Enhanced stereoselectivity of α-mannosylation under thermodynamic control using trichloroacetimidates

Article abstract of DOI:10.1016/j.carres.2010.03.025

O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of α-(1→2)-linked N-(3-deoxy-l-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-d-mannose). The blockwise synthesis of larger fragments of such O-PSs involves oligosaccha

Full text of DOI:10.1016/j.carres.2010.03.025

Carbohydrate Research 345 (2010) 999–1007
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Enhanced stereoselectivity of
a-mannosylation under thermodynamic
control using trichloroacetimidates
*
ˇ
Shu-jie Hou, Pavol Kovác
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of a-(1?2)-linked N-
(3-deoxy-L-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-D-mannose). The blockwise synthesis of
larger fragments of such O-PSs involves oligosaccharide glycosyl donors that contain a nonparticipating
2-O-glycosyl group at the position vicinal to the anomeric center where the new glycosidic linkage is
Received 4 February 2010
Received in revised form 17 March 2010
Accepted 18 March 2010
Available online 21 March 2010
formed. Such glycosyl donors may bear at C-4 either a latent acylamino (e.g., azido) or the 3-deoxy-
cero-tetronamido group. While monosaccharide glycosyl donors, even those bearing a nonparticipating
group at O-2 (e.g., methyl), and the 4-N-(3-deoxy- -glycero-tetronyl) side chain form -linked oligosac-
charides with excellent stereoselectivity, -mannosylation with analogous oligosaccharide donors in this
L-gly-
Keywords:
O-Specific polysaccharide
Cholera
Vibrio cholerae O1
Oligosaccharide synthesis
Glycosylation
L
a
a
series is adversely affected by the presence of the side chain. Consequently, the unwanted b-product is
formed in a considerable amount. Conducting the reaction at elevated temperature under thermody-
namic control substantially enhances formation of the a-linked oligosaccharide. This effect is much more
b-Mannosylation
pronounced when glycosyl trichloroacetimidates, rather than thioglycosides or glycosyl chlorides, are
used as glycosyl donors.
Published by Elsevier Ltd.
1. Introduction
Our recent blockwise synthesis11 of the Ogawa hexasaccharide
from disaccharide glycosyl donors bearing the 4-(3-deoxy-L-glyce-
Cholera in humans is caused by three strains of Vibrio cholerae—
O1 Inaba, O1 Ogawa, and O139.1 Progress in the development and
practical relevance of a conjugate vaccine for cholera from a syn-
thetic antigen depends, among other things, on the availability of
oligosaccharides that mimic the O-specific polysaccharides (O-
PSs) of the bacterial pathogens involved. The O-PSs of O1 Inaba
ro-tetronamido) group in place (fully assembled glycosyl donors,
as opposed to donors bearing 4-azido groups) has definite advan-
tages over previous approaches, despite less than optimum stere-
oselectivity in the formation of the
linkages. There,11 we were able to improve the yield of the desired,
-linked product by reacting a disaccharide thioglycoside donor
a-(1?2)-interglycosidic
a
and O1 Ogawa are very similar and consist of a (1?2)-
perosamine (4-amino-4,6-dideoxy- -mannose) whose amino
group is acylated with 3-deoxy- -glycero-tetronic acid. They dif-
a
-linked
under thermodynamic control. Here we report on further improve-
ment of the synthesis of the hexasaccharide sequence, which was
accomplished by the use of relevant mono- and disaccharide
D
L
fer2,3 in that the Ogawa O-PS has a methyl group at O-2 of the up-
stream,4 terminal perosamine residue (Fig. 1). We have been
involved in the synthesis of oligosaccharides that mimic the O-PS
of V. cholerae O1 and O139 for more than a decade.5 The chemical
syntheses are challenging and, despite several attempts to improve
early approaches,6–9 there is still a need to optimize the synthetic
strategy. We have shown10 that immunization of mice with a con-
jugate made from the synthetic hexasaccharide that mimics the
upstream terminus of the Ogawa O-PS conferred protection. There-
fore, we have recently focused our efforts on improving the synthe-
sis of that segment in the V. cholerae O1 series.
OMe
O
OH
O
H3C
H3C
O
O
NH
NH
HO
HO
HO
HO
HO
HO
O
O
O
H3C
H3C
O
O
O
NH
NH
HO
HO
HO
HO
O
O
O
O
HO
HO
H3C
HO
H3C
HO
O
O
n
n
NH
NH
HO
HO
HO
HO
O
O
Inaba
Ogawa
* Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 480 5703.
ˇ
E-mail address: kpn@helix.nih.gov (P. Kovác).
Figure 1. Structure of the O-PSs of the two strains of Vibrio cholerae O1.
0008-6215/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.carres.2010.03.025

ˇ
S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1000
trichloroacetimidates as glycosyl donors. Under thermodynamic
control, the stereoselectivity of formation of the -mannosyl link-
size
a
-mannopyranosyl linkages with high stereoselectivity (the
a
pure a-products were obtained in >80% yields). Following their
age markedly increased, compared to the use of thioglycosides as
strategy, we have been able to prepare various oligosaccharides
in the V. cholerae O1 series, including a dodecasaccharide.7,18–20
Encouraged by the high stereoselectivity of formation of the
donors.11
a
-mannopyranosyl linkage in the absence of anchimeric assis-
2. Results and discussion
tance,7,16,17 and by the precedence21 for highly stereoselective for-
mation of
a-mannosyl linkage from the fully assembled donor 2,
Generally, it is more efficient to synthesize higher oligosaccha-
rides using a convergent (blockwise) strategy than by a linear
(stepwise) approach. Also, in the assembly of large N-acyl-hexosa-
mine-containing oligosaccharides, it is preferable to use intermedi-
ates where the N-acyl group is already installed (e.g., 3–5) than
those containing latent acylamino groups (e.g., 1, where the azido
group can be converted to an acylamino function at a later stage of
the synthesis). Examples of such strategies can be found in the two
different approaches to the tetrasaccharide side chain of the major
glycoprotein of the Bacillus anthracis exosporium.12,13 In our initial
attempt to synthesize oligosaccharides in the V. cholerae O1 ser-
ies,14 we explored the feasibility of using fully assembled interme-
diates (cf., our synthesis of the Inaba disaccharide).14 There, the
we used11 donors 3–5 for glycosylation in the assembly of the Oga-
wa hexasaccharide 27. It turned out11 that, unlike with glycosyl
donors 1 and 2, the presence of the side chain in the oligosaccharide
donors 3–5 resulted in loss of the ability of these donors to form a-
mannosyl linkage with high stereoselectivity. For example,11 the
reaction of thioglycoside 4 with methyl 6-hydroxyhexanoate in
DCM at ꢀ20 °C gave mainly the unwanted b glycoside. Thus, para-
doxically, although the synthesis of the b-mannosyl linkage is one
of the most difficult glycosidic linkages to synthesize, we faced the
uncommon task to minimize formation of that linkage. Conducting
the glycosidation of methyl 6-hydroxyhexanoate at higher temper-
atures changed the situation considerably, as it resulted in in-
creased relative amount of the desired
a product formed, with
formation of the
a-mannopyranosyl linkage was highly stereose-
the latter slightly predominating when the reaction was conducted
lective due to anchimeric assistance from a participating acyl
group at O-2 in the monosaccharide glycosyl donor. This
approach,14 however, could not be extended to the synthesis of
at the temperature of refluxing toluene.11 With oligosaccharide
glycosyl acceptors 6 and 7 and donor 4, the
a:b ratio of products
could be increased from 2:1 (DCM as solvent, room temperature)
higher
a-(1?2)-linked oligosaccharides because of the absence
to 5:1 (refluxing toluene).11
of selectively removable protecting group in the product
a
A high yield of the
theses of V. cholerae O1 antigens. Guided by the increase of
reoselectivity of glycosylation under thermodynamic control using
thioglycosides as glycosyl donors, we deemed it important to
examine how trichloroacetimidate 14, which is analogous to
a
product is the prerequisite for efficient syn-
-ste-
disaccharide. We have subsequently made a series of Inaba
oligosaccharides by a stepwise approach from a fully assembled
monosaccharide donor.15 There, again, the stereoselectivity of gly-
cosylation was not an issue because of the presence of the partic-
ipating 2-C-acetyloxy group in the donor.
