Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

Article abstract of DOI:10.1016/S0008-6215(03)00313-6

The chemo-enzymatic synthesis is described of tetrasaccharide β-D-Galp-(1→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)] -β-D-GlcpNAc-(1→O(CH₂)₆NH₂ (1) and octasaccharide β-D-Galp-(1→4)-β-D-Glcp-(1→6)-[β-D-Galp- (1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4) -β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]-β-D-GlcpNAc- (1→O(CH₂)₆NH₂ (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were β-(1→4)-connected to the internal N-acetyl-β-D-glucosamine residues by using bovine milk β-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.

Full text of DOI:10.1016/S0008-6215(03)00313-6

Carbohydrate Research 338 (2003) 2611Á2627
/
www.elsevier.com/locate/carres
Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment
of the capsular polysaccharide of Streptococcus pneumoniae type 14
John A.F. Joosten, Johannis P. Kamerling,* Johannes F.G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH
Utrecht, The Netherlands
Received 16 April 2003; accepted 17 June 2003
Abstract
The chemo-enzymatic synthesis is described of tetrasaccharide b-
GlcpNAc-(10O(CH2)6NH2 (1) and octasaccharide b- -Galp-(104)-b-
b- -Galp-(104)-b- -Glcp-(106)-[b- -Galp-(104)]-b- -GlcpNAc-(10O(CH2)6NH2 (2), representing one and two tetrasacchar-
ide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate
linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were b-(104)-connected to the internal N-acetyl-b-
-glucosamine residues by using bovine milk b-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier
D
-Galp-(10
/
4)-b-
D
-Glcp-(10
/
6)-[b-
D
-Galp-(10
/
4)]-b-
D-
/
D
/
D-Glcp-(106)-[b-
/
D
-Galp-(10
/
4)]-b-
D
-GlcpNAc-(10
/
3)-
D
/
D
/
D
/
D
/
/
D
proteins to be tested as potential vaccines in animal models.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Carbohydrates; Streptococcus pneumoniae; Oligosaccharide synthesis
1. Introduction
cell dependent neoglycoconjugate antigen of which it
has been proven that it gives an efficient immune
response in the high-risk groups.11 Currently, neoglyco-
conjugate vaccines against S. pneumoniae serotypes,
prepared by conjugation of isolated CPSs or of a
mixture of polysaccharide-derived oligosaccharides to
a protein carrier, have been introduced.11 The presence
of oligosaccharide mixtures can complicate the product
analysis, especially in the case of pneumococcal con-
jugate vaccines where many serotypes have to be
included.
Gram-positive Streptococcus pneumoniae are members
of the normal microflora of the human nasopharynx.
Carriage of S. pneumoniae is more common in young
children than in adults, and varies by geographic region
and time.1,2 S. pneumoniae is still a leading cause of life-
threatening diseases such as otitis media, pneumonia,
meningitis, bacteraemia, and septicaemia,3,4 mainly due
to a growing resistance towards antibiotics.5,6 Vaccina-
tion with the available 23-valent capsular polysaccharide
(CPS) vaccines7 offers protection for healthy adults to
invasive pneumococcal diseases.8 However, these vac-
cines are ineffective in the most important high-risk
groups, such as infants, small children, immuno-com-
promised patients, and the elderly,9 because they do not
respond adequately to the T-cell independent polysac-
charides as antigens.10 Conjugation of S. pneumoniae
carbohydrate antigens to a protein carrier results in a T-
By using well-defined oligosaccharides for conjuga-
tion, it is possible to investigate the influence of different
parameters originating from the carbohydrate part of
the neoglycoproteins on their immunogenicity. Studies
with oligosaccharideÁprotein conjugates related to the
/
CPS of S. pneumoniae serotypes 3 and 6B showed that,
via this approach, the minimal size of the carbohydrate
antigen could be determined.12,13
Recently, we have reported the chemo-enzymatic
synthesis of (spacered mimics of) fragments of the CPS
of S. pneumoniae type 14, comprising a branched
tetrasaccharide and alkyl-bridged hexa- and octasac-
charides.14,15 These oligosaccharides, together with
* Corresponding author. Tel.: ꢀ
30-2540980.
E-mail address: j.p.kamerling@chem.uu.nl (J.P. Kamerling).
/
31-30-2533479; fax: ꢀ31-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00313-6

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J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
tetra- and octasaccharide fragments of the type 14 CPS
obtained by partial N-deacetylation/deamination, were
investigated for their immunological behavior.16 Based
on the promising results of the branched tetrasacchar-
ide, representing one repeating unit, it was decided to
synthesize longer intact oligosaccharide fragments of the
type 14 CPS. So far, we have reported the synthesis of 6-
aminohexyl-spacered penta- and hexasaccharide frag-
ments,17 and here we report the chemo-enzymatic
synthesis of 6-aminohexyl-spacered tetra- and octasac-
charide fragments of the CPS (Fig. 1).
approach to 1 and 2, first the intermediate linear
trisaccharide 3 (Scheme 1) and hexasaccharide 4
(Scheme 2), respectively, were synthesized. Galactose
residues were b-(10
/
4)-attached to the internal N-
acetyl-b- -glucosamine residues by using bovine milk
D
b-1,4-galactosyltransferase (EC 2.4.1.22) and UDP-ga-
lactose.
2.2. Synthesis of tetrasaccharide fragment 1
In an earlier report,17 we have shown that by using the
trisaccharide donor (2,3,4,6-tetra-O-acetyl-b-
pyranosyl)-(104)-(2,3,6-tri-O-acetyl-b- -glucopyrano-
syl)-(106)-2-deoxy-3,4-di-O-p-methylbenzoyl-2-phtha-
limido-b- -glucopyranosyl trichloroacetimidate (20)
(Scheme 2) a linear tetra- and pentasaccharide could
be synthesized. However, coupling of donor 20 with the
spacer 6-azido-1-hexanol (6) could not be successfully
realized. Therefore, an alternative route was followed to
synthesize trisaccharide 3 (Scheme 1). To this end, two
building blocks, 6-azidohexyl 2-deoxy-3,4-di-O-p-
D-galacto-
/
D
2. Results and discussion
/
D
2.1. Retrosynthetic strategy
The CPS of S. pneumoniae type 14 is built up from the
tetrasaccharide repeating unit 0
-Glcp-(106)-[b- -Galp-(104)]-b-
18 The title oligosaccharides (Fig. 1) represent either one
/
3)-b-
D
-Galp-(10
/
4)-b-
D
/
D
/
D
-GlcpNAc-(10
/
.
(1) or two (2) tetrasaccharide-repeating units in which
each repeating unit is built up from a linear backbone
and a branched galactose residue. In a chemical
methylbenzoyl-2-phthalimido-b-
ceptor (11) and (2,3,4,6-tetra-O-acetyl-b-
anosyl)-(104)-2,3,6-tri-O-acetyl-b- -glucopyranosyl
D
-glucopyranoside ac-
D
-galactopyr-
/
D
Fig. 1. Overview of 6-aminohexyl-spacered oligosaccharides, representing fragments varying in length between one and two
repeating units of the capsular polysaccharide of S. pneumoniae type 14. (a) Synthesis described in this paper (compounds 1 and 2).
(b) Synthesis described by Michalik and co-workers.17

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2613
Scheme 1. Synthesis of tetrasaccharide 1: (a) 0.5 equiv BF3×Et2O, 0 8C, CH2Cl2, 89%; (b) NaOMe (pH 8), MeOH, CH2Cl2,
/
quantitative; (c) t-BdPhSiCl, DMAP, Et3N, pyridine, CH2Cl2, 88%; (d) p-mBzCl, 0 8C, pyridine, CH2Cl2, 69%; (e) AcCl, 0 8C,
MeOH, toluene, 75%; (f) 10% TMSOTf, 0 8C, CH2Cl2, 45%; (g) NaOMe (pH 10), MeOH, CH2Cl2; (h) NH2CH2CH2NH2, 90 8C, 1-
BuOH; (i) pyridine, Ac2O, 86% over three steps; (j) NaOMe (pH 10), MeOH, CH2Cl2, 90%; (k) 10% PdÁC, H2, aq 25% NH3, t-
/
BuOH, water, 71%; (l) 1.8 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH 7.5), 2.5 U b-1,4-galactosyltransferase, 12 U
alkaline phosphatase, 37 8C, 91%.
trichloroacetimidate donor (12)19 were designed
(Scheme 1). As a first step in the synthesis of 11, 3,4,6-
trisaccharide 13 in a moderate yield (45%). The main
side-product was the 6-O-acetylated acceptor, which was
isolated in 47% yield. This product, probably formed via
the orthoester intermediate, could be converted back
into acceptor 11 by treatment with acetyl chloride in dry
methanol (data not shown). No attempts were made to
improve the yield of this condensation reaction. O-De-
acylation of 13 using sodium methoxide at pH 10,
followed by N-dephthaloylation by treatment with 1,2-
diaminoethane in 1-butanol at 90 8C, and subsequent
N,O-acetylation using acetic anhydride in pyridine
afforded 14 in 86% yield over three steps. The O-
acetylation step was carried out to facilitate chromato-
graphic purification. O-Deacetylation of 14 using so-
dium methoxide at pH 10 gave 15 (90%). Finally,
catalytic hydrogenation of the azido group of 15 using
10% palladium on charcoal and H2 in the presence of
ammonia, yielded linear backbone 3 (71%). Tetrasac-
charide 1 was synthesized in 91% yield by transfer of
galactose from UDP-galactose to O-4 of the N-acetyl-b-
tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-glucopyranosyl
trichloroacetimidate (5)20 was coupled to 6-azido-1-
hexanol (6) using 0.5 equiv boron trifluoride ethyl
etherate (BF3 ×
dichloromethane, giving 7 in 89% yield. After O-de-
acetylation of 7 using sodium methoxide at pH 8 (08),
/
Et2O) as a catalyst at 0 8C in dry
/
a tert-butyldiphenylsilyl group (tBdPhSi) was selectively
introduced at the primary hydroxyl function of 8 using
tert-butyldiphenylsilyl chloride in pyridine in the pre-
sence of a catalytic amount of 4-dimethylaminopyridine
(DMAP), affording 9 in 88% yield over two steps. The
remaining free hydroxyl functions at O-3 and O-4 of 9
were protected with a toluoyl group (mBz) by using p-
methylbenzoyl chloride in pyridine at 0 8C (0
/10, 69%).
The toluoyl group was chosen to avoid migration of an
ester group from O-4 to O-6 after removal of the silyl
group at O-6.21 Finally, removal of the tert-butyldiphe-
nylsilyl ether group by using acetyl chloride in dry
methanol yielded monosaccharide acceptor 11 (75%).
Coupling of 12 and 11, at 0 8C, using 10% trimethylsilyl
trifluoromethanesulfonate (TMSOTf) as a catalyst gave
D
-glucosamine residue of 3 by using bovine milk b-1,4-
galactosyltransferase as a catalyst (Scheme 1). Alkaline
phosphatase was added to the incubation mixture to

