Immunogens from a synthetic hexasaccharide fragment of the O-SP of Vibrio cholerae O:1, serotype Ogawa

Article abstract of DOI:10.1016/j.tetasy.2004.11.021

The known 5-(methoxycarbonyl)pentyl α-glycoside of the hexasaccharide of Vibrio cholerae O:1, serotype Ogawa 24 was newly prepared. The efficiency of construction of the hexasaccharide from the disaccharide glycosyl acceptor 6 and each of the two tetrasaccharide glycosyl donors 16, and 18, as an alternative to the iterative coupling of the disaccharide glycosyl donor 7 with 6, was evaluated. Compound 24 was treated with each of hydrazine hydrate, ethylenediamine, and hexamethylenediamine to give ligands 25, 27, and 29, respectively, equipped with different linkers. Reaction of the foregoing compounds with squaric acid diethyl ester, and the reactions of the thus formed monoesters 26, 28, and 30 with BSA were evaluated. The rate of formation of the corresponding monoester was higher with hydrazide 25 than with amines 27 and 29, whose reaction rates were virtually the same. Reactions of squaric acid derivatives 26, 28, and 30 with BSA were conducted under the same conditions (reaction temperature, ligand-BSA ratio, and concentration with respect to the hapten) and targeted for neoglycoconjugates with 24-BSA ratio of ～5. Monitoring the conversions by SELDI-TOF mass spectrometry in conjunction with the ProteinChip system made it possible to conduct the conjugations in controlled fashion, and to terminate the reactions when the desired carbohydrate-protein ratios were reached. Products from 26, 28, and 30, neoglycoconjugates 31-33, containing 4.9, 5.0, and 5.1 moles of 24/BSA, respectively, were obtained in 87-93% yields. When the conjugation started with the initial molar carbohydrate-protein ratio of 15:1, the final molar carbohydrate-BSA ratio reached after 14 days with ligands 26, 28, and 30 was 9.0, 11.0, and 9.1, respectively.

Full text of DOI:10.1016/j.tetasy.2004.11.021

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 187–197
Immunogens from a synthetic hexasaccharide fragment of the
O-SP of Vibrio cholerae O:1, serotype Ogawa
´ˇ*
Rina Saksena, Jian Zhang and Pavol Kovac
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 30 October 2004; accepted 10 November 2004
Available online 24 December 2004
Abstract—The known 5-(methoxycarbonyl)pentyl a-glycoside of the hexasaccharide of Vibrio cholerae O:1, serotype Ogawa 24 was
newly prepared. The efficiency of construction of the hexasaccharide from the disaccharide glycosyl acceptor 6 and each of the two
tetrasaccharide glycosyl donors 16, and 18, as an alternative to the iterative coupling of the disaccharide glycosyl donor 7 with 6, was
evaluated. Compound 24 was treated with each of hydrazine hydrate, ethylenediamine, and hexamethylenediamine to give ligands
25, 27, and 29, respectively, equipped with different linkers. Reaction of the foregoing compounds with squaric acid diethyl ester,
and the reactions of the thus formed monoesters 26, 28, and 30 with BSA were evaluated. The rate of formation of the correspond-
ing monoester was higher with hydrazide 25 than with amines 27 and 29, whose reaction rates were virtually the same. Reactions of
squaric acid derivatives 26, 28, and 30 with BSA were conducted under the same conditions (reaction temperature, ligand–BSA
ratio, and concentration with respect to the hapten) and targeted for neoglycoconjugates with 24–BSA ratio of ꢀ5. Monitoring
the conversions by SELDI-TOF mass spectrometry in conjunction with the ProteinChipÒ system made it possible to conduct the
conjugations in controlled fashion, and to terminate the reactions when the desired carbohydrate–protein ratios were reached. Prod-
ucts from 26, 28, and 30, neoglycoconjugates 31–33, containing 4.9, 5.0, and 5.1 moles of 24/BSA, respectively, were obtained in
87–93% yields. When the conjugation started with the initial molar carbohydrate–protein ratio of 15:1, the final molar carbo-
hydrate–BSA ratio reached after 14 days with ligands 26, 28, and 30 was 9.0, 11.0, and 9.1, respectively.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
choice for conjugation of synthetic oligosaccharides to
proteins is that developed by Tietze et al.,5,6 which uti-
lizes the unique reactivity of squaric acid diesters. When
treated with an amine at pH7, a substance of this class
selectively forms a monoamide ester. The latter can be
then converted to a diamide by reaction with another
amine at pH9. When the two amines are an oligosacchar-
ide and a protein, the product will be a neoglycoconju-
gate. To make synthetic oligosaccharides suitable for
conjugation to proteins by squaric acid chemistry, they
are usually prepared in the form of glycosides, such as
24, whose linker can be transformed to contain a pri-
mary amino group. This has been most frequently
accomplished by aminolysis with ethylenediamine.7
Squaric acid diesters have also been derivatized with
hydrazides8,9 but, to our knowledge, a direct compari-
son of the effect of the nature of the linker upon conju-
gation using squaric acid chemistry has not been made.
The nature of the linker is an important variable in con-
jugate vaccine development. The linker (spacer) sepa-
rates the specific antigen (the B-cell epitope) from the
protein carrier (the T-cell epitope), thereby minimizing
the steric influence of the protein, which can affect both
During the past few years, this laboratory has been
working toward developing a conjugate vaccine for
cholera from synthetic fragments of O-specific polysac-
charides (O-SP) of Vibrio cholerae O:1. We have already
been able to show1 that a potent immunogen for protec-
tive anti V. cholerae O:1 antibodies can be obtained
from the upstream2 terminal hexasaccharide of the O-
SP and bovine serum albumin (BSA). Development of
conjugate vaccines from synthetic oligosaccharides is a
very young field and structural requirements for a
potent, medically acceptable synthetic vaccine are not
known. Within our efforts to develop a clinically useful
vaccine, we not only want to make conjugate immuno-
gens following established protocols, but we also want
to look at the effect of some fundamental variables upon
conjugation and the immunogenicity. Because of its high
efficiency3,4 and experimental simplicity, our method of
*
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402
0589; e-mail: kpn@helix.nih.gov
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.021

188
R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
chemistry of conjugation and immune response. Here
we describe conjugation to BSA of the upstream, termi-
nal hexasaccharide of the O-SP of V. cholerae O:1, sero-
type Ogawa equipped with three different linkers, and
evaluate the effect of the nature of the linker upon the
chemistry of conjugation.
7, to give the hexasaccharide intermediate 10. Deacetyl-
´
ation (Zemplen) of 10, followed by methylation of the
formed 11 with the MeI/Ag2O/Me2S reagent17 then gave
12. In the other two approaches, alcohol 6 was con-
densed with each of tetrasaccharide glycosyl donors 16
and 18, to give 10 and 12, respectively.
To increase the potential of an Ogawa hexasaccharide–
protein construct as a clinically useful vaccine for chol-
era,1 we are continuously striving to improve the synthe-
sis of the linker-equipped hexasaccharide. Here, we are
comparing the block-wise assembly of the hexasaccha-
ride intermediate 12 using different glycosyl donors.
Synthesis of the foregoing tetrasaccharide glycosyl
donors (Scheme 2) utilized the known18 hemiacetal 13,
which was now obtained in a more straightforward
way by hydrolysis of thioglycoside 7. Thus, all three ap-
proaches employ common intermediates 6 and 7. Com-
pound 13 was converted to trichloroacetimidate 14, and
treated with alcohol 15, obtained by deacetylation of 7,
to give 2IV-O-acetate 16, which was readily transformed
into the 2IV-methyl ether 18, through intermediate 17.
Results of serological evaluation of the immune re-
sponses in small laboratory animals, challenged with
products of conjugations here described, will be pub-
lished in due time.
When starting with the glycosyl acceptor 6, hexasacchar-
ide 12 was obtained in 59% (6 + 16 ! 10 ! 11 ! 12)
and 55% yield (6 + 18 ! 12) when tetrasaccharide gly-
cosyl donors were used, and in 78.5% yield
(6 + 7 ! 8 ! 9 ! 12) with the disaccharide glycosyl
donor (see Experimental). Thus, there is no advantage in
using the tetrasaccharide synthons 16 and 18 to assem-
ble the hexasaccharide 12. It appears that, in this series,
larger oligosaccharides are not as efficient glycosyl
donors as their less bulky counterparts.
2. Results and discussion
2.1. Synthesis of the linker-equipped hexasaccharides
The synthesis of hapten 24 was first carried out in this
laboratory almost 10 years ago.10 The finding that a
neoglycoconjugate based on that hexasaccharide has po-
tential as a vaccine for cholera1 prompted us to revise
the original synthesis. Several improvements have been
implemented in the newer variants,11–14 but each has
its advantages and drawbacks. The linker-equipped
oligosaccharide 24 was now newly synthesized. The
purpose of this new approach was to compare
construction of hexasaccharide intermediate 12 from
the disaccharide glycosyl acceptor 6 and tetrasaccharide
glycosyl donors 16 and 18, with the assembly from 6 by
iterative extension of the oligosaccharide chain with the
disaccharide glycosyl donor 7.15 Once assembled, com-
pound 12 was further transformed to the known hexa-
saccharide 24. Treatment of the latter with each of
hydrazine hydrate, ethylenediamine, and hexamethylene-
diamine produced the upstream, terminal fragment of
the O-SP of V. cholerae O:1, serotype Ogawa, equipped
with different linkers. We expect that comparison of
conjugations of these materials to BSA, and future eval-
uation of sera resulting from immunization of small lab-
oratory animals with the resulting immunogens, will
identify the most suitable derivative to be conjugated
to a medically acceptable carrier.
To introduce the 3-deoxy-L-glycero-tetronic acid side
chain amine 19 (Scheme 3), prepared by selective reduc-
tion15,19 of the azido functions in 12, was treated with
acid 21, obtained by simple acetylation of 2020 with ace-
tic anhydride in pyridine (Scheme 4), to give the fully
protected hexasaccharide 22. Preliminary experiments
(not described in Experimental) showed that the product
of amidation with the fully protected acid 21, was easier
to purify than the one resulting from amidation with 20,
or from acetylation of the crude product resulting from
the reaction of 20 + 19.
