Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide

Article abstract of DOI:10.1016/j.carres.2008.03.037

The title rhamnooligosaccharides [α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-1-O-(CH₂)₅COOMe, α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-1-O-(CH₂)₅COOMe, α-d-Rhap4NAc-(1→2)-α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→3)-α-d

Full text of DOI:10.1016/j.carres.2008.03.037

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1693–1706
Synthesis of spacer-equipped di-, tri-, and the
tetrasaccharide fragments of the deacetylated O-PS1 of
Citrobacter gillenii O9a,9b, and a related pentasaccharide
´ˇ*
Rina Saksena, Anatoli Chernyak and Pavol Kovac
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 29 February 2008; received in revised form 20 March 2008; accepted 30 March 2008
Available online 4 April 2008
Abstract—The title rhamnooligosaccharides [a-D-Rhap4NAc-(1?3)-a-D-Rhap4NAc-1-O-(CH2)5COOMe, a-D-Rhap4NAc-(1?3)-
a-D-Rhap4NAc-(1?3)-a-D-Rhap4NAc-1-O-(CH2)5COOMe, a-D-Rhap4NAc-(1?2)-a-D-Rhap4NAc-(1?3)-a-D-Rhap4NAc-(1?3)-
a-D-Rhap4NAc-1-O-(CH2)5COOMe, and a-D-Rhap4NAc-(1?3)-a-D-Rhap4NAc-(1?2)-a-D-Rhap4NAc-(1?3)-a-D-Rhap4NAc-
(1?3)-a-D-Rhap4NAc-1-O-(CH2)5COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,
6-dideoxy-2-O-benzyl-a-D-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final
products were fully characterized as corresponding per-O-acetyl derivatives.
Published by Elsevier Ltd.
Keywords: Citrobacter gillenii; Oligosaccharide synthesis; D-4-NAc-rhamnooligosaccharides
1. Introduction
O9a,9b LPS yielded PS1 and PS2 in 11.4% and 18%
yield, respectively.
Citrobacter gillenii O9a,9b is one of the Citrobacter
strains that are found in the intestinal tract of some ver-
tebrates, and may be associated with meningitis, brain
abscesses, and neonatal sepsis in humans.1 A character-
istic feature2 of C. gillenii O9a,9b is the presence of two
different O-polysaccharides (PS) as parts of the lipopoly-
saccharide (LPS). One of them (PS2) is a homopolymer
composed of a-(1?2)-linked 4-acetamido-4,6-dideoxy-
a-D-mannopyranose (N-acetyl-D-perosamine, D-Rha
p4NAc) residues. The other one (PS1) is also built up
of N-acetyl-D-perosamine, and its tetrasaccharide
repeating unit has the structure: ?3)-a-D-Rhap4NAc-
(1?2)-a-D-Rhap4NAc-(1?2)-a-D-Rhap4NAc-(1?3)-
a-D-Rhap4NAc-(1?. The NMR data suggest2 that the
O-PS1 is partially 2-O-acetylated, and that the degree
of acetylation of the ?3-linked N-acetyl-D-perosamine
residues is ꢀ70%. Gel-permeation chromatography of
products of mild acid hydrolysis of the C. gillenii
Rabbit antiserum raised against C. gillenii O9a,9b
bacteria reacted with PS1 in Ouchterlony double immu-
nodiffusion test and immunoblotting. PS2 and LPS from
Vibrio cholerae O:1, which has a structurally related O-
PS, were not reactive with the same sera. It was rea-
soned2 that the unreactivity was due to the small molec-
ular size of PS2, and to the different N-acyl group
present in the O-PS from V. cholerae O:1. The structure
of the tetrasaccharide repeating unit of PS1 lacking the
O-acetyl group is the same as the tetrasaccharide part of
a pentasaccharide repeating unit from the O-chain PS of
Escherichia hermanii,3 the bacteria often isolated from
infected wounds. It is noteworthy, in this context, that
the serological reactivity of the LPS of C. gillenii
O9a,9b was not affected by O-deacetylation.2
2. Results and discussion
This laboratory has been involved in developing a con-
jugate vaccine for cholera using synthetic fragments of
*
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301496
1685; e-mail: kpn@helix.nih.gov
0008-6215/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.carres.2008.03.037

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R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
the O-PS of V. cholerae O:1 as the antigenic compo-
nent.4–9 The considerable structural similarity between
the O-PS of V. cholerae O:1 and C. gillenii O9a,9b lies
in the presence of the (1?2)-a-linked D-perosamine in
both O-PSs. The two polysaccharides differ in the N-acyl
groups present, that is, acetyl in C. gillenii O9a,9b as
compared to 3-deoxy-L-glycero-tetronyl group in V.
cholerae O:1, and in absence of the (1?3)-interglycosi-
dic linkages and O-acetyl groups in V. cholerae O:1.
The usefulness of pure, well-defined fragments of the
O-PS of C. gillenii O9a,9b as probes for inhibition stud-
ies and studies of cross-reactivity as well as our experi-
ence in the chemical synthesis of perosamine-
containing oligosaccharides10–15 prompted us to synthe-
size fragments that mimic partial structures of the O-PS
of C. gillenii O9a,9b.
by combining advantages of each of the previous two
protocols leading to it.20,21 We used mesylate 1, recom-
mended by Eis and Ganem20 but prepared by an im-
20
proved procedure,19 and treated it with NaN3 in the
presence of a crown ether, as used by Bundle et al.21
with methyl 6-deoxy-1,2-O-isopropylidene-4-O -trifluo-
romethanesulfonyl-a-D-mannopyranoside. In this way,
we were able to readily introduce the azido function
while bypassing the need to use the better, trif-
luoromethanesulfonyloxy leaving group,21 which would
be cost prohibitive when working on a large scale. Un-
like with the conversion in the absence of the crown
ether, only slight discoloration of the reaction mixture
occurred during the displacement reaction, and the crys-
talline azide 2 was obtained consistently in 80–90%
yield.
Because O-acetyl groups are not present in the O-PS
of V. cholerae O:1, and their presence in the O-PS of C.
gillenii O9a,9b was found non-essential for serology,2 we
designed the synthetic schemes to yield non-acetylated
oligosaccharides (Schemes 1–4). To obtain the tetrasac-
charide repeating unit and the two oligosaccharide frag-
ments from which it is formed, we chose a stepwise
synthetic strategy starting with the linker (spacer)-
equipped glycosyl acceptor 12. In this way, as the linker
chosen can be readily transformed into derivatives suit-
able for conjugation, the final oligosaccharides were ob-
tained in form amenable to both inhibition/binding
studies and preparation of neoglycoconjugates by a vari-
ety of protocols.16–18
The hitherto unknown compound 12 was obtained
from mesylate 119 following a series of known conver-
sions, where a number of minor modifications led to im-
proved yields of desired intermediates and/or made the
individual steps experimentally less demanding. Conse-
quently, the known20,21 azido compound 2 was prepared
Precursors to glycosyl donors bearing selectively
removable protecting groups at position O-2 or O-3,
benzyl ethers 3 and 4, which were required for the syn-
thesis of oligosaccharides containing (1?2)- and
(1?3)-linkages, were obtained by selective benzylation
of 2 byphase-transfer catalysis and benzylation via stan-
nylation, respectively. The reaction time of the phase-
transfer mediated alkylation could be shortened, and
the yield of the conversion22 2?3 + 4 could be im-
proved by using a larger relative amount of the phase-
transfer catalyst. The mixture was resolved by column
chromatography, and the 2-benzyl ether was now ob-
tained crystalline. Some amount of the 3-benzyl ether
4 was obtained from this reaction but the bulk of this
compound was obtained by benzylation of the 2,3-
stannylene acetal of 2. However, instead of applying
the original procedure20 leading to 4, which required re-
moval of large amount of unchanged BnBr, we em-
ployed the benzylation protocol by Nashed23 (cf.
Section 3). Acetolysis of 3 gave the hitherto unknown
1,3-di-O-acetyl derivatives 5 (a) and 6 (b). The 1,3-di-
O-acetyl derivative 5 and its 1,2-O-acetylated analog 7,
prepared as described,14,21 were subsequently trans-
formed to the corresponding 1-thioglycosides 8 and
10,14,21 respectively. The b anomer 9, formed when 5
was treated with ethanethiol and isolated by chromatog-
raphy along with 8, was also fully characterized.
OR
H3C
O
R1
R2O
OR
O
8
+ 12
H3C
R1
O
5-Methoxycarbonylpentyl glycoside 12 was prepared
COOMe
O
´
from acetate 11 by Zemplen deacetylation. The latter
was obtained from 6 and 5-methoxycarbonylpentanol24
by SnCl4-mediated25 glycosylation, essentially as de-
scribed for the preparation of the similar 3-O-benzyl
derivative.7 It is worth noting that the conversion
6?11 was fast and afforded a high yield (ꢀ80%) of
the 1,2-trans glycoside, even though the reaction could
not involve the acetoxonium ion intermediate, which
was the case in previous situations involving a partici-
pating group at O-2.7,25
R1
R2
R
N3
N3
13
Bn
Ac
14
15
16
17
Bn
Bn
H
H
NHAc
NHAc
NHAc
H
H
Ac
Ac
Oligosaccharides 16, 21, and 26 were synthesized
(Schemes 1–3) by NIS and silver triflate-mediated con-
Scheme 1.

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1695
OR2
O
´
(13, 18, and 23) were deacetylated (Zemplen), to afford
glycosyl acceptors (14, 19, and 24) for further extension
of the oligosaccharide chain. It is noteworthy that the
fast-atom-bombardment (FAB) mass-spectrum of com-
pound 13 showed a peak at m/z 685.4, instead of the ex-
pected peak at m/z 710([M+H] +). It reflected reduction
of one azido group to an amino group during the course
of mass-spectrometric analysis.26 The same phenome-
non was observed with all compounds containing more
than one azido group.
H3C
R4
R3O
R
R1
R1
R2
R3
R4
R
1
2
3
4
5
6
7
H
H
OMe
OMe
OMe
OMe
OAc
H
H
H
H
H
OMs
N3
While the one-pot conversion of a single azide group
to the N-acetamido group by catalytic hydrogenolysis
in the presence of Ac2O and MeOH works well,27 similar
conversions described here with compounds containing
multiple azido groups were achieved more cleanly using
a two-step process. The azido groups in 14, 19, and 24
were first reduced with H2S in aqueous pyridine,21,28
and subsequent N-acetylation with acetic anhydride in
the presence of methanol gave the acetamido derivatives
15, 20, and 25. Catalytic hydrogenolysis of the foregoing
substances then gave the target oligosaccharide frag-
ments 16, 21, and 26.
The purpose of making the related pentasaccharide 31
was its use in inhibition and other immunochemical
studies with antibodies specific for the O-PS of C. gillenii
O9a,9b. It was obtained by coupling of donor 8 with tet-
rasaccharide acceptor 24, to give fully protected penta-
saccharide 28, followed by conversions (Scheme 4)
similar to those described above.
N3
H
Bn
H
H
N3
H
Bn
Ac
Ac
Bn
N3
H
Bn
Bn
Ac
N3
OAc
H
N3
OAc
N3
8
H
SEt
Bn
Ac
SEt
H
H
Ac
Bn
N3
N3
N3
N3
Bn
Ac
9
10
SEt
11
12
H
H
COOMe
COOMe
Bn
Bn
Ac
H
O
O
The molecular mass of the deprotected oligosaccha-
rides was verified by mass spectrometry and their
structures follow from the mode of synthesis. The
densations of thioglycoside donors 8 or 1014,21 with
acceptors 12, 14, and 19. The products thus obtained
OR2
O
H3C
RO
R1
OR
O
H3C
R1
10
+ 14
O
OR
O
H3C
R1
O
COOMe
O
R1
R2
R
N3
18
Bn
Ac
N3
19
20
Bn
Bn
H
H
NHAc
21
22
H
NHAc
NHAc
H
Ac
Ac
Scheme 2.

