A highly convergent synthesis of a complex oligosaccharide derived from group B type III Streptococcus

Article abstract of DOI:10.1021/jo001477w

An efficient synthesis of a heptasaccharide derived from group B type III Streptococcus carrying an artificial spacer (1) is described. Rapid assembly of a protected heptasaccharide (16a) is accomplished from readily available building blocks 2-5 without a single protecting group manipulation between glycosylation steps. The synthetic strategy may be applied to the assembly of other branched complex oligosaccharides. The deprotected heptasaccharide 1 was coupled to a poly[N-(acryloyloxy)succinimide, and the resulting material will be used for the development of an ELISA assay to detect antibodies against GBS, type III in pregnant women.

Full text of DOI:10.1021/jo001477w

VOLUME 66, NUMBER 8
APRIL 20, 2001
© Copyright 2001 by the American Chemical Society
Articles
A High ly Con ver gen t Syn th esis of a Com p lex Oligosa cch a r id e
Der ived fr om Gr ou p B Typ e III Str eptococcu s
Alexei V. Demchenko and Geert-J an Boons*
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road,
Athens, Georgia 30602
gjboons@ccrc.uga.edu
Received October 13, 2000
An efficient synthesis of a heptasaccharide derived from group B type III Streptococcus carrying
an artificial spacer (1) is described. Rapid assembly of a protected heptasaccharide (16a ) is
accomplished from readily available building blocks 2-5 without a single protecting group
manipulation between glycosylation steps. The synthetic strategy may be applied to the assembly
of other branched complex oligosaccharides. The deprotected heptasaccharide 1 was coupled to a
poly[N-(acryloyloxy)succinimide, and the resulting material will be used for the development of an
ELISA assay to detect antibodies against GBS, type III in pregnant women.
In tr od u ction
Baker and co-workers4 in which purified GBS capsular
polysaccharide was administered to women during the
third trimester of pregnancy. Among women with low or
undetectable preexisting levels of specific antibodies, 57%
developed a rise in antibody titer. The low response rate
and the absence of T-lymphocyte stimulation of vaccina-
tion with GBS capsular polysaccharides were addressed
by conjugation of the capsular polysaccharide to a carrier
protein. Successful preclinical trials with these conjugates
led to phase-1 and phase-2 trials that assessed safety,
immunogenicity, and dosing of GBS conjugated vaccines.5
The Gram-negative bacteria, group B type III Strep-
tococcus (GBS, type III), is the most prevalent cause of
neonatal sepsis.1 Mortality and morbidity rates from
these infections continue to be substantial, and a method
to prevent this illness is urgently needed. Active im-
munization of newborn babies is not practical, since most
cases occur within the first month of life. However,
newborns may be protected by vaccination of pregnant
mothers who are deficient in antibodies specific for the
native, type III GBS polysaccharides.2,3 Antibodies raised
by these women will pass the placenta and supply the
newborns with protective levels of antibodies. The fea-
sibility of this approach was supported by a study of
Polysaccharide-protein conjugates have been prepared
by several different coupling procedures. In general, these
methods suffer from low reproducibility, protein cross-
linking, or destruction of vital immunological domains.
(1) Wald, E. R.; Bergman, I.; Taylor, H. G.; Chiponis, D.; Porter, C.;
Kubek, K. Pediatrics 1986, 77, 217-221.
(2) Kasper, D. L.; Baker, C. J .; Baltimore, R. S.; Crabb, J . H.;
Schiffman, G.; J ennings, H. J . J . Exp. Med. 1979, 149, 327-339.
(3) Baker, C. J .; Rench, M. A.; Edwards, M. S.; Carpenter, R. J .;
Hays, B. M.; Kasper, D. L. New Eng. J . Med. 1988, 319, 1180-1185.
(4) Baker, C. J .; Kasper, D. L. New Engl. J . Med. 1976, 294, 753-
756.
(5) (a) Paoletti, L. C.; Kasper, D. L.; Michon, F.; Difabio, J .; Holme,
K.; J ennings, H. J .; Wessels, M. R. J . Biol. Chem. 1990, 265, 18278-
18283. (b) Lagergard, T.; Shiloach, J .; Robbins, J . B.;. Schneerson, R.
Infect. Immun. 1990, 58, 687-694.
10.1021/jo001477w CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/22/2001

2548 J . Org. Chem., Vol. 66, No. 8, 2001
Sch em e 1. Retr osyn th etic An a lysis of 1
Demchenko and Boons
On the other hand, synthetic oligosaccharides that are
equipped with an artificial spacer that has a unique
chemical functionality can be coupled to proteins in a
more controlled and reproducible manner.6
ethers as glycosyl acceptors. Trityl ethers are often used
as protecting groups, but they can also act as efficient
glycosyl acceptors. More importantly, secondary trityl
ethers are much more reactive in glycosylations than
primary ones.11 These features permitted first glycosy-
lation of the secondary trityl ether at C-4 of 4 with sialic
acid containing thioglycosyl donor 3 followed by lactosy-
lation of the primary trityl group with cyanoethylidene
glycosyl donor 2. In general, it is attractive to first
glycosylate a hydroxyl of low reactivity followed by
glycosylation of more reactive (primary) ones. A critical
aspect was the exploitation of the finding that thiogly-
cosides can be activated with methyl trifluoromethane-
sulfonate (methyl triflate, MeOTf) without affecting an
n-pentenyl functionality. This feature made it possible
that the n-pentenyl glycoside of 4 could act as a glycosyl
acceptor in the glycosylation with thioglycoside 3. An-
other key aspect was that the acetamido group at C-5 of
the sialic acid moiety of 3 was protected as an N-
acetylacetamido group. This protection prevented the
undesirable N-methylation of the acetamido group at C-5′
of the glycosyl donor 3 during the MeOTf-mediated
glycosylation. In addition, as it was found, this function-
ality facilitates the synthesis of 3. Finally, the cheaply
available disaccharide D-lactose was utilized for the
preparation of the building blocks 2 and 5, therefore
reducing the number of required glycosylations. These
strategic issues facilitated the assembly of 16a in a
convergent manner form the building blocks 2-5 without
a single protecting group manipulation between glyco-
sylation steps.
We report here a highly convergent synthesis of a
heptasaccharide (1, Scheme 1) derived from type III GBS
oligosaccharide. The compound 1, which represents the
smallest saccharide structure that can raise relevant
antibodies,7 is equipped with an artificial aminopropyl
spacer. This spacer allows selective coupling to carrier
proteins to give highly immunogenic conjugates. In this
study, the heptasaccharide was coupled to an activated
polymer for the preparation of a material that can be
used for the development of an ELISA assay. Such an
assay will make it possible to detect antibody levels in
pregnant women.
Resu lts a n d Discu ssion
Ideally, complex oligosaccharides are synthesized by
a highly convergent strategy whereby glycosyl donors and
acceptors are assembled into oligomeric structures in-
volving a minimal number of synthetic steps.8 The
strategic plan for a convergent synthesis for 1 that meets
this requirement is summarized in retrosynthetic Scheme
1. Thioalkyl and n-pentenyl glycosides 3 and 4 were
selected as key saccharide building blocks. The anomeric
constituents of these compounds are stable under many
protecting group manipulations, therefore offering ef-
ficient protection during the preparation of targeted
glycosyl donors and acceptors. In the presence of an
appropriate activating reagent, however, thioglycosides9
and n-pentenyl glycosides10 can be activated and act as
highly reactive glycosyl donors. The second important
strategic issue was the use of triphenylmethyl (trityl, Tr)
The preparation of building blocks 3-5 is shown in
Scheme 2. Compound 3 was synthesized by the following
sequence of manipulations. Coupling of 6b12 with 713 in
the presence of N-iodosuccinimide (NIS) and a catalytic
(6) Verheul, A. F. M.; Boons, G. J .; van der Marel, G. A.; van Boom,
J . H.; J ennings, H. J .; Snippe, H.; Verhoef, J .; Hoogerhout, P.; Poolman,
J . T. Infect. Immun. 1991, 59, 3566-3573.
(11) (a) Tsvetkov, Y. E.; Kitov, P. I.; Backinowsky, L. V.; Kochetkov,
N. K. Tetrahedron Lett. 1993, 34, 7977-7980. (b) Tsvetkov, Y. E.; Kitov,
P. I.; Backinowsky, L. V.; Kochetkov, N. K. J . Carbohydr. Chem. 1996,
15, 1027-1050. (c) Demchenko, A.; Boons, G. J . Tetrahedron Lett. 1997,
38, 1629-1632.
(7) Wessels, M. R.; Pozsgay, V.; Kasper, D. L.; J ennings, H. J . J .
Biol. Chem. 1987, 262, 8262-8267.
(8) Boons, G. J . Tetrahedron 1996, 52, 1095-1121.
(9) Fugedi, P.; Garegg, P. J .; Lohn, H.; Norberg, T. Glycoconjugate
J . 1987, 4, 97-108.
(10) Fraser-Reid, B.; Udodong, U. E.; Wu, Z. F.; Ottosson, H.;
Merritt, J . R.; Rao, C. S.; Roberts, C.; Madsen, R. Synlett 1992, 927-
942.
