Studies on vaccines against cholera. Synthesis of neoglycoconjugates from the hexasaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, by single-point attachment or by attachment of the hapten in the form of clusters

Article abstract of DOI:10.1016/S0008-6215(99)00185-8

The terminal hexasaccharide of the O-antigen of Vibrio cholerae O:1, serotype Ogawa, has been synthesized in the form of a glycoside whose aglycon (linker) allows conjugation to carrier proteins by reductive amination. The conjugate obtained from direct, single-point attachment of the linker- equipped hapten to chicken serum albumin (CSA) contained seven hapten residues/CSA. A neoglycoconjugate containing the carbohydrate antigen in the form of clusters was obtained using, as a hapten subcarrier, an oligopeptide containing 16 amino groups. It was treated with a limited amount of hapten, to give a hapten-carrying subcarrier (HCS). Subsequent conjugation of HCS to CSA, using squaric acid diethyl ester as a conjugation reagent, gave a cross- linked, glycocluster conjugate containing 51% (w/w) of the carbohydrate.

Full text of DOI:10.1016/S0008-6215(99)00185-8

www.elsevier.nl/locate/carres
Carbohydrate Research 321 (1999) 157–167
Studies on vaccines against cholera. Synthesis of
neoglycoconjugates from the hexasaccharide determinant
of Vibrio cholerae O:1, serotype Ogawa, by single-point
attachment or by attachment of the hapten in the form
of clusters
Jian Zhang, Pavol Kova´cˇ *
National Institutes of Health, NIDDK, Laboratory of Medicinal Chemistry, 8 Center Dri6e, Bethesda,
MD 20892-0815, USA
Received 20 May 1999; accepted 21 June 1999
Abstract
The terminal hexasaccharide of the O-antigen of Vibrio cholerae O:1, serotype Ogawa, has been synthesized in the
form of a glycoside whose aglycon (linker) allows conjugation to carrier proteins by reductive amination. The
conjugate obtained from direct, single-point attachment of the linker-equipped hapten to chicken serum albumin
(CSA) contained seven hapten residues/CSA. A neoglycoconjugate containing the carbohydrate antigen in the form
of clusters was obtained using, as a hapten subcarrier, an oligopeptide containing 16 amino groups. It was treated
with a limited amount of hapten, to give a hapten-carrying subcarrier (HCS). Subsequent conjugation of HCS to
CSA, using squaric acid diethyl ester as a conjugation reagent, gave a cross-linked, glycocluster conjugate containing
51% (w/w) of the carbohydrate. Published by Elsevier Science Ltd.
Keywords: Glycoconjugate; Oligosaccharide; Glycocluster; Subcarrier; Synthetic vaccine
there is a need for more potent and safe
immunogens free from such drawbacks. It is
expected that conjugate vaccines, resulting
from chemical linking of immunologically
dominant epitopes present in LPSs to proteins,
will alleviate these problems [2–4].
Part of our effort towards synthetic vaccines
is directed towards developing a potent im-
munogen for protective anti-V. cholerae O:1
antibodies using synthetic fragments of the
O-polysaccharide (O-PS) as antigen. The inter-
nal part of the O-PS of the two main strains of
V. cholerae O:1, Inaba and Ogawa, consists
1. Introduction
Despite impressive progress that has been
made in vaccine development, cholera is one of
the diseases for which a satisfactory vaccine is
not available. The existing cellular vaccines, as
well as those based on the lipopolysaccharide
(LPS) of Vibrio cholerae O:1, have serious
age-related and/or protection limitations. In
addition, regardless of the mode of administra-
tion, they elicit adverse reactions [1]. Clearly,
* Corresponding author. Tel.: +1-301-496-3569; fax: +1-
301-402-0589.
E-mail address: kpn@helix.nih.gov (P. Kova´cˇ)
[5–7] of a-(1“2)-linked
D
-perosamine (4-
amino-4,6-dideoxy- -mannose) whose amino
D
0008-6215/99/$ - see front matter Published by Elsevier Science Ltd.
PII: S0008-6215(99)00185-8

158
J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
While other approaches can be envisioned,
from the practical point of view it is advanta-
geous to carry out the initial synthetic steps
as for the preparation of analogous methyl
glycosides [13,17,18]. The blockwise construc-
tion of higher oligosaccharides suitable for
linking to proteins dictates that the aglycon
exchange (from methyl to a linker/spacer, or
to a group that could be more readily ex-
changed for the spacer molecule than the
methyl group) be effected at the disaccharide
stage. We have demonstrated one such strat-
egy in our synthesis of the hexa- and the
dodecasaccharide of the V. cholerae O:1,
serotype Ogawa [19,20]. There, a glycosyl
chloride derived from a disaccharide was used
as the key glycosyl donor, and the principal
material was carried through the synthesis in
the form of the trimethylsilylethyl glycoside
until an oligosaccharide of the desired length
was obtained. Subsequently, the trimethylsi-
lylethyl aglycon was exchanged for the spacer
molecule that allowed linking to carrier
proteins using a carbodiimide-type conjuga-
tion. Here, larger haptens are prepared using
thioglycoside 1 [15,21] as the key glycosyl
donor, and they are linked to carriers by
reductive amination [22]. Due to their stabil-
ity during many chemical manipulations,
thioglycosides can be used as both glycosyl
donors and acceptors, which reduces the total
number of synthetic steps towards the target
products. Accordingly, the glycosyl donor 1
and the linker-acceptor 2 [23] were condensed
to give the fully protected, linker-equipped
disaccharide 3 together with a large propor-
tion of the b anomer 15 (see Schemes 1 and
2). Anomeric configurations for the two disac-
charides formed were assigned based on the
diagnostically significant differences in the JC-
1,H-1 coupling constants [24,25]. The lack of
stereoselectivity observed during this glycosyl-
ation was unexpected, since analogous cou-
pling [8] of the same glycosyl acceptor with
thioglycosides 16, which also carry a non-par-
ticipating group at O-2, gave the correspond-
ing a-glycoside stereospecifically in high yield.
Attempts to introduce the hexanamide linker
with higher a-stereoselectivity failed. When
compound 2 was glycosylated (these unsuc-
Fig. 1. Structure of the O-PS of Vibrio cholerae O:1, serotype
Ogawa.
group is acylated with 3-deoxy- -glycero-
L
tetronic acid. The upstream terminal perosa-
mine moiety of the O-PS of the Ogawa strain
is methylated at O-2 (Fig. 1). We have previ-
ously reported [8,9] on syntheses of neoglyco-
conjugates from the terminal monosaccharide
determinant [10] of the O-PS of V. cholerae
O:1, serotype Ogawa. Here, we describe syn-
theses of neoglycoconjugates derived from the
terminal hexasaccharide. In addition to conju-
gating hapten 14 to a carrier protein accord-
ing to the single-point attachment model [3],
we have also prepared a glycoconjugate con-
taining the same hapten in the form of multi-
valent clusters. The latter was prepared
following the concept we have recently intro-
duced [11] and applied to making a conjugate
from the corresponding monosaccharide. En-
hanced antigenicity resulting from multivalent
ligands, compared with their counterparts re-
sulting from single-point attachment of hap-
tens, has been well documented [12]. Our
future evaluation of immunogenicity of the
two types of conjugates, which differ from
each other only in this one fundamental ar-
chitectonic element, will provide information
concerning the merit of this concept.
2. Results and discussion
Synthesis of perosamine-containing oligo-
saccharides was originally developed by
Bundle and co-workers [13–16] in connection
with the preparation of methyl glycosides of
antigenic determinants of the Brucella A anti-
gen. Syntheses of similar but linker-equipped
haptens, glycosides whose aglycons allow
linking to proteins, is a more formidable task.