The aforementioned, fully protected hexasaccharide 22
was deprotected (22 ! 23 ! 24), and ester 24, thus
obtained, was treated with each of hydrazine hydrate,
ethylenediamine, and hexamethylenediamine, to give
synthons 25, 27, and 29. Conversion of these materials
to the corresponding monoethyl esters 26, 28, and 30,
respectively, was uneventful, but the reaction of the
hydrazide derivative 25 with squaric acid diethyl ester
was considerably faster. Contrary to the observations
by others,9 we have not observed violet color develop-
ment during formation of squaric acid monoesters from
hydrazides.
The assembly of 12 (Scheme 1) started with the hitherto
unknown glycosyl acceptor 3, whose precursor 2 was
now obtained from 1 in a higher yield (83% using BF3Æ
Et2O as a catalyst) than previously reported14 (75%,
with SnCl4 as a catalyst). Reaction of 3 with thioglyco-
2.2. Conjugation
side 416 (!5), followed by Zemplen deacetylation gave
Serological evaluation1 of neoglycoconjugates from
BSA and the hexasaccharide determinant of V. cholerae
O:1, serotype Ogawa, having carbohydrate–BSA ratio
ꢀ5, ꢀ10, and ꢀ15 has shown that the construct with
the lowest degree of substitution was most useful as an
experimental vaccine. To examine the effect of the nat-
ure of linker upon the conjugation by squaric acid chem-
istry and, perhaps, immunogenicity, the purpose of this
´
the key, linker-equipped disaccharide glycosyl acceptor
6. The construction of the hexasaccharide 12 from 6
was achieved in three different ways. Following the
route that led to the analogous methyl glycoside,11,16
condensation of 6 with 7 gave the fully protected tetra-
saccharide 8, which was deacetylated, and the alcohol 9
thus formed was treated (Scheme 2) with the same donor

R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
189
OAc
O
OR
O
H3C
H3C
N3
N3
BnO
BnO
4
SEt
O
O
H3C
OAc
O
OR
O
H3C
N3
H3C
N3
N3
HO
COOCH3
BnO
BnO
BnO
O
COOCH3
R
1
OAc
O
COOCH3
5
6
Ac
OR
O
H3C
OH
N3
R
BnO
2
3
Ac
OH
O
O
H3C
N3
BnO
2
OAc
O
H3C
N3
O
O
H3C
N3
BnO
BnO
O
O
H3C
O
COOCH3
N3
R
BnO
8
9
Ac
OH
SEt
7
OR
O
H3C
N3
16 or 18
BnO
R
O
O
10
11
12
Ac
OH
H3C
N3
BnO
OMe
4
O
O
H3C
6
N3
BnO
O
COOCH3
Scheme 1.
OAc
O
OAc
O
H3C
H3C
N3
N3
BnO
BnO
O
O
O
O
OAc
O
OH
O
OMe
O
H3C
H3C
H3C
H3C
H3C
N3
N3
N3
N3
N3
BnO
BnO
BnO
BnO
BnO
13
OH
14
O
CCl3
O
O
O
H3C
H3C
H3C
O
O
O
NH
N3
N3
N3
BnO
BnO
BnO
2
2
2
O
O
O
O
O
O
H3C
H3C
H3C
OH
O
N3
H3C
N3
N3
N3
BnO
BnO
BnO
BnO
16
SEt
17
SEt
18
SEt
7
O
O
H3C
N3
BnO
SEt
15
Scheme 2.
work was to prepare constructs similar to the one previ-
ously examined1 and showing the best immunogenicity
(the same hexasaccharide, carbohydrate–BSA ratio
ꢀ5) but involving different linkers. Previous work13
where the conjugation was conducted with an initial
molar carbohydrate–BSA ratio of 75 indicated that the
desired carbohydrate density (carbohydrate–BSA ratio
ꢀ5) should be possible to achieve starting with much
smaller excess of the carbohydrate, while maintaining
an acceptable reaction rate. Conjugation under such
conditions was expected to be slower and, thus, easier
to control, and also more economical, in view of the
labor-intensive hexasaccharide involved. Accordingly,
conjugations with the three ligands 26, 28, and 30 were
carried out at the initial molar hapten–BSA ratio of
15:1, and the increasing molecular mass of the product
was monitored by SELDI-TOF mass spectrometry in
conjunction with the ProteinChipÒ system.8 When the

190
R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
OMe
O
OMe
O
OMe
O
H3C
H3C
H3C
C NH
R1O
O
N3
H2N
RO
BnO
BnO
OR1
O
O
O
O
O
O
H3C
H3C
H3C
C NH
R1O
21
O
N3
H2N
RO
BnO
BnO
4
4
OR1
4
O
O
O
O
O
O
H3C
H3C
H3C
O
C
NH
R1O
RO
N3
H2N
BnO
BnO
OR1
O
COR2
O
COOCH3
O
COOCH3
R1 R2
12
19
R
OCH3
OCH3
OCH3
22
23
24
25
Ac Bn
OH Bn
OH OH
O
OH OH NHNH2
O
26
27
OH OH NHHN
OC2H5
O
NHCH2CH2NH2
OH OH
O
NHCH2CH2HN
28
29
OH OH
OH OH
OC2H5
O
O
NH(CH2)6NH2
NH(CH2)6HN
OC2H5
NH
30
31
OH OH
O
O
OH
OH
NHHN
BSA
4.9
O
O
O
O
OH
OH
OH
OH
32
33
NHCH2CH2HN
NH(CH2)6HN
NH
NH
BSA
BSA
5
5.1
Scheme 3.
O
OH
O
OH
was withdrawn, and the high molecular mass material
was isolated, and freeze-dried, to give conjugates 31–
33. It was of interest to compare the highest carbohy-
drate–BSA ratio attainable with haptens containing
different spacers. Therefore, the rest of the material
was allowed to react further and periodical monitoring
of the reaction was continued. After 14 days of reaction
time, virtually no additional increase in the carbo-
hydrate–protein ratio was noted with either of the
conjugates 31–33. This agreed with our previous obser-
vation4 that after that period of time virtually all active
monoester was consumed, due to conjugation or hydro-
lysis under the basic reaction conditions. The results in
Table 1 show that the fastest and also most efficient con-
jugation was that with the monoethyl ester prepared
from the pentanoic acid 2-(aminoethyl)amide.
C
C
HO
AcO
OBn
OBn
21
20
Scheme 4.
targeted carbohydrate–BSA ratio in each of the reac-
tions was reached (Table 1), 90% of the reacting material
Table 1. Effect of the nature of the linker upon conjugation of ligands
26, 28, and 30 to BSAa
Ligand
Ligand–BSA
ratio/time
required
Final molar
ligand–BSA
ratio after 14 days
Conjugation
efficiency [%]b
26
28
30
4.9/4 days
5/24 h
5.1/39 h
9
11
8.9
60
73
59
It is noteworthy that the molar carbohydrate–protein
ratios found for all three conjugates prepared are virtu-
ally the same as those targeted (hapten–BSA = 5). This
confirms the utility8 of SELDI-TOF mass spectrometry
in conjunction with the ProteinChipÒ system as an
important tool in glycoconjugate chemistry. Monitoring
a All reactions have been carried out at an initial, molar ligand–BSA
ratio of 15:1, at 15mmol concentration of the ligand.
b After 14 days, based on the ratio of ligand linked to BSA over the
initial amount of ligand used.

R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
191
conjugation of synthetic oligosaccharides to carriers by
this technique allows the reaction to be carried out in
a predictable way, and match the desired and achieved
carbohydrate–carrier ratios in neoglycoconjugates with
unprecedented accuracy. Also, when squaric acid chem-
istry is applied, the reaction rate can be controlled by
adjusting the initial molar hapten–carrier ratio.3 Due
to the efficiency of the conjugation observed with squa-
rate 26, ethylenediamine appears to be the reagent of
choice for converting ester groups in spacers into an
amide/amine, to make the parent ligand amenable to
conjugation by squaric acid chemistry, unless future
serological studies with constructs 31–33 would dictate
otherwise.
esters.6 Liquid chromatography–electron spray-ioniza-
tion mass spectrometry (LCESI MS) was performed
with a Hewlett–Packard 1100 MSD spectrometer.
Matrix assisted laser desorption-ionization time-of-
flight mass spectrometry (MALDI-TOF MS) was per-
formed with a Shimadzu AXIMA/CFR spectrometer.
Surface-enhanced laser desorption-ionization time-of-
flight mass spectrometry (SELDI-TOF MS) was done
by PBS-II Mass ReaderÒ (Ciphergen Biosystems, Inc),
calibrated with All in One Protein Standards (Ciphergen
Biosystems, Inc). The H4 or NP20 Protein Chip ArraysÒ
were used as sample carriers. The accuracy of molecular
mass determination was increased by using the carrier
protein (BSA, m/z 66,433Da) as an internal standard.13
Attempts have been made to obtain correct analytical
data for all new compounds. However, some com-
pounds tenaciously retained traces of solvents, despite
exhaustive drying, and analytical figures for carbon
could not be obtained within 0.3%. Structures of these
compounds follow unequivocally from the mode of syn-
thesis, NMR data, and m/z values found in their low-
and high-resolution mass spectra. TLC and NMR spec-
troscopy verified the purity of all compounds. BSA
(Fraction V, Prod. No. A-4503) was purchased from
Sigma Chemical Company, and purified as described
by Chen.24 Ethylenediamine (Aldrich Chemical Co.)
was freshly distilled, bp 115–116°C. Solid hexamethyl-
enediamine, a product of DuPont, was freshly distilled,
bp 92°C/2kPa. Palladium-on-charcoal catalyst (5%,
ESCAT 103) was a product of Engelhard Industries.
{N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-pyridin-
1-ylmethylene]-N-methylmethanaminium hexafluoro-
phosphate N-oxide} HATU was purchased from
Applied Biosystems. 3,4-Diethoxy-3-cyclobutene-1,2-
dione (squaric acid diethyl ester) was purchased from
Aldrich Chemical Co., and used as supplied. Solutions
in organic solvents were dried with anhydrous Na2SO4,
and concentrated at 40°C/2kPa.