a
OAc
O
OLev
O
OMe
O
OLev
O
H3C
N3
BnO
H3C
C NH
BnO
H3C
C NH
BnO
H3C
C NH
BnO
O
O
O
AcO
AcO
AcO
OMe
O
H3C
C NH
AcO
O
AcO
O
OAc
OAc
OAc
O
O
O
H3C
H3C
H3C
H3C
O
O
O
O
O
O
O
N3
BnO
C NH
BnO
C NH
C NH
BnO
AcO
AcO
AcO
SEt
OAc
BnO
SEt
SEt
Cl
OAc
OAc
OAc
SEt
1
2
3
4
5
OH
O
H3C
O
C NH
AcO
BnO
OAc
AcO
OMe
O
O
O
H3C
C NH
BnO
H3C
O
O
C NH
AcO
BnO
n
OAc
OAc
AcO
O
O
SEt
H3C
O
C
NH
BnO
32
OAc
O
COOMe
n
6
7
0
2
Seminal work by Peters and Bundle, within their synthetic work
toward oligosaccharides that mimic the Brucella A polysaccharide,
thioglycoside 3,11 would perform under similar conditions. To
our knowledge, glycosylation under thermodynamic control with
trichloroacetimidates has not been attempted, lest decomposition
of the highly reactive donor might preclude glycosylation.
showed that large
a-linked, perosamine-containing oligosaccha-
rides can by synthesized in very good yields from donors that lack
a participating moiety at C-2.16,17 They used the C-4 azido group-
containing (1?2)-linked disaccharide glycosyl donor 1 to synthe-
To compare the stereoselectivity of formation of an
a-perosami-
nyl linkage from thioglycoside 311 with that using the correspond-

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S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1001
ing trichloroacetimidate, we synthesized imidate 14 (Scheme 1)
and treated it with alcohols 15 and 18 (Scheme 2). When the reac-
tion of 14 and 15 was carried out in DCM at ꢀ35 °C, a considerable
to thermodynamic control became evident when the same syn-
thons were allowed to react either at room temperature or at
100 °C, in toluene. Thus, while at room temperature the trisaccha-
rides were formed in the a:b ratio of 10:1, the reaction in hot tol-
uene led to compounds 16 and 17 in the ratio of 20:1. It should be
amount of the b-linked trisaccharide 17 was formed (
a
:b ꢁ2.5:1,
NMR, unpublished results). The proclivity of this transformation
OLev
O
OLev
O
OLev
H3C
H3C
H3C
O
O
O
O
a
d
b
e
C NH
BnO
C NH
BnO
C NH
BnO
AcO
AcO
AcO
OH
OCNHCCl3
OAc
OAc
OAc
OAc
10
8
9
c
OLev
O
OLev
O
OLev
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
H3C
O
AcO
AcO
C NH
AcO
OH
O
H3C
C NH
BnO
BnO
O
f
OAc
OAc
O
O
AcO
O
O
OAc
AcO
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
H3C
O
O
AcO
AcO
C NH
BnO
OAc
OAc
OCNHCCl3
OH
11
OAc
OAc
OAc
OAc
14
13
12
Scheme 1. Reagents and conditions: (a) piperidine, THF, rt, 95%; (b) DBU, Cl3CCN, DCM, 0 °C, 71%; (c) hydrazine acetate, DCM–MeOH (10:1, v/v), rt, 86%; (d) 10, TMSOTf, 4 Å
MS, toluene–DCM (3:1, v/v), rt, 76%; (e) piperidine, THF, 0 °C to rt, 75%; (f) DBU, Cl3CCN, DCM, 0 °C to rt, 84%.
OLev
O
OLev
O
H3C
NH
BnO
H3C
NH
BnO
O
O
C
AcO
C
AcO
OAc
AcO
O
O
OAc
AcO
O
O
OH
O
H3C
NH
BnO
H3C
H3C
NH
BnO
O
O
O
a
C
C
NH
BnO
C
AcO
14
+
+
O
OAc
O
COOMe
H3C
NH
BnO
OAc
OAc
O
O
H3C
NH
BnO
15
O
O
H3C
O
C
AcO
NH
BnO
C
AcO
OH
O
OAc
O
COOMe
O
COOMe
OAc
O
C
AcO
17
16
OAc
AcO
O
O
H3C
14
c
O
b
NH
C
BnO
OAc
O
O
H3C
O
C
NH
AcO
BnO
OAc
O
COOMe
18
OLev
O
OH
O
OH
O
H3C
H3C
O
H3C
O
C
NH
BnO
O
AcO
C NH
AcO
NH
C
AcO
BnO
BnO
OAc
AcO
O
O
OAc
O
O
OAc
AcO
O
O
H3C
H3C
O
O
H3C
C
NH
BnO
O
NH
BnO
C
AcO
C NH
BnO
O
OAc
OAc
AcO
O
O
d
O
O
OAc
H3C
H3C
H3C
NH
BnO
O
O
+
O
O
C
NH
BnO
C NH
AcO
AcO
C
BnO
OAc
AcO
O
O
OAc
O
O
OAc
O
O
H3C
H3C
NH
BnO
H3C
O
O
O
C
NH
BnO
C
AcO
C
NH
BnO
AcO
OAc
AcO
OAc
OAc
O
O
O
O
O
O
H3C
H3C
H3C
C NH
BnO
O
O
O
C
NH
C
NH
AcO
AcO
BnO
BnO
O
COOMe
OAc
OAc
O
COOMe
O
COOMe
OAc
19
α,β
20
21
Scheme 2. Reagents and conditions: (a) TMSOTf, 4 Å MS, toluene, 100 °C, 86%; (b) hydrazine acetate, DCM–MeOH (10:1, v/v), rt, 87%; (c) 14, TMSOTf, 4 Å MS, toluene, 100 °C,
75%; (d) hydrazine acetate, DCM–MeOH (10:1, v/v), rt, 91%.

ˇ
1002
S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
OMe
O
OMe
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
a
AcO
AcO
OAc
OMe
OAc
OAc
23
22
b
OMe
O
OMe
O
H3C
H3C
C NH
BnO
O
O
c
C NH
AcO
AcO
OCNHCCl3
OH
BnO
OAc
OAc
25
24
Scheme 3. Reagents and conditions: (a) Ac2O–HOAc–H2SO4 (10:4:0.1, v/v), rt, 91%; (b) piperidine, THF, rt, 75%; (d) DBU, Cl3CCN, DCM, 0 °C to rt, 86%.
noted that the glycosylation yields in these three reactions were
high (86–95%). Reaction of 14 and alcohol 18 under thermody-
tuent. Thirdly, and in contrast to their monosaccharide counter-
parts, the stereoselectivity of glycosylation conducted at
conventional reaction conditions is poor when the glycosyl donor,
a thioglycoside, glycosyl chloride, or trichloroacetimidate, is an
namic control gave pentasaccharide 19
a,b with the same high ste-
reoselectivity (
a
:b ratio, ꢁ20:1, NMR). Separation of the two
pentasaccharides was difficult and the mixture of anomers was re-
solved after removal of the levulinoyl group to give 20 and 21.