(12) (a) Demchenko, A. V.; Boons, G. J . Chem. Eur. J . 1999, 5, 1278-
1283. (b) Demchenko, A. V.; Boons, G. J . Tetrahedron Lett. 1998, 39,
3065-3068.
(13) J ansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J .; Magnusson, G.;
Dahmen J .; Noori, G., Stenvall, K. J . Org. Chem. 1988, 53, 5629-5647.

Convergent Synthesis of a Complex Oligosaccharide
J . Org. Chem., Vol. 66, No. 8, 2001 2549
with trityl perchlorate16 in the presence of γ-collidine.
After silica gel column chromatography, compound 4 was
isolated in a yield of 87%.
Sch em e 2. Syn th esis of th e Bu ild in g Block s 3-5a
Spacer-containing lactosyl acceptor 5 was synthesized
by starting from the known (3-chloropropyl) glycoside
1217 (Scheme 2) by treatment with sodium azide to give
(3-azidopropyl) glycoside 13, which was deacetylated
under Zemple´n conditions. The 2,3-cis diol of the result-
ing compound was selectively protected as an isopropyli-
dene acetal by treatment with acetone and TMSCl, and
the remaining hydroxyls were benzylated with benzyl
bromide and NaH in DMF. Finally, the isopropylidene
acetal of the fully protected derivative was removal by
treatment with TFA in DCM/water to give 5 in 67%
overall yield from 12.
With significant quantities of glycosyl acceptors and
donors in hand, the assembly of the protected heptasac-
charide 16a was undertaken. Thus, coupling of thiogly-
coside 3 with di-O-tritylated acceptor 4 in the presence
of MeOTf 18 proceeded with absolute regio- and stereo-
selectivity and trisaccharide 14 was obtained in an
excellent yield of 96% (Scheme 3). The coupling reaction
was performed in the presence of a relatively large
amount of activated molecular sieves (3 Å) to prevent
cleavage of the secondary trityl ether. The prevention of
hydrolysis is very important since the analogous saccha-
ride having a free 4-hydroxyl group could not be glyco-
sylated under the described reaction conditions. This
observation indicates that the C-4 hydroxyl is activated
by tritylation and the increase of reactivity probably
arises from steric repulsion between the two trityl ethers
resulting in lengthening and therefore polarization of the
C-O bond of the secondary trityl ether.11 Next, the less
reactive primary trityl ether of 14 was glycosylated with
the cyanoethylidene derivative 219 in the presence of a
catalytic amount of trityl perchlorate to furnish pen-
tasaccharide 15 in a good yield of 82%. The use of ethyl
per-O-acetyl thiolactoside instead of 2 was only modestly
successful when MeOTf was used as the promoter in the
absence of molecular sieves. Probably, this glycosylation
route requires cleavage of the trityl group prior to
glycosylation, which may be achieved with the strong
triflic acid formed in situ. The proposed reaction path is
supported by the fact that in the presence of molecular
sieves (acid scavenger) no glycosylation occurred and only
destruction of the thioglycosyl donor was observed.20
a
Reagents: (a) NIS/TfOH, DCM; (b) Ac2O/pyridine; (c) Ac2O,
BF3‚Et2O, DCM; (d) TMSSMe, TMSOTf, DCM; (e) TFA, DCM/H2O;
(f) TrClO4, γ-collidine, DCM; (g) NaN3, KI, DMF; (h) MeONa/
MeOH; (i) TMSCl, acetone; (j) BnBr, NaH, DMF.
amount of trifluoromethanesulfonic (triflic) acid in ace-
tonitrile at -35 °C gave disaccharide 8b in an exceptional
yield of 81%. On the other hand, glycosylation of 7 with
the popular methyl thio sialyl donor 6a 14 with 7 under
similar conditions gave a much lower yield (62%) of the
corresponding disaccharide 8a . In addition, the reaction
time for the preparation of 8b was significantly shorter
than that for 8a (5 min vs 2.5-3 h). These featureshigh-
light the fact that the N-acetylacetamido moiety of 6b
significantly improves its glycosyl donor properties. O-
Acetylation of 8a with acetic anhydride in pyridine (8c)
followed by acetolysis of the anomeric TMSEt group and
benzylidene acetal with acetic anhydride in the presence
of BF3-etherate gave anomeric acetate 9 mainly as a
â-anomer (R/â ) 1/15). O-Acetylation prior to the acetoly-
sis was found to be the most efficient way to direct the
latter reaction toward stereoselective formation of a
â-acetate. This is an important requirement since the
corresponding R-acetate showed poor reactivity in a
subsequent thiomethylation reaction. Thus, conversion
of 9 into a methyl thioglycosyl donor 3 was accomplished
by treatment with TMSSMe in the presence of a catalytic
amount of TMSOTf. Thioglycoside 3 was isolated only
as a â-anomer in 85% overall yield from 8b.
(14) (a) Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H. Carbo-
hydr. Res. 1991, 212, 277-281. (b) Marra, A.; Sinay, P. Carbohydr.
Res. 1989, 187, 35-42. (c) Komba, S.; Ishida, H.; Kiso, M.; Hasegawa,
A. Carbohydr. Res. 1996, 285, c1-c8. (d) Komba, S.; Ishida, H.; Kiso,
M.; Hasegawa, A. Bioorg. Med. Chem. 1996, 4, 1833-1847.
(15) Madsen, R.; Udodong, U. E.; Roberts, C.; Mootoo, D. R.;
Konradsson, P.; Fraser-Reid, B. J . Am. Chem. Soc. 1995, 117, 7, 1554-
1565.
(16) Dauben, H. P., J r.; Honnen, L. R.; Harmon, K. M. J . Org. Chem.
1960, 25, 1442-1445.
(17) Coles, H. W.; Dodds, M. L.; Bergeim, F. H. J . Am. Chem. Soc.
1938, 60, 1020.
(18) (a) Lo¨nn H., Carbohydr. Res. 1985, 139, 105-113. (b) Several
other promoters were examined including DMTST, IDCP, NIS/TfOH,
and NIS/TMSOTf, but these reagents proved uncompatible with the
n-pentenyl moiety of glycosyl acceptor 14.
(19) (a) Kochetkov, N. K.; Nifant′ev, N. E.; Backinowsky, L. V.
Tetrahedron 1987, 43, 3109-3122. (b) Betaneli, V. I.; Ovchinnikov,
M. V.; Backinowsky, L. V.; Kochetkov, N. K. Carbohydr. Res. 1979,
68, c11-c13.
Key compound 4 was prepared from the known n-
pentenyl glycoside 1015 by trifluoroacetic acid mediated
cleavage of the benzylidene acetal to give intermediate
11 in a 96% yield, which was di-O-tritylated by treatment
(20) Presumably, harsh reaction conditions required for the glyco-
sylation of primary trityl functionalities (NIS/ TMSOTf) are not
appropriate in this particular case, since it is expected that the
n-pentenyl moiety would be activated: Boons, G. J .; Bowers S.; Coe
D. M. Tetrahedron Lett. 1997, 38, 3773-3776.

2550 J . Org. Chem., Vol. 66, No. 8, 2001
Demchenko and Boons
Sch em e 3. Assem bly of a n d Deblock in g of a
Hep ta sa cch a r id e fr om GBSa
Sch em e 4. Syn th esis of P olya cr yla m id e-Ba sed
Con ju ga te w ith GBSa
a
Reagents: (a) Et3N, DMF; (b) aq NH4OH.
The structure of 1 was confirmed by COSY, TOCSY,
HSQC, HSQC-TOCSY, and ROESY NMR experiments.
Large coupling constants (J 1,2 ) 7.7-8.6 Hz) were
detected for the anomeric protons (H-1A-D, -1F, and -1G)
in 1, which proofed unambiguous the â-configuration of
these glycosidic linkages. The anomeric assignment of the
R-linked N-acetylneuraminic acid (unit E) was based on
empirical rules for the H-3Eeq signals (δ ) 2.73 ppm),
which is downshifted in comparison to that of a â-anomer
(δ ) 2.25-2.40 ppm).21
Compound 1 was coupled to a poly(N-acryloyloxy)-
succinimide (pNAS) polymer,22,23 which was prepared by
a procedure described by Whitesides and co-workers.
Thus, 17 was coupled with 0.17 equiv of 1 in DMF/Et3N
for 20 h (Scheme 4). The remaining N-hydroxysuccinim-
ide esters were converted into amides by treatment with
20% aqueous ammonia solution. The resulting glycopoly-
mer 18 was dialyzed exhaustively and then lyophilized
to give a pure glycopolymer.
a
Reagents: (a) MeTOf, MS 3 Å, DCM; (b) TrCO4, DCM; (c) NIS/
TMSOTf, MS 3 Å, CM; (d) LiI, pyridine; (e) (NH2CH2)2, n-BuOH;
(f) Ac2O/MeOH; (g) Pd/C, EtOH/H2O; (h) 1 N aq NaOH.
