Synthesis of four spacer-containing 'tetrasaccharides' that represent four possible repeating units of the capsular polysaccharide of Streptococcus pneumoniae type 6B

Article abstract of DOI:10.1016/S0008-6215(97)10013-1

In the framework of studies towards oligosaccharide-conjugate based vaccines against Streptococcus pneumoniae, the synthesis is reported of four spacer-containing 'tetrasaccharides' that each can be conceived as representing a repeating unit of the capsular polysaccharide of S. pneumoniae serotype 6B, namely, 3-aminopropyl D-ribityl-(5 → hydrogen phosphate → 2)- α-D-galactopyranosyl-(1 → 3)-α-D-glucopyranosyl-(1 → 3)-α-L- rhamnopyranoside, 3-aminopropyl α-L-rhamnopyranosyl-(1 → 4)-D-ribityl-(5 → hydrogen phosphate → 2)-α-D-galactopyranosyl-(1 → 3)-α-D-glucopyranoside, 3-aminopropyl α-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 4)-D- ribityl-(5 → hydrogen phosphate → 2)-α-D-galactopyranoside, and α-D- galactopyranosyl-(1 → 3)-α-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl- (1 → 4)-5-O-(3-aminopropyl hydrogen phosphate)-D-ribitol. Phosphorylations were carried out using the H-phosphonate method.

Full text of DOI:10.1016/S0008-6215(97)10013-1

Ž
.
Carbohydrate Research 306 1998 111–125
Synthesis of four spacer-containing
‘tetrasaccharides’ that represent four possible
repeating units of the capsular polysaccharide of
Streptococcus pneumoniae type 6B
)
Mark-J.L. Thijssen, Menno H.G. Bijkerk, Johannis P. Kamerling ,
Johannes F.G. Vliegenthart
BijÕoet Center, Department of Bio-Organic Chemistry, Utrecht UniÕersity, P.O. Box 80P075, NL-3508
TB, Utrecht, Netherlands
Received 11 April 1997; accepted 2 September 1997
Abstract
In the framework of studies towards oligosaccharide-conjugate based vaccines against
Streptococcus pneumoniae, the synthesis is reported of four spacer-containing ‘tetrasac-
charides’ that each can be conceived as representing a repeating unit of the capsular
Ž
polysaccharide of S. pneumoniae serotype 6B, namely, 3-aminopropyl D-ribityl- 5™hydrogen
.
Ž
.
Ž
.
phosphate ™ 2 -a-D-galactopyranosyl- 1 ™ 3 -a-D-glucopyranosyl- 1 ™ 3 -a-L-
Ž
.
Ž
rhamnopyranoside, 3-aminopropyl a-L-rhamnopyranosyl- 1 ™ 4 -D-ribityl- 5 ™ hydrogen
.
Ž
.
phosphate™2 -a-D-galactopyranosyl- 1™3 -a-D-glucopyranoside, 3-aminopropyl a-D-glu-
Ž
.
Ž
.
Ž
.
copyranosyl- 1™3 -a-L-rhamnopyranosyl- 1™4 -D-ribityl- 5™hydrogen phosphate™2 -
Ž
.
Ž
.
a-D-galactopyranoside, and a-D-galactopyranosyl- 1™3 -a-D-glucopyranosyl- 1™3 -a-L-
Ž
.
Ž
.
rhamnopyranosyl- 1™4 -5-O- 3-aminopropyl hydrogen phosphate -D-ribitol. Phosphoryla-
tions were carried out using the H-phosphonate method. q 1998 Elsevier Science Ltd. All
rights reserved.
Keywords: Phosphorylated oligosaccharide; Capsular polysaccharide; Streptococcus pneumoniae 6B
1. Introduction
such as penicillin in the battle against bacterial infec-
tions. However, when several bacterial strains be-
Infection with the gram-positive bacterium Strep-
tococcus pneumoniae is still one of the leading causes
w x
came resistant against antibiotic treatment 5 , the
need for an adequate preventive vaccination program
w x
w
x
of death 1 . Early experiments 2–4 with pneumo-
coccal capsular polysaccharide-constituted vaccines
were overshadowed by the introduction of antibiotics
again became clear. Nowadays, 90 serotypes of S.
w x
pneumoniae are known 6 , and can be recognised by
the composition of their respective capsular poly-
saccharides. A 23-valent pneumococcal vaccine has
) Corresponding author.
w x
been available since 1983 7 , consisting of the puri-
0008-6215r98r$19.00 q 1998 Elsevier Science Ltd All rights reserved.

(
)
112
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
fied capsular polysaccharides of 23 serotypes of S.
pneumoniae and accounting for 90% of bacteremic
infections in the United States. However, on inocula-
tion, this vaccine does not evoke an immunological
memory because the immune system reacts towards
the polysaccharide antigens via a Thymus-Indepen-
shown that they can convert the TI response into a
Ž
.
Thymus-Dependent TD response, with the benefi-
w
x
cial development of immunological memory 9–16 .
However, antibodies produced against this kind of
conjugate are often inefficient in binding components
w
x
of the immune system after opsonisation 17 .
Oligosaccharide-conjugate based vaccines are under
Ž
.
dent TI mechanism, thereby, not creating memory
cells, and furthermore, the induction of tolerance is a
w
x
investigation 18–21 for their abilities to overcome
these problems.
w x
problem 8 . Polysaccharide–protein conjugates have
w
x
In earlier reports 22,23 , we have described the
synthesis of several spacer-containing oligosaccha-
ride fragments of the capsular polysaccharide of S.
2. Results and discussion
In earlier studies, we have presented the synthesis
Ž .
pneumoniae type 6B 1 for the preparation of neo-
of compound 49 in a modest yield via a blockwise
w
x
glycoconjugates. The polysaccharide consists of re-
peating units of four monosaccharides, linked by a
phosphate function. From this structure, four in
chemical as well as in immunological sense different
repeating units can be envisaged as synthetic targets.
A requirement for the preparation of neoglycoconju-
gates with these repeating units is the presence of a
spacer group in the molecule. Here, the synthesis of
these four possible spacer-containing repeating units,
namely, 34, 40, 44, and 49, is reported.
coupling 22 . In this report, we describe an improved
strategy for the preparation of this compound, making
use of the carbohydrate moiety 29 which is synthe-
sised in a stepwise manner, and then introducing the
spacer-phosphate function by selective phosphoryla-
tion. Compound 29 is well suited for future prepara-
tion of structures larger than one repeating unit. The
other spacer-containing repeating units 34, 40, and 44
are each prepared from two carbohydrate building
blocks, which are linked through a phosphate func-
Scheme 1.

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
113
Scheme 2.
. Ž
.
tion. In the following, the synthesis of these building
blocks is described first.
benzyl-a-D-glucopyranosyl - 1™3 -2-O-acetyl-4-O-
Ž
.
benzyl-a-L-rhamnopyranoside 15 , rhamnosyl accep-
tor 9 was prepared as follows Scheme 1 . Ethyl
2-O-acetyl-4-O-benzyl-3-O-monochloroacetyl-1-thio-
Ž
.
Preparation of building blocks.—In order to syn-
Ž
thesise 3-N-benzyloxycarbonylaminopropyl 3,4,6-tri-
. Ž
. Ž
Ž .
w x
O-acetyl-a-D-galactopyranosyl - 1™3 - 2,4,6-tri-O-
a-L-rhamnopyranoside 2 22 was linked to 3-N-
Scheme 3.

(
)
114
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
Ž .
w
x
Ž
.
benzyloxycarbonylaminopropanol
3
24 in
deacetylated ™7 , and converted into orthoester 8
with trimethyl orthoacetate, followed by an in situ
ring opening by addition of water to give 9 in a yield
of 71% over 3 steps.
Ž
.
dichloromethane using N-iodosuccinimide NIS –tri-
flic acid TfOH as a promoter system, to give 4
78% , which was demonochloroacetylated with hy-
drazine dithiocarbonate HDTC 25 in acetic acid–
2,6-lutidine to give 9 in a yield of 69%. Alterna-
tively, spacer 3 was glycosylated with ethyl 2,3-di-
O-acetyl-4-O-benzyl-1-thio-a-L-rhamnopyranoside
Ž
.
Ž
.
Ž
. w x
Glycosylation of 9 with ethyl 3-O-allyl-2,4,6-tri-
Ž
. w x
O-benzyl-1-thio-b-D-glucopyranoside 10 27 in
dichloromethane–diethyl ether using methyl triflate
as a promoter resulted in a non-stereoselective cou-
Ž .
w
x
Ž
.
5 26 in dichloromethane using methyl triflate as a
pling reaction a:b 1.9:1 , from which the desired
compound 11 could be isolated in a yield of 34%
Ž
.
promoter to give 6 81% . This compound was then
Scheme 4.

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
115
Scheme 5.
Ž
.
Ž
Scheme 2 and 3 . Deallylation of 11 Wilkinson
ride derivative 14. Thus, 13 was coupled with 12 in
diethyl ether using trimethylsilyl triflate as a pro-
moter, giving the desired compound with a complete
a-stereoselectivity in a yield of 52%. Finally, re-
.
catalyst, then mercuric oxide–mercuric chloride
yielded acceptor 12 54% .
Ž
.
Because of the successful glycosylation of a simi-
lar acceptor with 3,4,6-tri-O-acetyl-2-O-allyl-arb-
D-galactopyranosyl trichloroacetimidate 13 23 , this
donor was also tested in the preparation of trisaccha-
moval of the allyl group at C-2Y Wilkinson catalyst,
Ž
Ž
.
w
x
.
then mercuric oxide–mercuric chloride gave the first
Ž .
building block 15 37% .
Scheme 6.

(
)
116
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
Ž
Ž
In the synthesis of the last building block 3,4,6-tri
In the synthesis of 2,3,4,6-tetra-O-benzyl-a-D-
. Ž . Ž
. Ž
. Ž
glucopyranosyl - 1™3 - 2,4 - di - O - benzyl - a - L -
-O-acetyl-a-D-galactopyranosyl - 1™3 - 2,4,6-tri-O-
. Ž
.
. Ž . Ž
rhamnopyranosyl - 1™4 -1,2,3-tri-O-benzyl-D-ribitol
benzyl-a-D-glucopyranosyl - 1™3 - 2,4-di-O-benzyl-
Ž
.
Ž
. Ž
.
21 , the previously prepared 2-O-acetyl-4-O-benzyl-
a-L-rhamnopyranosyl - 1™4 -1,2,3-tri-O-benzyl-D-
. Ž
.
Ž .
ribitol 29 , first 23 was prepared from ethyl 2,4,6-tri-
a-L-rhamnopyranosyl - 1™4 -5-O-allyl-1-O-benzoyl-
Ž
.
w
x
Ž
.
.
2,3-di-O-benzyl-D-ribitol 17 22 was glycosylated
O-benzyl-1-thio-b-D-glucopyranoside 22 and p-
Ž
. Ž
with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-b-D-gluco-
methoxybenzyl chloride 81% Scheme 4 . Then, 23
Ž
.
w
x
w x
pyranoside 16 28,29 in 1,2-dichloroethane–diethyl
was condensed with 17 22 in 1,2-dichloroethane–
diethyl ether using iodonium dicollidineperchlorate
ether using iodonium dicollidineperchlorate as a pro-
w
x
moter to yield a mixture of 18 and the b-coupled
30 , to yield a mixture of 24 and its b-analogue 24b
Ž
1
1
product 18b 62%; a:b 6:1, as determined by H
62%; a:b 2:1, as established by H and 13C NMR
Ž
13
.
.
analysis . The mixture could only be separated after
and C NMR analysis . Complete separation of the
Ž
.
mixture could only be achieved after deacylation
deacylation ™25r25b and subsequent benzylation
Ž
.
Ž
.
™19r19b, 82% and subsequent benzylation of the
with benzyl bromide ™26, 43% over 2 steps .
Ž
resulting deprotected hydroxyl functions ™ 20,
Removal of the p-methoxybenzyl function using am-
.
Ž
.
83% . Compound 20 was deallylated by treatment
monium cerium IV nitrate gave trisaccharide 27 in a
yield of 72%.
Coupling of compounds 13 and 27 in diethyl ether
using trimethylsilyl triflate as a promoter yielded
‘tetrasaccharide’ derivative 28 with a complete a-
with potassium tert-butoxide in N, N-dimethylform-
amide at 80 8C followed by cleavage of the resulting
1-propenyl function in acetone–0.1 M hydrochloric
Ž
.
acid, to afford building unit 21 49% .
Scheme 7.