J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
159
Scheme 1.
cessful experiments are not described in Sec-
tion 3) with the monosaccharide thioglycoside
ethyl 2 - O - acetyl - 3 - O - benzyl - 4 - azido - 4,6-
treatment of a solution of 8 in Me2SO with
MeI in the presence of KOH [27], gave the
expected product 9 together with the N-
methyl derivative 17 (Schemes 1 and 2). The
latter structure of the byproduct was readily
dideoxy-a,b- -mannopyranoside [14], the cor-
D
responding orthoester was the sole product.
Acid-catalyzed isomerization of the latter to
the desired glycoside failed because of the
presence of the acid-labile dimethyl acetal
function in the aglycon.
1
indicated by the comparison of its H NMR
spectrum with that of 9. In the spectrum of
17, the typical broad, low-field triplet for NH
was no longer present, and the spectrum
showed the presence of an additional singlet
for the N-methyl group. As expected, com-
pound 17 was obtained as a mixture of two
isomeric forms (NMR). A clean conversion
8“9 was eventually achieved by methylation
with neat MeI and Ag2O.
To extend the oligosaccharide chain, disac-
charide 3 was deacetylated, and product 4 was
treated with the thioglycoside glycosyl donor 1
(“5) (Scheme 1). This sequence of reactions
was repeated (5“6“7), and the hexasaccha-
ride so obtained was deacetylated to give 8.
The latter compound is an important interme-
diate for the synthesis of hexasaccharide frag-
ments of V. cholerae O:1 in both the Ogawa
and Inaba series, as well as for syntheses of
higher members, since it allows chemical ma-
nipulation (methylation or chain extension) at
position O-2 in the upstream terminal moiety.
For making the title hexasaccharide, introduc-
tion of the methyl group at that position was
explored. Although this appears to be a simple
conversion, the following observations are
worth mentioning. No reaction was observed
when a solution of 8 in CH2Cl2 was treated
with MeI and Ag2O for 24 h. Addition of
(CH3)2S, known to render [26] MeI and Ag2O
a more powerful reagent for methylation, or
To introduce the 3-deoxy- -glycero-
L
tetronamido side-chain, the azido group in 9
was reduced with H2S [28], and the amine 10
thus obtained was treated with acid 18 [29]
(Scheme 2) in the presence of O-(7-azabenzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium hex-
afluorophosphate (HATU) [30,31] to give the
fully protected hexasaccharide 11. Simulta-
neous removal of the benzyl and benzylidene
protecting groups by catalytic hydrogenolysis
(“12), followed by hydrolysis with dilute
trifluoroacetic acid, then gave the aldehyde 14.
The latter reaction gave rise to a single
1
product as shown by H NMR spectroscopy
(see Section 3).

160
J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
For conjugation according to the single-
[11] (Scheme 3). It involved coupling, by re-
ductive amination, of a limited amount of
hapten 14 to the subcarrier molecule 19 con-
taining 16 amino groups. The hapten-carrying
subcarrier (HCS) thus obtained was then
treated (in phosphate buffer, pH 7) with ex-
cess of squaric acid diethyl ester to derivatize
[33–35] the amino groups that remained un-
changed upon the preceding treatment with
14. The derivatized HCS (DHCS) thus formed
was then treated (in borate buffer, pH 9) with
CSA to give a cross-linked, glycocluster conju-
gate 20. Owing to the putative polymolecular
nature and high molecular weight of the
product obtained, and in view of its intended
use as an immunogen, the most meaningful
characteristic of this material is the content of
the antigen 14. This was determined by the
phenol–sulfuric acid colorimetric assay. It
showed that the material contained 51% of
carbohydrate (expressed as the methyl gly-
coside analog of 14 [21], which was used as
standard material).
point attachment model [3], aldehyde 14 was
treated with chicken serum albumin (CSA) in
the presence of borane–pyridine complex, to
reduce the intermediate Schiff base [32]. The
reaction conditions applied were such that,
based on the study [8] involving the analogous
monosaccharide determinant of V. cholerae
O:1, serotype Ogawa, incorporation of ten
hapten residues was expected. The molecular
mass value of 78,650 Da found for the neogly-
coconjugate obtained (MALDI-TOF mass
spectrometry) corresponds, on the average, to
incorporation of seven hapten residues/CSA
(based on 66,973 Da found for CSA). This
suggests, together with our results [8] with the
analogous monosaccharide equipped with
same linker, that efficiency of conjugation de-
creases with the size of haptens.
Preparation of the neoglycoconjugate con-
taining the same hapten but in the form of
clusters 20 was carried out following our re-
cently described, conceptually new protocol
Scheme 2.

J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
161
Scheme 3.
ber of
D-perosamine residues in the molecule.
3. Experimental
When reporting assignments of NMR signals
in oligosaccharides, sugar residues are serially
numbered, beginning with the one bearing the
aglycone, and are identified by a Roman nu-
meral superscript in listings of signal assign-
General methods.—Unless stated otherwise,
optical rotations were measured at ambient
temperature for solutions in chloroform (c
ꢀ1), with a Perkin–Elmer automatic polar-
imeter, model 341. All reactions were moni-
tored by thin-layer chromatography (TLC) on
glass slides (Whatman or Analtech) coated
ments. Nuclei assignments without
a
superscript 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. Nuclei associated with
,
with silica gel (60 A pore size). Column chro-
matography was performed by gradient elu-
tion from columns of silica gel. Solvent
mixtures slightly less polar than those used for
TLC were used at the onset of development.
Assignments of NMR signals (300 and 75
the 3-deoxy- -glycero-tetronyl group are de-
L
noted with a prime and those associated with
the spacer-arm aglycon are denoted with a
double prime. When reporting NMR spectral
data for 17 (an isomeric mixture), only struc-
turally significant resonances reflecting the
presence of the N-methyl group in the
molecule are given. HATU was purchased
from PerSeptive Biosystems, Inc. Palladium-
on-charcoal catalyst (5%, ESCAT 103) was a
product of Engelhard Industries. The solution
of BH3 in pyridine (8 M) was purchased from
Aldrich Chemical Co. The subcarrier 19 was
custom synthesized by AnaSpec, Inc. (San
Jose, CA). All glycosylations were carried out
in the dark under argon. Unless specified oth-
erwise, solutions were concentrated at 40 °C/2
kPa.
1
13
MHz for H and C, respectively) were made
by first-order analysis of the spectra, and
when feasible, the assignments were confirmed
by homonuclear and heteronuclear 2D corre-
lation spectroscopy, run with the software
supplied with the spectrometers. In the case of
higher oligosaccharides the assignments were
aided by comparison with spectra of related
substances previously reported. When the lat-
ter approach was used, to assist in the 13C
NMR signal-nuclei assignments, advantage
was taken of variations of line intensity ex-
pected for oligosaccharides belonging to the
same homologous series [36–38]. Thus, spec-
tra showed close similarity of chemical shifts
of equivalent carbon atoms of the internal
residues, and an increase in the relative inten-
sity of these signals with the increasing num-
N-(2,2-Dimethoxyethyl)-6-[2-O-acetyl-4-
azido-3-O-benzyl-4,6-dideoxy-h- -mannopyr-
D
anosyl - (1“2) - (4 - azido - 3 - O - benzyl - 4,6-

162
J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
(t, 2 H, J 5.3 Hz, H-7%%a,b), 4.28 (s, 1 H, H-1I),
4.16–4.07 (m, 2 H, H-2I,5II), 3.3 (dd, 1 H, J2,3
3.3, J3,4 10.0 Hz, H-3II), 3.91–3.83 (m, 1 H,
H-1%%a), 3.47–3.34 (m, 12 H, H-3I,4I,II,1%%b,6%%-
a,b, 2 OCH3), 3.16–3.08 (m, 1 H, H-5I), 2.18
(t, 2 H, J 7.5 Hz, H-5%%), 2.03 (s, 3 H, COCH3),
1.70–1.55 (m, 4 H, H-2%%a,b,4%%a,b), 1.42–1.34
(m, 4 H, H-3%%, incl d at 1.38, J5,6 6.0 Hz,
H-6I), 1.27 (d, 3 H, J5,6 6.2 Hz, H-6II); 13C
NMR (CDCl3): 102.60 (C-7%%), 99.68 (C-1I,
JC,H 154.1 Hz), 98.20 (C-1II, JC,H 171.9 Hz),
80.74 (C-3I), 75.99 (C-3II), 71.72, 701.65 (2
CH2Ph), 71.16 (C-5I), 70.69 (C-2I), 69.62 (C-
1%%), 67.22 (C-2II), 66.71 (C-5II), 63.96 (2 C,
C-4I,II), 54.33, 54.29 (2 OCH3), 40.80 (C-6%%),
36.37 (C-5%%), 29.26 (C-2%%), 25.65 (C-3%%), 25.24
(C-4%%), 20.86 (COCH3), 18.41 (C-6I), 18.27
(C-6II); CIMS: m/z 784 [M+1]+, 801 [M+
18]+. Anal. Found: C, 58.33; H, 6.91; N,
12.40.