The n-pentenyl moiety of the pentasaccharide 15 has
remained intact throughout the above-discussed syn-
thetic steps leaving it available to serve as an efficient
anomeric leaving group.10 Thus, coupling of 15 with 5 in
the presence of NIS/TMSOTf gave the requisite hep-
tasaccharide 16a in a high yield of 62%.
Con clu sion s
In conclusion, we have described a highly convergent
synthesis of a heptasaccharide derived from the group B
type III Streptococcus polysaccharide. This saccharide,
which is equipped with an artificial aminopropyl spacer,
was coupled to an activated pNAS polymer. Currently,
the resulting material is used for the development of an
ELISA assay to detect antibodies against the capsular
polysaccharide of group B type III Streptococcus in
pregnant women. Important strategic aspects of the
synthesis of 1 include the use of a thioglycosyl donor and
n-pentenyl acceptor/donor. Furthermore, tritylation of the
C-4 and C-6 of a glycosamine derivative allows first
glycosylation of the secondary position (C-4) followed by
glycosylation of the primary one (C-6). Another important
aspect was the use of the neuraminic acid donor 6b that
has an N-acetylacetamido functionality at C-5. The
additional acetyl moiety increases the reactivity of the
glycosyl donor and prevents methylation of the acetamido
group during a MeOTf-promoted glycosylation. It is to
be expected that the strategic principles used for the
Compound 16a was deprotected by a five-step proce-
dure. The methyl ester of the sialic acid moiety of 16a
was saponified by treatment with LiI in pyridine to give
a lithium carboxylate 16b, which was purified by silica
gel column chromatography. Under these conditions, the
secondary N-acetyl functionality of the sialic acid moiety
of 16a was cleaved. Next, reaction of 16b with ethylene-
diamine in n-butanol removed the O-acetyl esters and
phthalimido protecting groups to give a free amine 16c,
which was immediately N-acetylated by the treatment
with acetic anhydride in methanol to give 16d . Next,
catalytic hydrogenation over Pd/charcoal removed the
benzyl ethers with concomitant reduction of the azido
moiety to an amine. Finally, the deprotected compound
was treated with 1 N aqueous NaOH and purified by size-
exclusion column chromatography on Sephadex G-25 to
afford 1 in 31% overall yield from 16a . A similar sequence
of deprotection reactions was performed with a heptasac-
charide that had the amino group of the spacer protected
as benzyloxycarbonyl group. However, this functionality
was cleaved when the phthalimido group was removed
under a variety of different reaction conditions.
(21) Dabrowski, U.; Friebolin, H.; Brossmer, R.; Supp, M. Tetrahe-
dron Lett. 1979, 20, 4637-4640.
(22) Bovin, N. V. Glycoconjugate J . 1998, 15, 431-446 and refer-
ences therein.
(23) Mammen, M.; Dahmann, G.; Whitesides, G. M. J . Med. Chem.
1995, 38, 4179-4190.

Convergent Synthesis of a Complex Oligosaccharide
J . Org. Chem., Vol. 66, No. 8, 2001 2551
r a n osid e (3). A solution of 8b (698 mg, 0.79 mmol) in a
mixture of Ac2O (4 mL) and pyridine (6 mL) was kept for 16 h
at room temperature. The reaction was quenched with MeOH
(15 mL) and the resulting solution concentrated in vacuo. The
residue was coevaporated from toluene (3 × 15 mL) and then
dried under high vacuum to give pure 2-(trimethylsilyl)ethyl
2-O-acetyl-4,6-O-benzylidene-3-O-[methyl 4,7,8,9-tetra-O-acetyl-
5-(N-acetylacetamido)-3,5-dideoxy-D-glycero-R-D-galacto-non-
2-ulopyranosylonate]-â-D-galactopyranoside (8c) as a white
foam: Rf 0.40 (acetone/toluene, 1/4, v/v). Compound 8c was
dissolved in a mixture of DCM (16 mL), and Ac2O (521 µL,
5.53 mmol) was added. The solution was placed under an
atmosphere of argon and cooled (0 °C), and BF3‚Et2O (250 mL,
1.98 mmol) was added dropwise. After being stirred for 3.5 h
at room temperature, the reaction mixture was diluted with
DCM (55 mL) and washed with water (20 mL), saturated
aqueous NaHCO3 (2 × 20 mL), and water (3 × 20 mL). The
organic phase was dried (MgSO4) and filtered and the filtrate
concentrated in vacuo. The residue was purified by silica gel
column chromatography (5% gradient acetone in toluene) to
afford 3-O-[methyl 4,7,8,9-tetra-O-acetyl-5-(N-acetylacetamido)-
3,5-dideoxy-D-glycero-R-D-galacto-non-2-ulopyranosylonate]-
1,2,4,6-tetra-O-acetyl-R/â-D-galactopyranose [9, R/â ) 1/15, Rf
0.25 (ethyl acetate/ toluene, 1/1, v/v)]. Ratio of the anomers
was established by comparison of integral intensities of the
corresponding signals 6.29 (d, 1H, J 1,2 ) 3.6 Hz, H-1R) and
synthesis of 1 will be used for the assembly of other
oligosaccharides.
Exp er im en ta l Section
Column chromatography was performed on silica gel 60 (EM
Science, 70-230 mesh), HPLC chromatography was performed
on Prodigy 5 µm silica 100 Å column (250 × 10 mm), and
reactions were monitored by TLC on Kieselgel 60 F254 (EM
Science). The compounds were detected by examination under
UV light and by charring with 10% sulfuric acid in methanol.
Solvents were removed under reduced pressure at <40 °C.
DCM, MeNO2, and MeCN were distilled from CaH2 (twice) and
stored over molecular sieves (3 Å). Anhydrous DMF and
benzene (EM Science) were used without further purification.
Methanol was dried by refluxing with magnesium methoxide,
distilled, and stored under argon. Pyridine was dried by
refluxing with CaH2 and then distilled and stored over
molecular sieves (3 Å). Molecular sieves (3 Å), used for
reactions, were crushed and activated in vacuo at 390 °C
during 8 h in the first instance and then for 2-3 h at 390 °C
directly prior to application. Melting points were measured
with an Electrothermal 9200 melting point apparatus, optical
rotations with a J ASCO P-1020 polarimeter. The assignments
of pairs of signals marked with * or # in 13C NMR spectra may
be interchanged.
1
5.84 (d, 1H, J 1,2 ) 8.4 Hz, H-1â) in the H NMR spectrum. A
2-(Tr im eth ylsilyl)eth yl 4,6-O-Ben zylid en e-3-O-[m eth yl
4,7,8,9-tetr a -O-a cetyl-5-(N-a cetyla ceta m id o)-3,5-d id eoxy-
D-glycer o-r-D-ga la cto-n on -2-u lop yr a n osylon a t e]-â-D-ga -
la ctop yr a n osid e (8b). A mixture of 2-(trimethylsilyl)ethyl
4,6-O-benzylidene-â-D-galactopyranoside13 (7, 89 mg, 0.24
mmol), methyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-acetylacet-
amido)-3,5-dideoxy-2-thio-D-glycero-R,â-D-galacto-non-2-ulopy-
ranosid]onate12 (6b, 157 mg, 0.28 mmol), and activated mo-
lecular sieves (3 Å, 600 mg) in MeCN (3.5 mL) was stirred for
3 h under an atmosphere of argon. The mixture was cooled to
-40 °C, and NIS (95 mg, 0.42 mmol) and TfOH (3.5 µL, 0.04
mmol) were added. TLC analysis after stirring for 10 min
indicated that the reaction had gone to completion. The
reaction mixture was diluted with DCM (20 mL), the solid was
filtered off, and the residue was washed with DCM (3 × 10
mL). The combined filtrates (∼50 mL) were washed with