2614
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
Scheme 2. Synthesis of octasaccharide 2: (a) 66% TMSOTf, 0 8C, CH2Cl2, 62%; (b) TFA, 0 8C, water, CH2Cl2, 84%; (c) trimethyl
orthoacetate, p-TsOH, CH3CN; (d) AcOH, water, 85% over two steps; (e) 10% TMSOTf, ꢁ20 8C, CH2Cl2, 12%; (f) NaOMe (pH
10), MeOH, CH2Cl2; (g) NH2CH2CH2NH2, 80 8C, 1-BuOH; (h) pyridine, Ac2O, 71% over three steps; (i) NaOMe (pH 10), MeOH,
89%; (j) 10% PdÁC, H2, aq 25% NH3, t-BuOH, water, 67%; (k) 3 equiv UDP-Gal, aq 50 mM sodium cacodylate buffer (pH 7.5), 1
/
/
U b-1,4-galactosyltransferase, 10 U alkaline phosphatase, 37 8C, 22%.
prevent feedback inhibition by released UDP, thereby
facilitating a high conversion of 3. Chemical and chemo-
enzymatic syntheses of structure 1, containing other
2.3. Synthesis of octasaccharide fragment 2
Coupling of (2,6-di-O-acetyl-3,4-di-O-isopropylidene-b-
D
-galactopyranosyl)-(10
/
4)-2,3,6-tri-O-acetyl-b-
D-glu-
functionalities at the anomeric center have been de-
scribed and reviewed by Kamerling.22 1H NMR data of
copyranosyl trichloroacetimidate donor (16)23 to gluco-
samine acceptor 11 (Scheme 2) at 0 8C, using 66%
15, 3, and 1 are presented in Tables 1Á3, respectively.
/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2615
Table 1
500 MHz H NMR data (TOCSY, ROESY) of 15 at 300 K (in ppm)
1
Proton
dH
GlcNAcI
GlcII
GalIII
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.51
3.68
3.52
3.52
3.62
4.21
3.89
4.55
3.38
3.67
3.67
3.61
3.98
3.82
4.45
3.53
3.66
3.93
a
n.d.
n.d.
n.d.
O(CH2)2(CH2)2(CH2)2N3
OCH2CH2(CH2)2CH2CH2N3
CH2N3
1.34Á
1.54Á
3.32
/
1.37 (4 H)
/1.61 (4 H)
OCH2(CH2)5N3
NDCOCH3
3.61, 3.89
2.03
a
n.d., not determined.
trimethylsilyl trifluoromethanesulfonate, gave trisac-
charide 17 (62%). O-Deisopropylidenation of 17 with
10% trimethylsilyl trifluoromethanesulfonate as a cata-
lyst, gave hexasaccharide 31 in only 12% yield (Scheme
2). The low yield is probably due to the low reactivity of
the free O-3 position, since the acceptor could be
isolated after the reaction. Other coupling attempts, at
different temperatures and with different catalysts, did
not improve the yield of the reaction.
Therefore, in a second approach, an attempt was
made to synthesize hexasaccharide 31 via a slightly
different route in which acetyl ester groups were
replaced by benzyl ether functions. O-Deacylation of
17, followed by benzylation with benzyl bromide in dry
N,N-dimethylformamide in the presence of sodium
hydride resulted also in the removal of the phthalimido
protective group. Introduction of benzyl groups via
aqueous 90% trifluoroacetic acid (0
/
18, 84%), followed
by selective acetylation at O-4 of the galactose residue
via orthoester formation using trimethyl orthoacetate
and p-toluenesulfonic acid, and subsequent ring opening
with aqueous 80% acetic acid afforded 19 (85% over two
steps).24 A small amount of side-product (6%) was
formed in which O-3 of the galactose residue was
acetylated, either due to acetyl migration or ring open-
ing to O-3. Coupling of 19 with (2,3,4,6-tetra-O-acetyl-
b-
glucopyranosyl)-(10
zoyl-2-phthalimido-b-
midate (20)17 in dichloromethane at ꢁ
D
-galactopyranosyl)-(10
6)-2-deoxy-3,4-di-O-p-methylben-
-glucopyranosyl trichloroaceti-
20 8C, using
/
4)-(2,3,6-tri-O-acetyl-b-
D-
/
D
/
Table 2
500 MHz H NMR data (TOCSY, ROESY) of 3 at 300 K (in ppm)
1
Proton
dH
GlcNAcI
GlcII
GalIII
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.51
3.68
3.53
3.52
3.62
4.21
3.89
4.55
3.37
3.67
3.67
3.62
3.99
3.82
4.45
3.55
3.67
3.93
a
n.d.
n.d.
n.d.
O(CH2)2(CH2)2(CH2)2ND2
OCH2CH2(CH2)2CH2CH2ND2
CH2ND2
1.32Á
1.53Á
2.98
/
1.34 (4 H)
1.56 (2 H), 1.62Á1.68 (2 H)
/
/
OCH2(CH2)5ND2
NDCOCH3
3.58, 3.92
2.03
a
n.d., not determined.

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J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
Table 3
500 MHz H NMR data (TOCSY, ROESY) of 1 at 300 K (in ppm)
1
Proton
dH
a
GlcNAcI
4.54
3.74
GlcII GalIII
GalVII b
4.53
3.53
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.56
3.38
3.67
3.67
3.62
3.98
3.82
4.45
3.55
3.68
3.92
n.d. c
3.73
3.78
3.69
3.82
3.68
3.92
3.72
4.29
n.d.
3.73
3.96
3.78
O(CH2)2(CH2)2(CH2)2ND2
OCH2CH2(CH2)2CH2CH2ND2
CH2ND2
1.36Á
/
1.38 (4 H)
1.55Á
/
1.58 (2 H), 1.62Á1.67 (2 H)
/
2.98
3.60, 3.92
2.03
OCH2(CH2)5ND2
NDCOCH3
a
Gal(b1-4)Glc.
Gal(b1-4)GlcNAc.
n.d., not determined.
b
c
coupling of the benzyl-protected lactose donor, with
acceptor 11, yielded a/b-mixtures, which could not be
fractionated. In an alternative approach to obtain 31,
trisaccharide donor 28 was designed to be combined
with 11 and 12 (Scheme 3). Coupling of 3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl tri-
Scheme 3. Alternative synthesis of hexasaccharide backbone 31: (a) 8% TMSOTf, ꢁ
MeOH, CH2Cl2, 82%; (c) t-BdPhSiCl, DMAP, Et3N, pyridine, CH2Cl2, 89%; (d) p-mBzCl, 0 8C, pyridine, CH2Cl2, 74%; (e) 10%
PdÁC, H2, EtOH, EtOAc, 90%; (f) pyridine, Ac2O, quantitative; (g) hydrazinium acetate, DMF, 65%; (h) CCl3CN, DBU, 0 8C,
CH2Cl2, 69%; (i) 10% TMSOTf, ꢁ40 8C, CH2Cl2, 38%; (j) 1.0 M TBAF in THF, AcOH, 0 8C, 90%; (k) TMSOTf, ꢁ40 8C,
CH2Cl2, 60%.
/
70 8C, CH2Cl2, 86%; (b) NaOMe (pH 9),
/
/
/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2617
chloroacetimidate (5)20 to (4-O-acetyl-2,6-di-O-benzyl-
b- -galactopyranosyl)-(104)-1,2,3,6-tetra-O-benzyl-b-
-glucopyranose (21)25 in dichloromethane at ꢁ
70 8C,
3. Experimental
3.1. General methods
D
/
D
/
using 8% trimethylsilyl trifluoromethanesulfonate as a
catalyst gave 22 (86%). Mild O-deacetylation of 22,
using sodium methoxide (pH 9), afforded 23 (82%), with
retention of the acetyl group at galactose O-4. Selective
tert-butyldiphenylsilylation of O-6 of the glucosamine
residue of 23 using tert-butyldiphenylsilyl chloride and a
catalytic amount of 4-dimethylaminopyridine in pyri-
All chemicals were of reagent grade, and were used
without further purification. Reactions were monitored
by TLC on Silica Gel 60 F254 (E. Merck); after
examination under UV light, compounds were visua-
lized by heating with 10% (v/v) ethanolic H2SO4, orcinol
(2 mg/mL) in 20% (v/v) methanolic H2SO4, or ninhydrin
(1.5 mg/mL) in 1-BuOHÁ
/
waterÁAcOH (38:1.75:0.25).
/
dine (0/24, 89%), followed by toluoylation of O-3 and
In the work-up procedures of reaction mixtures, organic
solns were washed with appropriate amounts of the
indicated aq solns, then dried (MgSO4), and concen-
trated under diminished pressure at 40 8C. Column
chromatography was performed on Silica Gel 60 (E.
O-4 using p-methylbenzoyl chloride in pyridine, gave 25
(74%). O-Debenzylation of 25 using 10% palladium on
charcoal and H2 and subsequent O-acetylation using
acetic anhydride in pyridine yielded 26 in 90% over two
steps. Selective removal of the anomeric O-acetyl group
by treatment with hydrazinium acetate in dry N,N-
Merck, 0.063Á/0.200 mm). Optical rotations were mea-
sured with a PerkinÁ
/
Elmer 241 polarimeter, using a 10
cm, 1 mL cell. 1H NMR spectra were recorded at 300 K
with a Bruker AC 300 (300 MHz) or a Bruker AMX 500
(500 MHz) spectrometer; the dH values are given in ppm
relative to the signal for internal Me4Si (dH 0, CDCl3
and CD3OD) or internal acetone (dH 2.225, D2O). 13C
NMR spectra (APT, 75 MHz) were recorded at 300 K
with a Bruker AC 300 spectrometer; dC values are given
in ppm relative to the signal of CDCl3 (dC 76.9, CDCl3)
or for internal acetone (dC 30.89, D2O). Two-dimen-
dimethylformamide (0
/
27, 65%), followed by imidation
using trichloroacetonitrile in the presence of 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) gave donor 28 in an
a:b ratio of 9:1 (69%). Coupling of 28 to glucosamine
acceptor 11 (Scheme 3) at ꢁ40 8C using 10% trimethyl-
/
silyl trifluoromethanesulfonate as a catalyst afforded
tetrasaccharide 29 (38%). O-De-tert-butyldiphenylsily-
lation of 29 in a 1:1 mixture of 1.0 M tetrabutylammo-
nium fluoride (TBAF) in tetrahydrofuran and acetic
acid (pH 6) yielded 30 (90%). It should be noted that
removal of the tert-butyldiphenylsilyl group by using
acetyl chloride in dry methanol at 0 8C also lead to the
removal of acetyl groups. Coupling of tetrasaccharide
sional 1HÁ1
/
H TOCSY (mixing times 7 and 100 ms),
1
ROESY (mixing time 300 ms), and HÁ13
/
C correlated
HSQC NMR spectra (500 MHz) were recorded at 300 K
with a Bruker AMX 500 spectrometer. Exact masses
were measured by nano electrospray time-of-flight mass
spectrometry (positive-ion mode) using a Micromass
LCToF mass spectrometer at a resolution of 5000
FWHM. Gold-coated capillaries were loaded with 1
mL of sample (conc 20 mM) dissolved in a 1:1 (v/v)
acceptor 30 with 3 equiv of (2,3,4,6-tetra-O-acetyl-b-
D-
galactopyranosyl)-(10
/
4)-2,3,6-tri-O-acetyl-b-
D-gluco-
pyranosyl trichloroacetimidate (12),19 using 10% tri-
methylsilyl trifluoromethanesulfonate as a catalyst at
ꢁ
/
40 8C, gave hexasaccharide 31 (60%). O-Deacylation
mixture of MeCNÁwater with 0.1% formic acid. Penta-
/
of 31 with sodium methoxide in a methanolÁdichlor-
/
fluorophenylalanine was added as internal standard.
The capillary voltage was set at 1500 V and the cone
voltage was set at 30 V. Elemental analyses were carried
out at the Department of Organic Chemistry of the
University of Nijmegen (The Netherlands).
omethane mixture, followed by N-dephthaloylation
by treatment with 1,2-diaminoethane in 1-butanol at
80 8C, and subsequent N,O-acetylation using acetic
anhydride in pyridine gave 32 (71% over three steps).
O-Deacetylation of 32 using sodium methoxide at pH
10 (0
/
33; 89%), and catalytic hydrogenation of the
3.2. 6-Azidohexyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranose (7)
azido group using 10% palladium on charcoal and H2
gave 4 in 67% yield. Finally, octasaccharide 2 was
synthesized in 22% yield by transfer of galactose from
A soln of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-
glucopyranosyl trichloroacetimidate (5)20 (2.45 g, 4.23
mmol) and 6-azido-1-hexanol (6) (1.3 g, 9.08 mmol) in
UDP-galactose to O-4 of the two N-acetyl-b-
D-gluco-
samine residues of 4 by using bovine milk b-1,4-
galactosyltransferase as a catalyst (Scheme 2). The
low yield of the reaction was due to purification
˚
dry CH2Cl2 (50 mL), containing molecular sieves 4 A
(400 mg), was stirred for 2 h under Ar. After cooling to
1
problems. H NMR data of 33, 4, and 2 are presented
0 8C, BF3×
the mixture was stirred for 1.5 h, after which a second
portion of BF3×Et2O (0.1 mL, 0.79 mmol) was added,
/Et2O (0.28 mL, 2.19 mmol) was added and
in Tables 4Á6, respectively.
/
Conjugation of the oligosaccharides 1 and 2 to
CRM197 (cross reactive material) and immunological
studies are in progress.
/
and the stirring was continued for 1.5 h. The mixture
was neutralized with Et3N, washed with aq satd