3. Experimental
3.1. General methods
Unless stated otherwise, optical rotations were mea-
sured at ambient temperature for solutions in chloro-
form (c ꢀ 1), with
a
Perkin–Elmer automatic
polarimeter, Model 341. All reactions were monitored
by thin-layer chromatography (TLC) on Silica gel 60
coated glass slides. Column chromatography was per-
formed by gradient elution from columns of silica gel
with the high performance rapid flash chromatography
system (RT Scientific). Solvent mixtures less polar than
those used for TLC were used at the onset of separation.
Nuclear magnetic resonance (NMR) spectra were
measured at 300MHz (1H) and 75MHz (13C) with a
Varian Gemini or Varian Mercury spectrometer. Assign-
ments of NMR signals were made by first-order analysis
of the spectra, and by comparison with spectra of
related substances.11,15,16,21 When the latter approach
was used, advantage was taken of variations of line
intensity expected for oligosaccharides belonging to
the same homologous series to aid in the 13C NMR sig-
nal-nuclei assignments.22,23 Thus, spectra showed close
similarity of chemical shifts of equivalent carbon atoms
of the internal residues, and an increase in the relative
intensity of these signals with the increasing number of
D-perosamine residues in the molecule. When feasible,
the assignments were supported by homonuclear decou-
pling experiments or homonuclear and heteronuclear
2-dimensional correlation spectroscopy, run with the
software supplied with the spectrometers. When report-
ing assignments of NMR signals, sugar residues in oligo-
saccharides are serially numbered, beginning with the
one bearing the aglycone, and are identified by a Roman
numeral superscript in listings of signal assignments.
Nuclei-assignments without a superscript notation indi-
cate that those signals have not been individually
assigned. Thus, for example, in a spectrum of a tetrasac-
charide, a resonance denoted H-3 (as opposed to, for
example, H-3II), can be that of H-3 of either sugar resi-
due. Of two geminal protons showing distinctly different
chemical shifts the one resonating at a lower field is des-
ignated Ha, and the one resonating at a higher field is
designated Hb. Some signals in the 13C NMR spectra
of squaric acid derivatives appeared as doublets, the
splitting of these signals being due to the vinylogous
amide group, characteristic of squaric acid amide
3.2. General method for glycosylation with N-iodosuc-
cinimide/silver trifluoromethanesulfonate (NIS/AgOTf)
A mixture of the thioglycoside glycosyl donor
(1.3mmol), the glycosyl acceptor (1mmol) and finely
˚
powdered 4A molecular sieves (0.5g) in CH2Cl2 (10–
15mL) was stirred under argon for 15min. Solid NIS
(1.4mmol) was added at 0°C, followed by a solution
of AgOTf (0.4mmol) in dry toluene (4mL), and the mix-
ture was stirred at the same temperature for 3min. Cool-
ing was terminated and, when TLC showed that the
reaction was complete (ꢀ15min), the mixture was
neutralized with Et3N, washed successively with aq
NaHCO3 and water, dried, and concentrated. Chroma-
tography gave the desired product.
3.2.1. Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside 3. Boron trifluoride eth-
erate (8mL, 62mmol) was added to a stirred solution of
1
16 (15g, 41.3mmol) and methoxycarbonylpentanol (9g,
61.95mmol) in CH2Cl2 (400mL) and the stirring was
continued for 24h at room temperature, when TLC
(4:1 toluene–EtOAc) showed that the reaction was
complete. After neutralization with aqueous NaHCO3

192
R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
solution, the mixture was partitioned between water and
CH2Cl2, the organic phase was dried, and concentrated.
Chromatography of the residue gave methoxycarbonyl-
pentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranoside (2, 83%), which was indistinguishable
(TLC, NMR) from the substance obtained previously in
75% yield, when SnCl4 was used as a Lewis acid catalyst.
CH2Ph), 3.96 (m, 1H, H-2II), 3.87 (br t, 1H, H-2I),
3.72 (dd, partially overlapped, J2,3 3.0, J3,4 9.6Hz, H-
3I), 3.70 (dd, partially overlapped, J2,3 3.2, J3,4 9.4Hz,
H-3II), 3.65 (s, 3H, COOCH3), 3.62–3.54 (m, 2H, H-
5II, 10a), 3.49–3.25 (m, 4H, H-5I, 10b, incl. 3.43, 3.28, 2
t, J ꢀ 10.1Hz, H-4I, H-4II, respectively), 2.56 (br d,
1H, OH), 2.30 (t, 2H, J 7.5Hz, H-50a,b), 1.68–1.50 (m,
4H, H-20a,b,40a,b), 1.38–1.26 (m, 8H, H-30a,b, H-6I,II);
13C NMR (CDCl3): d 100.70 (C-1II), 98.41 (C-1I),
77.49 (C-3I), 77.23 (C-3II), 73.56 (C-2I), 71.71, 71.57 (2
CH2Ph), 67.17 (C-10), 67.03 (C-5II), 66.78 (C-2II),
66.75 (C-5I), 63.99 (C-4II), 63.46 (C-4I), 51.19
(COOCH3), 33.58 (C-50), 28.72 (C-20), 25.38 (C-30),
24.34 (C-40), 18.32, 18.13 (C-6I,II). LCESI MS: m/z 691
([M+Na]+). Anal. Calcd for C33H44N6O9: C, 59.27; H,
6.63; N, 12.57. Found: C, 59.11; H, 6.36; N, 12.55.
´
Deacetylation (Zemplen) of the foregoing acetate 2 gave
the title alcohol 3 in virtually theoretical yield. A small
amount of material was eluted from a silica gel column
to afford the analytical sample, [a]D = +66 (c 0.4,
1
CHCl3). H NMR (CDCl3): d 4.79 (d, 1H, J1,2 1.6Hz,
H-1), 4.71, 4.66 (2 d, 4H, 2 CH2Ph), 3.96 (m, 1H, H-
2), 3.72 (dd, 1H, J2,3 3.2, J3,4 9.4Hz, H-3), 3.67 (s, par-
tially overlapped, COOCH3), 3.64, 3.61 (2 t, partially
overlapped, J 6.7Hz, H-10a), 3.56–3.47 (m, 1H, H-5),
3.41 (t, partially overlapped, H-4), 3.41, 3.36 (2 t, par-
tially overlapped, H-10b), 2.49 (br d, 1H, OH), 2.32 (t,
2H, J 7.3Hz, H-50a,b), 1.73–1.52 (m, 4H, H-20a,b,40a,b),
1.40–1.30 (m, 5H, H-30a,b, incl. d, 1.31, J5,6 6.0Hz, H-
6); 13C NMR (CDCl3): d 98.87 (C-1), 78.38 (C-3),
71.94 (CH2Ph), 67.45 (C-10), 67.21 (C-2), 66.48 (C-5),
63.92 (C-4), 51.46 (COOCH3), 33.88 (C-50), 28.96 (C-
20), 25.64 (C-30), 24.60 (C-40), 18.38 (C-6). LCESI MS:
m/z 430 ([M+Na]+). Anal. Calcd for C20H29N3O6: C,
58.95; H, 7.17; N, 10.31. Found: C, 58.74; H, 7.22; N,
10.31.
3.2.4. Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1!2)-bis[4-azido-3-O-benz-
yl-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-4-azido-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranoside 9. Reaction
of the foregoing glycosyl acceptor 6 (4.0g, 6.0mmol)
with donor 7,11,15 gave methoxycarbonylpentyl 2-O-ace-
tyl-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyrano-
syl-(1!2)-bis[4-azido-3-O-benzyl-4,6-dideoxy-a-D-man-
nopyranosyl]-(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranoside 8 (6.9g, 95.8%). 1H NMR (CDCl3):
d 5.40 (dd, 1H, J1,2 1.9, J2,3 3.2Hz, H-2IV), 4.93 (d, 1H,
J1,2 1.9Hz, H-1III), 4.87 (d, 1H, J1,2 1.9Hz, H-1II), 4.84
(d, 1H, J1,2 1.9Hz, H-1IV), 4.73–4.51 (m, 9H, 4 CH2Ph,
incl. d, ꢀ4.60, H-1I), 3.87 (br t, 1H, H-2III), 3.83 (br t,
1H, H-2II), 3.79–3.75 (m, 2H, H-2I, 3IV), 3.71–3.65 (m,
6H, H-3I–III, incl. s, 3.67, COOCH3), 3.65–3.15 (m,
10H, H-4I–IV, 5I–IV, 10a,b), 2.32 (t, 2H, J 7.7Hz, H-
50a,b), 2.10 (s, 3H, COCH3), 1.69–1.50 (m, 4H, H-
40a,b,20a,b, in that order), 1.37–1.28 (m, 2H, H-30a,b),
1.26, 1.20, 1.16 (3 d, partially overlapped, 12H, H-6I–
IV); 13C NMR (CDCl3): d 100.37 (C-1II), 100.02 (C-
1III), 99.05 (C-1IV), 98.55 (C-1I), 77.42, 76.77, 76.58
(C-3I–III), 75.41 (C-3IV), 73.93 (C-2I), 73.40, 73.35 (C-
3.2.2. Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl-(1!2)-4-azido-
3-O-benzyl-4,6-dideoxy-a-D-mannopyranoside 5. Fol-
lowing the general procedure, the title disaccharide
was obtained in 95% yield, when starting from 3
(12.9g, 31.6mmol), [a]D = +48 (c 0.9, CHCl3). 1H
NMR (CDCl3): d 5.44 (dd, 1H, J1,2 1.9, J2,3 3.2Hz, H-
2II), 4.84 (d, 1H, H-1II), 4.73, 4.72, 4.64, 4.55 (4 d, par-
2
tially overlapped, J 11.1Hz, 2 CH2Ph), 4.68 (d, par-
tially overlapped, J1,2 1.9Hz, H-1I), 3.85 (dd, partially
overlapped, J2,3 3.1Hz, H-2I), 3.82 (dd, partially over-
lapped, J3,4 9.9Hz, H-3II), 3.76 (dd, 1H, J3,4 9.8Hz, H-
3I), 3.68 (s, 3H, COOCH3), 3.65–3.57 (m, 2H, H-5II,
10a), 3.52–3.29 (m, 4H, H-5I, 10b, incl. 3.41, 3.35, 2 t,
J ꢀ 9.8Hz, H-4I, H-4II, respectively), 2.34 (t, 2H, J
7.5Hz, H-50a,b), 2.09 (s, 3H, COCH3), 1.71–1.53 (m,
4H, H-20a,b,40a,b), 1.41–1.30 (m, 8H, H-30a,b, incl.
