Syntheses of anomerically phosphodiester-linked oligomers of the repeating units of the Haemophilus influenzae types c and f capsular polysaccharides

Article abstract of DOI:10.1021/jo001302m

Spacer-equipped dimers and trimers of the repeating units of the capsular polysaccharide of Haemophilus influenzae type c, -4)-3-O-Ac-β-D-GlcpNAc-(1→3)-α-D-Galp-(1-OPO₃ ^--, and type f, -3)-β-D-GalpNAc-(1→4)-3-O-Ac-α-D-GalpNAc-(1-OPO_{3 -}-, have been synthesized for use in immunological studies. H-Phosphonate chemistry was used for the formation of the interglycosidic phosphate diester linkages. Two types of building blocks, a spacer glycoside disaccharide starting monomer (15 and 22) and an anomeric monoester α-H-phosphonate disaccharide elongating monomer (12 and 27), were built up for each serotype structure from properly protected monosaccharide precursors using mainly thioglycosides as glycosyl donors. Stereospecificity in the formation of the α-linked monoester H-phosphonate was possible in type c through crystallization of the pure α-anomer of the precursor hemiacetal from an α/β-mixture, whereas in type f, the hemiacetal was isolated directly as exclusively the α-anomer. Subsequent phosphonylation using triimidazolylphosphine was performed without anomerization. Formation of the anomeric phosphate diester linkages was performed using pivaloyl chloride as coupling reagent followed by I₂/H₂O oxidation of the formed diester H-phosphonates. Original experiments afforded no diester product at all, but optimization of the oxidation conditions (lowering the temperature and dilution with pyridine prior to I₂ addition) gave the dimers in good yields (71% and 81%) and, subsequently, after removal of a temporary silyl protecting group in the dimers, the trimers in fair yields (36% and 37%), accompanied by hydrolysis of the dimer phosphate linkage. One-step deprotection through catalytic hydrogenolysis efficiently afforded the target dimer (30 and 36) and trimer structures (32 and 39). The synthetic scheme allows for further elongation to give higher oligomers.

Full text of DOI:10.1021/jo001302m

6234
J . Org. Chem. 2001, 66, 6234-6243
Syn th eses of An om er ica lly P h osp h od iester -Lin k ed Oligom er s of
th e Rep ea tin g Un its of th e Ha em oph ilu s in flu en za e Typ es c a n d f
Ca p su la r P olysa cch a r id es
J onas Hansson, Per J . Garegg, and Stefan Oscarson*
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
S-106 91 Stockholm, Sweden
s.oscarson@organ.su.se
Received August 28, 2000
Spacer-equipped dimers and trimers of the repeating units of the capsular polysaccharide of
Haemophilus influenzae type c, -4)-3-O-Ac-â-D-GlcpNAc-(1f3)-R-D-Galp-(1-OPO3--, and type f,
-3)-â-D-GalpNAc-(1f4)-3-O-Ac-R-D-GalpNAc-(1-OPO3--, have been synthesized for use in im-
munological studies. H-Phosphonate chemistry was used for the formation of the interglycosidic
phosphate diester linkages. Two types of building blocks, a spacer glycoside disaccharide starting
monomer (15 and 22) and an anomeric monoester R-H-phosphonate disaccharide elongating
monomer (12 and 27), were built up for each serotype structure from properly protected
monosaccharide precursors using mainly thioglycosides as glycosyl donors. Stereospecificity in the
formation of the R-linked monoester H-phosphonate was possible in type c through crystallization
of the pure R-anomer of the precursor hemiacetal from an R/â-mixture, whereas in type f, the
hemiacetal was isolated directly as exclusively the R-anomer. Subsequent phosphonylation using
triimidazolylphosphine was performed without anomerization. Formation of the anomeric phosphate
diester linkages was performed using pivaloyl chloride as coupling reagent followed by I2/H2O
oxidation of the formed diester H-phosphonates. Original experiments afforded no diester product
at all, but optimization of the oxidation conditions (lowering the temperature and dilution with
pyridine prior to I2 addition) gave the dimers in good yields (71% and 81%) and, subsequently,
after removal of a temporary silyl protecting group in the dimers, the trimers in fair yields (36%
and 37%), accompanied by hydrolysis of the dimer phosphate linkage. One-step deprotection through
catalytic hydrogenolysis efficiently afforded the target dimer (30 and 36) and trimer structures
(32 and 39). The synthetic scheme allows for further elongation to give higher oligomers.
In tr od u ction
structures corresponding to the type c and type f CPSs
are described.
Haemophilus influenzae is a Gram-negative bacterium
causing, among others, meningitis, pneumoniae, and
otitis. The bacteria are divided into six serotypes, a-f,
all corresponding to a specific capsular polysaccharide
(CPS) structure.1 Noncapsulated bacteria are referred to
as nontypable H. influenzae (NTHi). There are already
commercial glycoconjugate vaccines based on processed
native CPS coupled to a carrier protein against type b,2
which is the serogroup causing the most severe H.
influenzae infections. In a program directed toward a
more complete understanding of the immunological
properties of the carbohydrate surface antigens of H.
influenzae, well-defined synthetic structures from all the
CPS serotypes and also the lipopolysaccharide (LPS) were
a prerequisite. We have earlier reported the synthesis
of structures from serotypes a,3 b,4 d,5 and e,6 as well as
LPS structures.7 In this paper, the syntheses of oligomer
Of the six different CPSs, four, types a, b, c, and f, are
built up by repeating oligosaccharide units bridged by
phosphate diester linkages.1 In types c and f, one of these
phosphate diester bonds is anomeric (Figure 1).8-11 The
synthesis of anomeric phosphodiesters is severely com-
plicated, as compared to that of nonanomeric ones, by
the requirement of stereospecificity in the linkage com-
bined with the inherent instability of the formed diesters.
The instability is due to the ability of the glycosyl ring
to form a stabilized anomeric carbocation through the
expulsion of an anomeric leaving group, e.g., a monoester
phosphate, and is recognized by the fact that anomeric
phosphotriesters are used as effective glycosyl donors.12,13
The ease of formation of an anomerically pure phos-
phodiester as well as the stability of it varies greatly
depending on the substrate. However, the yields are
invariantly much lower than those obtained in nucleotide
(1) Kenne, L.; Lindberg, B. In The Polysaccharides; Aspinall, G. O.,
Ed.; Academic Press: New York, 1983; Vol. 2, p 287.
(2) Lindberg, A. A.; Pillai, S. In New approaches to stabilisation of
vaccines potency; Brown, F., Ed.; Karger: Basel, Switzerland, 1996;
Vol. 87, p 59.
(3) Classon, B.; Garegg, P. J .; Lindh, I. Carbohydr. Res. 1988, 179,
31.
(7) Bernlind, C.; Oscarson, S. J . Org. Chem. 1998, 63, 7780.
(8) Branefors-Helander, P.; Classon, B.; Kenne, L.; Lindberg, B.
Carbohydr. Res. 1979, 76, 197.
(9) Egan, W.; Tsui, F.-P.; Climenson, P. A.; Schneerson, R. Carbo-
hydr. Res. 1980, 80, 305.
(10) Branefors-Helander, P.; Kenne, L.; Lindqvist, B. Carbohydr.
Res. 1980, 79, 308.
(4) Garegg, P. J .; J ohansson, R.; Lindh, I.; Samuelsson, B. Carbo-
hydr. Res. 1986, 150, 285.
(5) Classon, B.; Garegg, P. J .; Oscarson, S.; Tide´n, A.-K. Carbohydr.
Res. 1991, 216, 187.
(11) Egan, W.; Tsui, F.-P.; Schneerson, R. Carbohydr. Res. 1980,
79, 271.
(12) Hashimoto, S.-I.; Honda, T.; Ikegami, S. J . Chem. Soc., Chem.
Commun. 1989, 685.
(6) Garegg, P. J .; Oscarson, S.; Tide´n, A.-K. Carbohydr. Res. 1992,
225, 163.
(13) Plante, O. J .; Andrade, R. B.; Seeberger, P. H. Org. Lett. 1999,
1, 211.
10.1021/jo001302m CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/22/2001

Syntheses of H. influenzae Capsular Polysaccharide
J . Org. Chem., Vol. 66, No. 19, 2001 6235
Sch em e 1. Syn th esis of Typ e f Mon osa cch a r id e
In ter m ed ia tesa
F igu r e 1. Structures of the repeating units of the capsular
polysaccharides of H. influenzae types c8,9 and f.10,11
synthesis, and only few examples of successful synthesis
of anomerically linked oligomers of oligosaccharides have
been reported.14-18
The repeating units of the CPS of H. influenzae types
c (Hic) and f (Hif) contain added complexity in the form
of aminosugars and acetyl substituents, the latter ex-
cluding the use of most ester-protecting groups and too
basic reaction conditions. Also, the diester formation to
give Hic structures was found to be unexpectedly prob-
lematic, because initial attempts to form a disaccharide
phosphodiester using published conditions failed com-
pletely,19 indicating a severe instability of the desired
products.
In this article, the syntheses of a dimer and a trimer
of the repeating unit of types c and f are presented. The
phosphodiesters are formed using the H-phosphonate
method,20 and all structures are synthesized as spacer
glycosides to facilitate the coupling to proteins and
formation of immunogenic glycoconjugates.
a
Key: (i) TBDMS-Cl, imidazole, DMF; (ii) Ph3P, CH2Cl2; (iii)
phthalic anhydride, n-Bu4NCN, toluene, reflux; (iv) Br2, CH2Cl2;
(v) Ac2O, pyridine; (vi) NaCNBH3, HCl/Et2O, THF; (vii)
pNO2PhCH2CH2OH, Et4NBr, DMF, CH2Cl2.
tures, which increased the yield in the deprotection step
considerably.
The synthesis of the starting and the elongating
monomer of type f is summarized in Schemes 1 and 2.
Because the repeating unit of H. influenzae type f is built
up by two GalNAc residues, all of the necessary monosac-
charide intermediates could be synthesized from a single
precursor, ethyl 2-azido-4,6-O-benzylidene-2-deoxy-1-
thio-â-D-galactopyranoside (1) (Scheme 1), obtained via
azidonitration of galactal.21 Initially, attempts were made
to use an even more convergent strategy, among others,
utilizing the azido group as the sole amino group equiva-
lent. However, glycosylations between the R-trichloro-
acetimidate analogue of the azido donor 2 and acceptor
6 gave exclusively the R-linked disaccharide, despite
earlier results showing SN2-type reactions and inverted
anomeric configuration with this type of donor.22 This
result incited the introduction of a participating, â-direct-
ing N-protecting group in the donor, and consequently,
donors 3 and 4 were synthesized. Also, the use of a
common thiodisaccharide for the formation of both the
starting and the elongating monomer was planned, but
once more, stereoselectivity problems were encountered,
this time in the introduction of the spacer moiety. Thus,
coupling between donor 9 and the spacer, 2-(p-nitrophe-
nyl)ethanol, using dimethyl(methylthio)sulfonium tri-
Resu lts a n d Discu ssion
Because of the instability of the anomeric phosphodi-
ester linkage, it was decided to introduce the spacer as
a stable O-glycoside, and accordingly, two disaccharide
precursors were designed and constructed for each sero-
type, a spacer disaccharide starting monomer acceptor
(15 and 22) and an anomeric monoester hydrogen phos-
phonate disaccharide elongating monomer (12 and 27)
containing a temporary protecting group to allow con-
tinued elongations. As a spacer, a p-aminophenylethyl
group was originally chosen, the phenyl group allowing
easy UV detection and also the possibility of radioactive
labeling by 135I. However, hydrogenolysis to deprotect the
synthesized type f structures also reduced the aromatic
system in the spacer. Consequently, a nonaromatic
spacer, 2-aminoethanol, was chosen for the type c struc-
(14) Nikolaev, A. V.; Rutherford, T. J .; Ferguson, M. A. J .; Brima-
combe, J . S. Bioorg. Med. Chem. Lett. 1994, 4, 785.