Synthesis of the hexasaccharide fragments of the O-PS of V.
cholerae O1, serotypes Inaba and Ogawa, required extension of
pentasaccharide 20 at O-2 with glycosyl donor 10 and its 2-O-
methyl analog 25, respectively.
oligosaccharide made from
the 4-(3-deoxy- -glycero-tetronamido) side chain already in place.
In such situations, the stereoselectivity can be markedly im-
a-(1?2)-linked perosamine having
L
a
proved by conducting the reaction under thermodynamic control.
The benefit of the thermodynamic control is more pronounced
when glycosylation is effected by the use of trichloroacetimidate,
which are preferred for this purpose, to the corresponding thiogly-
cosides or glycosyl chlorides.11
To obtain imidate 25, the known8 glycoside 22 was subjected
sequentially to acetolysis (?23), anomeric deacetylation, and con-
version of the free sugar 24 thus formed trichloroacetimidate 25.
We have previously observed21 high stereoselectivity of formation
4. Experimental
of the
a-perosaminyl linkage under conventional, not thermody-
namically controlled conditions (room temperature, CH2Cl2 as sol-
vent) using a different fully assembled monosaccharide glycosyl
donor, namely thioglycoside 32. Therefore, alcohol 20 was treated
with each of imidates 10 and 25 (Scheme 4) under the same con-
ditions. Both reactions were very slow, and some unchanged glyco-
syl donor was still present in both reaction mixtures after 16 h of
reaction time. The b-linked hexasaccharides could not be found
among the reaction products (NMR spectra of isolated, minor by-
products), indicating excellent stereoselectivity of formation of
26 and 27, which were isolated in 73% and 69% yield, respectively.
Hydrogenolysis of the foregoing fully protected intermediates gave
alcohols 28 and 30, respectively. Treatment of the former with
NaOMe in MeOH effected simultaneous removal of protecting acet-
yl and levulinoyl groups to give the fully deprotected hexasaccha-
ride 29. Treatment of the Ogawa compound 30 with
ethylenediamine effected simultaneous deacetylation of the side
chains and amidation of the spacer to give the final hexasaccharide
31, which is amenable for conjugation by squaric acid chemistry
(Scheme 3).22
4.1. General methods
Unless stated otherwise, optical rotations were measured at
ambient temperature with a Perkin–Elmer automatic polarimeter,
Model 341. All reactions were monitored by thin-layer chromatog-
raphy (TLC) on Silica Gel 60 coated glass slides. Column chroma-
tography was performed by elution from columns of silica gel
with the CombiFlash Companion Chromatograph (Isco, Inc.) or Isol-
era Flash Chromatograph (Biotage). Solvent mixtures less polar
than those used for TLC were used at the onset of separation. Nu-
clear magnetic resonance (NMR) spectra were measured at
300 MHz (1H) and 75 MHz (13C) with a Varian Gemini or Varian
Mercury spectrometer, or at 600 MHz (1H) and 150 MHz (13C) with
a Bruker Avance 600 spectrometer. Assignments of NMR signals
were made by homonuclear and heteronuclear two-dimensional
correlation spectroscopy, run with the software supplied with
the spectrometers. Assignment of 13C NMR spectra of some higher
oligosaccharides was aided by comparison with spectra of related
substances reported previously from this laboratory. When the lat-
ter approach was used, to aid in the 13C NMR signal-nuclei assign-
ments, advantage was taken of variations of line intensity expected
for oligosaccharides belonging to the same homologous series.25,26
Thus, spectra showed close similarity of chemical shifts of equiva-
lent carbon atoms of the internal residues, and an increase in the
relative intensity of these signals with the increasing number of
3. Conclusions
Results of this study confirm our previous11 observation that
the presence of the 3-deoxy-L-glycero-tetronamido side chain in
glycosyl donors derived from perosamine significantly affects the
stereoselectivity of glycosylation. When such glycosyl donors bear
a nonparticipating group (O-alkyl or O-glycosyl) at the position vic-
inal to the active glycosidic center, the stereochemical outcome of
perosaminylations—conducted by us7,11,15,21,23 and else-
where16,17,24—can be summarized as follows. Firstly, the formation
D-perosamine residues in the molecule. Nevertheless, often only
incomplete assignment was achieved because of the overlap of res-
onances. When reporting assignment of NMR signals, nuclei asso-
ciated with the 4-amido side chain are denoted with a prime and
those with the spacer (linker) are denoted with a double prime.
When reporting assignments of NMR signals, sugar residues in oli-
gosaccharides are serially numbered, beginning with the one bear-
ing the aglycon, and are identified by a Roman numeral superscript
in listings of signal assignments. Signals for sugar ring nuclei lack-
ing Roman numeral assignment may belong to any of the rings.
of
charide glycosyl donors having azido group at O-4 are employed.
Secondly, the glycosidic linkage can also be stereoselectively
a-glycosidic linkage is highly favored when mono- and oligosac-
a
formed from monosaccharide glycosyl donors where the 4-N-side
chain is already in place (e.g., 2 or 32), regardless of the O-2 substi-

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S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1003
10, a
25, a
20
OLev
O
OMe
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
OAc
OAc
O
O
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
OAc
OAc
O
O
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
OAc
OAc
O
O
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
OAc
O
O
OAc
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
OAc
OAc
O
O
O
O
H3C
C NH
BnO
H3C
C NH
BnO
O
O
AcO
AcO
O
COOMe
O
COOMe
OAc
OAc
26
b
27
c
OR
O
OMe
O
H3C
H3C
O
O
R2O
RO
RO
C NH
C NH
R1O
R1O
R2O
R2O
O
O
O
O
H3C
H3C
O
O
C NH
C NH
RO
R1O
R1O
R2O
RO
O
O
O
O
4
4
H3C
H3C
O
O
R2O
R2O
RO
RO
NH
NH
C
C
R1O
R1O
O
COOMe
O
COR2
R2
Ac
R
H
H
R1 R2
R
R1
H
28 Lev
30
31
OMe
Ac
H
29
NHCH2CH2NH2
H
H
H
Scheme 4. Reagents and conditions: (a) TMSOTf, 4 Å MS, DCM, rt, 73% for 27 and 69% for 28; (b) (i) 5% Pd/C, H2, DCM–MeOH (1:10, v/v), rt; (ii) NaOMe, MeOH, rt, 89% over
two steps; (c) (i) 5% Pd/C, H2, DCM–MeOH (1:10, v/v), rt, 87%; (ii) H2NCH2CH2NH2, 50 °C, 75%.