dideoxy-h- (3) and i- -mannopyranosyl)oxy]-
D
hexanamide (15).—Solid N-iodosuccinimide
(NIS, 1.24 g, 5.51 mmol), followed by a solu-
tion of silver trifluoromethanesulfonate
(AgOTf, 0.2 g, 0.78 mmol) in toluene (8 mL),
was added at 5–10 °C to a mixture of 1 (2.46
g, 3.92 mmol), 2 (1.03 g, 4.7 mmol) and
,
pulverized 4 A molecular sieves (1 g) in
CH2Cl2 (50 mL), which had been stirred at the
same temperature for 15 min. The stirring was
continued until TLC (2:1 hexane–acetone)
showed that all of 1 was consumed. Cooling
was terminated, the mixture was neutralized
with Et3N and filtered, and the filtrate
was washed successively with aq NaHCO3 and
water, dried, and concentrated. Chroma-
tography gave first the desired a isomer 3
1
(1.29 g, 42%), [h]D +64° (c 1.5). H NMR
(CDCl3): 5.67 (m, 1 H, NH), 5.40 (dd, 1 H,
J1,2 1.9, J2,3 3.0 Hz, H-2II), 4.85 (d, 1 H, H-1II),
4.70, 4.68, 4.63, 4.53 (4 d, partially over-
N-(2,2-Dimethoxyethyl)-6-[4-azido-3-O-
2
lapped, 4 H total, J 11.0 and 11.6 Hz, respec-
benzyl - 4,6 - dideoxy - h -
D
- mannopyranosyl -
tively, 2 CH2Ph), 4.64 (d, partially overlapped,
J1,2 1.8 Hz, H-1I), 4.37 (t, 1 H, J 5.1 Hz,
H-7%%), 3.84 (bdd, 1 H, H-2I), 3.79 (dd, 1 H,
J3,4 9.9 Hz, H-3II), 3.73 (dd, 1 H, J3,4 9.7 Hz,
H-3I), 3.63–3.53 (m, 2 H, H-5II,1%%a), 3.49–
3.28 (m, 13 H, H-4I,II,5I,1%b,6%%a,b, incl s at
3.39 for 2 OCH3), 2.18 (t, 2 H, J 7.3 Hz,
H-5%%a,b), 2.07 (s, 3 H, COCH3), 1.70–1.58 (m,
partially overlapped, H-4%%a,b), 1.62–1.51 (m,
partially overlapped, H-2%%a,b), 1.38–1.24 (m,
8 H, H-3%%a,b, incl 2 d at 1.30 and 2.28, J5,6
ꢀ6.1 Hz, H-6II and H-6I, respectively); 13C
NMR (CDCl3): 102.58 (C-6%%), 99.31 (C-1II,
JC,H 171.6 Hz), 98.44 (C-1I, JC,H 169.2 Hz),
77.67 (C-3I), 75.27 (C-3II), 73.93 (C-2I), 71.91,
71.47 (2 CH2Ph), 67.47 (C-5II), 67.43 (C-1%%),
67.14 (C-2II), 66.95 (C-5I), 64.09 (C-4I), 63.76
(C-4II), 54.30 (2 C, 2 OCH3), 40.76 (C-7%%),
36.37 (C-5%%), 29.04 (C-2%%), 25.72 (C-3%%), 25.25
(C-4%%), 20.86 (OCH3), 18.50 (C-6I), 18.42 (C-
6II); CIMS: m/z 784 [M+1]+, 801 [M+18]+.
Anal. Calcd for C38H53N7O11: C, 58.24; H,
6.77; N, 12.52. Found: C, 57.98; H, 6.77; N,
12.35.
(1“2)-(4-azido-3-O-benzyl-4,6-dideoxy-h-
D
-
mannopyranosyl)oxy]hexanamide (4).—Con-
ventional deacetylation (Zemple´n) of 3 gave 4
in virtually theoretical yield, [h]D +76° (c
1
1.5). H NMR (CDCl3): 5.75 (m, 1 H, NH),
4.93 (d, 1 H, J1,2 1.6 Hz, H-1II), 4.72–4.59 (m,
5 H, 2 CH2Ph, incl d, 4.66, J1,2 1.8 Hz, H-1I),
4.36 (t, 1 H, J 5.3, H-7%%), 3.98 (m, 1 H, H-2II),
3.88 (bt, 1 H, H-2I), 3.72 (dd, partially over-
lapped, J2,3 3.0, J3,4 9.9 Hz, H-3I), 3.70 (dd,
partially overlapped, J2,3 3.2, J3,4 9.7 Hz, H-
3II), 3.65–3.54 (m, 2 H, H-1%%a, H-5II), 3.44–
3.24 (m, 12 H, H-4I,4II,5I,1%%b,6%%a,b, incl s,
3.38 for 2 OCH3), 2.58 (d, 1 H, J2,OH 2.1 Hz,
OH), 2.17 (t, 2 H, J 7.5 Hz, H-5%%), 1.70–1.51
(2 m, partially overlapped, H-4%%,2%% in that
order), 1.38–1.24 (m, 8 H, H-3%%, incl 2 d, 1.29
and 1.28, partially overlapped, J5,6 ꢀ6.1 Hz,
H-6I,II); 13C NMR (CDCl3): 102.59 (C-7%%),
100.82 (C-1II), 98.56 (C-1I), 77.71 (C-3I), 77.43
(C-3II), 73.78 (C-2II), 71.95, 71.85 (2 CH2Ph),
67.39 (C-1%%), 67.14 (C-5II), 67.00 (C-2II), 66.89
(C-5II), 64.20 (C-4II), 63.68 (C-4II), 54.25 (2 C,
2 OCH3), 40.72 (C-6%%), 36.30 (C-5%%), 28.99
(C-2%%), 25.67 (C-3%%), 25.21 (C-4%%), 18.48 (C-
6I), 18.31 (C-6II), CIMS: m/z 742 [M+1]+,
759 [M+18]+. Anal. Calcd for C36H51N7O10:
C, 58.30; H, 6.88; N, 13.23. Found: C, 58.21;
H, 6.91; N, 13.10.