(15) Nikolaev, A. V.; Rutherford, T. J .; Ferguson, M. A. J .; Brima-
combe, J . S. J . Chem. Soc., Perkin Trans. 1 1995, 1977.
(16) Nikolaev, A. V.; Rutherford, T. J .; Ferguson, M. A. J .; Brima-
combe, J . S. J . Chem. Soc., Perkin Trans. 1 1996, 1559.
(17) Nilsson, M.; Norberg, T. J . Chem. Soc., Perkin Trans. 1 1998,
1699.
(18) Hansson, J .; Oscarson, S. Curr. Org. Chem. 2000, 4, 535.
(19) Helland, A.-C. Doctoral Thesis, Stockholm University, 1991.
(20) Stawinski, J . In Handbook of Organophosphorus Chemistry;
Engel, R., Ed.; Marcel Dekker Inc.: New York, 1992; p 377.
(21) Lemieux, R. U.; Ratcliffe, R. M. Can. J . Chem. 1979, 57, 1244.
(22) Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.
1994, 50, 21.

6236 J . Org. Chem., Vol. 66, No. 19, 2001
Hansson et al.
Sch em e 2. Syn th esis of Typ e f Sta r tin g a n d
Elon ga tin g Disa cch a r id e Mon om er sa
corresponding bromo sugar donor 4 by treatment with
bromine. Acetylation of 1 inserted the native acetyl
substituent and gave 5 (99%), which was processed into
two different 4-OH acceptors, 6 and 8. Reductive opening
of the benzylidene acetal26 yielded the thioglycoside
acceptor 6 (83%), whereas coupling with the spacer, 2-(4-
nitrophenyl)ethanol, using halide-assisted conditions
(f7, 70%), followed by benzylidene opening, gave the
R-linked spacer glycoside acceptor 8 (89%). Attempts to
use 6 as donor precursor instead of 5 in a halide-assisted
glycosylation with the spacer were accompanied by acetyl
migration, and a mixture of 8 and its 4-O-acetyl analogue
(2:1) was obtained.
These monosaccharide intermediates were now utilized
in the formation of the disaccharide monomers (Scheme
2). Silver triflate-promoted orthogonal glycosylation be-
tween the bromo sugar donor 4 and the thioglycoside
acceptor 6 gave the â-linked disaccharide 9 (76%). The
coupling between the two thioglycosides, 3 and 6, was
also tried. The azido group is more deactivating than the
N-phthaloyl group,27 and hence, 6 should act as the
acceptor in such a glycosylation, but the reactivity
difference was found to be too small to ensure an effective
orthogonal coupling. At the best, 31% of 9 could be
isolated using N-iodosuccinimide/trimethylsilyl triflate
(NIS/TMSOTf) 28 as promoter at -50 °C. The glycosyla-
tion between the thioglycoside donor 3 and the spacer
glycoside acceptor 8 with DMTST as promoter was
straightforward, and disaccharide 13 was obtained in
79% yield. Because no more glycosylations were to be
performed, the stereodirecting qualities of the azido
(nonparticipating) and N-phthaloyl (participating) groups
were not needed any more, and these were accordingly
changed into the acetamido groups present in the target
molecules. In a five-step sequence, 9 and 13 were
converted to 10 (74%) and 14 (78%), respectively. Hy-
drolysis of the thioglycoside in 10 gave the hemiacetal
11 (81%). No stereoselectivity problems in the formation
of the anomeric H-phosphonate elongating monomer were
encountered, because 11 was isolated as the pure R-ano-
mer, from which the R-monoester H-phosphonate 12
could easily be formed in 87% yield by treatment with
triimidazolylphosphine followed by basic hydrolysis.29
Removal of the tert-butyldimethylsilyl (TBDMS) group
from 14 provided the starting monomer acceptor 15
(80%).
a
Key: (i) AgOTf, DTBMP, CH2Cl2, -50 °C; (ii) H2NCH2CH2NH2,
toluene/EtOH, reflux; (iii) Ac2O, pyridine; (iv) Ph3P, CH2Cl2; (v)
H2O, THF, reflux; (vi) NIS, H2O, acetone; (vii) PCl3, imidazole,
Et3N, MeCN; (viii) Et3NHHCO3, H2O; (ix) DMTST, DTBMP,
CH2Cl2; (x) Et3N(HF)3, THF.
flate23 (DMTST) as promoter in diethyl ether gave mainly
the â-glycoside (R/â 2:5). The use of the corresponding
bromo sugar donor and halide-assisted conditions24 did
not yield any product at all. Therefore, it was decided to
introduce the spacer at the monosaccharide level, where
the halide-assisted glycosylation was found to work well
(see below).
Silylation, with tert-butyldimethylsilyl chloride, of the
3-OH group in 1 to give 2 (94%) introduced a temporary
protecting group to allow later elongation at this position
(Scheme 1). The transformation of the azido group into
a N-phthaloyl group, using a slightly modified version
of the procedure published by Garcia et al.,25 yielded
thioglycoside donor 3 (88%) with a participating group
to ascertain â-linkage formation in subsequent glycosy-
lations. This donor can also effectively be turned into the
The syntheses of the starting and the elongating
monomer of type c are summarized in Schemes 3 and 4.
Originally also, the two type c monomers were planned
to be synthesized from the same thioglycoside disaccha-
ride, but the less efficient tin-promoted allylation of ethyl
1-thiogalactopyranosides30 as compared to the corre-
sponding trimethylsilylethyl (TMSE) O-glycoside,31 in
combination with problems of stereoselectivity in the
introduction of the spacer at the disaccharide level, made
(26) Garegg, P. J .; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982,
108, 97.
(27) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.;
Wong, C.-H. J . Am. Chem. Soc. 1999, 121, 734.
(28) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J . H.
Tetrahedron Lett. 1990, 31, 1331.
(29) Garegg, P. J .; Regberg, T.; Stawinski, J .; Stro¨mberg, R. Chem.
Scr. 1986, 26, 59.
(30) Das, S. K.; Ghosh, R.; Roy, N. J . Carbohydr. Chem. 1993, 12,
693.
(23) Fu¨gedi, P.; Garegg, P. J . Carbohydr. Res. 1986, 149, C9.
(24) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; J ames, K. J . Am.
Chem. Soc. 1975, 97, 4056.
(25) Garcia, J .; Vilarrasa, J .; Bordas, X.; Banaszek, A. Tetrahedron
Lett. 1986, 27, 639.
(31) Nilsson, U.; Ray, A. K.; Magnusson, G. Carbohydr. Res. 1994,
252, 117.

Syntheses of H. influenzae Capsular Polysaccharide
J . Org. Chem., Vol. 66, No. 19, 2001 6237
Sch em e 3. Syn th esis of a Typ e c Sp a cer
Mon osa cch a r id e Accep tor a
obtained as an R/â-mixture (R/â 8:1), which could not be
separated at this stage (Scheme 3). Deacetylation and
methyl triflate-promoted glycosylation35 with the thiogly-
coside donor 19 gave in good yield and with stereospeci-
ficity the â-(1f3)-linked disaccharide 20 (75%), isolated
now as the pure R-galactoside (Scheme 4). Use of DMTST23
as promoter in this coupling gave the elimination product
of the donor as a main byproduct, but this formation
could be suppressed to a large extent by the use of methyl
triflate as promoter. Transformation of the phthalimido
group into an acetamido group was accomplished using
ethylenediamine, followed by acetylation to give 21 (49%).
A major side reaction was base-promoted O-4fO-3 silyl
migration36,37 to give the 4′-O-acetyl-3′-O-TBDMS isomer
of 21 as a byproduct in 30% yield, which explains the
rather low yield of 21 in this transformation. However,
the choice of suitable temporary protecting groups is
rather limited, and the excellent properties shown by the
TBDMS group later in the synthesis more than compen-
sated for this loss of material at this early stage. Finally,
removal of the silyl protecting group by treatment with
Et3N(HF)3 yielded the starting monomer 22 (80%).
a
Key: (i) Br2, CH2Cl2; (ii) HOCH2CH2N3, Et4NBr, DMF,
CH2Cl2; (iii) NaOMe, MeOH/CH2Cl2.
Sch em e 4. Syn th esis of Typ e c Sta r tin g a n d
Elon ga tin g Disa cch a r id e Mon om er sa
Coupling between the same donor as above, 19, and
the known TMSE glycoside acceptor 23,31 this time using
DMTST-promotion (without elimination byproducts),
gave disaccharide 24 (86%), which was transformed into
the acetamido analogue 25 (63%), as discussed for
derivative 21 above. Also here, silyl migration was a
major side reaction (27%). The TMSE glycoside in 25 was
hydrolyzed38 to give the free hemiacetal and allow the
introduction of the anomeric H-phosphonate. Fortu-
nately, the pure R-anomer 26 (58%) could be crystallized
out from the obtained R/â-mixture, which directly solved
the steroselectivity problem because 26 could be phos-
phonylated29 without epimerization to give the R-linked
hydrogenphosphonate monoester 27 (89%).
With all of the starting and elongating monomers in
hand, the formation of phosphodiester linkages was now
attempted (Schemes 5 and 6). The H-phosphonate ap-
proach was chosen because earlier experiences showed
this method to have advantages over the phopshoramid-
ite method in the construction of anomeric phosphodi-
ester linkages.39 Early studies by Garegg and Helland
indicated that it was not possible to form the Hic (1f4)-
phosphodiester-linked disaccharide by using the normal
approach with an anomeric H-phosphonate monoester as
electrophile.19 To try to solve this problem, a new
methodology, in which a 4-O-linked nonanomeric H-
phosphonate monoester was used as nucleophile in
glycosylation-type couplings to glycosyl donors, was
developed to give good yields of diester phosphates but
as R/â-mixtures.40 Initial experiments with disaccharide
monomers gave exclusively the R-anomer, but these
results were not reproducible. This prompted us to
investigate the original reaction more thoroughly by 31P
NMR. It was then found that the phosphonate diester
a
Key: (i) MeOTf, DTBMP, CH2Cl2; (ii) H2NCH2CH2NH2,
toluene/EtOH, reflux; (iii) Ac2O, pyridine; (iv) Et3N(HF)3, THF;
(v) DMTST, DTBMP, CH2Cl2; (vi) TFA/CH2Cl2 2:1; (vii) PCl3,
imidazole, Et3N, MeCN; (viii) Et3NHHCO3, H2O.
the change to a less convergent synthesis of these two
blocks necessary.32
Although it is a galactopyranose, which normally is
quite R-selective in glycosylations because of the axial
4-substituent covering the â-side,33 the chlorosugar of 1634
gave mainly the â-glycoside (R/â 1:2) in a silver triflate-
promoted glycosylation with the spacer 2-azidoethanol.
Application of halide-assisted glycosylation conditions24
using the bromo sugar gave, as expected, much better
R-selectivity, but still, the spacer glycoside 17 was
(35) Lo¨nn, H. Carbohydr. Res. 1985, 139, 105.
(36) J ones, S. S.; Reeves, C. B. J . Chem. Soc., Perkin Trans. 1 1979,
2762.