Only resonances that could be confidently assigned are listed in
signal assignments. When reporting preparation of known
substances, their identity was confirmed by comparison of NMR
characteristics with those published previously. Liquid chromatog-
raphy–electron spray-ionization mass spectrometry (ESI-MS) was
performed with a Hewlett–Packard 1100 MSD spectrometer. At-
tempts have been made to obtain correct combustion analysis data
for all new compounds. However, some compounds tenaciously re-
tained traces of solvents, despite exhaustive drying, and analytical
figures for carbon could not be obtained within 0.4%. Structures of
these compounds follow unequivocally from the mode of synthe-
sis, NMR spectroscopic data, and m/z values found in their mass
spectra, and their purity was verified by TLC and NMR spectros-
copy. Five percent palladium-on-charcoal catalyst (Escat|103)
was purchased from Engelhard Industries. 1-(3-Dimethylamino-
propyl)-3-ethyl-carbodiimide (EDAC) was purchased from ACROS
Organics. Rubber septa used to close reaction flasks containing
organic solvents were protected with a thin Teflon| sheet to avoid
leaching. Solutions in organic solvents were dried with anhydrous
Na2SO4 and concentrated at 40 °C/2 kPa.
4.2. 1-O-Acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
L-glycero-
tetronamido)-4,6-dideoxy- -mannopyranose (11)
a-D
A solution of hydrazine acetate (4.10 g, 44.6 mmol) in MeOH
(50 mL) was added to a stirred mixture of 811 (18.4 g, 31.9 mmol)
in DCM (500 mL). The stirring was continued until TLC (3:1 tolu-
ene–acetone) showed that the starting material was completely
consumed (ꢁ6 h). After concentration, chromatography (3:1 hex-
ane–EtOAc) afforded 11 (13.2 g, 86%), [a]
+16 (c 0.5, CHCl3); 1H
D
NMR (600 MHz, CDCl3) d: 7.40–7.26 (m, 5H, Ph), 6.14 (d,
J1,2 = 1.2 Hz, H-1), 6.03 (d, J = 8.3 Hz, 1H, NH), 5.14 (dd, J = 8.0 Hz
and 4.9 Hz, 1H, H-20), 4.67–4.51 (ABq, J = 12.0 Hz, 2H, PhCH2),
4.18–3.99 (m, 6H, 2-OH, H-3, H-4, H-5, H-40), 3.85 (m, 1H, H-2),

ˇ
S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1004
2.12–2.02 (m, 11H, 3COCH3, H-40), 1.24 (d, J5,6 = 6.4 Hz, 3H, H-6);
13C NMR (150 MHz, CDCl3) d: 95.4 (C-1), 137.2 (Cq), 92.8 (C-1),
74.9 (C-3), 71.4 (PhCH2), 71.1 (C-20), 68.9 (C-5), 66.3 (C-4), 59.9
(C-40), 52.7 (C-2), 30.9 (C-30), 21.0, 20.9, 20.8 (3CH3CO), 17.9 (C-
6); TOF–MS m/z: 504 [M+Na]+. Anal. Calcd for C23H31NO10: C,
57.37; H, 6.49. Found: C, 57.11; H, 6.55.
(C-1II), 92.5 (C-1I), 74.2 (C-3I), 73.3 (C-3II), 72.6 (C-2I), 71.9 (PhCH2),
71.3 and 71.2 (C-20I,II), 70.7 (PhCH2), 69.6 (C-5II), 69.0 (C-5I), 67.2
(C-2II), 60.1 and 60.0 (C-40I,II), 53.4 (C-4I), 51.9 (C-4II), 38.0 (CH2CO),
31.0 and 30.9 (C-30I,II), 29.8 (COCH3), 28.2 (OCOCH2), 21.0, 20.9,
20.8 (5C, 5COCH3), 18.1 and 17.9 (C-6I,II); TOF-HRMS m/z: [M+H]+
calcd for C49H65N2O20: 1001.4131. Found 1001.4139. Anal. Calcd
for C49H64N2O20: C, 58.79; H, 6.44. Found: C, 58.60; H, 6.48.
4.3. 1-O-Acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
tetronamido)-4,6-dideoxy-2-O-levulinoyl- -mannopyranosyl-
(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-tetro-
namido)-4,6-dideoxy- -mannopyranose (12)
L-glycero-
a-D
4.4. 5-(Methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-
L
3-deoxy-
-mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
-glycero-tetronamido)-4,6-dideoxy- -mannopyranosyl-(1?2)-
3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-tetronamido)-
4,6-dideoxy- -mannopyranoside (16)
L-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-a-
a-
D
D
L
a-D
Piperidine (54 mL, 549 mmol) was added with stirring to a solu-
tion of 827 (15.7 g, 27.1 mmol) in THF (250 mL) and the stirring was
continued at room temperature until TLC (2:1 hexane–acetone)
showed that the reaction was complete. The mixture was diluted
with DCM (1000 mL) and washed successively with ice-cooled
0.5 M HCl (2 ꢂ 250 mL), satd NaHCO3 (200 mL), and brine
(200 mL), and the organic phase was dried and concentrated.
Chromatography (2:1, hexane–acetone) gave 3-O-benzyl-4-(2,4-di-O-
L
a
-D
Piperidine (3.6 mL, 36 mmol) was added with stirring at 0 °C to
a solution of 12 (1.32 g, 1.3 mmol) in THF (20 mL) and the mixture
was allowed to warm up to room temperature. After 24 h, the mix-
ture was diluted with DCM (200 mL) and washed with 0.5 M HCl
(50 mL) and saturated NaHCO3 aq (50 mL), dried, concentrated,
and chromatography (3:1?95:5 EtOAc–hexane) gave 3-O-benzyl-
acetyl-3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-
:b
product, 300 MHz, CDCl3) d: 7.35–7.26 (m, 5H,
a
,b-
D
-mannopyranose (9, 13.8 g, 95%) as a mixture of anomers (
a
4-(2,4-di-O-acetyl-3-deoxy-
2-O-levulinoyl- -mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-
O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -manno-
L-glycero-tetronamido)-4,6-dideoxy-
ꢁ6:1). 1H NMR (
a
a-D
Ph), 5.82 (d, J = 7.9 Hz, 1H, NH), 5.40 (t, J = 2.06 Hz, 1H, H-2),
5.20–5.16 (m, 2H, H-1 and H-20), 4.66–4.33 (ABq, J = 12.0 Hz, 2H,
PhCH2), 4.16–3.90 (m, 5H, H-3, H-4, H-5, H-40), 2.76–2.64 (m, 4H,
L
a-D
pyranose (13, 940 mg, 75%). 1H NMR (600 MHz, CDCl3) d: 7.36–
7.27 (m, 10H, 2Ph), 5.91 (d, J = 8.3 Hz, 1H, NHI), 5.82 (d,
J = 9.2 Hz, 1H, NHII), 5.49 (t, J = 2.5 Hz, H-2II), 5.20–5.15 (m, 3H,
H-1I, H-20I,II), 4.87 (d, J1,2 = 1.9 Hz, H-1II), 4.65–4.38 (m, 4H,
2PhCH2), 4.20–3.90 (m, 9H, H-2I, H-3I, H-4I, H-5I, H-4II, H-40I,II),
3.81–3.74 (m, 2H, 3II, H-5II), 3.10 (br s, 1H, 1-OH), 2.76–2.60 (m,
4H, CH2CH2), 2.10–1.92 (m, 19H, 5COCH3, H-30I,II), 1.18 (d,
J5,6 = 5.8 Hz, 3H, H-6I), 1.15 (d, J5,6 = 6.3 Hz, 3H, H-6II); 13C NMR
(150 MHz, CDCl3) d: 137.8 (Cq), 137.6 (Cq), 99.5 (C-1II), 93.4 (C-
1I), 74.5 (C-2I, C-3I), 72.9 (C-3II), 71.3 (PhCH2), 71.2 and 71.0 (C-
20I,II), 70.4 (PhCH2), 68.5 (C-5II), 67.9 (C-5I), 67.1 (C-2II), 60.0 (C-
CH2CH2), 2.20–2.00 (m, 11H, 3COCH3 and H-30), 1.21 (d, J5,6
=
6.0 Hz, H-6); 13C NMR (75 MHz, CDCl3) d: 172.2 (CO), 169.8 (CO),
169.6 (CO), 137.9 (Cq), 92.5 (C-1), 73.2 (C-3), 71.3 (C-20), 70.8
(PhCH2), 67.8 (C-2 and C-5), 60.2 (C-40), 53.0 (C-4), 38.2 (CH2CO),
31.1 (C-30), 30.0 (OCOCH2), 28.3 (COCH3), 21.0 (2CH3CO), 18.3 (C-
6); TOF-HRMS m/z: [M+H]+ calcd for C26H36NO11: 538.2288. Found
538.2278.
