One-pot preparation of a series of glycoconjugates with predetermined antigen-carrier ratio from oligosaccharides that mimic the O-PS of Vibrio cholerae O:1, serotype Ogawa

Article abstract of DOI:10.1016/S0008-6215(03)00273-8

Di-through the pentasaccharide that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa were synthesized in the form of 5-methoxycarbonylpentyl glycosides and linked to BSA using squaric acid diester chemistry. The conjugation reactions were monitored by surface-enhanced laser-desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS), which allowed conducting the conjugation of the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten-BSA ratio had been reached. This made it possible to prepare, from one hapten in a one-pot reaction, a series of neoglycoconjugates having different, predetermined carbohydrate-carrier ratios. The accuracy of molecular mass determination in SELDI-TOF MS analysis could be increased by using the carrier protein as the internal standard.

Full text of DOI:10.1016/S0008-6215(03)00273-8

Carbohydrate Research 338 (2003) 2591ꢀ2603
/
www.elsevier.com/locate/carres
One-pot preparation of a series of glycoconjugates with
predetermined antigenꢀcarrier ratio from oligosaccharides that mimic
the O-PS of Vibrio cholerae O:1, serotype Ogawa
/
Rina Saksena, Xingquan Ma, Pavol Kova´cˇ*
Laboratory of Medicinal Chemistry (LMC), NIDDK, National Institutes of Health, Building 8, Rm B1A24, Bethesda, MD 20892-0815, USA
Received 25 March 2003; accepted 22 May 2003
Abstract
Di-through the pentasaccharide that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1,
serotype Ogawa were synthesized in the form of 5-methoxycarbonylpentyl glycosides and linked to BSA using squaric acid diester
chemistry. The conjugation reactions were monitored by surface-enhanced laser-desorption/ionization-time-of-flight mass spectro-
metry (SELDI-TOF MS), which allowed conducting the conjugation of the synthetic oligosaccharides in a controlled way and
termination of the reaction when the desired molar haptenꢀ/BSA ratio had been reached. This made it possible to prepare, from one
hapten in a one-pot reaction, a series of neoglycoconjugates having different, predetermined carbohydrateꢀ
/carrier ratios. The
accuracy of molecular mass determination in SELDI-TOF MS analysis could be increased by using the carrier protein as the internal
standard.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Vibrio cholerae O:1; Synthetic oligosaccharides; Vaccine; Neoglycoconjugate immunogens; SELDI-TOF
1. Introduction
ogy,3 bacteriology,4 or bacterial polysaccharides,5,6 our
efforts, as well as those of others,7 have been focused
towards developing a conjugate vaccine for cholera from
synthetic fragments of the O-PS. We have previously
reported8 that neoglycoconjugates from the hexasac-
charide that mimics the upstream terminus of the O-PS
of Vibrio cholerae O:1, serotype Ogawa linked to bovine
serum albumin (BSA) showed vibriocidal activity and
protective capacity in neonatal mice and, thus, potential
as vaccines for cholera. In order to establish the effect of
the size of the antigenic oligosaccharide upon immuno-
genicity and protective capacity, we have now synthe-
sized a large series of similar neoglycoconjugates from
mono-through the pentasaccharide fragments of the O-
PS, and have characterized them by the carbohydrate/
BSA ratio (Scheme 1). The serological evaluation of the
materials reported herein will be communicated else-
where in due time.
The O-specific polysaccharides (O-PSs) of the two main
strains of Vibrio cholerae, Ogawa and Inaba, consist of a
chain of a-(10
nopyranose ( -perosamine), the amino group of which
is acylated with 3-deoxy- -glycero-tetronic acid. Only
/
2)-linked 4-amino-4,6-dideoxy-D-man-
D
L
the Ogawa strain has the O-2 of its upstream, terminal
end moiety of perosamine methylated (Fig. 1).1,2 Con-
tinued occurrence of cholera in the third, as well as in
the developed world, augments the need for an effective
and safe vaccine for this serious enteric disease. In
contrast to recent approaches utilizing molecular biol-
* Corresponding author. Tel.: ꢁ
301-4020589.
E-mail address: kpn@helix.nih.gov (P. Kova´cˇ).
/
1-301-4963569; fax: ꢁ1-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00273-8

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R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
Fig. 1. Structure of the O-PS of V. cholerae O1, serotypes Inaba and Ogawa.
2. Results and discussion
Previously,18 when a hexasaccharide similar to 3ꢀ
/
6
but lacking the tetronamido side chain was methylated
under the above conditions the reaction was a clean, one
product reaction. On the contrary, methylation of each
The O-PSs of V. cholerae O:1, serotypes Inaba and
Ogawa are the protective antigens for the disease caused
by these pathogens.9,10 Our previous solution11 and X-
ray crystallographic12 studies identified the upstream
terminus of the O-PS of V. cholerae O:1, serotype
Ogawa as the dominant determinant epitope. That
structural motif was included in the neoglycoconjugates
prepared from the synthetic hexasaccharide,13 which
showed potential as vaccines for cholera.8 The linker
equipped monosaccharide hapten 1 required during this
work was prepared as described previously.14 The
of the oligosaccharides 3ꢀ5 at the above conditions was
/
accompanied by formation of variable amounts of a by-
product. Based on the NMR and high-resolution mass
spectral data, the compound formed during methylation
of the disaccharide 3 was identified as the methoxycar-
bonyl derivative 29. The molecular formula,
C57H70N2O17, was deduced from the HRMS data.
Compared to the 1H NMR spectrum of the desired
product 7, the spectrum of 29 lacked the signal for the
ethereal Me group at d 3.28 but showed an additional
singlet (OCH3, d 3.74), and the signal of H-2II was
shifted downfield (d 5.30), indicating the presence of an
electron-withdrawing (CH3OCOO) group at C-2. The
13C NMR spectrum of 29 showed the signal of the
OCH3 group at d 51.46, and the signal for C-2II
appeared at d 70.90, showing that O-2 was not alkylated
(c.f. the chemical shift for C-2 in the spectrum of the 2-
O-methyl derivative 7, d 75.72). Taking into considera-
tion the foregoing structurally significant spectral data,
the structure 29 was further substantiated when treat-
ment of 29 with NaOMe in methanol regenerated the
starting alcohol 3. The same product 29 was formed
when either freshly made or commercial Ag2O was used.
The details concerning the formation of 29 during
methylation of 3 or, presumably, analogous products
when oligosaccharides 4 and 5 were methylated in the
requisite di-through the pentasaccharides 7ꢀ
2) were obtained from the intermediate 2-OH deriva-
tives (3ꢀ5), which were obtained during syntheses of
substances in the Inaba series (3ꢀ
6),15 namely by
methylation or glycosylation using the 2-O-methylated
glycosyl donor 28. Thus, methylation16,17 of alcohols 3ꢀ
5 with MeI and Ag2O gave the fully protected sub-
stances 7ꢀ9, which were subjected to hydrogenolysis to
afford the deprotected methyl esters 11ꢀ13. We also
explored preparation of the desired, fully protected
oligosaccharides 7ꢀ10 through the alternative route
involving glycosylation of 3ꢀ5 with 28 because the
/
10 (Scheme
/
/
/
/
/
/
/
methylation, especially of higher oligosaccharides, was
impractically slow, and was also accompanied by a side
reaction. Therefore, methylation of 6 was not pursued,
and the pentasaccharide 10 was only obtained by
glycosylation of 5 with 28.
Scheme 1.

R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ
/2603
2593
same way, were not investigated further. However, it
appears that, among other factors, a carbonate must
have played role in this side reaction. A plausible source
of a carbonate could be a small amount of BaCO3
present in the Ag2O used. The former was likely to have
been present in Ba(OH)2, which is the common reagent
used in preparation16 of Ag2O. Since BaCO3 is insoluble
in water, once present in the Ag2O formed from AgNO3
and Ba(OH)2, it is virtually impossible to wash it out.
Thus, BaCO3 could have been carried over to the
product Ag2O, and could conceivably contribute to
the occurrence of the side reaction. It is noteworthy that
the formation of side products was not observed when
the same batch of reagents were used in the conversion
values and for characteristic resonances present in the
1H and 13C NMR spectra of 15ꢀ
18 and 19ꢀ22).
/
/
For conjugation, we chose the method originally
described by Tietze, which is based on the selective
reaction of amines with squaric acid diesters.19,20 The
method is simple, it requires inexpensive, commercially
available reagents, and it has gained popularity in
synthetic vaccine development because it allows pre-
paration of neoglycoconjugates with a wide range of
haptenꢀ
carrier ratios.14,18,21ꢀ24 Further advantages of
/
the method are that its use does not result in cross-
linked, poorly defined, lattice type products, and that a
some of the excess hapten used at the onset of the
reaction can be recovered.25 We have done some
fundamental work on conjugation of synthetic oligo-
saccharides using squaric acid chemistry14,18,26,27 and,
with the positive results of our own and of others at
hand,8,28,29 we are confident that this method of
conjugation will be useful in synthetic vaccine prepara-
tion. With only minor modifications, conjugations here
described were performed applying the two stage pro-
tocol involving purification of the squaric acid monoe-
sters,23 (c.f. for a simplified procedure24) as it is more
likely to consistently afford well defined products,
clearly a requirement when such materials would be
used as vaccines.
270
benzylidene-b-
To form substances amenable to conjugation by the
squaric acid chemistry, each of the methyl esters 11ꢀ14
was subjected to aminolysis with excess of ethylenedia-
mine, and the amines formed (15ꢀ18, respectively) were
subsequently converted to the squaric acid monoesters
19ꢀ22. Purification of amines and squaric acid deriva-
/28 or in the methylation of methyl 3-O-benzyl-4,6-
D-glucopyranoside.
/
/
/
tives was most conveniently done by solid phase
extraction and, if necessary, followed by preparative
TLC on normal phase silica gel. The NMR spectra
showed remarkable purity of materials thus obtained.
The same technique and mass spectrometry fully con-
firmed the expected structures (c.f. Section 3 for the m/z
We targeted our carbohydrateꢀ
and 23aꢀ26c) to have a haptenꢀBSA ratio of ꢀ
and ꢀ15. To maintain an acceptable reaction rate, the
/
BSA constructs (2aꢀ
/c
/
/
/
5, ꢀ10
/
/
Scheme 2.