2618
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
Table 4
500 MHz H NMR data (TOCSY, ROESY) of 33 at 300 K (in ppm)
1
Proton
dH
a
GlcNAcI
GlcII b
GaIII c
GlcNAcIV d
GlcV e GalVI f
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.52
3.68
3.52
n.d. g
3.60
4.21
3.88
4.55
3.37
3.66
n.d.
n.d.
3.98
3.81
4.44
3.59
3.72
4.16
n.d.
n.d.
n.d.
4.70
3.76
3.56
n.d.
3.61
4.22
3.89
4.55
3.37
3.66
n.d.
n.d.
3.98
3.81
4.46
3.57
3.68
3.92
n.d.
n.d.
n.d.
O(CH2)2(CH2)2(CH2)2N3
OCH2CH2(CH2)2CH2CH2N3
CH2N3
1.33Á
1.53Á
3.32
/
1.37 (4 H)
/1.60 (4 H)
OCH2(CH2)5N3
NDCOCH3
3.61, 3.89
2.03 (2)
a
GlcNAc(b1-O(CH2)6N3).
b
Glc(b1-6)GlcNAc(b1-O(CH2)6N3).
c
Gal(b1-4)Glc(b1-6)GlcNAc(b1-O(CH2)6N3).
GlcNAc(b1-3)Gal.
Glc(b1-6)GlcNAc(b1-3)Gal.
Gal(b1-4)Glc(b1-6)GlcNAc(b1-3)Gal.
n.d., not determined.
d
e
f
g
1
NaHCO3 and water, dried, filtered, and concentrated.
Column chromatography (9:1 CH2Cl2ÁEtOAc) of the
residue gave 7, isolated as a slightly yellow syrup (2.10 g,
89%); Rf 0.66 (9:1 CH2Cl2Á 198 (c 1,
EtOAc); [a]2D0
CHCl3); H NMR (300 MHz, CDCl3): d 1.12Á
/
1.17 (m,
1.45 (m, 2
/
4 H, 2 CH2), 1.23Á
H, CH2), 1.86, 2.03, and 2.11 (3 s, each 3 H, 3 COCH3),
/
1.29 (m, 2 H, CH2), 1.43Á
/
/
ꢀ
/
3.06 (t, 2 H, CH2N3), 3.44 (m, 1 H, OCHH), 4.17 (dd, 1
Table 5
500 MHz H NMR data (TOCSY, ROESY) of 4 at 300 K (in ppm)
1
Proton
dH
a
GlcNAcI
GlcII b GalIII c GlcNAcIV d
GlcV e GalVI f
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.51
3.68
3.52
n.d. g
n.d.
4.56
3.37
3.67
n.d.
n.d.
n.d.
n.d.
4.43
3.60
3.72
4.16
n.d.
n.d.
n.d.
4.70
3.76
3.55
n.d.
n.d.
4.22
3.89
4.56
3.37
3.67
n.d.
n.d.
n.d.
n.d.
4.45
3.55
3.67
3.93
n.d.
n.d.
n.d.
4.21
3.88
O(CH2)2(CH2)2(CH2)2ND2
OCH2CH2(CH2)2CH2CH2ND2
CH2ND2
1.35Á
/
1.39 (4 H)
1.57 (2 H), 1.64Á1.66 (2 H)
1.55Á
/
/
2.99
3.59, 3.91
2.03 (2)
OCH2(CH2)5ND2
NDCOCH3
a
GlcNAc(b1-O(CH2)6NH2).
b
Glc(b1-6)GlcNAc(b1-O(CH2)6NH2).
c
d
e
f
Gal(b1-4)Glc(b1-6)GlcNAc(b1-O(CH2)6NH2).
GlcNAc(b1-3)Gal.
Glc(b1-6)GlcNAc(b1-3)Gal.
Gal(b1-4)Glc(b1-6)GlcNAc(b1-3)Gal.
n.d., not determined.
g

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2619
Table 6
500 MHz H NMR data (TOCSY, ROESY) of 2 at 300 K (in ppm)
1
Proton
dH
GlcNAcI GlcII b GalIII c GlcNAcIV d
a
GlcV e GalVI f GalVII g GalVIII h
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.53
3.72
n.d. i
n.d.
n.d.
4.29
3.95
4.56
3.37
3.67
n.d.
3.62
3.99
3.83
4.43
3.59
3.72
4.16
n.d.
n.d.
n.d.
4.70
3.81
n.d.
n.d.
3.73
4.28
3.97
4.56
3.37
3.67
n.d.
3.62
3.99
3.83
4.46
3.55
n.d.
3.94
n.d.
n.d.
n.d.
4.54
3.54
3.67
3.93
n.d.
n.d.
n.d.
4.54
3.54
3.67
3.93
n.d.
n.d.
n.d.
O(CH2)2(CH2)2(CH2)2ND2
OCH2CH2(CH2)2CH2CH2ND2
1.38Á
/
1.42 (4 H)
1.54Á
/
1.57 (2 H), 1.63Á1.66 (2
/
H)
2.98
CH2ND2
OCH2(CH2)5ND2
NDCOCH3
3.68, 3.92
2.03 (2)
a
GlcNAc(b1-O(CH2)6NH2).
b
Glc(b1-6)GlcNAc(b1-O(CH2)6NH2).
c
d
e
f
Gal(b1-4)Glc(b1-6)GlcNAc(b1-O(CH2)6NH2).
GlcNAc(b1-3)Gal.
Glc(b1-6)GlcNAc(b1-3)Gal.
Gal(b1-4)Glc(b1-6)GlcNAc(b1-3)Gal.
Gal(b1-4)GlcNAc(b1-O(CH2)6NH2).
Gal(b1-4)GlcNAc(b1-3)Gal.
n.d., not determined.
g
h
i
H, J5,6a 2.3, J6a,6b 12.3 Hz, H-6a), 4.31 (dd, 1 H, J1,2 8.5,
J2,3 10.8 Hz, H-2), 4.33 (dd, 1 H, J5,6b 4.7 Hz, H-6b),
5.17 (dd, 1 H, J3,4 9.1, J4,5 9.8 Hz, H-4), 5.36 (d, 1 H, H-
H-3), 5.13 (d, 1 H, H-1), 7.81Á
7.90 (m, 4 H, Phth); 13C
/
NMR (75.5 MHz, CD3OD): d 26.2, 26.9, 29.3, and 29.8
(4 CH2), 51.8 (CH2N3), 58.2 (C-2), 62.4 and 70.0 (C-6,
OCH2), 72.2, 72.3, and 77.9 (C-3, C-4, C-5), 99.3 (C-1),
123.8, 132.6, and 135.3 (Phth); HRMS of C20H26N4O7
1), 5.79 (dd, 1 H, H-3), 7.75Á
7.89 (2 m, 4 H, Phth); 13C
/
NMR (75.5 MHz, CDCl3): d 20.2, 20.4, and 20.5
(COCH3), 25.1, 26.0, 28.4, and 28.8 (4 CH2), 50.9
(CH2N3), 54.5 (C-2), 61.9 and 69.6 (C-6, OCH2), 68.9,
70.6, and 71.6 (C-3, C-4, C-5), 98.0 (C-1), 123.3, 131.1,
and 134.2 (Phth), 169.3, 169.9, and 170.5 (COCH3);
(M, 434.180): [Mꢀ
/
NH4]ꢀ found 452.212, calcd
452.214.
HRMS of C26H32N4O10 (M, 560.211): [Mꢀ
NH4]ꢀ
found 578.250, calcd 578.254.
/
3.4. 6-Azidohexyl 6-O-tert-butyldiphenylsilyl-2-deoxy-2-
phthalimido-b- -glucopyranoside (9)
D
To a soln of 8 (2.01 g, 4.64 mmol) in CH2Cl2 (30 mL)
were added Py (3.0 mL), a catalytic amount of 4-
dimethylaminopyridine, Et3N (0.86 mL), and tert-bu-
tyldiphenylsilylchloride (1.7 mL, 6.54 mmol). The mix-
ture was stirred for 16 h, then poured into cold water,
washed with aq satd NaHCO3, dried, filtered, and
3.3. 6-Azidohexyl 2-deoxy-2-phthalimido-b-D-
glucopyranoside (8)
To a soln of 7 (2.60 g, 4.64 mmol) in MeOH (20 mL) and
CH2Cl2 (5 mL) was added NaOMe (pH 8). The mixture
was stirred for 5 h, then neutralized with Dowex 50ꢂ
(Hꢀ), filtered, and concentrated giving 8 as a white solid
(2.01 g, quantitative); Rf 0.42 (4:1 CH2Cl2ÁMeOH);
[a]2D0 158 (c 1, CHCl3); 1H NMR (300 MHz, CD3OD):
d 1.08Á1.10 (m, 4 H, 2 CH2), 1.14Á1.20 (m, 2 H, CH2),
1.38Á1.42 (m, 2 H, CH2), 3.01 (t, 2 H, CH2N3), 3.73 (dd,
/
8
concentrated. Column chromatography (1:1 tolueneÁ
EtOAc) of the residue gave 9, isolated as a white foam
[3.12 g, 88% (starting from 7)]; Rf 0.53 (1:1 tolueneÁ
/
/
/
1
ꢁ
/
EtOAc); [a]2D0
CDCl3): d 1.06 [s, 9 H, (CH3)3CSi], 1.06Á
CH2), 1.25Á1.27 (m, 2 H, CH2), 1.39Á
ꢁ
/
168 (c 1, CHCl3); H NMR (300 MHz,
1.13 (m, 4 H, 2
1.41 (m, 2 H,
/
/
/
/
/
/
1 H, J5,6b 4.9, J6a,6b 11.8 Hz, H-6b), 3.84 (m, 1 H,
OCHH), 3.92 (dd, 1 H, J5,6a 1.5 Hz, H-6a), 3.96 (dd, 1
H, J1,2 8.5, J2,3 10.6 Hz, H-2), 4.25 (dd, 1 H, J3,4 7.7 Hz,
CH2), 3.02 (t, 2 H, CH2N3), 3.38 (m, 1 H, OCHH), 3.58
(m, 1 H, H-5), 3.67 (t, 1 H, H-4), 3.76 (m, 1 H, OCHH),
3.96 (d, 2 H, J5,6 4.9 Hz, H-6a, H-6b), 4.09 (dd, 1 H, J1,2