A solution of compound 9 (600 mg, 1.19 mmol) in DCM (10 mL)
was treated at 0 °C with Cl3CCN (1.2 mL, 12 mmol) in the presence
of DBU (0.09 mL, 0.6 mmol) for 2 h and concentrated. Chromatog-
raphy (3:2 hexane–EtOAc) gave 3-O-benzyl-4-(2,4-di-O-acetyl-
I,II
40 ), 52.6 (C-4I), 51.9 (C-4II), 38.0 (CH2CO), 31.0 and 30.9 (C-30I,II),
29.8 (COCH3), 28.2 (OCOCH2), 20.9 (2COCH3), 20.8 (2COCH3), 18.1
3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-
a
-
D
-
and 17.9 (C-6I,II); TOF-HRMS m/z: [M+H]+ calcd for C47H63N2O19
:
mannopyranose 1-O-trichloroacetimidate (10, 540 mg, 71%). 1H
NMR (300 MHz, CDCl3) d: 8.71 (s, 1H, NH), 7.35–7.28 (m, 5H, Ph),
6.23 (d, J1,2 = 1.92 Hz, H-1), 5.80 (d, J = 7.4 Hz, 1H, NH), 5.51 (t,
J = 2.2 Hz, 1H, H-2), 5.17 (dd, J = 7.9 Hz and 4.8 Hz, 1H, H-20),
4.66–4.37 (ABq, J = 12.0 Hz, 2H, PhCH2), 4.15–3.93 (m, 5H, H-3, H-
4, H-5, H-40), 2.79–2.71 (m, 4H, CH2CH2), 2.18–2.05 (m, 11H,
3COCH3 and H-30), 1.25 (d, J5,6 = 5.6 Hz, H-6); 13C NMR (75 MHz,
CDCl3) d: 95.4 (C-1), 72.5 (C-3), 71.3 (C-20), 71.09, 70.6 (PhCH2),
66.25 (C-2 and C-5), 60.1 (C-40), 52.7 (C-4), 38.1 (CH2CO), 31.1
(C-30), 30.0 (OCOCH2), 28.2 (COCH3), 21.0 (2CH3CO), 18.3 (C-6);
TOF-HRMS m/z: [M+H]+ calcd for C28H36N2O11Cl3: 681.1385. Found
681.1388.
A mixture of 10 (9.8 g, 14.4 mmol), 11 (6.29 g, 13.0 mmol), and
4 Å MS (2.1 g) in toluene–DCM (3:1, v/v 150 mL) was stirred under
N2 at room temperature for 30 min. TMSOTf (16 ll, 0.65 mmol)
was added, and the stirring was continued for 4 h when TLC (3:2
hexane–acetone) showed that the donor was completely con-
sumed. After addition of Et3N (1.0 mL), the mixture was filtered
through Celite pad, the filtrate was concentrated, and chromatog-
959.4052. Found 959.4014.
DBU (0.18 mL, 0.9 mmol) was added with stirring at 0 °C to a
mixture of 13 (9.0 g, 9.4 mmol), CNCCl3 (4.7 mL, 47.0 mmol), and
DCM (150 mL), the mixture was allowed to warm up to room
temperature and stirred overnight. After concentration, chroma-
tography (3:1?3:2 hexane–acetone) gave 3-O-benzyl-4-(2,4-di-
O-acetyl-3-deoxy-
noyl- -mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-
3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -mannopyranose-
L-glycero-tetronamido)-4,6-dideoxy-2-O-levuli-
a
-D
L
a-D
1-O-trichloroacetimidate (14, 8.7 g, 84%). 1H NMR (600 MHz,
CDCl3) d: 8.62 (s, 1H, OCNHCCl3), 7.37–7.27 (m, 10H, 2Ph), 6.15
(d, J1,2 = 2.0 Hz, H-1I), 5.89 (d, J = 7.8 Hz, 1H, NHI), 5.74 (d,
J = 9.0 Hz, 1H, NHII), 5.51 (br t, J = ꢁ2.4 Hz, H-2II), 5.23–5.17 (m,
2H, H-20I,II), 4.91 (d, J1,2 = 1.7 Hz, H-1II), 4.68–4.40 (m, 4H, 2PhCH2),
4.20–3.97 (m, 9H, H-40I,II, H-4II, H-2I, H-5I, H-3I, H-4I), 3.78–3.76 (m,
2H, 3II, H-5II), 2.75–2.65 (m, 4H, CH2CH2), 2.10–1.92 (m, 19H,
5COCH3, H-30I,II), 1.24 (d, J5,6 = 6.3 Hz, 3H, H-6I), 1.19 (d,
J5,6 = 6.3 Hz, 3H, H-6II); 13C NMR (150 MHz, CDCl3) d: 137.7 (Cq),
137.4 (Cq), 99.8 (C-1II), 96.5 (C-1I), 74.2 (C-3I), 73.1 (C-3II), 72.6
(C-2I), 71.9 (PhCH2), 71.3 and 71.2 (C-20I,II), 70.8 (C-5I), 70.7
(PhCH2), 69.1 (C-5II), 67.2 (C-2II), 60.1 (C-40I,II), 52.7 (C-4II), 51.9
(C-4I), 38.2 (CH2CO), 31.2 and 31.0 (C-30I,II), 30.0 (COCH3), 28.3
(OCOCH2), 21.1 (2COCH3), 21.0 (2COCH3), 18.3 and 18.0 (C-6I,II);
TOF–MS m/z: 1127 [M+Na]+.