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R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
molar haptenꢀ
/
BSA ratio at the onset of the conjugation
later stages of the reaction, when these larger molecules
have to penetrate deeper into the three-dimensional
structure of the protein, to reach the less accessible
amino groups present in the protein. We cannot explain
the anomalous, apparently somewhat higher rate of the
conjugation of the tetrasaccharide, compared to that of
the trisaccharide.
This laboratory was the first to show the utility of
surface-enhanced laser desorption/ionization time-of-
flight mass spectrometry (SELDI-TOF MS) in combi-
nation with the ProteinChip† System for monitoring
the conjugation of synthetic oligosaccharides to pro-
reaction was 75, which was fivefold excess based on the
highest targeted loading. All reactions were carried out
under the same conditions, including the 15 mmolar
concentration of the hapten.14 The targeted haptenꢀ
BSA ratios and those observed in the final products,
and the reaction times required to reach the final
loading are in Table 1. Fig. 2 shows the overall course
of the conjugations. It can be seen that there is virtually
no difference between the rate of conjugation of the
mono- and the disaccharide. With the higher oligosac-
charides, the reaction rate decreases markedly at the
/

R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ
/2603
2595
teins.25,26 The ability to monitor conjugation reactions
and obtain virtually real time information about
the increasing molecular mass of products being formed
constitutes a new milestone in the preparation of
neoglycoconjugates. As we show below, a series of
such materials can be efficiently prepared from one
hapten in a one-pot reaction. According to the protocol
described in the Experimental, when the conjugation
reaction is monitored by SELDI-TOFMS, a portion
of the reaction mixture can be withdrawn and
Table 1
The targeted and observed haptenꢀ
/
BSA ratios in the product
conjugates, and the respective reaction time a
Hapten/Product Loading (hapten/BSA) Time required (h)
Targeted
Observed
1/2a
1/2b
5
10
15
5
5.3
9.7
1.25
3.75
1/2c
15.2
4.7
11.0
19/23a
19/23b
19/23c
20/24a
20/24b
20/24c
21/25a
21/25b
21/25c
22/26a
22/26b
22/26c
1.0
4.0
processed when the desired carbohydrateꢀprotein
/
10
15
5
9.7
ratio is reached. The rest of the material is allowed to
react until the conjugate shows the next desired,
predetermined molecular weight. Following this proto-
col, we have been able to prepare three different
neoglycoconjugates with very close to predetermined
14.5
4.9
11.5
3.0
10
15
5
9.8
7.0
16.75
3.0
13.9
5.9
10
15
5
9.0
5.75
15.0
3.0
haptenꢀ/carrier ratio from each of the haptens 1 and
13.9
5.0
19ꢀ22. The use of the carrier protein as an internal
/
standard (see Section 3) increased the accuracy and
reliability of molecular mass determination by SELDI-
TOFMS. This was particularly helpful with conjugates
10
15
10.5
13.9
9.0
21.5
a
2aꢀ2c made from the monosaccharide hapten.
/
All reaction were carried out at ambient temperature (23ꢀ
/
With these materials, the molecular mass deviation
from the actual value allowed by the manufacturer of
the instrument (0.3%) can amount to a considerable
fraction of one monosaccharide hapten. The extra
operation involved in the protocol is simple, and
the method can be also useful in routine matrix-assisted
LDI-TOFMS.
26 8C) at the initial molar haptenꢀ
hapten concentration of 15 mmol.
/
BSA ratio of 75, and at the
where.34 When the latter approach was used to aid in the
13C NMR signal-nuclei assignments, advantage was
taken of variations of line intensity expected for
oligosaccharides belonging to the same homologous
series.35,36 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 N-
3. Experimental
3.1. General methods
acylated
D-perosamine residues in the molecule. When
Instruments and laboratory techniques used were the
same as those described previously.25,30 Unless stated
otherwise, optical rotations were measured at ambient
feasible, the assignments were supported by homo-
nuclear decoupling experiments or homonuclear
(1H{1H}COSY) and heteronuclear (13C{1H}HETCOR)
2-dimensional correlation spectroscopy, run with the
software supplied with the spectrometers. When report-
ing assignments of NMR signals of oligosaccharides,
sugar residues in oligosaccharides are serially numbered,
beginning with the one bearing the aglycon, and are
identified by a Roman numeral superscript in listings of
signal assignments. Nuclei-assignments without a super-
script notation indicate that those signals have not been
individually assigned. Thus, for example, in a spectrum
of a pentasaccharide, a resonance denoted H-3 could be
that of H-3 of either sugar residue. When reporting
temperature for solutions in chloroform (c ꢀ
/
1), with a
PerkinꢀElmer automatic polarimeter, Model 341. All
/
reactions were monitored by thin-layer chromatography
(TLC) on Silica Gel 60 coated glass slides (Whatman or
Analtech). Preparative TLC was performed using Silica
Gel GF-coated Analtech Uniplates (1500 mm). Column
chromatography was performed by gradient elution
from columns of silica gel, using PurChrom 35G High
Performance Rapid Chromatography System (RT
Scientific, Inc.). Solvent mixtures less polar than those
used for TLC were used at the onset of development.
NMR spectra were measured at 300 (1H) and 75 MHz
(13C), with a Varian Mercury spectrometer. Solid Phase
Extraction was performed using Strata SPE Tubes
(Phenomenex, Inc.). Assignments of NMR signals
were made by first-order analysis of the spectra, and
by comparison with spectra of related substances
reported previously from this laboratory13,30ꢀ33 or else-
NMR data, the nuclei belonging to the 3-deoxy-L-
glycero-tetronic acid side chain are referred to as primed
(?), and those of the spacer aglycon as double primed (ƒ).
Some signals on 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 esters.19 Surface-

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R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
Fig. 2. Rate of conjugation for the mono- through the pentasaccharide. For reaction conditions, see Table 1.
enhanced laser desorption/ionization time-of-flight mass
spectrometry (SELDI-TOF MS) was done using PBS-II
Mass Reader† (Ciphergen Biosystems, Inc), calibrated
using Ciphergen’s All in One Protein Standards, and H4
or NP20 Protein Chip Arrays†. The accuracy of
molecular mass determination was increased by using
the carrier protein (BSA, m/z 66,430 Da) as an internal
standard. This eliminated errors originating from spot-
to-spot variations in the SELDI-TOFMS, and made the
fine calibration of the instrument virtually unnecessary
when used for monitoring the conjugations or molecular
mass determination of final products here described.
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
evaporation of water from the residue (ꢂ
solution of the crude product in water (ꢀ2 mL) was
applied on a SPE column (2ꢀ10 g sorbent mass, as
required), which had been conditioned by washing with
MeOH (30ꢀ80 mL), followed by water (50ꢀ100 mL).
The elution was effected with water (20ꢀ50 mL),
followed by a gradient of water (25 mL)050% aq
MeOH (25 mL). Fractions of 2 mL were collected,
analyzed by TLC, and those containing the desired
material were concentrated to a small volume. Filtration
through a syringe filter (0.45 mm porosity) and freeze-
drying, gave products as white solids.
/
3), the
/
/
/
/
/
/
3.3. General procedure for preparation of squaric acid
monoesters 19
/
22
could not be obtain within 90.3%. Structures of these
/
ꢀ
compounds follow unequivocally from the mode of
synthesis and m/z values found in their low- and high-
resolution mass spectra, and TLC and NMR spectro-
scopy verified their purity. BSA (Fraction V, Prod. No.
A-4503) was purchased from Sigma Chemical Com-
pany, and purified as described by Chen.37 Ethylene-
Diethyl squarate (0.015 mmol) was added to a soln of
each of the above amines 15ꢀ18 (0.01 mmol) in a
potassium phosphate buffer pH 7.0 (3 mL), and the
mixture was kept at room temperature overnight, when
/
TLC (1:1 CH2Cl2ꢀMeOH) showed that all the amine
/
diamine was freshly distilled, bp 115ꢀ116 8C. Palla-
/
had been consumed and that a less polar, UV positive
product was formed. Without any work-up, the mixture
was subjected to SPE, using a column (sorbent mass, 5
g), which had been conditioned by washing with MeOH
(40 mL), followed by water (60 mL). The elution was
effected with water (20 mL), followed by a gradient of
dium-on-charcoal catalyst (5%, ESCAT 103) was a
product of Engelhard Industries. Silver oxide was
purchased from Fluka Chemical Company, or prepared
as described.16
water (25 mL)0
/
50% aq MeOH (25 mL). Fractions of 2
3.2. General procedure for preparation of amines 15
/
18
ꢀ
A solution of the ester (0.1 mmol) in ethylenediamine
mL were collected and analyzed by TLC. Fractions
containing the desired material were concentrated and
purified by preparative TLC, if necessary. An aq soln of
the pure material, thus obtained, was filtered through a
syringe filter (0.45 mm porosity) and freeze-dried, to give
white or light-yellow solids.
from esters 11
/
14
ꢀ
(20 mmol) was heated at 60ꢀ
(1.3:1:0.1 CH2Cl2ꢀMeOHꢀ25% NH4OH) showed that
the reaction was complete. After concentration and
/
708 overnight, when TLC
/
/