1.32, 1.31, 2 d, J5,6 6.2Hz, H-6II, H-6I, respectively);
13C NMR (CDCl3): d 99.02 (C-1II), 98.38 (C-1I), 77.58
(C-3I), 75.18 (C-3II), 73.82 (C-2I), 71.81, 71.34 (2
CH2Ph), 67.36 (C-5II), 67.32 (C-10), 67.00 (C-2II),
66.86 (C-5I), 63.94 (C-4I), 63.63 (C-4II), 51.38
(COOCH3), 33.85 (C-50), 28.80 (C-20), 25.36 (C-30),
24.40 (C-40), 20.75 (COC3), 18.35, 18.25 (C-6I,II). LCESI
MS: m/z 733 ([M+Na]+). Anal. Calcd for C35H46N6O10:
C, 59.14; H, 6.52; N, 11.82. Found: C, 59.27; H, 6.66; N,
11.387.
2
II,III), 72.16, 72.09, 71.99, 71.51 (4 C H2Ph), 67.75,
67.70, 67.60, 67.06 (C-5I–IV), 67.46 (C-10), 67.02 (C-
2IV), 64.29, 64.17, 63.97, 63.75 (C-4I–IV), 51.50
(COOC3), 33.87 (C-50), 28.98 (C-20), 25.63 (C-30),
24.61 (C-40), 20.96 (COCH3), 18.56, 18.45, 18.34 (2C,
C, C; C-6I–IV). LCESI MS: m/z 1255 ([M+Na]+).
The foregoing acetate 8 (6.9g) was deacetylated conven-
tionally, to give the title alcohol 9 (5.65g, 91%),
1
[a]D = +102 (c 2.7, CHCl3). H NMR (CDCl3): d 4.97
(d, 1H, J1,2 1.8Hz, H-1IV), 4.94 (d, 1H, J1,2 1.8Hz, H-
1III), 4.87 (d, 1H, J1,2 1.8Hz, H-1II), 4.75–4.55 (m, 9H,
4 CH2Ph, incl. d, ꢀ4.59, H-1I), 3.99 (m, 1H, H-2IV),
3.93 (br t, 1H, H-2III), 3.82 (br t, 1H, H-2II), 3.77 (br
t, 1H, H-2I), 3.73–3.68 (m, 7H, H-3I–IV, incl. s, 3.67,
COOCH3), 3.60–3.15 (m, 10H, H-4I–IV, 5I–IV, 10a,b),
2.34–2.29 (m, 3H, OH, incl. t, 2.31, H-50a,b), 1.68–1.49
(m, 4H, H-40a,b,20a,b, in that order), 1.37–1.13 (m,
14H, H-6I–IV, 30a,b); 13C NMR (CDCl3): d 100.40 (C-
1IV), 100.34 (C-1III), 100.16 (C-1II), 98.53 (C-1I), 77.61,
77.39, 76.90, 76.51 (C-3I–IV), 73.90 (C-2I), 73.46 (C-
2II), 73.13 (C-2III), 72.14, 72.08, 72.05, 71.99 (4 CH2Ph),
67.72, 67.70, 67.29, 67.00 (C-5I–IV), 67.43 (C-10), 67.05
3.2.3. Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1!2)-4-azido-3-O-benzyl-
´
4,6-dideoxy-a-D-mannopyranoside 6. Zemplen deacetyl-
ation of 5 gave 6 in virtually theoretical yield, [a]D =
+96 (c 1.3, CHCl3). 1H NMR (CDCl3): d 4.92 (d,
1H,J1,2 1.7Hz, H-1II), 4.70–4.58 (m, 5H, H-1I, 2

R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
193
(C-2IV), 64.27, 64.12, 63.75 (C, 2C, C; C-4I–IV), 51.48
(COOCH3), 33.85 (C-50), 28.95 (C-20), 25.60 (C-30),
24.59 (C-40), 18.55, 18.45, 18.25 (2C, C, C; C-6I–IV);
LCESI MS: m/z 1191.7 ([M+H]+). Anal. Calcd for
C59H74N12O15: C, 59.48; H, 6.26; N, 14.11. Found: C,
59.60; H, 6.36; N, 14.10.
73.14 (C-2V), 72.10, 71.99 (5C, C; 6 CH2Ph), 67.75,
67.65, 67.29, 66.99 (3C, C, C, C; C-5I–VI), 67.43 (C-10),
67.05 (C-2IV), 64.27, 64.11, 63.75 (C, 4C, C; C-4I–VI),
51.47 (COOCH3), 33.84 (C-50), 28.95 (C-20), 25.60 (C-
30), 24.58 (C-40), 18.54, 18.45, 18.26 (2C, 3C, C;
C-6I–IV). Anal. Calcd for C85H104N18O21: C, 59.57; H,
6.12; N, 14.71. Found: C, 59.28; H, 6.11; N, 14.53.
3.2.5. Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl-(1!2)-tetrakis-
[4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl]-
(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyrano-
side 10. (a) Condensation of 7 with 9 (5.1g, 4.2mmol),
as described in the general procedure for glycosylation,
gave the title hexasaccharide 10 (6.4g, 85%), [a]D =
+50 (c 7.6, CHCl3). Structurally significant signals
3.2.7. Methoxycarbonylpentyl 2-O-methyl-4-azido-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl-(1!2)-tetrakis-
[4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl]-
(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyrano-
side 12. (a) Me2S (catalytic amount, ꢀ0.1mL) was
added to a suspension of alcohol 11 (0.5g), Ag2O (1g)
and MeI (2.5mL) in 1,2-dimethoxyethane (10mL), and
the mixture was stirred overnight at room temperature.
TLC (4:1 hexane–EtOAc) then showed that the starting
material virtually completely disappeared. After filtra-
tion, the solids were washed with CH2Cl2, the combined
filtrates were concentrated, and chromatography of the
residue gave 12 (0.45g, 90%), [a]D = +103 (c 2.2,
CHCl3). Structurally significant signals in the 1H
NMR (CDCl3) spectrum were at: d 4.93–4.90 (m, 3H,
H-1II,V,VI), 4.85, 4.84 (2 d, partially overlapped, 2H,
H-1III,IV), 4.76–4.59 (m, 13H, 6 CH2Ph, incl. d, ꢀ4,59,
H-1I), 3.96, 3.85, 3.82, 3.79 (4 br t, 4H, H-2II–V), 3.76
(br t, 1H, H-2I), 3.67 (s, partially overlapped,
COOCH3), 3.33, 3.30 (2 t, 2H, J 6.5Hz, H-10a,b), 3.21
(s, partially overlapped, OCH3-2), 2.32 (t, 2H, J
7.3Hz, H-500a,b), 1.68–1.49 (m, 4H, H-400a,b,200a,b, in
that order), 1.37–1.12 (m, 20H, H-300a,b, 6I–VI); 13C
NMR (CDCl3): d 100.33, 100.19, 100.10, 100.07 (C-
1
in the H NMR (CDCl3) spectrum were at: d 5.41 (dd,
1H, J1,2 1.9, J2,3 3.2Hz, H-2VI), 4.95 (d, 1H, J1,2
1.6Hz, H-1V), 4.87 (d, 1H, J1,2 1.6Hz, H-1II), 4.86–
4.84 (m, 3H, H-1III,IV,VI), 4.75–4.52 (m, 13H, 6 CH2Ph,
incl. d, ꢀ4.59, H-1I), 3.88 (br dd, 1H, H-2V), 3.84 (br dd,
1H, H-2II), 3.82–3.72 (m, 4H, H-2I,III,IV, 3VI), 3.67 (s,
partially overlapped, COOCH3), 2.31 (t, 2H, J 7.7Hz,
H-50a,b), 2.11 (s, 3H, COCH3), 1.69–1.49 (m, 4H, H-
40a,b,20a,b, in that order), 1.37–1.12 (m, 20H, H-6I–VI
,
30a,b,); 13C NMR (CDCl3): d 100.32 (C-1II), 100.09
(2C, C-1III,IV), 100.01 (C-1V), 99.05 (C-1VI), 98.55 (C-
1I), 77.42, 76.80, 76.62, 76.51 (C, C, C, 2C; C-3I–V),
75.41 (C-3VI), 73.96 (C-2I), 73.53 (C-2II), 73.38 (2C; C-
2
III,IV), 73.29 (C-2V), 72.13, 72.03, 72.00, 71.52 (3C, C,
C, C; 6 CH2Ph), 67.78, 67.70, 67.61, 67.02 (3C, C, C,
C; C-5I–VI), 67.46 (C-10), 67.08 (C-2IV), 64.30, 64.19,
64.14, 63.99, 63.76 (C, C, 2C, C, C; C-4I–VI), 51.50
(COOCH3), 33.87 (C-50), 28.98 (C-20), 25.63 (C-30),
24.61 (C-40), 18.56, 18.46, 18.35 (2C, 3C, C; C-6I–IV).
Anal. Calcd for C87H106N18O22: C, 59.51; H, 6.08; N,
14.36. Found: C, 59.75; H, 6.14; N, 14.25.
1
II–V), 98.73 (C-1VI), 98.54 (C-1I), 77.42, 77.12, 76.57,
76.50 (2C, C, C, 2C; C-3I–VI), 76.32 (C-2VI), 74.00 (C-
2I), 73.53, 73.38, 73.17, 73.10 (C-2II–V), 72.34, 72.11,
72.08, 72.00 (C, 3C, C, C; 6 CH2Ph), 67.80, 67.71,
67.02 (3C, 2C, C; C-5I–VI), 67.43 (C-10), 64.23, 64.14,
64.05 (2C, 3C, C; C-4I–VI), 58.87 (OCH3-2), 51.45
(COOCH3), 33.85 (C-50), 28.96 (C-20), 25.61 (C-30),
24.59 (C-40), 18.54, 18.45, 18.37 (2C, 3C, C; C-6I–IV).