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
117
stereoselectivity in a yield of 81%. Removal of the
subsequent debenzylationrdebenzyloxycarbonylation
allyl functions on both C-5 and C-2Z Wilkinson
Ž
Ž
.
Pd–C, H2 gave, after purification on Bio-Gel P-2,
.
Ž
.
catalyst, then mercuric oxide–mercuric chloride gave
compound 40 73% over 2 steps .
Ž
.
building block 29 61% .
In the preparation of the target compound 44
Ž
.
Preparation and deprotection of phosphorylated
Scheme 7 , trisaccharide derivative 21 was phospho-
compounds.—The preparation of target compound 34
nylated with 3-N-benzyloxycarbonylaminopropyl
Ž
.
Ž
Scheme 5 started with the coupling of 1,2,3,4-tetra-
3,4,6-tri-O-benzyl-2-O- triethylammonium H-phos-
Ž
.
.
Ž
. w x
O-benzyl-5-O- triethylammonium H-phosphonate -
D-ribitol 30 23 to HO-2 of trisaccharide deriva-
tive 15 in pyridine–acetonitrile using pivaloyl chlo-
phonate -a-D-galactopyranoside 41 23 in pyri-
dine–acetonitrile using pivaloyl chloride to give 42,
which was oxidised in situ with iodine in pyridine–
water, yielding 43 in 49% over 2 steps. Debenzyla-
Y
Ž
. w x
w
x
Ž
.
ride 31 ™31 , and subsequent mild in situ oxida-
Ž
.
tion of the resulting phosphonate diester using iodine
tionrdebenzyloxycarbonylation of 43 Pd–C, H2
Ž
.
in pyridine–water ™ 32, 78% over 2 steps .
gave after desalting on Bio-Gel P-2, compound 44 in
Ž
.
Deacetylation methanol–ammonia, ™33 and sub-
a yield of 72% over 2 steps.
Ž
.
sequent debenzylationrdebenzyloxycarbonylation
As final target structure, compound 49 Scheme 8
Ž
.
Pd–C, H2 gave 34 after purification on Bio-Gel P-2
was prepared along an analogous route as described
for 44. To this end, compound 29 was phosphony-
lated selectively at HO-5 with 3-N-benzyl-
oxycarbonylaminopropyl triethylammonium H-phos-
in a yield of 76% over 2 steps.
Ž
For the synthesis of target compound 40 Scheme
6 , as a first step 3-N-benzyloxycarbonylaminopropyl
.
Ž
. Ž
.
w x
3,4,6-tri-O-acetyl-a-D-galactopyranosyl - 1 ™ 3 -
phonate 45 22 in pyridine–acetonitrile using pival-
oyl chloride to give 46, which was converted into 47
by mild in situ oxidation using iodine in pyridine–
Ž
. w x
2,4,6-tri-O-benzyl-a-D-glucopyranoside 35 23 was
Ž
condensed with 2,3,4-tri-O-acetyl-a-L-rhamnopyr-
. Ž
.
Ž .
anosyl - 1™4 -1-O-benzoyl-2,3-di-O-benzyl-5-O-
water 53% over 2 steps . Then, deacetylation
Ž
.
Ž
.
w
Ž
x
Ž
.
triethylammonium H-phosphonate -D-ribitol 36 23
methanol–ammonia, ™48 , followed by debenzyla-
Ž
.
in pyridine–acetonitrile using pivaloyl chloride ™
tionrdebenzyloxycarbonylation Pd–C, H2 , gave 49
after purification on Bio-Gel P-2, in a yield of 76%
over 2 steps.
.
37 . Subsequent mild in situ oxidation using iodine in
Ž
.
pyridine–water yielded 38 50% over 2 steps . Treat-
Ž
.
ment of 38 with methanol–ammonia ™39 and
Conjugation of the free products to carrier proteins
Scheme 8.

(
)
118
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
Ž
.
Ž
and the results of immunological tests with the neo-
glycoconjugates will be published elsewhere.
d 7.46–7.16 m, 10 H, 2 Ph , 5.281 dd, 1 H, J2,3
.
Ž
3.5, J3,4 9.5 Hz, H-3 , 5.230 dd, 1 H, J1,2 1.8 Hz,
H-2 , 5.08 bs, 2 H, COOCH2 Ph , 4.687 and 4.626
2 d, each 1 H, OCH2 Ph , 4.672 d, 1 H, H-1 , 3.760
m, 1 H, H-5 , 3.490 t, 1 H, J4,5 9.5 Hz, H-4 ,
3.47–3.42 m, 2 H, OCH2 CH2 2 N , 3.34–3.25 m,
O CH2 2CH2 N , 2.133 and 1.961 2 s, each 3 H, 2
Ac , 1.84–1.76 m, 2 H, OCH2CH2CH2 N , 1.332
d, 3 H, J5,6 6.2 Hz, 3 H-6 . Anal. Calcd for
.
Ž
.
Ž
.
Ž
Ž
.
3. Experimental
Ž
.
.
Ž
Ž
.
.
Ž
General methods.—For a survey of general meth-
w
x
Ž
.
.
Ž
ods used in this study, see Ref. 23 .
.
Ž
.
3-N-Benzyloxycarbonylaminopropyl 2-O-acetyl-4-
Ž
.
O-benzyl-3-O-monochloroacetyl-a-L-rhamnopyran-
Ž .
oside 4 .—A mixture of ethyl 2-O-acetyl-4-O-ben-
C28H35NO9: C, 63.50; H, 6.66. Found: C, 63.63; H,
zyl-3-O-monochloroacetyl-1-thio-a-L-rhamnopyran-
6.69.
Ž .
w x Ž .
oside
2
22 2.0 g, 4.8 mmol , 3-N-benzyl-
3-N-Benzyloxycarbonylaminopropyl 2-O-acetyl-4-
Ž .
3
w
x
Ž
Ž .
Ž .
oxycarbonylaminopropanol
mmol and 4 A molecular sieves in CH2Cl2 12.5
mL was stirred for 30 min at 0 8C. Then, a soln of
NIS 1.08 g, 4.80 mmol and TfOH 36 mL, 0.41
mmol in 1:1 CH2Cl2–Et2O 37.6 mL was added.
After 1 min, TLC 7:3 hexane–EtOAc showed the
appearance of a single product 4, Rf 0.25 , and the
mixture was neutralised with Et3N, filtered through
Celite, diluted with CH2Cl2, washed with aq 10%
24 1.16 g, 5.54
O-benzyl-a-L-rhamnopyranoside 9 .— a To a soln
˚
.
Ž
Ž
Ž
.
of 4 263 mg, 0.466 mmol in 1:3 HOAC–2,6-luti-
dine 6.4 mL was added a freshly prepared hydra-
.
.
Ž
.
Ž
Ž
.
w
x
Ž
.
zine dithiocarbonate HDTC soln 25 4 mL . TLC
.
Ž
.
Ž
.
9:1 CH2Cl2–EtOAc, Rf 0.39 indicated the de-
Ž
.
monochloroacetylation to be completed in 15 min.
Then, the mixture was diluted with CH2Cl2, and
Ž
.
Ž
.
Ž
.
washed with water 3= , dried MgSO4 , filtered,
Ž
and concentrated. Column chromatography 92:7:1
CH2Cl2–EtOAc–HOAC of the residue gave 9, iso-
lated in 69% as a foam 158 mg . b To a soln of 6
Ž
.
Ž
.
Ž
.
.
Na2S2O3 2= and water 3= , dried MgSO4 ,
filtered, and concentrated. Column chromatography
6:4 hexane–EtOAc of the residue afforded 4, iso-
Ž
. Ž .
Ž
.
Ž
.
Ž
Ž
.
1.70 g, 3.21 mmol in MeOH 20 mL was added
1
Ž
.
Ž
w x
Ž
.
Ž
.
.
lated as a syrup 2.11 g, 78% ; a y238 c 1 ; H
NaOMe pH 9 . After 2 h, TLC 1:1 hexane–EtOAc
D
Ž
.
.
NMR CDCl3 : d 7.35–7.25 m, 10 H, 2 Ph , 5.312
indicated a complete conversion into 7. The mixture
q
Ž
.
Ž
Ž
.
dd, 1 H, J2,3 3.4, J3,4 9.5 Hz, H-3 , 5.258 dd, 1 H,
J1,2 1.8 Hz, H-2 , 5.09 bs, 2 H, COOCH2 Ph , 4.700
and 4.648 2 d, each 1 H, OCH2 Ph , 4.683 d, 1 H,
H-1 , 3.909 and 3.845 2 d, each 1 H, 2 COCH2Cl ,
was neutralised with Dowex-50 H resin, filtered,
concentrated, and co-concentrated with CH2Cl2.
.
Ž
.
Ž
Ž
.
Ž
Crude 7 was dissolved in 6:1 toluene–CH2Cl2 21
.
Ž
.
.
Ž
.
mL , and trimethyl orthoacetate 1.2 mL, 9.4 mmol
Ž
.
Ž
Ž
3.524 t, 1 H, J4,5 9.5 Hz, H-4 , 3.50–3.45 m, 2 H,
and a catalytic amount of p-toluenesulfonic acid 60
Ž
.
.
Ž
.
Ž
O C H 2 C H 2 N , 3.32 – 3.26
m ,
2
H ,
mg were added. After 30 min, TLC 1:1 hexane–
EtOAc indicated a complete conversion of 7 into 8.
Then, water 1 mL was added, and the soln was
stirred for 20 min, when TLC 1:1 hexane–EtOAc
2
Ž
.
.
Ž
.
Ž
.
O CH2 2C H2 N , 2.136 s, 3 H, Ac , 1.84–1.77 m, 2
.
Ž
Ž
.
H, OCH2CH2CH2 N , 1.347 d, 3 H, J5,6 6.2 Hz, 3
H-6 . Anal. Calcd for C28H34ClNO9: C, 59.63; H,
.
Ž
.
6.08. Found: C, 59.94; H, 5.95.
3 - N - Benzyloxycarbonylaminopropyl 2, 3 - di - O -
acetyl-4-O-benzyl-a-L -rhamnopyranoside 6 .—A
showed the disappearance of 7 and the appearance of
Ž .
a single new spot 9 . The mixture was diluted with
Ž .
Ž
.
Ž
.
EtOAc, washed with water 2= , dried MgSO4 ,
mixture of ethyl 2,3-di-O-acetyl-4-O-benzyl-1-thio-a-
filtered, and concentrated. Column chromatography
Ž .
w
x
Ž
.
Ž
.
L-rhamnopyranoside 5 26 4.29 g, 11.2 mmol ,
86:13:1 CH2Cl2–acetone–HOAC of the residue af-
Ž
Ž
.
3-N-benzyloxycarbonylaminopropanol 3; 3.29 g,
forded 9, isolated as a foam 1.11 g, 71% from 6 ;
w x
Ž
Ž
Ž
Ž
OCH2 CH2 2 N , 3.441 m, 1 H, H-5 , 3.341 t, 1 H,
J4,5 9.4 Hz, H-4 , 3.31–3.24 m,
O CH2 2CH2 N , 2.144 s, 3 H, Ac , 1.83–1.70 m, 2
H, OCH2CH2CH2 N , 1.336 d, 3 H, J5,6 6.3 Hz, 3
H-6 . Anal. Calcd for C26H33NO8: C, 64.05; H, 6.82.
Found: C, 63.89; H, 6.90.
1
˚
.
Ž . Ž .
15.7 mmol and 4 A molecular sieves in CH2Cl2
a
y308 c 1 ; H NMR CDCl3 : d 7.34–7.32
D
Ž
.
Ž
.
Ž
.
150 mL was stirred for 30 min at room temperature.
MeOTf 6.1 mL, 54 mmol was added, and after 18 h
TLC 6:4 hexane–EtOAc showed the disappearance
of 5, and the appearance of a new product with Rf
0.27 6 . The mixture was neutralised with Et3N,
filtered through Celite, diluted with CH2Cl2, washed
with water 3= , dried MgSO4 , filtered, and con-
centrated. Column chromatography 6:4 hexane–
m, 10 H, 2 Ph , 5.080 s, 2 H, COOCH2 Ph , 5.072
.
.
.
dd, 1 H, J1,2 1.6, J2,3 3.7 Hz, H-2 , 4.825 and 4.702
Ž
.
Ž
.
2 d, each 1 H, OCH2 Ph , 4.699 d, 1 H, H-1 , 4.073
dd, 1 H, J3,4 9.4 Hz, H-3 , 3.76–3.64 m, 2 H,
.
Ž
Ž .
Ž
.
.
Ž
.
Ž
.
Ž
2
Ž
H,
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
.
.
EtOAc of the residue afforded 6, isolated as a syrup
4.79 g, 81% ; a y118 c 1 ; H NMR CDCl3 :
1
Ž
.
w x
Ž
.
Ž
.
D