R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ
/2603
2597
3.4. General procedure for the preparation of
neoglycoconjugates 2a 2c, 23a 26c
bonyl-a-
O-benzylidene-3-deoxy-
deoxy-a-
-mannopyranoside (29, 13 mg, 21%). 1H
NMR (CDCl3): d 6.37, 6.22 (2 d, 1 H each, J4,NH 9.4
Hz, 2 NH), 5.56, 5.54 (2 s, 1 H each, 2 CHPh), 5.30
(broad t, 1 H, H-2II), 4.99 (d, 1 H, J1,2 1.9 Hz, H-1II),
D
-mannopyranosyl-(10
/
2)-3-O-benzyl-4-(2,4-
/
/
L-glycero-tetronamido)-4,6-di-
ꢀ
ꢀ
D
BSA (20 mg, 0.3 mmol) was suspended in a borate
buffer, pH 9.0 (400 mL) and homogenized with the aid of
a vortexer. The respective haptenic monoamide (0.0226
mmol) was transferred from its weighing container,
using 1.1 mL of phosphate buffer pH 9.0 divided into
three portions (400, 400 and 300 mL), into the above fine
suspension of BSA. The clear soln thus formed was
stirred at room temperature, while the progression of the
conjugation was periodically monitored by SELDI-TOF
MS, following the protocol recommended by Ciphergen
for NP20 ProteinChips†. For monitoring the conjuga-
tion using BSA as the internal standard, a sample (1 mL)
was withdrawn and mixed with pH 7 phosphate buffer
(9 mL), giving soln A. A 1 mL sample of a stock soln of
BSA (20 mg/1.5 mL) was diluted with 9 mL of water,
yielding solution B (internal standard). A portion of A
(2 mL) was mixed with B (1 mL) and 1mL of the resulting
soln was applied on the ProteinChip†. The chip was air-
dried, washed with water (5 mL, twice), with drying in
between the washes and, finally, a soln (0.5 mL, twice) of
the energy-absorbing molecule (satd solution of sinapi-
nic acid in 1% TFA) was applied. The chip was air-
dried, followed by reading the molecular masses. Two
peaks corresponding to single-charged molecules were
observed. The average hapten/BSA ratio was calculated
from the difference between the molecular masses shown
for BSA and the glycoconjugate formed. When the
required hapten/BSA ratio was reached, 500 mL of the
soln was withdrawn, diluted with phosphate buffer pH
7.0 (3 mL), and subjected to ultra filtration using the
Amicon cell equipped with PM-10 membrane (Millipore
Copporation, Bedford, MA). The retained material was
4.66 (d, 1 H, J1,2 1.7 Hz, H-1I), 4.73ꢀ
2J 11.8 Hz, 2 CH2Ph), 4.38ꢀ
2?I,II,4?aI,II), 4.12ꢀ3.88 (m, 5 H, H-2I,4I,II,4?bI,II), 3.86ꢀ
3.71 (m, 7 H, H-3I,II,5I,II, incl. s, 3.74, OCOOCH3),
3.62ꢀ3.54 (m, 4 H, H-1ƒa, incl. s, 3.60 COOCH3), 3.32,
3.29 (2 t, 1 H, J 6.2 Hz, H-1ƒb), 2.77 (t, 2 H, J 7.4 Hz,
H-5ƒ), 2.10ꢀ 1.50 (m,
1.83 (2 m, 2 H, each, H-3?I,II), 1.66ꢀ
4 H, H-4ƒ,2ƒ in that order), 1.39ꢀ1.28 (m, 2 H, H-3ƒ),
/
4.47 (4 d, 1 H each,
/
4.21 (m, 4 H, H-
/
/
/
/
/
/
1.19, 1.17 (2 d, partially overlapped, 6 H, J5,6 6.0 Hz, H-
6I,II); 13C NMR (CDCl3): d 101.35, 101.19 (2 CHPh),
99.45 (C-1II), 98.85 (C-1I), 76.58 (2 C, C-2?I,II), 75.69 (C-
3I), 74.27 (C-2I), 73.29 (C-3II), 71.98, 71.12 (2 CH2Ph),
70.90 (C-2II), 68.42, 67.42 (C-5I,II), 67.34 (3 C, C-
4?I,II,1ƒ), 54.84 (OCOOCH3), 52.62, 51.67 (C-4I,II),
51.46 (COOCH3), 33.89 (C-5ƒ), 28.86 (C-2ƒ), 28.62 (C-
3?), 25.26 (C-3ƒ), 24.56 (C-4ƒ), 18.00, 17.95 (C-6I,II).
FABMS: m/z 1055.41 ([Mꢁ
/
1]ꢁ), 1077.39 ([MꢁNa]ꢁ);
/
HRMS: m/z 1187.3727. C57H70CsN2O17 requires
1187.3661.
Eluted next was the desired methyl ether 7 (41 mg,
1
70%), [a]D
6.22 (2 d, 1 H each, J4,NH 9.8 Hz, 2 NH), 5.57, 5.54 (2 s,
ꢃ
/
15.18 (c 0.5). H NMR (CDCl3): d 6.40,
1 H each, 2 CHPh), 5.01 (broad doublet, 1 H, H-1II),
4.71ꢀ
partially overlapped, J1,2
4 H, H-2?I,II,4?aI,II), 4.18ꢀ
3.80ꢀ
3.70 (m, 5 H, H-2II,3I,II,5I,II), 3.63ꢀ
/
4.50 (4 d, partially overlapped, 4 CH2Ph), 4.66 (d,
1.7 Hz, H-1I), 4.42ꢀ
4.29 (m,
4.92 (m, 5 H, H-2I,4I,II,4?bI,II),
3.55 (m, 4 H,
ꢀ
/
/
/
/
/
H-1ƒ, incl s at 3.60, COOCH3), 3.36 (m, 1 H, H-1ƒb),
3.28 (s, 3 H, OCH3-2), 2.28 (t, 2 H, J 7.4 Hz, H-5ƒ),
freeze-dried, to give conjugates in 80ꢀ/95% yields. The
2.11ꢀ
/
1.82 (m, 4 H, H-3?a,bI,II), 1,67ꢀ
1.51 (m, 4 H, H-
/
molecular mass of the final products was verified by
SELDI-TOFMS.
2ƒa,bI,II,4ƒa,bI,II), 1.38 (m, 2 H, H-3ƒa,bI,II), 1.19 (m, 6
H, J5,6 6.3 Hz, H-6I,II). 13C NMR (CDCl3): d 101.29,
101.22 (2 CHPh), 99.58 (C-1II), 99.06 (C-1II), 76.50 (2 C,
C-2?I,II), 76.47, 75.38 (C-3I,II), 75.72 (C-2II), 73.34 (C-2I),
72.33, 71.06 (CH2Ph), 68.55, 67.52 (C-5I,II), 67.33 (C-
1ƒ), 67.27 (C-4?I,II), 59.13 (OCH3-2), 52.44, 51.81 (C-
4I,II), 33.86 (C-5ƒ), 28.85 C-2ƒ), 28.58 (C-3?), 25.55 (C-
3ƒ), 24.52 (C-4ƒ), 18.04, 17.95 (C-6I,II). FABMS: m/z
3.5. 5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-
dideoxy-2-O-methyl-a-
benzyl-4-(2,4-O-benzylidene-3-deoxy-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (7)
L
-glycero-tetronamido)-4,6-
-mannopyranosyl-(102)-3-O-
-glycero-
D
/
L
D
1011.42 ([Mꢁ
/
1]ꢁ), 1033.40 ([MꢁNa]ꢁ). HRMS: m/z
/
(a) A solution of the disaccharide 315 (58 mg, 0.058
mmol) in MeI (2 mL) was treated with Ag2O (137 mg,
1143.3843. C56H70CsN2O15 requires 1143.3831. Anal.
Calcd for C56H70N2O15: C, 66.52; H, 6.98; N 2.77.
Found: C, 66.64; H, 6.83; N, 2.62.
(b) A mixture of thioglycoside 28 (1.25 g, 2.50 mmol),
5-methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzyli-
ꢀ
/
10 equiv), and the mixture was stirred at room
temperature with exclusion of direct light overnight,
when TLC (2:1 tolueneꢀacetone) showed that nearly no
/
starting material was present. After filtration, the solids
were washed with CH2Cl2, the combined filtrate was
concentrated, and the residue was chromatographed
dene-3-deoxy-
L
-glycero-tetronamido)-4,6-dideoxy-a-
D-
mannopyranoside15 (1.2 g, 2.10 mmol) and 4 A mole-
cular sieves (6 g) in dry CH2Cl2 (30 mL) was stirred for
15 min at room temperature. NIS (0.75 g, 3.33 mmol)
was added, followed by solid AgOTf (0.23 g, 0.92
mmol). The mixture turned red after few minutes, the
˚
(5:10
bonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-
-glycero-tetronamido)-4,6-dideoxy-2-O-methoxycar-
/
2:1 tolueneꢀ
/
acetone), to give first 5-methoxycar-
L