LCESI MS: m/z 1749 ([M+Na]+). Anal. Calcd for
C86H106N18O21: C, 59.78; H, 6.18; N, 14.59. Found: C,
59.86; H, 6.28; N, 14.59.
(b) Condensation of alcohol 6 (4.7g, 7mmol) with the
tetrasaccharide glycosyl donor 16, as described in the
general procedure for glycosylation gave, after chroma-
tography, material (8.0g, 65%), which was indistinguish-
able (TLC, NMR) from the above described substance.
3.2.6. Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1!2)-tetrakis[4-azido-3-
O-benzyl-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-4-azido-
3-O-benzyl-4,6-dideoxy-a-D-mannopyranoside 11. Con-
(b) Condensation of glycosyl acceptor
6 (0.7g,
1.08mmol) with donor 18, following the general proto-
col for glycosylation gave, after chromatography (4:1
hexane–acetone)material (0.96g, 55%), which was indis-
tinguishable from the above-described substance.
´
ventional deacetylation (Zemplen) of 10 gave alcohol
11 in 84% yield, [a]D = +108 (c 1.7, CHCl3). Structurally
1
significant signals in the H NMR (CDCl3) spectrum
were at: d 4.99 (d, 1H, J1,2 1.7Hz, H-1VI), 4.97 (d, 1H,
J1,2 2.0Hz, H-1V), 4.89 (d, 1H, J1,2 2.0Hz, H-1II), 4.85
(br d, 2H, H-1III,IV), 4.75–4.59 (m, 13H, 6 CH2Ph, incl.
d, ꢀ4.59, H-1I), 4.00 (m, 1H, H-2VI), 3.95 (br dd, 1H,
H-2V), 3.84 (br t, 1H, H-2II), 3.81, 3.79 (2 br t, 2H, H-
3.2.8. 2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranosyl-(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-
a,b-D-mannopyranose 13. Water (0.1mL) was added at
0°C to a stirred solution of ethyl 2-O-acetyl-4-azido-3-
O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-(1!2)-4-azi-
do-3-O-benzyl-4,6-dideoxy-1-thio-a-D-mannopyranoside
2
III,IV), 3.77 (br dd, 1H, H-2I), 3.67 (s, partially over-
lapped, COOCH3), 2.36 (d, 1H, J2,OH 2.0Hz, OH),
2.32 (t, 2H, J 7.3Hz, H-50a,b), 1.69–1.50 (m, 4H, H-
40a,b,20a,b, in that order), 1.38–1.13 (m, 20H, H-
30a,b,6I–VI); 13C NMR (CDCl3): d 100.41 (C-1VI),
100.31, 100.15, 100.06 (C, C, 2C; C-1II–V), 98.52 (C-
1I), 77.61, 77.37, 76.91, 76.52, 76.46 (C, C, C, C, 2C;
C-3I–VI), 73.95 (C-2I), 73.53 (C-2II), 73.35 (2C; C-2III,IV),
7
11,15 (313mg, 0.5mmol) in acetone (10mL), followed by
NIS (135mg, 0.6mmol). The cooling was removed after
5min, and the solution was stirred at room temperature
until TLC (hexane–EtOAc) showed that the reaction
was virtually complete (2h). One major product, show-
ing slower mobility than the starting material, was
formed. The mixture was neutralized with solid

194
R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
NaHCO3, acetone was evaporated, and the residue
was extracted with CH2Cl2. After concentration, chro-
matography gave material (232mg, 83%), which was
indistinguishable from the previously, independently
synthesized substance.18
nate (catalytic amount, ꢀ0.1mL) was added, and stirring
at room temperature was continued for 15min, when
TLC showed almost complete disappearance of the start-
ing materials. After neutralization with triethylamine,
the mixture was filtered, the filtrate was concentrated,
and the residue was chromatographed to give 16
(13.1g, 84%), [a]D = +119 (c 0.7, CHCl3). 1H NMR
(CDCl3): d 5.41 (dd, 1H, J1,2 2.0, J2,3 3.1Hz, H-2IV),
5.09 (d, 1H, J1,2 1.2Hz, H-1I), 4.94 (d, 1H, J1,2 1.6Hz,
H-1III), 4.85 (d, 1H, J1,2 2.0, H-1IV), 4.83 (d, 1H, J1,2
1.6Hz, H-1II), 4.74–4.51 (4 d, 8H, 4 CH2Ph), 3.87 (br t,
1H, H-2III), 3.84–3.81 (m, 2H, H-2I,II), 3.79–3.73 (m,
2H, H-3IV, 5I), 3.70 (dd, partially overlapped, J2,3 3.2,
J3,4 ꢀ 9.5Hz, H-3III), 3.67 (dd, partially overlapped,
J2,3 3.2, J3,4 9.8Hz, H-3II), 3.60 (dd, 1H, J2,3 2.8,
J3,4 9.8Hz, H-3I), 3.56–3.18 (m, 7H, H-4I–IV, 5II–IV),
2.64–2.46 (m, 2H, CH2CH3), 2.10 (s, 3H, COCH3),
1.27–1.18 (m, 15H, H-6I–IV, incl. t, 1.24, J 7.8Hz,
CH2CH3); 13C NMR (CDCl3): d 100.55 (C-1II), 100.02
(C-1III), 99.07 (C-1IV), 83.33 (C-1I), 77.75 (C-3I), 76.79
(C-3III), 76.59 (C-3II), 75.02 (C-2I), 75.42 (C-3IV), 73.37
(2C, C-2II,III), 72.17, 72.02, 71.53 (2C, C, C; 4 CH2Ph),
67.87, 67.80, 67.63 (C-5II–IV), 67.51 (C-5I), 67.10 (C-
2IV), 64.53 (C-4I), 64.21 (C-4II), 64.00 (C-4III), 63.80
(C-4IV), 25.60 (CH2CH3), 18.63, 18.58, 18.50, 18.43 (C-
3.2.9. Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-a-D-manno-
pyranosyl-(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-man-
´
nopyranoside 15. Zemplen deacetylation of ethyl 2-O-
acetyl-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyrano-
syl-(1!2)-4-azido-3-O-benzyl-4,6-dideoxy-1-thio-a-D-
mannopyranoside 711,15 gave 15 in ꢀ90% yield,
[a]D = +191. 1H NMR (CDCl3): d 5.17 (d, 1H, J1,2
1.5Hz, H-1I), 4.90 (d, 1H, J1,2 1.7Hz, H-1II), 4.75–4.58
(4 d, 4H, J ꢀ 11.5Hz, 2 CH2Ph), 3.98 (ddd, 1H, J2,3
3.3, J2,OH 1.8Hz, H-2II), 3.95 (dd, 1H, J2,3 2.8Hz,
H-2I), 3.87–3.77 (m, 1H, H-5I), 3.72 (dd, partially over-
lapped, J3,4 9.8Hz, H-3II), 3.65 (dd, partially over-
lapped, J3,4 ꢀ 9.9Hz, H-3I), 3.69–3.59 (m, partially
overlapped, H-5II), 3.43 (t, 1H, J 9.9Hz, H-4II), 3.32
(t, 1H, J 10.0Hz, H-4I), 2.66–2.48 (m, 2H, CH2CH3),
2.36 (d, 1H, OH), 1.30 (d, partially overlapped, H-
6
I,II), 1.26 (t, partially overlapped, J 7.5Hz, CH2CH3);
13C NMR (CDCl3): d 100.89 (C-1II), 83.39 (C-1I),
78.13 (C-3I), 71.60 (C-3II), 75.86 (C-2I), 72.14, 72.06 (2
CH2Ph), 67.52 (C-5I), 67.33 (C-5II), 64.47 (C-4I), 63.76
(C-4II), 25.60 (C2CH3), 18.52, 18.45 (C-6I,II), 14.92
(CH2C3); CIMS: m/z 602 ([M+NH4]+). Anal. Calcd for
C28H36N6O6S: C, 57.52; H, 6.21; N, 14.37; S, 5.48.
Found: C, 57.38; H, 6.14; N, 14.28; S, 5.53.
6
I–IV), 14.93 (CH2CH3); CIMS: m/z 1149 ([M+H]+),
1171 ([M+Na]+). Anal. Calcd for C56H68N12O13S: C,
58.52; H, 5.96; N, 14.62; S, 2.79. Found: C, 58.67; H,
6.08; N, 14.36; S, 2.68.
3.2.11.
Ethyl
4-azido-3-O-benzyl-4,6-dideoxy-a-D-
3.2.10. Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-
a-D-mannopyranosyl-(1!2)-bis(-4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl)-4-azido-3-O-benzyl-4,6-
dideoxy-1-thio-a-D-mannopyranoside 6. To a stirred
solution of compound 13 (8.7g, 14.95mmol) and tri-
chloroacetonitrile (6mL, 60mmol) in CH2Cl2 (200mL)