aqueous Na2S2O3 (20%, 20 mL) and water (3 × 20 mL). The
organic phase was dried (MgSO4) and filtered and the filtrate
concentrated in vacuo. The residue was purified by silica gel
column chromatography (5% gradient acetone in toluene) to
afford 8b as a white foam (172 mg, 81%): Rf 0.5 (acetone/
toluene, 3/7, v/v); [R]26D ) +51.8 (c ) 1.00, DCM); mp +174.4-
175.1 °C (diethyl ether-hexane); MALDI-TOF m/z ) 906.5 [M
+ Na]+; 1H NMR δ 7.25-7.55 (m, 5H, aromatic), 5.49 (m, 1H,
J 4′,5′ ) 10.0 Hz, H-4′), 5.40 (s, 1H, >CHPh), 5.37 (m, 1H, J 8′,9′a
) 2.6 Hz, J 8′,9′b ) 5.5 Hz, H-8′), 5.17 (dd, 1H, J 7′,8′ ) 8.5 Hz,
H-7′), 4.96 (dd, 1H, J 6′,7′ ) 1.5 Hz, H-6′), 4.46 (d, 1H, J 1,2 ) 7.5
Hz, H-1), 4.33 (dd, 1H, J 9′a,9′b ) 12.5 Hz, H-9′a), 4.28 (dd, 1H,
J 6a,6b ) 12.5 Hz, H-6a), 4.26 (dd, 1H, J 3,4 ) 3.6 Hz, H-3), 4.17
(dd, 1H, J 5′,6′ ) 10.0 Hz, H-5′), 4.09 (dd, 1H, H-9′b), 4.08 (dd,
solution of 9 in DCM (12 mL) was placed under an atmosphere
of argon. TMSSMe (410 µL, 2.90 mmol) and TMSOTf (157 µL,
0.87 mmol) were added, and the reaction mixture was stirred
for 60 h at room temperature. It was then diluted with DCM
(50 mL) and washed with water (25 mL), saturated aqueous
NaHCO3 (2 × 20 mL), and water (3 × 25 mL). The organic
phase was dried (MgSO4) and filtered and the filtrate concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (5% gradient ethyl acetate in toluene) to
afford 3 as a white foam (17.0 g, 85% over three steps) as a
mixture of anomers (R/â ) 17/1) that were used without further
purification. Analytical data for â-3: Rf 0.40 (toluene/acetone,
3/1, v/v); [R]26 ) +6.2 (c ) 1.10, DCM); MALDI-TOF m/z )
D
1
874.4 [M + Na]+; H NMR δ 5.50-5.60 (m, 1H, J 4′,5′ ) 11.2
Hz, H-4′), 5.45-5.50 (m, 1H, J 8′,9′ ) 2.6 Hz, J 8′9′b ) 5.1 Hz,
H-8′), 5.17 (dd, 1H, J 7′,8′ ) 8.8 Hz, H-7′), 5.08 (dd, 1H, J 2,3
)
9.7 Hz, H-2), 5.03 (dd, 1H, J 4,5 ) 0.8 Hz, H-4), 4.68 (dd, 1H,
J 3,4 ) 3.3 Hz, H-3), 4.61 (dd, 1H, J 6′,7′ ) 2.3 Hz, H-6′), 4.55 (d,
1H, J 1,2 ) 10.0 Hz, H-1), 4.31 (dd, 1H, J 5′,6′ ) 10.3 Hz, H-5′),
4.30 (dd, 1H, J 9′a,9′b ) 12.5 Hz, H-9′a), 3.98-4.10 (m, 3H, H-6a,
6b, 9′b), 3.90-3.93 (m, 1H, H-5), 3.89 (s, 3H, -COOCH3), 2.65
(dd, 1H, J 3′e,4′ ) 5.0 Hz, J 3′e,3′a ) 12.0 Hz, H-3′e), 2.28, 2.35
[2s, 6H, -N(COCH3)2], 1.94, 2.02, 2.03, 2.03, 2.08, 2.17, 2.18,
2.22 (8 s, 24H, 7 × -OCOCH3, -SCH3), 1.59 (dd, 1H, J 3′a,4′
)
10.0 Hz, H-3′a); 13C NMR δ 166.00-175.12 (>CdO), 96.94
(C-2′), 83.14 (C-1), 74.57 (C-5), 72.59 (C-3), 69.78 (C-6′), 68.32
(C-4), 68.06 (C-2), 67.95 (C-8′), 67.36 (C-7′), 67.28 (C-4′), 62.55
(C-6), 62.47 (C-9′), 56.27 (H-5′), 53.38 (-COOCH3), 26.83, 27.90
[-N(COCH3)2], 20.05-21.18 (7 × -OCOCH3), 12.22 (-SCH3).
Anal. Calcd for C35H49NO21Si (851.25): C, 49.35; H, 5.80; N,
1.64. Found: C, 49.56; H, 5.92; N, 1.70.
1H, H-6b), 4.02-4.09 (m, 1H, -OCH2a-), 3.97 (dd, 1H, J 4,5
)
1.0 Hz, H-4), 3.83 (dd, 1H, J 2,3 ) 9.8 Hz, H-2), 3.72 (s, 3H,
-COOCH3), 3.62-3.68 (m, 1H, -OCH2b-), 3.45 (m, 1H, H-5),
2.88 (dd, 1H, J 3′e,4′ ) 5.1 Hz, J 3′e,3′a ) 13.0 Hz, H-3′e), 2.73 (br
s, 1H, 2-OH), 2.29, 2.37 [2s, 6H, -N(COCH3)2], 1.96, 2.03, 2.13,
2.17 (4s, 12H, 4 × -OCOCH3), 1.95 (dd, 1H, J 3′e,4′ ) 10.6 Hz,
H-3′a), 0.96-1.15 (m, 2H, -CH2TMS), 0.00 (s, 9H, TMS); 13C
NMR δ 168.18-174.41 (7 × >CdO), 126.03-128.87 (aromatic),
102.30 (C-1), 100.96 (>CHPh), 97.25 (C-2′), 75.39 (C-3), 74.13
(C-4), 70.03 (C-6′), 69.21 (C-6), 68.66 (C-2), 68.42 (C-8′), 66.88
(C-7′), 66.78 (C-4′), 66.60 (-OCH2-), 66.17 (C-5), 62.03
(C-9′), 56.86 (C-5′), 52.96 (-COOCH3), 39.10 (C-3′), 25.97,
27.92 [-N(COCH3)2], 20.69-21.22 (4 × -OCOCH3), 18.11
(-CH2TMS), -1.386 (TMS); HR-FAB MS [M + Na]+ calcd
for C40H57NO19NaSi 906.319178, found 906.321120.
P en t-4-en yl 3-O-Ben zyl-2-d eoxy-2-p h th a lim id o-â-D-glu -
cop yr a n osid e (11). A solution of pent-4-enyl 3-O-benzyl-4,6-
O-benzylidene-2-deoxy-2-phthalimido-â-D-glucopyranoside15 (10,
640 mg, 1.15 mmol) in a mixture of TFA (5 mL), DCM (25 mL)
and water (400 µL) was kept for 10 min at room temperature
and then diluted with DCM (100 mL) and washed with water
(50 mL), saturated aqueous NaHCO3 (2 × 30 mL), and water
(3 × 40 mL). The organic phase was dried (MgSO4) and
filtered, and the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (15%
gradient ethyl acetate in toluene) to afford 11 as a white
foam (115 mg, 96%): Rf 0.25 (ethyl acetate/toluene, 1/1, v/v);
26
[R]
) +27.2 (c ) 1.00, DCM); MALDI-TOF m/z ) 489.8
D
[M + Na]+; 1H NMR δ 6.96-7.90 (m, 9H, aromatic), 5.48-
5.64 (m, 1H, -CHd), 5.17 (d, 1H, J 1,2 ) 8.7 Hz, H-1), 4.66-
4.81 (m, 2H, CH2d), 4.62 (dd, 2H, J 2 ) 12.5 Hz, -CH2Ph),
4.28 (dd, 1H, J 3,4 ) 8.7 Hz, H-3), 4.15 (dd, 1H, J 2,3 ) 10.0,
Meth yl 2,4,6-Tr i-O-a cetyl-3-O-[m eth yl 4,7,8,9-tetr a -O-
a cetyl-5-(N-a cetyla ceta m id o)-3,5-d id eoxy-D-glycer o-r-D-
ga la cto-n on -2-u lop yr a n osylon a te]-1-th io-â-D-ga la ctop y-

2552 J . Org. Chem., Vol. 66, No. 8, 2001
Demchenko and Boons
H-2), 3.96 (dd, 1H, J 6a,6b ) 12.5 Hz, H-6a), 3.84 (dd, 1H, H-6b),
3.73-3.85 (m, 2H, H-4, -OCH2a-), 3.49-3.58 (m, 1H, H-5),
3.35-3.45 (m, 1H, -OCH2b-), 2.46 (br s, 1H, 4-OH), 2.07
(br s, 1H, 6-OH, 1.74-1.92 (m, 2H, -CH2CHd), 1.38-1.60
concentrated in vacuo to afford 3-azidopropyl 4-O-(â-D-galac-
topyranosyl)-â-D-glucopyranoside as white solid (Rf 0.15, MeOH/
DCM, 1/4, v/v), which was dissolved in a mixture of TMSCl (4
mL) and acetone (10 mL). The reaction mixture was kept for
3 h at room temperature, and then hexane (10 mL) was added
and the mixture was concentrated in vacuo. The residue was
coevaporated from acetone/toluene (1/2, v/v, 3 × 15 mL), dried
(MgSO4), and then dissolved in DMF (5 mL). Benzyl bromide
(884 mL, 7.43 mmol) and NaH (360 mg, 8.93 mmol) were added
to the solution, and the reaction mixture was vigorously stirred
for 1 h at room temperature and then poured into ice-water
(50 mL). The aqueous layer was extracted with ethyl acetate/
Et2O (1/1, v/v, 3 × 25 mL). The combined organic layers were
washed with water (3 × 30 mL), dried (MgSO4), and concen-
trated in vacuo. The residue was dissolved in a mixture of TFA
(5 mL), DCM (20 mL), and water (400 µL). After being stirred
for 5 min at room temperature, the reaction mixture was
diluted with DCM (50 mL) and washed with water (30 mL),
saturated aqueous NaHCO3 (2 × 25 mL), and water (3 × 30
mL). The organic phase was dried (MgSO4) and filtered, and
the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (10% gradient
ethyl acetate in hexane) to afford 5 as a white solid (733 mg,
67% over four steps): Rf 0.50 (ethyl acetate/hexane, 1/1, v/v);
(m, 2H, -OCH2CH2-); 13C NMR
δ 134.10 (-CHd),
127.96-128.51 (aromatic), 114.89 (CH2d), 98.69 (C-1), 79.65
(C-3), 75.34 (C-5), 74.73 (-CH2Ph), 72.57 (C-4), 69.31
(-OCH2-), 62.89 (C-6), 55.91 (C-2), 30.14 (-CH2-CHd), 28.87
(-CH2-CH2O-). Anal. Calcd for C26H29NO7 (467.19): C, 66.80;
H, 6.25; N, 3.00. Found: C, 66.54; H, 6.31; N, 3.08.