2598
R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
stirring was continued for another 15 min, when the
reaction was quenched by the addition of Et3N (1 mL).
3.29 (t, 2 H, J 6.3 Hz, H-6ƒa,b), 2.78 (t, 2 H, J 7.4 Hz,
H-7ƒa,b), 2.23 (t, J 7.8 Hz, H-5ƒa,b), 2.08ꢀ1.77 (2 m, 4
H, H-3?I,IIa,b), 1.70ꢀ
1.57 (m, 4 H, H-4ƒa,b,2ƒa,b), 1.46ꢀ
/
The mixture was partitioned between aq 1:1 Na2S2O3ꢀ
NaHCO3, the organic layer was dried with MgSO4, and
2:1 tolueneꢀace-
/
/
/
1.34 (m, 2 H, H-3ƒa,b), 1.17, 1.14 (2 d, J5,6 6.2 Hz, H-
concentrated. Chromatography (5:10
/
/
6I,II). The 1H NMR spectrum taken in pyridine-d5
tone) gave material (1.8 g, 85%), which was identical
(TLC, NMR) with compound 7 described above.
showed a 3-proton multiplet (d 8.1ꢀ7.90) for the three
/
NH groups; 13C NMR (CD3OD): d 100.79 (C-1II),
100.25 (C-1I), 80.69 (C-2II), 79.68 (C-2I), 70.64 (2 C, C-
2?I,II), 69.64, 69.57 (C-3I,II), 69.31, 68.73 (C-5I,II), 68.46
(C-1ƒ), 59.40, 59.38 (C-4?I,II), 59.19 (OCH3), 54.72, 54.48
(C-4I,II), 42.07 (C-6ƒ), 41.78 (C-7ƒ), 38.25 (2 C, C-3?I,II),
36.94 (C-5ƒ), 30.15 (C-2ƒ), 26.87 (C-3ƒ), 26.16 (C-4ƒ),
3.6. 5-Methoxycarbonylpentyl 4-(3-deoxy-
tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (11)
L-glycero-
D-
/
L
D
18.36, 18.27 (C-6I,II); FABMS: m/z 683.38 ([Mꢁ
/
1]ꢁ),
A mixture of 7 (1.0 g, 0.99 mmol) and 5% palladium-on-
carbon catalyst (600 mg) in methanol (50 mL) was
stirred under hydrogen overnight, when TLC (4:1
705.38
([Mꢁ
/
Na]ꢁ);
C29H54N4O14Li requires 689.3797.
HRMS:
m/z
689.3768.
CH2Cl2ꢀ
plete. Conventional processing and elution from a small
column of silica gel (6:1 to 3:1 CH2Cl2ꢀMeOH) gave
amorphous, deprotected methyl ester 11 (592 mg, 92%);
/
MeOH) showed that the reaction was com-
3.8. 1-{(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside}-2-
ethoxycyclobutene-3,4-dione (19)
L
D-
/
/
L
D
1
[a]D
ꢃ15.18 (c 0.5, CH3OH). Definite signals in the H
/
NMR (D2O) spectrum were at: d 5.17 (d, 1 H, J1,2 1.6
Hz, H-1II), 4.92 (d, 1 H, J1,2 1.5 Hz, H-1I), 4.30, 4.29 (2
dd, partially overlapped, 2 H, H-2?I,II), 4.10 (dd,
partially overlapped, J2,3 3.3, J3,4 10.3 Hz, H-3II), 4.06
(dd, partially overlapped, J2,3 3.3, J3,4 10.3 Hz, H-3I),
Following the general procedure described above, con-
version of 15 (50 mg) gave the ethyl ester 19 (57 mg,
96%). Definite signals in the 1H NMR spectrum
(CD3OD) were at d 5.09 (d, 1 H, J1,2 1.6 Hz, H-1II),
3.96 (broad t, partially overlapped, H-2I), 3.95ꢀ
/
3.67 (m,
13 H, H-4I,II,5I,II,4?I,II,1ƒa, incl dd, at 3.77, partially
4.83 (d, 1 H, J1,2 1.6 Hz, H-1I), 4.78ꢀ
OCH2CH3), 4.21ꢀ
4.16 (m, 2 H, H-2?I,II), ꢀ
partially overlapped, H-2I), ꢀ
3.74 (m, partially over-
lapped, 4?I,IIa,b), 3.68 (broad, partially overlapped, H-
2II), 3.48 (s, 3 H, OCH3), 2.23ꢀ
2.15 (2 t, partially
overlapped, 2 H, H-5ƒa,b), 2.08ꢀ1.77 (m, 4 H, H-
3?I,IIa,b), 1.66ꢀ
1.54 (m, 4 H, H-4ƒa,b,2ƒa,b), 1.49ꢀ1.34
/
4.68 (m, 2 H,
3.82 (broad,
/
/
overlapped, for H-2II, and s, at 3.69 for COOCH3),
/
3.58ꢀ
(t, 2 H, J 7.5 Hz, H-5ƒa,b), 2.10ꢀ
3?I,IIa,b), 1.69ꢀ
1.58 (m, 4 H, H-2ƒa,b,4ƒa,b), 1.44ꢀ
/
3.50 (m, 1 H, H-1ƒb), 3.49 (s, 3 H, OCH3-2), 2.41
1.80 (2 m, 4 H, H-
1.35
/
/
/
/
/
(H-3ƒa,b), 1.20, 1.17 (2 d, partially overlapped, 6 H, H-
6I,II); 13C NMR (D2O): d 99.22 (C-1II), 98.47 (C-1I),
79.06 (C-2II), 78.36 (C-2I), 69.09 (2 C, C-2?I,II), 68.30 (2
C, C-5,1ƒ), 67.71, 67.68, 67.58 (3 C, C-5,3I,II), 58.86
(OCH3-2), 57.94 (2 C, C-4?I,II), 53.29, 53.18 (C-4I,II),
52.22 (COOCH3), 36.09, 36.06 (C-3?I,II), 33.70 (C-5ƒ),
28.19 (C-2ƒ), 24.98 (C-3ƒ), 24.10 (C-4ƒ), 16.97. 16.92 (C-
/
/
/
(m, 5 H, CH3CH2, H-3ƒa,b), 1.17, 1.14 (2 d, J5,6 6.2 Hz,
H-6I,II); 13C NMR (CD3OD): d 100.80 (C-1II), 100.24
(C-1I), 80.68 (C-2II), 79.64 (C-2I), 70.70 (d, CH2CH3),
70.69 (2 C, C-2?I,II), 69.66, 69.61 (C-3I,II), 69.33, 68.73
(C-5I,II), 68.44 (C-1ƒ), 59.44, 59.41 (C-4?I,II), 59.21
(OCH3), 54.73, 54.50 (C-4I,II), 45.20 (d, C-6ƒ), 40.75
(d, C-7ƒ), 38.24, 38.21 (C-3?I,II), 36.92 (C-5ƒ), 30.18 (C-
2ƒ), 26.82 (d, C-3ƒ), 26.55 (d, C-4ƒ), 18.34, 18.26 (C-6I,II),
6I,II); FABMS: m/z 655.4 ([Mꢁ
/
1]ꢁ), 677.4 ([Mꢁ
Na]ꢁ); HRMS: m/z 661.3367. C28H50N2O15Li requires
661.3371.
16.17 (d, CH3CH2); FABMS: m/z 807 ([Mꢁ
1]ꢁ).
/
3.7. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (15)
L
-
3.9. 5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy-2-O-methyl-a- -mannopyranosyl-(102)-3-O-
benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl-(10
O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (8)
D-
L
/
L
D
/
D
L
D
/2)-3-
Following the general procedure described above, con-
version of 11 (270 mg) gave the amine 15 (229 mg, 85%);
1H NMR (CD3OD): d 5.09 (d, 1 H, J1,2 1.6 Hz, H-1II),
L
D
4.82 (d, 1 H, J1,2 1.6 Hz, H-1I), 4.22ꢀ
/
4.15 (m, 2 H, H-
2?I,II), 3.98 (dd, partially overlapped, J2,3 3.1, J3,4 10.2
Hz, H-3I), 3.98ꢀ3.65 (m, 12 H, H-3II,4I,II,5I,II
4?a,bI,II,1ƒa, incl. broad, ꢀ3.82, H-2I, broad, ꢀ
3.68,
H-2II), 3.48 (s, 3 H, OCH3), 3.45ꢀ
3.38 (m, 1 H, H-1ƒb),
(a) The trisaccharide alcohol 4 (320 mg) was treated for
3 days with MeI (7 mL) and Ag2O (5 g), as described for
the preparation of 7 (a). Chromatography (5:1 to 2:1
/
,
/
/
tolueneꢀ/acetone) gave the desired, fully protected
/
methyl ether 8 (290 mg, 90%), [a]D
ꢃ
/
23.38 (c 0.45);