(37) Krog-J ensen, C.; Oscarson, S. Carbohydr. Res. 1998, 308, 287.
(38) J ansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J .; Magnusson, G.;
Dahme´n, J .; Noori, G. J . Org. Chem. 1988, 53, 5629.
(39) Westerduin, P.; Veeneman, G. H.; van der Marel, G. A.; van
Boom, J . H. Tetrahedron Lett. 1986, 27, 6271.
(32) Hansson, J .; Doctoral Thesis, Stockholm University, 2000.
(33) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155.
(34) Kong, F.; Lu, D.; Zhou, S. Carbohydr. Res. 1990, 198, 141.
(40) Garegg, P. J .; Hansson, J .; Helland, A.-C.; Oscarson, S. Tetra-
hedron Lett. 1999, 40, 3049.

6238 J . Org. Chem., Vol. 66, No. 19, 2001
Hansson et al.
Sch em e 5. Syn th esis of a Dim er a n d a Tr im er of th e Rep ea tin g Un it of H. in flu en za e Typ e f CP Sa
a
Key: (i) Piv-Cl, pyridine; (ii) I2, H2O, -40 °C; (iii) Et3N(HF)3, THF; (iv) H2, Pd/C, EtOH/HOAc/H2O.
Sch em e 6. Syn th esis of a Dim er a n d a Tr im er of th e Rep ea tin g Un it of H. in flu en za e Typ e c CP Sa
a
Key: (i) Piv-Cl, pyridine; (ii) I2, H2O, -40 °C; (iii) Et3N(HF)3, THF; (iv) H2, Pd/C, EtOH/HOAc/H2O.
was formed almost quantitatively but that this interme-
phosphodiester. In the type f series, coupling of H-
phosphonate 12 with acceptor 15, employing pivaloyl
chloride as coupling reagent, followed by I2/H2O oxidation
yielded the phosphate diester 28 (81%) (Scheme 5).
Removal of the TBDMS group without any cleavage of
diate was lost in the consecutive oxidation. Optimization
of the oxidation step with monosaccharide monomers,
mainly through lowering the temperature, then led to a
working methodology for the construction of the desired

Syntheses of H. influenzae Capsular Polysaccharide
J . Org. Chem., Vol. 66, No. 19, 2001 6239
was performed under reduced pressure at less than 40 °C (bath
temperature). NMR spectra were recorded at 25 °C and 300
or 400 MHz (1H) and 75 or 100 MHz (13C) in CDCl3 with Me4-
Si as the internal standard (δ ) 0), unless otherwise stated.
Mass spectra were recorded in the negative ion mode (ESI).
TLC was performed on silica gel 60 F254 with detection by UV
light and charring with 8% sulfuric acid. Silica gel (0.040-
0.063 mm) was used for column chromatography.
Eth yl 2-Azid o-4,6-O-ben zylid en e-2-d eoxy-1-th io-â-D-ga -
la ctop yr a n osid e (1). A catalytic amount of 1 M NaOMe was
added to a solution of ethyl 2-azido-3,4,6-tri-O-acetyl-2-deoxy-
1-thio-â-D-galactopyranoside41 (2.56 g, 6.8 mmol) in MeOH/
CH2Cl2 (7:3, 50 mL). After being stirred at room temperature
for 1.5 h, the mixture was treated with Dowex 50 (H+) resin,
filtered, and concentrated. The residue was dissolved in MeCN
(45 mL) after which R,R-dimethoxytoluene (2.0 mL, 14 mmol)
and p-toluenesulfonic acid (44 mg) were added, and the
resulting mixture was stirred at room temperature for 20 min.
Neutralization with Et3N, concentration, and purification on
silica gel (toluene/EtOAc, 2:1) gave 1 as a solid (2.10 g, 6.2
mmol, 91%). [R]D -45 (c 1.0, CHCl3). mp 106-109 °C. 13C
NMR: δ 14.9, 23.9, 63.2, 69.0, 69.7, 73.1, 74.6, 83.3, 101.3,
126.2-137.1. 1H NMR: δ 1.34 (t, 3H), 2.72-2.87 (m, 2H), 3.43
(d, 1H), 3.62-3.65 (m, 2H), 4.00 (dd, 1H), 4.19 (dd, 1H), 4.27
(d, 1H, J ) 9.6 Hz), 4.32 (dd, 1H), 5.54 (s, 1H,), 7.37-7.51 (m,
5H). Anal. Calcd for C15H19N3O4S: C, 53.40; H, 5.68; N, 12.45.
Found: C, 53.55; H, 5.70; N, 12.42.
Eth yl 4,6-O-Ben zylid en e-3-O-(ter t-bu tyld im eth ylsilyl)-
2-d eoxy-2-p h th a lim id o-1-th io-â-D-ga la ctop yr a n osid e (3).
Imidazole (1.17 g, 17 mmol) was added to a solution of 1 (2.91
g, 8.6 mmol) and tert-butyldimethylsilyl chloride (2.34 g, 16
mmol) in DMF (23 mL). The resulting mixture was stirred at
70 °C for 1.5 h and then taken up in toluene, washed with
H2O, NaHCO3(aq), and H2O, dried, filtered, and concentrated.
Purification on silica gel using toluene/EtOAc (10:1) as eluent
afforded 3.65 g (8.1 mmol, 94%) of 2 as a white solid. [R]D -22
(c 1.1, CHCl3).; mp 106-108 °C. 13C NMR: δ -4.7, -4.6, 15.0,
18.0, 23.6, 25.7, 63.0, 69.1, 69.8, 74.3, 75.7, 83.3, 100.6, 125.9-
137.6. Compound 2 (3.65 g, 8.1 mmol) and triphenylphosphine
(2.54 g, 9.7 mmol) were dissolved in dry CH2Cl2 (30 mL) under
N2 and stirred for 20 h at room temperature, after which the
reaction mixture was concentrated. Phthalic anhydride (1.80
g, 12 mmol) and n-tetrabutylammonium cyanide (217 mg, 0.81
mmol) were added to the residue dissolved in toluene (40 mL),
and the mixture was refluxed for 44 h. An additional amount
of n-tetrabutylammonium cyanide (217 mg, 0.81 mmol) was
then added, and the mixture was concentrated and purified
on silica gel (toluene/EtOAc, 10:1) to give 3 in 88% yield (3.97
g, 7.1 mmol). Crystallization from EtOH gave the title com-
pound as a white solid. mp 156-158 °C. [R]D +50 (c 0.85,
CHCl3). 13C NMR: δ -5.0, -4.4, 14.8, 17.6, 22.8, 25.2, 52.4,
69.2, 69.5, 70.0, 76.1, 80.3, 122.7-137.7, 167.4, 168.5. 1H
NMR: δ 0.24 (s, 3H), 0.25 (s, 3H), 1.47 (t, 3H), 2.86-3.17 (m,
2H), 3.88 (d, 1H, J ) 1.1 Hz), 4.30 (dd, 1H, J ) 1.9, 12.4 Hz),
4.39 (d, 1H, J ) 1.9 Hz), 4.63 (dd, 1H, J ) 1.7, 12.4 Hz), 4.99
(m, 2H, J ) 6.6 Hz), 5,56 (m, 1H), 5.79 (s, 1H), 7.60-8.11 (m,
9H). Anal. Calcd for C29H37NO6SSi: C, 62.67; H, 6.71; N, 2.52.
Found: C, 62.74; H, 6.80; N, 2.48.
2-(4-Nitr op h en yl)eth yl 3-O-Acetyl-2-a zid o-4,6-O-ben -
zylid en e-2-d eoxy-r-D-ga la ctop yr a n osid e (7). Compound 1
(1.56 g, 4.6 mmol) was dissolved in pyridine (30 mL) and acetic
anhydride (6 mL). After 2.5 h, the reaction mixture was
coconcentrated with toluene and purified on silica gel (toluene/
EtOAc, 5:1) to give 1.74 g (4.59 mmol, 99%) of 5. [R]D -0.95 (c
0.95, CHCl3). 13C NMR: δ 15.0, 20.9, 23.7, 59.5, 69.0, 69.5,
72.7, 73.9, 83.6, 100.8, 126.1-137.3, 170.1. To a solution of 5
(614 mg, 1.6 mmol) in dry CH2Cl2 (20 mL) under argon was
added bromine (0.1 mL, 1.9 mmol). After 5 min, the solution
was coconcentrated with toluene and dried in a vacuum. The
residue was dissolved in dry CH2Cl2 (4 mL), and 2-(p-
nitrophenyl)ethanol (812 mg, 4.9 mmol), 9 drops of DMF, and
the phosphate was achieved by treatment with Et3N(HF)3
to give the dimer acceptor 29 (88%).
As was found also for the continued formation of higher
oligomers of the H. influenzae type c structure, the yield
in the formation of the trimer was lowered quite drasti-
cally as compared to that of the dimer formation. In the
first attempt, using the same conditions as for the
formation of 28, no trimer at all could be isolated; only a
product, 33 (85%, Scheme 3), resulting from the cleavage
of the phosphate diester bond in 29 was obtained. Once
more, it was mainly the conditions during the oxidation
that caused decomposition. Through dilution of the reac-
tion mixture with pyridine before the iodine and water
was added, less hydrolytic conditions were established,
and the trimer 31 could be isolated in an acceptable yield
of 37%, still accompanied by the formation of 33 (40%).
Finally, deprotection by catalytic hydrogenolysis of
dimer 29 and desilylation and subsequent hydrogenolysis
of trimer 31 were performed. These steps also reduce the
nitro group to give an amino function, which is essential
for the conjugation of the structures to a protein. Some-
times, hydrogenolysis of phosphate-containing deriva-
tives is ineffective; however, with these compounds, the
reaction proceeded most effectively, even reducing the
aromatic ring system to give the target products as their
cyclohexylamino derivatives, 30 (56%) and 32 (64%),
respectively.
In the type c series, monomer H-phosphonate 27 was
coupled to the starting monomer 22 to give the dimeric
phosphodiester 34 in 71% yield (Scheme 6). Desilylation
then gave the 4′′′-OH intermediate 35 (88%), which now,
as in the type f synthesis, could be either elongated or
deprotected.
One-step deprotection by catalytic hydrogenolysis both
removed all the benzyl-protecting groups and concomi-
tantly reduced the spacer azido group to give 36 (92%).
In the continued elongation of 35, the same elongating
monomer 27 and the optimized coupling conditions for
the formation of the type f trimer were used. Although
this appeared to be most promising initially on TLC,
materials were lost, as discussed, during oxidation and
workup. However, the trimer 37 could be isolated in a
fair 36% yield.
Once more, the removal of the silyl-protecting group
was high-yielding to give the 4′′′′′-OH derivative 38 with
the possibility of further elongation. Catalytic hydro-
genolysis then smoothly gave the deprotected amino-
containing trimer 39 in 77% overall yield.
In conclusion, the syntheses of complex spacer glyco-
sides of the dimers and trimers of the repeating units of
the CPSs of H. influenzae type c and f have been
accomplished in a stereospecific manner. The structures
contain the natively found 3-O-acetyl substituents. In the
formation of the anomeric interglycosidic phosphodiester
linkages, care has to be taken because of the instability
of the products, as was evident by initial failures. With
the oxidation step performed at low temperatures and
after dilution, the formation of the dimers could be
obtained in 71% and 88% yields, respectively, and the
subsequent elongation to the trimers could be performed
in 36% and 37% yields. The synthetic scheme also allows
for further elongation.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. Organic
solutions were dried over MgSO4 before concentration, which
(41) Paulsen, H.; Rauwald, W.; Weichert, U. Liebigs Ann. Chem.