P en t-4-en yl 3-O-Ben zyl-2-d eoxy-2-p h th a lim id o-4,6-d i-
O-tr ityl-â-D-glu cop yr a n osid e (4). A solution of γ-collidine
(580 µL, 4.40 mmol) in DCM (12 mL) was added to a flask
containing 11 (515 mg, 1.10 mmol) and triphenylmethyl
perchlorate13 (1.13 g, 3.30 mmol), and the resulting mixture
was stirred until all solid dissolved. After the reaction mixture
was stirred in the dark for 20 h at room temperature, pyridine
(1 mL) was added and the mixture was diluted with DCM (20
mL) and washed with water (4 × 15 mL). The organic phase
was dried (MgSO4) and filtered, and the filtrate was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (5% gradient of ethyl acetate in hexane) and
crystallized from Et2O/hexane to afford 4 as colorless needless
(910 mg, 87%): Rf 0.50 (ethyl acetate/hexane, 3/7, v/v); [R]26
D
) +54.1 (c ) 1.18, DCM); mp 196.1-197.0 °C (diethyl ether-
hexane); MALDI-TOF m/z ) 975.0 [M + Na]+; 1H NMR, δ,
6.50-7.68 (m, 39H, aromatic), 5.52-5.61 (m, 1H, -CH2d), 5.18
(s, 1H, J 1,2 ) 8.6 Hz, H-1), 4.66-4.75 (m, 2H, CH2d), 4.58 (dd,
1H, J 3,4 ) 7.9 Hz, H-3), 4.20 (m, 1H, H-5), 3.90-3.95 (m, 1H,
-OCH2a-), 3.90 (dd, 1H, J 2,3 ) 10.3 Hz, H-2), 3.86 (dd, 2H, J 2
) 11.7 Hz, -CH2Ph), 3.49-3.56 (m, 1H, -OCH2b-), 3.29 (dd,
[R]26 ) +17.3 (c ) 1.39, DCM); MALDI-TOF m/z ) 898.8
D
[M + Na]+; 1H NMR δ 7.16-7.36 (m, 25H, aromatic), 4.85
(dd, 2H, J 2 ) 11.0 Hz, -CH2Ph), 4.76 (dd, 2H, J 2 ) 11.2 Hz,
-CH2Ph), 4.72 (dd, 2H, J 2 ) 11.5 Hz, -CH2Ph), 4.49 (dd, 2H,
J 2 ) 12.2 Hz, -CH2Ph), 4.41 (d, 1H, J 1′,2′ ) 6.8 Hz, H-1′), 4.39
(dd, 2H, J 2 ) 12.0 Hz, -CH2Ph), 4.30 (d, 1H, J 1,2 ) 7.8 Hz,
H-1), 3.97 (dd, 1H, J 4,5 ) 9.5 Hz, H-4), 3.91-3.96 (m, 1H,
-OCH2a-), 3.91 (ps, 1H, H-4′), 3.78 (dd, 1H, J 5,6a ) 4.2 Hz,
J 6a,6b ) 11.0 Hz, H-6a), 3.71 (dd, 1H, J 5,6b ) 1.7 Hz, H-6b),
3.59 (dd, 1H, J 5′,6′a ) 6.4 Hz, H-6′a), 3.57-3.62 (m, 1H,
-OCH2b-), 3.56 (dd, 1H, J 3,4 ) 9.0 Hz, H-3), 3.48 (dd, 1H, J 5′,6′b
) 5.1 Hz, J 6′a,6′b ) 9.8 Hz, H-6′b), 3.32-3.42 (m, 7H, H-2, 2′,
3′, 5, 5′, -CH2N3), 2.38, 2.45 (2d, 2H, 3′-OH, 4′-OH), 1.80-
1.91 (m, 2H, -CH2-); 13C NMR, δ 127.45-128.69 (aromatic),
103.80 (C-1), 102.80 (C-1′), 83.11 (C-3), 82.04 (C-3′), 80.32 (C-
2), 76.88 (C-4), 75.18, 75.28, 75.40, 75.51 (3 × -CH2Ph, C-5*),
73.22, 73.52, 73.79, 73.79 (2 × -CH2Ph, C-2′, 5′*), 68.54, 69.00,
69.10 (C-4′, 6, 6′), 66.79 (-OCH2-), 48.67 (-CH2N3), 29.65
(-CH2-). Anal. Calcd for C50H57N3O11 (875.40): C, 68.55; H,
6.56; N, 4.80. Found: C, 68.39; H, 6.48; N, 4.89.
1H, J 5,6a ) 1.4 Hz, J 6a,6b ) 9.6 Hz, H-6a), 2.81 (dd, 1H, J 4,5
)
9.6 Hz, H-4), 2.56 (dd, 1H, J 5,6b ) 9.3 Hz, H-6b), 1.79-1.89
(m, 2H, -CH2CHd), 1.46-1.61 (m, 2H, -CH2CH2O-); 13C
NMR, δ, 144.71 (-CHd), 123.40-138.68 (aromatic), 114.89
(CH2d), 97.91 (C-1), 86.56, 88.91 (2 × -CPh3), 81.05 (C-3),
76.31 (C-5), 74.58 (-CH2Ph), 73.88 (C-4), 68.84 (-OCH2-),
66.33 (C-6), 57.27 (C-2), 29.01, 30.33 (-CH2CH2CHd). Anal.
Calcd for C64H57NO7 (951.41): C, 80.73; H, 6.03; N, 1.47.
Found: C, 81.00; H, 5.99; N, 1.43.
3-Azid op r op yl 4-O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la cto-
p yr a n osyl)-2,3,6-tr i-O-a cetyl-â-D-glu cop yr a n osid e (13). A
mixture of 3-chloropropyl 4-O-(2,3,4,6-tetra-O-acetyl-â-D-ga-
lactopyranosyl)-2,3,6-tri-O-acetyl-â-D-glucopyranoside17 (12, 1.08
g, 1.52 mmol) and KI (505 mg, 3.04 mmol) in DMF was stirred
for 30 min at 50 °C. After addition of NaN3 (988 mg, 15.2 mg),
the reaction mixture was refluxed for another 30 min and then
poured into ice-water (30 mL). The aqueous layer was
extracted with ethyl acetate/Et2O (1/1, v/v, 3 × 15 mL). The
combined organic layers were washed with water (3 × 20 mL),
dried (MgSO4), and concentrated in vacuo to dryness. The
residue was purified by silica gel column chromatography (10%
gradient ethyl acetate in toluene) to afford 13 as a white foam
P en t-4-en yl O-[Meth yl 4,7,8,9-tetr a -O-a cetyl-5-(N-a ce-
t yla cet a m id o)-3,5-d id eoxy-D-glycer o-r-D-ga la cto-n on -2-
u lop yr a n osylon a te]-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la c-
top yr a n osyl)-(1f4)-3-O-ben zyl-2-d eoxy-2-p h th a lim id o-6-
O-tr ityl-â-D-glu cop yr a n osid e (14). A suspension of 3 (192.4
mg, 0.226 mmol), 4 (165.4 mg, 0.174 mmol), and activated
molecular sieves (3 Å, 800 mg) in DCM (3 mL) was stirred for
3 h under an atmosphere of argon. MeOTf (98 µL, 0.87 mmol)
was added, and the reaction mixture was stirred for 2.5 h at
room temperature. Then, Et3N (1 mL) was added, and the
mixture was diluted with DCM (30 mL), the solid filtered off,
and the residue was washed with DCM (3 × 20 mL). The
combined filtrates (∼ 90 mL) were washed with water (3 × 40
mL), the organic phase was dried (MgSO4) and filtered, and
the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (5% gradient
acetone in toluene) to afford 14 as a white foam (mg, 96%): Rf
0.45 (acetone/toluene, 3/7, v/v); [R]26D ) + 16.6 (c ) 1.30, DCM);
mp 176.0-177.2 °C (diethyl ether); MALDI-TOF m/z ) 1537.4
[M + Na]+;1H NMR δ 6.78-7.80 (m, 24 H, aromatic), 5.49-
5.62 (m, 2H, J 4′′,5′′ ) 10.7 Hz, H-4′′, -CHd), 5.38-5.44 (m, 1H,
(929 mg, 85%): Rf 0.55 (ethyl acetate/toluene, 3/2, v/v), [R]26
D
) -4.7 (c ) 1.59, DCM); MALDI-TOF m/z ) 741.8 [M + Na]+;
1H NMR δ 5.34 (dd, 1H, J 4′,5′ ) 0.5 Hz, H-4′), 5.19 (dd, 1H, J 3,4
) 9.3 Hz, H-3), 5.10 (dd, 1H, J 2′,3′ ) 10.4 Hz, H-2′), 4.95 (dd,
1H, J 3′,4′ ) 3.5 Hz, H-3′), 4.88 (dd, 1H, J 2,3 ) 9.3 Hz, H-2), 4.49
(dd, 1H, J 5′,6′a ) 1.9 Hz, H-6′a), 4.48 (d, 1H, J 1′,2′ ) 8.0 Hz,
H-1′), 4.46 (d, 1H, J 1,2 ) 8.0 Hz, H-1), 4.03-4.17 (m, 3H, H-5′,
6a, 6′b), 3.81-3.92 (m, 2H, H-6b, -OCH2a-), 3.79 (dd, 1H, J 4,5
) 9.6 Hz, H-4), 3.53-3.64 (m, 2H, H-5, -OCH2b-), 3.30-3.40
(m, 2H, -CH2N3), 1.96, 2.04, 2.04, 2.04, 2.06, 2.12, 2.14 (7s,
21H, 7 × -OCOCH3), 1.74-1.90 (m, 2H, -CH2-); 13C NMR δ
168.04-170.42 (7 × >CdO), 101.29 (C-1′), 100.80 (C-1), 76.50
(C-4), 73.05 (C-3), 72.97 (C-5), 71.94 (C-2), 71.26 (C-5′), 70.99
(C-3′), 69.42 (C-2′), 66.91 (C-4′), 66.74 (-OCH2-), 62.23
(C-6′), 61.10 (C-6), 48.28 (-CH2N3), 29.33 (-CH2-), 20.85-
21.19 (7 × -OCOCH3). Anl. Calcd for C29H41N3O18 (719.24):
C, 48.40; H, 5.74; N, 5.84. Found: C, 48.56; H, 5.77; N, 5.80.