R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ
/2603
2599
1H NMR (CDCl3): d 6.31, 6.25 (d, 2 H, d, 1 H, J4,NH 9.3
Hz, 3 NH), 5.56, 5.53, 5.52 (3 s, 1 H each, 3 CHPh), 5.00
(d, 1 H, J1,2 1.7 Hz, H-1III), 4.95 (d, 1 H, J1,2 1.8 Hz, H-
3I), 69.40 (C-5), 69.32 (2 C, C-3II,5), 68.72 (C-5), 68.37
(C-1ƒ), 59.41, 59.37 (C, 2 C, C-4?IꢀIII), 59.13 (OCH3-2),
54.69, 54.47 (C, 2 C, C-4?IꢀIII), 52.07 (COOCH3), 38.21
(3 C, C-3?IꢀIII), 34.59 (C-5ƒ), 30.00 (C-2ƒ), 26.67 (C-3ƒ),
25.56 (C-4ƒ), 18.29, 18.14 (2 C, C, C-6IꢀIII). FABMS: m/
1II), 4.70ꢀ
overlapped, H-1I), 4.19 (broad t, 1 H, H-2II), 4.50ꢀ
(m, 6 H, H-4IꢀIII,4?IꢀIII), 3.87 (broad t, 1 H, H-2I), 3.78ꢀ
3.67 (m, 7 H, H-2II,3IꢀIII,5IꢀIII), 3.60ꢀ
3.53 (m, 4 H, H-
1ƒa, incl s at 3.59, COOCH3), 3.33ꢀ3.25 (m, 4 H, H-1ƒb,
incl s at 3.59, OCH3-2), 2.27 (t, 2 H, J 7.5 Hz, H-5ƒa,b),
2.10ꢀ 1.48 (m, 4 H, H-
1.79 (m, 6 H, H-3IꢀIIIa,b), 1.65ꢀ
2ƒa,b,4ƒa,b), 1.35ꢀ1.24 (m, 2 H, H-3ƒa,b), 1.18, 1.16,
1.07 (3 d, partially overlapped, J5,6 6.2 Hz, H-6IꢀIII); 13
/
4.27 (m, 13 H, 3 CH2Ph, 2?I-III,4?aI-III, incl. d,
/
3.92
/
z 1034.3 ([Mꢁ
Cs]ꢁ).
/
/
/
3.11. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl-(10
(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (16)
L-
D
-
/
/
/
L
/
D
/
2)-4-
C
L
D-
NMR (CDCl3): d 101.36, 101.19 (C, 2 C, 3 CHPh),
101.11 (C-1II), 99.14 (C-1III), 98.86 (C-1I), 76.57, 76.50
(2 C, C, C-2?IꢀIII), 75.72 (2 C, C-2III,3), 75.35, 75.28 (2
C-3), 74.33 (C-2I), 72.71 (C-2II), 71.98, 71.43, 70.98 (3
CH2Ph), 68.45, 68.42, 67.48 (C-5IꢀIII), 67.31, 67.27,
67.22 (C, C, 2 C, C-1ƒ,4?IꢀIII), 59.03 (OCH3-2), 52.25,
51.94, 51.83 (C-4IꢀIII), 51.38 (COOCH3), 33.81 (C-5ƒ),
28.78 (C-2ƒ), 28.52 (3 C, C-3?IꢀIII), 25.49 (C-3ƒ), 24.48
(C-4ƒ), 18.02, 17.91 (C, 2 C, C-6IꢀIII); FABMS: m/z
Following the general procedure described above, con-
version of 12 (50 mg) gave the amine 16 (45 mg, 87%).
Definite signals in the 1H NMR spectrum (CD3OD)
were at d 5.12 (bs, 2 H, H-1IIꢀIII), 4.82 (bs, 1 H, H-1I),
4.22ꢀ
4.16 (m, 3 H, H-2?IꢀIII), 4.12 (broad t, 1 H, H-2II),
/
4.04 (dd, 1 H, J2,3 3.1, J3,4 10.0 Hz, H-3II), 3.67 (broad t,
1 H, H-2III), 3.48 (s, partially overlapped, OCH3-2), 3.28
(t, 1 H, J 6.5 Hz, H-6ƒa,b), 2.77 (t, 2 H, H-7ƒa,b), 2.23 (t,
1436.74 ([Mꢁ
/
1]ꢁ), 1458.72 ([MꢁNa]ꢁ); HRMS: m/z
/
1568.5715. C80H97N3O21Cs requires 1568.5669.
A small amount of by product, analogous to com-
pound 29, which was formed during methylation of 3,
was also obtained.
(b) A mixture of the glycosyl donor 28 (90 mg, 0.18
mmol) and glycosyl acceptor 315 (120 mg, 0.12 mmol) in
dry CH2Cl2 (10 mL) was treated with NIS and AgOTf
(21 mg, 0.084 mmol), as described for the preparation of
2 H, J 7.2 Hz, H-5ƒ), 2.08ꢀ
/
1.77 (2 m, 6 H, H-3?IꢀIII),
1.70ꢀ1.57 (m, 4 H, H-4ƒa,b,2ƒa,b in that order), 1.46ꢀ
/
/
1.36 (m, 2 H, H-3ƒa,b), 1.17, 1.16, 1.15 (3 d, 9 H, J5,6 5.8
Hz, H-6IꢀIII); 13C NMR (CD3OD): d 102.68 (C-1II),
100.44 (C-1III), 100.17 (C-1I), 80.68 (C-2III), 79.87 (C-
2I), 78.91 (C-2II), 70.63 (3 C, C-2?IꢀIII), 69.65 (C-3III),
69.52 (C-3I), 69.43 (C-5), 69.34 (C-3II,5), 68.74 (C-5),
68.45 (C-1ƒ), 59.38 (3 C, C-4?IꢀIII), 59.17 (OCH3), 54.76,
54.49 (C, 2 C, C-4?IꢀIII), 42.20 (C-6ƒ), 41.80 (C-7ƒ), 38.23
(3 C, C-3?IꢀIII), 36.94 (C-5ƒ), 30.11 (C-2ƒ), 26.84 (C-3ƒ),
26.51 (C-4ƒ), 18.34, 18.19 (2 C, C, C-6IꢀIII); FABMS: m/
7 (b). Chromatography (5:1 to 2:1 tolueneꢀacetone)
/
gave 8 (133 mg, 77%), which was identical (TLC, NMR)
with the compound described above.
z 930.6 ([Mꢁ
1]ꢁ).
/
3.10. 5-Methoxycarbonylpentyl 4-(3-deoxy-
tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl-(10
(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (12)
L
-glycero-
D-
3.12. 1-[(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl-(10
(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
/
L
L
D-
D
/2)-4-
/
L
L
D-
D
/2)-4-
L
D-
mannopyranoside]-2-ethoxycyclobutene-3,4-dione (20)
Treatment of 8 (900 mg) with hydrogen, as described for
the preparation of 11, gave 12 (540 mg, ꢀ100%).
Definite signals in the 1H NMR spectrum (CD3OD)
were at d 5.13ꢀ
5.11 (m, 2 H, H-1II,III), 4.82 (d, 1 H, J1,2
1.6 Hz, H-1I), 4.22ꢀ4.16 (m, 3 H, H-2?IꢀIII), 4.12 (broad
doublet, 1 H, H-2II), 4.04 (dd, 1 H, J2,3 3.2, J3,4 10.2 Hz,
H-3II), 3.68ꢀ
3.65 (m, 5 H, partially overlapped, H-
2III,1ƒa, incl s at 3.67 for OCH3-2), 3.48 (s, 3 H,
COOCH3), 3.45ꢀ3.38 (m, 1 H, H-1ƒb), 2.35 (t, 2 H, J
7.3 Hz, H-5ƒa,b), 2.08ꢀ1.77 (2 m, 6 H, H-3?a,b), 1.69ꢀ
1.38 (m, 2 H, H-
3ƒa,b), 1.17, 1.16, 1.13 (3 d, 9 H, J5,6 6.1 Hz, H-6IꢀIII);
13C NMR (CD3OD): d 102.68 (C-1II), 100.43 (C-1III),
100.15 (C-1I), 80.65 (C-2III), 79.86 (C-2I), 78.90 (C-2II),
70.66, 70.61 (C, 2 C, C-2?IꢀIII), 66.67 (C-3III), 69.55 (C-
/
Following the general procedure described above, con-
version of 16 (50 mg) gave the ethyl ester 20 (36 mg,
65%). Definite signals in the 1H NMR spectrum
(CD3OD) were at d 5.12 (2 d, partially overlapped, 2
/
/
H, H-1II,III), 4.82 (broad doublet, 1 H, H-1I), 4.22ꢀ
4.16
/
/
(m, 3 H, H-2?IꢀIII), 4.12 (dd, 1 H, J1,2 1.7, J2,3 3.0 Hz, H-
2II), 4.04 (dd, 1 H, J3,4 10.1 Hz, H-3II), 3.67 (m, H-
/
2III,6ƒa,b), 3.54ꢀ
2), 3.44ꢀ
partially overlapped, 2 H, H-5ƒ), 2.08ꢀ
H-3?IꢀIII), 1.67ꢀ
1.54 (m, 4 H, H-4ƒa,b,2ƒa,b in that
order), 1.49ꢀ
/
3.48 (m, 4 H, H-1ƒa, incl s, 3.48, OCH3-
3.36 (m, 3 H, H-1ƒb,7ƒa,b), 2.22ꢀ2.14 (2 t,
1.77 (2 m, 6 H,
/
/
/
/
1.57 (m, 4 H, H-2ƒa,b,4ƒa,b), 1.46ꢀ
/
/
/
/
1.35 (m, 5 H, CH3CH2, H-3ƒa,b), 1.17,
1.15, 1.13 (3 d, 9 H, J5,6 6.0 Hz, H-6IꢀIII); 13C NMR
(CD3OD): d 102.69 (C-1II), 100.49 (C-1III), 100.19 (C-