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
119
(
3-N-Benzyloxycarbonylaminopropyl 3-O-allyl-2,4,
) (
into 12 to be completed, and the mixture was diluted
with CH2Cl2, filtered, washed with water, aq 5% Kl,
)
6-tri-O-benzyl-a-D-glucopyranosyl - 1™3 -2-O-
Ž
.
Ž
.
acetyl-4-O-benzyl-a-L-rhamnopyranoside 11 .—A
mixture of ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-
water, aq 10% NaHCO3, and water, dried MgSO4 ,
filtered, and concentrated. Column chromatography
Ž
.
w
x
Ž
.
Ž
.
b-D-glucopyranoside 10 27 184 mg, 0.372 mmol ,
6:4 hexane–EtOAc of the residue afforded 12, iso-
1
˚
Ž
.
Ž
.
Ž
w x
Ž
.
9 140 mg, 0.287 mmol and 4 A molecular sieves in
1:4 CH2Cl2–Et2O 10 mL was cooled to 0 8C, and
stirred for 30 min. Then, MeOTf 162 mL, 1.43
mmol was added. After 4 h, TLC 95:5 CH2Cl2–
acetone indicated the disappearance of 9 and the
appearance of two spots 11r11b, Rf 0.39 and 0.46,
a:b 1.9:1 as was estimated from a sample taken for
H NMR , and the mixture was neutralised with
Et3N, diluted with CH2Cl2, washed with water 3= ,
dried MgSO4 , filtered, and concentrated. Column
chromatography 65:35 hexane–EtOAc of the
lated in 54% as a glass 21 mg ; a D q498 c 1 ; H
NMR CDCl3 : d 7.37–7.24 m, 25 H, 5 Ph , 5.319
Ž
.
Ž
.
.
Ž
Ž
Ž
Ž
.
dd, 1 H, J1,2 1.9, J2,3 3.3 Hz, H-2 , 5.179 d, 1 H,
X
.
.
Ž
.
X
X
J1 ,2 3.5 Hz, H-1 , 5.08 bd, 2 H, COOCH2 Ph ,
.
4.848, 4.812, 4.720, 4.543, 4.498, 4.460, 4.303, and
4.196 8 d, each 1 H, 4 OCH2 Ph , 4.673 d, 1 H,
Ž
Ž
.
Ž
.
.
Ž
H-1 , 3.504 t, 1 H, J3,4 sJ4,5 s9.5 Hz, H-4 , 3.421
1
.
Ž
.
Ž
Ž
.
.
dd, 1 H, H-3 , 3.26–3.19 m, 2 H, O CH2 2CH2 N ,
X
Ž
.
Ž
.
Ž
.
2.38 bs, 1 H, HO-3 , 1.964 s, 3 H, Ac , 1.81–1.68
Ž
Hz, 3 H-6 .
Ž
.
.
Ž
m, 2 H, OCH2C H2CH2 N , 1.352 d, 3 H, J5,6 6.2
Ž
.
.
Ž
.
(
residue afforded 11, isolated as a glass 93 mg, 34% ;
3-N-Benzyloxycarbonylaminopropyl 3,4,6-tri-O-
1
w x
Ž . Ž .
) ( ) (
a
q468 c 1 ; NMR CDCl3 : H, d 7.35–7.24
acetyl-2-O-allyl-a-D-galactopyranosyl - 1™3 - 2,4,6-
D
Ž
.
Ž
.
) (
)
m, 25 H, 5 Ph , 5.974 m, 1 H, OCH2CH5CH2 ,
tri-O-benzyl-a-D-glucopyranosyl - 1™3 -2-O-acetyl-
X
Ž
.
Ž
X
X
Ž
.
5.105 d, 1 H, J1 ,2 3.3 Hz, H-1 , 5.07 bd, 2 H,
COOC H2 Ph , 4.885, 4.826, 4.556, 4.534, 4.435, and
4.298 6 d, each 1 H, 3 OCH2 Ph , 4.67 bd, 2 H,
OCH2 Ph , 4.644 d, 1 H, J1,2 1.8 Hz, H-1 , 4.137
4-O-benzyl-a-L-rhamnopyranoside 14 .—A mixture
of 3,4,6-tri-O-acetyl-2-O-allyl-arb-D-galactopyrano-
.
Ž
.
Ž
Ž
.
w
x
Ž
.
syl trichloroacetimidate 13 23 98 mg, 0.20 mmol ,
˚
.
Ž
.
Ž
.
12 107 mg, 0.116 mmol and 4 A molecular sieves
in Et2O 11 mL was cooled to 0 8C, and stirred for 1
h. Then, TMSOTf 45 mL, 0.23 mmol was added.
After 1 h, TLC 92.5:7.5 CH2Cl2–acetone indicated
the disappearance of 12 and the appearance of a
single product 14, Rf 0.51 , and the mixture was
neutralised with Et3N, diluted with CH2Cl2, washed
with water 3= , dried MgSO4 , filtered, and con-
X
Ž
.
Ž
.
X
X
X
X
Ž
.
dd, 1 H, J2 ,3 9.5 Hz, H-2 , 3.920 t, 1 H, J3 ,4 9.4
X
.
Ž
Ž
.
Ž
.
Hz, H-3 , 3.24–3.17 m, 2 H, O CH2 2CH2 N , 1.908
Ž
.
Ž
.
Ž
.
s, 3 H, Ac , 1.79–1.66 m, 2 H, OCH2CH2CH2 N ,
1.351 d, 3 H, J5,6 6.2 Hz, 3 H-6 ; 13C, d 170.3
Ž
.
Ž
Ž
.
Ž .
Ž
.
C OCH 3
,
156.2
NCOOCH 2 Ph , 135.2
.
Ž
.
OCH2CH5CH2 , 116.3 OCH2CH5CH2 , 97.4
and 92.5 C-1,1 , 81.7, 79.4, 78.9, 77.6, 71.9, 69.8,
67.9, and 67.5 C-2,3,4,5,2 ,3 ,4 ,5 , 76.0, 74.8, 74.1,
73.0, 72.8, 68.1, 66.4, and 65.2 4 OCH2 Ph,
NCOOCH2 Ph, OCH2 CH2 2 N, OCH2CH5CH2,
and C-6 , 38.1 O CH 2 2 C H 2 N , 29.4
OCH2CH2CH2 N , 20.7 COCH3 , 17.8 C-6 . Anal.
Calcd for C56H65NO13 PH2O: C, 68.77; H, 6.90.
X
Ž
.
Ž
.
Ž
.
X
X
X
X
Ž
.
Ž
centrated. Column chromatography 93:7 CH2Cl2–
acetone of the residue afforded 14, isolated as a
Ž
.
1
Ž
.
Ž
.
w x
Ž
.
.
syrup 76 mg, 52% ; a y368 c 1 ; H NMR
D
X
.
w
Ž
.
x
Ž
Ž
.
Ž
CDCl3 : d 7.40–7.04 m, 25 H, 5 Ph , 3.26–3.19
m, 2 H, O CH2 2CH2 N , 2.059, 2.015, and 1.934 3
Ž
.
Ž
.
Ž
.
Ž
.
.
.
Ž
Ž
.
s, 3,6,3 H, 4 Ac , 1.373 d, 3 H, J5,6 6.2 Hz, 3 H-6 .
Anal. Calcd. for C68H81NO21: C, 65.42; H, 6.54.
Found: C, 65.30; H, 6.46.
Found: C, 69.10; H, 7.07.
(
3-N-Benzyloxycarbonylaminopropyl 2,4,6-tri-O-
) (
)
(
benzyl-a-D-glucopyranosyl - 1™3 -2-O-acetyl-4-O-
3-N-Benzyloxycarbonylaminopropyl 3,4,6-tri-O-
Ž
.
) (
) (
benzyl-a-L-rhamnopyranoside 12 .—To a soln of 11
acetyl-a-D-galactopyranosyl - 1™3 - 2,4,6-tri-O-
Ž
Ž
.
w
x
) (
)
41 mg, 43 mmol and 1,4-diazabicyclo 2.2.2 octane
DABCO 15 mg, 0.13 mmol in 8:3:1 EtOH–
benzyl-a-D-glucopyranosyl - 1™3 -2-O-acetyl-4-O-
. Ž
.
Ž
.
benzyl-a-L-rhamnopyranoside 15 .—To a soln of 14
Ž
Ž .
.
Ž
Ž
.
Ž
.
toluene–water 5 mL was added tris triphenylphos-
70 mg, 56 mmol and DABCO 15 mg, 0.13 mmol
.
Ž
.
Ž
.
phine rhodium I chloride 10 mg . After boiling un-
der reflux for 3 h, TLC 95:5 CH2Cl2–acetone
showed the reaction to be completed. The mixture
was cooled, diluted with CH2Cl2, washed with 0.1 M
HCl and water 2= , and concentrated. To a soln of
the residue in 9:1 acetone–water 5 mL were added
HgCl2 60 mg, 0.22 mmol and a catalytic amount of
HgO 1.1 mg . After stirring the mixture for 1 h,
in 8:3:1 EtOH–toluene–water 6 mL was added
tris triphenylphosphine rhodium I chloride 10 mg .
After boiling under reflux for 90 min, TLC 9:1
CH2Cl2–EtOAc indicated the reaction to be com-
pleted. The mixture was cooled, diluted with CH2Cl2,
washed with 0.1 M HCl and water 2= , and con-
centrated. To a soln of the residue in 9:1 acetone–
water 5 mL were added HgCl2 200 mg, 0.737
Ž
.
Ž
.
Ž .
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
Ž
.
Ž
.
Ž
.
.
Ž
.
TLC 6:4 hexane–EtOAc showed the conversion
mmol and a catalytic amount of HgO 1.0 mg . After