3-Azid op r op yl 4-O-(2,6-Di-O-ben zyl-â-D-ga la ctop yr a n o-
syl)-2,3,6-tr i-O-ben zyl-â-D-glu cop yr a n osid e (5). A mixture
of 13 (899 mg, 1.25 mmol) and NaOMe (3 mg, 0.05 mmol) in
MeOH was kept for 16 h at room temperature and then
neutralized (Dowex, H+) and filtered, and the filtrate was
H-8′′), 5.13 (dd, 1H, J 7′′,8′′ ) 8.8 Hz, H-7′′), 5.03 (d, 1H, J 1,2
)
8.5 Hz, H-1), 5.00 (dd, 1H, J 4′,5′ ) 0.3 Hz, H-4′), 4.86 (dd, 1H,
J 2′,3′ ) 9.7 Hz, H-2′), 4.87 (d, 1H, J 1′,2′ ) 7.6 Hz, H-1′), 4.68-
4.75 (m, 2H, CH2d), 4.61 (dd, 1H, J 2 ) 12.5 Hz, -CH2Ph),
4.60 (dd, 1H, J 6′′,7′′ ) 2.5 Hz, H-6′′), 4.36 (dd, 1H, J 3′,4′ ) 3.7
Hz, H-3′), 4.30 (dd, 1H, J 5′′,6′′ ) 10.4 Hz, H-5′′), 4.28 (dd, 1H,
J 3,4 ) 9.0 Hz, H-3), 4.23 (dd, 1H, J 8′′,9′′a ) 2.5 Hz, J 9′′a,9′′b
)
12.6 Hz, H-9′′a), 4.16 (dd, 1H, J 2,3 ) 9.0 Hz, H-2), 4.13 (dd,
1H, J 4,5 ) 9.0 Hz, H-4), 4.00 (dd, 1H, J 8′′,9′′b ) 4.9 Hz, H-9′′b),
3.95 (pd, 2H, J 6′a,6′b ) 6.4 Hz, H-6′a, 6′b), 3.78-3.99 (m, 1H,

Convergent Synthesis of a Complex Oligosaccharide
J . Org. Chem., Vol. 66, No. 8, 2001 2553
-OCH2a-), 3.80 (s, 3H, -COOCH3), 3.78 (m, 1H, J 5′,6′a ) J 5′,6′b
) 6.4 Hz, H-5′), 3.55-3.60 (m, 2H, H-5, 6a), 3.40-3.47 (m, 1H,
-OCH2b-), 3.18 (dd, 1H, J 5,6b ) 5.8 Hz, J 6a,6b ) 10.1 Hz, H-6b),
2.57 (dd, 1H, J 3′′e,4′′ ) 5.5 Hz, J 3′′e,3′′a )12.8 Hz, H-3′′e), 2.30,
2.35 [2s, 6H, -N(COCH3)2], 1.82-1.90 (m, 2H, -CH2-CH2d
), 1.78, 1.93, 1.95, 1.96, 1.96, 2.03, 2.10 (7s, 21H, 7 ×
-OCOCH3), 1.61 (dd, 1H, J 3′′a,4′′ )12.8 Hz, H-3′′a), 1.49-1.59
(m, 2H, -OCH2CH2-); 13C NMR, δ, 167.78-174.09 (>CdO),
144.24 (-CHd), 123.38-138.82 (aromatic), 114.88 (CH2d),
99.42 (C-1′), 98.32 (C-1), 97.63 (C-2′′), 86.88 (-CPh3), 77.97
(C-3), 77.73 (C-4), 74.52 (C-5, -CH2Ph), 72.04 (C-3′), 71.60 (C-
2′), 70.81 (C-5′), 70.16 (C-6′′), 68.78 (-OCH2-), 68.15 (C-4′,
8′′), 67.22 (C-4′′, 7′′), 62.98 (C-6), 62.26 (C-6′, 9′′), 56.53 (C-
5′′), 56.34 (C-2), 53.41 (-COOCH3), 38.50 (C-3′′), 30.32 (-CH2-
CHd), 30.05 (-CH2CH2O-), 27.06, 28.42 [-N(COCH3)2],
21.10-21.63 (7 × -OCOCH3). Anal. Calcd for C79H88N2O28
(1512.55): C, 62.69; H, 5.86; N, 1.85. Found: C, 62.54; H, 6.00;
N, 1.91.
(-OCOCH3). Anal. Calcd for C86H108N2O45 (1888.62): C, 54.66;
H, 5.76; N, 1.48. Found: C, 54.48; H, 7.81; N, 1.53.
3-Azid op r op yl O-[Meth yl 4,7,8,9-tetr a -O-a cetyl-5-(N-
a ce t yla ce t a m id o)-3,5-d id e oxy-D -glycer o-r-D -ga la ct o-
n on -2-u lop yr a n osylon a te]-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-
D-glu cop yr a n osyl)-(1f4)-O-[(2,3,4,6-Te t r a -O-a ce t yl-â-
D-ga la ct op yr a n osyl)-(1f4)-(2,3,6-t r i-O-a cet yl-â-D-glu co-
p yr a n osyl)-(1f6)]-O-(3-O-ben zyl-2-d eoxy-2-p h th a lim id o-
â-D-glu cop yr a n osyl)-(1f3)-O-(2,6-Di-O-ben zyl-â-D-ga la c-
t op yr a n osyl)-(1f4)-2,3,6-t r i-O-b en zyl-â-D-glu cop yr a n o-
sid e (16a ). A mixture of 5 (48 mg, 0.054 mmol), 15 (51.1 mg,
0.027 mmol), and activated molecular sieves (3 Å, 300 mg) in
DCM (1.5 mL) was stirred for 3 h under argon. NIS (18.3 mg,
0.081 mmol) and TMSOTf (1.5 µL, 0.008 mmol) were added.
After the reaction mixture was stirred for 24 h at room
temperature, it was diluted with DCM (20 mL), the solid
filtered off, and the residue washed with DCM (3 × 10 mL).