was added diazabicyclo[5.4.0]undec-7-ene (0.9mL,
6mmol) and the mixture was stirred for 10min, when
TLC showed that the reaction was complete. The
mixture was concentrated and chromatography gave
mannopyranosyl-bis(1!2)-(-4-azido-3-O-benzyl-4,6-dide-
oxy-a-D-mannopyranosyl)-4-azido-3-O-benzyl-4,6-dide-
oxy-1-thio-a-D-mannopyranoside
17. Deacetylation
´
(Zemplen) of the foregoing acetylated tetrasaccharide
gave alcohol 17 in virtually theoretical yield,
[a]D = +121. 1H NMR (CDCl3): d 5.09 (d, 1H, J1,2
1.2Hz, H-1I), 4.97 (d, 1H, J1,2 1.7Hz, H-1IV), 4.95 (d,
1H, J1,2 1.7Hz, H-1III), 4.84 (d, 1H, J1,2 1.7Hz,
H-1II), 4.74–4.57 (m, 8H, 4 CH2Ph), 4.00–3.97 (m, 1H,
H-2IV), 3.93 (br t, 1H, H-2III), 3.83 (br t, 1H, H-2I),
3.80 (br t, 1H, H-2II), 3.80–3.74 (m, partially over-
lapped, H-5I), 3.72–3.67 (m, 3H, H-3II–IV), 3.60 (dd,
1H, J2,3 2.9, J3,4 9.9Hz, H-3I), 3.56–3.38 (m, 4H, H-
4,5II–IV), 3.34–3.18 (m, 3H, 2 H-4, incl. t, partially over-
lapped, J 9.9Hz, H-4I), 2.61–2.47 (m, 2H, CH2CH3),
2.39 (d, 1H, J2,OH 2.1Hz, OH), 1.28–1.18 (m, 15H, 4
H-6, CH2CH3); 13C NMR (CDCl3): d 100.48 (C-1II),
100.39 (C-1IV), 100.12 (C-1III), 83.26 (C-1I), 77.71 (C-
3I), 77.56, 76.85, 76.45 (C-3II–IV), 75.95 (C-2I), 73.39
(C-2II), 73.12 (C-2III), 72.05, 71.95 (3C, C; 4 CH2Ph),
67.82, 67.72, 67.28 (C-5II–IV), 67.45 (C-5I), 67.03 (C-
2IV), 64.45 (C-4I), 64.10, 63.72 (2C, C; C-4II–IV), 25.54
(C2CH3), 18.58, 18.54, 18.44, 18.28 (C-6I–IV), 14.88
(CH2CH3); CIMS: m/z 1129 ([M+Na]+). Anal. Calcd
for C54H66N12O12S: C, 58.58; H, 6.01; N, 15.18; S,
2.90. Found: C, 58.36; H, 5.99; N, 14.98; S, 2.86.
amorphous
2-O-acetyl-4-azido-3-O-benzyl-4,6-dide-
oxy-a-D-mannopyranosyl-(1!2)-4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl trichloroacetimidate 14
1
(10.6g, 95%). H NMR (CDCl3): d 6.11 (d, 1H, J1,2
1.9Hz, H-1I), 5.44 (dd, 1H, J1,2 1.7, J2,3 3.0Hz,
H-2II), 4.89 (d, 1H, H-1II), 4.77–4.55 (4 d, partially over-
lapped, 2 CH2Ph), 3.89 (br t, 1H, H-2I), 3.80 (dd,
partially overlapped, J3,4 9.9Hz, H-3II), 3.76 (dd, par-
tially overlapped, J2,3 3.0, J3,4 9.9Hz, H-3I), 3.72–3.58
(m, 2H, H-5I,II), 3.45, 3.42 (2 t, 2H, J ꢀ 10Hz,
H-4I,II), 2.12 (s, 3H, COCH3), 1.33, 1.32 (2 d, partially
overlapped, 6H, J5,6 ꢀ 6Hz, H-6I,II); 13C NMR
(CDCl3): d 99.43 (C-III), 96.22 (C-II), 90. 70 (CN),
76.72 (C-3I), 75.35 (C-3II), 72.37, 71.61 (2 CH2Ph),
72.10 (C-2I), 70.05 (C-5I), 67.81 (C-5II), 67.08 (C-2II),
63.63, 63.39 (C-4I,II), 20.84 (COCH3), 18.52, 18.26 (C-
6
I,II); CIMS: m/z 743 ([M+NH4]+).
3.2.12. Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-meth-
yl-a-D-mannopyranosyl-(1!2)-bis(4-azido-3-O-benzyl-
4,6-dideoxy-a-D-mannopyranosyl)-4-azido-3-O-benzyl-
A mixture of the foregoing imidate (10.6g, 14.6mmol),
˚
alcohol 15 (7.9g, 13.5mmol), and powdered 4A mole-
cular sieves (7.5g) in CH2Cl2 (250mL) was stirred under
argon for 15min. Trimethylsilyl trifluoromethanesulfo-
4,6-dideoxy-1-thio-a-D-mannopyranoside
18. Methyl
iodide (0.225mL, 36mmol) was added to a stirred mix-

R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
195
ture of 17 (2.67g, 24mmol) and powdered KOH (4.1g,
72mmol) in Me2SO (30mL). After 1h TLC showed that
the reaction was complete and that one product was
formed. Water was added, to dissolve the solids, and
the mixture was neutralized with aqueous AcOH
(ꢀ10%, v/v). The mixture was partitioned between
CH2Cl2 and brine, the organic phase was concentrated,
and chromatography of the residue gave 18 (2.4g, 89%),
mp 79–80°C (from EtOH), [a]D = +140 (c 0.8, CHCl3).
1H NMR (CDCl3): d 5.10 (d, 1H, J1,2 1.3Hz,
H-11), 4.91 (2 d, overlapped, 2H, J1,2 1.7Hz, H-1III,IV),
4.86 (d, 1H, J1,2 1.7Hz, H-1II), 4.75–4.56 (m, 8H, 4
CH2Ph), 3.95 (br t, 1H, H-2III), 3.85 (br t, 1H, H-2I),
3.82 (br t, partially overlapped, H-2II), 3.82–3.73 (m,
partially overlapped, H-5I), 3.72–3.66 (m, 3H, H-
(COOCH3), 33.75 (C-50), 29.93 (C-20), 25.58 (C-
30), 24.52 (C-40), 18.10, 18.07, 18.03, 17.92 (3C, C, C,
C; C-6I–VI). Anal. Calcd for C86H118N6O21: C, 65.71;
H, 7.57; N, 5.35. Found: C, 65.43; H, 7.56; N, 5.19.
3.2.14. Methoxycarbonylpentyl 3-O-benzyl-2-O-methyl-
4-(2-O-acetyl-4-O-benzyl-3-deoxy-L-glycero-tetronamido)-
4,6-dideoxy-a-D-mannopyranosyl-(1!2)-tetrakis[4-(2-O-
acetyl-4-O-benzyl-3-deoxy-L-glycero-tetronamido)-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-4-(2-O-
acetyl-4-O-benzyl-3-deoxy-L-glycero-tetronamido)-3-O-
benzyl-4,6-dideoxy-a-D-mannopyranoside 22. A solu-
tion of crystalline 4-O-benzyl-L-glycero-tetronic acid20
20 (3.0g) in 2:1 Ac2O–pyridine [2:1, (v/v), 3.0mL] was
kept overnight at room temperature. TLC (15:1:0.1
CH2Cl2–MeOH–AcOH) showed the reaction to be com-
plete. After concentration, and evaporation of several
portions of water from the residue, to remove volatiles,
the residue (3.3g, 92%) contained pure (NMR, TLC) 2-
O-acetyl-3-O-benzyl-L-glycero-tetronic acid 21, [a]D =
3
II–IV), 3.61 (dd, 1H, J2,3 2.7Hz, J3,4 9.9Hz, H-3I),
3.59–3.48 (m, partially overlapped, H-5IV), 3.48–3.41
(m, 3H, H-4IV, 5II,III), 3.38 (br t, 1H, 2IV), 3.34–3.20
(m, partially overlapped, H-4I–III), 3.20 (s, partially
overlapped, OCH3), 2.64–2.44 (m, 2H, CH2CH3),
1.28–1.17 (m, 15H, H-6I–IV, CH2CH3). 13C NMR
1
ꢁ34 (c 2.4, CHCl3). H NMR (CDCl3): d 9.87 (br s,
(CDCl3):
d
100.47 (C-1II), 100.10 (C-1III),
1H, COOH), 7.35–7.24 (m, 5H, aromatic), 5.22 (dd,
1H, J2,3a 5.0, J2,3b 8.5Hz, H-2), 4.53, 4.45 (2 d, 2H, J
11.7Hz, CH2Ph), 3.65–3.53 (m, 2H, H-4a,b), 2.31–2.06
(m, 5H, H-3a,b, incl. s, 2.09, COCH3). 13C NMR
(CDCl3): d 72.97 (CH2Ph), 69.04 (br, C-2), 65.15 (C-
4), 31.17 (C-3), 20.50 (COC3). LCESI MS: m/z 275
([M+Na]+).
98.63 (C-1IV), 83.23 (C-1I), 77.68 (C-3I), 77.28, 77.00
(C-3III,IV), 76.48 (C-3II), 76.21 (C-2IV), 75.92 (C-2I),
73.17 (C-2II), 73.01 (C-2III), 72.22, 72.02, 71.88 (2C,
C, C; 4 CH2Ph), 67.77, 67.69, 67.60 (C-5II–IV), 67.39
(C-5I), 64.40 (C-4I), 64.15, 64.01 (C-4II,III),
63.95 (C-4IV), 58.76 (COC3), 25.43 (C2CH3), 18.49,
18.42, 18.36, 18.31 (C-6I–IV), 14.78 (CH2C3); FAB MS:
m/z 1253 ([M+Cs]+). Anal. Calcd for C56H68N12O13S:
C, 58.91; H, 6.11; N, 14.99; S, 2.86. Found: C, 58.63;
H, 6.08; N, 14.90; S, 2.80.
To a solution of 19 (100mg, 0.064mmol) and 21
(161mg, 0.64mmol) in CH2Cl2 (3mL) was added
HATU (243mg, 0.64mmol) followed by N-ethyldiiso-
propylamine (0.08mL, 0.64mmol), and the mixture
was stirred overnight at room temperature. TLC (20:1
CH2Cl2–MeOH) showed that the reaction was complete
and that one major product was formed. The mixture
was partitioned between CH2Cl2 and water, which was
kept slightly acidic (pHꢀ6) by addition of 1M HCL.
The organic phase was washed with a mixture (1:1,
v/v) of brine and NaHCO3 solution, dried, and concen-
trated. Chromatography of the residue gave 22 (122mg,
80%), [a]D = ꢁ37. Structurally significant signals in the
1H NMR (CDCl3) spectrum were at: d 6.40–5.83 (m, 6
NH), 5.30–5.21 (m, 6H, H-20I–VI), 5.03, 4.98 (br d, br
s, 4H total, H-1II–V), 4.87 (br s, 1H, H-1VI), 4.68 (br s,
1H, H-1I), 3.61 (s, overlapped, COOCH3), 3.54–3.47
(m, 12H, 40I–VIa,b), 3.19 (s, overlapped, OCH3-2), 2.29
(t, 2H, J 7.4Hz, H-500a,b), 1.99, 1.95, 1.94, 1.92, 1.91
3.2.13. Methoxycarbonylpentyl 2-O-methyl-4-amino-3-
O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-(1!2)-tetra-
kis[4-amino-3-O-benzyl-4,6-dideoxy-a-D-mannopyrano-
syl]-(1!2)-4-amino-3-O-benzyl-4,6-dideoxy-a-D-manno-
pyranoside 19. Hydrogen sulfide gas was passed
through a solution of the azido derivative 18 (0.7g) in
pyridine–water [2:1 (v/v), 36mL], for 30min at 40°C.