1988, 75.

6240 J . Org. Chem., Vol. 66, No. 19, 2001
Hansson et al.
4 Å molecular sieves were added. The mixture was cooled to 0
°C, and Et4NBr (510 mg, 2.4 mmol) was added. Stirring under
argon was continued at room temperature for 23 h. Then, the
mixture was filtered through Celite, concentrated, and purified
on a silica gel column (toluene/EtOAc, 9:1) to give 545 mg (1.1
mmol, 70%) of 7. [R]D +179 (c 0.90, CHCl3). 13C NMR: δ 20.9,
35.7, 57.0, 62.6, 68.1, 68.8, 69.1, 73.2, 98.3 (J C,H ) 171 Hz),
acetic anhydride (6 mL) at room temperature for 1 day,
coconcentrated with toluene, and purified on silica gel (gradi-
ent of CHCl3/MeOH, 30:1-25:1) to give 597 mg (0.74 mmol,
84%) of 10. [R]D -15 (c 0.67, CHCl3). 13C NMR: δ -4.7, -4.6,
14.7, 18.0, 20.7, 23.1, 23.7, 24.1, 25.6, 49.1, 56.8, 65.7, 67.5,
68.9, 68.9, 71.9, 73.0, 74.0, 76.2, 77.5, 84.4, 98.0, 100.4, 125.9-
138.1, 169.6, 171.1, 171.4. To a stirred solution of 10 (547 mg,
0.68 mmol) in acetone (12 mL) containing water (0.12 mL) was
added N-iodosuccinimide (230 mg, 1.0 mmol). After 40 min,
the reaction was diluted with CH2Cl2, washed twice with 1 M
Na2S2O3 and once with water, dried, filtered, and concentrated.
Silica gel chromatography (CHCl3/MeOH 16:1) gave 11 (425
mg, 0.56 mmol, 81%) as its pure R-anomer. [R]D +32 (c 0.87,
CHCl3). 13C NMR: δ -4.8, -4.6, 18.0, 20.7, 23.2, 23.7, 25.5,
48.0, 56.7, 65.7, 67.6, 69.0, 69.3, 69.6, 70.3, 72.8, 73.0, 76.2,
91.9 (J C,H ) 171 Hz), 98.1, 100.4, 125.8-138.1, 169.9, 171.4.
To a stirred solution of imidazole (163 mg, 2.4 mmol) in MeCN
(4.5 mL) at 0 °C was added PCl3 (64 µL, 0.74 mmol) and Et3N
(358 µL, 2.6 mmol). Stirring was continued at 0 °C for 15 min.
Compound 11 (154 mg, 0.17 mmol) in MeCN (4.5 mL) was then
added dropwise during 30 min. After that time, the reaction
mixture was stirred at room temperature for 10 min, quenched
with 1 mL of 1 M triethylammonium bicarbonate (TEAB), and
after an additional 5 min, concentrated. From the residue was
evaporated pyridine/Et3N (4:1, 15 mL). The residue was then
dissolved in CHCl3 and washed four times with 0.5 M TEAB;
the organic layer was dried by filtration through cotton,
concentrated, and passed through a silica gel column (CHCl3
containing 1% Et3N, gradient of 0-9% MeOH) to give 158 mg
(0.15 mmol, 87%) of 12. [R]D +35 (c 0.68, CHCl3). 13C NMR: δ
1
100.5, 123.4-146.2, 170.2. H NMR: δ 2.16 (s, 1H), 3.05 (dt,
2H), 3.39 (d, 1H, J ) 1.1 Hz), 3.78-4.0 (m, 4H), 4.16 (dd, 1H,
J ) 1.7, 12.6 Hz), 4.36 (dd, J ) 1.1, 3.6 Hz), 5.05 (d, 1H, J )
3.3 Hz), 5.25 (dd, 1H, J ) 3.3, 11.3 Hz), 5.49 (s, 1H), 7.34-
8.20 (m, 9H). Anal. Calcd for C23H24N4O8: C, 57.02; H, 4.99;
N, 11.56. Found: C, 56.78; H, 4.93; N, 11.37.
Eth yl 4,6-O-Ben zylid en e-3-O-(ter t-bu tyld im eth ylsilyl)-
2-d eoxy-2-p h th a lim id o-â-D-ga la ctop yr a n osyl-(1f4)-3-O-
a cetyl-2-a zid o-6-O-ben zyl-2-d eoxy-1-th io-â-D-ga la ctop y-
r a n osid e (9). A solution of 5 (2.11 g, 5.6 mmol), NaCNBH3
(3.54 g, 56 mmol), and 3 Å molecular sieves in THF (50 mL)
was stirred under N2 for 30 min. HCl in diethyl ether was
added until the gas evolution ceased (7 mL). After 10 min,
when TLC indicated complete conversion of the starting
material into one single product, the reaction mixture was
filtered through a layer of Celite, washed with THF, concen-
trated, and purified on silica gel (toluene/EtOAc, 4:1) to give
a colorless oil. Another silica gel column (light petroleum/
EtOAc, 2:1) was necessary in order to get pure 6 (1.75 g, 4.6
mmol, 83%). [R]D -27 (c 0.93, CHCl3). 13C NMR: δ 15.1, 21.0,
24.6, 60.2, 67.6, 69.7, 73.7, 75.4, 76.4, 84.4, 127.6-137.1, 169.8.
To a solution of 3 (1.20 g, 2.16 mmol) in dry CH2Cl2 (20 mL)
under N2 was added bromine (0.12 mL, 2.4 mmol). After 5 min,
the solution was coconcentrated with toluene and dried in a
vacuum. The residue (4) was dissolved together with 6 (633
mg, 1.66 mmol), 2,6-di-tert-butyl-4-methylpyridine (DTBMP)
(409 mg, 2.0 mmol), and 4 Å molecular sieves in dry CH2Cl2
(30 mL) and stirred under N2 at -50 °C for 40 min. Silver
triflate (640 mg, 2.5 mmol) was then added in small portions
during 5 min. After 15 min, Et3N was added, and the reaction
mixture was filtered through Celite, concentrated, and purified
on a silica gel column (toluene/EtOAc, 8:1) to give 9 (1.10 g,
1.26 mmol) in 76% yield. [R]D +31 (c 1.0, CHCl3). 13C NMR: δ
-4.8, -4.5, 14.9, 17.7, 20.6, 22.4, 25.3, 54.2, 60.6, 66.3, 67.7,
68.2, 68.8, 72.2, 73.0, 75.2, 75.7, 77.1, 82.9, 98.3, 100.7, 122.5-
138.1, 167.1, 168.8, 170.0. 1H NMR: δ 0.19 (s, 3H), 0.22 (s,
3H), 0.86 (s, 9H), 1.44 (t, 3H), 2.30 (s, 3H), 2.64-2.93 (m, 2H),
3.32 (dd, 1H, J ) 10.2, 10.2 Hz), 3.69 (s, 1H), 3.77 (m, 2H),
4.00 (dd, 1H), 4.18 (dd, 1H), 4.25 (d, 1H, J ) 9.9 Hz), 4.27-
4.35 (m, 3H), 4.76 (q, 2H), 4.79 (dd, 1H, J ) 8.2, 11.0 Hz),
4.86 (dd, 1H, J ) 2.5, 10.2 Hz), 5.07 (dd, 1H, J ) 3.8, 11.0
Hz), 5.30 (d, 1H, J ) 8.2 Hz), 5.73 (s, 1H), 7.41-8.09 (m, 14H).
Anal. Calcd for C44H54N4O11SSi: C, 60.39; H, 6.22; N, 6.40.
Found: C, 60.29; H, 6.33; N, 6.28.
Tr ieth yla m m on iu m 2-Aceta m id o-4,6-O-ben zylid en e-3-
O-(ter t-bu tyldim eth ylsilyl)-2-deoxy-â-D-galactopyr an osyl-
(1f4)-2-a ceta m id o-3-O-a cetyl-6-O-ben zyl-2-d eoxy-r-D-ga -
la ctop yr a n osyl Hyd r ogen p h osp h on a te (12). Compound 9
(740 mg, 0.85 mmol) was dissolved in EtOH/toluene (3:1, 15
mL). Addition of ethylenediamine (2.8 mL, 42 mmol) followed
by reflux for 19 h gave the free amino compound. Concentra-
tion left a syrup which was subsequently treated with pyridine
(15 mL) and acetic anhydride (5 mL) for 4 h at room
temperature. Coconcentration with toluene followed by silica
gel chromatography (toluene/EtOAc, 3:1) gave 588 mg (0.75
mmol, 88%) of the 2′-acetamido derivative as a white foam.
[R]D +16 (c 0.91, CHCl3). 13C NMR: δ -4.7, -4.6, 14.9, 18.0,
20.8, 23.6. 25.0, 25.5, 56.8, 61.6, 65.9, 67.6, 68.8, 68.9, 71.5,
73.0, 75.3, 76.0, 77.5, 83.8, 97.9, 100.4, 125.1-138.1, 170.1,
170.5. This compound (695 mg, 0.88 mmol) in CH2Cl2 (7 mL)
was treated overnight with triphenylphosphine (278 mg, 1.1
mmol) to give the intermediate phosphazene. Concentration
left a white foam which was dissolved in THF/water (5:1, 15
mL) and refluxed for 2 days. The reaction mixture was then
diluted with CHCl3 and water, the organic layer was separated
off, and the aqueous layer was washed with additional CHCl3.
The combined organic phases were dried, filtered, and con-
centrated. The residue was treated with pyridine (20 mL) and
-4.7, -4.6, 9.0, 18.0, 20.7, 23.2, 23.8, 25.6, 45.5, 47.9 (3J C,P
)
5.2), 56.8, 65.8, 67.6, 69.0, 69.2, 70.7, 70.8, 72.6, 72.8, 76.3,
93.5 (2J C,P ) 4.9), 98.2, 100.4, 125.9-138.3, 170.0, 171.2, 171.4.
1H NMR: δ 0.05 (s, 3H), 0.09 (s, 3H), 0.91 (s, 9H), 1.21 (t, 9H),
1.91 (s, 3H), 2.05 (s, 3H), 2.13 (s, 3H), 3.23 (m, 1H), 3.44 (s,
1H), 3.56 (dd, 1H), 3.81 (dd, 1H), 3.95 (dd, 1H), 3.99 (d, 1H, J
) 3.6 Hz), 4.10 (dd, 1H), 4.19 (d, 1H, J ) 1.9 Hz), 4.28 (dd,
1H), 4.54 (dd, 2H), 4.73 (dt, 1H), 4.98 (dd, 1H, J ) 3.6, 10.4
Hz), 5.19-5.23 (m, 2H, J ) 8.2 Hz), 5.51 (s, 1H), 5.56 (dd, 1H,
J ) 3.3, 8.5 Hz), 6.12 (d, 1H), 6.50 (d, 1H), 6.96 (d, 1H, J )
629 Hz), 7.20-7.57 (m, 10H). 31P NMR (CH2Cl2): δ 0.54. MS:
calcd for C44H70N3O14PSi [M - Et3NH]-, 821.3; found, 821.3.