2620
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
8.4, J2,3 10.9 Hz, H-2), 4.36 (dd, 1 H, J3,4 8.4 Hz, H-3),
5.17 (d, 1 H, H-1); 13C NMR (75.5 MHz, CDCl3): d 19.1
[(CH3)3CSi], 25.3, 26.0, 28.4, and 29.0 (4 CH2), 26.7
[(CH3)3CSi], 51.0 (CH2N3), 56.3 (C-2), 64.8 and 69.1 (C-
6, OCH2), 71.7, 74.1, and 74.4 (C-3, C-4, C-5), 98.0 (C-
28.4, and 28.9 (4 CH2), 50.9 (CH2N3), 54.7 (C-2), 61.2
and 69.5 (C-6, OCH2), 69.8, 70.5, and 74.3 (C-3, C-4, C-
5), 98.1 (C-1), 165.4 and 165.9 (2 CH3C6H4CO); HRMS
of C36H38N4O9 (M, 670.263): [Mꢀ
/
NH4]ꢀ found
688.294, calcd 688.298.
1); HRMS of C36H44N4O7Si (M, 672.297): [Mꢀ
/
NH4]ꢀ
found 690.332, calcd 690.330.
3.7. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b- -glucopyranoside (13)
D-
/
D-
3.5. 6-Azidohexyl 6-O-tert-butyldiphenylsilyl-2-deoxy-
3,4-di-O-p-methylbenzoyl-2-phthalimido-b-
glucopyranoside (10)
/
D-
D
A soln of (2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl)-
D-glucopyranosyl trichlor-
To a soln of 9 (2.75 g, 4.09 mmol) in Py (35 mL) was
added, at 0 8C, dropwise a soln of p-methylbenzoyl
chloride (1.35 mL, 10.2 mmol) in dry CH2Cl2 (10 mL).
The mixture was stirred for 20 h, then, washed with cold
water and aq satd NaHCO3, dried, filtered, and
(10
/
4)-2,3,6-tri-O-acetyl-b-
oacetimidate (12)19 (0.26 g, 0.33 mmol) and 11 (0.17 g,
0.26 mmol) in dry CH2Cl2 (5 mL), containing molecular
˚
sieves 4 A (0.2 g), was stirred for 1 h under Ar. The
mixture was cooled to 0 8C and TMSOTf (6 mL, 33
mmol) was added. The mixture was stirred for 1 h,
during which period the temperature was allowed to
reach room temperature (rt), then neutralized with
Et3N, washed with aq satd NaHCO3 and water, dried,
filtered, and concentrated. Column chromatography
concentrated. Column chromatography (19:1 tolueneÁ
EtOAc) of the residue gave 10, isolated as a colourless
syrup (3.25 g, 69%); Rf 0.65 (7:1 tolueneÁ
EtOAc); [a]2D0
68 (c 1, CHCl3); 1H NMR (300 MHz, CDCl3): d 1.03
[s, 9 H, (CH3)3CSi], 1.16Á1.19 (m, 4 H, 2 CH2), 1.25Á
1.29 (m, 2 H, CH2), 1.49Á1.55 (m, 2 H, CH2), 2.27 and
/
/
ꢁ
/
/
/
/
(1:2 tolueneÁ/EtOAc) of the residue afforded 13, isolated
2.35 (2 s, each 3 H, 2 CH3C6H4CO), 3.05 (t, 2 H,
CH2N3), 3.48 (m, 1 H, OCHH), 4.51 (dd, 1 H, J1,2 8.5,
J2,3 10.8 Hz, H-2), 5.48 (d, 1 H, H-1), 5.57 (t, 1 H, H-4),
6.17 (dd, 1 H, J3,4 9.3 Hz, H-3), 7.04 and 7.13 (2 d, each
2 H, Phth); 13C NMR (75.5 MHz, CDCl3): d 19.1
[(CH3)3CSi], 21.5 (2 CH3C6H4CO), 25.4, 26.2, 28.5, and
29.1 (4 CH2), 26.6 [(CH3)3CSi], 51.1 (CH2N3), 55.0 (C-
2), 62.9 and 69.3 (C-6, OCH2), 69.7, 71.3, and 75.1 (C-3,
C-4, C-5), 98.0 (C-1), 164.9 and 165.0 (2 CH3C6H4CO);
Anal. Calcd for C52H56N4O9Si: C, 68.70; H, 6.21; N,
6.16. Found: C, 68.58; H, 6.26; N, 6.02.
as a white foam (148 mg, 45%); Rf 0.55 (1:2 tolueneÁ
/
1
EtOAc); [a]2D0
CDCl3): d 1.14Á
H, CH2), 1.45Á1.51 (m, 2 H, CH2), 1.95, 2.04, 2.05, 2.07,
ꢁ
/
68 (c 1, CHCl3); H NMR (300 MHz,
/
1.16 (m, 4 H, 2 CH2), 1.22Á1.30 (m, 2
/
/
and 2.14 (5 s, 3,3,9,3,3 H, 7 COCH3), 2.27 and 2.34 (2 s,
each 3 H, 2 CH3C6H4CO), 3.05 (t, 2 H, CH2N3), 3.49
(m, 1 H, OCHH), 3.60 (m, 1 H, H-5II), 4.64 (d, 1 H, J1,2
7.7 Hz, H-1II), 4.90 (t, 1 H, J2,3 9.2 Hz, H-2II), 4.95 (dd,
1 H, J2,3 10.3, J3,4 3.4 Hz, H-3III), 5.10 (dd, 1 H, J1,2 7.8
Hz, H-2III), 5.15 (t, 1 H, H-4I), 5.46 (d, 1 H, J1,2 8.4 Hz,
H-1I), 6.17 (dd, 1 H, J2,3 10.7, J3,4 9.2 Hz, H-3I), 7.04
and 7.15 (2 d, each 2 H, Phth), 7.61 and 7.78 (2 d, each 4
H, 2 CH3C6H4CO); 13C NMR (75.5 MHz, CDCl3): d
3.6. 6-Azidohexyl 2-deoxy-3,4-di-O-p-methylbenzoyl-2-
phthalimido-b-
D-glucopyranoside (11)
20.3Á20.6 (COCH3), 21.4 (2 CH3C6H4CO), 25.3, 26.0,
/
28.4, and 28.9 (4 CH2), 50.9 (CH2N3), 54.8 (C-2I), 60.6,
62.0, 68.1, and 69.6 (C-6I, C-6II, C-6III, OCH2), 66.5,
68.9, 69.8, 70.5, 70.7, 70.8, 71.5, 72.4, 72.7, 74.0, and
76.2 (C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-2III, C-
3III, C-4III, C-5III), 98.0, 100.3, and 100.9 (C-1I, C-1II, C-
To a soln of 10 (0.72 g, 0.79 mmol) in dry toluene (5.0
mL) was added, at 0 8C, a soln of acetyl chloride (0.93
mL, 13.05 mmol) in dry MeOH (25 mL). The mixture
was stirred under Ar for 4 h, then neutralized with Et3N,
washed with water, dried, filtered, and concentrated.
1III), 165.1 and 165.4 (2 CH3C6H4CO), 168.8Á
170.1
/
Low-pressure column chromatography (5:1 tolueneÁ
EtOAc) of the residue gave 11, isolated as a colourless
syrup (0.40 g, 75%); Rf 0.28 (4:1 tolueneÁ
EtOAc); [a]2D0
38 (c 1, CHCl3); 1H NMR (300 MHz, CDCl3): d
1.15Á1.13 (m, 4 H, 2 CH2), 1.24Á1.28 (m, 2 H, CH2),
1.43Á1.49 (m, 2 H, CH2), 2.23 and 2.30 (2 s, each 3 H, 2
CH3C6H4CO), 3.04 (t, 2 H, CH2N3), 3.51 (m, 1 H,
OCHH), 3.75 (dd, 1 H, J5,6b 4.0, J6a,6b 12.7 Hz, H-6b),
4.55 (dd, 1 H, J1,2 8.5, J2,3 10.7 Hz, H-2), 5.50 (t, 1 H, H-
4), 5.55 (d, 1 H, H-1), 6.31 (dd, 1 H, J3,4 9.3 Hz, H-3),
7.02 and 7.13 (2 d, each 2 H, Phth), 7.66 and 7.83 (2 d,
each 4 H, 2 CH3C6H4CO); 13C NMR (75.5 MHz,
CDCl3): d 21.3 and 21.4 (2 CH3C6H4CO), 25.2, 26.0,
/
(COCH3); Anal. Calcd for C62H72N4O26: C, 57.76; H,
5.63; N, 4.35. Found: C, 57.65; H, 5.81; N, 4.19.
/
ꢁ
/
3.8. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-2-acetamido-3,4-di-O-acetyl-2-
deoxy-b- -glucopyranoside (14)
D-
/
/
/
D-
/
/
D
To a soln of 13 (141 mg, 0.11 mmol) in MeOH (20 mL)
and CH2Cl2 (3 mL) was added NaOMe (pH 10), and the
mixture was stirred for 24 h. After neutralization with
Dowex 50ꢂ
/
8 (Hꢀ) and filtration, the solution was
concentrated. To a solution of the residue in 1-BuOH