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R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
OR
H3C
O
R1
R2O
O
H3C
O
R1
RO
OR
H3C
O
R1
O
8
+ 19
OR
H3C
O
R1
O
COOMe
O
R1
R2
R
N3
N3
23
24
25
Bn
Bn
Bn
Ac
H
NHAc
H
26
27
H
NHAc
NHAc
H
Ac
Ac
Scheme 3.
OR
O
H3C
R1
R2O
OR
O
H3C
R1
O
O
H3C
RO
O
R1
OR
O
8
+ 24
H3C
R1
O
OR
O
H3C
R1
O
COOMe
O
R1
R2
R
N3
N3
Bn
Bn
Ac
H
28
29
30
31
32
Bn
H
NHAc
NHAc
NHAc
H
H
Ac
Ac
Scheme 4.

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1697
compounds were fully characterized as per-O-acetates
17, 22, 27, and 32 whose NMR data were fully consis-
tent with their structures.
solutions in water (HPLC quality) were passed through
the Anotop syringe filter (Whatman, 0.2 lm porosity).
Solutions in organic solvents other than alcohols were
dried with anhydrous Na2SO4, and concentrated at
<40 °C/2 kPa.
3. Experimental
3.2. General procedure for the NIS/AgOTf-mediated
glycosylations
3.1. General methods
Unless stated otherwise, optical rotations were measured
at ambient temperature for solutions in chloroform
(c ꢀ 1), with a Perkin–Elmer automatic polarimeter,
Model 341 or with a Jasco automatic polarimeter, Model
P-2000. All reactions were monitored by thin-layer chro-
matography (TLC) on Silica Gel 60 coated glass slides
(Whatman or Analtech). Column chromatography was
performed by gradient elution from columns of silica
gel. Solvent mixtures less polar than those used for
TLC were used at the onset of development. NMR spec-
tra of monosaccharide derivatives were measured at
22 °C, at 300 MHz (1H) and 75 MHz (13C), with a Varian
Mercury spectrometer. NMR spectra of oligosaccharides
were measured at 300 or 600 MHz (1H) and 75 or
150 MHz (13C), with a Varian Mercury or Bruker
Avance spectrometers. All assignments were supported
by homonuclear and heteronuclear 2-dimensional corre-
lation spectroscopy, run with the software supplied with
the spectrometers. When reporting assignments of NMR
signals of oligosaccharides, sugar residues in oligosac-
charides are serially numbered, beginning with the one
bearing the aglycon, and are identified by a Roman nu-
meral superscript in listings of signal assignments. When
reporting NMR data, the nuclei belonging to the spacer
aglycon are denoted with a prime (0). 1H NMR spectra of
some compounds containing NHAc groups showed pres-
ence of isomers and/or considerable broadening of sig-
nals, and coupling constants in such cases are not
reported. When presence of isomers was evident, only
resonances characteristic of the more abundant isomer
are reported. Liquid chromatography–electron spray-
ionization mass spectrometry (ESI-MS) was performed
with a Hewlett–Packard 1100 MSD spectrometer. At-
tempts have been made to obtain correct analytical data
for all new compounds. However, some compounds
tenaciously retained traces of solvents, despite exhaustive
drying, and analytical figures for carbon could not be ob-
tained within 0.4%. The [a]D values reported for such
materials may not be quite accurate. Structures of these
compounds follow unequivocally from the mode of syn-
thesis and m/z values found in their mass spectra, and
TLC and NMR spectroscopy verified their purity. Palla-
dium-on-charcoal catalyst (5%, ESCAT 103) was a prod-
uct of Engelhard Industries. 1,2-Di-O-acetyl-4-azido-3-
O-benzyl-4,6-dideoxy-a-D-mannopyranoside (7) was
prepared as described.21 Before final freeze-drying of
the deprotected oligosaccharides 16, 21, 26, and 31, their
A mixture of glycosyl donor (1.3 mmol), acceptor
(1 mmol) and finely powdered 4 A molecular sieves
˚
(0.5 g) in CH2Cl2 (10–15 mL) was stirred under argon
for 15 min. The mixture was cooled to ꢀ10 °C, and solid
NIS (1.4 mmol) was added, followed by a solution of
AgOTf (0.4 mmol) in toluene (4 mL). The stirring was
continued for 3 min at the same temperature, cooling
was terminated, and when TLC showed that the reac-
tion was complete (ꢀ15 min) the mixture was neutral-
ized with Et3N, partitioned between CH2Cl2 and satd
aq NaHCO3, concentrated, and the residue was purified
by chromatography.
3.3. Methyl 4-azido-4,6-dideoxy-a-D-mannopyranoside
(2)
A mixture of methyl 4-O-methanesulfonyl-6-deoxy-
a-D-mannopyranoside19 (1, 142.2 g, 0.555 mol), NaN3
(87 g, 1.338 mol), and dicyclohexano-15-crown-5 (3.7 g,
16.8 mmol) in DMF (1 L) was heated with vigorous stir-
ring at 100 °C until TLC (3:1 hexane–acetone) showed
that all starting material was consumed. DMF was evap-
orated from the yellow reaction mixture, the residue was
treated with CH2Cl2, and the mixture was filtered through
a Celite pad. The filtrate was concentrated and the residue
was purified by chromatography to give, after crystalliza-
tion from isopropyl ether–hexane, 95.5 g (85%) of pure 2,
mp 84–85 °C, Ref. 20 mp 83–84 °C, 72% yield. CIMS m/z
221 [M+NH4]+.
3.4. Methyl 4-azido-2-O-benzyl-4,6-dideoxy-a-D-manno-
pyranoside (3) and methyl 4-azido-3-O-benzyl-4,6-dide-
oxy-a-D-mannopyranoside (4)
3.4.1. A mixture of 2 (20 g, 98.4 mmol) and Bu4NBr
(2.7 g, 8.375 mmol), CH2Cl2 (300 mL), aqueous 20%
NaOH (300 mL), and BnBr (12 mL, 98.4 mmol) was vig-
orously stirred overnight, when TLC (3:1 hexane–
EtOAc) showed almost complete conversion of the start-
ing material. The phases were separated, the aqueous
phase was extracted with CH2Cl2, and the combined or-
ganic phase was washed with water and concentrated.
Chromatography gave first methyl 4-azido-2,3-di-O-benz-
yl-4,6-dideoxy-a-D-mannopyranoside (2.95 g, 7.8%),
[a]D +60.4 (c 0.9), Ref. 22 [a]D +65.9 (c 2.5). CIMS:
m/z 401 [M+NH4]+.

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R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
Eluted next was the 2-O-benzyl derivative 3 (18.97 g,
2.11, 2.00
(2s, 3H each, 2COCH3), 1.40 (d, 3H,
65%), mp 35–36 °C (from hexane), [a]D +6.4 (c 0.7),
Ref. 22 [a]D +8.54 (c 5.2), for amorphous 3 obtained in
56% yield. CIMS: m/z 311 [M+NH4]+. Anal. Calcd for
C14H19N3O4: C, 57.33; H, 6.53; N 14.33. Found: C,
57.08; H, 6.46; N, 14.36.
Eluted last was the 3-O-benzyl derivative 4, (4.75 g,
16.5%), CIMS: m/z 311 [M+NH4]+. 1H NMR data
recorded for 3 and 4 agreed with those reported.20–22
J5,6 = 6.0 Hz, H-6). 13C NMR (75 MHz, CDCl3): d 92.14
(C-1), 74.94 (CH2Ph), 74.32 (C-3), 73.91 (C-2), 71.84 (C-
5), 61.81 (C-4), 20.79, 20.63 (2CO CH3), 18.20 (C-6).
3.6. Ethyl 3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-1-
thio-a-(8) and b-D-mannopyranoside (9)
Ethanethiol (7.8 mL, 105 mmol) followed by BF3ꢂEt2O
(10.3 mL, 83.7 mmol) was added to a solution of 514,21
(30 g, 82.6 mmol) in dry CH2Cl2 (660 mL), which had
3.4.2. Bu2SnO (40.0 g, 164.7 mmol) was added to a solu-
tion of 2 (33 g, 162.4 mmol) in MeOH (ꢀ1 L) and the mix-
ture was heated under reflux until a clear solution was
obtained (ꢀ1–2 h). Toluene (ꢀ100 mL) was added and
solvents were evaporated. After the residue had been dried
in a vacuum oven at 40 °C for 2 h, it was dissolved in DMF
(200 mL), benzyl bromide (38 mL, 317 mmol) and Bu4NI
(1 g) was added, and the mixture was heated at ꢀ100 °C
until TLC showed complete disappearance of the starting
material (4–6 h). After concentration, excess of BnBr was
evaporated at 60 °C/133 Pa, and the residue was purified
by chromatography to give the desired compound 4 as a
light-yellow thick oil (40 g, 84%), which was identical with
the material described above.
˚
been stirred with molecular sieves (4 A, 3 g) for
15 min. The stirring was continued for 20 min at room
temperature, when TLC (10:1 hexane–EtOAc) showed
that the reaction was complete. After neutralization
with satd aq NaHCO3, the product was extracted with
CH2Cl2, the organic layer was washed with water and
concentrated. The residue was purified by chromatogra-
phy to give first the a-anomer 8 (25 g), [a]D +123 (c 1.6).
1H NMR (300 MHz, CDCl3): d 5.30 (d, 1H, J1,2
1.2 Hz, H-1), 5.04 (dd, 1H, J2,3 = 3.3, J3,4 = 10.3 Hz,
=
2
H-3), 4.71, 4.54 (2d, 1H each, J = 12.3 Hz, CH2Ph),
4.04–3.96 (m, 2H, H-2,5), 3.68 (t, 1H, J = 10.2 Hz, H-
4), 2.70–2.51 (m, 2H, CH2CH3), 2.07 (s, 3H, COCH3),
1.37 (d, 3H, J5,6 = 6.2 Hz, H-6), 1.27 (t, 3H, J =
7.3 Hz, CH2CH3). 13C NMR (75 MHz, CDCl3): d
81.53 (JC-1,H-1 = 164.2 Hz, C-1), 76.34 (C-2), 72.71 (C-
3), 72.49 (CH2Ph), 67.22 (C-5), 62.84 (C-4), 25.22
(CH2CH3), 20.78 (COCH3), 18.25 (C-6), 14.74
(CH2CH3). CIMS: m/z 383 [M+NH4]+. Anal. Calcd
for C17H23N3O4S: C, 55.87; H, 6.34; N, 11.50; S, 8.77.
Found: C, 55.99; H, 6.23; N, 11.54; S, 8.75.
3.5. 1,3-Di-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-a-
(5) and b-D-mannopyranose (6)
A solution of 3 (19.86 g, 67.7 mmol) in Ac2O–AcOH–
H2SO4 (50:20:0.01, 480 mL) was kept at room tempera-
ture for 45 min, when TLC (40:1 toluene–acetone)
showed that the reaction was complete. NaOAc trihy-
drate wasadded to neutralize H2SO4, and the pH was ad-
justed to ꢀ7 with aqueous Na2CO3 and NaHCO3. The
mixture was extracted with dichloromethane to give,
after concentration, crystalline residue 5 (23 g, 93%),
mp 60–62 °C (from ethanol), [a]D +49.5 (c 0.7). 1H
NMR (300 MHz, CDCl3): d 6.12 (d, 1H, J1,2 = 2.0 Hz,
H-1), 5.05 (dd, 1H, J2,3 = 3.2, J3,4 = 10.4 Hz, H-3),
Eluted later was the b-anomer 9 (2.2 g), [a]D ꢁ95 (c 1.6).
1H NMR (300 MHz, CDCl3): d 4.79 (dd, partially over-
lapped, J2,3 = 3.2, J3,4 = 10.4 Hz, H-3), 4.80, 4.62 (2d,
partially overlapped, 2J ꢀ 12.0 Hz, CH2Ph), 4.60 (d, par-
tially overlapped, J1,2 ꢀ 1.0 Hz, H-1), 4.03 (dd, 1H, H-2),
3.65 (t, 1H, H-4), 3.31–3.22 (m, 1H, H-5), 2.71 (q, 2H, J =
7.4 Hz, CH2CH3), 1.98 (s, 3H, COCH3), 1.40 (d, 3H,
J5,6 = 6.0 Hz, H-6), 1.29 (t, 3H, J = 7.5 Hz, CH2CH3).
2
4.73, 4.55 (2d, 1H each, J = 12.5 Hz, CH2Ph), 3.81
(dd, 1H, H-2), 3.74–3.63 (m, 2H, H-4,5), 2.11, 2.06 (2s,
3H each, 2COCH3), 1.36 (d, 3H, J5,6 = 6.0 Hz, H-6).
13C NMR (75 MHz, CDCl3): d 90.93 (C-1), 73.41 (C-2),
72.90 (CH2Ph), 71.85 (C-3), 69.44 (C-5), 62.04 (C-4),
20.95, 20.77 (2CO CH3), 18.39 (C-6). CIMS: m/z 381
[M+NH4]+. Anal. Calcd for C17H21N3O6: C, 56.19; H,
5.83; N, 11.56. Found: C, 56.43; H, 5.82; N, 11.62.
The mother liquor was purified by chromatography,
to give first 400 mg of the a-anomer 5, total yield, 95%.
Eluted next was a small amount of material that was
identified by NMR spectroscopy as the b-anomer 6,
13C NMR (75 MHz, CDCl3): d 84.31 (JC-1,H-1
=
150.6 Hz, C-1), 77.41 (C-2), 76.18 (C-3), 76.08 (CH2Ph),
75.65 (C-5), 62.60 (C-4), 27.00 (CH2CH3), 21.03
(COCH3), 18.95 (C-6), 15.34 (CH2CH3). CIMS: m/z 383
[M+NH4]+. Anal. Calcd for C17H23N3O4S: C, 55.87; H,
6.34; N, 11.50; S, 8.77. Found: C, 56.17; H, 6.26; N,
11.52; S, 8.76.
The intermediate, mixed fraction (1.85 g) was purified
by chromatography, to give more 8 (665 mg, total yield,
85%) and 9 (625 mg, total yield, 9.4%).
1
[a]D ꢁ12.7 (c, 5.1). H NMR (300 MHz, CDCl3): d 5.65
3.7. 5-Methoxycarbonylpentyl 4-azido-2-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside (12)
(d, 1H, J1,2 = 0.9 Hz, H-1), 4.83, 4.65 (2d, partially over-
2
lapped, J = 12.2 Hz, CH2Ph), 4.78 (dd, partially over-
lapped, J2,3 = 3.2, J3,4 = 10.5 Hz, H-3), 4.01 (br d, 1-H,
H-2), 3.64 (t, J = 10.0 Hz, H-4), 3.44–3.34 (m, 1H, H-5),
SnCl4 (3.5 mL, 29.5 mmol) was added to a solution of 5
(8.25 g, 22.7 mmol) in dry CH2Cl2 (200 mL) and the