2600
R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
1I), 80.71 (C-2III), 79.84 (C-2I), 78.93 (C-2II), 70.76 (d,
CH2CH3), 70.72, 70.65 (2 C, C, C-2?IꢀIII), 69.71, 69.60,
69.45, 69.38, 68.75 (C, C, C, 2 C, C, C-3IꢀIII,5IꢀIII),
68.38 (C-1ƒ), 59.45, 59.42, 59.41 (C-4?IꢀIII), 59.16
(OCH3-2), 54.79, 54.52 (C, 2 C, C-4?IꢀIII), 45.18 (d, C-
6ƒ), 40.75 (d, C-7ƒ), 38.26, 38.22 (2 C, C, C-3?IꢀIII), 36.91
(C-5ƒ), 30.17 (C-2ƒ), 26.80 (d, C-3ƒ), 26.47 (C-4ƒ), 18.35,
18.19 (2 C, C, C-6IꢀIII), 16.15 (d, CH3CH2); FABMS:
chromatography (5:10
/
2:1 tolueneꢀacetone) gave ma-
/
terial (455 mg, 79%), which was identical (TLC, NMR)
with the fully protected tetrasaccharide 9 described
above.
3.14. 5-Methoxycarbonylpentyl 4-(3-deoxy-
tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-bis-[(102)-4-(3-deoxy-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (13)
L-glycero-
D-
/
L-glycero-
m/z 1054.4 ([Mꢁ
/
1]ꢁ).
D
/2)-
L
D-
3.13. 5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy-2-O-methyl-a- -mannopyranosyl-bis-[(10
O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (9)
L
D
/2)-(3-
Hydrogenolysis of the fully protected tetrasaccharide 9
(700 mg), as described for the preparation of 8, gave 13
L
D
/
2)-
(420 mg, ꢀ
spectrum (CD3OD) were at d 5.14ꢀ
1IIꢀIV), 4.82 (d, 1 H, J1,2 1.5 Hz, H-1I), 4.22ꢀ
H, H-2?IꢀIV), 4.12ꢀ4.08 (m, 2 H, H-2II,III), 4.06ꢀ
2 H, H-3II,III), 3.68ꢀ
3.65 (m, partially overlapped, H-
2IV,1ƒa, incl. s at 3.66 for COOCH3), 3.48 (s, 3 H,
OCH3-2), 3.45ꢀ3.38 (m, 1 H, H-1ƒb), 2.34 (t, 2 H, J 7.2
Hz, H-5ƒa,b), 2.08ꢀ1.76 (2 m, 6 H, H-3?a,b), 1.69ꢀ1.57
(m, 4 H, H-2ƒa,b,4ƒa,b), 1.46ꢀ1.38 (m, 2 H, H-3ƒa,b),
1.18ꢀ
1.12 (m, 12 H, H-6IꢀIV); 13C NMR (CD3OD): d
/
100%). Definite signals in the 1H NMR
L
/
5.11 (m, 3 H, (H-
4.16 (m, 4
4.01 (m,
D
/
/
/
(a) The trisaccharide alcohol 4 (50 mg) was treated with
MeI (2.5 mL) and Ag2O (1.5 g), as described for the
preparation of 7 (a). The reaction time had to be
extended to 5 days in order for the starting alcohol to
be almost completely consumed, as shown by TLC.
/
/
/
/
/
Chromatography (5:1 to 2:1 tolueneꢀ
desired, fully protected methyl ether 9 (45 mg, 90%),
[a]D
298 (c 0.4). Definite signals in the 1H NMR
/
acetone) gave the
/
102.69, 102.38 (C-1II,III), 100.44 (C-1IV), 100.16 (C-1I),
80.64 (C-2IV), 79.84 (C-2I), 78.99 (2 C, C-2II,III), 70.61 (4
C, C-2?IꢀIV), 69.67 (C-3IV), 69.57 (C-3I), 69.42, 69.34 (2
C, 3 C, C-3II,III,5,5,5), 68.71 (C-5), 68.38 (C-1ƒ), 59.38 (4
C, C-4?IꢀIV), 59.12 (OCH3-2), 54.70, 54.48 (C, 3 C, C-
4?IꢀIV), 52.09 (COOCH3), 38.21 (4 C, C-3?IꢀIV), 34.60
(C-5ƒ), 30.00 (C-2ƒ), 26.68 (C-3ƒ), 25.57 (C-4ƒ), 18.31,
18.24, 18.18 (2 C, C, C, C-6IꢀIV); FABMS: m/z 1281.4
ꢃ
/
spectrum (CDCl3) of 9 were at d 6.34, 6.28, 6.27, 6.19 (4
d, partially overlapped, 4 H, J4,NH 9.4 Hz, 4 NH), 5.55,
5.54, 5.53, 5.52 (4 s, 4 CHPh), 4.99 (d, 1 H, J1,2 1.8 Hz,
H-1IV), 4.97, 4.93 (2 d, 1 H each, J1,2 1.8 Hz, H-1IIꢀIII),
ꢀ
4.62 (part of a multiplet, H-1I), 4.20, 4.10 (2 broad t,
/
partially overlapped, H-2II,III), 3.84 (broad t, partially
overlapped, H-2I), 3.78 (broad t, partially overlapped,
H-2IV), 3.59 (s, partially overlapped, COOCH3), 3.26 (s,
partially overlapped, OCH3-2), 2.26 (t, 2 H, J 7.5 Hz, H-
([Mꢁ
Cs]ꢁ).
/
3.15. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-bis-[(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
(4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (17)
L-
5ƒa,b), 2.06ꢀ
/
1.84 (m, 8 H, H-3?IꢀIVa,b), 1.64ꢀ
/1.47 (m, 4
D-
H, H-2ƒ,4ƒa,b), 1.34-1.25 (m, 2 H, H-3ƒa,b), 1.15, 1.13.
1.08. 1.05 (4 d, 12 C, H-6IꢀIV); 13C NMR (CDCl3): d
101.35, 101.24, 101.13 (C, C, 2 C, 4 CHPh), 100.90,
100.50 (C-1II,III), 99.12 (C-1IV), 98.78 (C-1I), 76.64,
76.58, 76.50 (C, 2 C, C, C-2?IꢀIV), 75.79 (C-2IV), 75.68,
75.44, 75.24, 74.60 (C-3IꢀIV), 74.16 (C-2I), 72.92, 72.86
(C-2II,III), 71.88, 71.34, 71.24, 71.00 (4 CH2Ph), 68.59,
68.51, 68.42, 67.52 (C-5IꢀIV), 67.27 (5 C, C-1ƒ,4?IꢀIV),
59.01 (OCH3-2), 52.16, 52.00, 51.90, 51.63 (C-4IꢀIV),
51.41 (COOCH3), 33.82 (C-5ƒ), 28.79 (C-2ƒ), 28.54 (4 C,
C-3?IꢀIV), 25.49 (C-3ƒ), 24.49 (C-4ƒ), 18.03, 17.93 (C, 3
/
L
D
/2)-
L
D-
Treatment of the methyl ester 13 (80 mg) as described
for preparation of amine 16, gave the title compound 17
(73 mg, 90%). Definite signals in the 1H NMR spectrum
(CD3OD) were at d 5.15 (d, 1 H, J1,2 1.5 Hz, H-1), 5.12
(m, 2 H, H-1), 4.81 (d, 1 H, J1,2 1.4 Hz, H-1I), 4.22ꢀ
(m, 4 H, H-2?IꢀIV), 4.12ꢀ4.08 (m, 2 H, H-2II,III), 4.06ꢀ
4.01 (m, partially overlapped, 2 H, H-3II,III), ꢀ
3.67 (m,
partially overlapped, H-2IV), 3.48ꢀ
3.38 (m, 4 H, H-1ƒb,
/4.15
/
/
C, C-6IꢀIV). FABMS: m/z 1861.93 ([Mꢁ
([Mꢁ
Na]ꢁ); HRMS: m/z 1993.7477. C104H124CsN4O27
requires 1993.7507.
/
1]ꢁ), 1883.94
/
/
/
incl s, 3.48, OCH3-2), 3.26 (t, 2 H, J 6.7 Hz, H-6ƒa,b),
2.73 (t, 2 H, H-7ƒa,b), 2.22 (t, 2 H, J 7.3 Hz, H-5ƒa,b),
A small amount of by product, analogous to com-
pound 29, which was formed during methylation of 3,
was also obtained.
(b) Reaction of the glycosyl donor 28 (240 mg, 0.48
mmol), and alcohol 415 (440 mg, 0.31 mmol), as
described for the preparation of 7 (b), followed by
2.08ꢀ
/
1.76 (2 m, 8 H, H-3?IꢀIV), 1.69ꢀ
/1.57 (m, 4 H, H-
4ƒa,b,2ƒa,b in that order), 1.46ꢀ
/
1.36 (m, 2 H, H-3ƒa,b),
1.18ꢀ C
1.13 (4 d, partially overlapped, 12 H, H-6IꢀIV); 13
/
NMR (CD3OD): d 102.71, 102.41 (C-1II,III), 100.49 (C-
1IV), 100.19 (C-1I), 80.68 (C-2IV), 79.87 (C-2I), 79.02 (2

