3OH (50 mL) was treated with a catalytic amount of LiOCH3
(50 mg) and the solution heated at reflux (7 d). The solution was
then neutralized with Amberlite IR 120 (H+), filtered, and subjected
to flash chromatography (EtOAc–hexanes, 1:3) to afford the alco-
CH2@CH), 4.85 (1H, dd, J2 ,3 10.2 Hz, J3 ,4 3.8 Hz, H30), 4.68 (1H, d,
0
0
0
0
J1 ,2 7.7 Hz, H10), 4.50 (1H, d, J3,4 3.1 Hz, H4), 4.40 (1H, dd, J3 ,4
0
0
0
0
3.8 Hz, J4 ,5 0.7 Hz, H40), 4.32 (1H, dd, J6 ,6 12.4 Hz, J5 ,6 1.4 Hz, H60),
4.25 (1H, dd, J6,6 12.8 Hz, J5,6 1.6 Hz, H6), 4.23 (1H, dd, J2,3 10.6 Hz,
J3,4 3.1 Hz, H3), 4.13–4.01 (3H, m, H20, H6, H60), 3.90 (1H, dd, J2,3
10.6 Hz, J1,2 3.4 Hz, H2), 3.75–3.68 (2H, m, H5, CH@CH2(CH2)5CH2O),
3.58–3.50 (2H, H50, CH@CH2(CH2)5CH2O), 2.56 (1H, d, J 2.1 Hz, OH),
2.10–2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.59 (2H, m,
CH@CH2(CH2)5CH2O), 1.45–1.28 (6H, m, CH@CH2(CH2)5CH2O),
1.22 (9H, s, (CH3)3C); 13C NMR (125.7 MHz): dC 178.3 (C@O), 138.9
(CH2@CH), 137.8 (Ph), 137.6 (Ph), 128.8 (Ph), 128.1 (2C, Ph), 128.1
(Ph), 126.3 (Ph), 125.9 (Ph), 114.3 (CH2@CH), 104.3 (C10), 100.9
(PhCH), 100.5 (PhCH), 98.4 (C1), 76.2 (C4), 75.0 (C3), 73.15, 73.10
(C30, C40), 69.2, 69.1 (C6, C60), 68.7 (C20), 68.5 (CH@CH2(CH2)5CH2O),
66.5 (C50), 63.1 (C5), 59.1 (C2), 39.0 ((CH3)3C), 33.7 (CH@CH2(CH2)5-
CH2O), 29.3 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O),
28.78 (CH@CH2(CH2)5CH2O), 27.1 ((CH3)3C), 25.9 (CH@CH2(CH2)5-
CH2O). ESIMS: m/z Calcd [C39H51N3O11]Na+: 760.3416. Found:
760.3410.
0
0
0
0
0
0
hol 26 as a colorless oil (480 mg, 78%). [a] +12.6 (c 0.4, CH2Cl2);
Rf 0.11 (EtOAc–hexanes, 3:7); 1H NMR (500 MHz): dH 7.54–7.48
(4H, m, Ph), 7.38–7.22 (21H, m, Ph), 5.88–5.77 (1H, m, CH2@CH),
00 00
5.61 (1H, s, PhCH), 5.56 (1H, s, PhCH), 5.37 (1H, d, J1 ,2 2.4 Hz,
H100), 5.07 (1H, d, J1,2 3.4 Hz, H1), 5.04–4.99 (1H, m, CH2@CH),
4.98–4.94 (1H, m, CH2@CH), 4.92 (1H, A of AB, J 11.6 Hz, PhCH2),
4.82–4.78 (3H, m, PhCH2), 4.94 (1H, d, J1 ,2 7.2 Hz, H10), 4.72, 4.58
(2H, AB, J 11.7 Hz, PhCH2), 4.50 (1H, d, J3,4 3.2 Hz, H4), 4.33–4.20
(5H, m, H3, H40, H500, H6, H60), 4.12–4.02 (4H, m, H200, H300, H6,
0
0
H60), 3.95 (1H, dd, J2 ,3 9.5 Hz, J1 ,2 7.2 Hz, H20), 3.93–3.87 (1H, m,
H30), 3.77–3.70 (2H, m, H5, CH@CH2(CH2)5CH2O), 3.67 (1H, dd,
J2,3 10.9 Hz, J1,2 3.4 Hz, H2), 3.58–3.52 (2H, m, H400, CH@CH2-
0
0
0
0
(CH2)5CH2O), 3.50 (1H, s, H50), 3.41 (1H, d,
J 6.5 Hz, OH),
2.10–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.62 (2H, m,
CH@CH2(CH2)5CH2O), 1.44–1.32 (6H, m, CH@CH2(CH2)5CH2O),
00
600
0.95 (3H, d, J5
6.5 Hz, H600); 13C NMR (125.7 MHz): dC 139.0
(CH2@CH), 138.9 (Ph), 138.2 (Ph), 137.8 (Ph), 137.6 (Ph), 129.2
(Ph), 128.8 (Ph), 128.31 (2C, Ph), 128.27 (Ph), 128.2 (Ph), 128.11
(Ph), 128.08 (Ph), 128.04 (Ph), 127.6 (Ph), 127.41 (2C, Ph), 127.39
(Ph), 127.38 (Ph), 126.4 (Ph), 126.3 (Ph), 114.4 (CH2@CH), 102.0,
101.4 (C10, PhCH), 100.7 (PhCH), 98.9, 98.7 (C1, C100), 79.7, 78.1,
77.0 (C200, C300, C400), 76.7, 76.4 (2C), 75.5, 73.7, 72.2 (C20, C3, C30,
C4, C40, C500), 74.8 (PhCH2), 73.4 (PhCH2), 72.8 (PhCH2), 69.2, 69.1,
68.7 (C6, C60, CH@CH2(CH2)5CH2O), 66.6, 63.3 (C5, C50), 59.2
(C2), 33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O),
28.82 (CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 25.9
(CH@CH2(CH2)5CH2O), 16.4 (C600). ESIMS: m/z Calcd [C61H71N3O14]-
Na+: 1092.4828. Found: 1092.4831.
3.10. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-
pivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-
galactopyranosyl)-2-deoxy- -galactopyranoside (25)
a
-D
A solution of the alcohol 23 (640 mg, 0.870 mmol) in dry Et2O
(10 mL) was stirred over 4 Å molecular sieves (500 mg) (rt, 1 h).