(
)
120
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
Ž
Ž
Ž
Ž
.
Ž
.
stirring for 3 h, the mixture was diluted with CH2Cl2,
filtered, washed with water, aq 5% Kl, water, aq 10%
C OCH 3 , 18 b , 166.2
C OPh , 134.6
.
Ž
.
OCH2CH5CH2 , 116.7 OCH2CH5CH2X , 103.1
C-1 , 18b , 97.3 C-1 , 18 , 92.2 C-1 , 21.0
COCH3, 18b , 20.7 COCH3, 18 , 17.7 C-6 , 18 ,
Y
Y
Ž
.
.
Ž
.
Ž
.
NaHCO3, and water, dried MgSO4 , filtered, and
X
Ž
.
Ž
.
Ž
.
concentrated. Column chromatography 9:1
CH2Cl2–EtOAc of the residue afforded 15, isolated
X
.
Ž
.
17.6 C-6 , 18b .
1
Ž
.
w x
Ž
.
Ž
.
in 37% as a glass 25 mg ; a y128 c 1 ; H
To a soln of 18r18b 202 mg, 0.158 mmol in 1:5
D
Ž
.
Ž
.
Ž
.
Ž
.
NMR CDCl3 : d 7.70–7.03 m, 25 H, 5 Ph , 5.571
CH2Cl2–MeOH 5 mL was added NaOMe pH 11 .
After 1 h, TLC 8:2 toluene–EtOAc showed a com-
plete conversion into 19r19b Rf 0.26 . The mix-
ture was neutralised with Dowex-50 H resin, fil-
Y
Ž
.
Ž
Y
Y
Ž
.
d, 1 H, J1 ,2 3.8 Hz, H-1 , 5.306 dd, 1 H, J1,2 1.7,
J2,3 3.1 Hz, H-2 , 5.270 dd, 1 H, J3 ,4 3.1, J4 ,5 -1
X
.
Ž
Y
Y
Y
Y
Ž
.
Y
X
q
.
Ž
.
X
X
Ž
.
Hz, H-4 , 5.215 d, 1 H, J1 ,2 3.5 Hz, H-1 , 5.134
Y
Ž
.
.
Ž
Ž
Y
Y
Ž
dd, 1 H, J2 ,3 10.5 Hz, H-3 , 5.07 bs, 2 H,
COOCH2 Ph , 4.693 d, 1 H, H-1 , 4.160 dd, 1 H,
J2 ,3 9.6 Hz, H-2 , 3.27–3.21 m,
O CH2 2CH2 N , 2.619 d, 1 H, HO-2 , 2.070, 2.027,
tered, and concentrated. Column chromatography 8:2
.
Ž
.
toluene–EtOAc of the residue afforded 19r19b,
X
isolated in 82% as a glass 146 mg ; 1H NMR
.
Ž
Ž
X
X
Ž
.
2
H,
Y
Ž
.
.
.
Ž
.
Ž
,
.
Ž
2
CDCl3 : d 7.33–7.11 m, 35 H, 7 Ph , 5.865 m, 1
Ž
.
Ž
.
Ž
1.955, and 1.931 4 s, each 3 H, 4 Ac , 1.372 d, 3 H,
H, OCH 2C H5CH 2
OCH2CH5CH2 , 5.170 d, 1 H, J1 ,2 -1 Hz, H-1,
19 , 5.128 d, 1 H, J1 ,2 1.8 Hz, H-1, 19b , 4.919 d,
5.30–5.10 m,
H,
X
.
.
Ž
X
X
J5,6 6.2 Hz, 3 H-6 .
X
(
)
.
Ž
.
Ž
X
X
2,3,4,6-Tetra-O-benzyl-arb-D-glucopyranosyl -
Y
(
) (
)
.
Ž
Y
Y
X
X
1™3 - 2-O-acetyl-4-O-benzyl-a-L-rhamnopyranosyl
1 H, J1 ,2 2.1 Hz, H-1 , 19 , 1.242 d, 3 H, J5 ,6 6.2
X
Hz, 3 H-6 , 19 , 1.191 d, 3 H, J5 ,6 6.2 Hz, 3 H-6X,
(
)
.
Ž
X
X
- 1™4 -5-O-allyl-1-O-benzoyl-2,3-di-O-benzyl-D-
Ž
. Ž
) ( ) (
.
ribitol 18r18b , 2,3,4,6-tetra-O-benzyl-arb-D-
19b .
Ž
.
glucopyranosyl - 1™3 - 4-O-benzyl-a-L-rhamnopy-
A soln of 19r19b 207 mg, 0.183 mmol and
) (
)
Ž
.
Ž
ranosyl - 1™4 -5-O-allyl-2,3-di-O-benzyl-D-ribitol
benzyl bromide 75 mL, 0.63 mmol in DMF 3.5
mL was added dropwise to a stirred, cooled 0 8C
suspension of NaH 50 mg, 2.1 mmol in DMF 1
mL . After 3 h, TLC 8:1 toluene–EtOAc, Rf 0.59
Ž
. Ž
) ( ) (
.
Ž
.
19r19b , 2,3,4,6-tetra-O-benzyl-a-D-glucopyra-
)
Ž
.
Ž
nosyl - 1™3 - 2,4-di-O-benzyl-a-L-rhamnopyranosyl
(
)
Ž
) (
.
.
.
w
- 1™4 -5-O-allyl-1,2,3-tri-O-benzyl-D-ribitol 20 , and
Ž
(
)
Ž
Ž
.x
2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl - 1™3 -
20 , 0.66 20b showed the benzylation to be com-
) (
)
2,4-di-O-benzyl-a-L-rhamnopyranosyl - 1™4 -1,2,3-
pleted. The excess of NaH was destroyed with MeOH,
the mixtur e was diluted with EtOAc, washed with
water 3= , dried MgSO4 , filtered, and concen-
trated. Column chromatography 8:1 toluene–EtOAc
Ž
.
tri - O - benzyl - D - ribitol 21 .—A mixture of ethyl
2,3,4,6-tetra-O-benzyl-1-thio-b-D-glucopyranoside
Ž
.
Ž
.
Ž
.
w
x
Ž
. Ž
Ž
.
16 28,29 266 mg, 0.455 mmol , 2-O-acetyl-4-
. Ž
.
Ž
.
Ž
O-benzyl-a-L-rhamnopyranosyl - 1™4 -5-O-allyl-1-
of the residue gave 20 199 mg, 83% and 20b 37
Ž
. w x Ž
.
w x
O-benzoyl-2,3-di-O-benzyl-D-ribitol 17 22 232
mg, 15% , both isolated as a syrup; for 20: a D q58
1
˚
Ž
.
Ž
.
Ž
.
Ž
mg, 0.307 mmol and 4 A molecular sieves in 1:5
1,2-dichloroethane–Et2O 10.2 mL was stirred for
30 min, then IDCP 426 mg, 0.909 mmol was added.
After 1 h, TLC 7:3 hexane–EtOAc indicated the
c 1 ; NMR CDCl3 : H, d 7.28–7.18 m, 45 H, 9
.
.
Ž
.
Ž
Ph , 5.775 m, 1 H, OCH2CH5CH2 , 5.21–5.06 m,
2 H, OCH2CH5CH2 , 5.189 d, 1 H, J1 ,2 3.6 Hz,
H-1 , 5.152 d, 1 H, J1 ,2 1.8 Hz, H-1 , 1.188 d, 3
H, J5 ,6 6.2 Hz,
Ž
Ž
75.7, 75.2, 75.1, 70.2, and 68.7
2,3,4,2 ,3 ,4 ,5 ,2 ,3 ,4 ,5 , 75.4, 74.7, 73.5, 73.2,
73.0, 72.8, 72.7, 72.3, 71.9, 70.3, 70.1 2 C , and
68.2 C-1,5,6 , 9 OCH2 Ph, and OCH2CH5CH2 ,
Ž
.
.
Ž
Y
Y
Y
X
Ž
.
.
Ž
.
Ž
X
X
H-6 ; 13C,
d
134.7
X
.
X
X
disappearance of 17 and the appearance of a new spot
3
Ž
.
.
.
Ž
.
Rf 0.43 . The mixture was diluted with CH2Cl2 ,
filtered through Celite, washed with aq 10% Na2S2O3
OCH2CH5CH2 , 116.5 OCH2CH5CH2 , 97.6
C-1 , 94.0 C-1 , 82.1, 80.1, 79.4 2 C , 78.3, 77.7,
Y
X
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
2= and water 2= , dried MgSO4 , filtered, and
C-
X
X
X
X
Y
Y
Y
Y
Ž
.
concentrated. Column chromatography 8:2 hexane–
EtOAc of the residue afforded 18r18b, isolated as a
glass 243 mg, 62%, a:b 6:1 ; NMR CDCl3 : H, d
7.95–7.05 m, 40 H, 8 Ph , 5.865 m, 1 H,
OCH2C H5CH2 , 5.486 dd, 1 H, J1 ,2 2.0, J2 ,3 2.6
Hz, H-2 , 18 , 5.342 dd, 1 H, J1 ,2 1.8, J2 ,3 3.4 Hz,
H-2 , 18b , 5.243 d, 1 H, J1 ,2 3.4 Hz, H-1 , 18 ,
.
Ž
.
1
Y
Ž
.
Ž
.
Ž
Ž
.
Y
Ž
.
Ž
.
18.0 C-6 .
.
Ž
X
X
X
X
Ž
.
Ž
.
A soln of 20 85 mg, 65 mmol in DMF 5 mL
X
.
Ž
X
X
X
X
Ž
was heated at 80 8C, and KOtBu 120 mg, 1.07
mmol was added, giving the solution a deep black
colour. After 30 min, TLC 30:1 toluene–acetone
indicated a complete conversion of the allyl Rf
0.46 into the 1-propenyl function Rf 0.66 . The
mixture was cooled, diluted with CH2Cl2, washed
with aq 5% NaCl and water, and concentrated. The
X
Y
.
Ž
.
Y
Y
.
X
Ž
.
Ž
Ž
.
5.240 d, 1 H, H-1, 18 , 5.20–5.11 m, 2 H,
OCH2CH5CH2 , 5.135 d, 1 H, H-1, 18b , 2.144
X
.
Ž
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
s, 3 H, Ac, 18b , 1.935 s, 3 H, Ac, 18 , 1.240 d, 3
X
.
Ž
X
X
X
X
H, J5 ,6 6.2 Hz, 3 H-6 , 18 , 1.156 d, 3 H, J5 ,6 6.2
X
13
.
Ž
.
Hz, 3 H-6 , 18b ; C, d 170.2 COCH3, 18 , 169.9