raphy (3:1?3:2 hexane–acetone) gave 12 (10.0 g, 76%). [a]
D
ꢀ120 (c 0.92, CHCl3); 1H NMR (600 MHz, CDCl3) d: 7.30–7.17 (m,
10H, 2Ph), 6.02 (d, J = 8.7 Hz, 1H, NHI), 5.99 (d, J1,2 = 2.2 Hz, 1H,
H-1I), 5.68 (d, J = 9.0 Hz, 1H, NHII), 5.41 (t, J = 2.5 Hz, H-2II), 5.09
(m, 2H, H-20I,II), 4.85 (d, J1,2 = 1.8 Hz, H-1II), 4.60–4.30 (m, 4H,
2PhCH2), 4.04–3.95 (m, 7H, H-3I, H-5I, H-4II, H-40I,II), 3.85 (t,
J = 2.5 Hz, 1H, H-2I), 3.71–3.67 (m, 3H, H-4I, 3II, H-5II), 2.62 (m,
4H, CH2CH2), 2.10–1.92 (m, 22H, 6COCH3, H-30I,II), 1.15 (m, 6H,
H-6I,II); 13C NMR (150 MHz, CDCl3) d: 137.6 (Cq), 137.5 (Cq), 99.4
A solution of TMSOTf in toluene (0.03 M, 10 mL) was added at
100 °C under N2 to a stirred mixture of 14 (7.5 g, 6.79 mmol), 5-
(methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-a-D
-mannopyranoside (15,11

ˇ
S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1005
3.5 g, 6.17 mmol), 4 Å molecular sieves (10 g), and toluene
(300 mL). After 5 h, when TLC (3:2 hexane–acetone) showed that
donor 14 was consumed, the mixture was cooled to room temper-
ature and Et3N (0.8 mL) was added. The mixture was filtered
through a Celite pad, the filtrate was concentrated, and chromatog-
raphy (3:1?3:2 hexane–acetone) gave the unchanged glycosyl
(110 mL). After 3 h, when TLC (1:1 hexane–acetone) showed that
the reaction was virtually complete, the mixture was cooled to
room temperature and Et3N (0.8 mL) was added. The mixture
was filtered through Celite pad, the filtrate was concentrated,
and chromatography (6:1?3:1 hexane–acetone) gave an anomeric
mixture of fully protected pentasaccharides 19a,b (6.0 g, total yield
acceptor 15 (0.83 g) and a mixture of known8
16 and 5-(methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-
a
-linked compound
75%). TOF–MS m/z: 2374 [M+Na]+. The foregoing anomeric mixture
(6.0 g, 2.5 mmol) was treated with hydrazine acetate (202 mg,
3.0 mmol) in MeOH (400 mL) overnight. Work-up, as described
above for a similar reaction, and chromatography (5:1 DCM–ace-
tone), gave first the b-linked product, 5-(methoxycarbonyl)pentyl
3-deoxy-
mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
glycero-tetronamido)-4,6-dideoxy-b-
O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
dideoxy- -mannopyranoside (17) (combined yield, 7.8 g, glyco-
L
-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-
a
-
D
-
L
-
D
-mannopyranosyl-(1?2)-3-
-glycero-tetronamido)-4,6-
L
3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
4,6-dideoxy- -mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-
acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -manno-
pyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glyce-
ro-tetronamido)-4,6-dideoxy-b- -mannopyranosyl-(1?2)-3-O-
benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy- -mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-
acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -manno-
L-glycero-tetronamido)-
a
-D
a-D
sylation yield 84%, 16:17 ꢁ20:1). The mixture was rechromato-
graphed (12:1 toluene–2-propanol) to give 16 (6.22 g) and 17
(0.21 g). A small amount of unresolved mixture of 16 and 17 was
also obtained.
L
a-D
L
D
L
Compound 17: 1H NMR (600 MHz, CDCl3) d: 7.92 (d, J = 9.0 Hz,
1H, NHIII), 7.36–7.08 (m, 15H, 3Ph), 6.43 (d, J = 10.0 Hz, 1H, NHI),
6.25 (d, J = 7.4 Hz, 1H, NHII), 5.47 (d, J1,2 = 1.3 Hz, 1H, H-1III), 5.44
(dd, J1,2 = 1.3 Hz, J2,3 = 3.1 Hz, 1H, H-2III), 5.26 (dd, J = 5.4 Hz,
J = 7.6 Hz, 1H, H-20), 5.02 (m, 2H, H-20), 4.80 (d, J = 10.3 Hz, 1H,
PhCH2), 4.75 (d, J1,2 = 1.6 Hz, 1H, H-1I), 4.67 (d, J = 11.3 Hz, 1H,
PhCH2), 4.56 (s, 1H, H-1II), 4.36 (d, J2,3 = 2.2 Hz, H-2II), 4.28 (d,
J = 11.3 Hz, 1H, PhCH2), 4.24–4.21 (m, 2H, H-2I, H-4III), 4.10–3.98
(m, 13H, PhCH2, H-4I, H-3II, H-5II, H-5III, H-40I,II,III), 3.77 (dd,
a-D
L
a-D
pyranoside (21, 220 mg, 4%). 1H NMR (600 MHz, CDCl3) d: 7.74 (br
s, 1H, NH), 7.37–7.21 (m, 25H, 5Ph), 6.40–6.12 (m, 4H, 4NH), 5.45
(br s, 1H, H-1), 5.28 (dd, J = 4.8, 8.2 Hz, 1H, H-20), 5.18–5.12 (m, 4H,
4 ꢂ H-20), 5.04 (br s, 1H, H-1), 4.90 (br s, 1H, H-1), 4.78 (d, over-
lapped with CHPh, H-1), ꢁ4.30 (m, H-1IV, overlapped), 2.30 (t,
J = 7.2 Hz, H-500a,b), 2.17–1.92 (m, 40H, H-30I–V, 10COCH3), 1.67–
1.63 (m, 4H, H-200a,b, H-400a,b), 1.38 (m, 2H, H-300a,b), 1.26–1.08 (m,
15H, H-6I-V); 13C NMR (150 MHz, CDCl3) d: 100.9 (C-1, JC-1,H-1
172 Hz), 99.6 (br, C-1, JC-1,H-1 not determined), 98.8(C-1, JC-1,H-1
169.6 Hz), 98.6 (C-1, JC-1,H-1 172 Hz), 98.1(C-1, JC-1,H-1 154 Hz),
20.9 and 20.8 (5C, C-6I-V); TOF–MS m/z: 2275 [M+Na]+.
J2,3 = 3.1 Hz, J3.4 = 10.9 Hz, H-3III), 3.64–3.61 (m, 6H, OCH3, H-100
,
a
H-3I and H-5I), 3.37–3.34 (m, 2H, H-4II, H-100b), 2.56–2.51 (m, 4H,
CH2CH2), 2.29 (t, J = 7.5 Hz, 2H, H-500a,b), 2.02–1.97 (m, 27H,
5COCH3, H-30I,II,III), 1.62–1.54 (m, 4H, H-200a,b, H-400a,b), 1.28 (m, 2H,
H-300a,b), 1.26 (d, J5,6 = 6.3 Hz, 3H, H-6I), 1.19 (d, J5,6 = 6.3 Hz, 3H,
H-6II); 13C NMR (150 MHz, CDCl3) d: 97.4 (2C, C-1I, C-1II), 96.9 (C-
1III), 77.3 (C-3III), 74.2 (C-3I), 71.7 (C-20), 71.6 (PhCH2), 71.5 (PhCH2),
71.1 (C-20), 70.8 (C-20), 69.6 (C-2I, C-5III), 69.4 (PhCH2), 69.3 (C-5II),
68.8 (C-2III), 67.2 (C-5I), 76.1 (C-100), 66.9 (C-2II), 60.9 (C-40), 60.1
(C-40), 59.9 (C-40), 55.7 (C-4II), 51.8 (C-4I, C-4II), 38.4 (2C, CH2CH2),
34.0 (C-500), 31.3(C-30), 31.2 (C-30), 30.6 (C-30), 28.8 (C-200), 25.6 (C-
300), 24.4 (C-400), 21.0 (2COCH3), 20.9 (2COCH3), 20.8 (2COCH3),
18.4, 18.1 and 18.0 (C-6I,II,III); TOF-HRMS m/z: [M+Na]+ calcd for
C75H101N3O29Na: 1530.6418. Found 1530.6324.