The mixture was then cooled (ꢀ10 °C), treated with TMSOTf
(50 lL, 0.28 mmol) followed by dropwise addition of the trichloro-
acetimidate26 24 (1.50 g, 2.67 mmol) in dry Et2O (5 mL). The mix-
ture was stirred (1 h), treated with Et3N (0.5 mL), filtered, and
subjected to flash chromatography (EtOAc–hexanes, 1:3) to yield
the trisaccharide 25 as a colorless oil (720 mg, 72%). [a] +19.6 (c
0.6, CH2Cl2); Rf 0.75 (EtOAc–hexanes, 1:1); 1H NMR (500 MHz):
dH 7.52–7.44 (4H, m, Ph), 7.38–7.22 (21H, m, Ph), 5.88–5.77 (1H,
m, CH2@CH), 5.59 (1H, s, PhCH), 5.48 (1H, s, PhCH), 5.39 (1H, d,
3.12. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-2-O-
(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-
J1 ,2 3.4 Hz, H100), 5.09 (1H, d, J1,2 3.3 Hz, H1), 5.07 (1H, dd, J2 ,3
deoxy- -galactopyranoside (27)
a-D
00 00
0
0
9.4 Hz, J3 ,4 3.7 Hz, H30), 5.04–4.99 (2H, m, CH2@CH), 4.94 (1H, d,
0
0
J1 ,2 7.8 Hz, H10), 4.90, 4.54 (2H, AB, J 11.5 Hz, PhCH2), 4.77, 4.60
(2H, AB, J 11.5 Hz, PhCH2), 4.73, 4.67 (2H, AB, J 11.6 Hz, PhCH2),
A solution of the azide 26 (500 mg, 0.468 mmol) in pyridine
(4 mL) was treated with AcSH (2 mL) and the solution stirred (10
d). The solution was then concentrated and the residue subjected
to flash chromatography (CH2Cl2–MeOH, 9:1) to afford predomi-
nantly 27 as a colorless oil. The oil was then taken up in MeOH
and treated with a catalytic amount of NaOMe in MeOH and the
solution stirred (rt, 5 h). The solution was neutralized with Amber-
lite IR 120 (H+), filtered, and the residue subjected to flash chroma-
tography (EtOAc–hexanes, 1:1) to afford 27 as a colorless oil
0
0
4.52–4.46 (2H, m, H4, H40), 4.45 (1H, q, J5 ,6 6.5 Hz, H500),
00 00
4.35–4.23 (4H, m, H20, H3, H6, H60), 4.09–4.03 (4H, m, H200, H300,
H6, H60), 3.79–3.71 (2H, m, H5, CH@CH2(CH2)5CH2O), 3.60–3.52
(3H, m, H2, H50, CH@CH2(CH2)5CH2O), 3.49–3.47 (1H, m, H400),
2.12–2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.72–1.64 (2H, m,
CH@CH2(CH2)5CH2O), 1.47–1.34 (6H, m, CH@CH2(CH2)5CH2O),
1.10 (9H, s, (CH3)3C), 0.92 (3H, d, J5 ,6 6.5 Hz, H600); 13C NMR
00 00

1422
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
(400 mg, 78%). [
a
] +13.4 (c 0.6, CH2Cl2); Rf 0.73 (EtOAc–hexanes,
was stirred (rt, 1 h). The mixture was then cooled (ꢀ10 °C), treated
with TMSOTf (50
L, 0.29 mmol); the trichloroacetimidate 1525
3:1); 1H NMR (500 MHz): dH 7.64–7.58 (4H, m, Ph), 7.43–7.18
(19H, m, Ph), 7.14–7.04 (3H, m, Ph, NH), 5.84–5.75 (1H, m,
CH2@CH), 5.58 (1H, s, PhCH), 5.54 (1H, s, PhCH), 5.20 (1H, d, J1,2
l
(1.38 g, 2.92 mmol) in dry Et2O (15 mL) was then added dropwise
and the mixture was allowed to stand (20 min). The mixture was
neutralized with Et3N (0.5 mL), filtered, concentrated and subjected
to flash chromatography (EtOAc–hexanes 1:3) to afford the near
pure tetrasaccharide 29 as a colorless oil (950 mg, 71%). A portion
of the tetrasaccharide (900 mg, 0.65 mmol) was taken up in pyri-
dine (6 mL) and treated with AcSH (3 mL) and the solution was stir-
red (7 d). A further addition of pyridine (2 mL) and AcSH (1 mL) was
conducted and the solution stirred another 7 d. The solution
was concentrated and subjected to flash chromatography
(CH2Cl2ꢀCH3OH, 10:1) to afford 30 as a colorless oil (700 mg,
3.2 Hz, H1), 5.07 (1H, d, J1 ,2 3.7 Hz, H100), 5.02–4.91 (3H, m, PhCH2,
CH2@CH), 4.82–4.75 (3H, m, PhCH2), 4.68 (1H, A of AB, J 11.4 Hz,
00 00
0
0
PhCH2), 4.60 (1H, A of AB, J 11.6 Hz, PhCH2), 4.56 (1H, d, J1 ,2
7.4 Hz, H10), 4.50 (1H, ddd, J2,3 11.1 Hz, JNH 6.4 Hz, J1,2 3.2 Hz, H2),
4.38 (1H, d, J3,4 2.5 Hz, H4), 4.31 (1H, d, J6,6 11.7 Hz, H6), 4.23
(1H, d, J3 ,4 3.4 Hz, H40), 4.20 (1H, d, J6 ,6 11.4 Hz, H60), 4.12 (1H,
0
0
0
0
q, J5 ,6 6.4 Hz, H500), 4.08–4.00 (4H, m, H200, H3, H6, H60), 3.94–
00 00
3.88 (2H, m, H20, H300), 3.77 (1H, dd, J2 ,3 9.8 Hz, J3 ,4 3.4 Hz, H30),
0
0
0
0
3.70 (1H, d, J3 ,4 2.1 Hz, H400), 3.67–3.59 (2H, m, H5, CH@CH2-
00 00
(CH2)5CH2O), 3.45 (1H, s, H50), 3.40 (1H, ddd, J 9.9 Hz, 6.5 Hz,
6.5 Hz, CH@CH2(CH2)5CH2O), 2.07–2.01 (2H, m, CH@CH2-
(CH2)5CH2O), 1.81 (3H, s, CH3CO), 1.56–1.46 (2H, m, CH@CH2-
(CH2)5CH2O), 1.41–1.24 (6H, m, CH@CH2(CH2)5CH2O), 1.18 (3H, d,
76%). [
a
] +61.0 (c 0.2, CHCl3); Rf 0.3 (CH2Cl2ꢀCH3OH, 20:1); 1H
NMR (500 MHz): dH 7.63–7.59 (2H, m, Ph), 7.55–7.51 (2H, m, Ph),
7.45–7.20 (17H, m, Ph), 7.19–7.12 (4H, m, Ph), 5.85–5.75 (1H, m,
000,4000
CH2@CH), 5.51 (1H, s, PhCH), 5.46 (1H, s, PhCH), 5.43 (1H, d, J3
J5 ,6 6.4 Hz, H600); 13C NMR (125.7 MHz): dC 170.5 (C@O), 138.9
1.7 Hz, H4000), 5.29–5.24 (2H, m, H100, NH), 5.14 (1H, d, J1,2 3.2 Hz,
H1), 5.12–5.07 (2H, m, H1000, H3000), 5.06–4.92 (4H, m, PhCH2,
CH2@CH), 4.75 (1H, A of AB, J 11.6 Hz, PhCH2), 4.72 (1H, A of AB, J
11.4 Hz, PhCH2), 4.65–4.60 (1H, m, H5000) 4.59–4.46 (6H, m, H10,
00 00
(CH2@CH), 138.7 (Ph), 138.6 (Ph), 138.0 (Ph), 137.7 (Ph), 137.1
(Ph), 129.01 (Ph), 129.00 (Ph), 128.48 (Ph), 128.47 (Ph), 128.4
(Ph), 128.2 (Ph), 128.15 (Ph), 128.07 (Ph), 128.0 (Ph), 127.6 (Ph),
127.44 (Ph), 127.38 (Ph), 127.32 (Ph), 127.26 (Ph), 126.7 (Ph),
126.5 (Ph), 126.2 (Ph), 114.4 (CH2@CH), 102.9 (C10), 101.3 (PhCH),
100.8 (PhCH), 99.2 (C100), 97.6 (C1), 79.7, 79.1 (C20, C300), 77.5 (C400),
75.6 (C200, C3), 75.0, 74.7 (C4, C40), 74.7 (PhCH2), 74.4 (PhCH2), 72.6
(PhCH2), 72.4 (C30), 69.6, 69.3, 68.3 (C6, C60, CH@CH2(CH2)5CH2O),
68.5 (C500), 66.5 (C50), 63.0 (C5), 49.4 (C2), 33.7 (CH@CH2(CH2)5-
CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.87 (CH@CH2(CH2)5CH2O),
28.86 (CH@CH2(CH2)5CH2O), 26.0 (CH@CH2(CH2)5CH2O), 22.6
(CH3CO), 16.5 (C600). ESIMS: m/z Calcd [C63H75NO15]Na+:
1108.5029. Found: 1108.5035.
H2, H2000, H4, PhCH2), 4.34–4.27 (2H, m, H40, H6), 4.23 (1H, dd, J6 ,6
0
0
12.4 Hz, J5 ,6 1.1 Hz, H60), 4.12 (1H, q, J5 ,6 6.3 Hz, H500), 4.10–3.90
0
0
00 00
(7H, m, H20, H200, H3, H6, H60, H6000), 3.85 (1H, dd, J2 ,3 9.5 Hz, J3 ,4
0
0
0
0
3.8 Hz, H30), 3.81 (1H, dd, J2 ,3 10.1 Hz, J3 ,4 2.5 Hz, H300), 3.74
00 00
00 00
(1H, d, J3 ,4 2.5 Hz, H400), 3.66–3.61 (2H, m, H5, CH@CH2-
00 00
(CH2)5CH2O), 3.44 (1H, s, H50), 3.41–3.35 (1H, m, CH@CH2-
(CH2)5CH2O), 2.14 (3H, s, CH3CO), 2.07–2.01 (2H, m,
CH@CH2(CH2)5CH2O), 1.96 (3H, s, CH3CO), 1.88 (3H, s, CH3CO),
1.59 (3H, s, CH3CO), 1.56–1.46 (2H, m, CH@CH2(CH2)5CH2O),
00 00
1.41–1.25 (9H, m, CH3CO, CH@CH2(CH2)5CH2O), 1.18 (1H, d, J5 ,6
6.3 Hz, H600). 13C NMR (125.7 MHz): dC 170.34 (C@O), 170.31
(C@O), 170.2 (C@O), 170.0 (C@O), 169.9 (C@O), 138.9 (CH2@CH),
138.62 (Ph), 138.57 (Ph), 137.7 (2C, Ph), 129.5 (Ph), 128.5 (Ph),
128.35 (Ph), 128.32 (2C, Ph), 128.2 (Ph), 128.1 (Ph), 128.0 (Ph),
127.9 (2C, Ph), 127.7 (Ph), 127.5 (Ph), 127.2 (Ph), 126.9 (Ph),
126.7 (Ph), 126.4 (Ph), 114.4 (CH2@CH), 104.2 (C10), 101.6 (PhCH),
101.1 (PhCH), 97.7 (2C, C1, C100), 92.2 (C1000), 79.9, 77.8 (2C), 76.3,
76.0 (2C), 75.5, 73.2, 69.8, 67.73, 67.66, 66.9 (C20, C200, C3, C30, C300,
C3000, C4, C40, C400, C4000, C500, C5000), 75.0 (PhCH2), 73.5 (PhCH2), 73.0
(PhCH2), 69.6, 69.3, 62.7 (C6, C60, C6000), 68.2 (CH@CH2(CH2)5CH2O),
65.5 (C50), 63.0 (C5), 49.2, 47.1 (C2, C2000), 33.7 (CH@CH2(CH2)5-
CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.86 (CH@CH2(CH2)5CH2O),
28.84 (CH@CH2(CH2)5CH2O), 26.0 (CH@CH2(CH2)5CH2O), 22.6
(CH3CO), 22.4 (CH3CO), 20.8 (CH3CO), 20.7 (CH3CO), 20.6 (CH3CO),
16.7 (C600). ESIMS: m/z Calcd [C77H94N2O23]Na+: 1437.6140. Found:
1437.6116.
3.13. 7-Octyl 2-acetamido-3-O-(2-O-(
a-L-fucopyranosyl)-b-D-
galactopyranosyl)-2-deoxy- -galactopyranoside (28)
a
-D
The octenyl glycoside 11 (140 mg, 0.219 mmol) was treated with
Pd–C (10%, 20 mg), taken up in MeOH (20 mL) and stirred under a H2
atmosphere (rt, 1 d). The mixture was then filtered, concentrated,
and the residue was subjected to C-18 chromatography (CH3OH–
H2O, 1:1) to afford the octyl glycoside 28 as a colorless oil (130 mg,
93%). [a
] +41.4 (c 0.4, CH3OH); 1H NMR (CD3OD, 500 MHz): dH 5.12
(1H, d, J1 ,2 3.8 Hz, H100), 4.96 (1H, J1,2 3.5 Hz, H1), 4.60 (1H, d, J1 ,2
00 00
0
0
7.2 Hz, H10), 4.25 (1H, dd, J2,3 11.2 Hz, J1,2 3.5 Hz, H2), 4.23 (1H, q,
J5 ,6 6.6 Hz, H500), 4.18 (1H, d, J3,4 2.5 Hz, H4), 3.98 (1H, dd, J2,3
00 00
11.2 Hz, J 3,4 2.5 Hz, H3), 3.86–3.80, 3.78–3.63, 3.57–3.52 (13H,
3 ꢁ m, H20, H200, H30, H300, H40, H400, H5, H50, H6, H60, CH3(CH2)6CH2O),
3.39–3.33 (1H, m, CH3(CH2)6CH2O), 1.98 (3H, s, CH3C@O), 1.62–1.52
(2H, m, CH3(CH2)6CH2O), 1.41–1.23 (13H, m, H600, CH3(CH2)6CH2O),
0.90 (3H, t, J 6.8 Hz, CH3(CH2)6CH2O). 13C NMR (125.7 MHz, CD3OD):
dC 173.4 (C@O), 103.7 (C10), 101.9 (C100), 98.0 (C1), 80.8, 76.6, 74.9,
73.3, 71.8, 71.6, 70.7, 70.1, 69.2, 69.0 (C20, C200, C30, C300, C4, C40,
C400, C5, C50, C500), 77.8 (C3), 69.0 (CH3(CH2)6CH2O), 62.8, 62.5 (C6,
C60), 51.1 (C2), 33.1 (CH3(CH2)6CH2O), 30.64 (CH3(CH2)6CH2O),
30.59 (CH3(CH2)6CH2O), 30.5 (CH3(CH2)6CH2O), 27.4 (CH3(CH2)6-
CH2O), 23.7 (CH3(CH2)6CH2O), 22.7 (CH3CO), 16.9 (C600), 14.5
(CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+: 664.3151.
Found: 664.3145.
3.15. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-3-O-
(2,3,4,6-tetra-O-benzyl-
benzyl- -fucopyranosyl)-b-
galactopyranoside (34)
a
-
D
-galactopyranosyl)-2-O-(2,3,4-tri-O-
a-
L
D-galactopyranosyl)-2-deoxy-a-D-
A solution of the acceptor 26 (928 mg, 0.868 mmol) in dry Et2O
(20 mL) was treated with 4 Å molecular sieves (0.5 g) and the mix-
ture stirred (rt, 1 h). The mixture was then cooled (ꢀ10 °C), treated
with TMSOTf (30 l
L, 0.17 mmol); the trichloroacetimidate 3228
(1.80 g, 2.60 mmol) in dry Et2O (15 mL) was then added dropwise
and the mixture was allowed to stand (20 min). The mixture was
neutralized with Et3N (0.5 mL), filtered, concentrated, and sub-
jected to flash chromatography (EtOAc–hexanes 1:4) to afford
the near pure tetrasaccharide 33 (922 mg, 68%) as a colorless oil.