Eluted next was the b isomer 15 (1.23 g,
1
40%), [h]D +18°. H NMR (CDCl3): 5.68 (m,
1 H, NH), 5.50 (dd, 1 H, J1,2 1.8, J2,3 3.2 Hz,
H-2II), 5.04 (d, 1 H, H-1II), 4.73, 4.68, 4.55 (d,
2
1 H, J 10.5 Hz; s 2 H, d, 1 H, 2 CH2Ph), 4.36

J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
163
(m, 9 H, 4 CH2Ph, incl d at ꢀ4.6 for H-1I),
4.37 (t, 1 H, J 5.0 Hz, H-7%%), 4.0 (m, 1 H,
H-2IV), 3.93, 3.82, 3.78 (3 bt, 1 H each, H-
2I–III), 3.72–3.65 (m, 4 H, H-3I–IV), 3.62–3.14
(m, 18 H, H-4I–IV,5I–IV,1%%a,b,6%%a,b, incl s at
3.38 for 2 OCH3), 2.56 (d, 1 H, J 2.0 Hz, OH),
2.17 (t, 2 H, J 7.3 Hz, H-5%%), 1.69–1.59 (m,
partially overlapped, H-4%%a,b), 1.59–1.50 (m,
partially overlapped, H-2%%), 1.38–1.23 (m,
partially overlapped, H-3%%), 1.25–1.15 (m,
partially overlapped, H-6I–IV); 13C NMR
(CDCl3): 102.51 (C-7%%), 100.41 (C-1IV), 100.26,
100.09 (C-1II,III), 98.43 (C-1I), 77.51, 77.35,
76.81, 76.42 (C-3I–IV), 73.80 (C-2I), 73.40,
73.00 (C-2II,III), 72.05, 71.98, 71.95, 71.83 (4
CH2Ph), 67.37 (C-1%%), 67.65 (2 C), 67.23,
66.92 (2 C, C-5I–IV,2IV), 64.21, 64.02 (2 C),
63.68 (C-4I–IV), 54.28 (2 C, 2 OCH3), 40.71
(C-6%%), 36.31 (C-5%%), 29.00 (C-2%%), 25.65 (C-
3%%), 25.21 (C-4%%), 18.50 (2 C), 18.40, 18.19
(C-6I–IV); CIMS: m/z 1281 [M+18]+,
FABMS: m/z 1286 [M+Na]+. Anal. Calcd
for C62H81N13O16: C, 58.90; H, 6.46; N, 14.40.
Found: C, 58.66; H, 6.44; N, 14.26.
N-(2,2-Dimethoxyethyl)-6-[2-O-acetyl-4-
azido-3-O-benzyl-4,6-dideoxy-h- -mannopyr-
anosyl - (1“2) - bis(4 - azido - 3 - O - benzyl - 4,6 -
dideoxy - h - - mannopyranosyl) - (1“2) - (4 -
azido-3-O-benzyl-4,6-dideoxy-h- -mannopyr-
D
D
D
anosyl)oxy]hexanamide (5).—Compounds 1
and 4 (0.25–3.0 mmol) were treated with NIS
and AgOTf, as described for the preparation
of 3. Yields of 5 in several experiments varied
from 56 to 72%, [h]D +78°. 1H NMR
(CDCl3): 5.64 (m, 1 H, NH), 5.40 (dd, 1 H,
J1,2 1.8, J2,3 3.0 Hz, H-2IV), 4.93, 4.87 (2 d, 1 H
each, J1,2 1.6 Hz, H-1II,III), 4.84 (d, 1 H, J1,2
1.5 Hz, H-1IV), 4.73–4.51 (m, 9 H, 4 CH2Ph,
incl d, ꢀ4.60, H-1I), 4.37 (t, 1 H, J 5.2, H-7%%),
3.87, 3.83 (2 bt, 1 H each, H-2II,III), 3.79–3.75
(m, 2 H, H-2I,3IV), 3.71–3.65 (m, 3 H, H-
3I–III), 3.63–3.14 (m, 18 H, H-4I–IV
,
5I–IV,1%%a,b,6%%a,b, incl at 3.39, 2 OCH3), 2.18
(t, 2 H, J 7.7 Hz, H-5%%a,b), 2.01 (s, 3 H,
COCH3), 1.70–1.57 (m, partially overlapped,
2 H, H-4%%a,b), 1.59–1.50 (m, partially over-
lapped, 2 H, H-2%%a,b), 1.37–1.23 (m, partially
overlapped, H-3%%a,b, incl 3 d at 1.26, 1.20,
and 1.15, the one at 1.26 showing ꢀdouble
intensity compared to those at higher fields,
N-(2,2-Dimethoxyethyl)-6-[2-O-acetyl-4-
azido-3-O-benzyl-4,6-dideoxy-h-
anosyl-(1“2)-tetrakis(4-azido-3-O-benzyl-
4,6-dideoxy-h- -mannopyranosyl)-(1“2)-(4-
azido-3-O-benzyl-4,6-dideoxy-h- -mannopyr-
D
-mannopyr-
13
H-6I–IV); C NMR (CDCl3): 172.81, 169.74 (2
CO), 102.62 (C-7%%), 100.37, 100.03 (C-1II,III),
99.06 (C-1IV), 98.53 (C-1I), 77.43, 76.76, 76.58
(C-3I–III), 75.39 (C-3IV), 73.96 (C-2I), 73.46 (2
C, C-2II,III), 72.17, 72.08, 71.99, 71.50 (4
CH2Ph), 67.76 (2 C), 67.62, 67.10, 67.02 (C-
2IV,5I–IV), 67.48 (C-1%%), 64.34, 64.21, 64.01 (C-
4I–III), 63.80 (C-4IV), 54.36 (2 C, 2 OCH3),
40.81 (C-6%%), 36.42 (C-5%%), 29.08 (C-2%%), 25.74
(C-3%%), 25.29 (C-4%%), 20.93 (COCH3), 18.57 (2
C), 18.45, 18.34 (C-6I–IV), FABMS: m/z 1328
[M+Na]+; Anal. Calcd for C64H83N13O17: C,
58.84; H, 6.40; N, 13.94. Found: C, 58.67; H,
6.47; N, 13.78.
D
D
anosyl)oxy]hexanamide (7).—A mixture of 1
(1.7 g, 2.7 mmol) and 6 (2.0 g, 1.6 mmol) was
treated with NIS and AgOTf, as described for
the preparation of 3, to give 7 (1.47 g, 51%),
after isolation of the main product by chro-
1
matography, [h]D +76°. H NMR (CDCl3):
5.66 (bt, 1 H, NH), 5.41 (dd, 1 H, J1,2 1.8, J2,3
3.2 Hz, H-2VI), 4.95 (d, 1 H, J1,2 1.8 Hz,
H-1VI), 4.88, 4.86–4.84 (d, 1 H, J1,2 1.8 Hz, m,
3 H, H-1II–V), 4.74–4.51 (m, 13 H, 6 CH2Ph,
incl d at ꢀ4.60 for H-1I), 4.36 (t, 1 H, J 5.8
Hz, H-7%%), 3.88, 3.84, 3.81–3.76 (2 bt, 1 H
each, m, 4 H, H-2I–VI,), 3.74–3.62 (m, 6 H,
N-(2,2-Dimethoxyethyl)-6-[4-azido-3-O-
benzyl - 4,6 - dideoxy - h -
(1“2)-bis(4-azido-3-O-benzyl-4,6-dideoxy-h-
- mannopyranosyl) - (1“2) - (4 - azido - 3 - O -
benzyl-4,6-dideoxy-h- -mannopyranosyl)oxy]-
D
- mannopyranosyl -
H-3I–VI), 3.54–3.14 (22 H, H-4I–VI,5I–VI
,
D
1%%a,b,6%%a,b, incl s at ꢀ3.38 for 2 OCH3), 2.17
(t, 2 H, J 7.3 Hz, H-5%%a,b), 2.09 (S, 3 H,
COCH3), 1.69–1.61 (m, partially overlapped,
H-4%%a,b), 1.62–1.50 (m, partially overlapped,
H-2%%a,b), 1.38–1.12 (m, 20 H, H-6I–VI,3%%a,b);
13C NMR (CDCl3): 102.52 (C-7%%), 102.24 (C-
1VI), 100.01 (2 C), 99.93, 98.98 (C-1II–V), 98.45
D
hexanamide (6).—Conventional deacetylation
(Zemple´n) of 5 gave 6 in virtually theoretical
1
yield, [h]D +84°. H NMR (CDCl3): 5.71 (bt,
1 H, NH), 4.98 (d, 1 H, J1,2 1.3 Hz, H-1IV),
4.94, 4.88 (2 d, J1,2 1.3 Hz, H-1II,III), 4.74–4.55

164
J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
(C-1I), 77.36, 76.72, 76.57, 76.42 (2 C, C-3I–V),
75.32 (C-3VI), 73.86, 73.48, 73.32 (2 C), 73.23
(C-2I–V), 72.05 (3 C), 71.98, 71.92, 71.43 (6
CH2Ph), 67.39 (C-1%%), 67.70 (3 C), 67.62,
67.54, 67.01, 66.92 (C-2VI,5I–VI), 64.24, 64.15,
64.06 (2 C), 63.92, 63.69 (C-4I–VI), 54.29 (2 C,
2 OCH3), 40.72 (C-6%%), 36.33 (C-5%%), 29.01
(C-2%%), 25.67 (C-3%%), 25.22 (C-4%%), 20.88
(COCH3), 18.51 (2 C), 18.40 (3 C), 18.28
(C-6I–VI); FABMS: m/z 1828 [M+1]+. Anal.