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2621
(45 mL) was added 1,2-diaminoethane (1 mL, 15 mmol),
and the mixture was stirred overnight at 90 8C, then, co-
concentrated with toluene. A soln of the residue in Py
(50 mL) and Ac2O (40 mL) was stirred overnight, then
co-concentrated with toluene, EtOH and CH2Cl2.
3.10. 6-Aminohexyl b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranoside (3)
D
-galactopyranosyl-(10
/
4)-b-D-
/
D-
To a soln of 15 (60 mg, 89.4 mmol) in t-BuOH (25 mL)
and water (20 mL) were added 10% PdÁC (240 mg) and
Column chromatography (3:1 CH2Cl2Á
residue yielded 14, isolated as a colourless glass (98 mg,
86%); Rf 0.61 (9:1 CH2Cl2Á 128 (c 1,
MeOH); [a]2D0
CHCl3); 1H NMR (500 MHz, CDCl3; 2D TOCSY,
ROESY, HSQC): d 1.37Á1.38 (m, 4 H, 2 CH2), 1.58Á
/
acetone) of the
/
three drops aq 25% NH3, and the mixture was stirred for
24 h under H2, then filtered over hyflo, and co-
concentrated with toluene. Column chromatography of
the residue on a Toyopearl HW-40S column, eluted with
aq 0.1 M NH4OAc, and subsequent lyophilization gave
3, isolated as a white powder (41 mg, 71%); Rf 0.30
/
ꢁ
/
/
/
1.59 (m, 4 H, 2 CH2), 1.93 (s, 3 H, NHCOCH3), 1.96,
2.01, 2.03, 2.04, 2.06, 2.12, and 2.14 (7 s, 3,6,3,6,3,3,3 H,
9 COCH3), 3.26 (t, 2 H, CH2N3), 3.46 (m, 1 H, OCHH),
3.70 (m, 1 H, H-5I), 4.13 (dd, 1 H, J5,6b 6.4, J6a,6b 11.3
Hz, H-6bIII), 4.47 (dd, 1 H, J5,6a 2.2, J6a,6b 12.2 Hz, H-
6aII), 4.49 (d, 1 H, J1,2 8.0 Hz, H-1III), 4.58 (d, 1 H, J1,2
7.6 Hz, H-1II), 4.65 (d, 1 H, J1,2 8.3 Hz, H-1I), 4.95 (dd,
1 H, J2,3 10.4, J3,4 3.4 Hz, H-3III), 5.10 (dd, 1 H, H-2III),
5.14 (t, 1 H, J2,3 9.2, J3,4 9.2 Hz, H-3II), 5.26 (dd, 1 H,
(2:1:1 AcOHÁ
/
1-BuOHÁ
/
water); [a]2D0
ꢁ28 (c 1, water);
/
13C NMR (75.5 MHz, D2O): d 22.8 (NDCOCH3), 25.2,
25.8, 27.3, and 29.0 (4 CH2), 40.0 (CH2ND2), 56.1
(C-2I), 60.7 (C-6II), 61.6 (C-6III), 69.1 (C-4III), 69.2
(C-6I), 71.5 (OCH2), 73.4 (C-2II), 70.3, 71.5, 73.1,
74.3, 74.9, 75.4, 75.5, and 75.9 (C-3I, C-4I, C-5I, C-3II,
C-5II, C-2III, C-3III, C-5III), 79.0 (C-4II), 101.8 (C-1I),
103.2 (C-1II), 103.5 (C-1III), 175.0 (NDCOCH3);
J2,3 10.4, J3,4 9.1 Hz, H-3I), 5.34 (d, 1 H, J4,5
4III), 5.79 (d, 1 H, J2,NH 8.9 Hz, NHCOCH3); 13C NMR
(75.5 MHz, CDCl3): d 20.2Á20.5 (COCH3), 22.9
B
/
1 Hz, H-
HRMS of C26H48N2O16 (M, 644.300): [Mꢀ
H]ꢀ
/
/
1
found 645.306, calcd 645.308. For H NMR data, see
Table 2.
(NHCOCH3), 25.2, 26.1, 28.5, and 29.0 (4 CH2), 51.0
(CH2N3), 54.5 (C-2I), 60.6 (C-6III), 61.8 (C-6II), 66.4 (C-
4III), 67.9 (C-6I), 68.8 (C-2III), 69.1 (C-4I), 69.2 (OCH2),
70.3 (C-5III), 70.7 (C-3III), 71.3 (C-2II), 72.1 (C-3I), 72.4
(C-5II), 72.6 (C-3II), 73.1 (C-5I), 75.9 (C-4II), 100.0 (C-
1II), 100.2 (C-1I), 100.8 (C-1III), 168.7Á
170.4 (COCH3,
/
3.11. 6-Aminohexyl b-
glucopyranosyl-(106)-[b-
acetamido-2-deoxy-b- -glucopyranoside (1)
D
-galactopyranosyl-(10
/
4)-b-D-
4)]-2-
/
D
-galactopyranosyl-(10
/
NHCOCH3); Anal. Calcd for C44H64N4O25: C, 50.38;
H, 6.15; N, 5.34. Found: C, 50.24; H, 6.06; N, 5.35;
D
HRMS of C44H64N4O25 (M, 1048.386): [Mꢀ
/
H]ꢀ found
To a soln of 3 (8.4 mg, 13.0 mmol) in aq 50 mM sodium
cacodylate buffer pH 7.5 (600 mL), containing 5 mM
MnCl2, bovine serum albumin (BSA) (0.5 mg), and
NaN3 (0.02%), were added alkaline phosphatase (12 U),
UDP-galactose (15 mg, 24.5 mmol), and b-1,4-galacto-
syltransferase (2.5 U). The reaction mixture was incu-
bated for 20 h at 37 8C, then, water (150 mL) was added.
1049.397, calcd 1049.393.
3.9. 6-Azidohexyl b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranoside (15)
D-galactopyranosyl-(10
/
4)-b-D-
/
D-
UDP-Galactose was removed on a Dowex 1ꢂ
8 (Clꢁ)
/
To a soln of 14 (50 mg, 47.6 mmol) in MeOH (8 mL) and
CH2Cl2 (1 mL) was added NaOMe (pH 10), and the
mixture was stirred for 2.5 h. After neutralization with
column, eluted with water. The eluate was concentrated,
and the residue applied on a Bio Gel P-2 column, eluted
with aq 0.1 M NH4HCO3 at a flow rate of 40 mL/h. The
appropriate fractions were freeze-dried to give 1 (9.6 mg,
Dowex 50ꢂ
concentrated. Column chromatography (2:1 MeOHÁ
CH2Cl2) of the residue yielded 15, isolated as a colour-
less glass (29 mg, 90%); Rf 0.63 (4:1 MeOHÁCH2Cl2);
[a]2D0 88 (c 1, water); 13C NMR (75.5 MHz, D2O): d
/
8 (Hꢀ) and filtration, the solution was
/
91%); Rf 0.15 (2:1:1 AcOHÁ
/
1-BuOHÁ
/
water); [a]2D0
ꢁ38
/
(c 0.8, water); 13C NMR (75.5 MHz, D2O): d 22.7
(NDCOCH3), 25.2, 25.9, 27.3, and 29.0 (4 CH2), 40.1
(CH2ND2), 55.8 (C-2I), 60.7, 61.7 (2 C), 68.1, and 71.2
(C-6I, C-6II, C-6III, C-6IV, OCH2), 69.2 (2 C), 71.6 (3 C),
73.0, 73.2, 73.3, 74.2, 75.0, 75.4, 75.9, 76.0, 78.7, and
79.1 (C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-2III, C-
3III, C-4III, C-5III, C-2IV, C-3IV, C-4IV, C-5IV), 101.8,
103.0, 103.4, and 103.6 (C-1I, C-1II, C-1III, C-1IV), 175.0
/
ꢁ
/
22.8 (NDCOCH3), 25.3, 26.2, 28.6, and 29.1 (4 CH2),
51.8 (CH2N3), 56.2 (C-2I), 60.7 (C-6II), 61.7 (C-6III),
69.3 (C-6I), 71.1 (OCH2), 69.2 (C-4III), 70.4, 71.6, 73.2,
73.4, 74.4, 75.0, 75.4, 75.5, 76.0, and 79.1 (C-3I, C-4I, C-
5I, C-2II, C-3II, C-4II, C-5II, C-2III, C-3III, C-5III), 101.8
(C-1I), 103.2 (C-1II), 103.6 (C-1III), 175.0 (NDCOCH3);
HRMS of C26H46N4O16 (M, 670.290): [Mꢀ
NH4]ꢀ
found 688.330, calcd 688.333. For H NMR data, see
Table 1.
(NDCOCH3); HRMS of C32H58N2O21 (M, 806.353):
1
/
H]ꢀ found 807.359, calcd 807.361. For H NMR
1
[Mꢀ
/
data, see Table 3.

2622
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
3.12. 6-Azidohexyl (2,6-di-O-acetyl-3,4-di-O-
isopropylidene-b- 4)-(2,3,6-tri-
-galactopyranosyl)-(10
O-acetyl-b- -glucopyranosyl)-(106)-2-deoxy-3,4-di-O-
p-methylbenzoyl-2-phthalimido-b- -glucopyranoside (17)
3.24 (bs, 1 H, OH), 3.48 (m, 1 H, OCHH), 3.72 (dd, 1 H,
J3,4 9.3, J4,5 9.7 Hz, H-4II), 3.78 (dd, 1 H, J5,6b 7.7, J6a,6b
11.6 Hz, H-6bI), 3.83 (bs, 1 H, H-4III), 4.05 (m, 1 H, H-
5I), 4.13 (dd, 1 H, J5,6b 5.7, J6a,6b 11.9 Hz, H-6bII), 4.22
(dd, 1 H, J5,6a 6.4, J6a,6b 11.4 Hz, H-6bIII), 4.42 (dd, 1 H,
J5,6a 1.5 Hz, H-6aII), 4.46 (dd, 1 H, J1,2 8.5, J2,3 10.7 Hz,
H-2I), 4.62 (d, 1 H, J1,2 7.9 Hz, H-1II), 4.81 (dd, 1 H, J1,2
8.0, J2,3 9.1 Hz, H-2III), 4.92 (dd, 1 H, J2,3 9.2 Hz, H-2II),
5.13 (t, 1 H, H-3II), 5.34 (dd, 1 H, J3,4 9.3, J4,5 9.9 Hz, H-
4I), 5.45 (d, 1 H, H-1I), 6.16 (dd, 1 H, H-3I), 7.04 and
7.15 (2 d, each 2 H, Phth), 7.62 and 7.78 (2 d, each 4 H, 2
D
/
D
/
D
A soln of (2,6-di-O-acetyl-3,4-di-O-isopropylidene-b-
D-
galactopyranosyl)-(10
/
4)-2,3,6-tri-O-acetyl-b-
D-gluco-
pyranosyl trichloroacetimidate donor (16)23 (0.51 g, 0.69
mmol) and 11 (0.31 g, 0.48 mmol) in dry CH2Cl2 (5 mL),
˚
containing powdered molecular sieves 4 A (1 g), was
stirred for 1 h under Ar. After cooling to 0 8C, TMSOTf
(83 mL, 0.46 mmol) was added. The mixture was stirred
for 20 min, during which period the temperature was
allowed to reach rt, then neutralized with Et3N, filtered
over hyflo, and concentrated. Column chromatography
CH3C6H4CO); 13C NMR (75.5 MHz, CDCl3): d 20.4Á
/
20.6 (COCH3), 21.2 and 21.3 (2 CH3C6H4CO), 25.1,
25.9, 28.3, and 28.8 (4 CH2), 50.8 (CH2N3), 54.6 (C-2I),
62.0, 62.3, 68.1, and 69.4 (C-6I, C-6II, C-6III, OCH2),
68.3, 69.8, 70.7, 71.2, 72.1, 72.2, 72.5, 72.7, 73.7, 76.1,
and 77.1 (C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-
2III, C-3III, C-4III, C-5III), 97.8, 100.4, and 100.7 (C-1I,
(3:1 tolueneÁ
/
EtOAc with 1% Et3N) of the residue
afforded 17, isolated as a white foam (0.36 g, 62%); Rf
1
0.57 (1:1 tolueneÁ
NMR (300 MHz, CDCl3): d 1.15Á
1.26Á1.30 (m, 2 H, CH2), 1.30 and 1.52 [2 s, each 3 H,
C(CH3)2], 1.47Á1.52 (m, 2 H, CH2), 2.04, 2.05, 2.07, and
/
EtOAc); [a]2D0
ꢀ
/
38 (c 1, CHCl3); H
/
1.16 (m, 4 H, 2 CH2),
C-1II, C-1III), 165.0 and 165.3 (2 CH3C6H4CO), 169.1Á
170.7 (COCH3); HRMS of C58H68N4O24 (M, 1204.422):
[Mꢀ
H]ꢀ found 1205.399, calcd 1205.430.
/
/
/
/
2.11 (4 s, 3,3,6,3 H, 5 COCH3), 2.25 and 2.32 (2 s, each 3
H, 2 CH3C6H4CO), 3.04 (t, 2 H, CH2N3), 3.49 (m, 1 H,
OCHH), 3.60 (m, 1 H, H-5II), 4.47 (dd, 1 H, J1,2 8.4, J2,3
10.7 Hz, H-2I), 4.63 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.85 (t, 1
H, H-2III), 4.93 (t, 1 H, H-2II), 5.15 (t, 1 H, H-3II), 5.36
(t, 1 H, H-4I), 5.47 (d, 1 H, H-1I), 6.18 (dd, 1 H, J3,4 9.2
Hz, H-3I), 7.03 and 7.13 (2 d, each 2 H, Phth), 7.62 and
7.78 (2 d, each 4 H, 2 CH3C6H4CO); 13C NMR (75.5
3.14. 6-Azidohexyl (2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b- -glucopyranoside (19)
D-
/
D-
/
D
To a soln of 18 (86 mg, 72.8 mmol) in dry MeCN (1 mL)
were added trimethyl orthoacetate (23 mL, 182 mmol)
and a catalytic amount of p-toluenesulfonic acid. After
stirring for 3.5 h, aq 80% AcOH (50 mL) was added, and
the stirring was continued for 1 h. The mixture was
diluted with CH2Cl2, washed with cold aq satd NaHCO3
and cold water, dried, filtered, and concentrated.
MHz, CDCl3): d 20.5Á20.6 (COCH3), 21.3 and 21.4 (2
/
CH3C6H4CO), 25.2 and 26.0 (2 CH2), 25.9 and 27.1
[(CH3)2C], 28.4 and 28.9 (2 CH2), 50.9 (CH2N3), 54.7
(C-2I), 62.1, 62.9, 68.1, and 69.6 (C-6I, C-6II, C-6III
,
OCH2), 69.6, 70.7 (2 C), 71.4, 72.3, 72.6 (2 C), 72.9, 74.0,
75.9, and 76.7 (C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II,
C-2III, C-3III, C-4III, C-5III), 97.9, 100.3, and 100.5 (C-1I,
C-1II, C-1III), 110.6 [(CH3)2C], 165.1 and 165.4 (2
Column chromatography (1:4 tolueneÁ
residue afforded 19, isolated as a white foam (78 mg,
85%); Rf 0.49 (1:4 tolueneÁ 108 (c 1,
EtOAc); [a]2D0
CHCl3); H NMR (300 MHz, CDCl3): d 1.14Á1.16 (m,
4 H, 2 CH2), 1.23Á1.28 (m, 2 H, CH2), 1.45Á1.53 (m, 2
/EtOAc) of the
/
ꢁ
/
1
CH3C6H4CO), 168.9Á
C61H72N4O24 (M, 1244.453): [Mꢀ
/
170.5 (COCH3); HRMS of
NH4]ꢀ found
/
/
/
/
1262.475, calcd 1262.488.
H, CH2), 2.03, 2.04, 2.06, 2.07, 2.11, and 2.15 (6 s, each 3
H, 6 COCH3), 2.27 and 2.34 (2 s, each 3 H, 2
CH3C6H4CO), 2.76 (bd, 1 H, OH), 3.05 (t, 2 H,
CH2N3), 3.49 (m, 1 H, OCHH), 3.61 (m, 1 H, H-5II),
4.47 (dd, 1 H, J1,2 8.4, J2,3 10.7 Hz, H-2I), 4.63 (d, 1 H,
J1,2 7.8 Hz, H-1II), 4.85 (dd, 1 H, J1,2 7.8, J2,3 9.9 Hz, H-
2III), 4.92 (dd, 1 H, J2,3 9.3 Hz, H-2II), 5.14 (t, 1 H, H-
3.13. 6-Azidohexyl (2,6-di-O-acetyl-b-D-
galactopyranosyl)-(10
glucopyranosyl)-(106)-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b- -glucopyranoside (18)
/
4)-(2,3,6-tri-O-acetyl-b-
D-
/
D
To a soln of 17 (155 mg, 0.13 mmol) in CH2Cl2 (2.5 mL)
at 0 8C, was added aq 90% TFA (0.25 mL). The mixture
was stirred for 1.5 h, then co-concentrated with toluene
to give 18, isolated as a colourless glass (127 mg, 84%);
3II), 5.28 (d, 1 H, J3,4 3.3, J4,5
B
1 Hz, H-4III), 5.35 (t, 1
/
H, H-4I), 5.46 (d, 1 H, H-1I), 6.18 (dd, 1 H, J3,4 9.1 Hz,
H-3I), 7.04 and 7.14 (2 d, each 2 H, Phth), 7.62 and 7.78
(2 d, each 4 H, 2 CH3C6H4CO); 13C NMR (75.5 MHz,
Rf 0.20 (1:2 tolueneÁ
1H NMR (500 MHz, CDCl3; 2D TOCSY, ROESY): d
1.11Á1.17 (m, 4 H, 2 CH2), 1.25Á1.30 (m, 2 H, CH2),
1.44Á1.50 (m, 2 H, CH2), 2.04, 2.07, 2.10, and 2.12 (4 s,
/
EtOAc); [a]2D0
ꢁ
/
68 (c 1, CHCl3);
CDCl3): d 20.5Á20.6 (COCH3), 21.4 (2 CH3C6H4CO),
/
25.3, 26.0, 28.4, and 28.9 (4 CH2), 50.9 (CH2N3), 54.8
(C-2I), 61.3, 62.2, 68.2, and 69.6 (C-6I, C-6II, C-6III,
OCH2), 69.1, 69.9, 70.7, 70.8, 71.4 (2 C), 72.5, 72.6, 72.8,
74.0, and 76.2 (C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II,
C-2III, C-3III, C-4III, C-5III), 165.1 and 165.4 (2
/
/
/
6,3,3,3 H, 5 COCH3), 2.28 and 2.34 (2 s, each 3 H, 2
CH3C6H4CO), 2.81 (bs, 1 H, OH), 3.04 (t, 2 H, CH2N3),