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
121
Ž
. Ž
mg, 0.163 mmol , 2-O-acetyl-4-O-benzyl-a-L -
residue was dissolved in 9:1 acetone–0.1 M HCl 10
.
. Ž
.
mL and boiled under reflux for 40 min, when TLC
30:1 toluene–acetone indicated a complete conver-
sion of the 1-propenyl-containing compound into a
lower moving spot Rf 0.09 . The mixture was neu-
tralised with aq 25% NH4OH, concentrated, diluted
with CH2Cl2, washed with aq 10% NaHCO3 and
water, dried MgSO4 , filtered, and concentrated.
Column chromatography 7:3 hexane–EtOAc of the
rhamnopyranosyl - 1™4 -5-O-allyl-1-O-benzoyl-2,3-
Ž
.
Ž
.
w
x
Ž
.
di-O-benzyl-D-ribitol 17 22 98 mg, 0.13 mmol ,
˚
and 4 A molecular sieves in 1:5 1,2-dichloroethane–
Ž
.
Ž
.
Et2O 3.8 mL was stirred for 30 min, then IDCP
Ž
Ž
.
152 mg, 0.324 mmol was added. After 1 h, TLC
6:4 hexane–EtOAc indicated the disappearance of
17 and the appearance of a new spot 24r24b, Rf
0.34 . The mixture was diluted with CH2Cl2, filtered
.
Ž
.
Ž
Ž
.
.
Ž
residue afforded 21, isolated in 49% as a glass 40
through Celite, washed with aq 10% Na2S2O3, wa-
1
.
w x
Ž . Ž .
q28 c 0.25 ; H NMR CDCl3 : d
Y Y
Ž
.
mg ;
a
ter, aq 10% NaHCO3, and water, dried MgSO4 ,
filtered, and concentrated. Column chromatography
DŽ
.
Ž
7.34–7.09 m, 45 H, 9 Ph , 5.207 d, 1 H, J1 ,2 3.4
Y
X
.
Ž
.
X
X
Ž
.
Hz, H-1 , 4.964 d, 1 H, J1 ,2 -1 Hz, H-1 , 2.43
m, 1 H, HO-5 , 1.245 d, 3 H, J5 ,6 6.1 Hz, 3 H-6 .
8:2 hexane–EtOAc of the residue afforded 24r24b,
X
Ž
.
Ž
.
Y
Y
Ž
.
isolated as a syrup 106 mg, 62%, a:b 2:1 ; NMR
Ž
C6 H4OCH3 , 5.871 m, 1 H, OCH2CH5CH2 , 5.478
1
.
Ž
Ethyl 2,4,6-tri-O-benzyl-3-O-p-methoxybenzyl-1-thio
CDCl3 : H, d 7.95–6.68 m, 39 H, 7 Ph and
Ž
.
.
Ž
.
-b-D-glucopyranoside 23 .—A soln of ethyl 2,4,6-
. Ž
X
Ž
Ž
.
Ž
X
X
X
X
tri-O-benzyl-1-thio-b-D-glucopyranoside 22 1.01 g,
dd, 1 H, J1 ,2 2.0, J2 ,3 2.9 Hz, H-2 , 24 , 5.328 dd,
X
.
Ž
.
Ž
X
X
X
X
2.04 mmol and p-methoxybenzyl chloride 0.35 mL,
2.6 mmol in DMF 5 mL was added dropwise to a
stirred, cooled 0 8C suspension of NaH 150 mg,
6.25 mmol in DMF 5 mL . TLC 7:3 hexane–
EtOAc showed the formation of 23 Rf 0.56 to be
completed in 2 h. After destroying the excess of NaH
with MeOH, the mixture was diluted with CH2Cl2,
washed with water 3= , dried MgSO4 , filtered,
concentrated, and co-concentrated with toluene 2= ,
1 H, J1 ,2 1.8, J2 ,3 3.4 Hz, H-2 , 24b , 5.234 d, 1
Y
.
Ž
.
.
Ž
.
Y
Y
H, J1 ,2 3.5 Hz, H-1 , 24 , 5.159 d, 1 H, H-1, 24b ,
5.126 d, 1 H, H-1, 24 , 3.746 s, 3 H, C6H4OC H3,
24b , 3.704 s, 3 H, C6H4OCH3, 24 , 2.141 s, 3 H,
Ac, 24b , 1.928 s, 3 H, Ac, 24 , 1.234 d, 3 H, J5 ,6
X
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž
X
X
X
.
Ž
X
X
6.2 Hz, 3 H-6 , 24 , 1.150 d, 3 H, J5 ,6 6.2 Hz, 3
H-6 , 24b ; 13C, d 170.2 COCH3, 24 , 169.9
X
.
Ž
.
Ž
.
Ž
.
Ž
Ž
Ž
Ž
.
Ž
.
COC H 3 , 24 b , 166.2
C O Ph , 134.6
Ž
.
.
Ž
.
OCH2CH5CH2 , 116.8 OCH2CH5CH2 , 103.2
C-1 , 24b , 97.3 C-1, 24b , 96.5 C-1 , 24 , 92.4
C-1, 24 , 55.1 C6H4OCH3X, 21.0 COCH3, 24b ,
Y
X
Y
Ž
.
Ž
.
.
Ž
.
.
Ž
Ž
.
EtOH 2= , and CH2Cl2 2= to give a white
solid. Crystallisation of the solid from EtOH gave 23
X
.
Ž
.
.
1
X
Ž
.
Ž
.
Ž
.
Ž
Ž
.
Ž
1.02 g, 81% as white crystals; H NMR CDCl3 : d
20.7 COCH3, 24 , 17.8 C-6 , 24 , 17.7 C-6 , 24b .
Ž
.
.
7.33–6.81 m, 19 H, 3 Ph and C6 H4OCH3 , 4.918,
4.841, 4.824, 4.765, 4.748, 4.595, 4.562, and 4.533
8 d, each 1 H, 3 OC H2 Ph and OCH2C6H4OCH3 ,
4.455 d, 1 H, J1,2 9.7 Hz, H-1 , 3.781 s, 3 H,
C6H4OCH3 , 3.740 dd, 1 H, J5,6a 2.3, J6a,6b 10.9
Hz, H-6a , 3.664 dd, 1 H, J5,6b 4.8 Hz, H-6b , 3.665
t, 1 H, J2,3 sJ3,4 s8.7 Hz, H-3 , 3.581 t, 1 H, J4,5
8.9 Hz, H-4 , 3.457 m, 1 H, H-5 , 3.421 dd, 1 H,
To a soln of 24r24b 361 mg, 0.276 mmol in 1:3
Ž
.
Ž
.
CH2Cl2–MeOH 8 mL was added NaOMe pH 12 .
Ž
.
After 18 h, the pH of the mixture had decreased, and
Ž
.
Ž
Ž
.
an extra amount of NaOMe was added pH 12 . After
.
Ž
Ž
.
another 18 h, TLC 3:1 toluene–EtOAc indicated a
complete conversion of 24r24b into 25r25b Rf
0.34 . The mixture was neutralised with Dowex-50
resin, filtered, and concentrated. Column chro-
matography 65:35 hexane–EtOAc of the residue
.
Ž
.
Ž
Ž
.
Ž
.
.
q
.
Ž
.
Ž
Ž
H
.
Ž
.
Ž
Ž
.
H-2 , 2.88–2.64 m, 2 H, SCH2CH3 , 1.323 t, 3 H,
.
Ž
SCH2CH3 .
afforded 25r25b, isolated in 85% as a glass 272
(
.
Ž
.
2,4,6-Tri-O-benzyl-3-O-p-methoxybenzyl-arb-D-
mg . A soln of 25r25b 73 mg, 63 mmol and
) (
) (
Ž
.
Ž
.
glucopyranosyl - 1™3 - 2-O-acetyl-4-O-benzyl-a-L-
benzyl bromide 25 mL, 0.21 mmol in DMF 1 mL
) (
)
Ž
.
rhamnopyranosyl - 1™4 -5-O-allyl-1-O-benzoyl-2,3-
was added dropwise to a stirred, cooled 0 8C sus-
pension of NaH 13 mg, 0.54 mmol in DMF 1 mL .
Ž
. Ž
Ž
.
Ž
Ž
Ž
.
.x
di-O-benzyl-D-ribitol 24r24b , 2,4,6-tri-O-benzyl-3-
) (
)
w
.
O-p-methoxybenzyl-arb-D-glucopyranosyl - 1™3 -
TLC 8:2 hexane–EtOAc, Rf 0.59 26 , 0.66 26b
(
) (
)
4-O-benzyl-a-L-rhamnopyranosyl - 1™4 -5-O-allyl-
showed the benzylation to be completed in 1 h. After
destroying the excess of NaH with MeOH, the mix-
ture was diluted with EtOAc, washed with water
Ž
. Ž
2,3-di-O-benzyl-D-ribitol 25r25b , 2,4,6-tri-O-benzyl
) (
)
-3-O-p-methoxybenzyl-a-D-glucopyranosyl - 1™3 -
(
) (
)
Ž
.
Ž
.
2,4-diyOybenzyl-a-L-rhamnopyranosyl - 1™4 -5-
3= , dried MgSO4 , filtered, and concentrated.
(
.
Ž
Ž
.
O-allyl-1,2,3-tri-O-benzyl-D-ribitol 26 , and 2,4,6-tri-
) ( ) (
Column chromatography 24:1 toluene–EtOAc of
the residue gave 26 43 mg, 51% and 26b 22 mg,
26% , both isolated as a syrup; for 26: a q38 c
1 ; NMR CDCl3 : H, d 7.32–6.73 m, 44 H, 8 Ph
Ž
.
Ž
O-benzyl-a-D-glucopyranosyl - 1™3 - 2,4-di-O-
) (
)
.
w x
Ž
benzyl-a-L-rhamnopyranosyl - 1™4 -5-O-allyl-1,2,3-
D
1
Ž
.
Ž
.
Ž
.
Ž
tri-O-benzyl-D-ribitol 27 .—A mixture of 23 100