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1699
mixture was stirred for 20 min with exclusion of
atmospheric moisture. A solution of 5-(methoxycar-
bonyl)pentanol24 (4.3 g, 29.5 mmol) in dry CH2Cl2
(30 mL) was added to a slightly yellow reaction mixture
and, when the reaction was complete (ꢀ40 min, TLC in
40:1 toluene–acetone), the mixture was neutralized with
satd aq NaHCO3. The product was extracted with
CH2Cl2 and, after concentration, chromatography gave
5-methoxycarbonylpentyl 3-O-acetyl-4-azido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranoside (11, 8.28 g, 80%).
Section 3.2, to give after chromatography (10:1?5:1
hexane–EtOAc) 13 (9.385 g, 89%), [a]D +65 (c 0.8).
1H NMR (300 MHz, C6D6):
d
5.63 (dd, 1H,
J2,3 = 3.2, J3,4 = 10.0 Hz, H-3II), 5.28 (d, 1H,
J1,2 = 1.7 Hz, H-1II), 4.81 (d, 1H, J1,2 = 1.7 Hz, H-1I),
4.61, 4.48 (2dd, 1H each, 2J = 11.8 and 12.1 Hz,
2CH2Ph), 4.22 (dd, 1H, H-2II), 4.14 (dd, 1H,
J2,3 = 3.2, J3,4 = 9.8 Hz, H-3I), ꢀ3.87 (m, partially
overlapped, H-5II), 3.81 (t, partially overlapped,
H-4II), 3.76 (dd, 1H, H-2I), 3.71–3.55 (m, 2H,
H-4I,5I), 3.47, 3.44 (2t, 1H, J = 6.4 Hz, H-10a), 3.38
(s, 3H, COOCH3), 3.13, 3.09 (2t, 1H, H-10b), 2.06 (t,
2H, J = 7.4 Hz, H-50), 1.74 (s, 3H, COCH3), 1.52–
1.42 (m, 2H, H-40a,b), 1.35–1.09 (m, 10H, H-20a,
b,30a,b, incl. 2d at 1.24, 1.22, J5,6 = 6.1 Hz, H-6I,II).
13C NMR (75 MHz, C6D6): d 100.34 (C-1II), 97.88
(C-1I), 78.39 (C-3I), 78.15 (C-2I), 76.25 (C-2II), 73.72,
73.43 (2CH2Ph), 72.85 (C-3II), 68.64 (C-5II), 68.20 (C-
5I), 68.05 (C-10), 65.53 (C-4I), 63.55 (C-4II), 51.38
(COOCH3), 34.20 (C-50), 29.60 (C-20), 26.25 (C-30),
25.19 (C-40), 20.79 (COCH3), 18.97 (C-6I), 18.84
(C-6II). FABMS: m/z 685.4 [M+Hꢁ2N+2H]+,
843.3 [M+Cs]+, 717.1 [M+Li]+. Anal. Calcd for
C35H46N6O10: C, 59.14; H, 6.52; N, 11.82. Found: C,
58.95; H, 6.52; N, 11.72.
1H NMR (300 MHz, CDCl3): d 5.09 (dd, 1H, J2,3
=
3.3, J3,4 = 10.3 Hz, H-3), 4.73 (d, 1H, J1,2 = 1.8 Hz, H-
2
1), 4.66, 4.57 (2d, 1H each, J = 12.1 Hz, CH2Ph), 3.80
(dd, 1H, H-2), 3.67 (s, partially overlapped, COOCH3),
3.67–3.43 (m, partially overlapped, H-4,5,10a), 3.37–3.34
(2t, 1H, J = 6.5 Hz, H-10b), 2.31 (t, 2H, J = 7.3 Hz, H-
50a,b), 2.07 (s, 3H, COCH3), 1.71–1.51 (m, 4H, H-
40a,b,20a,b), 1.39–1.31 (m, 5H, H-30a,b, incl. d, 1.35,
J5,6 ꢀ 5.8 Hz, H-6). 13C NMR (75 MHz, CDCl3): d
97.57 (JC-1,H-1 = 169.1 Hz, C-1), 75.00 (C-2), 73.14
(CH2Ph), 72.53 (C-3), 67.53 (C-10), 66.82 (C-5), 62.74
(C-4), 51.43 (COOCH3), 33.84 (C-50), 28.92 (C-20),
25.58 (C-30), 24.57 (C-40), 20.89 (COCH3), 18.38 (C-6).
FABMS: m/z 450 [M+H]+, 472 [M+Na]+.
A solution of the foregoing acetylated compound 11
(5.66 g, 15.58 mmol) in MeOH (150 mL) was treated
with 1 M NaOMe (ꢀ5 mL). After 18 h, TLC (5:1 hex-
ane–EtOAc) showed that the deacetylation of 12 was
complete. The solution was neutralized with Amberlite
IR-120 (H+) cation-exchange resin, concentrated, and
chromatography of the residue gave 12 (4.38 g, 85%),
3.9. 5-Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-4-azido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranoside (14)
´
De-O-acetylation (Zemplen) of 13 and chromatogra-
1
[a]D +19.4 (c 4). H NMR (300 MHz, CDCl3): d 4.80
phy (15:1 toluene–EtOAc) gave 14 (4.23 g, 94%), [a]D
+54 (c 1.2). 1H NMR (C6D6, 300 MHz): d 5.22 (d,
1H, J1,2 = 1.8 Hz, H-1II), 4.81 (d, 1H, J1,2 = 1.8 Hz,
H-1I), 4.50, 4.40 (2dd, 2H each, 2J = 11.9 Hz,
2CH2Ph), 4.12 (dd, partially overlapped, 1H,
J3,4 = 3.1, J2,3 = 9.7 Hz, H-3I), ꢀ4.11 (ddd, partially
overlapped, H-3II), 3.90 (dd, 1H, H-2II), 3.81–3.75
(m, partially overlapped, H-2I,5II), 3.72 (t, partially
overlapped, J = 9.7 Hz, H-4I), 3.66–3.56 (m, 1H,
H-5I), 3.34 (t, partially overlapped, J = 9.9 Hz,
H-4II), 3.48, 3.45 (2t, 1H, J = 6.2 Hz, H-10a), 3.37 (s,
3H, COOCH3), 3.12, 3.09 (2t, 1H, H-10b), 2.17 (d,
1H, J3,OH = 10.1 Hz, OH), 2.06 (t, 2H, J = 7.4 Hz,
H-50a,b), 1.52–1.42 (m, 2H, H-40a,b), 1.37–1.09 (m,
10H, H-20a,b, 30a,b, incl. 2d at 1.28, 1.23,
(d, 1H, J1,2 = 1.5 Hz, H-1), 4.73, 4.57 (2d, 1H each,
2J = 11.7 Hz, CH2Ph), 3.84 (ddd, 1H, J2,3 = 3.7,
J3,4 = 10.1 Hz, H-3), 3.67 (dd, partially overlapped,
1H, H-2), 3.66 (s, partially overlapped, COOCH3),
3.63, 3.60 (2t, partially overlapped, J = 6.8 Hz, H-10a),
3.53–3.44 (m, 1H, H-5), 3.36, 3.33 (2t, 1H, H-10b),
3.26 (t, 1H, J = 9.9 Hz, H-4), 2.60 (d, 1H, J3,OH
=
10.3 Hz, OH), 2.30 (t, 2H, J = 7.4 Hz, H-50a,b), 1.68–
1.50 (m, 4H, H-40a,b,20a,b), 1.39–1.26 (m, 5H, H-30a,b,
incl. d, 1.31, J5,6 = 6.2 Hz, H-6). 13C NMR (75 MHz,
CDCl3): d 96.53 (C-1), 77.36 (C-2), 72.84 (CH2Ph),
70.22 (C-3), 67.28 (C-10), 66.39 (2C, C-4,5), 51.36
(COOCH3), 33.73 (C-50), 28.85 (C-20), 25.51 (C-30),
24.47 (C-40), 18.24 (C-6). FABMS: m/z 408 [M+H]+,
430 [M+Na]+. Anal. Calcd for C20H29N3O6: C, 58.95;
H, 7.17; N, 10.31. Found: C, 59.10; H, 7.19; N,
10.23.
J5,6 = 6.2 Hz, H-6I,II
,
in that order). 13C NMR
(75 MHz, C6D6): d 100.05 (C-1II), 97.69 (C-1I), 78.96
(C-3I), 78.37 (C-2II), 78.13 (C-2I), 73.56, 73.16
(2CH2Ph), 71.25 (C-3II), 68.17 (C-5I), 68.13 (C-10),
68.09 (C-5II), 67.03 (C-4II), 65.69 (C-4I), 51.37
(COOCH3), 34.20 (C-50), 29.59 (C-20), 26.25 (C-30),
25.19 (C-40), 18.04 (C-6I), 18.88 (C-6II). FABMS:
m/z 643.4 [M+Hꢁ2N+2H]+, 691.4 [M+Na]+. Anal.
Calcd for C33H44N6O9: C, 59.57; H, 6.63; N, 12.57.
Found: C, 59.45; H, 6.76; N, 12.76.
3.8. 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-4-azido-
2-O-benzyl-4,6-dideoxy-a- D-mannopyranoside (13)
The glycosyl donor 8 (7.18 g, 19.65 mmol) and acceptor
12 (6.16 g, 15.12 mmol) were coupled as described in