R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ
/2603
2601
C, C-2II,III), 70.63 (4 C, C-2?IꢀIV), 69.69 (C-3IV), 69.57
(C-3I), 69.46, 69.38, 68.74 (2 C, 3 C, C, C-3II,III,5IꢀIV),
68.47 (C-1ƒ), 59.40 (4 C, C-4?IꢀIV), 59.15 (OCH3-2),
54.78, 54.51 (C, 3 C, C-4?IꢀIV), 42.74 (C-6ƒ), 41.95 (C-
7ƒ), 38.24 (C-3?IꢀIV), 36.96 (C-5ƒ), 30.12 (C-2ƒ), 26.84
(C-3ƒ), 26.54 (C-4ƒ), 18.34, 18.26, 18.19 (2 C, C, C, C-
(CDCl3) of 10 were at d 6.44ꢀ
/
6.18 (5 d, partially
overlapped, 5 H, 5 NH), 5.55, 5.54, 5.51 (3 s, 5 H, 5
CHPh), 4.98 (d, 1 H, J1,2 1.8 Hz, H-1V), 4.96, 4.89 (2 d,
3 H, J1,2 1.8 Hz, H-1IIꢀIV), ꢀ
/
4.61 (m, partially over-
lapped, H-1I), 4.17ꢀ
/4.05 (m, partially overlapped, H-
2IIꢀIV), 3.80 (broad doublet, partially overlapped, H-2I),
3.77 (m, partially overlapped, H-2V), 3.58 (s, partially
overlapped, COOCH3), 3.25 (s, partially overlapped,
6IꢀIV); FABMS: m/z 1177 ([Mꢁ1]ꢁ).
/
OCH3-2), 2.26 (t, 2 H, J 7.5 Hz, H-5ƒa,b), 2.07ꢀ
10 H, H-3?IꢀVa,b), 1.64ꢀ
1.47 (m, 4 H, H-2ƒ,4ƒa,b),
1.34ꢀ
/
1.84 (m,
3.16. 1-{(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-bis-[(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
/
L
D-
/
1.25 (m, 2 H, H-3ƒa,b), 1.12, 1.07, 1.06, 1.03 (4 d,
partially overlapped, 15 H, H-6IꢀIV); 13C NMR
(CDCl3): d 101.32, 101.23 (3 C, 2 C, 5 CHPh), 100.99,
100.45 (C, 2 C, C-1IIꢀIV), 99.18 (C-1V), 98.82 (C-1I),
76.65, 76.60 (3 C, 2 C C-2?IꢀV), 75.84 (2 C, C-2IV,3),
/
L
D
/
2)-
L
D-
mannopyranoside}-2-ethoxycyclobutene-3,4-dione (21)
75.57, 75.21, 74.70, 74.50 (4ꢂ
C-3), 74.26 (C-2I), 72.90,
/
Treatment of amine 17 (70 mg) with squaric acid diethyl
ester, as described above for similar conversions, gave
the monoester 21 (76 mg, 96%). Definite signals in the
1H NMR spectrum (CD3OD) were at d 5.15 (d, 1 H, J1,2
1.5 Hz, H-1II), 5.12 (m, 2 H, 2 H-1III,IV), 4.81 (d, 1 H,
72.78 (2 C, C, C-2IIꢀIV), 72.00, 71.30, 71.23, 71.10, 71.06
(5 CH2Ph), 68.68, 68.63, 68.58, 67.62 (C, C, 2 C, C, C-
5IꢀV), 67.34 (6 C, C-1ƒ,4?IꢀV), 59.01 (OCH3-2), 52.04,
51.98, 51.51, 51.40 (C, C, 2 C, C, C-4IꢀV), 51.40
(COOCH3), 33.88 (C-5ƒ), 28.85 (C-2ƒ), 28.61 (5 C, C-
3?IꢀV), 25.56 (C-3ƒ), 24.55 (C-4ƒ), 18.09, 18.01 (C, 4 C,
J1,2 1.4 Hz, H-1I), 4.78ꢀ
4.16 (m, 4 H, H-2?IꢀIV), 4.12ꢀ
4.01 (m, 2 H, H-3II,III), ꢀ
3.67 (broad doublet of
doublets, partially overlapped, H-2IV), 3.54ꢀ
3.47 (m, 4
H, H-1ƒa, incl s, 3.47, OCH3-2), 3.43ꢀ3.37 (m, 3 H, H-
1ƒb,7ƒa,b), 2.20, 2.17 (2 t, partially overlapped, 2 H, J
7.5 Hz, H-5ƒa,b), 2.08ꢀ
1.76 (2 m, 8 H, H-3?IꢀIV), 1.66ꢀ
1.54 (m, 4 H, H-4ƒa,b,2ƒa,b in that order), 1.49ꢀ1.35 (m,
5 H, H-3ƒa,b, incl q at 1.46, CH2CH3), 1.18ꢀ1.13 (4 d,
/
4.68 (m, 2 H, CH2CH3), 4.22ꢀ
/
/
4.08 (m, H-2II,III), 4.06ꢀ
/
C-6IꢀIV); FABMS: m/z 2288.59 ([Mꢁ
([Mꢁ
requires 2418.9346.
/
1]ꢁ), 2310.81
/
/
Na]ꢁ); HRMS: m/z 2418.9312. C128H151CsN5O33
/
/
/
/
/
3.18. 5-Methoxycarbonylpentyl 4-(3-deoxy-
tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-tris-[(102)-(4-(3-deoxy-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (14)
L
-glycero-
/
D
-
partially overlapped, 12 H, H-6IꢀIV); 13C NMR
(CD3OD): d 102.63, 102.36 (C-1II,III), 100.43 (C-1IV),
100.15 (C-1I), 80.65 (C-2IV), 79.71 (C-2I), 78.94 (2 C, C-
2II,III), 70.77 (d, CH2CH3), 70.63 ( 4 C, C-2?IꢀIV), 69.66
(C-3IV), 69.56 (C-3I), 69.45, 69.34, 68.67 (2 C, 3 C, C, C-
3II,III,5IꢀIV), 68.37 (C-1ƒ), 59.41, 59.39 (2 C each, C-
4?IꢀIV), 59.13 (OCH3-2), 54.75, 54.48 (C, 3 C, C-4?IꢀIV),
45.16 (d, C-6ƒ), 40.72 (d, C-7ƒ), 38.20 (4 C, C-3?IꢀIV),
36.88 (C-5ƒ), 30.13 (C-2ƒ), 26.79 (d, C-3ƒ), 26.45 (d, C-
4ƒ), 18.34, 18.26, 18.19 (2 C, C, C, C-6IꢀIV), 16.17 (d,
/
L
-glycero-
D
/2)-
L
D-
Hydrogenolysis of the foregoing, fully protected penta-
saccharide 10 (1 g), as described for the preparation of
11, gave the methyl ester 14 (0.6 g, ꢀ100%). Definite
/
1
signals in the H NMR spectrum (CD3OD) were at d
5.13, 5.11 (2 m, 4 H, (H-1IIꢀV), 4.81 (bs, 1 H, Hz, H-1I),
3.67 (s, partially overlapped, COOCH3), 3.47 (s, 3 H,
CH3CH2); FABMS: m/z 1301.6 ([Mꢁ
1]ꢁ).
/
OCH3-2), 3.45ꢀ
Hz, H-5ƒa,b), 2.08ꢀ
(m, 4 H, H-2ƒa,b,4ƒa,b), 1.46ꢀ
1.18ꢀ
1.13 (m, 15 H, H-6IꢀV); 13C NMR (CD3OD): d
/
3.38 (m, 1 H, H-1ƒb), 2.35 (t, 2 H, J 7.2
1.76 (2 m, 6 H, H-3?a,b), 1.69ꢀ1.57
1.38 (m, 2 H, H-3ƒa,b),
/
/
3.17. 5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy-2-O-methyl-a- -mannopyranosyl-tris-[(10
O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a- -mannopyranoside (10)
/
L
/
D
/
2)-3-
2)-
102.64, 102.36 (C, 2 C, C-1IIꢀIV), 100.38 (C-1V), 100.14
(C-1I), 80.60 (C-2V), 79.70 (C-2I), 79.03, 78.90 (2 C, C-
2IIꢀIV), 70.59 (5 C, C-2?IꢀV), 69.29 (C-3V), 69.55 (C-3I),
69.38, 69.34 (2 C, 4 C, C-3IIꢀIV,5,5,5,5), 68.69 (C-5),
68.37 (C-1ƒ), 59.37 (5 C, C-4?IꢀV), 59.12 (OCH3-2),
54.65, 54.50, 54.44 (C, 2 C, 2 C, C-4?IꢀV), 52.10
(COOCH3), 38.16 (5 C, C-3?IꢀV), 34.59 (C-5ƒ), 29.97
(C-2ƒ), 26.65 (C-3ƒ), 25.55 (C-4ƒ), 18.31, 18.27, 18.23,
L
D
/
L
D
Reaction of the glycosyl donor 28 (252 mg, 0.50 mmol)
with the tetrasaccharide glycosyl acceptor 6,15 as de-
scribed for the preparation of 7 (b) gave the fully
protected pentasaccharide 10 (575 mg, 75%), [a]D
18.19 (2 C, C, C, C, C-6IꢀV); FABMS: m/z 1528.1 ([Mꢁ
Cs]ꢁ).
/
ꢃ
298 (c 0.5). Definite signals in the 1H NMR spectrum
/