(
)
122
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
X
Y
Z
.
Ž
.
.
Ž
.
and C6 H4OCH3 , 5.801 m, 1 H, OCH2CH5CH2 ,
OCH2CH5CH2 , 97.7, 97.0, and 91.9 C-1,1 ,1 ,
X
Ž
.
Ž
Ž
.
Ž
.
Ž
.
5.24–5.08 m, 2 H, OCH2CH5CH2 , 5.20–5.18 m,
2 H, H-1,1 , 3.714 s, 3 H, C6H4OCH3 , 1.241 d, 3
H, J5 ,6 6.1 Hz,
20.7 2 C and 20.5 3 COCH3 , 18.0 C-6 . Anal.
Calcd for C91H104O22: C, 70.52; H, 6.76. Found: C,
70.48; H, 6.65.
X
Y
.
Ž
.
Ž
3
H-6 ; 13C,
d
134.7
X
.
X
X
Ž
.
.
Ž
.
(
) (
)
OCH2CH5CH2 , 116.5 OCH2CH5CH2 , 97.7
3,4,6-Tri-O-acetyl-a-D-galactopyranosyl - 1™3 -
X
Y
Ž
Ž
.
C-
(
) ( ) (
and 94.0 C-1, 1 , 81.7, 80.1, 79.5, 79.4, 78.3 2 C ,
2,4,6-tri-O-benzyl-a-D-glucopyranosyl - 1™3 - 2,4-
Ž
) (
)
77.7, 75.7, 75.2, 75.1, and 68.7
di-O-benzyl-a-L-rhamnopyranosyl - 1™4 -1,2,3-tri-O-
X
X
X
X
Y
Y
Y
Y
.
Ž
.
Ž
2,3,4,2 ,3 ,4 ,5 ,2 ,3 ,4 ,5 , 75.4, 75.0, 74.7, 73.6,
benzyl-D-ribitol 29 .—To a solution of 28 76 mg,
Ž
.
8
.
Ž
.
73.2 2 C , 73.0, 72.9, 72.7, 72.3, 71.9, 70.1, and
49 mmol and DABCO 50 mg, 0.44 mmol in 8:3:1
EtOH–toluene–water 12 mL was added tris tri-
Ž
Ž
.
Ž
68.2
O C H Ph, O C H C 6 H O C H ,
2 2 4 3
Y
.
Ž
.
.
Ž .
Ž
.
OCH2CH5CH2, and C-1,5,6 , 55.1 C6H4OCH3 ,
phenylphosphine rhodium I chloride 29 mg . After
X
Ž
.
Ž
.
18.0 C-6 .
boiling under reflux for 3 h, TLC 6:4 hexane–EtOAc
showed the reaction to be completed. The mixture
was cooled, diluted with CH2Cl2, washed with 0.1 M
HCl and water 2= , and concentrated. To a solution
of the residue in 9:1 acetone–water 6 mL were
added HgCl2 96 mg, 0.35 mmol and a catalytic
amount of HgO 4.8 mg . After stirring the mixture
for 2 h, TLC 6:4 hexane–EtOAc showed the con-
Ž
.
To a soln of 26 180 mg, 0.134 mmol in 3:6:1
toluene–acetonitrile–water 10 mL was added am-
Ž
.
Ž
.
Ž
Ž
.
monium cerium IV nitrate CAN; 300 mg, 0.547
.
Ž
.
Ž
.
mmol . After 1 h, TLC 8:2 hexane–EtOAc showed
the conversion of the starting compound into one new
spot 27, Rf 0.36 . The reaction mixture was diluted
with CH2Cl2, washed with water, aq 5% NaHSO3,
aq 10% NaHCO3, and water, dried MgSO4 , fil-
tered, and concentrated. Column chromatography 8:2
hexane–EtOAc of the residue afforded 27, isolated
as a syrup 118 mg, 72% ; a D q88 c 1 ; H NMR
CDCl3 : d 7.32–7.09 m, 40 H, 8 Ph , 5.809 m, 1
H, OCH2CH5CH2 , 5.25–5.22 m, 2 H, H-1,1 ,
1.223 d, 3 H, J5 ,6 6.1 Hz, 3 H-6 . Anal. Calcd for
C76H84O14: C, 74.73; H, 6.93. Found: C, 74.55; H,
6.84.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
version of 28 into 29 Rf 0.16 to be completed. The
mixture was diluted with CH2Cl2, filtered, washed
with water, aq 5% Kl, water, aq 10% NaHCO3, and
Ž
.
1
Ž
.
w x
Ž
.
Ž
.
water, dried MgSO4 , filtered, and concentrated.
Ž
.
Ž
.
Ž
X
Ž
.
Column chromatography 6:4 hexane–EtOAc of the
Y
.
Ž
.
Ž
residue afforded 29, isolated in 61% as a glass 44
mg ; a q588 c 1 ; H NMR CDCl3 : d 7.29–
7.05 m, 40 H, 8 Ph , 5.533 d, 1 H, J1 ,2 3.8 Hz,
H-1 , 5.286 d, 1 H, J1 ,2 3.4 Hz, H-1 , 5.204 dd,
1 H, J3 ,4 3.3, J4 ,5 f1 Hz, H-4 , 5.118 dd, 1 H,
J2 ,3 10.5 Hz, H-3 , 5.065 d, 1 H, J1 ,2 2.3 Hz,
X
1
Ž
.
X
X
.
w x
Ž
.
Ž
.
D
Ž
.
Ž
Z
Z
Z
Y
.
Ž
.
Ž
Y
Y
Z
(
)
)
)
.
Ž
Z
Z
Z
Z
3,4,6-Tri-O-acetyl-2-O-allyl-a-D-galactopyranosyl -
) (
Z
(
(
(
.
Ž
Z
Z
X
X
1™3 - 2,4,6-tri-O-benzyl-a-D-glucopyranosyl -
X
) (
)
.
Ž
.
1™3 - 2,4-di-O-benzyl-a-L -rhamnopyranosyl -
H-1 , 2.059, 2.035, and 1.860 3 s, each 3 H, 3 Ac ,
Y
Ž
Ž
.
Ž
.
X
X
1™4 -5-O-allyl-1,2,3-tri-O-benzyl-D-ribitol 28 .—A
1.271 d, 3 H, J5 ,6 6.2 Hz, 3 H-6 . Anal. Calcd for
C85H96O22: C, 69.47; H, 6.58. Found: C, 69.40; H,
6.48.
Ž
.
mixture of 13 110 mg, 0.224 mmol , 27 123 mg,
˚
.
Ž
0.101 mmol and 4 A molecular sieves in Et2O 10
mL was cooled to 0 8C, and stirred for 1 h. Then,
TMSOTf 42 mL, 0.22 mmol was added. After 20
min, TLC 8:2 toluene–EtOAc showed the disap-
pearance of acceptor 27 and the appearance of a
single product 28, Rf 0.48 , and the mixture was
neutralised with Et3N, diluted with CH2Cl2, washed
.
(
3-N-Benzyloxycarbonylaminopropyl 1,2,3,4-tetra-
Ž
.
) (
O-benzyl-D-ribityl - 5™triethylammonium phosphate
Ž
.
) (
)
)
™2 - 3,4,6-tri-O-acetyl-a-D -galactopyranosyl -
(
(
Ž
) (
)
1™3 - 2,4,6-tri-O-benzyl-a-D-glucopyranosyl -
Ž
.
1™3 -2-O-acetyl-4-O-benzyl-a-L-rhamnopyranoside
.
(
32 and 3 - aminopropyl - D - ribityl - 5™hydrogen
Ž
.
Ž
.
)
(
)
with water 3= , dried MgSO4 , filtered, and con-
phosphate™2 -a-D-galactopyranosyl- 1™3 -a-D-
Ž
(
)
Ž
.
centrated. Column chromatography 6:1 toluene–
EtOAc of the residue afforded 28, isolated as a syrup
126 mg, 81% ; a q668 c 1 ; NMR CDCl3 :
H, d 7.33–7.11 m, 40 H, 8 Ph , 5.820 and 5.613 2
m, each 1 H, 2 OCH2CH5CH2 , 5.596 d, 1 H,
J1 ,2 3.5 Hz, H-1 , 5.33–5.31 m, 2 H, H-1,1 ,
5.166 dd, 1 H, J3 ,4 3.2, J4 ,5 1.1 Hz, H-4 ,
glucopyranosyl- 1™3 -a-L-rhamnopyranoside 34 .
.
Ž
w
—A mixture of 1,2,3,4-tetra-O-benzyl-5-O- triethyl-
Ž
.
w x
Ž
.
Ž
.
Ž
.
Ž
.
x
Ž
ammonium H-phosphonate -D-ribitol 30 23 41
mg, 61 mmol and pivaloyl chloride 60 mL, 0.49
mmol in 2:5 pyridine–acetonitrile 0.7 mL was
stirred for 60 min, then a soln of 15 20 mg, 17
mmol in acetonitrile 1.0 mL was added. After 90
min, TLC 9:1 CH2Cl2–acetone revealed the disap-
pearance of 15 and the appearance of a new spot
31 . A 0.5 M soln of iodine in 95:5 pyridine–water
D
1
Ž
.
.
Ž
.
Ž
.
Ž
.
Z
X
Y
.
Ž
.
.
Z
Z
Ž
Z
Ž
Z
Z
Z
Z
.
Ž
.
Ž
.
Ž
.
2.026, 1.997, and 1.845 3 s, each 3 H, 3 Ac , 1.265
d, 3 H, J5 ,6 6.1 Hz, 3 H-6 ; 13C, d 170.3, 170.0,
Y
Ž
.
X
X
Ž
.
Ž
Ž
Ž
.
and 169.6 3 COCH3 , 134.7 and 134.3
O CH 2 C H 5 CH 2
2
.
Ž
.
Ž
,
117.5 and 116.6
2
150 mL was added, and after 18 h, TLC 9:1