1700
R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
3.10. 5-Methoxycarbonylpentyl (4-acetamido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-4-acetam-
ido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranoside (15)
due was eluted from a small column of silica gel using
5:1 CHCl3–MeOH as eluent to give, after freeze-drying
and additional drying at 40 °C/133 Pa, 16 (99.5 mg,
1
96%), [a]D +8.2 (c 1.7, H2O). Definite signals in the H
A gentle stream of H2S was passed through a solution of
14 (500 mg, 0.75 mmol) in 2:1 pyridine–water (5 mL) for
30 min at 40 °C. The mixture was stirred at this temper-
ature for 16 h, when TLC (10:1:0.1 CH2Cl2–MeOH–
concd NH4OH) showed that the reaction was complete.
A stream of nitrogen was passed through the reaction
mixture for 15 min, and the solution was co-concen-
trated with several co-evaporations of water to remove
pyridine. After co-evaporation with toluene, to remove
water, the residue was dissolved in MeOH (20 mL)
and Ac2O (1 mL) was added. After 16 h, TLC (3:2
CH2Cl2–acetone) showed that the reaction was com-
plete. The mixture was concentrated and chromatogra-
phy of the residue (2:1?1:1 CH2Cl2–acetone) gave 15
(390 mg, 75%), mp 176.5–177 °C (from acetone), [a]D
+35.4 (c 0.8). 1H NMR (600 MHz, ꢀ1:1 CDCl3–
C6D6): d 5.87 (d, 1H, J4,NH = 9.8 Hz, NHI), 5.18 (d,
1H, J4,NH = 9.2 Hz, NHII), 5.00 (d, 1H, J1,2 = 1.3 Hz,
H-1II), 4.80 (d, 1H, J1,2 = 2.0 Hz, H-1I), 4.78–4.50 (4d
partially overlapped, 2CH2Ph), 4.25 (q, 1H, J =
9.8 Hz, H-4I), 4.00 (q, 1H, J = 9.8 Hz, H-4II), 3.94 (dd,
NMR spectrum (600 MHz, D2O, 50 °C) were at d 5.18
(d, 1H, J1,2 = 1.7 Hz, H-1II), 5.08 (d, 1H, J1,2 = 1.8 Hz,
H-1I), 4.23 (dd, 1H, J2,3 = 3.0 Hz, H-2I), 4.19 (t,
J = 10.3 Hz, H-4I), ꢀ4.12 (dd, overlapped, H-3I),
ꢀ4.11 (m, overlapped, H-3II,5II), ꢀ4.09 (overlapped,
H-4II), ꢀ4.08 (overlapped, H-2II), ꢀ4.07 (m, H-5I),
3.98–3.94 (m, 4H, H-10a, incl. s at 3.96 for COOCH3),
3.80, 3.78 (2t, 1H, J = 6.0 Hz, H-10b), 2.67 (t, 2H,
J = 7.5 Hz, H-50a,b), 2.30, 2.29 (2s, 6H, 2NHCOCH3),
1.92–1.85 (m, 4H, H-40a,b,20a,b), 1.69–1.62 (m, 2H,
H-30a,b), 1.46 (d, 3H, J5,6 = 6.2 Hz, H-6I), 1.44 (d, 3H,
J5,6 = 5.8 Hz, H-6II). 13C NMR (D2O, 50 °C): d 180.41
(COOCH3), 177.67, 177.41 (2NHCOCH3), 105.26
(C-1II), 102.75 (C-1I), 79.99 (C-3I), 72.68 (C-2II), 72.33
(C-2I) 71.34, 71.27 (C-3II,5II), 70.96 (C-10), 70.60 (C-5I),
56.04 (C-4II), 55.20 (COOCH3), 55.07 (C-4I,II), 36.72
(C-50), 31.26 (C-20), 28.07 (C-30), 27.13 (C-40), 25.33,
25.25 (2COCH3), 19.96, 19.90 (C-6I,II). FABMS: m/z
543 [M+Na]+. The corresponding per-O-acetyl deriva-
tive 17 showed [a]D +58 (c 0.9, CHCl3). 1H NMR
(600 MHz, CDCl3): d 6.09 (d, 1H, J4,NH = 9.5 Hz,
NHI), 5.68 (d, 1H, J4,NH = 10.0 Hz, NHII), 5.18 (dd,
1H, J2,3 = 3.1 Hz, H-2I), 5.09 (dd, 1H, J2,3 = 3.3,
J3,4 = 10.9 Hz, H-3II), 4.99 (d, 1H, J1,2 = 2.0 Hz,
H-1II), 4.90 (dd, 1H, H-2II), 4.70 (d, 1H, H-1I), 4.18 (q,
partially overlapped, J = 10.1 Hz, H-4II), 4.13 (q, par-
tially overlapped, J = 10.9 Hz, H-4I), 4.07 (dd, 1H,
H-3I), 3.88 (m, 1H, H-5II), 3.77 (m, 1H, H-5I), 3.69–
3.66 (m, 4H, H-10a, incl. 3.67, s, COOCH3), 3.42, 3.40
(2t, 1H, J = 5.1 Hz, H-10b), 2.36 (m, 2H, H-50a,b), 2.23,
2.12, 2.01, 2.06, 1.96 (5s, 15H, 5COCH3), 1.67 (m, 3H,
H-20a,40a,b), 1.58 (m, 1H, H-20b), 1.49 (m, 1H, H-30a),
1.35 (m, 1H, H-30b), 1.26 (d, 3H, J5,6 = 6.2 Hz, H-6I),
1.23 (d, 3H, J5,6 = 6.3 Hz, H-6II). 13C NMR (150 MHz,
CDCl3): d 97.97 (C-1II), 97.25 (C-1I), 73.49 (C-3I),
70.45 (C-2I), 69.95 (C-2II), 68.84 (C-5II), 67.95 (C-3II),
67.63 (C-5I), 66.86 (C-10), 52.61 (C-4I), 51.58 (COOCH3),
51.34 (C-4II), 33.86 (C-50), 28.55 (C-20), 25.88 (C-30),
24.29 (C-40), 23.40, 23.33 (2 NHCOCH3), 21.20, 20.96,
20.81 (3COCH3), 17.92 (C-6II), 17.75 (C-6I). ESI-MS
(m/z): 647.3023; calcd for [M+H]+ 6747.3027. Anal.
Calcd for C29H46N2O14: C, 53.86; H, 7.17; N, 4.33.
Found: C, 54.04; H, 7.26; N, 4.29.
J2,3 = 3.1, J3,4 = 10.4 Hz, H-3I), 3.70 (dd, J2,3
=
3.4 Hz, H-2II), 3.69 (br t, H-2I), 3.67 (m, 2H, H-3II,5I),
3.60 (m, 1H, 10a), 3.45 (m, 1H, H-5II), 3.42 (s, 3H,
COOCH3), 3.27 (m, 1H, H-10b), 2.66 (br d, 1H, OH),
2.21–2.12 (m, 2H, H-50a,b), 1.80, 1.79 (2s, 6H,
2COCH3), 1.59–1.52 (m, 3H, H-40a,b,20a), 1.48–1.42
(m, 1H, H-20b), 1.40–1.29 (m, 1H, H-30a), 1.27 (d, 3H,
J5,6 = 6.0 Hz, H-6I), 1.25–1.19 (m, 1H, H-30b), 1.10 (d,
3H, J5,6 = 6.2 Hz, H-6II). 13C NMR (150 MHz, ꢀ1:1
CDCl3–C6D6): d 99.26 (C-1II), 97.12 (C-1I), 77.52 (C-
2II), 77.33 (C-2I), 76.66 (C-3I), 72.70, 72.01 (2CH2Ph),
69.41 (C-3II), 67.99 (C-5II), 67.84 (C-5I), 66.85 (C-10),
53.99 (C-4II), 52.68 (C-4I), 51.22 (COOCH3), 33.82
(C-50), 28.62 (C-20), 26.00 (C-30), 24.44 (C-40), 23.13,
23.08 (2COCH3), 18.09 (C-6I), 17.94 (C-6II). FABMS:
m/z 701 [M+H]+, 723 [M+Na]+. Anal. Calcd for
C37H52N2O11: C, 63.41; H, 7.49; N, 4.00. Found: C,
63.44; H, 7.39; N, 3.97.
3.11. 5-Methoxycarbonylpentyl (4-acetamido-4,6-dide-
oxy-a-D-mannopyranosyl)-(1?3)-4-acetamido-4,6-dide-
oxy-a-D-mannopyranoside (16) and 5-methoxycarbonyl-
pentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-a-D-
mannopyranosyl)-(1?3)-4-acetamido-2-O-acetyl-4,6-
dideoxy-a-D-mannopyranoside (17)
3.12. 5-Methoxycarbonylpentyl (2-O-acetyl-4-azido-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-
azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-4-azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyran-
oside (18)
A mixture of compound 15 (140 mg) and 5% palladium-
on-charcoal catalyst (140 mg) in MeOH (40 mL) was
stirred under hydrogen overnight at 45 °C. TLC (5:1
CHCl3–MeOH) showed that the reaction was complete.
After filtration and evaporation of the solvent, the resi-
Reaction of 1014,21 (2.2 g, 6.02 mmol) and 14 (3.125 g,
4.67 mmol) as described in Section 3.2 gave, after chro-