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2623
CH3C6H4CO), 169.2Á
C60H70N4O25 (M, 1246.432): [Mꢀ
1264.443, calcd 1264.467.
/
170.8 (COCH3); HRMS of
50ꢂ
column chromatography (9:10
the residue gave 23, isolated as a white solid (5.1 g, 82%);
Rf 0.10 (1:1 tolueneÁ
EtOAc); [a]2D0 18 (c 1, CHCl3); 1H
/
8 (Hꢀ), filtered, and concentrated. Low-pressure
1:1 tolueneÁEtOAc) of
/
NH4]ꢀ found
/
/
/
ꢀ
/
3.15. Benzyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
NMR (500 MHz, CDCl3; 2D TOCSY, ROESY,
HSQC): d 2.07 (s, 3 H, COCH3), 2.97 (m, 1 H, H-5I),
3.23 (dd, 1 H, J5,6b 7.4, J6a,6b 9.6 Hz, H-6bII), 3.68 (t, 1
H, H-4III), 3.81 (dd, 1 H, J5,6b 3.1, J6a,6b 12.3 Hz, H-
6bIII), 3.87 (dd, 1 H, H-4I), 3.92 (dd, 1 H, J5,6a 2.0 Hz,
H-6aIII), 4.04 (dd, 1 H, J1,2 8.1, J2,3 10.6 Hz, H-2III), 4.08
and 4.22 (2 d, each 1 H, OCH2C6H5), 4.24 and 4.38 (2 d,
each 1 H, OCH2C6H5), 4.24 and 4.45 (2 d, each 1 H,
OCH2C6H5), 4.66 and 4.83 (2 d, each 1 H, OCH2C6H5),
4.55 and 4.85 (2 d, each 1 H, OCH2C6H5), 4.63 and 4.87
(2 d, each 1 H, OCH2C6H5), 5.39 (d, 1 H, H-1III), 5.53
b-
b-
D
-glucopyranosyl)-(10
/
3)-(4-O-acetyl-2,6-di-O-benzyl-
D-
D-galactopyranosyl)-(10
/
4)-2,3,6-tri-O-benzyl-b-
glucopyranoside (22)
A soln of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl trichloroacetimidate (5)20 (2.62 g, 4.52
mmol) and (4-O-acetyl-2,6-di-O-benzyl-b- -galactopyr-
-glucopyra-
D-
D
anosyl)-(10
/
4)-1,2,3,6-tetra-O-benzyl-b-
D
nose (21)25 (3.39 g, 3.66 mmol) in dry CH2Cl2 (80 mL),
˚
containing powdered molecular sieves 4 A (2 g), was
stirred for 30 min under Ar. The mixture was cooled to
(d, 1 H, J3,4 3.5, J4,5
B
1 Hz, H-4II); 13C NMR (75.5
/
ꢁ
/
70 8C, and TMSOTf (0.1 mL, 0.54 mmol) was added.
MHz, CDCl3): d 20.6 (COCH3), 56.6 (C-2III), 61.0 (C-
6III), 67.3 and 67.7 (C-6I, C-6II), 70.4, 72.7, 73.1, 74.0,
74.6, and 74.7 (6 OCH2C6H5), 70.2 (2 C), 70.6, 71.8,
74.3, 75.5 (2 C), 78.2, 80.1, 81.2, and 82.3 (C-2I, C-3I, C-
4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-3III, C-4III, C-5III),
99.0 (C-1III), 101.4 and 101.9 (C-1I, C-1II), 170.9
(COCH3); HRMS of C70H73NO18 (M, 1215.482):
The mixture was stirred for 2.5 h, during which period
the temperature was allowed to reach rt, then neutra-
lized with Et3N, and filtered over hyflo. The filtrate was
washed with water, dried, filtered, and concentrated.
Low-pressure column chromatography (9:1 tolueneÁ
EtOAc) of the residue gave 22, isolated as a white solid
EtOAc); [a]2D0
(4.26 g, 86%); Rf 0.37 (2:1 tolueneÁ 128
/
/
ꢀ
/
[Mꢀ
NH4]ꢀ found 1233.497, calcd 1233.517.
/
(c 1, CHCl3); 1H NMR (500 MHz, CDCl3; 2D TOCSY,
ROESY, HSQC): d 1.81, 2.01, 2.02, and 2.06 (4 s, each 3
H, 4 COCH3), 3.02 (m, 1 H, H-5I), 3.46 (t, 1 H, H-5II),
3.52 (dd, 1 H, J5,6a 3.6, J6a,6b 10.9 Hz, H-6I), 3.60 (dd, 1
H, J2,3 9.6, J3,4 3.3 Hz, H-3II), 3.81 (m, 1 H, H-5III), 3.92
(m, 1 H, H-4I), 4.22 (dd, 1 H, J5,6b 4.1, J6a,6b 12.3 Hz, H-
6bIII), 4.32 (d, 1 H, J1,2 7.4 Hz, H-1II), 4.33 (d, 1 H, J1,2
7.7 Hz, H-1I), 4.15 and 4.34 (2 d, each 1 H, OCH2C6H5),
4.42 (d, 1 H, OCHHC6H5), 4.51 (d, 1 H, OCHHC6H5),
4.68 and 4.84 (2 d, each 1 H, OCH2C6H5), 4.57 and 4.86
(2 d, each 1 H, OCH2C6H5), 4.65 and 4.89 (2 d, each 1
H, OCH2C6H5), 5.17 (t, 1 H, J4,5 9.5 Hz, H-4III), 5.40 (d,
3.17. Benzyl (6-O-tert-butyldiphenylsilyl-2-deoxy-2-
phthalimido-b- 3)-(4-O-acetyl-2,6-
-glucopyranosyl)-(10
di-O-benzyl-b- -galactopyranosyl)-(104)-2,3,6-tri-O-
benzyl-b- -glucopyranoside (24)
D
/
D
/
D
To a soln of 23 (4.25 g, 3.49 mmol) in dry CH2Cl2 (35
mL) was added dry Py (1.67 mL), 4-dimethylaminopyr-
idine (0.14 g, 1.1 mmol), tert-butyldiphenylsilylchloride
(0.61 mL, 4.35 mmol), and Et3N (1.20 mL). The mixture
was stirred for 48 h, washed with cold water, cold aq
satd NaHCO3, and cold water, dried, filtered, and
concentrated. Low-pressure column chromatography
1 H, J4,5
5.80 (dd, 1 H, J2,3 10.7, J3,4 9.5 Hz, H-3III); 13C NMR
20.2 (COCH3), 54.5 (C-
B
/
1 Hz, H-4II), 5.55 (d, 1 H, J1,2 8.5 Hz, H-1III),
(17:30
/
3:1 tolueneÁEtOAc) of the residue gave 24,
/
(75.5 MHz, CDCl3): d 19.9Á
/
isolated as a white solid (4.51 g, 89%); Rf 0.49 (1:1
1
2III), 61.1 (C-6III), 67.3 and 67.9 (C-6I, C-6II), 70.4, 72.7,
73.0, 74.0, 74.5, and 74.7 (6 OCH2C6H5), 68.4, 69.3,
70.1, 71.3, 72.2, 74.3, 75.2, 78.4, 78.9, 81.2, and 82.2 (C-
2I, C-3I, C-4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-3III, C-
4III, C-5III), 97.8 (C-1III), 101.3 and 101.9 (C-1I, C-1II),
168.9, 169.1, 169.5, and 170.1 (4 COCH3); HRMS of
tolueneÁ
/
EtOAc); [a]2D0
ꢁ158 (c 1, CHCl3); H NMR
/
(500 MHz, CDCl3; 2D TOCSY, ROESY, HSQC): d
1.05 [s, 9 H, (CH3)3CSi], 1.97 (s, 3 H, COCH3), 3.03 (m,
1 H, H-5I), 3.51 (dd, 1 H, J5,6b 4.0, J6a,6b 10.8 Hz, H-
6bI), 3.63 (dd, 1 H, J2,3 9.6, J3,4 3.5 Hz, H-3II), 3.89 (m, 1
H, H-4I), 3.98 (d, 2 H, H-6aIII, H-6bIII), 4.05 (dd, 1 H,
J1,2 8.3, J2,3 11.0 Hz, H-2III), 4.28 (d, 1 H, J1,2 7.2 Hz, H-
1II), 4.12 and 4.28 (2 d, each 1 H, OCH2C6H5), 4.34 (d, 1
H, J1,2 7.4 Hz, H-1I), 4.16 and 4.36 (2 d, each 1 H,
OCH2C6H5), 4.40 (dd, 1 H, J3,4 8.2 Hz, H-3III), 4.25 and
4.46 (2 d, each 1 H, OCH2C6H5), 4.66 and 4.83 (2 d,
each 1 H, OCH2C6H5), 4.56 and 4.86 (2 d, each 1 H,
OCH2C6H5), 4.61 and 4.87 (2 d, each 1 H, OCH2C6H5);
13C NMR (75.5 MHz, CDCl3): d 19.0 [(CH3)3CSi], 20.5
(COCH3), 26.6 [(CH3)3CSi], 56.6 (C-2III), 64.8 (C-6III),
67.6 and 68.1 (C-6I, C-6II), 70.7, 72.9, 73.2, 74.3, 74.8,
and 74.9 (6 OCH2C6H5), 70.1, 70.9, 72.5, 74.0, 74.4,
C76H79NO21 (M, 1341.514): [Mꢀ
NH4]ꢀ found
1359.546, calcd 1359.549.
/
3.16. Benzyl (2-deoxy-2-phthalimido-b-D-
glucopyranosyl)-(10
galactopyranosyl)-(10
glucopyranoside (23)
/
3)-(4-O-acetyl-2,6-di-O-benzyl-b-
D-
/
4)-2,3,6-tri-O-benzyl-b-
D-
To a soln of 22 (6.88 g, 5.12 mmol) in CH2Cl2 (13.5 mL)
and MeOH (54 mL) was added NaOMe (pH 9). The
mixture was stirred for 2 h, then neutralized with Dowex