A solution of the tetrasaccharide 33 (550 mg, 0.345 mmol) in dry
pyridine (6 mL) was treated with AcSH (3 mL) and the solution
was stirred (rt, 4 d). Further pyridine (4 mL) and AcSH (2 mL)
3.14. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-deoxy-
3,4,6-tetra-O-acetyl-
O-(2,3,4-tri-O-benzyl-
2-deoxy- -galactopyranoside (30)
a
a
-
D
-galactopyranosyl)-4,6-O-benzylidene-2-
-
L
-fucopyranosyl)-b- -galactopyranosyl)-
D
a-D
A solution of the acceptor 26 (1.04 g, 0.973 mmol) in dry Et2O
(25 mL) was treated with 4 Å molecular sieves (1 g) and the mixture

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1423
was added and the solution was stirred further (rt, 4 d). The solu-
tion was then concentrated and subjected to flash chromatography
(CH3OH–CH2Cl2 1:9) to afford 34 (420 mg, 76%) as a colorless oil.
12.1 Hz, J5,6 1.6 Hz, H6), 4.02–3.97 (1H, m, CH@CH2(CH2)5CH2O),
0
0
3.91 (1H, dd, J2,3 10.6 Hz, J1,2 7.9 Hz, H2), 3.79 (1H, dd, J2 ,3
8.8 Hz, J1 ,2 7.6 Hz, H20), 3.71–3.66 (1H, m, H30), 3.60 (1H, dd, J2,3
10.6 Hz, J3,4 3.2 Hz, H3), 3.53 (1H, ddd, J 9.4 Hz, 6.9 Hz, 6.9 Hz,
CH@CH2(CH2)5CH2O), 3.42–3.40 (1H, m, H50), 3.37–3.36 (1H, m,
H5), 2.93 (1H, s, OH), 2.66 (1H, d, J 8.1 Hz, OH), 2.09–2.01 (2H,
m, CH@CH2(CH2)5CH2O), 1.72–1.61 (2H, m, CH@CH2(CH2)5CH2O),
1.48–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz):
dC 139.1 (CH2@CH), 137.7 (Ph), 137.6 (Ph), 129.2 (Ph), 129.0 (Ph),
128.3 (Ph), 128.2 (Ph), 126.5 (Ph), 126.3 (Ph), 114.2 (CH2@CH),
104.0 (C10), 102.4 (C1), 101.22 (PhCH), 101.16 (PhCH), 77.5 (C3),
75.4, 75.1 (C4, C40), 72.3, 71.5 (C20, C30), 70.1 (CH@CH2-
(CH2)5CH2O), 69.2, 69.1 (C6, C60), 66.8, 66.6 (C5, C50), 62.2 (C2),
33.7, (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.9
(CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2-
(CH2)5CH2O). ESIMS: m/z Calcd [C41H51N3O10]Na+: 676.2841.
Found: 676.2829.
0
0
[a
] +35.1 (c 0.5, CH2Cl2); Rf 0.50 (EtOAc–hexanes 3:7); 1H NMR
(500 MHz): dH 7.61–7.52 (4H, m, Ph), 7.36–7.00 (41H, m, Ph),
5.87–5.77 (1H, m, CH2@CH), 5.59 (1H, s, PhCH), 5.52 (1H, s, PhCH),
5.36–5.31 (2H, m, H100, H1000), 5.17 (1H, d, J1,2 3.1 Hz, H1), 5.05–4.99
(1H, m, CH2@CH), 4.98–4.95 (1H, m, CH2@CH), 4.88 (1H, A of AB,
J = 11.8 Hz, PhCH2), 4.80 (1H, A of AB, J = 11.1 Hz, PhCH2), 4.67–
4.61 (2H, m, PhCH2), 4.59–4.28 (15H, m, H10, H2, H4, H40, H5000,
H6, PhCH2), 4.27–4.22 (2H, m, H60, PhCH2), 4.18–4.13 (2H, m,
H20, H300), 4.12–4.06 (2H, m, H200, H60), 4.05–3.98 (3H, m, H3, H500,
H6), 3.94 (1H, dd, J2 ,3 9.7 Hz, J3 ,4 3.5 Hz, H30), 3.88 (1H, dd, J2
0
0
0
0
000,3000
4.0 Hz, H2000), 3.83–3.79 (2H, m, H3000, H400),
000,2000
10.2 Hz, J1
000,6000
3.68–3.61 (2H, m, H5, CH@CH2(CH2)5CH2O), 3.54 (1H, dd, J6
6.1 Hz, H6000), 3.45–3.36 (3H, m, H50, H6000,
000,6000
9.3 Hz, J5
CH@CH2(CH2)5CH2O), 3.29 (1H, br s, 1H, H4000), 2.10–2.03 (2H, m,
CH@CH2(CH2)5CH2O), 1.62 (3H, s, CH3CO), 1.56–1.48 (2H, m,
CH@CH2(CH2)5CH2O), 1.44–1.24 (6H, m, CH@CH2(CH2)5CH2O),
1.07 (3H, d, J5 ,6 6.3 Hz, H600). 13C NMR (125.7 MHz): dC 170.3
3.17. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-pival-
oyl-b-D-galactopyranosyl)-2-deoxy-b-D-galactopyranoside (37)
00 0000
(C@O), 139.1 (Ph), 138.9 (CH2@CH), 138.8 (Ph), 138.63 (Ph),
138.57 (Ph), 138.44 (Ph), 138.40 (Ph), 137.75 (Ph), 137.72 (Ph),
129.0 (Ph), 128.5 (Ph), 128.40 (Ph), 128.37 (Ph), 128.3 (Ph),
128.24 (Ph), 128.17 (Ph), 128.06 (2C, Ph), 128.05 (2C, Ph), 128.0
(Ph), 127.9 (2C, Ph), 127.8 (Ph), 127.7 (Ph), 127.65 (Ph), 127.62
(Ph), 127.41 (Ph), 127.38 (Ph), 127.34 (Ph), 127.12 (Ph), 127.06
(Ph), 127.0 (Ph), 126.9 (Ph), 126.52 (Ph), 126.49 (Ph), 126.4 (Ph),
114.4 (CH2@CH), 104.4 (C10), 101.15 (PhCH), 101.07 (PhCH), 97.7
(C1), 97.1 (C100), 91.8 (C1000), 79.3 (2C), 78.5 (2C), 77.6 (3C), 76.5
(2C), 75.6, 70.3, 69.58 (C20, C200, C2000, C3, C30, C300, C3000, C4, C40,
C400, C4000, C5000), 74.9 (PhCH2), 74.4 (PhCH2), 73.6 (PhCH2), 73.01
(PhCH2), 72.99 (PhCH2), 72.8 (PhCH2), 71.4 (PhCH2), 70.2, 69.63,
69.4 (C6, C60, C6000), 68.2 (CH@CH2(CH2)5CH2O), 67.5, 65.9,
63.0 (C5, C50, C500), 49.2 (C2), 33.8 (CH@CH2(CH2)5CH2O),
29.6 (CH@CH2(CH2)5CH2O), 28.91 (CH@CH2(CH2)5CH2O), 28.87
(CH@CH2(CH2)5CH2O), 26.0 (CH@CH2(CH2)5CH2O), 22.5 (CH3CO),
16.6 (C600). ESIMS: m/z Calcd [C97H109NO20]Na+: 1630.7435. Found:
1630.7441.