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1701
matography (20:1?5:1 hexane–EtOAc), the fully pro-
tected trisaccharide 18 (4.275 g, 95%), [a]D +79.6 (c 1.2).
1H NMR (600 MHz, C6D6): d 5.87 (dd, 1H, J1,2 = 1.8,
J2,3 = 3.1 Hz, H-2III), ꢀ5.25 (d, partially overlapped, H-
1III), ꢀ5.24 (d, partially overlapped, H-1II), 4.81 (d, 1H,
J1,2 = 1.7 Hz, H-1I), 4.74–4.39 (6d, partially overlapped,
6H, 3C H2Ph), 4.22 (dd, 1H, J2,3 = 3.1, J3,4 = 9.7 Hz,
H-3II), 4.13 (dd, partially overlapped, J2,3 = 3.3,
J3,4 = 9.2 Hz, H-3I), 4.12 (dd, partially overlapped, H-
2II), 4.10 (dd, 1H, J3,4 = 10.0 Hz, H-3III), 3.94 (m, 1H,
H-5III), 3.85–3.77 (m, 3H, H-5II,2I,4II in that order),
3.69 (t, 1H, J = 9.9 Hz, H-4I), 3.63 (t, 1H, J = 9.9 Hz,
H-4III), 3.59 (m, partially overlapped, H-5I), 3.47, 3.46
(2 t, J = 6.7 Hz, 1H, H-10a), 3.36 (s, 3H, COOCH3),
3.12, 3.11 (2t, 1H, H-10b), 2.05 (t, 2H, J = 7.3 Hz, H-
50a,b), 1.65 (s, 3H, COCH3), 1.49–1.44 (m, 2H, H-
40a,b), 1.36–1.29 (m, partially overlapped, H-20a,b), 1.31
(d, partially overlapped, J5,6 = 5.9 Hz, H-6III), 1.25 (d,
J5,6 = 6.1 Hz, H-6I), 1.20 (d, J5,6 = 6.0 Hz, H-6II), 1.18–
1.11 (m, 2H, H-30a,b). 13C NMR (150 MHz, C6D6): d
100.93 (C-1III), 100.04 (C-1II), 97.32 (C-1I), 80.17 (C-3I),
79.71 (C-3II), 78.34 (C-2I), 77.80 (C-2II), 76.93 (C-3III),
73.02, 72.98, 72.02 (3CH2Ph), 68.81 (C-5II) 68.54 (C-
5III), 68.22 (C-10), 67.99 (C-5I), 67.95 (C-2III), 65.26 (C-
4I), 65.19 (C-4II), 64.96 (C-4III), 51.39 (COOCH3), 34.18
(C-50), 29.61 (C-20), 26.28 (C-30), 25.18 (C-40), 20.73
(COCH3), 19.09, 19.06 (2C) (C-6I–III). FABMS: m/z 946
[M+Hꢁ2N+2H]+. Anal. Calcd for C48H61N9O13: C,
59.31; H, 6.33; N, 12.97. Found: C, 59.47; H, 6.34; N,
12.89.
C6D6): d 102.61 (C-1III), 100.01 (C-1II), 97.44 (C-1I),
80.01 (C-3I), 79.35 (C-3II), 79.01 (C-3III), 78.36 (C-2I),
77.96 (C-2II), 73.08, 73.00, 71.93 (3CH2Ph), 68.71 (C-
5II), 68.34 (C-5III), 68.19 (C-10), 68.05 (C-5I), 67.83 (C-
2III), 65.32 (C-4I), 65.27 (C-4II), 64.59 (C-4III), 51.38
(COOCH3), 34.19 (C-50), 29.61 (C-20), 26.27 (C-30),
25.19 (C-40), 19.08, 19.05, 19.04 (C-6I–III). FABMS:
m/z 904 [M+Hꢁ2N+2H]+, 952 [M+Na]+. Anal. Calcd
for C46H59N9O12: C, 59.41; H, 6.39; N, 13.55. Found:
C, 59.49; H, 6.55; N, 13.55.
3.14. 5-Methoxycarbonylpentyl (4-acetamido-3-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-acet-
amido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-4-acetamido-2-O-benzyl-4,6-dideoxy-a-D-manno-
pyranoside (20)
Reduction of 19 (1 g, 1.075 mmol) with H2S followed by
N-acetylation, as described for 14, gave 20 (0.9 g, 86%).
1H NMR (600 MHz, CD3OD at 45 °C): d 5.06 (d, 1H,
J1,2 = 1.8 Hz, H-1II), 4.93 (d, 1H, J1,2 = 1.8 Hz, H-1III),
4.80 (d, 1H, J1,2 = 1.8 Hz, H-1I), 4.79–4.41 (6d, partially
overlapped, 6H, 3CH2Ph), 4.18–4.13 (m, 2H, H-4I,II),
4.01–3.97 (m, 4H, H-2III,4III,3I,II in that order), 3.88
(m, 1H, H-5II), 3.82 (m, partially overlapped, H-5III),
3.79 (br t, partially overlapped, H-2I), 3.78 (br t, partially
overlapped, H-2II), 3.72 (m, 1H, H-5I), 3.69–3.63 (m, 5H,
H-10a, 3III, incl. s, 3.64, COOCH3), 3.42, 3.41 (2t, 1H,
J = 6.1 Hz, H-10b), 2.33 (t, 2H, J = 7.3 Hz, H-50a,b),
1.97, 1.96, 1.88 (3s, 3H each, 3COCH3), 1.66–1.61 (m,
partially overlapped, H-40a,b), 1.61–1.56 (m, partially
overlapped, H-20a,b), 1.43–1.38 (m, 2H, H-30a,b), 1.18,
1.15, 1.13 (3d, 3H each, J5,6 6.2 Hz, H-6I–III, in that or-
der). 13C NMR (150 MHz, CD3OD, 45 °C): d 103.45
(C-1III), 100.58 (C-1II), 98.93 (C-1I), 79.38 (C-2II), 78.85
(C-2I), 78.11, 77.97, 77.91 (C-3I–III), 74.05, 74.01, 72.07
(3CH2Ph), 69.74 (C-5II), 69.54 (C-5III), 68.96 (C-5I),
68.59 (C-10), 68.40 (C-2III), 53.90, 53.83 (C-4I,II), 53.12
(C-4III), 51.99 (COOCH3), 34.68 (C-50), 30.09 (C-20),
26.81 (C-30), 25.69 (C-40), 23.18, 22.99, 22.94
(3NHCOCH3), 18.54, 18.46, 18.28 (C-6I–III). FABMS:
m/z 984.5 [M+Li]+, 1000.50 [M+Na]+. FAB HRMS:
m/z 1000.4783 (100%); calcd for C52H71N3NaO15,
1000.4783.
3.13. 5-Methoxycarbonylpentyl (4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-(4-azido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-4-azido-2-
O-benzyl-4,6-dideoxy-a-D-mannopyranoside (19)
Deacetylation of 18 (5.45 g, 5.6 mmol) as described for
13 gave, after chromatography and crystallization, 19
(3.75 g, 98%), mp 37–40 °C (from isopropyl ether),
1
[a]D +76 (c 1.9). H NMR (600 MHz, C6D6): d 5.31
(d, 1H, J1,2 = 1.5 Hz, H-1III), 5.26 (d, 1H, J1,2
=
1.5 Hz, H-1II), 4.82 (d, 1H, J1,2 = 1.6 Hz, H-1I), 4.59–
4.34 (6d, partially overlapped, 6H, 3CH2Ph), 4.24 (dd,
1H, J2,3 = 3.1 Hz, H-2III), 4.21 (dd J2,3 = 3.2, J3,4
=
9.8 Hz, H-3II), ꢀ4.14 (dd, partially overlapped, H-2II),
4.13 (dd, partially overlapped, J2,3 = 3.1, J3,4 = 9.9 Hz,
H-3I), 3.90 (m, partially overlapped, H-5III), 3.88 (dd,
partially overlapped, J3,4 = 9.8 Hz, H-3III), 3.84–3.80
(m, 2H, H-2I,5II), 3.77 (t, 1H, J = 9.9 Hz, H-4II), 3.70
(t, J = 9.9 Hz, H-4I), 3.60 (m, 1H, H-5I), 3.51 (t, 1H,
J = 9.9 Hz, H-4III), 3.47, 3.45 (2t, 1H, J = 6.6 Hz, H-
10a), 3.36 (s, 3H, COOCH3), 3.12, 3.10 (2t, 1H, H-10b),
2.05 (t, 2H, J = 7.4 Hz, H-50a,b), 1.50–1.43 (m, 2H, H-
40a,b), 1.36–1.29 (m, 5H, H-20a,b,6III), 1.26 (d, 3H,
J5,6 = 6.1 Hz, H-6I), 1.20 (d, 3H, J5,6 = 6.0 Hz, H-6II),
1.17–1.12 (m, 2H, H-30a,b). 13C NMR (150 MHz,
3.15. 5-Methoxycarbonylpentyl (4-acetamido-4,6-di-
deoxy-a-D-mannopyranosyl)-(1?3)-(4-acetamido-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-4-acetamido-4,6-
dideoxy-a-D-mannopyranoside (21) and 5-methoxycar-
bonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-a-
D-mannopyranosyl)-(1?3)-(4-acetamido-2-O-acetyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-4-acetamido-2-O-
acetyl-4,6-dideoxy-a-D-mannopyranoside (22)
Compound 20 (0.75 g) was treated with hydrogen, as de-
scribed for 15. Elution of the crude product from a small