2-(4-Nitr op h en yl)eth yl 4,6-O-Ben zylid en e-3-O-(ter t-bu -
t yld im et h ylsilyl)-2-d eoxy-2-p h t h a lim id o-â-D-ga la ct op y-
r a n osyl-(1f4)-3-O-a cetyl-2-a zid o-6-O-ben zyl-2-d eoxy-r-D-
ga la ctop yr a n osid e (13). A solution of 7 (477 mg, 0.98 mmol),
NaCNBH3 (619 mg, 9.8 mmol), and 3 Å molecular sieves in
THF (20 mL) was stirred under argon for 20 min. HCl in
diethyl ether was added until the gas evolution ceased (3.5
mL). After 30 min, the reaction mixture was worked up as
described for compound 6. Two silica gel columns (toluene/
EtOAc, 3:1) gave 89% (426 mg, 0.88 mmol) of 8. [R]D +141 (c
0.99, CHCl3). 13C NMR: δ 20.9, 35.7, 57.0, 68.0, 68.5, 68.5,
70.0, 70.5, 73.7, 98.1, 123.4-146.2, 169.8. A solution of 8 (376
mg, 0.77 mmol), 3 (687 mg, 1.2 mmol), 2,6-di-tert-butyl-4-
methylpyridine (DTBMP) (159 mg, 0.77 mmol), and 4 Å
molecular sieves in dry CH2Cl2 (20 mL) was stirred at room
temperature under N2 for 30 min. DMTST (600 mg, 2.3 mmol)
was then added, and after 30 min, an additional amount of
DMTST (100 mg, 0.39 mmol) and donor 3 (86 mg, 0.15 mmol)
was added. After 1 h, the reaction was quenched with Et3N,
and the reaction mixture was filtered (Celite), concentrated,
and purified on silica gel (toluene/EtOAc, 9:1) to give 79% (599
mg, 0.61 mmol) of 13. [R]D +86 (c 0.58, CHCl3). mp 202-204
°C. 13C NMR: δ -4.9, -4.5, 17.7, 20.5, 25.2, 35.6, 54.1, 58.2,
66.1, 67.4, 67.6, 68.8, 69.0, 69.6, 70.8, 73.0, 73.5, 75.6, 97.1,
98.4, 100.7, 122.4-146.4, 167.3, 168.7, 170.1. 1H NMR: δ 0.22
(s, 3H), 0.25 (s, 3H), 0.89 (s, 9H), 2.29 (s, 3H), 3.18 (t, 2H),
3.45 (dd, 1H, J ) 3.3, 11.0 Hz), 3.70 (s, 1H), 3.74-3.95 (m,
4H), 4.09 (m, 1H), 4.20 (dd, 1H), 4.27 (d, 1H, J ) 3.0 Hz), 4.29
(d, 1H, J ) 4.1 Hz), 4.33 (dd, 1H), 4.73 (dd,), 4.77 (dd, 1H, J )
8.2, 10.7 Hz), 5.01 (d, 1H, J ) 3.8 Hz), 5.04 (dd, 1H, J ) 3.8,
11.0 Hz), 5.26 (dd, 1H, J ) 2.7, 11.3 Hz), 5.28 (d, 1H, J ) 8.5
Hz), 5.75 (s, 1H), 7.47-8.33 (m, 18H). Anal. Calcd for

Syntheses of H. influenzae Capsular Polysaccharide
J . Org. Chem., Vol. 66, No. 19, 2001 6241
C50H57N5O14Si: C, 61.27; H, 5.86; N, 7.15. Found: C, 61.16;
H, 5.97; N, 7.00.
4.71 (m, 6H), 4.84 (d, 1H, J ) 3.66 Hz), 5.27 (dd, 1H, J ) 3.8,
10.6 Hz), 7.23-7.34 (m, 15H). Anal. Calcd for C31H35N3O7: C,
66.30; H, 6.28; N, 7.48. Found: C, 66.17; H, 6.41; N, 7.46.
2-(4-Nitr oph en yl)eth yl 2-Acetam ido-4,6-O-ben zyliden e-
2-d eoxy-â-D-ga la ct op yr a n osyl-(1f4)-2-a cet a m id o-3-O-
a cetyl-6-O-ben zyl-2-d eoxy-r-D-ga la ctop yr a n osid e (15). A
solution of 13 (599 mg, 0.61 mmol) and ethylenediamine (2.0
mL, 30 mmol) in EtOH/toluene (3:2, 15 mL) was refluxed for
24 h and then coconcentrated with toluene. The residue was
treated with pyridine (15 mL) and Ac2O (5 mL) for 1 day.
Coconcentration with toluene left a solid, which was purified
on silica gel (toluene/EtOAc, 3:1) to give the 2′-NHAc derivative
in 86% yield (467 mg, 0.52 mmol). [R]D +78 (c 0.63, CHCl3).
13C NMR: δ -4.7, -4.6, 18.0, 20.8, 23.7, 25.5, 35.7, 56.8, 58.2,
65.9, 67.6, 67.7, 68.8, 69.2, 69.9, 70.4, 72.6, 73.0, 76.0, 97.6,
98.0, 100.5, 123.4-146.5, 170.1, 170.5. Triphenylphosphine
(155 mg, 0.59 mmol) was added to this compound (437 mg,
0.49 mmol) dissolved in dry CH2Cl2 (3 mL). The mixture was
stirred at room temperature for 20 h, concentrated, and dried
in a vacuum to give a white foam, which was refluxed in a
THF/water mixture (10:1, 9 mL) for 1 day and concentrated,
and the residue was partitioned between CHCl3 and water.
The organic phase was dried, filtered, and concentrated. The
residue was then stirred overnight in pyridine (9 mL) and
acetic anhydride (3 mL), and then the solution was coconcen-
trated with toluene. Purification on silica gel (CHCl3, gradient
of 0-3% MeOH) gave 14, slightly contaminated with triph-
enylphosphine oxide. 13C NMR: δ -4.8, -4.6, 18.0, 20.7, 23.1,
23.6, 25.5, 35.5, 47.9, 56.8, 65.8, 67.1, 67.5, 68.9, 69.1, 70.0,
70.5, 72.4, 73.0, 76.2, 97.4, 98.0, 100.4, 123.3-146.3, 169.4,
171.2, 171.5. Compound 14 was dissolved in THF (4 mL) and
treated with triethylamine tris(hydrogen fluoride) (Et3N(HF)3)
(0.96 mL, 5.9 mmol) for 2 days at room temperature. The
reaction mixture was then diluted with CHCl3, washed with
water, NaHCO3(aq), and water, dried, filtered, concentrated,
and purified on silica gel (two columns using a stepwise
gradient of CHCl3/MeOH, 30:1-2:1 and CHCl3/MeOH, 15:1,
respectively) which gave 15 (312 mg, 0.39 mmol, 80%). [R]D
+37 (c 0.62, CHCl3). mp 116-119 °C. 13C NMR: δ 20.7, 23.1,
23.4, 35.5, 47.9, 55.8, 66.2, 67.3, 68.6, 68.9, 69.8, 70.1, 71.1,
73.1, 74.8, 77.2, 97.4, 99.7, 100.9, 123.3-146.4, 169.3, 170.5,
Eth yl 3-O-Acetyl-6-O-ben zyl-4-O-(ter t-bu tyld im eth yl-
silyl)-2-d e oxy-2-p h t h a lim id o-1-t h io-â-D-glu cop yr a n o-
sid e (19). A mixture of ethyl 3-O-acetyl-6-O-benzyl-2-deoxy-
2-phthalimido-1-thio-â-D-galactopyranoside43 (4.49 g, 9.25 mmol),
TBDMS-Cl (2.51 g, 16.6 mmol), and imidazole (1.26 g, 18.5
mmol) in DMF (25 mL) was heated at 70 °C for 24 h, diluted
with toluene, washed with water, saturated NaHCO3(aq), and
water, dried, and concentrated. The residue was purified on
silica gel (toluene/EtOAc 10:1) to furnish 19 (97%, 5.38 g, 8.97
mmol). [R]D +14 (c 0.87, CHCl3). mp 36 °C. 13C NMR: δ -4.8,
-4.4, 15.1, 17.9, 20.9, 24.1, 25.6, 54.3, 68.2, 69.6, 73.3, 75.1,
1
80.4, 80.6, 123.4-138.3, 167.6, 167.9, 170.2. H NMR: δ 0.02
(s, 3H), 0.06 (s, 3H), 0.81 (s, 9H), 1.24 (t, 3H), 1.87 (s, 3H),
2.62-2.75 (m, 2H), 3.68-3.95 (m, 4H), 4.27 (dd, 1H, J ) 10.6,
10.3 Hz), 4.63 (dd, 2H), 5.52 (d, 1H, J ) 10.6 Hz), 5.62 (dd,
1H, J ) 10.3, 8.4 Hz), 7.29-7.88 (9H). Anal. Calcd for C31H41
-
NO7SSi: C, 62.07; H, 6.89; N, 2.34. Found: C, 61.89; H, 7.03;
N, 2.29.
2-Azid oeth yl 3-O-Acetyl-6-O-ben zyl-4-O-(ter t-bu tyld im -
et h ylsilyl)-2-d eoxy-2-p h t h a lim id o-â-D-glu cop yr a n osyl-
(1f3)-2,4,6-tr i-O-ben zyl-r-D-ga la ctop yr a n osid e (20). Com-
pound 17 (1.01 g, 1.8 mmol) was dissolved in CH2Cl2/MeOH
(3:2, 25 mL), and a catalytic amount of 1 M NaOMe in MeOH
was added. After being stirred at room temperature overnight,
the reaction mixture was neutralized with Dowex 50 (H+)
resin, filtered, concentrated, and purified on silica gel (toluene/
EtOAc, 8:1) which yielded 18 in 92% yield (860 mg, 1.66 mmol,
R/â 12:1). [R]D +32 (c 1.1, CHCl3). 13C NMR: δ 50.5, 66.7, 68.8,
69.4, 69.8, 72.7, 73.2, 74.9, 76.3, 77.1, 97.1, 127.4-138.1. To a
stirred solution of 18 (807 mg, 1.6 mmol), donor 19 (1.29 g,
2.2 mmol), DTBMP (319 mg, 1.6 mmol), and 4 Å molecular
sieves in dry CH2Cl2 (50 mL) under argon was added MeOTf
(230 µL, 2.0 mmol). After the mixture was stirred at room
temperature for 26 h, an additional amount of MeOTf (200
µL) was added, stirring was continued for another 18 h, and
then the mixture was quenched with Et3N (0.5 mL), filtered
through Celite, concentrated, and passed through a silica gel
column (light petroleum/EtOAc, 3:1) to give 20 (1.23 g, 1.2
mmol) in 75% yield. [R]D -24 (c 1.0, CHCl3). 13C NMR: δ -4.8,
-4.3, 17.9, 20.9, 25.6, 50.4, 55.8, 66.5, 68.5, 69.1, 69.3, 69.4,
72.8, 73.1, 73.2, 74.1, 74.9, 75.4, 77.4, 78.3, 97.7 (J C,H 169 Hz),
99.1 (J C,H 167 Hz), 122.8-138.6, 167.2, 167.4, 169.7. 1H
NMR: δ 0.02 (s, 3H), 0.06 (s, 3H), 0.81 (s, 9H), 1.86 (s, 3H),
3.22-3.34 (m, 2H,), 3.37-3.47 (m, 3H), 3.59-3.64 (m, 1H),
3.66-3.70 (m, 2H), 3.75 (d, 2H), 3.88 (d, 1H), 3.95 (dd, 1H),
3.98 (dd, 1H), 4.06 (d, 1H), 4.13 (dd, 1H, J ) 2.9, 10.3 Hz),
4.19 (d, 1H), 4.28 (dd, 1H, J ) 8.4, 10.6 Hz), 4.40 (d, 1H, J )
3.7 Hz), 4.40 (dd, 2H), 4.51 (d, 1H), 4.54 (dd, 2H), 4.98 (d, 1H),
5.67 (d, 1H, J ) 8.4 Hz), 5.67 (dd, 1H, J ) 8.4, 10.6 Hz), 6.98-
7.76 (m, 24H). Anal. Calcd for C58H68N4O13Si: C, 65.89; H,
6.48; N, 5.30. Found: C, 65.70; H, 6.49; N, 5.15.