A solution of the diol 36 (570 mg, 0.87 mmol) in dry pyridine
(10 mL) was treated with trimethylacetyl chloride (144 L,
l
1.2 mmol) and the solution was stirred (rt, 2 h). The solution was
concentrated and the residue was subjected to flash chromatogra-
phy (EtOAc–hexanes, 1:3) to afford 37 as a colorless oil (640 mg,
98%). [a
] +45.5 (c 1.3, CH2Cl2); Rf 0.31 (EtOAc–hexanes, 1:1); 1H
NMR (400 MHz): dH 7.59–7.48 (4H, m, Ph), 7.39–7.31 (6H, m, Ph),
5.90–5.77 (1H, m, CH2@CH), 5.58 (1H, s, PhCH), 5.44 (1H, s, PhCH),
0
0
0
0
5.05–4.93 (2H, m, CH2@CH), 4.87 (1H, dd, J2 ,3 10.2 Hz, J3 ,4 3.8 Hz,
H30), 4.70 (1H, d, J1 ,2 7.8 Hz, H10), 4.41 (1H, d, J3 ,4 3.7 Hz, H40),
4.37–4.29 (4H, m, H1, H4, H6, H60), 4.14–3.98 (4H, H20, H6, H60,
CH@CH2(CH2)5CH2O), 3.90 (1H, dd, J2,3 10.6 Hz, J1,2 7.9 Hz, H2),
3.63 (1H, dd, J2,3 10.6 Hz, J3,4 3.4 Hz, H3), 3.58–3.50 (2H, m, H50,
CH@CH2(CH2)5CH2O), 3.40 (1H, s, H5), 2.59 (d, J 2.4 Hz, OH),
2.11–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.75–1.62 (2H, m,
CH@CH2(CH2)5CH2O), 1.51–1.30 (6H, m, CH@CH2(CH2)5CH2O),
1.25 (9H, s, (CH3)3C); 13C NMR (100 MHz): dC 178.6 (C@O), 139.3
(CH2@CH), 138.0 (Ph), 137.9 (Ph), 129.15 (Ph), 129.08 (Ph), 128.4
(Ph), 128.3 (Ph), 126.7 (Ph), 126.1 (Ph), 114.4 (CH2@CH), 104.5
(C10), 102.7 (C1), 101.4 (PhCH), 100.7 (PhCH), 77.8 (C3), 75.5,
73.4 (C30, C40), 73.4 (C4), 70.4, 69.3, 69.2 (C6, C60, CH@CH2-
(CH2)5CH2O), 68.8 (C20), 66.9 (C5, C50), 62.4 (C2), 39.2 ((CH3)3C),
33.9 (CH@CH2(CH2)5CH2O), 29.7 (CH@CH2(CH2)5CH2O), 29.1
(CH@CH2(CH2)5CH2O), 29.0 (CH@CH2(CH2)5CH2O), 27.3 ((CH3)3C),
26.0 CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C39H51N3O11]Na+:
760.3416. Found: 760.3412.
0
0
0
0
3.16. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-b-
D-
galactopyranosyl)-2-deoxy-b- -galactopyranoside (36)
D
A solution of the acceptor 19 (949 mg, 2.35 mmol) in dry CH2Cl2
(20 mL) was stirred over 4 Å molecular sieves (1.5 g) and the mix-
ture was stirred (rt, 1 h). The solution was then cooled (ꢀ40 °C),
treated with TMSOTf (0.2 mL, 1.1 mmol) followed by dropwise
addition of the trichloroacetimidate34 20 (2.33 g, 4.71 mmol) and
then the mixture was allowed to warm (0 °C, 1 h). The mixture
was neutralized with Et3N (2 mL), concentrated, and subjected to
flash chromatography (EtOAc–hexanes, 1:1) to afford compound
35 as a colorless oil, which was immediately used in the next step.
The colorless oil was taken up in CH3OH (100 mL), treated with a
solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution
was neutralized with Amberlite IR 120 (H+), filtered, and subjected
to flash chromatography (EtOAc–hexanes, 1:1) to afford the diol 36
3.18. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-
pivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-
galactopyranosyl)-2-deoxy-b- -galactopyranoside (38)
D
A solution of the alcohol 37 (520 mg, 0.705 mmol) in dry Et2O
(15 mL) was treated with 4 Å molecular sieves (500 mg) and the
mixture stirred (rt, 1 h). The mixture was then cooled (ꢀ10 °C),
as a colorless oil (1.17 g, 76%). [a] +15.9 (c 1.1, CH2Cl2); Rf 0.33
(EtOAc–hexanes, 3:7); 1H NMR (500 MHz): dH 7.57–7.43 (2H, m,
Ph), 7.51–7.47 (2H, m, Ph), 7.37–7.31 (6H, m, Ph), 5.87–5.77 (1H,
m, CH2@CH), 5.57 (1H, s, PhCH), 5.53 (1H, s, PhCH), 5.03–4.98
treated with TMSOTf (50 lL, 0.226 mmol) followed by dropwise
addition of the trichloroacetimidate 2426 (1.50 g, 2.67 mmol) in
dry Et2O (5 mL). The mixture was stirred (1 h), was treated with
Et3N (0.5 mL), filtered, and subjected to flash chromatography
(EtOAc–hexanes, 1:3) to yield the trisaccharide 38 as a colorless
0
0
(1H, m, CH2@CH), 4.96–4.92 (1H, m, CH2@CH), 4.59 (1H, d, J1 ,2
7.6 Hz, H10), 4.35 (1H, d, J3,4 3.2 Hz, H4), 4.31 (1H, d, J1,2 7.9 Hz,
0
0
oil (605 mg, 75%). [
a
] ꢀ15.8 (c 0.8, CH2Cl2); Rf 0.52 (EtOAc–hex-
H1), 4.30 (1H, dd, J6,6 12.1 Hz, J5,6 1.5 Hz, H6), 4.27 (1H, dd, J6 ,6
12.4 Hz, J5 ,6 1.4 Hz, H60), 4.15 (1H, dd, J3 ,4 3.8 Hz, J4 ,5 0.7 Hz,
0
0
0
0
0
0
anes, 1:1); 1H NMR (500 MHz): dH 7.52–7.43 (4H, m, Ph), 7.35–
7.20 (21H, m, Ph), 5.87–5.75 (1H, m, CH2@CH), 5.55 (1H, s, PhCH),
H40), 4.05 (1H, dd, J6 ,6 12.4 Hz, J5 ,6 1.8 Hz, H60), 4.03 (1H, dd, J6,6
0
0
0
0

1424
P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
5.46 (1H, s, PhCH), 5.37 (1H, d, J1 ,2 3.4 Hz, H100), 5.07 (1H, dd, J2 ,3
3.20. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-2-O-
(2,3,4-tri-O-benzyl- -fucopyranosyl)-b- -galactopyranosyl)-2-
deoxy-b- -galactopyranoside (40)
00 00
0
0
9.6 Hz, J3 ,4 4.0 Hz, H30), 5.02–4.97 (1H, m, CH2@CH), 4.94–4.90
(2H, m, H10, CH2@CH), 4.88, 4.60 (2H, AB, J 11.5 Hz, PhCH2), 4.78,
4.53 (2H, AB, J 11.7 Hz, PhCH2), 4.71, 4.67 (2H, AB, J 11.4 Hz,
a-
L
D
0
0
D
PhCH2), 4.46 (1H, d, J3 ,4 4.0 Hz, H40), 4.37 (1H, q, J5 ,6 6.5 Hz,
H500), 4.34 (1H, d, J1,2 8.3 Hz, H1), 4.32–4.25 (4H, m, H20, H4, H6,
H60), 4.08–3.99 (5H, m, H200, H400, H6, H60, CH@CH2(CH2)5CH2O),
3.75 (1H, dd, J2,3 10.7 Hz, J1,2 7.8 Hz, H2), 3.62–3.58 (2H, m, H3,
H300), 3.53 (1H, ddd, J 9.3 Hz, 6.9 Hz, 6.9 Hz, CH@CH2(CH2)5CH2O),
3.48 (1H, s, H50) 3.37 (1H, s, H5), 2.09–2.02 (2H, m, CH@CH2-
(CH2)5CH2O), 1.76–1.62 (2H, m, CH@CH2(CH2)5CH2O), 1.47–1.30
(6H, m, CH@CH2(CH2)5CH2O), 1.07 (9H, s, (CH3)3C), 0.85 (3H, d,
A solution of the azide 39 (350 mg, 0.327 mmol) in pyridine
(4 mL) was treated with AcSH (2 mL) and the solution stirred (10
d). The solution was then concentrated and the residue was sub-
jected to flash chromatography (CH2Cl2–MeOH, 9:1) to afford pre-
dominantly 40 as a colorless oil. The oil was then taken up in
MeOH and treated with a catalytic amount of NaOMe in MeOH
and the solution was stirred (rt, 5 h). The solution was then neu-
tralized with Amberlite IR 120 (H+), filtered, and the residue was
subjected to flash chromatography (EtOAc–hexanes, 1:1) to afford
unreacted starting material 39 (100 mg, 28%), further elution
(EtOAc–hexanes, 1:1) afforded 40 as a colorless oil (210 mg, 60%).