Eluted next was the all-a-linked pentasaccharide 20 (5.0 g,
87%), [
a
]
ꢀ9.2 (c 1.08, CHCl3); 1H NMR (600 MHz, CDCl3) d:
D
7.40–7.24 (m, 25H, 5Ph), 6.53–6.13 (m, 4H, 4NH), 5.18 (m, 5H, H-
20I–V), 5.08 (d, J1,2 = 2.0 Hz, H-1), 4.09 (br s, 2H, 2H-1), 4.87 (br s,
1H, H-1), 4.71 (br s, 1H, H-1), 4.64–4.58 (m, 10H, 5PhCH2), 4.20–
4.08 (m, 20H, H-2I–V, H-4I–V, H-40I–V), 3.86–3.32 (m, 15H, H-3I–V
,
H-5I–V, OCH3, H-100a,b), 2.46 (br s, 1H, OH-2V), 2.32 (t, J = 7.2 Hz,
H-500a,b), 2.21–1.95 (m, 40H, H-30I–V, 10COCH3), 1.66–1.53 (m, 4H,
H-200a,b, H-400a,b), 1.44–1.32 (m, 2H, H-300a,b), 1.14–1.08 (m, 15H,
H-6I-V). 13C NMR (150 MHz, CDCl3) d: 100.7 (C-1), 99.9 (3C, 3C-1),
98.8 (C-1), 18.4 (C-6), 18.3 (C-6), 18.2 (2C, 2C-6), 17.9 (C-6). TOF-
HRMS m/z: [M+H]+ calcd for C112H150N5O43: 2252.9705. Found
2252.9724. Anal. Calcd for C112H149N5O43: C, 59.70; H, 6.66; N,
3.11. Found: C, 59.41; H, 6.70; N, 3.08.
4.5. 5-(Methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-3-
deoxy-
(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
mido)-4,6-dideoxy- -mannopyranosyl-(1?2)-3-O-benzyl-4-
(2,4-di-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy-
-mannopyranoside (18)
L-glycero-tetronamido)-4,6-dideoxy-a-D-mannopyranosyl-
L
-glycerotetrona-
a
-D
L
a-
4.7. 1-O-Acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
L-glycero-
D
tetronamido)-4,6-dideoxy-2-O-methyl-a-D-mannopyranose
(23)
Compound 16 (6.2 g, 4.1 mmol) was treated with hydrazine
acetate, as described for preparation of 11, to give trisaccharide
18 (5.0 g, 87%), which was identical (TLC, NMR) with the known,
independently synthesized11 substance.
Methyl 3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
L-glycero-tetro-
namido)-4,6-dideoxy-2-O-methyl-
a
-D
-mannopyranose (22,8 7.0 g,
15 mmol) was treated at room temperature with Ac2O–HOAc–
H2SO4 (10–4–0.1, 140 mL) for 1.5 h, and NaOAcꢃ3H2O (2.0 g) was
added to terminate the reaction. After concentration, chromatogra-
phy (1:1 hexane–EtOAc) gave 23 (6.7 g, 91%), mp 104–105 °C
4.6. 5-(Methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-
3-deoxy-
osyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
tetronamido)-4,6-dideoxy- -mannopyranosyl-(1?2)-3-O-
benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy- -mannopyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-
acetyl-3-deoxy- -glycero-tetronamido)4,6-dideoxy- -manno-
pyranosyl-(1?2)-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy- -mannopyranoside (20)
L
-glycero-tetronamido)-4,6-dideoxy-
a
-
D
-mannopyran-
L
-glycero-
(EtOH); [
a
]
ꢀ2 (c 1.1, CHCl3); 1H NMR (600 MHz, CDCl3) d:
D
a
-
D
7.36–7.27 (m, 5H, Ph), 6.13 (d, J1,2 = 1.9 Hz, 1H, H-1), 6.08 (d,
J = 8.1 Hz, 1H, NH), 5.16 (dd, J = 8.0 Hz and 4.7 Hz, 1H, H-20),
4.65–4.51 (ABq, J = 11.7 Hz, 2H, PhCH2), 4.18–4.07 (m, 4H, H-3, H-
5 and H-40), 3.80 (m, 1H, H-4), 3.50 (br s, 4H, H-2 and OCH3),
2.19 (m, 1H, H-3a ), 2.11–2.04 (m, 10H, 3CH3CO and H-3b ), 1.22
(d, J5,6 = 6.3 Hz, 3H, H-6). 13C NMR (150 MHz, CDCl3) d: 91.5 (C-
1), 75.7 (C-2), 74.4 (C-3), 71.6 (PhCH2), 71.3 (C-20), 69.5 (C-5),
60.1 (C-40), 59.3 (OCH3), 53.8 (C-4), 31.1 (C-30), 21.2, 21.0, 20.9
(3COCH3), 18.6 (C-6); TOF-HRMS m/z: [M+H]+ calcd for
L
a
-D
L
a-D
0
0
L
a-D
A solution of TMSOTf in toluene (0.03 M, 6 mL) was added with
stirring at 100 °C under N2 to a mixture of 1811 (5.0 g, 3.5 mmol),
14 (4.69 g, 4.25 mmol), and 4 Å molecular sieves (5.8 g) in toluene
C24H34NO10
:
496.2183. Found 496.2178. Anal. Calcd for

ˇ
S. Hou, P. Kovác / Carbohydrate Research 345 (2010) 999–1007
1006
C24H33NO10: C, 58.17; H, 6.71; N, 2.83. Found: C, 58.36; H, 6.76;
N 2.78.
H-700), 2.20 (t, J = 7.6 Hz, H-500), 1.98–1.92 (m, 6H, H-30a I–VI),
I–VI
1.80–1.72(m, 6H, H-30b ), 1.52 (m, 4H, H-200 and H-400), 1.27 (m,
2H, H-300), 1.12–1.08 (m, 18H, H-6I–VI). 13C NMR (100 MHz, D2O)
4.8. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
tetronamido)-4,6-dideoxy- -mannopyranosyl-(1?2)-tetra-
kis[-4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -manno-
pyranosyl]-4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-2-
O-methyl- -mannopyranoside (31)
L
-glycero-
d: 100.8– 98.3 (6C, C-1I–VI), 78.8, 77.7, 77.4, 77.1 (2C), 68.9 (6C,
I–VI
a
-
D
C-20 ), 57.8 (6C, C-40I–VI), 16.9–16.7 (6C, C-6I–VI); TOF–MS m/z:
L
a-D
1670.8 [M]+.
L
a-
D
4.9. 5-(Methoxycarbonyl)pentyl 3-O-benzyl-4-(2,4-di-O-acetyl-
3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-
a-
Piperidine (4.0 mL, 40 mL) was added dropwise to a solution of
23 (1.0 g, 2.0 mmol) in THF (10 mL). After 3 h, when TLC (3:1 DCM–
acetone) showed that the reaction was complete, the mixture was
concentrated and chromatography (10:1 DCM–acetone) gave the
D
-mannopyranosyl-(1?2)-tetrakis[-3-O-benzyl-4-(2,4-di-O-acetyl-
3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-
-glycero-tetrona-
-mannopyranoside (26)
a-D-mannopyra-
nosyl]-3-O-benzyl-4-(2,4-di-O-acetyl-3deoxy-
mido)-4,6-dideoxy-
L
a-D
intermediate 24, 3-O-Benzyl-4-(2,4-di-O-acetyl-3-deoxy-
ro-tetronamido)-4,6-dideoxy-2-O-methyl- ,b- -mannopyranose
(800 mg, 75%). TOF–MS: m/z: 476.1 [M+Na]+.