1702
R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
silica gel column (5:1?3:2 CH2Cl2–MeOH) gave com-
pound 21 (345 mg, 64%) [a]D +78.9 (c 3.7, H2O). Struc-
turally significant signals in the 1H NMR spectrum
(D2O at 50 °C, 600 MHz) were at d 5.13 (br d, 2H, H-
1II,III), 5.02 (br d, 1H, H-1I), 4.21 (br dd, 1H, H-2I),
3.93, 3.92, 3.79, 3.75 (4t, 2H, J = 6.5 Hz, H-10a,b), 2.62
(t, 2H, J = 7.4 Hz, H-50a,b), 2.27. 2.26, 2.25 (3br s,
12H, 3NHCOCH3), 1.43–1.41 (m, H-6I–III). Structurally
significant signals in the 13C NMR spectrum (D2O at
50 °C, 600 MHz) were at d 105.25, 105.13 (C-1II,III),
102.75 (C-1I), 52.11 (COOCH3), 36.86 (C-50), 31.31 (C-
20), 28.09 (C-30), 27.16 (C-40), 25.35, 25.32, 25.25
(3NHCOCH3), 20.03, 19.98, 19.95 (C-6I,III). FABMS:
m/z 708.5 [M+H]+, 730.5 [M+Na]+. The corresponding
per-O-acetyl derivative 22 showed [a]D +50 (c 1.2,
C6D6): d 5.53 (dd, 1H, J2,3 = 3.1, J3,4 = 10.1 Hz,
H-3IV), 5.36 (d, 1H, J1,2 = 1.6 Hz, H-1IV), 5.32 (d, 1H,
J1,2 = 1.8 Hz, H-1III), 5.26 (d, 1H, J1,2 = 1.4 Hz,
H-1II), 4.82 (d, 1H, J1,2 = 1.8 Hz, H-1I), 4.61–4.05 (8d,
partially overlapped, 4CH2Ph), 4.49 (br t, H-2III), 4.27
(dd, partially overlapped, H-3II), 4.15 (dd, 1H,
J2,3 = 3.0, J3,4 = 9.6 Hz, H-3I), ꢀ4.13 (m, 2H, H-2II,IV),
4.00 (dd, 1H, J2,3 = 2.8, J3,4 = 9.8 Hz, H-3III), 3.92–3.81
(m, partially overlapped, H-5IV,III,II in that order), 3.82
(m, H-2I,4IV), 4.81 (t, partially overlapped, H-4II), 4.76
(t, partially overlapped, H-4III), 3.74 (t, partially over-
lapped, H-4I), 3.60 (m, 1H, H-5I), 3.48, 3.46 (2t, 1H,
J = 6.4 Hz, H-10a), 3.36 (s, 3H, COOCH3), 3.13, 3.11
(2t, 1H, H-10b), 2.05 (t, 2H, J = 7.3 Hz, H-50a,b), 1.70
(s, 3H, COCH3), 1.50–1.45 (m, 2H, H-40a,b), 1.36–1.30
(m, 2H, H-20), 1.30 (d, partially overlapped, H-6IV),
1.29 (d, partially overlapped, H-6III), 1.25 (d, 3H,
J5,6 = 6.2 Hz, H-6I), 1.22 (d, 3H, J5,6 = 6.0 Hz, H-6II),
1.17–1.14 (m, 2H, H-30a,b). 13C NMR (150 MHz,
C6D6): d 102.19 (C-1III), 100.06 (C-1II), 99.59 (C-1IV),
97.36 (C-1I), 80.11 (C-3I), 79.43 (C-3III), 79.01 (C-3II),
78.53 (C-2I), 77.90 (C-2II), 75.52 (C-2IV), 73.29 (C-2III),
73.12, 73.09 (2CH2Ph), 73.01 (C-3IV), 72.97, 72.78
(2CH2Ph), 68.98 (C-5III), 68.77 (C-5II), 68.71 (C-5IV),
68.20 (C-10), 68.01 (C-5I), 65.51 (C-4II), 65.34 (C-4I),
65.10 (C-4III), 63.52 (C-4IV), 51.37 (COOCH3), 34.18
(C-50), 29.62 (C-20), 26.28 (C-30), 25.19 (C-40), 20.79
(COCH3), 19.05, 19.03 18.89 (4C, C-6I–IV). FABMS:
m/z 1207.6 [M+Hꢁ2N+2H]+, 1255 [M+Na]+. Anal.
Calcd for C61H76N12O16: C, 59.40; H, 6.21; N, 13.63.
Found: C, 59.43; H, 6.11, N, 13.54.
1
CHCl3). H NMR (600 MHz, CDCl3): d 6.38 (d, 1H,
J4,NH = 9.5 Hz, NHI), 5.83 (d, 1H, J4,NH = 9.9 Hz,
NHIII), 5.80 (d, 1H, J4,NH = 10.0 Hz, NHII), 5.14 (dd,
1H, J1,2 = 1.9 Hz, H-2I), 5.05 (dd, 1H, J2,3 = 2.6,
J3,4 = 10.8 Hz, H-3III), 5.02 (dd, 1H, J1,2 = 2.0,
J2,3 = 3.3 Hz, H-2II), 4.92–4.90 (m, 3H, H-2III,1III,II in
that order), 4.70 (d, 1H, J1,2 = 1.9 Hz, H-1I), 4.16 (t, par-
tially overlapped, J = 10.0 Hz, H-4III), 4.15–4.04 (m,
partially overlapped, H-4I,II,H-3I in that order), 3.87
(dd, partially overlapped, H-3II), 3.83 (m, partially over-
lapped, H-5I), ꢀ3.77 (m, partially overlapped, H-5III),
ꢀ3.75 (m, partially overlapped, H-5I), 3.69–3.65 (m,
4H, H-10a, incl. 3.68, s, COOCH3), 3.42, 3.40 (2t, 1H,
J = 5.4 Hz, H-10b), 2.36 (m, 2H, H-50a,b), 2.19, 2.17,
2.14 (3s, 9H, 3NHCOCH3), 2.05, 2.02, 2.00, 1.97 (4s,
12H, 4COCH3), 1.74–1.25 (4m, 6H, H-40a,b,20b,30a,30b
in that order), 1.25, 1.23, 1.21 (3d, 9H, J5,6 = 6.2 Hz,
H-6I,II,III in that order). 13C NMR (150 MHz, CDCl3):
d 98.44 (C-1II), 97.85 (C-1III), 97.05 (C-1I), 73.63 (C-
3I), 72.76 (C-3II), 70.53 (C-2I), 70.37 (C-2II), 69.49 (C-
2III), 68.82 (C-5 III), 68.44 (C-5II), 68.05 (C-3III), 67.55
(C-5I), 68.84 (C-10), 52.37 (C-4I), 52.14 (C-4II), 51.49
(COOCH3), 51.02 (C-4III), 33.78 (C-50), 28.44 (C-20),
25.76 (C-30), 24.24 (C-40), 23.26, 23.15, 21.12, 20.95,
20.88, 20.67 (7COCH3), 17.75, 17.66 (3C, C-6I–III).
ESI-MS (m/z): 914.3589; calcd for C39H61N3O19K:
914.3536. Anal. Calcd for C39H61N3O19: C, 53.48; H,
7.02; N, 4.80. Found: C, 53.76; H, 6.98; N, 4.56.
3.17. Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?2)-(4-azido-3-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-azido-2-
O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-4-
azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranoside (24)
Deacetylation of 23 (4.27 g, 3.46 mmol) as for 13, except
that the reaction was started at 50 °C, to solubilize the
starting material, gave pure 24 (4.03 g, 97%), which crys-
tallized on standing but could not be crystallized from
1
common solvents, mp 53–57 °C, [a]D +10.0 (c 0.4). H
NMR (600 MHz, C6D6): d 5.30 (br d, partially over-
lapped, H-1IV), 5.29 (d, partially overlapped, H-1III),
5.26 (d, 1H, J1,2 = 1.7 Hz, H-1II), 4.83 (d, 1H,
J1,2 = 1.8 Hz, H-1I), 4.63–4.01 (8d, partially overlapped,
4C H2Ph), 4.45 (br dd, 1H, H-2III), 4.25 (dd, 1H,
3.16. 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?2)-(4-
azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-(4-azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyr-
anosyl)-(1?3)-4-azido-2-O-benzyl-4,6-dideoxy-a-D-man-
nopyranoside (23)
J2,3 = 3.0, J3,4 = 9.8 Hz, H-3II), 4.14 (dd, 1H, J2,3
=
3.1, J3,4 = 10.0 Hz, H-3I), 4.11 (dd, 1H, J2,3 = 3.0 Hz,
H-2II), 4.05 (dd, 1H, J2,3 = 3.6, J3,4 = 9.9 Hz, H-3IV),
3.97 (dd, 1H, J2,3 = 3.5, J3,4 = 9.7 Hz, H-3III), 3.86–
3.81 (m, partially overlapped, H-5II,III), 3.82 (dd, par-
tially overlapped, H-2I), 3.80 (t, partially overlapped,
J = 9.8 Hz, H-4II), ꢀ3.74 (m, partially overlapped, H-
5IV), 3.72 (m, partially overlapped, H-4I,2IV), 3.62–3.56
Reaction of 8 (1.8 g, 5 mmol) and 19 (3.75 g, 3.86 mmol)
as described in Section 3.2 gave, after chromatography
(15:1?5:1 hexane–EtOAc), the protected tetrasaccha-
ride 23 (4.45 g, 94%), mp 112.5–113.5 °C (EtOH–
EtOAc), [a]D +37.2 (c 1.1). 1H NMR (600 MHz,

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1703
(m, 2H, H-5I,4III), 3.49, 3.47(2t, 1H, J = 6.5 Hz, H-10a),
3.37 (s, partially overlapped, COOCH3), 3.35 (t, par-
tially overlapped, J = 10.0 Hz, H-4IV), 3.15, 3.13 (2t,
1H, H-10b), 2.06 (t, 2H, J = 7.4 Hz, H-50a,b), 1.51–1.46
(m, 2H, H-40a,b), 1.38–1.32 (m, partially overlapped,
H-20a,b), 1.31–1.21 (4d, partially overlapped, H-6I–IV ),
1.20–1.13 (m, 2H, H-30a,b). 13C NMR (150 MHz,
C6D6): d 102.22 (C-1III), 100.10 (C-1II), 98.96 (C-1IV),
97.33 (C-1I), 80.16 (C-3I), 79.20 (C-3III), 78.86 (C-3II),
78.49 (C-2I), 77.88 (C-2II), 77.41 (C-2IV), 73.11, 73.06,
72.81, 72.66 (4CH2Ph), 72.97 (C-2III), 71.10 (C-3IV),
68.79. 68.77 (C-5II,III), 68.23 (C-10), 68.21 (C-5IV),
68.00 (C-5I), 67.03 (C-4IV), 65.67 (C-4II), 65.33
(C-4I), 65.10 (C-4III), 51.43 (COOCH3), 34.20
(C-50), 29.61 (C-20), 26.28 (C-30), 25.19 (C-20),
19.01, 18.95, 18.75 (4C, C-6I–IV). FABMS: m/z 1165.6
[M+Hꢁ2N+2H]+. Anal. Calcd for C59H74N12O15: C,
59.48; H, 6.26; N, 14.11. Found: C, 59.50; H, 6.27; N,
13.83.
(COOCH3), 23.30, 23.25, 22.91, 22.88 (4NHCOCH3),
18.68, 18.67, 18.57, 18.26 (C-6I–IV). FABMS: m/z 1278
[M+Na]+.
3.19. 5-Methoxycarbonylpentyl (4-acetamido-4,6-dide-
oxy-a-D-mannopyranosyl)-(1?2)-(4-acetamido-4,6-dide-
oxy-a-D-mannopyranosyl)-(1?3)-(4-acetamido-4,6-dide-
oxy- a-D-mannopyranosyl)-(1?3)-4-acetamido-4,6-
dideoxy-a-D-mannopyranoside (26) and 5-methoxycar-
bonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-a-
D-mannopyranosyl)-(1?2)-(4-acetamido-3-O-acetyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-(4-acetamido-2-O-
acetyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-4-acet-
amido-2-O-acetyl-4,6-dideoxy-a-D-mannopyranoside (27)
Hydrogenolysis of 25 (960 mg) and processing as de-
scribed for 16, gave the deprotected, amorphous tetra-
saccharide 26 in virtually theoretical yield, [a]D +62.4
1
(c 6.6, H2O). H NMR (600 MHz, D2O at 50 °C): d
5.27 (d, 1H, J1,2 = 1.6 Hz, H-1III), 5.20 (d, 1H, J1,2
=
3.18. 5-Methoxycarbonylpentyl (4-acetamido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?2)-(4-acet-
amido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-a-D-manno-
pyranosyl)-(1?3)-4-acetamido-2-O-benzyl-4,6-dideoxy-
a-D-mannopyranoside (25)
1.7 Hz, H-1II), 5.15 (d, 1H, J1,2 = 1.7 Hz, H-1IV), 5.05
(d, 1H, J1,2 = 1.7 Hz, H-1I), 4.33 (br dd, 1H, H-2II),
4.25 (br t, 1H, H-2I), ꢀ4.23 (m, partially overlapped,
H-3III), ꢀ4.19 (m, partially overlapped, H-3II), 4.12
(m, partially overlapped, H-3I), ꢀ4.09 (m, partially
overlapped, H-2IV), 4.03 (br dd, 1H, H-2III), 3.96 (m,
4H, H-10a, incl. s, 3.96 COOCH3), 3.80, 3.78 (2t, 1H,
J = 6.1 Hz, H-10b), 2.66 (t, 2H, J = 7.4 Hz, H-50a,b),
2.31, 2.30, 2.29, 2.28 (4s, 3H each, 4NHCOCH3),
1.92–1.85 (m, 4H, H-20a,b40a,b), 1.69–1.62 (m, 2H,
H-30a,b), 1.46–1.45 (m, 12H, H-6I–IV). 13C NMR
(150 MHz, D2O at 50°C): d 105.47 (C-1II), 105.21 (C-
11V), 103.90 (C-1III), 102.72 (C-1I), 81.60 (C-2III), 80.52
(C-3I), 80.02 (C-3II), 72.58 (C-2IV), 72.39 (C-2I), 72.20
(C-2II), 70.98 (C-10), 52.08 (COOCH3), 36.70 (C-50),
31.23 (C-20), 28.04 (C-30), 27.10 (C-40), 25.43, 25.34,
25.30, 25.27 (4NHCOCH3), 20.20, 20.05, 19.90, 19.86
(C-6I–IV). FABMS: m/z 917.5 [M+Na]+. The corre-
sponding per-O-acetyl derivative 27 showed [a]D +67
Treatment of 24 (1.42 g, 1.19 mmol) as for compound 19
but in pyridine and water (3:1), to assist solubility of the
starting material, gave the corresponding intermediate
tetrakis-amine (1 g). FABMS: m/z 1087.62 [M+H]+,
1109.61 [M+Na]+.
A solution of the foregoing amine was N-acetylated, as
described for preparation of 15, to give after chromatog-
raphy 25 (1.05 g, 70% from 24), [a]D +30.0 (c 0.2). Struc-
1
turally significant H NMR (600 MHz, C6D6) signals
were at d 7.62, 6.73, 5.86, 5.48 (4br s, NHI–IV), 5.21,
5.10, 5.07 (3br s, H-1II–IV), 4.80 (br s, H-1I), 4.37–4.28
(m, 4H, H-4I–IV), 3.60–3.58, 3.24–3.22 (2m, 2H,
H-10a,b), 3.38 (s, 3H, COOCH3), 2.12–2.09 (m, 2H,
H-50a,b), 2.00, 1.99, 1.87, 1.43 (4s, 4NHCOCH3), 1.46,
1.31, 1.24, 1.23 (4d, H-6I–IV). The spectrum taken in
CD3OD showed peaks at d 5.08, 5.01, 4.96 (3d, 1H each,
H-1II–IV), 4.80 (d, 1H, J1,2 = 1.9 Hz, H-1I), 4.82–4.36
(8d, 1H, each, 4CH2Ph), 4.15 (br t, partially overlapped,
H-4I), 3.78 (br dd, 1H, H-2I), 3.64 (s, partially over-
lapped, COOCH3), 3.42, 3.40 (2t, 1H, J = 6.2 Hz,
H-10b), 2.43 (t, 2H, J = 7.3 Hz, H-50a,b), 1.99, 1.98,
1.96, 1.93 (4s, 3H each, 4NHCOCH3), 1.18, 1.17, 1.12,
1.10 (4d, 12H, J5,6 ꢀ 6.3 Hz, H-6I–IV). Structurally sig-
nificant 13C NMR (150 MHz, C6D6) signals were at d
101.20, 99.40, 97.84 (br, C-1II–IV), 96.89 (br, C-1I),
51.03 (COOCH3), 23.27, 23.16. 23.09, 22.96
(NHCOCH3). The spectrum taken in CD3OD showed
peaks at d 102.19, 100.66 (3C) (C-1II–IV), 98.94 (C-1I),
74.25, 74.00, 73.92, 72.75 (4CH2Ph), 68.57 (C-10), 52.06
1
(c 1.1, CHCl3). H NMR (600 MHz, CDCl3): d 6.98
(d, 1H, J4,NH = 9.3 Hz, NHIV), 6.38 (d, 1H, J4,NH
=
9.8 Hz, NHI), 5.82 (br d, 2H, NHII,III), 5.16–5.13 (m,
3H, H-2I,2IV,3IV, in that order), 5.06 (dd, 1H,
J2,3 = 3.2 Hz, J3,4 = 8.7 Hz, H-3III), 4.95 (br dd, 1H,
H-2II), 4.90 (d, 1H, J1,2 = 2.0 Hz, H-1II), 4.88 (d, 1H,
J1,2 = 2.8 Hz, H-1III), 4.86 (br d, 1H, J1,2 ꢀ 1 Hz,
H-1IV), 4.71 (d, 1H, J1,2 = 1.7 Hz, H-1I), 4.20 (br q,
1H, H-4IV), 4.13 (br q, 1H, H-4I), 4.04–3.99 (m, 3H,
H-3II,4III,3I, in that order), 3.94–3.81 (m, 4H, H-4II,
5II,2III,5IV, in that order), 3.75 (m, partially overlapped,
H-5I), 3.73–3.65 (m, 5H, H-5III,10a, incl. 3.68, s,
COOCH3), 3.42, 3.40 (2t, 1H, J = 5.2 Hz, H-10b), 2.36
(t, 2H, J = 7.0 Hz, H-50), 2.19, 2.14, 2.13, 2.08, 2.07,
2.03, 2.01, 1.98, 1.97 (9s, 3H each, 9COCH3), 1.71–
1.61 (m, 3H, H-40a,b,20a), 1.60–1.55 (m, H-20b), 1.45,
1.38 (2 m, 2H, H-30a,b), 1.29, 1.25, 1.23, 1.20 (4d,