Calcd for C90H113N19O23: C, 59.10; H, 6.23; N,
14.55. Found: C, 59.15; H, 6.28; N, 14.5.
N-(2,2-Dimethoxyethyl)-6-[4-azido-3-O-
benzyl - 4,6 - dideoxy - h -
(1“2)-tetrakis(4-azido-3-O-benzyl-4,6-di-
deoxy-h- -mannopyranosyl)-(1“2)-(4-azido-
3-O-benzyl-4,6-dideoxy-h- -mannopyranosyl)-
D
- mannopyranosyl -
D
D
oxy]hexanamide (17).—(a) Methylation of 8
with the MeI–Ag2O–Me2S reagent [26] or
with MeI and KOH in Me2SO [27] gave, in
addition to the desired 2-O-methyl derivative
9, variable amounts of the N-methyl deriva-
tive 17. Chromatography (2:1 hexane–ace-
tone) gave first 17 (ꢀ10%), identified by
1
NMR spectroscopy. H NMR (CDCl3): 4.49,
4.41 (2 t, 1 H total, J 4.5 Hz, H-7%%), 3.06, 2.98
(2 s, 3 H total, NCH3), 2.38, 2.32 (2 t, 2 H
total, J 7.5 Hz, H-5%%); 13C NMR (CDCl3):
103.47, 103.12 (1 C total, C-7%%), 55.19, 54.62
(2 C total, 2 OCH3-7%%), 52.31, 50.02 (1 C total,
C-6%%), 37.11, 34.55 (1 C total, NCH3), 32.24,
32.74 (1 C total, C-5%%); FABMS: m/z 1946
[M+Cs]+.
benzyl - 4,6 - dideoxy - h -
(1“2)-tetrakis(4-azido-3-O-benzyl-4,6-di-
deoxy-h- -mannopyranosyl)-(1“2)-(4-azido-
3-O-benzyl-4,6-dideoxy-h- -mannopyranosyl)-
D
- mannopyranosyl -
D
D
oxy]hexanamide (8).—Conventional deacety-
lation (Zemple´n) of 7 gave 8 in virtually theo-
1
retical yield, [h]D 86°. H NMR (CDCl3): 5.62
(bt, 1 H, NH), 4.98 (d, 1 H, J1,2 1.4 Hz,
H-1VI), 4.96, 4.89, 4.84 (2 bd, 1 H each, bd, 2
H, H-1II–V), 4.74–4.58 (m, 13 H, 6 CH2Ph,
H-1I), 4.36 (t, 1 H, J 5.2 Hz, H-7%%), 4.00 (m, 1
H, H-2VI), 3.93, 3.84–3.76 (bt, 1 H, m, 4 H,
H-2I–V), 3.73–3.14 (m, 22 H, H-4I–VI,5I–
VI,1%%a,b,6%%a,b, incl s, at 3.86, 2 OCH3), 2.37
(d, 1 H, J 1.9 Hz, OH), 2.17 (t, 2 H, J 7.4 Hz,
H-5%%a,b), 1.69–1.59 (m, 2 H, H-4%%a,b), 1.59–
1.49 (m, 2 H, H-2%%a,b), 1.38–1.12 (2 m, par-
tially overlapped, H-3%%a,b, H-6I–VI); 13C NMR
(CDCl3): 102.62 (C-7%%), 100.46, 100.34,
100.19, 100.09 (2 C, C-1I–VI). 98.53 (C-1I),
77.61, 77.42, 76.92, 76.58, 76.47 (2 C, C-3I–VI),
73.39 (C-2I), 73.54, 73.37 (2 C), 73.14 (C-
2II–V), 72.08 (5 C), 71.98 (6 CH2Ph), 67.44
(C-1%%), 67.75 (3 C), 67.67, 67.29, 67.05, 66.96
(C-2VI,5I–VI), 64.29, 64.11 (4 C), 63.74 (C-
4I–VI), 54.32 (2 C, 2 OCH3), 40.72 (C-6%%),
36.35 (C-5%%), 29.02 (C-2%%), 25.66 (C-3%%), 25.22
(C-4%%), 18.49 (2 C), 18.39 (3 C), 18.19
(C-6I–VI); FABMS: m/z 1786 [M+1]+. Anal.
Calcd for C88H111N19O22: C, 59.15; H, 6.26; N,
14.89. Found: C, 59.22; H, 6.32; N, 14.88.
N-(2,2-Dimethoxyethyl)-6-[2-O-methyl-4-
Eluted next was the desired monomethyl
1
derivative 9 (ꢀ85%), [h]D +94° (c 0.4). H
NMR (CDCl3): 5.66 (bt, 1 H, NH), 4.93–5.84
(m, 5 H, H-1II–VI), 4.76–4.58 (m, 13 H, 6
CH2Ph, H-1I), 4.37 (t, 1 H, J 5.1 Hz, H-7%%),
3.96, 3.85, 3.82, 3.80–3.76 (3 bt, 1 H each, m
2 H, H-2I–V), 3.72–3.63 (m, 6 H, H-3I–VI),
3.60–3.16 (m, 26 H, H-2VI,4I–VI,5I–VI, H-1%%,6%%,
incl s at 3.37 for 2 OCH3-7%% and s at 3.20 for
OCH3-6), 2.18 (t, 2 H, J 7.4 Hz, H-5%%),
ꢀ1.70–1.59 (m, partially overlapped, H-4%%),
ꢀ1.60–1.5 (m, partially overlapped, H-2%%),
ꢀ1.38–1.26 (m, partially overlapped, H-3%%),
1.26–1.12 (m, 18 H, H-6I–VI); 13C NMR
(CDCl3): 102.56 (C-7%%), 100.30, 100.16, 100.05
(2 C, C-1II–V), 98.67 (C-1VI), 98.48 (C-1I),
77.42, 77.36, 77.04, 77.00, 76.58, 76.44 (C-
3I–VI), 76.25 (C-2VI), 73.89, 73.48, 73.32, 73.10,
73.02 (C-2I–V), 72.28, 72.06 (3 C), 71.97 (2 C,
6 CH2Ph), 67.71 (3 C), 67.64 (2 C), 66.91
(C-5I–VI), 67.38 (C-1%%), 64.21 (2 C), 64.06 (3
C), 63.98 (C-4I–VI), 58.80 (OCH3-2), 54.27 (2
C, 2 OCH3-7%%), 40.68 (C-6%%), 36.29 (C-5%%),
28.96 (C-2%%), 25.61 (C-3%%), 25.17 (C-4%%), 18.45
(2 C), 18.34 (3 C), 18.25 (C-6I–VI); FABMS:
m/z 1932 [M+Cs]+. Anal. Calcd for
C89H113N19O22: C, 59.36; H, 6.32; N, 14.78.