0
0
00 00
J5 ,6 6.5 Hz, H600); 13C NMR (125.7 MHz): dC 178.0 (C@O), 139.0
00 00
(CH2@CH), 138.92 (Ph), 138.89 (Ph), 138.5 (Ph), 137.91 (Ph),
137.89 (Ph), 137.6 (Ph), 129.0 (Ph), 128.9 (Ph), 128.3 (2C, Ph),
128.2 (2C, Ph), 128.0 (Ph), 127.51 (Ph), 127.45 (2C, Ph), 127.38
(Ph), 127.3 (Ph), 126.6 (Ph), 126.1 (Ph), 114.3 (CH2@CH), 102.8
(C1), 102.0 (C10), 101.2 (PhCH), 100.8 (PhCH), 97.2 (C100), 80.0, 76.3,
76.1, 75.6, 75.5 (C200, C30, C300, C4, C400), 78.2 (C3), 75.0 (PhCH2), 73.4
(PhCH2), 72.9 (PhCH2), 72.5 (C40), 70.6 (C20), 70.3
(CH@CH2(CH2)5CH2O), 69.0 68.9 (C6, C60), 66.8, 66.6, 66.1 (C5,
C50, C500), 62.8 (C2), 38.9 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O),
29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8
(CH@CH2(CH2)5CH2O), 27.0 ((CH3)3C), 25.8 (CH@CH2(CH2)5CH2O),
16.3 (C600). ESIMS: m/z Calcd [C66H79N3O15]Na+: 1176.5403. Found:
1176.5403.
[a
] ꢀ24.0 (c 0.4, CH2Cl2); Rf 0.62 (EtOAc–hexanes, 7:3); 1H NMR
(500 MHz): dH 7.54–7.46 (4H, m, Ph), 7.41–7.23 (18H, m, Ph),
7.17–7.08 (3H, m, Ph), 6.31 (1H, d, J 6.6 Hz, NH), 5.85–5.75 (1H,
m, CH2@CH), 5.60 (1H, s, PhCH), 5.55 (1H, s, PhCH), 5.49 (1H, d,
J1 ,2 3.5 Hz, H100), 5.25 (1H, d, J1,2 8.2 Hz, H1), 5.01–4.90 (3H, m,
00 00
CH2@CH, PhCH2), 4.78–4.67 (4H, m, PhCH2), 4.59–4.54 (2H, m,
H3, PhCH2), 4.45 (1H, d, J1 ,2 7.8 Hz, H10), 4.36 (1H, d, J3,4 3.4 Hz,
0
0
H4), 4.31–4.25 (2H, m, H6, H60), 4.15 (1H, d, J3 ,4 3.7 Hz, H40),
0
0
4.08–4.02 (4H, m, H200, H500, H6, H60), 3.97 (1H, dd, J2 ,3 9.6 Hz,
0
0
J1 ,2 7.8 Hz, H20), 3.93–3.87 (2H, m, H300, CH@CH2(CH2)5CH2O),
3.73–3.67 (1H, m, H30), 3.51–3.40 (5H, m, H2, H400, H5, H50,
CH@CH2(CH2)5CH2O), 3.37 (1H, d, J 8.1 Hz, OH), 2.06–2.00 (2H,
m, CH@CH2(CH2)5CH2O), 1.74 (3H, s, CH3CO), 1.63–1.53 (2H, m,
CH@CH2(CH2)5CH2O), 1.39–1.25 (6H, m, CH@CH2(CH2)5CH2O),
0
0
3.19. 7-Octen-1-yl 2-Azido-3-O-(4,6-O-benzylidene-2-O-(2,3,4-
a-L-fucopyranosyl)-b-D-galactopyranosyl))-2-
deoxy-b- -galactopyranoside (39)
tri-O-benzyl-
0.99 (3H, d, J5 ,6 6.4 Hz, H600); 13C NMR (125.7 MHz): dC 171.3
00 00
D
(C@O), 139.1 (CH2@CH), 138.6 (2C, Ph), 138.3 (Ph), 137.62 (Ph),
137.60 (Ph), 129.2 (Ph), 128.8 (Ph), 128.4 (Ph), 128.3 (Ph), 128.24
(Ph), 128.23 (Ph), 128.20 (Ph), 128.1 (Ph), 128.0 (Ph), 128.8 (Ph),
127.6 (Ph), 127.5 (2C, Ph), 126.5 (Ph), 126.3 (Ph), 114.2 (CH2@CH),
101.7 (C10), 101.4 (PhCH), 100.8 (PhCH), 99.1 (C1), 97.1 (C100), 79.4
(C300), 77.4 (C400), 77.1, 75.8, 74.8, 73.8, 73.5 (C20, C3, C30, C4, C40),
75.9 (C200), 74.8 (PhCH2), 73.8 (PhCH2), 73.0 (PhCH2), 69.5, 69.3,
69.2 (C6, C60, CH@CH2(CH2)5CH2O), 67.6 (C500), 66.6, 66.5 (C5,
C50), 55.7 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5-
CH2O), 28.90 (CH@CH2(CH2)5CH2O), 28.89 (CH@CH2(CH2)5CH2O),
25.8 (CH@CH2(CH2)5CH2O), 23.3 (CH3CO) 16.3 (C600). ESIMS: m/z
Calcd [C63H75NO15]Na+: 1108.5029. Found: 1108.5035.