2624
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
74.7, 75.5, 77.2, 79.1, 81.5, and 82.5 (C-2I, C-3I, C-4I, C-
5I, C-2II, C-3II, C-4II, C-5II, C-3III, C-4III, C-5III), 98.3
(C-1III), 101.7 and 102.2 (C-1I, C-1II), 169.7 (COCH3);
through. After filtration over hyflo and concentration,
column chromatography (19:1 CH2Cl2ÁMeOH) of the
residue gave a colourless syrup (0.94 g, 90%). A soln of
the syrup in Py (75 mL) and Ac2O (75 mL) was stirred
overnight, then co-concentrated with toluene, EtOH,
and CH2Cl2. Low-pressure column chromatography
/
HRMS of C86H91NO18Si (M, 1453.600): [Mꢀ
/
NH4]ꢀ
found 1471.598, calcd 1471.635.
(9:1 tolueneÁEtOAc) of the residue gave 26, isolated
/
3.18. Benzyl (6-O-tert-butyldiphenylsilyl-2-deoxy-3,4-di-
O-p-methylbenzoyl-2-phthalimido-b- -glucopyranosyl)-
(103)-(4-O-acetyl-2,6-di-O-benzyl-b-
galactopyranosyl)-(104)-2,3,6-tri-O-benzyl-b-
glucopyranoside (25)
as a colourless syrup (1.08 g, quantitatively). To a soln
of 26 (0.11 g, 78 mmol) in dry DMF (5 mL) was added
hydrazinium acetate (9 mg, 118 mmol). The mixture was
stirred for 4 h, washed with aq satd NaCl, dried, filtered,
and co-concentrated with toluene. Low-pressure column
D
/
D-
/
D-
chromatography (1:10
sidue gave 27, isolated as a white solid (69 mg, 65%); Rf
0.42 (1:1 tolueneÁ
EtOAc); 1H NMR (500 MHz, CDCl3;
/
2:3 tolueneÁ
/
EtOAc) of the re-
To a soln of 24 (1.17 g, 0.80 mmol) in dry Py (115 mL)
was added dropwise at 0 8C, a soln of p-methylbenzoyl
chloride (0.45 mL, 3.40 mmol) in dry CH2Cl2 (15 mL).
The mixture was stirred under Ar for 20 h at rt, diluted
with CH2Cl2 and poured into cold water. The organic
layer was washed with aq satd NaHCO3, dried, filtered,
and concentrated. Low-pressure column chromatogra-
/
2D TOCSY, ROESY): d 1.05 [s, 9 H, (CH3)3CSi], 1.88,
1.96, 2.03, 2.10, and 2.14 (5 s, 3,3,6,3,3 H, 6 COCH3),
2.25 and 2.34 (2 s, each 3 H, 2 CH3C6H4CO), 3.48 (m, 1
H, H-5II), 3.54 (m, 0.5 H, H-5Ib), 3.83 (dd, 1 H, J5,6a 1.3,
J6a,6b 11.0 Hz, H-6aIII), 4.25 (d, 1 H, J1,2 8.1 Hz, H-1II),
4.38 (dd, 1 H, J1,2 8.3, J2,3 10.8 Hz, H-2III), 4.66 (d, 0.5
H, J1b,2b 7.9 Hz, H-1Ib), 4.76 (dd, 0.5 H, J2b,3b 9.6 Hz,
H-2Ib), 4.77 (dd, 0.5 H, J1a,2a 3.5, J2a,3a 10.1 Hz, H-2Ia),
4.84 (dd, 1 H, J2,3 9.8 Hz, H-2II), 5.08 (t, 0.5 H, H-3Ib),
5.31 (d, 0.5 H, H-1Ia), 5.38 (t, 0.5 H, H-3Ia), 5.44 (t, 1 H,
H-4III), 6.20 (m, 1 H, H-3III), 7.02 and 7.12 (2 d, each 2
phy (24:1 tolueneÁ
isolated as a white solid (1.01 g, 74%); Rf 0.57 (9:1
tolueneÁ
EtOAc); [a]2D0 118 (c 1, CHCl3); 1H NMR
/EtOAc) of the residue gave 25,
/
ꢀ
/
(500 MHz, CDCl3; 2D TOCSY, ROESY, HSQC): d
1.03 [s, 9 H, (CH3)3CSi], 2.06 (s, 3 H, COCH3), 2.21 and
2.31 (2 s, each 3 H, 2 CH3C6H4CO), 3.08 (m, 1 H, H-5I),
3.33 (dd, 1 H, J5,6b 6.4, J6a,6b 9.9 Hz, H-6bII), 3.37 (dd, 1
H, J1,2 8.0, J2,3 9.5 Hz, H-2II), 3.46 (dd, 1 H, J5,6a
B1,
/
H, Phth); HRMS of C70H77NO25Si (M, 1359.455): [Mꢀ
/
H]ꢀ found 1360.424, calcd 1360.463.
J6a,6b 11.0 Hz, H-6aI), 3.53 (t, 1 H, H-5II), 3.58 (dd, 1 H,
J5,6b 4.0 Hz, H-6bI), 4.13 and 4.36 (2 d, each 1 H,
OCH2C6H5), 4.21 and 4.43 (2 d, each 1 H, OCH2C6H5),
4.31 and 4.50 (2 d, each 1 H, OCH2C6H5), 4.51 (dd, 1 H,
J1,2 8.1, J2,3 10.7 Hz, H-2III), 4.70 and 4.87 (2 d, each 1
H, OCH2C6H5), 4.59 and 4.88 (2 d, each 1 H,
OCH2C6H5), 4.65 and 4.93 (2 d, each 1 H, OCH2C6H5),
5.74 (d, 1 H, H-1III), 6.29 (dd, 1 H, J3,4 9.2 Hz, H-3III);
13C NMR (75.5 MHz, CDCl3): d 18.9 [(CH3)3CSi], 20.5
(COCH3), 21.3 and 21.4 (2 CH3C6H4CO), 26.5
[(CH3)3CSi], 55.4 (C-2III), 62.8 (C-6III), 67.6 (C-6I),
68.1 (C-6II), 70.6, 72.9, 73.2, 74.3, 74.8, and 74.9 (6
OCH2C6H5), 69.7 (C-4III), 70.2 (C-4II), 70.7 (C-3III),
72.7 (C-5II), 74.7 (C-5I), 74.8 and 75.4 (C-4I, C-5III), 77.2
(C-3II), 79.2 (C-2II), 81.5 and 82.5 (C-2I, C-3I), 98.4 (C-
1III), 101.8 and 102.2 (C-1I, C-1II), 164.8 and 165.4 (2
3.20. (6-O-Tert-butyldiphenylsilyl-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b-
3)-(2,4,6-tri-O-acetyl-b- -galactopyranosyl)-(10
2,3,6-tri-O-acetyl-a,b- -glucopyranosyl
trichloroacetimidate (28)
D
-glucopyranosyl)-(10
/
D
/
4)-
D
To a soln of 27 (75 mg, 55 mmol) in dry CH2Cl2 (0.5 mL)
were added, at 0 8C under Ar, trichloroacetonitrile (29
mL, 0.29 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(0.90 mL). After stirring at rt for 1.5 h, followed by
concentration, low-pressure column chromatography
(1:1 tolueneÁ
28, isolated as a slightly yellow foam (58 mg, 69%); Rf
0.63 (1:1 tolueneÁ
EtOAc); 1H NMR a-product (300
/
EtOAc with 1% Et3N) of the residue gave
CH3C6H4CO),
102H103NO20Si (M, 1689.684): [Mꢀ
1707.713, calcd 1707.718.
169.3
(COCH3);
HRMS
of
C
/
NH4]ꢀ found
/
MHz, CDCl3): d 1.06 [s, 9 H, (CH3)3CSi], 1.88, 1.97,
1.98, 2.05, 2.10, and 2.15 (6 s, each 3 H, 6 COCH3), 2.24
and 2.34 (2 s, each 3 H, 2 CH3C6H4CO), 3.50 (m, 1 H,
H-5II), 3.75 (t, 1 H, H-4I), 4.27 (d, 1 H, J1,2 8.0 Hz, H-
1II), 4.38 (dd, 1 H, J1,2 8.1, J2,3 10.8 Hz, H-2III), 4.44 (dd,
1 H, J5,6a 1.5, J6a,6b 11.7 Hz, H-6aI), 4.85 (dd, 1 H, J2,3
9.8 Hz, H-2II), 5.02 (dd, 1 H, J1,2 3.8, J2,3 10.2 Hz, H-2I),
6.24 (dd, 1 H, J3,4 9.2 Hz, H-3III), 6.45 (d, 1 H, H-1I),
7.01 and 7.11 (2 d, each 2 H, Phth), 8.63 (s, 1 H,
OC[NH]CCl3).
3.19. (6-O-Tert-butyldiphenylsilyl-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b-
-glucopyranosyl)-(10
3)-(2,4,6-tri-O-acetyl-b- -galactopyranosyl)-(104)-
2,3,6-tri-O-acetyl-a,b- -glucopyranose (27)
D
/
D
/
D
To a soln of 25 (1.46 g, 0.87 mmol) in EtOH (45 mL)
and EtOAc (45 mL) was added 10% PdÁC (1.78 g), and
the mixture was stirred for 8 h, while H2 was bubbled
/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2625
3.21. 6-Azidohexyl (6-O-tert-butyldiphenylsilyl-2-deoxy-
3,4-di-O-p-methylbenzoyl-2-phthalimido-b-
glucopyranosyl)-(103)-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b- -glucopyranoside (29)
trated. Column chromatography (1:1 tolueneÁ
/
EtOAc)
D
-
of the residue gave 30, isolated as a white solid (48 mg,
90%); Rf 0.42 (1:1 tolueneÁ 28 (c 1,
EtOAc); [a]2D0
CHCl3); 1H NMR (500 MHz, CDCl3; 2D TOCSY,
ROESY): d 1.10Á1.16 (m, 4 H, 2 CH2), 1.22Á1.29 (m, 2
H, CH2), 1.42Á1.46 (m, 2 H, CH2), 1.96, 1.97, 2.01, 2.04,
/
D
-
/
ꢀ
/
/
D-
/
/
/
D
/
2.07, and 2.21 (6 s, each 3 H, 6 COCH3), 2.27 and 2.33 (2
s, each 6 H, 4 CH3C6H4CO), 3.03 (t, 2 H, CH2N3), 3.96
(dd, 1 H, J5,6b 5.7, J6a,6b 11.9 Hz, H-6bII), 4.27 (dd, 1 H,
J5,6a 1.9 Hz, H-6aII), 4.28 (d, 1 H, J1,2 8.1 Hz, H-1III),
4.39 (dd, 1 H, J1,2 8.3, J2,3 10.9 Hz, H-2IV), 4.44 (dd, 1
H, J1,2 8.5, J2,3 10.8 Hz, H-2I), 4.57 (d, 1 H, J1,2 7.9 Hz,
H-1II), 4.86 (dd, 1 H, J2,3 9.4 Hz, H-2II), 4.90 (dd, 1 H,
J2,3 9.9 Hz, H-2III), 5.04 (t, 1 H, H-3II), 5.31 (dd, 1 H,
J3,4 9.0, J4,5 10.1 Hz, H-4I), 5.43 (d, 1 H, H-1I), 5.46 (t, 1
A soln of 28 (0.36 g, 0.24 mmol) and 11 (0.18 g, 0.29
mmol) in dry CH2Cl2 (5 mL), containing molecular
˚
sieves 4 A (0.1 g), was stirred for 30 min under Ar. The
mixture was cooled to ꢁ40 8C, and TMSOTf (4 mL, 24
/
mmol) was added. The mixture was stirred for 2 h,
during which period the temperature was allowed to
reach rt, then neutralized with Et3N, washed with aq
satd NaHCO3 and water, dried, filtered, and concen-
trated. Column chromatography (3:1 tolueneÁ
/EtOAc)
H, H-4IV), 5.56 (d, 1 H, J3,4 3.9, J4,5
(d, 1 H, H-1IV), 6.12 (dd, 1 H, J3,4 9.2 Hz, H-3IV), 6.16
(dd, 1 H, H-3I); 13C NMR (75.5 MHz, CDCl3): d 19.5Á
B
/
1 Hz, H-4III), 5.60
of the residue gave crude 29 after concentration. Gel-
filtration of the residue on a LH-20 column eluted with
/
CH2Cl2Á
(0.18 g, 38%); Rf 0.88 (1:1 tolueneÁ
/
MeOH (1:1) gave 29, isolated as a white solid
EtOAc); [a]2D0
48 (c
1, CHCl3); H NMR (500 MHz, CDCl3; 2D TOCSY,
ROESY): d 1.05 [s, 9 H, (CH3)3CSi], 1.11Á1.16 (m, 4 H,
2 CH2), 1.25Á1.29 (m, 2 H, CH2), 1.42Á1.50 (m, 2 H,
20.8 (COCH3), 21.4 (CH3C6H4CO), 25.3, 26.1, 28.4,
and 28.9 (4 CH2), 51.0 (CH2N3), 54.6 and 54.8 (C-2I, C-
2IV), 61.2, 62.0, 68.2, and 69.6 (2 C) (C-6I, C-6II, C-6III,
C-6IV, OCH2), 68.9, 69.0, 69.9, 70.2, 70.3, 70.8 (2 C),
71.4, 72.5 (2 C), 74.1, 74.9, 75.6, and 78.2 (C-3I, C-4I, C-
5I, C-2II, C-3II, C-4II, C-5II, C-2III, C-3III, C-4III, C-5III,
C-3IV, C-4IV, C-5IV), 98.0, 98.8, 100.4, and 100.5 (C-1I,
C-1II, C-1III, C-1IV), 165.1 and 165.4 (CH3C6H4CO),
/
ꢀ
/
1
/
/
/
CH2), 1.88, 1.94, 2.02, 2.03, 2.04, and 2.12 (6 s, each 3 H,
6 COCH3), 2.25, 2.26, and 2.33 (3 s, 3,3,6 H, 4
CH3C6H4CO), 3.02 (t, 2 H, CH2N3), 3.62 (t, 1 H, H-
4II), 3.76 (dd, 1 H, J5,6b 7.7, J6a,6b 11.6 Hz, H-6bIV), 3.83
(dd, 1 H, J5,6a 1.5, J6a,6b 11.2 Hz, H-6aI), 4.18 (d, 1 H,
J1,2 8.1 Hz, H-1III), 4.46 (dd, 1 H, J1,2 8.5, J2,3 10.8 Hz,
H-2IV), 4.57 (d, 1 H, J1,2 7.9 Hz, H-1II), 4.81 (dd, 1 H,
J2,3 9.8 Hz, H-2III), 4.87 (dd, 1 H, J2,3 9.0 Hz, H-2II),
5.01 (t, 1 H, H-3II), 5.33 (t, 1 H, H-4IV), 5.47 (d, 1 H, J1,2
8.1 Hz, H-1I); 13C NMR (75.5 MHz, CDCl3): d 19.1
168.1Á
1773.591): [Mꢀ
/
170.9 (COCH3); HRMS of C90H95N5O33 (M,
H]ꢀ found 1774.675, calcd 1774.599.
/
3.23. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-(2-deoxy-3,4-di-O-p-
methylbenzoyl-2-phthalimido-b-
-glucopyranosyl)-(10
3)-(2,4,6-tri-O-acetyl-b- -galactopyranosyl)-(104)-
(2,3,6-tri-O-acetyl-b- 6)-2-deoxy-
D-
/
D-
/
D
/
[(CH3)3CSi], 20.4Á
/
20.6 (COCH3), 21.4 and 21.5
D
/
(CH3C6H4CO), 25.3, 26.1, 28.4, and 29.0 (4 CH2),
26.6 [(CH3)3CSi], 51.0 (CH2N3), 54.8 and 55.0 (C-2I,
C-2IV), 61.3, 62.1, 63.0, 68.3, and 69.8 (C-6I, C-6II, C-
6III, C-6IV, OCH2), 68.8, 69.8, 69.9, 70.3, 70.8, 70.9,
71.4, 72.4, 72.7, 74.1, 74.4 (2 C), 75.3, and 75.6 (C-3I, C-
D
-glucopyranosyl)-(10
/
3,4-di-O-p-methylbenzoyl-2-phthalimido-b-D-
glucopyranoside (31)
(a) A soln of 19 (129 mg, 103 mmol) and (2,3,4,6-tetra-
O-acetyl-b- -galactopyranosyl)-(104)-(2,3,6-tri-O-ace-
tyl-b- -glucopyranosyl)-(106)-2-deoxy-3,4-di-O-p-
-glucopyranosyl tri-
4I, C-5I, C-2II, C-3II, C-4II, C-5II, C-2III, C-3III, C-4III
,
D
/
C-5III, C-3IV, C-4IV, C-5IV), 97.9, 98.0, 100.5, and 100.6
(C-1I, C-1II, C-1III, C-1IV), 164.9, 165.2, 165.4, and 165.5
(4 CH3C6H4CO), 168.7, 169.3, 169.4, 169.7, 170.1, and
170.2 (6 COCH3); HRMS of C106H113N5O33Si (M,
D
/
methylbenzoyl-2-phthalimido-b-
D
chloroacetimidate (20)17 (202 mg, 155 mmol) in dry
CH2Cl2 (2 mL), containing powdered molecular sieves 4
˚
A (0.2 g), was stirred for 30 min under Ar, then cooled
2011.708): [Mꢀ
/
H]ꢀ found 2012.795, calcd 2012.717.
to ꢁ20 8C, and TMSOTf (3 mL, 16 mmol) was added.
/
3.22. 6-Azidohexyl (2-deoxy-3,4-di-O-p-methylbenzoyl-
2-phthalimido-b- 3)-(2,4,6-tri-O-
-glucopyranosyl)-(10
acetyl-b- 4)-(2,3,6-tri-O-acetyl-
-galactopyranosyl)-(10
b- 6)-2-deoxy-3,4-di-O-p-
-glucopyranosyl)-(10
methylbenzoyl-2-phthalimido-b- -glucopyranoside (30)
The mixture was stirred for 1 h, during which period the
temperature was allowed to reach rt, then neutralized
with Et3N, filtered over hyflo, and concentrated. Col-
D
/
D
/
D
/
umn chromatography (1:2 tolueneÁEtOAc) of the
/
D
residue yielded after concentration crude 31. Gel-filtra-
tion of the residue on a LH-20 column, eluted with
A soln of 29 (60 mg, 29.8 mmol) in 1.0 M TBAF in THF
(0.7 mL) and AcOH (0.7 mL) was stirred for 1 h at pH 6,
at 0 8C, then for 3 h at rt. The mixture was washed with
water and aq 10% NaCl, dried, filtered, and concen-
CH2Cl2ÁMeOH (1:1), gave 31, isolated as white foam
/
(30 mg, 12%).
(b) A soln of 30 (84 mg, 47 mmol) and (12)19 (125 mg,
160 mmol) in dry CH2Cl2 (1 mL), containing powdered