The combined filtrates (∼50 mL) were washed with aqueous
Na2S2O3 (20%, 20 mL) and water (3 × 20 mL). The organic
phase was dried (MgSO4) and filtered and the filtrate concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (3% gradient acetone in toluene) to afford 16a
as a transparent film (45 mg, 62%): Rf 0.45 (acetone/toluene,
P en t-4-en yl O-[Meth yl 4,7,8,9-tetr a -O-a cetyl-5-(N-a ce-
t yla ce t a m id o)-3,5-d id e oxy-D-glycer o-r-D-ga la ct o-n on -
2-u lop yr a n osylon a te]-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-glu -
cop yr a n osyl)-(1f4)-[(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la c-
t op yr a n osyl)-(1f4)-(2,3,6-t r i-O-a cet yl-â-D-glu cop yr a n o-
syl)-(1f6)]-3-O-ben zyl-2-deoxy-2-ph th alim ido-â-D-glu copy-
r a n osid e (15). A solution of 14 (151 mg, 0.10 mmol) and 2
(64.5 mg, 0.10 mmol) in benzene (2 mL) was added in one of
the limbs of a tuning-fork-shaped tube and a solution TrClO4
(5.1 mg, 15 µmol) in MeNO2 (0.5 mL) in the other limb.19b The
tube was attached to a high-vacuum system, and the solutions
were lyophilized. DCM (∼2 mL) was distilled into the reaction
tube, and the solutions of the reactants were mixed. After 40
h at room temperature in the dark, the reaction was quenched
by the addition of a drop of pyridine and the resulting mixture
was diluted with DCM (50 mL) and washed with water (4 ×
15 mL). The organic phase was dried (MgSO4) and filtered and
the filtrate concentrated in vacuo. The residue was purified
by silica gel column chromatography (10% gradient acetone
in toluene) to afford 15 as a white foam (160 mg, 85%): Rf
0.45 (acetone/toluene, 3/7, v/v); [R]26D ) +18.2 (c ) 2.04, DCM);
1/3, v/v); [R]24 ) +12.9 (c ) 0.8, DCM); MALDI-TOF m/z )
D
2706.8 [M + Na]+; 1H NMR, δ, 6.75-7.45 (m, 34H, aromatic),
5.59 (m, 1H, J 8
) 2.6 Hz, J 8
) 5.8 Hz, H-8E), 5.53 (m,
E
E
E
E
,9a
,9b
E
E
C
1H, J 4
) 11.0 Hz, H-4E), 5.30 (d, 1H, J 1 C ) 8.4 Hz, H-1C),
,5
,2
5.28 (bs, 1H, H-4G), 5.19 (dd, 1H, J 3
) 9.3 Hz, H-3F), 5.15
G G
F
F
,4
(dd, 1H, J 7
) 9.2 Hz, H-7E), 5.06 (dd, 1H, J 2
) 10.4 Hz,
D D
E
E
,8
,3
H-2G), 5.00 (dd, 1H, J 2 D ) 10.0 Hz, H-2D), 4.94 (bd, 1H, J 4
D
,3
,5
G
G
) 2.9 Hz, H-4D), 4.87 (dd, 1H, J 3
) 4.8 Hz, H-3G), 4.86 (d,
F F
,4
1H, J 1 D ) 7.9 Hz, H-1D), 4.85 (dd, 1H, J 2
) 9.3 Hz, H-2F),
D
,2
,3
4.72 (dd, 2H, J 2 ) 10.4, CH2Ph), 4.68 (dd, 2H, J 2 ) 11.1 Hz,
F
F
CH2Ph), 4.68 (d, 1H, J 1
) 8.0 Hz, H-1F), 4.63 (dd, 2H, J 2
)
,2
D
D
12.2 Hz, CH2Ph), 4.61 (dd, 1H, J 3
) 2.9 Hz, H-3D), 4.61
,4
(dd, 1H, J 6
) 2.9 Hz, H-6E), 4.43 (dd, 2H, J 2 ) 12.5 Hz,
C C
E
E
,7
CH2Ph), 4.30-4.36 (m, 5H, J 3
) 9.5 Hz, H-1G, 3C, 5E, 6Fa,
,4
9Ea), 4.26 (d, 1H, J 1
) 7.6 Hz, H-1B), 4.18 (dd, 2H, J 2
)
A
B
B
,
2
11.7 Hz, CH2Ph), 4.17 (d, 1H, J 1 A ) 6.6 Hz, H-1A), 4.17 (dd,
,2
1H, J 2 C ) 9.5 Hz, H-2C), 4.11 (dd, 1H, J 5 b ) 3.8 Hz, J 6
C
F
F
F
F
,3
,6
a,6 b
1
MALDI-TOF m/z ) 1914.3 [M + Na]+; H NMR δ 6.77-7.82
) 11.8 Hz, H-6Fb), 4.01-4.16 (m, 4H, J 4
) 3.2 Hz, H-4B,
B
B
,5
(m, 9H, aromatic), 5.50-5.61 (m, 3H, H-4C, 8C, -CHd), 5.33
5C, CH2Ph), 3.91 (dd, 1H, J 9
) 12.4 Hz, H-9Eb), 3.80-
E
E
a,9
b
E
E
C C
(dd, 1H, J 4
) 0.4 Hz, H-4E), 5.21 (dd, 1H, J
) 9.3 Hz,
3.94 (m, 8H, H-5D, 6Ca, 6Da, 6Db, OCH2a, COOCH3), 3.73-3.80
,5
7 ,8
H-7C), 5.11 (dd, 1H, J 3 D ) 9.3 Hz, H-3D), 5.10 (dd, 1H, J 2
D
E E
(m, 7H, H-4A, 4C, 5G, 6Ga, 6Gb, 6Cb, 6Ba), 3.71 (dd, 1H, J 4
)
F
F
,4
,3
,5
) 8.8 Hz, H-2E), 5.07 (d, 1H, J 1
) 8.2 Hz, H-1A), 5.04 (dd,
A
A
9.1 Hz, H-4F), 3.62 (m, 1H, H-5F), 3.49 (m, 3H, H-5B, 6Bb,
,2
1H, J 2
) 8.8 Hz, H-2B), 4.94-4.98 (m, 2H, H-3E, 4B), 4.91
B B
B
B
OCH2b), 3.46 (dd, 1H, J 3
) 3.4 Hz, H-3B), 3.40 (dd, 1H,
A A
,3
,4
) 9.0 Hz, H-2D), 4.86 (d, 1H, J 1
) 7.1 Hz,
A
A
A
A
D
D
D D
(dd, 1H, J 2
,3
,2
J 5
) 4.7 Hz, J 6
b ) 11.0 Hz, H-6Aa), 3.36 (dd, 1H, J 3
,6
a
a,6
,4
H-1D), 4.82 (d, 1H, J 1
) 8.0 Hz, H-1B), 4.69-4.78 (m, 2H,
B
B
) 9.0 Hz, H-3A), 3.35 (dd, 1H, J 2
) 9.0 Hz, H-2B), 3.29-
B B
,2
,3
b ) 11.0 Hz, H-6C),
B B
C
C
C
C
3.35 (m, 3H, H-6Ab, CH2N3), 3.24 (dd, 1H, J 2
) 9.0 Hz,
H-2A), 3.00 (m,1H, H-5A), 2.72 (bs, 1H, 4B-OH), 2.63 (dd, 1H,
A A
CH2d), 4.64 (dd, 1H, J 5
) 1.5 Hz, J 6
,6
a
a,6
,3
4.62 (dd, 2H, J 2 ) 12.2 Hz, -CH2Ph), 4.62 (dd, 1H, J 3
)
,4
E
E
3.8 Hz, H-3B), 4.49 (d, 1H, J 1
) 7.7 Hz, H-1E), 4.38 (dd, 1H,
E
E
E
E
J 3
) 5.3 Hz, J 3
) 12.3 Hz, H-3Ee), 2.29, 2.35 [2s, 6H,
,2
e,4
e,3 a
D
D
D
D
J 5
) 1.5 Hz, J 6
b ) 11.5 Hz, H-6Da), 4.34 (m, 1H, H-5C),
D D
,6
a
a,6
N(COCH3)2], 1.85, 1.94, 1.94, 1.94, 1.96, 1.96, 1.97, 2.00, 2.01,
4.26-4.31 (m, 3H, H-3A, 6Aa, 9Ca), 4.16 (dd, 1H, J 5
) 6.0
,6
b
2.02, 2.10, 2.12, 2.18, 2.28 (14s, 42H, 14 × OCOCH3), 1.79 (m,
Hz, H-6Db), 4.12 (dd, 1H, J 5 ,6Ea ) 6.0 Hz, J 6
) 12.5 Hz,
E
E
E
E
E
2H, -CH2CH2CH2-), 1.60 (dd, 1H, J 3
) 12.3 Hz, H-3Ea);
a,6
b
a,4
H-6Ea), 4.11 (dd, 1H, J 5
) 6.5 Hz, H-6Eb), 4.06 (dd, 1H,
E
E
13C NMR δ 170.00-171.45 (>CdO), 122.00-136.85 (aro-
matic), 103.62 (C-1A), 102.25 (C-1B), 101.57 (C-1G), 101.19
(C-1F), 101.12 (C-1D), 98.95 (C-1C), 97.00 (C-2E), 83.23 (C-3B),
82.98 (C-3A), 82.02 (C-2A), 79.69 (C-4C)#, 78.46 (C-2B), 78.19
(C-3C), 77.07 (C-4F), 76.51 (C-4A)#, 75.48 (CH2Ph), 75.45
(C-6G), 75.25 (CH2Ph), 75.15 (C-5A), 74.71 (CH2Ph), 74.55
(CH2Ph), 73.38 (CH2Ph), 73.36 (CH2Ph), 73.32 (C-3F), 73.26
(C-5B), 72.90 (C-5F), 72.40 (C-2F), 71.65 (C-6E)*, 71.36 (C-3G),
71.27 (C-5D), 71.17 (C-2D), 70.85 (C-6C), 70.05 (C-5G), 69.63
(C-3D)*, 69.30 (C-2G), 69.08 (C-6B), 68.21 (C-6A), 68.01 (C-4B),
67.88 (C-4D), 67.61 (C-8E), 67.39 (C-7E), 67.27 (C-4E), 66.85
(C-4G), 66.60 (OCH2), 62.83 (C-9F), 62.53 (C-6F), 62.10 (C-6D),
60.96 (C-5C), 56.18 (C-5E), 55.98 (C-2C), 53.37 (COOCH3), 48.70