A solution of the trisaccharide 38 (510 mg, 0.44 mmol) in
CH3OH (50 mL) was treated with a catalytic amount of LiOCH3
(50 mg) and the solution was heated at reflux (7 d). The solution
was then neutralized with Amberlite IR 120 (H+), filtered, and sub-
jected to flash chromatography (EtOAc–hexanes, 1:3) to afford first
unreacted starting material (90 mg, 16%); further elution (EtOAc–
hexanes, 1:2) afforded the alcohol 39 as a colorless oil (380 mg,
80%). [
a
] ꢀ36.9 (c 0.4, CH2Cl2); Rf 0.26 (EtOAc–hexanes, 1:1); 1H
NMR (500 MHz): dH 7.54–7.48 (4H, m, Ph), 7.40–7.21 (21H, m,
Ph), 5.87–5.77 (1H, m, CH2@CH), 5.58 (1H, s, PhCH), 5.55 (1H, s,
PhCH), 5.35 (1H, s, H100), 5.03–4.98 (1H, m, CH2@CH), 4.96–4.91
(1H, m, CH2@CH), 4.90, 4.57 (2H, AB, J 11.6 Hz, PhCH2), 4.83–4.75
(4H, m, H10, PhCH2), 4.72 (1H, A of AB, J 11.6 Hz, PhCH2), 4.33–
3.21. 7-Octyl 2-acetamido-3-O-(2-O-(
a-L-fucopyranosyl)-b-D-
4.22 (5H, m, H1, H4, H500, H6, H60), 4.21 (1H, d, J3 ,4 2.5 Hz, H40),
galactopyranosyl)-2-deoxy-b- -galactopyranoside (41)
D
0
0
4.09 (1H, dd, J6 ,6 12.4 Hz, J5 ,6 1.7 Hz, H60), 4.07–3.98 (4H, m,
H200, H300, H6, CH@CH2(CH2)5CH2O), 3.94–3.87 (2H, m, H20, H30),
3.77 (1H, dd, J2,3 10.7 Hz, J1,2 7.9 Hz, H2), 3.67 (1H, br s, H400),
3.57 (1H, dd, J2,3 10.7 Hz, J3,4 3.3 Hz, H3), 3.55–3.49 (1H, m,
CH@CH2(CH2)5CH2O), 3.43 (1H, s, H50), 3.38–3.24 (2H, m, H5,
OH), 2.09–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.74–1.62 (2H, m,
CH@CH2(CH2)5CH2O), 1.50–1.29 (6H, m, CH@CH2(CH2)5CH2O),
0
0
0
0
The octenyl glycoside 12 (95 mg, 0.149 mmol) was treated with
PdꢀC (10%, 20 mg), taken up in MeOH (20 mL) and to a H2 atmo-
sphere (rt, 1 d). The mixture was then filtered, concentrated, and
the residue was subjected to C-18 chromatography (CH3OHꢀH2O,
1:1) to afford the octyl glycoside 41 as a colorless oil (70 mg,
73%). [
a
] ꢀ93.3 (c 0.2, MeOH); 1H NMR (CD3OD, 500 MHz): dH
0.94 (3H, d, J5 ,6 6.5 Hz, H600); 13C NMR (125.7 MHz): dC 139.1
5.21 (1H, d, J1 ,2 3.8 Hz, H100), 4.53 (1H, d, J1 ,2 7.2 Hz, H10), 4.25
00 00
00 00
0
0
(1H, q, J5 ,6 6.8 Hz, H500), 4.24 (1H, J1,2 8.8 Hz, H1), 4.09 (1H, dd,
00 00
(CH2@CH), 138.9 (Ph), 138.9 (Ph), 138.2 (Ph), 137.8 (Ph), 137.6
(Ph), 129.2 (Ph), 128.8 (Ph), 128.32 (Ph), 128.31 (Ph), 128.28 (3C,
Ph), 128.13 (Ph), 128.07 (Ph), 128.0 (Ph), 127.6 (Ph), 127.42 (Ph),
127.39 (Ph), 126.49 (Ph), 126.45 (Ph), 114.3 (CH2@CH), 102.7
(C1), 102.0 (C10), 101.4 (PhCH), 101.0 (PhCH), 98.9 (C100), 80.0,
78.0 (C300, C400), 77.3, 76.3, 75.5, 73.8 (6C, C20, C200, C3, C30, C4,
C40), 74.9 (PhCH2), 73.6 (PhCH2), 72.7 (PhCH2), 70.2 (CH@CH2-
(CH2)5CH2O), 69.2, 68.9 (C6, C60), 66.8, 66.74, 66.69 (C5, C50, C500),
63.1 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O),
28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8
(CH@CH2(CH2)5CH2O), 16.3 (C600). ESIMS: m/z Calcd [C61H71N3O14]-
Na+: 1092.4828. Found: 1092.4820.
J2,3 10.2 Hz, J1,2 8.8 Hz, H2), 4.06 (1H, d, J3,4 3.2 Hz, H4), 3.92–
3.64, 3.55–3.48 (15H, 2 ꢁ m, H20, H200, H3, H30, H300, H40, H400, H5,
H50, H6, H60, CH3(CH2)6CH2O), 3.47–3.40 (1H, m, CH3(CH2)6CH2O),
1.97 (3H, s, CH3C@O) 1.60–1.45 (2H, m, CH3(CH2)6CH2O), 1.40–
1.26 (10H, m, CH3(CH2)6CH2O), 1.24 (3H, d, J5 ,6 6.8 Hz, H600),
00 00
0.89 (3H, t, J 6.8 Hz, CH3(CH2)6CH2O). 13C NMR (125.7 MHz,
CD3OD): dC 173.3 (C@O), 104.1 (C1), 103.8 (C10), 101.2 (C100),
79.2, 78.5, 76.8, 76.4, 75.5, 73.5, 71.5, 70.6, 70.4 (C20, C200, C3,
C30, C300, C4, C40, C400, C500), 70.6 (CH3(CH2)6CH2O), 69.7, 68.3 (C5,
C50), 62.6 (C6, C60), 52.7 (C2), 33.1 (CH3(CH2)6CH2O), 30.7
(CH3(CH2)6CH2O), 30.55 (CH3(CH2)6CH2O), 30.52 (CH3(CH2)6CH2O),

P. J. Meloncelli et al. / Carbohydrate Research 346 (2011) 1406–1426
1425
27.2 (CH3(CH2)6CH2O), 23.7 (CH3(CH2)6CH2O), 23.4 (CH3C@O), 16.7
(C600), 14.5 (CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+:
664.3151. Found: 664.3146.
(200 lL), filtered, concentrated, and subjected to flash chromatog-
raphy (EtOAc–hexanes 1:1) to afford the tetrasaccharide 45 as a
colorless oil (1.77 g, 86%). A solution of the tetrasaccharide 45
(233 mg, 0.146 mmol) in dry pyridine (4 mL) was treated with
AcSH (2 mL) and stirred (rt, 14 d). Concentration followed by flash
chromatography (EtOAc–CH2Cl2 3:7) afforded the desired 46 as a
3.22. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-de-
oxy-3,4,6-tetra-O-acetyl-
idene-2-O-(2,3,4-tri-O-benzyl-
pyranosyl)-2-deoxy-b- -galactopyranoside (43)
a
-
D
-galactopyranosyl)-4,6-O-benzyl-
a-
L
-fucopyranosyl)-b- -galacto-
D
colorless oil (214 mg, 91%). Rf 0.18 (CH3OH–CH2Cl2 1:20); [a]
D
+4.3 (c 0.3, CH2Cl2); 1H NMR (500 MHz): dH 7.52–7.07 (45H, m,
Ph), 6.06 (1H, s, NH), 5.84–5.75 (1H, m, CH2@CH), 5.56 (1H, d,
J1 ,2 3.7 Hz, H100), 5.54 (1H, s,, PhCH), 5.39 (1H, s,, PhCH), 5.33
00 00
A solution of the acceptor 39 (1.42 g, 1.33 mmol) in dry Et2O
(25 mL) was treated with 4 Å molecular sieves (1 g) and the mix-
ture was stirred (rt, 1 h). The mixture was then cooled (ꢀ10 °C),
(1H, d, J1
3.6 Hz, H1000), 5.20 (1H, d, J1,2 7.9 Hz, H1) 5.02–4.96
000,2000
(1H, m, CH2@CH), 4.95–4.92 (1H, m, CH2@CH), 4.89 (1H, A of AB,