The mixture was kept overnight at 40°C in a loosely
closed flask, when TLC (CH2Cl2–MeOH) showed that
the starting material was no longer present, and that
one, largely predominating product was formed. After
concentration, the mixture was chromatographed to
give 19 (0.6g, 94%), [a]D = 0 (c 1.0, MeOH). Structur-
ally significant signals in the 1H NMR (CDCl3)
spectrum were at: d 5.10–5.06 (3 br d, 4H, H-1II–V),
5.00 (br d, 1H, H-1VI), 4.75 (br d, 1H, H-1I), 4.72–4.41
(m, 12H, 6 CH2Ph), 4.09, 4.05–4.03 (br t, 1H, m, 3H,
H-2II–V), 3.93 (br t, 1H, H-2I), 3.67 (s, partially over-
lapped, COOCH3), 3.38, 3.34 (dt, 1H, J 6.5Hz,
H-10b), 3.29 (s, 3H, OCH3-2VI), 2.93–2.80 (m,
6H, H-4I–VI), 2.33 (t, 2H, J 7.5Hz, H-50a,b), 1.70–1.53
(m, 4H, H-40a,b,20a,b, in that order), 1.41–1.14 (m,
32H, 6 NH2, H-30a,b,6I–VI); 13C NMR (CDCl3):
(5 s, one of them double intensity, partially overlapped
I–VI
with H-3b0
signals, 6 COCH3), 1.65–1.25 (2 m, H-
200a,b,300a,b,400a,b), 1.18–1.07 (6 d, partially overlapped,
H-6I–VI). Structurally significant signals in the 13C
NMR (CDCl3) spectrum were at: d 100.62, 99.74 (C,
3C, br, C-1II–V), 98.93 (C-1VI), 98.61 (C-1I), 72.94,
72.87, 72.79, 72.72 (C, C, 3C, C; 6 CH2Ph-3I–VI),
71.47, 71.45, 71.42, 71.36 (C, C, C, 3C; C-20I–VI),
I–
d
100.89, 100.81 (C, 3C, C-1II–V), 99.02, 98.99
71.34, 71.12, 71.04, 70.87, 70.63, 70.46 (6 CH2Ph-40
(C-1I,VI), 79.46, 78.97, 78.60, 78.52 (C, 2C, C, 2C;
C-3I–VI), 75.68 (C-2VI), 72.90, 72.46 (3C, C; C-2II–V),
72.81 (C-2I), 71.47, 71.12, 70.99, 70.95 (C, C, 2C,
2C; 6 CH2Ph), 70.13, 70.05, 69.50 (4C, C, C; C-5I–VI),
66.99 (C-10), 58.72 (OCH3-2VI), 53.67, 53.50,
53.46, 53,43 (C, 3C, C, C; C-4I–VI), 51.30
VI), 69.24, 68.93, 68.82, 68.72, 67.75 (C, C, 2C, C, C;
C-5I–VI), 67.05 (C-100), 65.93, 65.75, 65.70 (C, 3C, 2C;
C-40I–VI), 58.94 (OCH3-2VI), 51.99, 51.79, 51.73 (C,
2C, 3C; C-4I–VI), 51.52 (COOCH3), 33.74 (C-500), 31.99
(6C; C-30I–VI), 28.57 (C-200), 25.65 (C-300), 24.22 (C-400),
20.78, 20.75, 20.70, 20.62 (C, C, 3C, C; 6 COCH3),

196
R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
18.04, 17.96, 17.91 (4C, C, C; C-6I–VI). LCESI MS: m/z
2998 ([M+Na]+). Anal. Calcd for C164H202N6O45: C,
66.16; H, 6.84; N, 2.82. Found: C, 66.05; H, 6.87; N,
2.82.
ester 24 as described by Chernyak et al.8 Structurally
significant signals in the H NMR (CD3OD) spectrum
1
were at d 5.14, 5.12 (2 m, 5H, H-1II–VI), 4.81 (d, 1H,
J1,2 1.5Hz, H-1I), 4.22–4.16 (m, 6H, H-20I–VI), 3.48 (s,
partially overlapped, OCH3), 3.44, 3.41 (2, t, partially
overlapped, H-100b), 3.26 (t, 2H, J 6.4Hz, H-600), 2.74
(t, 2H, J 6.4Hz, H-700a,b), 2.22 (t, 2H, J 7.0Hz, H-
500a,b), 2.08–1.77 (2 m, 12H, H-30a,b), 1.70–1.58 (m,
4H, H-200a,b,400a,b), 1.47–1.36 (m, 2H, H-300a,b), 1.18–
3.2.15. Methoxycarbonylpentyl 4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-2-O-methyl-a-D-mannopyrano-
syl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-tetronamido)-4,6-
dideoxy-a-D-mannopyranosyl]-(1!2)-4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranoside 24. Com-
1.13 m,, 18H, H-6I–VI 13C NMR (CD3OD): d 102.70,
;
pound 22 (2.6g) was deacetylated (Zemplen), to give
102.44 (C, 3C, C-1II°V), 100.49 (C-1VI), 100.19 (C-1I),
80.68 (C-2VI), 79.81 (C-2I), 79.17, 79.00 (2C, 2C, C-
2II–V), 70.63 (C-20), 69.69, 69.59, 69.46, 69. 39, 68.75
(C, 2C, 3C, 6C; C-3I–V, 5I–VI), 68.47 (C-100), 59.42 (C-
´
methoxycarbonylpentyl
3-O-benzyl-4-(4-O-benzyl-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-
a-D-mannopyranosyl-(1!2)-tetrakis[4-(4-O-benzyl-3-
deoxy-L-glycero-tetronamido)-3-O-benzyl-4,6-dideoxy-
a-D-mannopyranosyl]-(1!2)-4-(4-O-benzyl-3-deoxy-L-
glycero-tetronamido)-3-O-benzyl-4,6-dideoxy-a-D-man-
nopyranoside 23 (1.95g, 81%). Structurally significant
20I–VI), 59.15 (OCH3), 54.74, 54.56, 54.50 (C, 3C, 2C,
I–VI
C-4I–VI), 42.58 (C-600), 41.91 (C-700), 38.23 (C-30
),
36.95 (C-500), 30.11 (C-200), 26.84 (C-300), 26.53 (C-400),
18.33, 18.28, 18.19 (2C, 3C, C; C-6I–VI).
1
signals in the H NMR (CDCl3) spectrum were at: d
6.95–5.60 (m, 6 NH), 5.06, 5.03, 5.01, 4.99 (4 br d, par-
tially overlapped, 4H, H-1II–V), 4.90 (br d, 1H, H-1VI),
4.69 (br d, 1H, H-1I), 4.66–4.39 (m, 24H, 12 CH2Ph),
3.91 (br t, 1H, H-2I), 3.62 (s, COOCH3), 3.33, 3.36 (2
t, 1H, J 6.4Hz, H-100 b), 3.23 (s, overlapped, OCH3-
2VI), 2.31 (t, 2H, J 7.5Hz, H-500a,b), 2.23–2.08, 1.96–
1.80 (2 m, 12H, H-30a,bI–VI), 1.69–1.53 (m, 4H, H-
200a,b,40a,b), 1.42–1.29 (m, 2H, H-300a,b), 1.20–1.07 (6
d, 18H, partially overlapped, H-6I–VI). Structurally sig-
nificant signals in the 13C NMR (CDCl3) spectrum were
at: d 100.52, 98.87, 99.60 (C, C, br, 2C, br; C-1II–V),
98.70 (C-1I), 98.56 (C-1VI), 73.72 (C-2I), 73.41, 73.36,
73.30, 73.26, 73.20, 73.14 (6 CH2Ph-3), 71.39, 71.14,
71.10, 70.95, 70.92, 70.60 (6 CH2Ph-40I–VI), 69.63,
69.50, 69.30, 69.18, 69.02, 68.93 (C-400I–VI), 68.88,
68.80, 67.91 (2C, 3C, C; C-5I–VI), 67.21 (C-100), 58.97
(OCH3-2VI), 51.77, 51.70, 51.59, 51.49 (2C, C, C, 2C;
C-4I–VI), 51.40 (COOCH3), 33.79, 33.60, 33.45 (4C,
2C, C; C-30I–VI, 500), 28.85 (C-200), 25.65 (C-300), 24.45
(C-400), 18.14, 18.09, 18.04, 17.97 (2C, 2C, C, C; C-6I–
VI). LCESI MS: m/z 2748 ([M+Na]+).
3.2.18. (6-Aminohexylamido)carbonylpentyl 4-(3-de-
oxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-D-
mannopyranosyl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-tet-
ronamido)-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-manno-
pyranoside 29. A mixture of ester 24 (0.05g) and hex-
amethylenediamine (0.5mL) was heated overnight
under argon at 65°C. Excess of the reagent was removed
by co-evaporation of water, and the residue was chro-
matographed. Lyophylization gave 29 (0.54mg, 83%)
as a white, amorphous solid.
1
Characteristic signals in the H NMR (CD3OD) spec-
trum were at d 5.14, 5.11 (2 m, 5H, H-1II–VI), 4.81 (d,
1H, J1,2 1.5Hz, H-1I), 4.22–4.16 (m, 6H, H-20I–VI),
3.67 (dd, 1H, J1,2 1.5, J2,3 3.3Hz, H-2), 3.48 (s, partially
overlapped, OCH3-2), 3.44, 3.41 (2, t, partially over-
lapped, H-100b), 3.17 (t, 2H, J 6.4Hz, H-600a,b), 2.70
(br t, 2H, H-1100a,b), 2.22 (t, 2H, J 7.0Hz, H-500a,b),
2.08–1.77 (2 m, 12H, H-30a,b), 1.68–1.32 (m, 14H, H-
200a,b,300a,b,400a,b,700a,b,800a,b,900a,b,1000a,b),
1.18–1.13
(m, 18H, H-6I–VI). 13C NMR (CD3OD): d 102.69,
102.42 (C, 3C; C-1II–V), 100.45 (C-1VI), 100.17 (C-1I),
80.68 (C-2VI), 79.76 (C-2I), 79.13, 78.98 (2C, 2C; C-2II–
V), 70.65 (C-20), 69.68, 69.59, 69. 41, 68.74 (C, 2C, 3C,
6C; C-3I–VI, 5I–VI), 68.50 (C-100), 59.42 (6C, C-40I–VI),
59.14 (OCH3-2), 54.77, 54.56, 54.50 (C, 3C, 2C;
C-4I–VI), 41.92 (C-600), 40.19 (C-1100), 38.22 (6C;
C-30I–VI), 36.97 (C-500), 32.27 (C-700), 30.29 (C-1000),
30.11 (C-200), 27.65, 27.44 (C-800, 900), 26.83 (C-300),
26.71 (C-400), 18.26, 18.21 (5C, C; C-6I–VI). LCESI MS:
m/z 1749 ([M+Na]+).