1
172.8. H NMR: δ 1.84 (s, 3H), 2.10 (s, 3H), 2.14 (s, 3H), 3.05
(dt, 2H), 3.40 (s, 1H), 3.57-3.80 (m, 5H), 3.95 (dd, 1H), 3.99-
4.23 (m, 5H), 4.55 (s, 2H), 4.63 (dt, 1H), 4.80 (d, 1H, J ′ ) 8.0
Hz), 4.81 (d, 1H, J ) 4.4 Hz), 5.12 (dd, 1H, J ) 2.5, 11.0 Hz),
5.39 (d, 1H), 5.55 (s, 1H), 6.23 (d, 1H), 7.25-8.20 (m, 14H).
MS: calcd for C40H47N3O14 [M + H]+, 794; found, 794.
2-Azid oeth yl 3-O-Acetyl-2,4,6-tr i-O-ben zyl-D-ga la cto-
p yr a n osid e (17). To a solution of ethyl 2,4,6-tri-O-benzyl-1-
thio-â-D-galactopyranoside42 (3.39 g, 6.9 mmol) in pyridine (50
mL) was added acetic anhydride (10 mL). The mixture was
stirred at room temperature overnight and then coconcen-
trated with toluene. The residue was purified by silica gel
chromatography (toluene/EtOAc, 15:1) to give 91% (3.33 g, 6.2
mmol) of ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio-D-galacto-
pyranoside (16). [R]D +30 (c 1.0, CHCl3). 13C NMR: δ 15.0,
20.8, 25.0, 68.2, 73.4, 74.6, 74.9, 75.4, 76.6, 76.6, 85.3, 127.6-
138.2, 170.2. This compound (1.16 g, 2.2 mmol) was treated
with bromine (122 µL, 2.4 mmol) in CH2Cl2 for 15 min, and
then the mixture was concentrated. After being dried in a
vacuum, the residue was dissolved in dry CH2Cl2 (6 mL),
2-azidoethanol (0.43 mL, 6.3 mmol), and DMF (12 drops), and
4 Å molecular sieves was added. After the mixture was cooled
to 0 °C under argon, Et4NBr (680 mg, 3.2 mmol) was added,
and the solution was stirred at room temperature for 40 h,
filtered through Celite, concentrated, and purified on silica gel
(toluene/EtOAc, 10:1) to give 17 as an R/â-mixure (8:1) (1.07
g, 1.9 mmol, 88%). [R]D +60 (c 1.0, CHCl3). 13C NMR: δ (R
product) 20.9, 50.5, 66.7, 68.3, 68.8, 72.1, 72.9, 73.2, 73.8, 75.0,
75.4, 97.6, 127.4-138.0, 169.9. 1H NMR: δ 1.95 (s, 3H), 3.41-
3.59 (m, 5H), 3.76-3.81 (m, 1H, Hb spacer), 4.03 (dd, 1H, J )
3.7, 10.6 Hz), 4.06 (d, 1H, J ) 2.93 Hz), 4.10 (t, 1H), 4.41-
2-Azid oeth yl
2-Aceta m id o-3-O-a cetyl-6-O-ben zyl-2-
d eoxy-â-D-glu cop yr a n osyl-(1f3)-2,4,6-t r i-O-b en zyl-r-D-
ga la ctop yr a n osid e (22). A solution of disaccharide 20 (1.18
g, 1.1 mmol) and ethylenediamine (3.7 mL, 56 mmol) in
ethanol/toluene (3:1, 24 mL) was refluxed for 18 h and then
coconcentrated with toluene. The residue was treated with
pyridine (20 mL) and acetic anhydride (6 mL) overnight, then
DMAP (50 mg) was added, and stirring was continued for 4
h. Coconcentration of the solvent with toluene and subsequent
purification on silica gel (toluene/EtOAc, 3:1) left the desired
product 21 (0.53 g, 0.55 mmol) in 49% yield together with the
4′-O-acetyl-3′-O-silyl compound (30%). [R]D -16 (c 1.1, CHCl3).
13C NMR: δ -4.8, -4.3, 17.9, 21.2, 23.0, 25.6, 50.5, 54.4, 66.7,
68.5, 68.7, 69.2, 69.7, 72.7, 73.1, 73.2, 74.7, 75.8, 76.0, 77.1,
78.6, 97.5, 102.6, 126.9-138.5, 169.3, 170.9. Disaccharide 21
(0.51 g, 0.53 mmol) was dissolved in THF (6 mL), and Et3N-
(HF)3 (1.03 mL, 6.3 mmol) was added. The mixture was stirred
under an argon atmosphere for 48 h. An additional amount of
(42) van Dorst, J . A. L. M.; van Heusden, C. J .; Tikkanen, J . M.;
Kamerling, J . P.; Vliegenthart, J . F. G. Carbohydr. Res. 1997, 297,
209.
(43) Buskas, T.; Konradsson, P. J . Carbohydr. Chem. 2000, 19, 25.

6242 J . Org. Chem., Vol. 66, No. 19, 2001
Hansson et al.
Et3N(HF)3 (1.03 mL, 6.3 mmol) was added, stirring was
continued for another 24 h, and then the mixture was diluted
with CH2Cl2, extracted with water, saturated NaHCO3(aq),
and water, dried, filtered, and concentrated. The residue was
passed through a silica gel column using toluene/EtOAc 2:3
as eluent, which gave 362 mg (0.42 mmol, 80%) of the
desilylated disaccharide 22 as a solid. [R]D -21 (c 0.55, CHCl3).
mp 43-46 °C. 13C NMR: δ 20.9, 23.0, 50.5, 53.9, 66.7, 69.2,
69.7, 70.2, 70.3, 72.7, 73.2, 73.5, 73.7, 74.6, 76.0, 76.8, 78.2,
97.5, 102.5, 127.1-138.4, 169.5, 171.4. 1H NMR: δ 1.69 (s, 3H),
2.04 (s, 3H), 3.29 (d, 1H), 3.31 (m, 6H), 3.72-3.79 (m, 4H),
3.93-3.97 (m, 2H), 3.99 (dd, 1H), 4.11 (dd, 1H), 4.15 (ddd, 1H),
4.35-4.70 (7H), 4.71 (d, 1H), 4.72 (d, 1H, J ) 3.7 Hz), 4.90
(dd, 1H), 4.94 (d, 1H), 5.49 (d, 1H, J ) 9.5 Hz), 7.23-7.36
(20H). Anal. Calcd for C46H54N4O12: C, 64.62; H, 6.37; N, 6.55.
Found: C, 64.50; H, 6.39; N, 6.45.
2-(4-Nitr oph en yl)eth yl 2-Acetam ido-4,6-O-ben zyliden e-
3-O-(tert-butyldimethylsilyl)-2-deoxy-â-D-galactopyranosyl-
(1f4)-2-a ce t a m id o-3-O-a ce t yl-6-O-b e n zyl-2-d e oxy-r-
D-ga la ctop yr a n osyl P h osp h a te-(1f3)-2-a ceta m id o-4,6-
O-ben zylid en e-2-d eoxy-â-D-ga la ctop yr a n osyl-(1f4)-2-a c-
eta m id o-3-O-a cetyl-6-O-ben zyl-2-d eoxy-r-D-ga la ctop yr a -
n osid e Tr iet h yla m m on iu m Sa lt (28). H-Phosphonate 12
(161 mg, 0.17 mmol) and acceptor 15 (126 mg, 0.16 mmol) were
dried by evaporation of pyridine (4 mL). The residue was then
dissolved in pyridine (2.9 mL) under argon, and pivaloyl
chloride (49 µL, 0.40 mmol) was added. After 15 min, the
reaction mixture was cooled to -40 °C, and water (160 µL)
and iodine (48 mg, 0.19 mmol) were added. When the mixture
had attained 0 °C (1.5 h), it was diluted with CHCl3 and
washed twice with 1 M Na2S2O3 and twice with water; the
organic phase was filtered through Na2SO4 and concentrated.
Purification on silica gel (CHCl3/MeOH, 5:1 containing 1%
Et3N) gave 81% (222 mg, 0.13 mmol) yield of phosphodiester
28. [R]D +40 (c 0.41, CHCl3). 13C NMR: δ -4.7, -4.6, 9.0, 18.0,
20.7, 21.0, 23.1, 23.2, 23.4, 23.7, 25.5, 35.6, 45.2, 48.0, 48.1,
51.3, 56.8, 65.8, 65.8, 67.2, 67.6, 68.6, 69.0, 69.4, 69.6, 70.0,
70.2, 71.1, 71.3, 72.7, 72.7, 73.1, 73.9, 76.2, 94.8 (J C,P ) 4.6
Hz), 97.3, 98.2, 100.4, 101.0, 101.9, 123.4-146.5, 169.1, 170.9,
2-(Tr im eth ylsilyl)eth yl 3-O-Acetyl-6-O-ben zyl-4-O-(ter t-
bu tyld im eth ylsilyl)-2-d eoxy-2-p h th a lim id o-â-D-glu cop y-
r a n osyl-(1f3)-2,4,6-t r i-O-b en zyl-â-D-ga la ct op yr a n osid e
(24). To a mixture of 19 (792 mg, 1.32 mmol), 2-(trimethylsi-
lyl)ethyl 2,4,6-tri-O-benzyl-â-D-galactopyranoside31 (23, 559
mg, 1.02 mmol), DTBMP (313 mg, 1.52 mmol), and 4 Å
molecular sieves in dry CH2Cl2 (20 mL) was added DMTST
(911 mg, 3.56 mmol). After the mixture was stirred at room
temperature for 15 min under nitrogen, Et3N was added, and
the mixture was filtered (Celite), concentrated, and subjected
to silica gel chromatography (toluene/EtOAc, 10:1) to give the
disaccharide 24 (86%, 955 mg, 0.88 mmol) as a white powder.
An aliqoute recrystallized from EtOH had a melting point of
150-151 °C. [R]D -9.1 (c 1.05, CHCl3). 13C NMR: δ -4.8, -4.3,
-1.6, 17.9, 18.3, 20.9, 25.6, 55.9, 67.3, 68.8, 69.1, 69.7, 73.3,
73.5, 73.8, 74.4, 74.8, 75.7, 76.3, 77.2, 78.5, 81.5, 98.9, 103.4,
123.1-139.1, 167.7, 167.8, 170.1. 1H NMR: δ 0.00 (s, 9H), 0.11
(s, 3H), 0.15 (s, 3H), 0.90 (m, 11H), 1.95 (s, 3H), 3.47 (m, 1H),
3.61-3.83 (7H), 3.89 (dd, J ) 9.89, 2.9 Hz), 3.96 (m, 1H), 4.03
(dd, 1H), 4.10 (d, 1H, J ) 2.9 Hz), 4.28-4.37 (3H), 4.47-5.06
(7H), 5.71 (dd, 1H), 5.83 (dd, 1H, J ) 8.1 Hz), 7.17-7.77 (24
H). Anal. Calcd for C61H77NO13Si2: C, 67.31; H, 7.13; N, 1.29.
Found: C, 67.06; H, 7.21; N, 1.23.