2602
R. Saksena et al. / Carbohydrate Research 338 (2003) 2591ꢀ2603
/
3.19. (2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-tris-[(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
(4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
mannopyranoside (18)
L
-
C-3ƒ), 26.45 (d, C-4ƒ), 18.35, 18.28, 18.21 (3 C, C, C, C-
/
D
-
6
IꢀV), 16.16 (d, CH3CH2); FABMS: m/z 1548.7 ([Mꢁ
/
L
1]ꢁ).
D
/
2)-
L
D-
3.21. Ethyl 3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-1-thio-a-
D
-mannopyranoside (28)
Treatment of the foregoing ester 14 (100 mg), as
described for the preparation of 15, gave amine 18 (85
Ethyl
deoxy-
2-O-acetyl-3-O-benzyl-4-(2,4-O-benzylidene-3-
D-
1
mg, 83%). Definite signals in the H NMR spectrum
L
-glycero-tetronamido)-4,6-dideoxy-1-thio-a-
(CD3OD) were at d 5.14, 5.11 (2 m, 4 H, H-1IIꢀV), 4.81
mannopyranoside15 (2.5 g) was deacetylated (Zemple´n).
Conventional processing afforded pure (TLC) ethyl 3-
(bs, 1 H, H-1I), 4.22ꢀ4.16 (m, 5 H, H-2?IꢀV), 3.67 (broad
/
t, partially overlapped, H-2V), 3.47ꢀ
3.38 (m, 4 H, H-1ƒb,
/
O-benzyl-4-(2,4-O-benzylidene-3-deoxy-
tronamido)-4,6-dideoxy-1-thio-a- -mannopyranoside
(27) (2.3 g, ꢀ
100%) as an oil; 1H NMR (CDCl3): d 6.27
(broad doublet, 1 H, J4,NH, NH), 5.56 (s, 1 H, CHPh),
2
5.34 (d, 1 H, J1,2 1.5 Hz, H-1), 4.65, 4.50 (2 d, 2 H, J
11.7 Hz, CH2Ph), 4.40ꢀ
3.27 (m, 4 H, H-2,4,5,4?b), 3.65 (dd, 1 H, J2,3 3.6, J3,4
10.2 Hz, H-3), 2.69ꢀ2.48 (m, 2 H, CH2CH3), 2.13ꢀ1.83
(2 m, 2 H, H-3?a,b), 1.27 (t, 1 H, J 7.5 Hz, CH3CH2),
1.23 (d, 3 H, J5,6 6.0 Hz, H-6); 13C NMR (CDCl3): d
101.18 (CHPh), 83.01 (C-1), 76.44 (2 C, C-2?,3), 71.38
CH2Ph), 68.53(C-5), 67.50 (C-2), 67.26 (C-4?), 51.90 (C-
4), 28.56 (C-3?), 24.93 (CH2CH3), 17.76 (C-6), 14.79
(CH2CH3).
Silver oxide (15 g) was added to a soln of 27 (5.0 g,
10.3 mmol) in MeI (25 mL), and the mixture was stirred
at r.t. with the exclusion of light. After 2 h, TLC (2:1
tolueneꢀacetone) showed that no starting material was
present. The reaction mixture was filtered, the filtrate
was concentrated, and the residue was chromatographed
L-glycero-te-
incl s, 3.47, OCH3-2), 3.26 (t, 1 H, J 6.7 Hz, H-6ƒa,b),
2.73 (t, 2 H, J 6.3 Hz, H-7ƒa,b), 2.22 (t, 2 H, J 7.3 Hz,
D
/
H-5ƒa,b), 2.08ꢀ
4 H, H-4ƒa,b,2ƒa,b in that order), 1.46ꢀ
3ƒa,b), 1.18ꢀ1.13 (5 d, partially overlapped, 15 H, H-
/
1.76 (2 m, 10 H, H-3?IꢀV), 1.70ꢀ
/1.57 (m,
/1.36 (m, 2 H, H-
/
/
4.32 (m, 2 H, H-2?,4?a), 4.13ꢀ
/
6IꢀV); 13C NMR (CD3OD): d 102.70, 102.44 (C, 2 C, C-
1IIꢀIV), 100.48 (C-1V), 100.20 (C-1I), 80.69 (C-2V), 79.80
(C-2I), 79.14, 79.01 (C, 2 C, C-2IIꢀIV), 70.64 (5 C, C-
2?IꢀV), 69.70 (C-3V), 69.59 (C-3I), 69.38, 68.74 (7 C, C,
C-3IIꢀIV,5IꢀV), 68.47 (C-1ƒ), 59.42 (5 C, C-4?IꢀV), 59.15
(OCH3), 54.76, 54.57, 54.51 (C, 2 C, 2 C, C-4IꢀV), 42.71
(C-6ƒ), 41.94 (C-7ƒ), 38.25 (5 C, C-3?IꢀV), 36.96 (C-5ƒ),
30.12 (C-2ƒ), 26.85 (C-3ƒ), 26.54 (C-4ƒ), 18.35, 18.29,
/
/
18.22 (2 C, 2 C, C, C-6IꢀV); FABMS: m/z 1425 ([Mꢁ
/
1]ꢁ).
3.20. 1-{(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
/
L
-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl-tris-[(102)-4-(3-deoxy- -glycero-
tetronamido)-4,6-dideoxy-a-
-mannopyranosyl]-(10
4-(3-deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
D-
/
L
D
/
2)-
(5:10
/
2:1 tolueneꢀ
/
acetone), to give 28 (4.8 g, 93%), m.p.
32.68 (c 0.5,
CHCl3); H NMR (CDCl3): d 6.29 (d, 1 H, J4,NH 9.0
L
D-
97.5ꢀ98.0 8C (from isopropyl ether), [a]D
/
ꢁ
/
mannopyranoside}-2-ethoxycyclobutene-3,4-dione (22)
1
Hz, NH), 5.55 (s, 1 H, CHPh), 5.36 (d, 1 H, J1,2 1.5 Hz,
Treatment of amine 18 (85 mg) with diethyl squarate, as
described for 15, afforded the squaric acid ethyl ester 22
(83 mg, 89%). Definite signals in the 1H NMR spectrum
(CD3OD) were at d 5.14, 5.11 (2 m, 4 H, H-1IIꢀV), 4.80
H-1), 4.65, 4.50 (2 d, 2 H, CH2Ph), 4.36ꢀ
H-2?,4?a), 4.19ꢀ3.95 (m, 3 H, H-4,5,4?b), 3.76 (dd, 1 H,
J2,3 2.8, J3,4 10.2 Hz, H-3), 3.63 (dd, 1 H, H-2), 3.49 (s, 3
H, OCH3), 2.70ꢀ2.51 (m, 2 H, CH2CH3), 2.09ꢀ1.81 (m,
/4.30 (m, 2 H,
/
/
/
(bs, 1 H, H-1I), 4.73 (m, 2 H, CH3CH2), 4.22ꢀ
H, H-2?IꢀV), 3.67 (broad t, partially overlapped, 2IV),
3.54ꢀ3.47 (m, 4 H, H-1ƒa, incl s, 3.47, OCH3-2), 3.43ꢀ
3.37 (m, 3 H, H-1ƒb,7ƒa,b), 2.20, 2.17 (2 t, partially
overlapped, 2 H, J 7.3 Hz, H-5ƒa,b), 2.08ꢀ1.76 (2 m, 10
H, H-3?IꢀV), 1.65ꢀ
1.53 (m, 4 H, H-4ƒa,b,2ƒa,b in that
order), 1.49ꢀ1.33 (m, 5 H, H-3ƒa,b, CH3CH2), 1.18ꢀ
/4.16 (m, 5
2 H, H-3?a,b), 1.27 (t, 3 H, J 7.4 Hz, CH2CH3), 1.22 (d,
3 H, J5,6 5.9 Hz, H-6); 13C NMR (CDCl3): d 101.02
(CHPh), 81.49 (C-1), 78.04 (C-2), 76.49 (C-2?), 76.27 (C-
3), 71.04 (CH2Ph), 67.84 (C-5), 67.25 (C-4?), 58.65
(OCH3-2), 52.72 (C-4), 28.54 (C-3?), 25.36 (CH2CH3),
17.88 (C-6), 14.87 (CH2CH3); FABMS: m/z 502.31
/
/
/
/
/
/
([Mꢁ
/
1]ꢁ), 524.31 ([MꢁNa]ꢁ). Anal. Calcd for
/
1.13 (5 d, partially overlapped, 15 H, H-6IꢀV); 13C
NMR (CD3OD): d 102.62 (C-1III), 102.39 (2 C, C-
1II,IV), 100.42 (C-1V), 100.16 (C-1I), 80.64 (C-2V), 79.65
(C-2I), 79.06, 78.92 (C, 2 C, C-2IIꢀIV), 70.79 (d,
CH3CH2), 70.63 (5 C, C-2?IꢀV), 69.66 (C-3V), 69.57
(C-3I), 69.38, 68.69 (7 C, C, C-3IIꢀIV,5IꢀV), 68.38 (C-1ƒ),
59.42 (5 C, C-4?IꢀV), 59.14 (OCH3-2), 54.73, 54.55, 54.48
(C, 2 C, 2 C, C-4?IꢀV), 45.17 (d, C-6ƒ), 40.72 (d, C-7ƒ),
38.20 (5 C, C-3?IꢀV), 36.88 (C-5ƒ), 30.13 (C-2ƒ), 26.75 (d,
C27H35NO6S: C, 64.65; H, 7.03; N, 2.79. Found: C,
64.55; H, 7.01; N, 2.86.
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