1704
R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
J5,6 = 1.2 Hz, H-6IV,I,III,II in that order). 13C NMR
(CDCl3, 150 MHz): d 99.55 (C-1III), 98.55 (C-1II),
97.85 (C-1IV), 97.06 (C-1I), 74.59 (C-3I), 74.21 (C-2III),
72.04 (C-3II), 70.95 (C-2II), 69.71 (C-3III), 70.55, 68.88
(C-2I,IV), 68.81 (C-3IV), 68.56 (C-5III), 68.50 (C-5IV),
68.19 (C-5II), 67.46 (C-5I), 66.81 (C-10), 52.74 (C-4II),
52.30 (2C,0 C-4I,III), 51.58 (COOCH3), 50.88 (C-4IV),
33.80 (C-5 ), 28.48 (C-20), 25.77 (C-30), 24.21 (C-40),
23.49, 23.36, 23.26, 23.16 (4NHCOCH3), 21.14, 21.01,
20.91, 20.79, 20.78 (5COCH3), 18.07, 17.78, 17.74,
17.73 (C-6I–IV). ESI-MS (m/z): 1105.4944 [M+H]+;
calcd for C49H77N4O24: 1105.4928. Anal. Calcd for
C49H76N4O24: C, 53.25; H, 6.93; N, 5.07. Found: C,
53.07; H, 6.97; N, 5.00.
(C-2III), 73.73, 73.10, 73.07 (3CH2Ph), 72.94 (2C,
CH2Ph, C-3V), 72.67 (CH2Ph), 69.27 (C-5IV), 68.82,
68.78 (C-5II,III), 68.70 (C-5V), 68.24 (C-10), 68.02 (C-5I),
65.62 (C-4II), 65.56 (C-4III), 65.37, 65.34 (C-4I,IV),
63.46 (C-4V), 51.38 (COOCH3), 34.19 (C-50), 29.62
(C-20), 26.30 (C-30), 25.20 (C-40), 20.79 (COCH3),
19.09 (C-6III), 19.05 (C-6IV), 19.03, 18.97 (C-6I,IV),
18.75 (C-6II). FABMS: m/z 1468.7 [M+Hꢁ2N+2H]+,
1516.6 [M+Na]+. Anal. Calcd for C74H91N15O19: C,
59.47; H, 6.14; N, 14.06. Found: C, 59.30; H, 6.21; N,
13.93.
3.21. 5-Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-(4-azido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?2)-(4-azido-3-
O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-
azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-4-azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyran-
oside (29)
3.20. 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-
azido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?2)-(4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyr-
anosyl)-(1?3)-(4-azido-2-O-benzyl-4,6-dideoxy-a-D-
mannopyranosyl)-(1?3)-4-azido-2-O-benzyl-4,6-dide-
oxy-a-D-mannopyranoside (28)
Deacetylation of 28 (2.25 g, 1.505 mmol) as described
for 12, except that 1:2 MeOH–toluene mixture was used
1
as solvent, gave 29 (2.05 g, 94%), [a]D +69.2 (c 2.4). H
Condensation of the glycosyl donor 8 (1 g, 2.73 mmol)
and acceptor 24 (2.61 g, 2.19 mmol), performed as
described in Section 3.2, afforded pure (TLC, NMR) 28
NMR (600 MHz, C6D6): d 5.37 (d, 1H, J1,2 = 1.7 Hz, H-
1III), 5.36 (d, 1H, J1,2 = 1.7 Hz, H-1IV), 5.24 (d, 1H,
J1,2 = 1.7 Hz, H-1II), 5.20 (d, 1H, J1,2 = 1.3 Hz, H-1V),
4.81 (d, 1H, J1,2 = 1.7 Hz, H-1I), 4.60–4.21 (10d, par-
tially overlapped, 5CH2Ph), 4.46 (br t, 1H, H-2III),
4.27 (dd, 1H, J2,3 = 3.1, J3,4 = 9.9 Hz, H-3II), 4.18 (dd,
1H, J2,3 = 3.0, J3,4 = 10.2 Hz, H-3IV), 4.12 (dd, 1H,
J2,3 = 3.2, J3,4 = 9.9 Hz, H-3I), 4.10 (dd, 1H, H-2II),
4.02 (dd, 1H, J2,3 = 3.6, J3,4 = 9.8 Hz, H-3V), 3.99 (dd,
1H, J3,4 = 9.9 Hz, H-3III), 3.88–3.82 (m, 5H, H-2IV,2-
V,5III,5IV,5II, in that order), 3.81 (br t, 1H, H-2I), 3.80
(t, 1H, J = 10.0 Hz, H-4II), 3.74 (t, 1H, J = 10.0 Hz,
H-4IV), 3.68 (t, partially overlapped, J = 10.0 Hz, H-
4I), 3.66 (t, partially overlapped, J = 10.0 Hz, H-4III),
3.59 (m, 2H, H-5V,5I, in that order), 3.48, 3.46 (2 t,
1H, J = 6.6 Hz, H-10a), 3.36 (s, 3H, COOCH3), 3.30 (t,
1H, J = 9.9 Hz, H-4V), 3.14, 3.12 (2t, 1H, H-10b), 2.05
(t, 2H, J = 7.2 Hz, H-50a,b), 1.50–1.45 (m, 2H, H-
40a,b), 1.37–1.31 (m, partially overlapped, H-20a,b),
1.31 (d, partially overlapped, J5,6 = 6.2 Hz, H-6III),
1.30 (d, 3H, J5,6 = 6.2 Hz, H-6IV), 1.23 (d, 3H,
J5,6 = 6.2 Hz, H-6I), 1.22 (d, 3H, J5,6 = 6.2 Hz, H-6V),
1.21 (d, 3H, J5,6 = 6.1 Hz, H-6II), 1.18–1.13 (m, 2H,
H-30a,b). 13C NMR (C6D6, 150 MHz): d 102.19 (C-
1III), 100.04 (C-1II), 99.86 (C-1V), 99.34 (C-1IV), 97.34
(C-1I), 80.34 (C-3I), 79.05 (C-3III), 78.97 (C-3II), 78.44
(C-21), 78.30 (C-2V), 77.92 (C-3IV), 77.83 (C-2II), 77.38
(C-2IV), 73.83 (C-2III), 73.54, 73.07, 73.02, 72.68, 72.62
(5CH2Ph), 71.28 (C-3V), 69.17 (C-5IV), 68.83, 68.76 (C-
5II,III), 68.25 (C-10), 68.10 (C-5V), 67.99 (C-5I), 66.86
(C-4V), 65.58 (C-4II), 65.52 (C-4IV), 65.50 (C-4III),
65.30 (C-4I), 51.41 (COOCH3), 34.19 (C-50), 29.62 (C-
20), 26.29 (C-30), 25.19 (C-40), 19.07, 19.04, 19.02 (2C),
1
(2.37 g, 73%), [a]D +49.8. H NMR (600 MHz, C6D6):
d 5.55 (dd, 1H, J2,3 = 3.3, J3,4 = 10.1 Hz, H-3V), 5.39
(d, 1H, J1,2 = 1.6 Hz, H-1III), 5.36 (d, 1H, J1,2
=
1.6 Hz, H-1IV), 5.28 (d, 1H, J1,2 = 1.6 Hz, H-1V), 5.24
(d, 1H, J1,2 = 1.6 Hz, H-1II), 4.81 (d, 1H, J1,2 = 1.6 Hz,
H-1I), 4.61–4.36 (m, 11H, 5CH2Ph, incl. br dd, 4.46,
H-2III), 4.27 (dd, 1H, J2,3 = 3.1, J3,4 = 9.9 Hz, H-3II),
4.22 (dd, J2,3 = 3.0, J3,4 = 10.1 Hz, H-3IV), 4.19 (dd,
H-2V), 4.13 (dd, partially overlapped, J2,3 = 3.2,
J3,4 = 9.9 Hz, H-3I), ꢀ4.11 (dd, partially overlapped,
H-2II), 4.00 (dd, 1H, J2,3 = 2.9, J3,4 = 9.9 Hz, H-3III),
3.89 (dd, partially overlapped, H-2IV), 3.87 (m, partially
overlapped, H-5III,II,IV, in that order), ꢀ3.82 (m, par-
tially overlapped, H-2I), 3.80 (t, partially overlapped,
H-4II), 3.79 (t, partially overlapped, H-4V), ꢀ3.73 (m,
partially overlapped, H-5V), 3.71 (t, partially overlapped,
H-4IV), 3.69 (t, partially overlapped, H-4I), 3.66 (t, par-
tially overlapped, H-4III), 3.58 (m, partially overlapped,
H-5I), 3.48, 3.46 (2t, 1H, J = 6.5 Hz, H-10a), 3.37 (s,
3H, COOCH3), 3.14, 3.12 (2t, 1H, H-10b), 2.05 (t, 2H,
J = 7.3 Hz, H-50a,b), 1.72 (s, 3H, COCH3), 1.50–1.45
(m, 2H, H-40a,b), 1.37–1.31 (m, partially overlapped,
H-20a,b, incl. 1.33, d, J5,6 = 6.2 Hz, H-6III), 1.28 (d,
J5,6 = 6.1 Hz, H-6IV), 1.24 (d, partially overlapped,
J5,6 = 6.2 Hz, H-6I), 1.23, (d, partially overlapped, H-
6V), 1.22 (d, partially overlapped, H-6II). 13C NMR
(150 MHz, C6D6): d 102.21 (C-1III), 100.26 (C-1V),
100.08 (C-1II), 99.60 (C-1IV), 97.38 (C-1I), 80.18 (C-3I),
79.04 (C-3III), 78.96 (C-3II), 78.47 (C-2I), 77.87 (C-2II),
77.45 (C-2IV), 77.43 (C-3IV), 76.21 (C-2V), 73.85