1
171.0, 171.2, 171.4, 171.8. H NMR (assorted signals): δ 0.05
(s, 3H), 0.09 (s, 3H), 0.87 (s, 9H), 1.06 (t, 9H), 1.80 (s, 3H),
1.89 (s, 3H), 2.01 (s, 3H), 2.10 (s, 3H), 2.11 (s, 3H), 2.13 (s,
3H), 3.01 (t, 2H), 4.82 (d, 1H, J ) 3.6 Hz), 4.97 (dd, 1H), 5.06
(dd, 1H), 5.48 (s, 1H), 5.53 (s, 1H). 31P NMR (CH2Cl2): δ -1.9.
MS: calcd for C84H115N6O28PSi [M - Et3NH]-, 1612.6; found,
1612.7.
2-(4-Am in ocycloh exyl)eth yl 2-Aceta m id o-2-d eoxy-â-D-
ga la ctop yr a n osyl-(1f4)-2-a ceta m id o-3-O-a cetyl-2-d eoxy-
r-D-ga la ct op yr a n osyl P h osp h a t e-(1f3)-2-a cet a m id o-2-
deoxy-â-D-galactopyr an osyl-(1f4)-2-acetam ido-3-O-acetyl-
2-deoxy-r-D-galactopyr an oside Sodiu m Salt (30). Phospho-
diester 28 (222 mg, 0.13 mmol) was treated with Et3N(HF)3
(340 µL, 2.1 mmol) in THF (2.4 mL). After being stirred at
room temperature for 41 h, the reaction mixture was parti-
tioned between CH2Cl2 and TEAB; the organic phase was
filtered through Na2SO4, concentrated, and purified on silica
gel (CHCl3/MeOH, 5:1 containing 1% Et3N), which gave 29 in
88% yield (183 mg, 0.11 mmol). [R]D +45 (c 0.67, CHCl3). 13C
NMR: δ 8.2, 20.8, 21.0, 23.2, 23.3, 23.5, 35.6, 45.4, 48.1, 48.4,
51.3, 54.8, 65.7, 66.4, 67.1, 68.6, 69.3, 69.5, 69.9, 70.7, 71.1,
71.3, 71.5, 72.9, 73.0, 73.1, 73.5, 73.9, 74.9, 77.2, 94.4, 97.3,
100.5, 100.9, 102.0, 123.3-146.3, 168.8, 170.0, 170.4, 170.8,
171.3, 173.4. 31P NMR (CH2Cl2): δ -2.0. Desilylated diester
29 (50 mg, 31 µmol) and palladium on activated carbon (10%,
50 mg) in EtOH/HOAc/water (2:1:1, 4 mL) were stirred under
H2 (110 psi) for 3 days. Additional Pd/C (25 mg) was added
after 17 h. Filtration (Celite), concentration, lyophilization, and
purification on a Biogel P2 column (water containing 1%
n-butanol) gave 30 (20.0 mg, 17.5 µmol, 56%). [R]D +122 (c
0.97, H2O). 13C NMR (D2O): δ 21.2, 22.6, 22.8, 23.1, 23.3, 27.1,
27.3, 30.8, 30.9, 33.5, 35.9, 48.9, 49.1, 49.8, 51.2, 52.2, 53.5,
61.0, 61.3, 61.8, 61.8, 67.1, 67.8, 68.6, 70.8, 70.9, 71.3, 72.0,
74.1, 74.6, 75.1, 75.5, 95.1 (J C,P ) 6.0 Hz), 97.7, 103.1, 173.8,
173.9, 174.4, 174.8, 175.3, 175.4. 1H NMR (assorted signals,
D2O): δ 1.00-1.91 (12H), 1.97 (s, 3H), 2.00 (s, 3H), 2.08 (s,
3H), 2.10 (s, 3H), 2.15 (s, 3H), 2.15 (s, 3H), 5.19 (dd, 1H), 5.20
(dd, 1H), 5.50 (dd, 1H, J ′ ) 3.7 Hz, J ) 7.3 Hz). 31P NMR
(D2O): δ -1.9. HRMS: calcd for C44H73N5O26P [M - Na]-,
1118.4281; found, 1118.4264.
Tr ieth yla m m on iu m 2-Aceta m id o-3-O-a cetyl-6-O-ben -
zyl-4-O-(ter t-bu tyld im eth ylsilyl)-2-d eoxy-â-D-glu cop yr a -
n osyl-(1f3)-2,4,6-tr i-O-ben zyl-r-D-ga la ctop yr a n osyl Hy-
d r ogen p h osp h on a te (27). Disaccharide 24 (740 mg, 0.68
mmol) was treated with ethylenediamine (2.0 mL) for 14 h as
described above. In the acetylation step, DMAP (25 mg) was
added after 7 h; stirring was then continued for another 20 h.
Workup and purification on silica gel (toluene/EtOAc, 4:1)
afforded 25 (63%, 431 mg, 0.43 mmol) and its regioisomer
(27%). [R]D -17 (c 1.0, CHCl3). 13C NMR: δ -4.8, -4.3, -1.5,
17.9, 18.4, 21.1, 22.7, 25.6, 54.3, 67.0, 68.7, 68.9, 69.0, 73.1,
73.3, 73.4, 74.1, 74.4, 75.8, 76.1, 76.3, 79.6, 80.8, 101.9, 103.1,
126.3-139.1, 169.4, 170.8. Compound 25 was dissolved in dry
CH2Cl2 (2.2 mL) under argon and cooled to 0 °C. Anhydrous
TFA (4.4 mL) was added. After 30 min, n-propyl acetate and
toluene were added, and the mixture was concentrated. The
residue was crystallized from diethyl ether/light petroleum to
afford the R-hemiacetal 26 (58%, 220 mg, 0.24 mmol). [R]D -23
(c 0.75, CHCl3). 13C NMR: δ -4.4, -3.9, 18.3, 21.6, 23.3, 26.0,
54.9, 69.1, 69.3, 69.3, 70.1, 73.0, 73.5, 75.0, 76.2, 76.3, 77.1,
78.1, 79.9, 91.8, 103.1, 126.9-139.2, 170.1, 171.2. Compound
26 (214 mg, 0.24 mmol) was phosphonylated as described for
derivative 11 to give 89% (225 mg, 0.21 mmol) yield of 27. [R]D
+6.3 (c 0.99, CHCl3). 13C NMR: δ -4.8, -4.3, 8.7, 17.9, 21.2,
22.8, 25.7, 45.3, 54.4, 68.4, 68.6, 69.3, 70.3, 71.6, 73.1, 74.9,
75.8 (J ) 5.4 Hz), 76.0, 76.6, 77.2, 78.5, 92.7 (J C,P ) 5.4 Hz),
102.5, 126.3-138.6, 169.6, 170.9. 1H NMR: δ 0.06 (s, 3H), 0.11
(s, 3H), 0.84 (s, 9H), 1.16 (t, 9H), 1.52 (s, 3H), 2.02 (s, 3H),
2.85 (q, 6H), 3.41-3.71 (5H), 3.91 (dd, 1H), 3.97 (ddd, 1H),
4.07 (d, 1H), 4.14 (ddd, 1H), 4.24 (dd, 1H, J ) 10.4, 3.0 Hz),
4.30 (t, 1H), 4.39-4.58 (6H), 4.68 (d, 1H, J ) 8.5 Hz), 4.82
(dd, 1H), 4.84-5.00 (2H), 5.27 (d, 1H), 5.81 (dd, 1 H, J ) 3.3,
8.8 Hz), 7.06 (d, 1H, J ) 638 Hz), 7.20-7.39 (20 H). 31P NMR
(CH2Cl2): δ 0.56. MS: calcd for C56H81N2O14PSi [M - Et3NH]-,
962.4; found, 962.4.
2-(4-Nitr oph en yl)eth yl 2-Acetam ido-4,6-O-ben zyliden e-
3-O-(tert-butyldimethylsilyl)-2-deoxy-â-D-galactopyranosyl-
(1f4)-2-a ce t a m id o-3-O-a ce t yl-6-O-b e n zyl-2-d e oxy-r-
D-ga la ctop yr a n osyl P h osp h a te-(1f3)-2-a ceta m id o-4,6-
O-ben zylid en e-2-d eoxy-â-D-ga la ctop yr a n osyl-(1f4)-2-a c-
eta m id o-3-O-a cetyl-6-O-ben zyl-2-d eoxy-r-D-ga la ctop yr a -
n osyl P h osp h a te-(1f3)-2-a ceta m id o-4,6-O-ben zylid en e-
2-d eoxy-â-D-ga la ct op yr a n osyl-(1f4)-2-a cet a m id o-3-O-
a cet yl-6-O-b en zyl-2-d eoxy-r-D-ga la ct op yr a n osid e Bis-
tr ieth yla m m on iu m Sa lt (31). Compounds 12 (29 mg, 0.031
mmol) and 29 (50 mg, 0.031 mmol) were dried by evaporation
of pyridine (3 × 1 mL). The residue was dissolved in the same

Syntheses of H. influenzae Capsular Polysaccharide
J . Org. Chem., Vol. 66, No. 19, 2001 6243
solvent (0.4 mL) under argon, and pivaloyl chloride (5.8 µL,
0.047 mmol) was added. After 15 min, the reaction mixture
was cooled to -40 °C, and a solution of pyridine/water (19:1,
1.64 mL) and iodine (10 mg, 0.037 mmol) were added. After
20 min (-30 °C), the reaction was worked up as described for
derivative 28. Silica gel chromatography (CHCl3/MeOH, 3:1
containing 1% Et3N) afforded 37% yield of 31 (29 mg, 0.011
mmol). [R]D +52 (c 1.0, CHCl3). 13C NMR: δ -4.7, -4,6, 8.7,
18.0, 20.7, 21.0, 21.1, 22.8, 23.3, 23.3, 23.5, 23.8, 25.5, 35.6,
45.5, 48.1, 48.4, 51.0, 56.7, 65.6, 65.9, 67.1, 67.6, 68.7, 69.0,
69.5, 69.8, 70.0, 70.3, 70.8, 71.0, 71.2, 71.4, 71.6, 72.4, 72.7,
73.1, 73.8, 74.0, 76.2, 77.1, 94.2, 94.7, 97.3, 98.2, 100.3, 101.0,
102.5, 102.6, 123.4-146.5, 168.9, 170.3, 170.5, 170.7, 171.0,
171.2, 171.3, 171.7. MS: calcd for C122H169N9O42P2Si [M -
2(Et3NH)]2-, 1158.9; found, 1159.2.
(D2O): δ 21.2, 21.7, 23.0, 23.0, 40.2, 55.0, 55.1, 61.1, 61.2, 61.9,
62.1, 64.7, 67.9, 68.4, 69.8, 71.8, 72.3, 72.5, 74.9, 75.8, 76.2,
76.3, 79.8, 79.9, 96.8 (J C,P ) 6.6 Hz), 99.5, 102.6, 102.9, 174.0,
1
174.2, 175.3, 175.4. H NMR (assorted signals, D2O): δ 1.98
(s, 3H), 1.98 (s, 3H), 2.12 (s, 3H), 2.22 (s, 3H, CH3CO), 3.27-
3.31 (m, 2H), 3.55 (1H), 3.64 (1H), 4.28 (ddd, 1H), 4.87 (d, 1H,
J ) 8.8 Hz), 4.91 (d, 1H, J ) 8.4 Hz), 5.06 (dd, 1H, J ) 10.3,
10.3 Hz), 5.23 (dd, 1H, J ) 10.3, 10.3 Hz), 5.49 (dd, 1H, J )
6.6 Hz). 31P NMR (D2O): δ -1.9. HRMS: calcd for C34H57N3O26-
PNa [M - Na]-, 954.2968; found, 954.3033.