Treatment of a solution of the foregoing compound 23
(1g) in methanol (50mL) with hydrogen in the presence
of 5% palladium-on-charcoal catalyst (ꢀ200mg) gave
the title compound 24 (0.9g, 88%), which was identical
(TLC, NMR) with the independently described10
substance.
3.2.16. Hydrazidocarbonylpentyl 4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-2-O-methyl-a-D-mannopyrano-
syl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-tetronamido)-4,6-
dideoxy-a-D-mannopyranosyl]-(1!2)-4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranoside 25. This
compound was prepared from ester 24 as described by
Ogawa et al.10 and was identical (TLC, NMR) with the
independently described10 substance.
3.2.19.
1-{(5-Hydrazidocarbonylpentyl)carbonylpentyl
4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-
methyl-a-D-mannopyranosyl-(1!2)-tetrakis[4-(3-deoxy-
L-glycero-tetronamido)-4,6-dideoxy-a-D-mannopyrano-
syl]-(1!2)-4-(3-deoxy-L-glycero-tetronamido)-4, 6-dide-
oxy-a-D-mannopyranoside}-2-ethoxycyclobutene-3,4 dione
26. Diethyl squarate (8lL, 55lmol) was added to a
solution of the hydrazide 25 (50mg, 30lmol) as de-
scribed,8 to give squaric acid monoethyl ester 26
(40mg, 83%). The NMR spectra (D2O) were very similar
to those of the analogous 2-methoxycyclobutene deriva-
3.2.17. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
L-glycero-tetronamido)-2-O-methyl-4,6-dideoxy-a-D-man-
nopyranosyl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-tetro-
namido)-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-manno-
pyranoside 27. This compound was prepared from

R. Saksena et al. / Tetrahedron: Asymmetry 16 (2005) 187–197
197
tive, prepared from the same hexasaccharide.8 Charac-
teristic of the structure of 26 was the cross-peak between
the multiplets at d 1.50 and ꢀ4.80 for the methyl and
methylene group of the squaric acid ethyl ester group,
concentration of the hapten to be 20mM. The mixture
was stirred at room temperature and monitored period-
ically by SELDI-TOF MS. When a molar carbohy-
drate–BSA ratio of 5 had been reached, 90% of the
solution was withdrawn and subjected to ultrafiltration
using Millipore cells (Amicon, Stirred Ultrafiltration
Cell, Model No. 8050), and filters (Ultrafiltration Mem-
brane, Amicon, Milipore) with a molecular mass cutoff
of 10,000Da. The solution of the high molecular mass
material was freeze-dried, to give the title conjugates
31 (9.6mg, 93%), 32 (9.0mg, 87%), and 33 (9.3mg,
90%) as white, fluffy solids. For details, see Table 1.
1
respectively, present in the H–1H NMR COSY spec-
trum. The 13C NMR spectrum showed signals character-
istic of the corresponding carbon nuclei at 15.17 (d) and
70.12 (d) ppm, respectively. Similar resonances were
present in the spectrum of the squaric acid monoethyl
ester derived from the Ogawa monosaccharide.3 LCESI
MS: m/2z 884.387 ([M/2+H]+).
3.2.20. 1-{(2-Aminoethylamido)carbonylpentyl 4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
D-mannopyranosyl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-
4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-man-
nopyranoside}-2-ethoxycyclobutene-3,4-dione 28. This
compound was prepared (245mg, 82%), from 27
(280mg, 0.16mmol), following the protocol described
in the preparation of 26. The NMR spectra (D2O) were
very similar to those of the analogous 2-methoxycyclo-
butene derivatives, prepared from the same hexasaccha-
ride.8 Characteristic of the structure of 28 was the cross-
peak between the multiplets at d 1.55 and ꢀ4.84 for the
methyl and methylene groups belonging to the squaric
acid ethyl ester group, respectively, present in the
1H–1H NMR COSY spectrum. The 13C NMR spectrum
showed signals characteristic of the corresponding car-
bon nuclei at 15.20 (d) and 70.75 (d) ppm, respectively.
Similar resonances were present in the spectrum of
the squaric acid monoethyl ester derived from the
Ogawa monosaccharide.3 LCESI MS: m/2z 898.4127
(2[M/2+1]+).
References
1. Chernyak, A.; Kondo, S.; Wade, T. K.; Meeks, M. D.;
´ˇ
Alzari, P. M.; Fournier, J.-M.; Taylor, R. K.; Kovac, P.;
Wade, W. F. J. Infect. Dis. 2002, 185, 950–962.
2. McNaught, A. D. Carbohydr. Res. 1997, 297, 1–92.
´ˇ
3. Zhang, J.; Yergey, A.; Kowalak, J.; Kovac, P. Carbohydr.
Res. 1998, 313, 15–20.
4. Saksena, R.; Chernyak, A.; Poirot, E.; Kovac, P. Conju-
´ˇ
gating Low Molecular Mass Carbohydrates to Proteins. 2.
Recovery of the Excess Ligand Used in the Conjugation
Reaction. In Methods in Enzymology; Lee, Y. C., Lee, R.,
Eds.; Academic, 2003; Vol. 362, pp 140–160.
5. Tietze, L. F.; Schro¨ter, C.; Gabius, S.; Brinck, U.;
Goerlach-Graw, A.; Gabius, H.-J. Bioconjugate Chem.
1991, 2, 148–153.
6. Tietze, L. F.; Arlt, M.; Beller, M.; Glu¨senkamp, K.-H.;
Ja¨hde, E.; Rajewsky, M. F. Chem. Ber. 1991, 124, 1215–
1221.
7. Kamath, V. P.; Diedrich, P.; Hindsgaul, O. Glycoconjugate
J. 1996, 13, 315–319.
8. Chernyak, A.; Karavanov, A.; Ogawa, Y.; Kovac, P.
´ˇ
Carbohydr. Res. 2001, 330, 479–486.
9. Pozsgay, V.; Dubois, E.; Pannell, L. J. Org. Chem. 1997,
62, 2832–2846.
3.2.21. 1-{(6-Aminohexylamido)carbonylpentyl 4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
D-mannopyranosyl-(1!2)-tetrakis[4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranosyl]-(1!2)-
4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-man-
nopyranoside}-2-ethoxycyclobutene-3,4-dione 30. This
compound was prepared (52mg, 80%), from 29
(61mg, 0.035mmol), following the protocol described
in the preparation of 26. The NMR spectra (D2O) were
very similar to those of the above 2-aminoethyl analog
28. Characteristic of the structure 30 was the cross-peak
between the multiplets at d 1.40 and ꢀ4.84 for the
methyl and methylene groups belonging to the
squaric acid ethyl ester group, respectively, present in
the 1H–1H NMR COSY spectrum. The 13C NMR spec-
trum showed signals characteristic of the corresponding
carbon nuclei at 15.20 (d) and 70.75 (d) ppm, respec-
tively. LCESI MS: m/2z 926 (2[M/2+H]+).
´ˇ
10. Ogawa, Y.; Lei, P.-s.; Kovac, P. Carbohydr. Res. 1996,
293, 173–194.
11. Zhang, J.; Kovac, P. Carbohydr. Res. 1997, 300, 329–339.
´ˇ
´ˇ
12. Zhang, J.; Kovac, P. Carbohydr. Res. 1999, 321, 157–167.
13. Saksena, R.; Ma, X.; Kovac, P. Carbohydr. Res. 2003, 338,
2591–2603.
14. Ma, X.; Saksena, R.; Chernyak, A.; Kovac, P. Org.
´ˇ
Biomol. Chem. 2003, 1, 775–784.
15. Peters, T.; Bundle, D. R. Can. J. Chem. 1989, 67, 497–502.
16. Peters, T.; Bundle, D. R. Can. J. Chem. 1989, 67, 491–496.
´ˇ
17. Kovac, P. Alkylation. In Handbook of Derivatives for
Chromatography; Blau, K., Halket, J. M., Eds.; John
Wiley & Sons Ltd: Chichester, 1993; pp 109–129.
´ˇ
18. Zhang, J.; Kovac, P. J. Carbohydr. Chem. 1999, 18, 461–
469.
19. Adachi, T.; Yamada, Y.; Inoue, I.; Saneyoshi, M.
Synthesis 1977, 45–46.
20. Lei, P.-s.; Ogawa, Y.; Kovac, P. J. Carbohydr. Chem.
´ˇ
1996, 15, 485–500.
21. Bundle, D. R.; Gerken, M.; Peters, T. Carbohydr. Res.
1988, 174, 239–251.
22. Gast, J. C.; Atalla, R. H.; McKelvey, R. D. Carbohydr.
Res. 1980, 84, 137–146.
3.2.22. Neoglycoconjugates from the hexasaccharide of V.
cholerae O:1 and BSA 31–33. Each of the squaric acid
derivatives 26 (4mg, 2.25lmol), 28 (4.05mg, 2.25lmol),
and 30 (4.16mg, 2.25lmol) was added to a solution of
BSA (10mg, 0.15lmol) in a pH9 borate buffer
(112lL). The amount of buffer used assured the initial
´ˇ
23. Kovac, P.; Hirsch, J. Carbohydr. Res. 1982, 100, 177–193.
24. Chen, R. F. J. Biol. Chem. 1967, 242, 173–181.