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
123
.
CH2Cl2–acetone showed the disappearance of 31
and the appearance of a new spot on the baseline.
The mixture was diluted with CH2Cl2, washed with
aq 5% Na2S2O3 and 1 M triethylammonium bicar-
on the baseline. The mixture was diluted with
CH2Cl2, washed with aq 5% Na2S2O3 and 1 M
Ž
.
Ž
.
triethylammonium bicarbonate 2= , dried MgSO4 ,
filtered, and concentrated. Column chromatography
Ž
.
Ž
.
Ž
bonate 2= , dried MgSO4 , filtered, and concen-
80:19:1 toluene–acetone–Et3N, then 80:19:1
Ž
.
trated. Column chromatography 90:9:1 CH2Cl2–
acetone–Et3N, then 90:9:1 CH2Cl2–MeOH–Et3N
of the residue and subsequent purification on
Sephadex LH-20 50:50:1 CH2Cl2–MeOH–Et3N
gave 32, isolated as a glass 32 mg, 78% . A soln of
32 16 mg, 8.5 mmol in 2:1 MeOH–aq 25% NH4OH
3 mL was heated for 48 h at 50 8C, then concen-
trated to yield crude 33, which was purified on
toluene–MeOH–Et3N of the residue and subsequent
purification on Sephadex LH-20 50:49:1 CH2Cl2–
MeOH–Et3N gave 38, isolated as a glass 9.7 mg,
50% ; NMR CDCl3 : H, d 7.98–7.15 m, 35 H, 7
Ph , 5.855 d, 1 H, J1 ,2 3.4 Hz, H-1 , 5.437 dd, 1
H, J2 ,3 10.7, J3 ,4 3.3 Hz, H-3 , 5.368 dd, 1 H,
J4 ,5 1.7 Hz, H-4 , 5.24–5.23 m, 2 H, H-1,1 ,
2.726 q, 6 H, N CH2CH3 3 , 2.097, 2.017, 1.978,
.
Ž
.
Ž
1
Ž
.
.
Ž
.
Ž
X
Ž
.
.
Ž
.
Ž
X
X
X
Ž
.
.
Ž
X
X
X
X
X
Z
Ž
.
.
Ž
.
X
X
Ž
Ž
. .
Ž
.
Ž
.
Sephadex LH-20 50:50:1 CH2Cl2–MeOH–Et3N .
To a soln of 33 in 1:2:2:2 water–EtOAc–2-pro-
1.961, 1.926, and 1.807 6 s, each 3 H, 6 Ac , 1.129
Ž
Ž
. .
Ž
Z
Z
t, 9 H, N CH2CH3 3 , 0.920 d, 3 H, J5 ,6 6.2 Hz,
3 H-6 ; 13C, d 170.2–169.6 COCH3 , 166.1
Z
Ž
.
Ž
.
.
Ž
.
panol–EtOH 3 mL was added 10% Pd–C 10 mg ,
and the mixture was hydrogenolysed at atmospheric
pressure for 24 h. After filtration, the hydrogenolysis
procedure was repeated. Then, the mixture was con-
centrated, and purified by Bio-Gel P-2 gel-permea-
tion chromatography using water as an eluent, afford-
ing 34, isolated as a white powder 4.9 mg, 76% ;
NMR D2O : H, d 5.643 d, 1 H, J1 ,2 4.2 Hz,
H-1 , 5.409 d, 1 H, J1,2 4.0 Hz, H-1 , 5.091 d, 1
Ž
Ž
w
Ž
.
Ž
.
COPh , 156.2 NCOOCH2 Ph , 97.1, 96.4, and 96.0
X
YY
.
w Ž . x
C-1,1 ,1
,
61.9
OCH 2 CH 2 2 N , 45.7
w Ž . x
38.6 O CH 2 2C H 2 N , 29.5
Z
Ž
.
x
N C H 2CH 3
,
3
.
Ž
.
w
Ž
.
x
OCH2CH2CH2 N , 17.0 C-6 , 9.8 N CH2CH3 3
P, d y2.39 PO4 .
A soln of 38 25 mg, 14 mmol in 2:1 MeOH–aq
;
31
Ž
.
Ž
.
Ž
.
1
Ž
.
Ž
Y
Y
Ž
.
25% NH4OH 6 mL was heated for 48 h at 50 8C,
then concentrated to yield crude 39, which was puri-
fied on Sephadex LH-20 50:50:1 CH2Cl2–MeOH–
Et3N . To a soln of 39 in 1:2:2:2 water–EtOAc–2-
Y
.
Ž
.
Ž
X
.
Ž
X
X
Ž
H, J1 ,2 3.7 Hz, H-1 , 3.465 t, 1 H, J3,4 sJ4,5 s9.6
Hz, H-4 , 2.03–1.96 m, 2 H, OCH2CH2CH2 N ,
.
Ž
.
.
.
31
Ž
.
Ž
Ž
.
Ž
1.317 d, 3 H, J5,6 5.9 Hz, 3 H-6 ; P, d 1.65 PO4 .
propanol–EtOH 5 mL was added 10% Pd–C 20
FABMSq Calcd for C26H50NO22 P: mrz 760.6 Mq
w
.
mg , and the mixture was hydrogenolysed at 4
xq
w
xq
kgrcm2 for 24 h. After filtration, the hydrogenolysis
procedure was repeated. Then, the mixture was con-
centrated, and purified by Bio-Gel P-2 gel-permea-
tion chromatography using water as an eluent, afford-
H . Found: mrz 760.6 MqH .
(
3-N-Benzyloxycarbonylaminopropyl 2,3,4-tri-O-
) (
) (
) (
acetyl-a-L-rhamnopyranosyl - 1™4 - 1-O-benzoyl-2,3
- di - O - benzyl - D - ribityl - 5™triethylammonium
) (
Ž
.
phosphate™2 - 3,4,6-tri-O-acetyl-a-D-galactopyrano-
ing 40, isolated as a white powder 7.7 mg, 73% ;
NMR D2O : H, d 5.618 d, 1 H, J1 ,2 4.0 Hz,
H-1 , 5.086 d, 1 H, J1 ,2 1.5 Hz, H-1 , 4.943 d, 1
H, J1,2 3.8 Hz, H-1 , 3.462 t, 1 H, J3 ,4 sJ4 ,5
9.7 Hz, H-4 , 3.21–3.10 m, 2 H, O CH2 2CH2 N ,
1
) (
)
Ž
.
Ž
X
X
syl - 1™3 -2,4,6-tri-O-benzyl-a-D-glucopyranoside
X
Z
Ž
(
.
.
Ž
.
Ž
Z
Z
38 and 3 - aminopropyl a - L - rhamnopyranosyl -
X
)
(
)
.
Ž
Z
Z
Z
Z
1™4 -D-ribityl- 5™hydrogen phosphate™2 -a-D-
s
Z
(
)
Ž
.
.
Ž
Ž
.
.
galactopyranosyl- 1™3 -a-D-glucopyranoside 40 .
Ž
Ž
.
Ž
—A mixture of 2,3,4-tri-O-acetyl-a-L-rhamnopyr-
2.05–1.96 m, 2 H, OCH2CH2CH2 N , 1.293 d, 3
H, J5 ,6 6.3 Hz, 3 H-6 ; 13C, d 101.4, 99.8, and
Z
. Ž
.
.
Z
Z
anosyl - 1™4 -1-O-benzoyl-2,3-di-O-benzyl-5-O-
X
Z
Ž
.
w x Ž
Ž
.
triethylammonium H-phosphonate -D-ribitol 23 36;
98.6 C-1,1,1 , 67.3, 65.6, 63.9, 62.4, and 61.8
Ž
OCH 2 CH 2 2 N and C-6,6X,1Y ,5Z
,
.
39.3
.
Ž
Ž
.
.
13 mg, 15 mmol and pivaloyl chloride 60 mL, 0.49
mmol in 2:5 pyridine–acetonitrile 1.2 mL was
stirred for 30 min, then 3 - N - benzyloxycarbonyl-
aminopropyl 3,4,6-tri-O-acetyl-a-D-galactopyranosyl
.
Ž
.
w
Ž
.
x
Ž
.
Ž
O CH2 2CH2 N , 28.2 OCH2CH2CH2 N , 17.9 C-
6 ; 31P: d 0.23 PO4 . FABMS Calcd for
Z
q
Ž
.
w
xq
Ž
.
C26H50NO22 P: mrz 760.6 MqH . Found: mrz
Ž
.
w x
w
xq
760.6 MqH .
- 1™3 -2,4,6-tri-O-benzyl-a-D-glucopyranoside 23
Ž
.
Ž
.
(
35; 10 mg, 11 mmol in acetonitrile 0.5 mL was
3-N-Benzyloxycarbonylaminopropyl 2,3,4,6-tetra-
) ( ) (
Ž
.
added. After 24 h, TLC 8:2 toluene–acetone re-
vealed the disappearance of 35 and the appearance of
a new spot 37 . To this mixture, a 0.5 M solution of
iodine in 95:5 pyridine–water 300 mL was added.
After 1 h, TLC 8:2 toluene–acetone indicated the
disappearance of 37 and the appearance of a new spot
O-benzyl-a-D-glucopyranosyl - 1™3 - 2,4-di-O-
) (
) (
benzyl-a-L-rhamnopyranosyl - 1™4 - 1,2,3-tri-O-
Ž
.
) (
benzyl-D -ribityl - 5™triethylammonium phosphate
Ž
.
)
Ž
.
™2 -3,4,6-tri-O-benzyl-a-D-galactopyrano-side 43
Ž
.
(
)
and 3-aminopropyl a-D-glucopyranosyl- 1™3 -a-L-
(
)
(
rhamnopyranosyl- 1™4 - D - ribityl - 5™hydrogen