Found: C, 59.26; H, 6.31; N, 14.61.
azido-3-O-benzyl-4,6-dideoxy-h-
nosyl - (1“2) - tetrakis(4 - azido - 3 - O - benzyl -
4,6-dideoxy-h- -mannopyranosyl)-(1“2)-(4-
azido-3-O-benzyl-4,6-dideoxy-h- -mannopyr-
D
-mannopyra-
D
D
(b) When compound 8 was treated with
Ag2O in MeI as a solvent and reagent
overnight, the byproduct 17 was not formed,
anosyl)oxy]hexanamide (9) and N-methyl-(2,2-
dimethoxyethyl)-6-[2-O-methyl-4-azido-3-O-

J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
165
After concentration, chromatography gave N-
(2,2-dimethoxyethyl)-6-{3-O-benzyl-4-(2,4-O-
and the methyl derivative 9 was obtained in
virtually theoretical yield.
benzylidene-3-deoxy-
4,6-dideoxy-2-O-methyl-a-
(1“2)-tetrakis[3-O-benzyl-4-(2,4-O-benzyl-
idene-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy - a - - mannopyranosyl - (1“2)] - [3 -
O-benzyl-4-(2,4-O-benzylidene-3-deoxy- -gly-
cero-tetronamido)-4,6-dideoxy-a- -manno-
L
-glycero-tetronamido)-
N-(2,2-Dimethoxyethyl)-6-[4-(3-deoxy- -
L
D
-mannopyranosyl-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-
h- -mannopyranosyl-(1“2)-tetrakis[4-(3-de-
oxy- -glycero-tetronamido)-4,6-dideoxy-h-
mannopyranosyl - (1“2)] - 4 - (3 - deoxy - - gly-
cero-tetronamido)-4,6-dideoxy-h- -mannopyr-
D
L
L
D
-
D
L
L
D
D
anosyl)oxy]hexanamide (12).—Hydrogen sul-
fide gas was passed for 30 min through a
solution of 9 (370 mg) in 2:1 pyridine–water
(6 mL) which was then kept at 40 °C
overnight. One product was formed, as shown
by TLC (10:1 CH2Cl2–MeOH). After concen-
tration, the residue was chromatographed to
give N-(2,2-dimethoxyethyl)-6-[4-amino-3-O-
pyranosyloxy}hexanamide (11). 1H NMR
(CDCl3): 6.42–6.08 (m, 6 H, NHI–VI), 5.7 (t, J
5.2, NH-7%%), 5.55, 5.54, 5.53, 5.52, 5.51, 5.50
(6 s, 6 CHPh), 4.99, 4.95, 4.29, 4.87 (bd, 1 H,
bs, 2 H, 2 bd 1 H each, H-1II–VI), 4.70–4.27
(m, 20 H, H-1I,2%I–VI,7%% 6 CH2Ph), 4.19–3.92
(m, 17 H, H-2I–V,4I–VI,4%aI–VI), 3.84–3.50 (m,
21 H, H-2VI,3I–VI,5I–VI,4%bI–VI,1%%a,b), 3.36,
3.35 (2 s, 6 H, 2 OCH3-7%%), 3.83, 3.30 (m, 2 H,
H-6%%a,b), 3.24 (s, 3 H, OCH3-2VI), 2.12 (t, 2
H, J 7.2 Hz, H-5%%), 2.09–1.80 (m, 12 H,
H-3%I–VI), 1.66–1.48 (2 m, 4 H, partially over-
lapped, H-4%%,2%% in that order), 1.35–1.25 (m,
benzyl-4,6-dideoxy-2-O-methyl-a-
ranosyl-(1“2)-tetrakis(4-amino-3-O-benzyl-
4,6-dideoxy-a- -mannopyranosyl)-(1“2)-(4-
-manno-
D-mannopy-
D
amino-3-O-benzyl-4,6-dideoxy-a-
D
pyranosyl)oxy]hexanamide (10, 270 mg, 81%).
1
Structurally diagnostic resonances in the H
13
2 H, H-3%%), 1.14–1.02 (m, 18 H, H-6I–VI); C
NMR (CDCl3) spectra: 5.74 (m, 1 H, NH%%),
5.09, 5.07, 5.00 (bd, m, bd, 5 H, H-1II–VI),
4.75–4.36 (m, 14 H, 6 CH2Ph, H-1I, incl t at
4.37, J 6.0 Hz, H-7%%), 4.12–3.90 (m, 6 H,
H-2I–VI), 3.39 (s, partially overlapped, 2
OCH3-7%%), 3.28 (s, OCH3-2), 2.19 (t, 1 H, J 7.2
Hz, H-5%%), 1.72–1.54 (2 m, partially over-
lapped, 4 H, H-4%%,2%% in that order), 1.40–1.14
(m, H-6I–VI,3%%); 13C NMR (CDCl3): 102.71
(C-7%%), 101.02 (4 C, C-1II–V), 99.18 (2 C, C-
1I,VI), 79.64, 79.16 (2 C), 78.76, 78.67 (2 C,
C-3I–VI), 75.86 (C-2VI), 73.04 (3 C), 72.87,
72.62 (C-2I–V), 71.65, 71.29, 71.13 (4 C, 6
CH2Ph), 70.31 (5 C), 69.62 (C-5I–VI), 67.11
(C-1%%), 58.89 (OCH3-2VI), 54.39 (2 C, 2 OCH3-
7%%), 53.79, 53.62 (3 C), 53.57 (2 C, C-4I–VI),
40.76 (C-6%%), 36.49 (C-5%%), 29.08 (C-2%%), 25.80
(C-3%%), 25.32 (C-4%%), 18.18 (4 C), 18.10, 17.99
(C-6I–VI); FABMS: m/z 1644 [M+1]+, 1776
[M+Cs]+.
Diisopropylethylamine (0.3 mL, 1.6 mmol)
followed by HATU (0.26 g, 1.6 mmol) was
added to a solution of the foregoing amine 10
(270 mg, 0.16 mmol) and the acid 18 (342 mg,
1.6 mmol) in CH2Cl2 (30 mL). The mixture
was stirred at room temperature (rt) for 30
min, when TLC (3:2 toluene–acetone) showed
that the starting amine was consumed and
that one faster-moving product was formed.
NMR (CDCl3): 102.56 (C-7%%), 101.30 (2 C),
101.21, 101.13 (2 C), 101.01 (6 CH2Ph),
100.40 (3 C), 100.27 (C-1II–V), 99.07 (C-1I),
98.73 (C-1VI), 76.58 (6 C, C-2%I–VI), 76.00,
75.75, 75.5975.04, 74.83, 74.60, 74.48, 74.40
(C-2I,VI,3I–VI), 72.73 (3 C), 72.58 (C-2II–V),
71.99, 71.33, 71.27, 70.97 (3 C, 6 CH2Ph),
68.66 (4 C), 68.57 (2 C, C-5I–VI), 67.25 (C-
4%I–VI,1%%), 59.02 (OCH3-2VI), 54.36 (2 C, 2
OCH3-7%%), 52.43, 51.95, 51.78 (2 C), 51.70,
51.42 (C-4I–VI), 40.76 (C-6%%), 36.44 (C-5%%),
28.82 (C-2%%), 28.55 (6 C, C-3%I–VI), 25.64 (C-
3%%), 25.19 (C-4%%), 17.90, 17.96 (5 C, C-6I–VI);
FABMS: m/z 2916 [M+Cs]+, 2784 [M+1]+.