J 11.6 Hz, PhCH2), 4.83 (1H, A of AB, J 11.4 Hz, PhCH2), 4.69–4.64
(3H, m, PhCH2), 4.62–4.39 (7H, m H10, H3, PhCH2), 4.25–4.19 (2H,
m, H20, H60), 4.13–4.07 (1H, m, H500), 4.06–3.99 (2H, m, H2000, H6),
3.97–3.90 (2H, m, CH@CH2(CH2)5CH2O, H60), 3.90–3.83 (2H, m,
H200, H5000), 3.81–3.69 (3H, m, H2, H4000, H5, CH@CH2(CH2)5CH2O),
treated with TMSOTf (50 lL, 0.29 mmol); the trichloroacetimidate
1525 (1.89 g, 3.98 mmol) in dry Et2O (25 mL) was then added drop-
wise and the mixture was allowed to stand (20 min). The mixture
was neutralized with Et3N (0.5 mL), filtered, concentrated, and
subjected to flash chromatography (EtOAc–hexanes 1:3) to afford
the near pure tetrasaccharide 42 as a colorless oil (1.46 g, 79%). A
solution of the tetrasaccharide 42 (630 mg, 0.455 mmol) in pyri-
dine (6 mL) was treated with AcSH (3 mL) and the solution was
stirred (7 d). The solution was concentrated and subjected to flash
chromatography (CH2Cl2–CH3OH, 10:1) to afford 43 as a colorless
oil (532 mg, 83%). Rf 0.37 (CH3OH–CH2Cl2 1:20); 1H NMR
(500 MHz): dH 7.53–7.49 (2H, m, Ph), 7.43–7.39 (2H, m, Ph),
7.37–7.24 (18H, m, Ph), 7.23–7.19 (3H, m, Ph), 5.93–5.86 (1H, s,
NH), 5.85–5.76 (1H, m, CH2@CH), 5.57 (1H, s, PhCH), 5.49 (1H, d,
7.2 Hz, H6000), 3.29 (1H, s, H400),
000,6000
000,6000
3.42 (1H, dd, J6
9.5 Hz, J5
3.20–3.14 (2H, m, H50, H6000), 2.07–1.98 (2H, m, CH@CH2(CH2)5-
CH2O), 1.63 (3H, s, CH3CO), 1.61–1.53 (2H, m, CH@CH2-
(CH2)5CH2O), 1.39–1.22 (6H, m, CH@CH2(CH2)5CH2O), 0.82 (3H, d,
J5 ,6 6.3 Hz, H600); 13C NMR (125.7 MHz): dC 171.3 (C@O), 139.1
00 00
(Ph), 139.0 (CH2@CH), 138.94 (Ph), 138.92 (Ph), 138.7 (Ph),
138.63 (Ph), 138.56 (Ph), 138.5 (Ph), 138.4 (Ph), 138.2 (Ph), 137.4
(Ph), 128.9 (Ph), 128.8 (Ph), 128.5 (Ph), 128.41 (Ph), 128.40 (Ph),
128.20 (Ph), 128.18 (Ph), 128.14 (Ph), 128.11 (Ph), 128.05 (Ph),
127.99 (Ph), 127.92 (Ph), 127.87 (Ph), 127.80 (Ph), 127.75 (Ph),
127.71 (Ph), 127.66 (Ph), 127.6 (Ph), 127.50 (Ph), 127.46 (Ph),
127.4 (Ph), 127.15 (Ph), 127.08 (Ph), 126.7 (Ph), 126.1 (Ph), 114.2
(CH2@CH), 102.5 (C10), 101.1 (PhCH), 100.7 (PhCH), 99.3 (C1),
96.1 (C100), 92.5 (C1000), 79.8, 77.8, 77.5 76.4, 76.3, 75.9, 75.7, 75.0,
73.5, 72.0, 70.6 (C20, C200, C2000, C3, C30, C300, C3000, C4, C40, C400, C4000,
C5000), 74.9 (PhCH2), 74.5 (PhCH2), 73.4 (PhCH2), 73.0 (PhCH2),
72.6 (PhCH2), 72.3 (PhCH2), 71.0 (PhCH2), 69.8, 69.7, 69.4, 69.2
(C6, C60, C6000, CH@CH2(CH2)5CH2O), 66.6, 66.5, 66.4 (C5, C50, C500),
55.7 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.6 (CH@CH2(CH2)5CH2O),
28.90 (CH@CH2(CH2)5CH2O), 28.89 (CH@CH2(CH2)5CH2O), 25.8
(CH@CH2(CH2)5CH2O), 23.1 (CH3CO), 16.3 (C600). ESIMS: m/z Calcd
[C95H107NO19]Na+: 1588.7330. Found: 1588.7356.
J1 ,2 3.9 Hz, H100), 5.47–5.43 (2H, m, PhCH, NH), 5.15–5.05 (4H,
00 00
m, H1, H1000, H4000, PhCH2), 5.02–4.97 (1H, m, CH2@CH), 4.96–4.92
(1H, m, CH2@CH), 4.91–4.82 (3H, m, H3000, PhCH2), 4.72–4.56 (4H,
m, H2000, H3, PhCH2), 4.51–4.46 (2H, m, H10, PhCH2), 4.37 (1H, d,
J3,4 3.9 Hz, H4), 4.33–4.28 (3H, m, H40, H6, H60), 4.23 (1H, dd, J2 ,3
0
0
9.6 Hz, J1 ,2 8.1 Hz, H20), 4.20–4.11 (2H, m, H200, H500), 4.09–4.03
(2H, m, H6, H60), 4.01–3.94 (2H, m, H5000, CH@CH2(CH2)5CH2O),
3.82–3.71 (3H, m, H30, H300, H6000), 3.55–3.38 (5H, m, H2, H400, H5,
0
0
H50, CH@CH2(CH2)5CH2O), 3.20 (1H, m, J6
8.9 Hz, H6000), 2.09
000,6000
(3H, s, CH3CO), 2.08–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.96 (3H,
s, CH3CO), 1.94 (3H, s, CH3CO), 1.76 (3H, s, CH3CO), 1.69–1.58
(2H, m, CH@CH2(CH2)5CH2O), 1.48 (3H, s, CH3CO), 1.44–1.29 (6H,
m, CH@CH2(CH2)5CH2O), 0.79 (3H, d, J5 ,6 6.3 Hz, H600); 13C NMR
00 00
(125.7 MHz): dC 170.4 (2C, C@O), 170.3 (C@O), 170.04 (C@O),
169.99 (C@O), 139.00 (CH2@CH), 138.6 (2C, Ph), 138.4 (Ph), 138.3
(Ph), 137.4 (Ph), 129.2 (Ph), 129.0 (Ph), 128.6 (Ph), 128.31 (Ph),
128.28 (Ph), 128.20 (Ph), 128.1 (Ph), 128.0 (Ph), 127.8 (Ph),
127.65 (Ph), 127.59 (Ph), 127.55 (Ph), 127.1 (Ph), 126.7 (Ph),
126.0 (Ph), 114.3 (CH2@CH), 102.3 (C10), 101.1 (PhCH), 100.8
(PhCH), 99.2, 97.0 (C1, C100) 92.1 (C1000), 79.6, 75.94, 75.87, 75.7,
73.89, 73.7, 70.5, 68.84, 68.82, 67.42, 67.36, 66.9, 66.5, 66.4 (C20,
C200, C3, C30, C300, C3000, C4, C40, C400, C4000, C5, C50, C500, C5000), 74.9
(PhCH2), 73.9 (PhCH2), 71.7 (PhCH2), 69.8, 69.3, 69.2 (C6, C60,
CH@CH2(CH2)5CH2O), 62.8 (C6000), 55.6, 46.3 (C2, C2000), 33.7
(CH@CH2(CH2)5CH2O), 29.6 (CH@CH2(CH2)5CH2O), 28.9 (2C,
CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 23.4 (CH3CO),
22.6 (CH3CO), 20.8 (CH3CO), 20.7 (CH3CO), 20.6 (CH3CO), 16.2
(C600). ESIMS: m/z Calcd [C77H94N2O23]Na+: 1437.6140. Found:
1437.6129.
Acknowledgments
Funding was provided by a Canadian Institutes of Health Re-
search (CIHR) team grant (RMF 92091), a Natural Sciences and
Engineering Research Council of Canada (NSERC) and Canadian
Institutes of Health Research (CIHR) CHRP grant (CHRPJ350946–
08), and the Alberta Ingenuity Centre for Carbohydrate Science.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.03.008.
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