(CH2N3), 38.49 (C-3E), 29.44, 29.92 [N(COCH3)2], 28.60
(-CH2CH2CH2-), 20.00-21.85 (OCOCH3). Anal. Calcd for
,6
b
J 2 A ) 9.9 Hz, H-2A), 4.03 (dd, 1H, J 8
b ) 4.6 Hz, J 9
) 6.0 Hz, J 6
)
A
C
C
C
C
,3
,9
a,9 b
12.5 Hz, H-9Cb), 3.97 (dd, 1H, J 5
) 10.5
B
B
B
B
,6
a
a,6 b
Hz, H-6Ba), 3.88-3.95 (m, 7H, H-4D, 5B, 5E, 6Bb, -COOCH3),
3.80-3.88 (m, 2H, H-6Ab, -OCH2a-), 3.60-3.71 (m, 3H, H-4A,
5A, 5D), 3.38-3.45 (m, 1H, -OCH2b-), 2.67 (dd, 1H, J 3
)
C
C
e,4
C
C
5.0 Hz, J 3
) 12.6 Hz, H-3Ce), 2.30, 2.36 [2s, 6H,
e,3
a
-N(COCH3)2], 1.92, 1.95, 1.96, 2.03, 2.03, 2.03, 2.03, 2.04, 2.04,
2.10, 2.11, 2.12, 2.20, 2.24 (14 s, 42H, 14 × -OCOCH3), 1.80-
C
C
1.89 (m, 2H, -CH2-CHd), 1.63 (dd, 1H, J 3
) 12.1 Hz,
a,4
H-3Ca), 1.48-1.57 (m, 2H, -OCH2CH2-); 13C NMR δ 167.87-
174.18 (>CdO), 138.59 (-CHd), 123.45-133.88 (aromatic),
115.03 (CH2d), 101.74 (C-1B), 101.49 (C-1E), 100.18 (C-1D),
98.20 (C-1A), 96.97 (C-2C), 80.71 (C-4A), 77.70 (C-3A), 77.28 (C-
5A), 76.85 (C-5E), 75.03 (-CH2Ph), 73.30 (C-5D), 72.82 (C-3D),
72.15 (C-2D), 71.67 (C-3B), 71.36 (C-5B), 71.19 (C-3E)*, 70.92
(C-4D), 70.79 (C-2B), 69.72 (C-6C), 69.36 (C-2E, -OCH2-), 67.98
(C-4B)*, 67.82 (C-4C, 8C), 67.23 (C-6A, 7C), 67.03 (C-4E), 62.67
(C-6D, 9C), 61.86 (C-6B), 61.13 (C-6E), 56.26 (C-5C), 55.93 (C-
2A), 53.36 (-COOCH3), 38.64 (C-3C), 30.09 (-CH2-CHd),
28.76 (-CH2CH2O-), 27.03, 28.44 [-N(COCH3)2], 20.98-21.89
C
131H155N5O55 (2677.95): C, 58.72; H, 5.83; N, 2.61. Found: C,
58.58; H, 6.01; N, 2.61.
3-Am in op r op yl O-(5-N-Aceta m id o-3,5-d id eoxy-D-glyc-
er o-r-D-ga la cto-n on -2-u lop yr a n osyl)-(2f3)-O-(â-D-glu -
cop yr a n osyl)-(1f4)-O-[(â-D-ga la ct op yr a n osyl)-(1f4)-(â-
D-glu cop yr a n osyl)-(1f6)]-O-(2-a cet a m id o-2-d eoxy-â-D-

2554 J . Org. Chem., Vol. 66, No. 8, 2001
Demchenko and Boons
glu cop yr a n osyl)-(1f3)-O-(â-D-ga la ct op yr a n osyl)-(1f4)-
â-D-glu cop yr a n osid e (1). A mixture of 16a (34 mg, 12.4 µmol)
and LiI (25 mg, 0.19 mmol) in pyridine (2 mL) was heated for
24 h at 85 °C. The reaction mixture was concentrated in vacuo,
and the residue was purified by silica gel column chromatog-
raphy (gradient 2% MeOH in DCM) to afford 16b. Compound
16b was dissolved in n-butanol (2 mL), and ethylenediamine
(400 µL) was added. After being stirred for 16 h at 90 °C, the
reaction mixture was then concentrated in vacuo, and the
residue was coevaporated from toluene (2 × 10 mL) and EtOH
(2 × 5 mL). The crude 16c was dissolved in MeOH (1.5 mL),
and acetic anhydride (400 µL) was added. After being stirred
for 6 h at room temperature, the reaction mixture was
concentrated under reduced pressure. The residue 16d was
dissolved in 50% aqueous EtOH (2 mL), and Pd/C (20 mg) was
added. The reaction mixture was stirred under an atmosphere
of H2 for 40 h. Then, the catalyst was filtered off and the
filtrate concentrated under reduced pressure. The residue was
dissolved in water (1 mL), and 1 M aqueous NaOH (200 µL)
was added. After being stirred for 15 min, the reaction mixture
was purified by Sephadex G-25 size-exclusion column chro-
matography (35 cm3, water elution) to afford 1 as an amor-
signals δ 104.26 (C-1B), 104.26 (C-1G), 104.02 (C-1C), 103.92
(C-1F), 103.53 (C-1D), 103.38 (C-1A), 83.35, 79.76, 78.81, 76.92,
76.59, 76.28, 76.01, 75.68, 74.70, 74.18, 74.04, 73.95, 73.78,
73.34, 73.02, 72.23, 71.29, 70.66, 69.87, 69.75, 69.56, 69.49,
69.31, 68.97, 68.83, 63.92, 62.23, 61.39, 59.80, 56.40, 52.95,
40.91 (C-3E), 24.17, 24.17.
P r ep a r a tion of a P olym er Con ta in in g GBS Hep ta sa c-
ch a r id e (18). The conjugation was performed in accordance
with a previously described procedure.23 A solution of 1 (1.16
mg, 0.828 µmol) in triethylamine (1.5 mL) was added to a
stirred solution of poly[N-(acryloxy)succinimide] (17, 0.828 mg,
4.87 mmol of N-hydroxysuccinimide ether, 17% theoretical
loading) in DMF (0.2 mL). The solution was stirred at room
temperature for 20 h, heated at 65 °C for 6 h, and then cooled
to room temperature. An aqueous solution of NH4OH (20%,
0.1 mL) was added, and the mixture was stirred at room
temperature for 24 h. The resulting mixture was dialyzed
against distilled water and then freeze-dried to afford 18 as a
white powder: selected 1H NMR data δ 4.68 (d, 1H, J ) 8.6
Hz), 4.58 (d, 1H, J ) 8.0 Hz), 4.51 (d, 1H, J ) 7.7 Hz), 4.45 (d,
1H, J ) 8.2 Hz), 4.42 (d, 1H, J ) 7.9 Hz), 4.40 (d, 1H, J ) 7.9
Hz).
phous powder (5.9 mg, 31%): [R]24 ) +2.09 (c ) 0.4, 70%
D
aqueous ethanol); selected 1H NMR data δ 4.68 (d, 1H, J 1
C
C
,2
Ack n ow led gm en t. This research was supported by
the University of Georgia Faculty start-up funds (to
G.J .B.). We thank Drs. E. Erranz, J . Glushka, and A.
Siriwardena for experimental assistance.
) 8.6 Hz, H-1C), 4.58 (d, 1H, J 1
) 8.0 Hz, H-1D), 4.51 (d,
D
D
,2
1H, J 1
) 7.7 Hz, H-1F), 4.45 (d, 1H, J 1 A ) 8.2 Hz, H-1A),
F
F
A
,2
,2
G
G
B B
4.42 (d, 1H, J 1
) 7.9 Hz, H-1G), 4.40 (d, 1H, J 1
) 7.9
,2
,2
Hz, H-1B), 4.26 (br d, 1H, H-5C), 4.14 (br s, 1H, H-4B), 4.07 (br
d, 1H, H-3D), 3.94 (br s, 1H, H-4D), 3.90 (br s, 1H, H-4G), 3.85
(dd, 1H, H-4C), 3.78 (dd, 1H, H-2C), 3.70 (dd, 1H, H-3C), 3.70
(dd, 1H, H-3B), 3.66 (m, 1H, H-4E), 3.65 (dd, 1H, H-3G), 3.64
(m, 2H, H-3F, 4F), 3.62 (dd, 1H, H-3A), 3.58 (dd, 1H, H-4A), 3.56
(dd, 1H, H-2B), 3.54 (dd, 1H, H-2D), 3.52 (dd, 1H, H-2G), 3.33
(dd, 1H, H-2F), 3.28 (dd, 1H, H-2A), 2.73 (dd, 1H, H-3Ee), 2.02
(s, 6H, NCOCH3 × 2), 1.79 (dd, 1H, H-3Ea); selected 13C NMR
Su p p or tin g In for m a tion Ava ila ble: Copies of NMR
spectra for compounds 1, 3-5, 8b, 11, 13-15, and 16a . This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O001477W