A mixture of the foregoing fully protected
compound 11 and 5% Pd–C catalyst in
MeOH (50 mL, Caution: Extreme fire hazard!)
was stirred for 48 h in a hydrogen atmosphere
at ambient pressure and temperature, when
TLC (2:1:0.4 EtOAc–MeOH–water) showed
that the reaction was essentially complete. Af-
ter filtration and concentration of the filtrate,
elution from a small column of silica gel gave
pure, hygroscopic dimethyl acetal, N-(2,2-
dimethoxyethyl) - 6 - {4 - (3 - deoxy -
tetronamido)-4,6-dideoxy-2-O-methyl-a-
mannopyranosyl - (1“2) - tetrakis[4 - (3 -
deoxy- -glycero-tetronamido)-4,6-dideoxy-a-
-mannopyranosyl-(1“2)]-4-[(3-deoxy-
L
- glycero -
D
-
L
D
L
-

166
J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
glycero-tetronamido)-4,6-dideoxy-a-
D
-man-
(2 C), 74.63 (C-2I–V), 71.12, 70.90 (2 C), 70.81
(4 C), 70.73, 70.35, 70.01, 69.75, 69.70, 69.37
nopyranosyl]oxy}hexanamide (12, 178 mg,
(2 C), 69.13, 68.92 (2 C), 68.61 (C-3I–VI
,
1
63% from 10). H NMR (D2O): 5.20, 5.18,
5I–VI,2%I–VI), 6948 (C-1%%), 59.96, 59.89 (5 C,
C-4%I–VI), 59.60 (OCH3-2VI), 54.32, 54.10 (2-
OCH3-7%%), 51.58 (4 C), 51.37, 51.05 (C-
4I–VI), 40.89 (C-6%%), 36.42 (C-5%%), 30.50 (C-
3%I–VI), 28.21 (C-2%%), 25.01 (C-3%%), 24.06 (C-
4%%), 17.83 (2 C), 17.77 (2 C), 17.71, 17.62
(C-6I–VI); FABMS: m/z 2604 [M+Cs]+. Anal.
Calcd for C107H161N7O58: C, 51.96; H, 6.56; N,
3.96. Found: C, 51.73; H, 6.59; N, 3.95.
5.15, 5.13 (bd, 1 H, bd 1 H, bs, 2 H, bs, 1 H,
H-1II–VI), 4.87 (bs, 1 H, H-1I), 4.51 (t, 1 H, J
5.4 Hz, H-7%%), 4.30–4.26 (m, 6 H, H-2%I–VI),
4.18–4.02 (m, 10 H, H-2II–V,3I–VI), 3.98–3.82
(m, 13 H, H-2I,4I–VI,5I–VI), 3.77–3.67 (m, 14
H, H-2VI,4%I–VI,1%%a), 3.56–3.50 (m, H-1%%b),
3.47 (s, 3 H, OCH3-2VI), 3.40 (s, 6 H, 2
OCH3-7%%), 3.32 (m, 2 H, H-6%%a,b), 2.25 (t, 2
H, J 7.2 Hz, H-5%%a,b), 2.08–1.78 (2 m, 12 H,
H-3%a,b), 1.65–1.56 (m, 4 H, H-2%%,4%%), 1.40–
1.30 (m, 2 H, H-3%%a,b), 1.22–1.12 (m, 18 H,
H-6I–VI); 13C NMR (D2O): 102.73 (C-7%%),
101.00, 100.92 (3 C, C-1II–V), 99.14 (C-1I),
98.59 (C-1VI), 79.02 (C-2VI), 77.84, 77.64,
77.31 (2 C), 77.26 (C-2I–V), 69.07 (C-2%I–VI),
68.38 (3 C), 68.03 (2 C), 67.84, 67.72, 67.62 (3
C), 67.55 (2 C), 67.47 (C-3I–VI,5I–VI,1%%), 58.85
(OCH3-2VI), 57.94 (C-4%I–VI), 54.62 (2 C, 2
OCH3-7%%), 53.26, 53.20, 53.04 (4 C, C-4I–VI),
40.81 (C-6%%), 36.09 (C-3%I–VI), 35.64 (C-5%%),
28.24 (C-2%%), 25.14 (C-3%%), 24.90 (C-4%%), 16.90
(6 C, C-6I–VI); FABMS: m/z 1848 [M+Cs]+.
The amorphous, hygroscopic compound 12
was characterized as the corresponding per-O-
acetyl derivative, N-(2,2-dimethoxyethyl)-6-
Single-point-attachment conjugation of the
terminal, hexasaccharide determinant of the O-
antigen of V. cholerae O:1, serotype Ogawa to
CSA.—A solution of 12 (1.8 mg, 1.05 mmol)
in 0.02 M TFA (1 mL) was kept at rt for 3
1
days. H NMR then showed that the reaction
was complete and that byproducts from cleav-
age of interglycosidic linkages were not
formed. Compared to the spectrum of 12, the
1H NMR spectrum of 14 (D2O) showed the
disappearance of the singlet at l 3.40
[CH(OCH3)2]. The triplet for H-7%% [CH(OD)2],
appearing in the spectrum of 12 [CH(OCH3)2]
at l 4.51, was shifted downfield to l 5.04. The
solution was freeze-dried, to give 14 as a white
solid. To a solution of the foregoing material
and CSA (3 mg, 0.045 mmol, corresponding to
a 1:2 ligand–NH2 ratio, based on 46 lysine
residues/CSA) in phosphate buffer (50 mL,
forming a solution concentration of the hap-
ten, 21 mM, which is well above the minimum
that is recommended [8]), was added borane–
pyridine complex (8 M, 1 mL, 8 mM). The
solution was kept at rt for 4 days. The mixture
was filtered through a 30 K Centricon device,
and the retained material was washed with
deionized water (3×2 mL). The retained ma-
terial was freeze-dried, to give a white, hygro-
scopic solid (3.2 mg). MALDI-TOF analysis
showed the molecular mass of the neoglyco-
conjugate thus obtained to be 78,650 Da, cor-
responding to an incorporation of approx-
imately seven residues of 14/CSA.
{3-O-acetyl-4-(2,4-di-O-acetyl-3-deoxy-
glycero-tetronamido)-4,6-dideoxy-2-O-methyl-
a - - mannopyranosyl - (1“2) - tetrakis[3 - O -
acetyl-4-(2,4-di-O-acetyl-3-deoxy- -glycero-
tetronamido) - 4,6 - dideoxy - a - - manno -
pyranosyl-(1“2)]-[3-O-acetyl-4-(2,4-di-O-
acetyl-3-deoxy- -glycero-tetronamido)-4,6-
dideoxy - a - - mannopyranosyl]oxy}hexan -
L
-
D
L
D
L
D
1
amide (13), [h]D +31° (c 0.7). H NMR
(CDCl3): 6.80–6.44 (m, 6 H, 6 NH%), 5.78 (t, 1
H, J 5.5, NH%%), 5.28–4.90 (m, 16 H, 4 H-1,
H-3I–VI,2%I–VI, incl d, 4.90, 1 H, J 2.1 Hz, H-1),
4.73 (bd, 1 H, H-1I), 4.42 (t, 1 H, J 5.2, H-7%%),
4.35–4.04 (m, 22 H, H-2II–V,4I–VI,4%I–VI), 3.93
(bt, 1 H, H-2I), 3.90–3.70 (m, 7 H, H-5I–VI
,
1%%a), 3.64 (bdd, 1 H, H-2VI), 3.54 (s, par-
tially overlapped, OCH3-2VI), 3.52–3.30 (m, 9
H, H-1%%b, 6%%a,b, incl s, 3.40, 2 OCH3-7%%),
2.20–2.05 (m, 7 H, H-5%%,3%aI–VI a, 6 COCH3),
1.86–1.67 (m, 6 H, 3%b), 1.65–1.15 (m, H-
Preparation of the glycocluster conjugate
20.—Subcarrier 19 (1.98 mg, 1 mmol) was
added to a solution of 14 [prepared from 12
(14 mg, 8.16 mmol)] in 0.1 M phosphate buffer
(pH 7.0, 400 mL), followed by boron–pyridine
complex (8.2 mL, 82 mmol). The mixture was
stirred at rt for 40 h, filtered, washed (thrice)
13
6I–VI,2%%,3%%,4%%); C NMR (CDCl3): 102.30 (C-
7%%), 100.24 (3 C), 99.90 (C-1II–V), 99.45 (C-1I),
98.45 (C-1VI), 77.77 (C-2VI), 75.38 (2 C), 75.18

J. Zhang, P. Ko6a´cˇ / Carbohydrate Research 321 (1999) 157–167
167
[8] J. Zhang, A. Yergey, J. Kowalak, P. Kova´cˇ, Tetrahedron,
54 (1998) 11783–11792.