R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
1705
18.78 (C-6I–V). FABMS: m/z 1426.6 [M+Hꢁ2N+2H]+,
1474.5 [M+Na]+. Anal. Calcd for C72H89N15O18: C,
59.53; H, 6.18; N 14.46. Found: C, 59.56; H, 6.25; N,
14.57.
50 °C) were at 5.26 (d, 1H, J1,2 = 1.4 Hz, H-1III),
5.22, 5.19, 5.15 (3d, J1,2 ꢀ 1.6 Hz, H-1II,IV,V), 5.05 (d,
1H, J1,2 = 1.5 Hz, H-1I), 4.24 (dd, 1H, J2,3 = 3.1 Hz,
H-2I), 4.04 (d, 1H, J2,3 = 3.0 Hz, H-2III), 3.95 (m,
4H, H-10a, incl. s, COOCH3), 3.80, 3.78 (2t, 1H,
H-10b), 2.66 (t, 2H, J = 7.6 Hz, H-50a,b), 2.31, 2.30,
2.28 (3s, 15H, 5NHCOCH3), 1.91–1.84 (m, 4H, H-
20a,b,40a,b), 1.68–1.60 (m, 2H, H-30a,b), 1.48–1.44
(5d, partially overlapped, H-6I–V). 13C NMR
(150 MHz, D2O at 50 °C): d 105.26, 105.22, 105.07
(C-1II,IV,V), 103.84 (C-1III), 102.71 (C-1I), 81.48
(C-2III), 80.56, 80.25, 79.67 (C-3I,II,IV), 70.99 (C-10),
52.06 (COOCH3), 36.69 (C-50), 31.21 (C-20), 28.01
(C-30), 25.08 (C-40), 25.41, 25.29, 25.28, 25.34, 25.22
(5NHCOCH3), 20.13, 20.04, 19.94, 19.87, 19.83 (C-6I–
V). FABMS: m/z 1082.6 [M+H]+, 1104.6 [M+Na]+.
The corresponding per-O-acetyl derivative 32 showed
[a]D +57 (c 1.1, CHCl3). ESI-MS (m/z): 1351.6146
[M+NH4]+, calcd for C59H95N6O29: 1351.6143. 1H
NMR (CDCl3, 600 MHz): d 7.33 (br d, 1H, NHIV),
6.39 (d, 1H, J4,NH = 10.1 Hz, NHI), 5.98 (br s, 1H,
NHII), 5.86 (br d, 1H, NHIII), 5.78 (d, 1H,
J4,NH = 9.9 Hz NHV), 5.18 (br dd, 1H, H-2IV), 5.15
(dd, 1H, J1,2 = 1.9, J2,3 = 3.2 Hz, H-2I), 5.07 (dd, par-
tially overlapped, H-3V), ꢀ5.07 (d, partially over-
lapped, H-1V), ꢀ5.06 (dd, partially overlapped,
H-3III), 4.95 (dd, 1H, J1,2 = 2.0, J2,3 = 3.1 Hz, H-2V),
4.92 (br t, 1H, H-2II), 4.90 (d, 1H, J1,2 = 2.0 Hz,
H-1II), 4.84 (d, 1H, J1,2 = 1.7 Hz, H-1IV), 4.83 (br d,
1H, J1,2 ꢀ 3 Hz, H-1III), 4.71 (d, 1H, J1,2 = 1.9 Hz,
H-1I), 4.16 (t, partially overlapped, J ꢀ 10.0 Hz,
H-4V), 4.14 (t, partially overlapped, H-4IV), 4.12 (t,
partially overlapped, J = 10.4 Hz, H-4I), 4.04–3.97
(m, 4H, H-3IV,3II,4III,3I, in that order), 3.96–3.89 (m,
3H, H-5V,4II,5II), 3.86 (br t, 1H, H-2III), 3.80 (m, 1H,
H-5IV), 3.74 (m, 1H, H-5I), 3.70–3.65 (m, 5H,
H-5III,10a, incl. 3.68, s COOCH3), 3.41, 3.40 (2 t, 1H,
J = 4.9 Hz, H-10b), 2.37 (t, 2H, J = 6.9 Hz, H-50a,b),
2.22–1.97 (10s, 33H, 11COCH3), 1.71–1.63 (m, 3H,
H-20a,40a,b), 1.61–1.54 (m, 1H, H-20b), 1.53–1.46 (m,
1H, H-30a), 1.37–1.30 (m, 1H, H-30b), 1.28–1.20 (6d,
15H, partially overlapped, J5,6 ꢀ 6.2 Hz, H-6I–V). 13C
NMR (CDCl3, 150 MHz): d 99.60 (C-1III), 98.58
(C-1II), 97.81 (br, C-1IV), 97.31 (C-1V), 97.02 (C-1I),
74.77 (br, C-3I), 73.64 (br, C-2III), 72.05 (br, C-3II),
71.47 (br, C-3IV), 71.03 (C-2II), 70.59 (C-2I), 69.96
(C-3III), 69.87 (C-2IV), 69.57 (C-2V), 68.57 (C-5V),
68.48 (C-5III), 68.39 (C-5IV), 68.09 (C-5II), 68.00
(C-3V), 67.45 (C-5I), 66.72 (C-10), 52.79 (C-4II), 52.40
(C-4III), 52.26 (2C, C-4I,IV), 51.56 (COCH3), 51.19
(C-4V), 33.79 (C-50), 28.47 (C-20), 25.72 (C-30), 24.16
(C-40), 23.46, 23.37, 23.33 (2C), 23.13 (5NHCOCH3),
21.07, 21.04, 20.99, 20.93, 20.73 (5COCH3), 18.08,
17.83, 17.76, 17.70, 17.64 (C-6I–V). Anal. Calcd for
C59H91N5O29: C, 53.11; H, 6.87; N, 5.25. Found: C,
52.99; H, 6.99; N, 5.01.
3.22. 5-Methoxycarbonylpentyl (4-acetamido-2-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-acet-
amido-2-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?2)-(4-acetamido-3-O-benzyl-4,6-dideoxy-a-D-manno-
pyranosyl)-(1?3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-
a-D-mannopyranosyl)-(1?3)-4-acetamido-2-O-benzyl-
4,6- dideoxy-a-D-mannopyranoside (30)
Reduction of 29 (1.935 g, 1.33 mmol) in 3:1 pyridine–
H2O was performed as described for 24. After work-
up and chromatography (EtOAc–MeOH 5:1 to 3:1),
the intermediate pentakis-amine {(: m/z 1322.67
[M+H]+) was N-acetylated, as described above for sim-
ilar conversions, and chromatography (CH2Cl2–acetone
1:1?2:3) gave 30 (1.5 g, 74%), [a]D +31.2 (c 1.8). FAB-
MS: m/z 1532.8 [M+H]+, 1554.8 [M+Na]+. Structurally
significant resonances in the 1H NMR (600 MHz,
CD3OD at 45 °C) were at d 5.15, 5.08, 5.07, 5.02 (4d,
1H each, J1,2 ꢀ 1.6 Hz, H-1II–V), 4.79 (d, 1H, J1,2
=
1.6 Hz, H-1I), 4.80–4.41 (10d, partially overlapped,
5CH2Ph), 3.64 (s, 3H, COOCH3), 2.33 (t, 2H, J =
7.5 Hz, H-50), 1.98, 1.97, 1.95, 1.92, 1.89 (5s, 3H each,
5NHCOCH3), 1.19, 1.18, 1.14, 1.30, 1.12 (5d, partially
overlapped, H-6I–V). Structurally significant resonances
in the 13C NMR (150 MHz, CD3OD at 45 °C) were
at d 102.20, 100.92, 100.28, 100.16 (C-1II–V), 99.12
(C-1I), 74.28, 74.15, 74.02, 73.96, 73.19 (5CH2Ph),
68.78 (C-10), 52.14 (COOCH3), 23.50, 23.44, 23.21,
23.06, 23.03 (5NHCOCH3), 18.90, 18.77, 18.73, 18.72,
18.46 (C-6I–V).
3.23. 5-Methoxycarbonylpentyl (4-acetamido-4,6-dide-
oxy-a-D-mannopyranosyl)-(1?3)-(4-acetamido-4,6-dide-
oxy-a-D-mannopyranosyl)-(1?2)-(4-acetamido-4,6-dide-
oxy- a-D-mannopyranosyl)-(1?3)-(4-acetamido-4,6-
dideoxy-a-D-mannopyranosyl)-(1?3)-4-acetamido-4,6-
dideoxy-a-D-mannopyranoside (31) and 5-methoxycar-
bonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-a-
D-mannopyranosyl)-(1?3)-(4-acetamido-2-O-acetyl-4,6-
dideoxy-a-D-mannopyranosyl)-(1?2)-(4-acetamido-3-O-
acetyl-4,6-dideoxy-a-D-mannopyranosyl)-(1?3)-(4-acet-
amido-2-O-acetyl-4,6-dideoxy-a-D-mannopyranosyl)-
(1?3)-4-acetamido-2-O-acetyl-4,6-dideoxy-a-D-manno-
pyranoside (32)
Hydrogenolysis of 30 (1.4 g, 0.91 mmol), performed as
described for 25 gave, after freeze-drying, the deprotec-
ted pentasaccharide 31 in virtually theoretical yield,
[a]D +79.2 (c 5.1, H2O). Structurally significant reso-
1
nances in the H NMR spectrum (600 MHz, D2O at

1706
R. Saksena et al. / Carbohydrate Research 343 (2008) 1693–1706
´ˇ
12. Ogawa, Y.; Lei, P.-S.; Kovac, P. Carbohydr. Res. 1996,
Acknowledgment
293, 173–194.
13. Ogawa, Y.; Lei, P.-S.; Kovac, P. Carbohydr. Res. 1996,
288, 85–98.
´ˇ
14. Zhang, J.; Kovac, P. Carbohydr. Res. 1997, 300, 329–339.
This research was supported by the Intramural Research
Program of the NIH, NIDDK.
´ˇ
15. Kovac, P. In Studies Toward a Rationally Designed
Conjugate Vaccine for Cholera Using Synthetic Carbohy-
drate Antigens; Bewley, C., Ed.; Royal Society of Chem-
istry: London, 2006; pp 175–220.
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