(
)
124
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
)
Ž
.
Ž
.
phosphate™2 - a - D - galactopyranoside 44 .—A
added, TLC 9:1 CH2Cl2–acetone revealed the dis-
mixture of 3-N-benzyloxycarbonylaminopropyl 3,4,6-
appearance of 29 and the appearance of a major
Ž
Ž
.
tri-O -benzyl-2-O - triethylam m onium H -
product 46 and some side-products. To the mixture,
.
Ž
. w x Ž
phosphonate -a-D-galactopyranoside 41 23 14 mg,
17 mmol and pivaloyl chloride 30 mL, 0.24 mmol
in 2:5 pyridine–acetonitrile 0.7 mL was stirred for
30 min, then 21 20 mg, 16 mmol in acetonitrile 1.5
mL was added. After 2 h, TLC 8:2 toluene–acetone
revealed the disappearance of 21 and the appearance
a 0.5 M solution of iodine in 95:5 pyridine–water
250 mL was added, and after 18 h, TLC 9:1
CH2Cl2–acetone showed the disappearance of 46
and the appearance of a new spot on the baseline.
The mixture was diluted with CH2Cl2, washed with
aq 5% Na2S2O3 and 1 M triethylammonium bicar-
.
Ž
.
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
of a new spot 42 . To this mixture, a 0.5 M soln of
bonate 2= , dried MgSO4 , filtered, and concen-
Ž
.
Ž
iodine in 95:5 pyridine–water 500 mL was added,
trated. Column chromatography 80:19:1 CH2Cl2–
MeOH–Et3N of the residue gave 47, isolated as a
glass 6.6 mg, 53% . A soln of 47 6.1 mg, 3.3
mmol in 2:1 MeOH–aq 25% NH4OH 2 mL was
heated for 24 h at 50 8C, and concentrated, to yield
crude 48, which was purified on Sephadex LH-20
50:50:1 CH2Cl2–MeOH–Et3N . To a soln of 48 in
1:2:2:2 water–EtOAc–2-propanol–EtOH 3 mL was
added 10% Pd–C 7 mg , and the mixture was
hydrogenolysed at atmospheric pressure for 8 h. After
filtration, the hydrogenolysis procedure was repeated.
Then, the mixture was concentrated, and purified by
Bio-Gel P-2 gel-permeation chromatography using
Ž
.
.
and after 24 h, TLC 8:2 toluene–acetone indicated
the disappearance of 42 and the appearance of a new
spot on the baseline. The mixture was diluted with
CH2Cl2, washed with aq 5% Na2S2O3 and 1 M
triethylammonium bicarbonate 2= , dried MgSO4 ,
filtered, and concentrated. Column chromatography
80:19:1 toluene–acetone–Et3N, then 80:19:1
toluene–MeOH–Et3N of the residue and subsequent
purification on Sephadex LH-20 50:49:1 CH2Cl2–
MeOH–Et3N gave 43, isolated as a glass 16 mg,
Ž
.
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
49% . To a solution of 43 16 mg, 7.7 mmol in
1:2:2:2 water–EtOAc–2-propanol–EtOH 3 mL was
Ž
.
Ž
.
added 10% Pd–C 10 mg , and the mixture was
hydrogenolysed at atmospheric pressure for 8 h. After
filtration, the hydrogenolysis procedure was repeated.
Then, the mixture was concentrated and purified by
Bio-Gel P-2 gel-permeation chromatography using
water as an eluent, affording 44, isolated as a white
powder 4.2 mg, 72% ; NMR D2O : H, d 5.164 d,
1 H, J1,2 3.6 Hz, H-1 , 5.147 bs, 1 H, J1 ,2 -1 Hz,
H-1 , 5.124 d, 1 H, J1 ,2 4.2 Hz, H-1 , 3.467 t, 1
water as an eluent, affording 49, isolated as a white
1
Ž
.
Ž
.
Ž
powder 1.9 mg, 76% ; NMR D2O : H, d 5.396 d,
Z
.
Ž
Z
Z
X
X
1 H, J1 ,2 4.1 Hz, H-1 , 5.134 d, 1 H, J1 ,2 1.7 Hz,
X
X
.
Ž
.
Ž
Y
Y
H-1 , 5.125 d, 1 H, J1 ,2 4 Hz, H-1 , 3.589 t, 1 H,
X
.
Ž
m ,
X
X
X
X
J3 ,4 s J4 ,5 s 9.8 Hz, H-4 , 3.161 t, 2 H,
Ž
.
.
Ž
O C H 2 C H 2 N , 2.05 – 2.00
2
H ,
2
1
Ž
.
.
Ž
.
Ž
.
Ž
X
X
OCH2CH2CH2 N , 1.318 d, 3 H, J5 ,6 6.2 Hz, 3
H-6 ; 31P, d 1.67 PO4 . FABMS Calcd for
X
q
Ž
Y
Y
.
Ž
.
Y
Z
Z
Z
w
xq
.
Ž
.
Ž
C26H50NO22 P: mrz 760.6 MqH . Found: mrz
760.6 MqH .
Y
Y
Y
Y
Y
w
xq
.
Ž
H, J3 ,4 sJ4 ,5 s9.6 Hz, H-4 , 3.21–3.13 m, 2 H,
Ž
.
.
Ž
O C H 2 C H 2 N , 2.22 – 2.15
m ,
2
H ,
2
.
Ž
Y
Y
OCH2CH2CH2 N , 1.315 d, 3 H, J5 ,6 6.6 Hz, 3
H-6 ; 31P, d 2.93 PO4 . FABMS Calcd for
Y
q
Acknowledgements
.
Ž
.
w
xq
C26H50NO22 P: mrz 760.6 MqH . Found: mrz
This investigation was supported with financial aid
from the Institute of Molecular Biology and Medical
w
xq
760.6 MqH .
(
) (
)
3,4,6-Tri-O-acetyl-a-D-galactopyranosyl - 1™3 -
Ž
.
Biotechnology IMB, Utrecht University . The au-
thors thank Drs. P.H. Kruiskamp and C.W.E.M. van
Zuylen for recording the NMR spectra of the depro-
tected products, Dr. G.H. Veeneman for valuable
discussion, and Mrs. A. van der Kerk-van Hoof for
recording the FAB mass spectra.
(
) (
) (
2,4,6-tri-O-benzyl-a-D-glucopyranosyl - 1™3 - 2,4-
) (
)
di-O-benzyl-a-L-rhamnopyranosyl - 1™4 -1,2,3-tri-O-
(
benzyl - 5 - O - 3 - N - benzyloxycarbonylaminopropyl
)
Ž
.
triethylammonium phosphate -D-ribitol 47 and a-D-
(
)
(
)
galactopyranosyl- 1™3 -a-D-glucopyranosyl- 1™3
(
)
(
-a-L-rhamnopyranosyl- 1™4 -5-O- 3-aminopropyl
)
Ž
.
hydrogen phosphate -D-ribitol 49 .—To a solution
Ž
.
.
of 29 10 mg, 6.8 mmol in 2:5 pyridine–acetonitrile
1 mL was added dropwise a solution of 3-N-benzyl-
References
Ž
oxycarbonylaminopropyl triethylammonium H-phos-
w x
Ž
1 Health and Public Policy Committee Am. Coll.
w
x
Ž
.
phonate 22 45; 10 mg, 37 mmol and pivaloyl
.
Ž
.
Phys. , Ann. Int. Med., 104 1986 pp. 118–120.
Ž
.
chloride 3.3 mL, 27 mmol in 2:5 pyridine–aceto-
w x
2 M. Heidelberger and O.T. Avery, J. Exp. Med., 38
Ž
.
Ž
.
nitrile 1.0 mL . When 300 mL of the mixture was
1923 73–79.

(
)
M.-J.L. Thijssen et al.rCarbohydrate Research 306 1998 111–125
125
w x
3 C.M. MacLeod, M. Heidelberger, R.G. Hodges, and
man, L.J.F. Gomes, J.H. van Boom, J. Verhoef, and
Ž
.
Ž .
H. Snippe, Vaccine, 11 1993 1429–1436.
W.G. Bernard, J. Exp. Med., 82 1945 445–465.
w x
4 M. Heidelberger, C.M. MacLeod, and M.M. Di Lapi,
w
w
x
19 R. Booy and E.R. Moxon, Arch. Dis. Child., 68
Ž
.
Ž
.
J. Exp. Med., 88 1948 369–372.
1993 440–441.
w x
Ž
.
x
5 M. Finland, ReÕ. Infect. Dis., 1 1979 4–21.
20 E. Alonso de Velasco, A.F.M. Verheul, A.M.P. van
Steijn, H.A.T. Dekker, R.G. Feldman, I.M. Fernan-
dez, J.P. Kamerling, J.F.G. Vliegenthart, J. Verhoef,
w x
Ž
.
6 J. Henrichsen, J. Clin. Microbiol., 33 1995 2759–
2762.
w x
Ž .
and H. Snippe, Infect. Immun., 62 1994 799–808.
21 C. Barrios, C. Tougne, B.S. Polla, P.H. Lambert, and
7 J.B. Robbins, R. Austrian, C.-J. Lee, S.C. Rastogi, G.
Schiffman, J. Henrichsen, P.H. Makela, C.V. Broome,
w
w
w
w
x
¨
¨
Ž
.
R.R. Facklam, R.H. Tiesjema, and J.C. Parke Jr., J.
G. del Guidice, Clin. Exp. Immunol., 98 1994 224–
228.
Ž
.
Infect. Dis., 148 1983 1136–1159.
w x
8 J.E.G. van Dam, A. Fleer, and H. Snippe, Anthonie
x
22 M.J.L. Thijssen, K.M. Halkes, J.P. Kamerling, and
Ž
.
Ž
.
Õan Leeuwenhoek, 58 1990 1–47.
J.F.G Vliegenthart, Bioorg. Med. Chem., 2 1994
1309–1317.
w x
9 D.M. Granoff, S.J. Holmes, M.T. Osterholm, J.E.
McHugh, A.H. Lucas, E.L. Anderson, R.B. Belshe,
J.L. Jacobs, F. Medley, and T.V. Murphy, J. Infect.
x
23 M.J.L. Thijssen, M.N. van Rijswijk, J.P. Kamerling,
and J.F.G. Vliegenthart, Carbohydr. Res., in press,
JD-291.
Ž
.
Dis., 168 1993 663–671.
w
w
w
w
x
x
24 P. Berntsson, A. Brandstrom, U. Junggren, L. Palmer,
¨ ¨
10 W.E. Paul, D.H. Katz, and B. Benacerraf, J. Im-
Ž
.
munol., 107 1971 685–688.
S.E. Sjostrand, and G. Sundell, Acta Pharm. Suec.,
¨
x
Ž
.
Ž .
14 1977 229–236.
11 H. Braley-Mullen, J. Immunol., 113 1974 1909–
Ž
.
w
x
25 C.A.A. van Boeckel and T. Beetz, Tetrahedron Lett.,
1920; Immunology, 40 1980 521–527.
x
Ž .
24 1983 3775–3778.
12 E.C. Beuvery, F. van Rossum, and J. Nagel, Infect.
Ž
.
w
x
Immun., 37 1982 15–22.
26 G.H. Veeneman, S.H. van Leeuwen, H. Zuurmond,
x
Ž
.
13 R. Schneerson, J.B. Robbins, C. Chu, A. Sutton, W.
Vann, J.C. Vickers, W.T. London, B. Curfman, M.C.
Hardegree, J. Shiloach, and S.C. Rastogi, Infect. Im-
and J.H. van Boom, J. Carbohydr. Chem., 9 1990
783–796.
w
x
27 T.M. Slaghek, M.J. van Vliet, A.A.M. Maas, J.P.
Ž
.
mun., 45 1984 582–591.
Kamerling, and J.F.G. Vliegenthart, Carbohydr. Res.,
w
w
x
Ž .
195 1989 75–86.
14 C.-J. Lee, Y. Takaoka, and T. Saito, ReÕ. Infect. Dis.,
Ž
.
w
w
w
w
x
9 1987 494–510.
28 F. Weygand and H. Ziemann, Justus Liebigs Ann.
x
Ž .
Chem., 657 1962 179–198.
15 S. Marburg, D. Jorn, R.L. Tolman, B. Arison, J.
McCauley, P.J. Kniskern, A. Hagopian, and P.P.
x
29 F. Dasgupta and P.J. Garegg, Acta Chem. Scand. Ser.
Ž
.
Ž .
B, 43 1989 471–475.
Vella, J. Am. Chem. Soc., 108 1986 5282–5287.
w
w
x
x
30 G.H. Veeneman and J.H. van Boom, Tetrahedron
16 M. Koskela, M. Harris, and G.S. Giebink, J. Clin.
Ž
.
Ž .
Lett., 31 1990 275–278.
Microbiol., 30 1992 1485–1490.
x
x
17 D.E. Moshier, A.J. Feeney, and P. Scherle, in R. Bell
31 J.E. Marugg, M. Tromp, E. Kuyl-Yeheskiely, G.A.
Ž
.
and G. Torriani Eds. , Towards better carbohydrate
Õaccines, Wiley, 1987, pp. 243–261.
van der Marel, and J.H. van Boom, Tetrahedron Lett.,
Ž
.
27 1986 2661–2664.
w
x
18 E. Alonso de Velasco, A.F.M. Verheul, G.H. Veene-