[9] J. Zhang, A. Yergey, J. Kowalak, P. Kova´cˇ, Carbohydr.
Res., 313 (1998) 15–20.
using a 3 K Centricon centrifugal filtering
device, and the retained material was freeze-
dried, to give the HCS.
[10] J. Wang, J. Zhang, C.E. Miller, S. Villenuve, Y. Ogawa,
P.-s. Lei, P. Lafaye, F. Nato, A. Karpas, S. Bystrick,
S.C. Szu, J.B. Robbins, P. Kova´cˇ, J.-M. Fournier, C.P.J.
Glaudemans, J. Biol. Chem., 273 (1998) 2777–2783.
[11] J. Zhang, P. Kova´cˇ, Bioorg. Med. Chem. Lett., 9 (1999)
487–490.
[12] Y.C. Lee, R.T. Lee, Neoglycoconjugates: Preparation and
Application, Academic, New York, 1994, p. 549.
[13] D.R. Bundle, M. Gerken, T. Peters, Carbohydr. Res., 174
(1988) 239–251.
[14] T. Peters, D.R. Bundle, Can. J. Chem., 67 (1989) 497–
502.
[15] T. Peters, D.R. Bundle, Can. J. Chem., 67 (1989) 491–
496.
Diethyl squarate (14 mL, 82 mmol) was
added to a solution of the above HCS in 0.1
M phosphate buffer (0.8 mL, pH 7.0), and the
solution was stirred overnight at rt. The mix-
ture was filtered, washed (thrice) using a 3 K
Centricon centrifugal filtering device, and the
retained material was freeze-dried to give the
DHCS as a white solid (8 mg). On MALDI-
TOF analysis, the material produced a broad
peak centered at ꢀm/z 8600.
A solution of the foregoing DHCS (1 mg)
in borate buffer (0.1 mL, pH 9.0) was treated
with CSA (0.49 mg) for 2 days at rt. The
mixture was processed as described for the
preparation of DHCS, except that a 30 K
Centricon centrifugal filtering device was used
to remove the low-molecular-mass material.
The retained material was freeze-dried to give
the target product 20 as an amorphous solid
(1.3 mg). The material, when analyzed by
phenol–sulfuric acid assay, was shown to con-
tain 51% of carbohydrate (expressed as the
methyl glycoside analog of 14, a synthetic
sample of which [21] was used as a reference).
[16] J. Kihlberg, E. Eichler, D.R. Bundle, Carbohydr. Res.,
211 (1991) 59–75.
[17] M.J. Eis, B. Ganem, Carbohydr. Res., 176 (1988) 316–
323.
[18] M. Gotoh, P. Kova´cˇ, J. Carbohydr. Chem., 13 (1994)
1193–1213.
[19] Y. Ogawa, P.-s. Lei, P. Kova´cˇ, Carbohydr. Res., 293
(1996) 173–194.
[20] Y. Ogawa, P. Kova´cˇ, Glycoconjugate J., 14 (1997) 433–
438.
[21] J. Zhang, P. Kova´cˇ, Carbohydr. Res., 300 (1997) 329–
339.
[22] G.R. Gray, B.A. Schwartz, B.J. Kamicker, Prog. Clin.
Biol. Res., 23 (1978) 583–594.
[23] J. Zhang, P. Kova´cˇ, Tetrahedron Lett., 39 (1998) 1091–
1094.
[24] K. Bock, C. Pedersen, J. Chem. Soc., Perkin Trans. 2,
(1974) 293–297.
[25] R. Kasai, M. Okihara, M. Asakawa, K. Mizutani, O.
Tanaka, Tetrahedron, 35 (1979) 1427–1432.
[26] P. Kova´cˇ, in K. Blau, J.M. Halket (Eds.), Handbook of
Deri6ati6es for Chromatography, Wiley, Chichester, 1993,
pp. 109–129.
Acknowledgements
[27] P. Fu¨gedi, P. Na´na´si, J. Carbohydr. Nucleosides Nucle-
otides, 8 (1981) 547–555.
[28] T. Adachi, Y. Yamada, I. Inoue, M. Saneyoshi, Synthe-
sis, 62 (1977) 45–46.
The authors thank A. Yergey and N. Whit-
taker for the mass spectral analyses.
[29] P.-s. Lei, Y. Ogawa, P. Kova´cˇ, J. Carbohydr. Chem., 15
(1996) 485–500.
[30] L.A. Carpino, J. Am. Chem. Soc., 115 (1993) 4397–4398.
[31] L.A. Caprino, A. El-Faham, C.A. Minor, F. Albericio,
J. Chem. Soc., Chem. Commun., (1994) 201–203.
[32] R.T. Lee, T.-C. Wong, R. Lee, L. Yue, Y.C. Lee,
Biochemistry, 28 (1989) 1856–1861.
[33] K.H. Glusenkamp, W. Drosdziok, G. Eberle, E. Jaehde,
M.F. Rajewsky, Z. Naturforsch., C: Biosci., 46 (1991)
498–501.
[34] L.F. Tietze, C. Schroter, S. Gabius, U. Brinck, A. Goer-
lach-Graw, H.-J. Gabius, Bioconjugate Chem., 2 (1991)
148–153.
[35] L.F. Tietze, M. Arlt, M. Beller, K.-H. Glu¨senkamp, E.
Ja¨hde, M.F. Rajewsky, Chem. Ber., 124 (1991) 1215–
1221.
[36] J.C. Gast, R.H. Atalla, R.D. McKelvey, Carbohydr.
Res., 84 (1980) 137–146.
[37] P. Kova´cˇ, J. Hirsch, Carbohydr. Res., 100 (1982) 177–
193.
References
[1] S.C. Szu, R. Gupta, J.B. Robbins, in K.I. Wachsmuth,
P.A. Blake, O. Olsvik Jr. (Eds.), Vibrio cholerae and
Cholera: Molecular to Global Perspectives, American
Society for Microbiology, Washington, DC, 1994, pp.
381–394.
[2] K.E. Stein, Int. J. Tech. Assess. Health Care, 10 (1994)
167–176.
[3] W.E. Dick Jr., M. Beurret Jr., in J.M. Cruse, R.E. Lewis
Jr. (Eds.), Conjugate Vaccines, Vol. 10, Karger, Basel,
1989, pp. 48–114.
[4] R.Z. Dintzis, Pediatr. Res., 32 (1992) 370–385.
[5] L. Kenne, B. Lindberg, P. Unger, B. Gustafsson, T.
Holme, Carbohydr. Res., 100 (1982) 341–349.
[6] K. Hisatsune, S. Kondo, Y. Isshiki, T. Iguchi, Y.
Haishima, Biochem. Biophys. Res. Commun., 194 (1993)
584.
[7] T. Ito, T. Higuchi, M. Hirobe, K. Hiramatsu, T. Yokota,
Carbohydr. Res., 256 (1994) 113–128.
[38] P. Kova´cˇ, J. Hirsch, J. Alfo¨ldi, V. Miha´lov, Carbohydr.
Res., 88 (1981) 146–152.