2-Azid oeth yl 2-Aceta m id o-3-O-a cetyl-6-O-ben zyl-4-O-
(ter t-b u t yld im et h ylsilyl)-2-d eoxy-â-D-glu cop yr a n osyl-
(1f3)-2,4,6-tr i-O-ben zyl-r-D-galactopyr an osyl P h osph ate-
(1f4)-2-a cet a m id o-3-O-a cet yl-6-O-b en zyl-2-d eoxy-â-D-
glu cop yr a n osyl-(1f3)-2,4,6-t r i-O-b e n zyl-r-D -ga la ct o-
p yr a n osyl P h osp h a te-(1f4)-2-a ceta m id o-3-O-a cetyl-6-O-
ben zyl-2-d eoxy-â-D-glu cop yr a n osyl-(1f3)-2,4,6-tr i-O-ben -
zyl-r-D-ga la ctop yr a n osid e Bis-tr ieth yla m m on iu m Sa lt
(37). Phosphonate 27 (33 mg, 31 µmol) and diester 35 (56 mg,
31 µmol) were dried by evaporation of pyridine (3 × 1 mL)
and then dissolved in the same solvent (0.4 mL) under an
argon atmosphere, and pivaloyl chloride (5.8 µL, 47 µmol) was
added. After 15 min, the mixture was cooled to -40 °C, and a
solution of pyridine/water (19:1, 1.6 mL) was added, im-
mediately followed by the addition of iodine (10 mg, 37 µmol).
When the reaction had attained 5 °C (2 h), it was worked up
as described for compound 28. Silica gel chromatography
(CHCl3 containing 1% Et3N, gradient of 0-9% MeOH) left the
diphosphate 37 (36%, 32 mg, 11 µmol), slightly contaminated
with starting phosphonate 27. [R]D -1.7 (c 0.69, CHCl3). 13C
NMR: δ -4.8, -4.2, 8.4, 17.8, 21.2, 21.2, 22.7, 22.9, 23.0, 25.7,
45.1, 50.5, 53.5, 53.8, 54.5, 66.6, 69.2, 69.4, 69.6, 69.8, 70.4,
71.8, 72.1, 72.7, 72.9, 73.1, 73.1, 73.2, 73.6, 73.9, 74.3, 74.7,
74.8, 75.5, 75.7, 76.0, 77.1, 77.3, 78.8, 80.2, 80.5, 94.8 (b), 95.5
(b), 97.5, 102.7, 103.0, 103.4, 126.9-138.6, 169.9, 170.0, 170.3,
171.1. 31P NMR (CDCl3): δ -2.7. MS: calcd for C152H198N8O40P2-
Si [M - 2(Et3NH)]2-, 1330.5; found, 1330.7.
2-(4-Am in ocycloh exyl)eth yl 2-Aceta m id o-2-d eoxy-â-D-
g a la c t o p y r a n o s y l-(1f4)-2-a c e t a m i d o -3-O -a c e t y l-2-
d eoxy-r-D-ga la ctop yr a n osyl p h osp h a te-(1f3)-2-a ceta m i-
d o-2-d eoxy-â-D-ga la ctop yr a n osyl-(1f4)-2-a ceta m id o-3-O-
a cetyl-2-d eoxy-r-D-ga la ctop yr a n osyl P h osp h a te-(1f3)-
2-a c e t a m id o -2-d e o x y -â-D -g a la c t o p y r a n o s y l-(1f4)-2-
a ce t a m id o-3-O-a ce t yl-2-d e oxy-r-D-ga la ct op yr a n osid e
Disod iu m Sa lt (32). Compound 31 (28 mg, 0.011 mmol) was
dissolved in THF (0.8 mL) and treated with Et3N(HF)3 (33 µL,
0.20 mmol) at room temperature for 2 days and then concen-
trated. The residue was hydrogenolyzed (110 psi) in EtOH/
HOAc/water (4:1:2, 2 mL) containing Pd/C (10%, 30 mg).
Additional catalyst was added after 7, 24, and 48 h (10 mg).
After 3 days, the reaction mixture was worked up and purified
as described for compound 30 to give 32 in 64% yield (12.0
mg, 7.1 µmol). [R]D +82 (c 0.93, H2O). 13C NMR (assorted
signals, D2O): δ 21.3, 22.7, 22.9, 23.3, 23.4, 49.2, 52.3, 53.6,
95.2 (b), 97.7, 103.2, 173.8, 173.9, 174.9, 175.4. 1H NMR
(assorted signals, D2O): δ 1.97 (s, 3H), 2.01 (s, 6H), 2.08 (s,
3H), 2.10 (s, 6H), 2.15 (s, 6H), 2.17 (s, 3H), 5.20 (m, 3H), 5.50
(2dd, 2H). 31P NMR: δ -1.7 (D2O). HRMS: calcd for
C
62H101N7O40P2 [M + H - 2Na]-, 1646.5638; found, 1646.5605.
2-Azid oeth yl 2-Aceta m id o-3-O-a cetyl-6-O-ben zyl-4-O-
2-Am in oeth yl 2-Aceta m id o-3-O-a cetyl-2-d eoxy-â-D-glu -
copyr an osyl-(1f3)-r-D-galactopyr an osyl P h osph ate-(1f4)-
2-acetam ido-3-O-acetyl-2-deoxy-â-D-glu copyr an osyl-(1f3)-
r-D-ga la ctop yr a n osyl P h osp h a te-(1f4)-2-a ceta m id o-3-
O-a cetyl-2-d eoxy-â-D-glu cop yr a n osyl-(1f3)-r-D-ga la cto-
p yr a n osid e Disod iu m Sa lt (39). Diphosphate 37 (30 mg, 10
µmol) in THF (0.8 mL) was treated with Et3N(HF)3 (41 µL,
250 µmol) at room temperature for 5 days and then concen-
trated with a stream of nitrogen and dried in a vacuum
overnight. The residue was dissolved in ethanol/acetic acid/
water (2:1:1, 2 mL) and hydrogenolyzed over Pd/C (30 mg) at
110 psi for 2 days. Additional catalyst (15 mg) was added after
24 h. Workup and purification as described for compound 30
gave the deprotected trimeric structure 39 (12.0 mg, 8.1 µmol,
77%). [R]D +38 (c 0.82, H2O). 13C NMR (D2O): δ 21.0, 21.4,
22.8, 22.8, 54.8, 54.9, 55.0, 60.9, 61.0, 61.6, 61.7, 61.9, 64.4,
67.7, 68.2, 69.6, 71.6, 72.1, 72.2, 72.3, 74.7, 75.5, 76.0, 76.1,
79.7, 96.5, 99.2, 102.3, 102.6, 102.7, 173.8, 173.9, 175.1, 175.2.
1H NMR (assorted signals, D2O): δ 1.98 (s, 9H), 2.12 (s, 3H),
2.15 (s, 6H), 3.27-3.31 (m, 2H), 4.21 (d, 1H, J ) 2.9 Hz), 4.23
(bd, 2H), 4.29 (ddd, 2H), 4.85-4.91 (3d, 3H, J ) 8.4 Hz), 4.98
(d, 1H, J ) 3.7 Hz), 5.05 (dd, 1H, J ) 9.5, 10.3 Hz), 5.22 (dd,
2H, J ) 9.5, 9.9 Hz), 5.49 (dd, 2H, J ) 6.2 Hz). 31P NMR
(D2O): δ -1.9. HRMS: calcd for C50H83N4O40P2 [M + H -
2Na]-, 1441.4059; found, 1441.4067.
(ter t-b u t yld im et h ylsilyl)-2-d eoxy-â-D-glu cop yr a n osyl-
(1f3)-2,4,6-tr i-O-ben zyl-r-D-galactopyr an osyl P h osph ate-
(1f4)-2-a cet a m id o-3-O-a cet yl-6-O-b en zyl-2-d eoxy-â-D-
glu cop yr a n osyl-(1f3)-2,4,6-t r i-O-b e n zyl-r-D -ga la ct o-
p yr a n osid e Tr ieth yla m m on iu m Sa lt (34). Compounds 22
(133 mg, 0.16 mmol) and 27 (100 mg, 0.094 mmol) were
coupled as described for the formation of compound 28. Silica
gel chromatography (CHCl3 containing 1% Et3N, gradient of
2.5-9% MeOH) gave diester 34 (71%, 127 mg, 0.66 mmol). [R]D
-8.3 (c 0.96, CHCl3). 13C NMR: δ -4.8, -4.2, 8.5, 17.9, 21.2,
21.2, 22.8, 22.9, 25.6, 45.2, 50.5, 53.7, 54.2, 66.6, 69.1, 69.4,
69.6, 69.8, 70.1, 70.5, 71.0, 72.1, 72.6, 73.0, 73.1, 73.2, 74.4,
74.7, 75.7, 75.8, 76.0, 77.0, 77.5, 78.8, 80.1, 94.8 (J C,P ) 6.1
Hz), 97.5, 102.7, 103.0, 126.8-138.7, 169.6, 169.7, 170.2, 171.0.
1H NMR (assorted signals): δ 4.02 (d, 1H), 4.13 (dd, 1H), 4.77
(d, 1H, J ) 3.7 Hz), 5.72 (dd, 1H, J ) 2.9, 8.1 Hz). 31P NMR
(CDCl3): δ -2.3. MS: calcd for C102H133N6O26PSi [M -
Et3NH]-, 1814.7; found, 1814.8.
2-Am in oeth yl 2-Aceta m id o-3-O-a cetyl-2-d eoxy-â-D-glu -
copyr an osyl-(1f3)-r-D-galactopyr an osyl P h osph ate-(1f4)-
2-acetam ido-3-O-acetyl-2-deoxy-â-D-glu copyr an osyl-(1f3)-
r-D-ga la ctop yr a n osid e Sod iu m Sa lt (36). Diester 34 (57
mg, 0.030 mmol) in THF (1.2 mL) was treated with Et3N(HF)3
(117 µL, 0.71 mmol) at room temperature for 4 days, diluted
with CH2Cl2, washed with TEAB, filtered through Na2SO4, and
concentrated. The residue was subjected to silica gel chroma-
tography (CHCl3/MeOH, 10:1 containing 1% Et3N) to give 35
(88%, 47 mg, 0.026 mmol). [R]D -5.4 (c 0.58, CHCl3). 13C
NMR: δ 8.4, 20.8, 21.1, 22.8, 23.0, 45.1, 50.5, 53.3, 53.8, 66.6,
69.2, 69.7, 69.8, 70.2, 70.4, 70.4, 71.1, 72.1, 72.5, 73.0, 73.1,
73.5, 74.3, 74.5, 74.6, 74.7, 75.7, 76.2, 77.0, 77.1, 78.4, 80.0,
94.7, 97.4, 102.6, 103.3, 126.8-138.7, 169.6, 169.9, 170.9, 171.4.
31P NMR (CH2Cl2): δ -3.0. Compound 35 (26 mg, 14 µmol)
was hydrogenolyzed as described for derivative 29 to yield 36
(13.0 mg, 13 µmol, 92%). [R]D +50 (c 0.90, H2O). 13C NMR
Ack n ow led gm en t. We thank EU (Contract No. BIO
4 CT 960158) and the Swedish Natural Science Re-
search Council for financial support.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of compounds 12, 15, 22, 27, 28, 30, 31, 32, 34, 36,
37, and 39. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O001302M