DBU (0.08 mL, 0.5 mmol) was added with stirring at 0° C to a
solution of 24 (2.2 g, 4.9 mmol) and CCl3CN (2.5 mL, 24 mmol) in
DCM (30 mL), and the mixture was allowed to warm up to room
temperature. After 3 h, the mixture was concentrated and chroma-
tography (3:1, hexane–acetone with 1% Et3N) afforded 3-O-benzyl-
L-glyce-
a
D
A solution of TMSOTf in toluene (0.03 M, 2 mL) was added with
stirring and exclusion of moisture to a mixture of 20 (500 mg,
0.22 mmol), 10 (300 mg, 0.44 mmol), and 4 Å molecular sieves
(300 mg) in dichloromethane (10 mL). The stirring was continued
overnight, when TLC (2:1 toluene–acetone) showed that only small
amount of the imidate remained unchanged. After neutralization
with Et3N (0.2 mL), filtration, and concentration of the filtrate,
the residue was chromatographed (3:1 toluene–acetone) to give
26 (450 mg, 73%). 1H NMR (600 MHz, CDCl3) d: 6.24–5.93 (m, 5H,
5NH), 5.40 (br s, 1H, H-2VI), 5.20–5.15 (m, 6H, H-20I–VI), 5.05–4.97
(3 s, 4H, H-1II–V), 4.69 (s, 2H, H-1I,VI), 4.63–4.43 (m, 12H, 6PhCH2),
4.30–4.01 (m, 22H, H-2II–V, H-4I–VI, H-40I–VI), 3.86 (br s, 1H, H-2I),
3.80–3.60 (m, 16H, H-3I–VI, H-5I–VI, OCH3, H-100a), 3.35 (m, 1H, H-
100b), 2.72–2.61 (m, 4H, CH2CH2), 2.33 (t, J = 7.2 Hz, H-500a,b), 2.26–
4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-
O-methyl- -mannopyranose-1-O-trichloroacetimidate (25, 2.5 g,
a
-D
86%). 1H NMR (600 MHz, CDCl3) d: 8.58 (s, 1H, NHCCl3), 7.36–7.27
(m, 5H, Ph), 6.27 (d, J1,2 = 2.0 Hz, 1H, H-1), 5.95 (d, J = 8.3 Hz, 1H,
NH), 5.16 (dd, J = 8.0 Hz and 4.7 Hz, 1H, H-20), 4.64–4.54 (ABq,
J = 12.0 Hz, 2H, PhCH2), 4.16–4.06 (m, 4H, H-3, H-5 and H-40),
3.93 (m, 1H, H-4), 3.63 (t, J = 2.5 Hz, 1H, H-2), 3.53 (s, 3H, OCH3),
2.18–2.03 (m, 8H, 2COCH3 and H-30), 1.23 (d, J5,6 = 6.4 Hz, 3H, H-
6); 13C NMR (150 MHz, CDCl3) d: 95.3 (C-1), 74.9 (C-2), 74.1 (C-
3), 71.5 (PhCH2), 71.0 (C-20), 70.2 (C-5), 59.2 (C-40), 59.2 (OCH3),
52.9 (C-4), 30.8 (C-30), 20.8 20.7 (2COCH3), 18.0 (C-6); TOF-HRMS,
m/z: calcd for C24H31N2O9NaCl3 [M+Na]+: 619.0993. Found
619.0969.
I–VI
1.99 (m, 51H, 13COCH3, H-30 ), 1.67–1.55 (m, 4H, H-200a,b, H-
400a,b), 1.43–1.31 (m, 2H, H-300a,b), 1.17–1.08 (m, 18H, H-6I-VI); 13C
NMR (150 MHz, CDCl3) d: 137.9 (Cq), 137.8 (Cq), 137.7 (Cq), 137.6
(Cq), 137.5 (Cq), 137.4 (Cq), 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.8, 127.6, 127.4, 100.5–99.6 (4C, C-1II–V), 98.6 (2C, C-
1
I,VI), 18.1–17.8 (6C, C-6I–VI); TOF–MS m/z: 1387 [M]2+, 2772
A solution of TMSOTf in toluene (0.03 M, 11 mL) was added with
exclusion of moisture to a stirred mixture of 25 (1.6 g, 2.68 mmol),
20 (4.0 g, 1.77 mmol), 4 Å molecular sieves (6.5 g) in DCM (100 mL),
and the stirring was continued overnight, when TLC (3:1 DCM–ace-
tone) showed that only small amount of the imidate 25 was pres-
ent. The mixture was neutralized with Et3N (1.0 mL), filtered, the
filtrate was concentrated, and chromatography (5:1 DCM–acetone)
gave the known11 hexasaccharide, 5-(methoxycarbonyl)pentyl 3-
[M+H]+, 2795 [M+Na]+.
4.10. 5-(Methoxycarbonyl)pentyl 4-(3-deoxy-L-glycero-tetrona-
mido)-4,6-dideoxy-
deoxy- -glycero-tetronamido)-4,6-dideoxy-
syl]-4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-
pyranoside (29)
a
-
D
-mannopyranosyl-(1?2)-tetrakis[-4-(3-
-mannopyrano-
-manno-
L
a-D
L
a-D
O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-
dideoxy- -mannopyranosyl-(1?2)-tetrakis[-3-O-benzyl-4-(2,4-
di-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy- -man-
nopyranosyl]-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-tetro-
-mannopyranoside 27 (3.3 g,
L-glycero-tetronamido)-4,6-
A solution of compound 26 (400 mg, 0.14 mmol) in DCM–MeOH
(1:10, 10 mL) was stirred under H2 with 5% Pd/C (400 mg) over-
night, then filtered and concentrated. Without further purification,
the residue was treated with 1.0 M NaOMe (1.0 mL) in MeOH
(10 mL) to complete deacylation. After neutralization with Amber-
lite IR-120, H+-resin, the mixture was filtered, the filtrate was con-
centrated, and the residue was chromatographed (11 DCM–MeOH)
to afford the known7 compound 29 (210 mg, 89%).
a
-D
L
a-D
L
namido)-4,6-dideoxy-2-O-methyl-a-D
yield 69%).
A stirred solution of 27 (3.3 g) in DCM–MeOH (1: 10, 70 mL) was
treated overnight with hydrogen in the presence of 5% Pd/C (2.0 g).
After filtration and concentration, the residue was chromato-
graphed (12:1 EtOAc–EtOH) to afford 5-(methoxycarbonyl)pen-
Acknowledgment
tyl-4-(2,4-di-O-acetyl-3-deoxy-
-mannopyranosyl-(1?2)-tetrakis[-4-(2,4-di-O-acetyl-3-deoxy-
glycero-tetronamido)-4,6-dideoxy- -mannopyranosyl]-4-(2,4-di-O-
acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy-2-O-methyl-
L-glycero-tetronamido)-4,6-dideoxy-
a-D
L-
This research was supported by the Intramural Research Pro-
gram of the NIH, NIDDK.
a-D
L
a-
D
-mannopyranoside 30 (2.25 g, 87%). TOF–MS m/z: 1074 [M]2+
,
References
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ted with ethylenediamine (17 mL) at 50 °C overnight. After concen-
tration, chromatography (1:1:0.1 MeOH–DCM–25% NH4OH)
afforded the known20 31 (1.3 g, 75%). 1H NMR (400 MHz, D2O) d:
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