2626
J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á2627
/
˚
molecular sieves 4 A (0.1 g), was stirred for 0.5 h under
Ar, then cooled to ꢁ40 8C, and TMSOTf (3 mL, 16
mmol) was added. The mixture was stirred for 30 min at
40 8C, then at rt for 3 h, and concentrated. Column
chromatography (1:1 tolueneÁEtOAc) of the residue
yielded crude 31. Gel-filtration of the residue on a LH-
20 column, eluted with 1:1 CH2Cl2ÁMeOH, gave 31,
isolated as a white foam (68 mg, 60%); Rf 0.74 (1:2
tolueneÁ
EtOAc); [a]2D0 48 (c 1, CHCl3); 1H NMR (500
MHz, CDCl3; 2D TOCSY, ROESY, HSQC): d 1.08Á
1.17 (m, 4 H, 2 CH2), 1.23Á1.30 (m, 2 H, CH2), 1.41Á
lated as a colourless glass (17 mg, 71%); Rf 0.30 (9:1
CH2Cl2Á
MeOH); [a]2D0 28 (c 1, CHCl3); 1H NMR
(500 MHz, CDCl3; 2D TOCSY, ROESY): d 1.37Á1.38
(m, 4 H, 2 CH2), 1.58Á1.61 (m, 4 H, 2 CH2), 1.90, 1.93,
1.96, 1.98, 1.99, 2.01, 2.02, 2.03, 2.06, 2.07, 2.08, 2.09,
2.10, 2.12, 2.13, 2.14 and 2.16 (17 s,
/
/
ꢁ
/
/
ꢁ
/
/
/
s
/
3,3,3,3,3,6,3,3,3,6,3,3,3,3,3,3,3 H, 2 NHCOCH3, 17
COCH3), 3.26 (t, 2 H, CH2N3), 3.31 (dd, 1 H, J1,2 8.2,
J2,3 10.6 Hz, H-2IV), 3.44 (m, 1 H, OCHH), 3.97 (dd, 1
H, J2,3 9.8, J3,4 3.8 Hz, H-3III), 4.53 (d, 1 H, J1,2 7.9 Hz,
H-1IV), 4.56 (d, 1 H, J1,2 8.0 Hz, H-1II), 4.57 (d, 1 H, J1,2
7.6 Hz, H-1V), 4.60 (d, 1 H, J1,2 8.3 Hz, H-1I), 4.97 (dd, 1
H, J2,3 10.4, J3,4 3.4 Hz, H-3IV), 5.02 (dd, 1 H, J1,2 8.0
Hz, H-2III), 5.08 (dd, 1 H, H-2IV), 5.12 (t, 1 H, J2,3 9.3,
J3,4 9.3 Hz, H-3II), 5.12 (dd, 1 H, J2,3 9.4, J3,4 9.5 Hz, H-
3V), 5.25 (dd, 1 H, J2,3 10.6, J3,4 9.3 Hz, H-3I), 5.40 (d, 1
H, J2,NH 7.8 Hz, NHCOCHI3V), 5.40 (dd, 1 H, J3,4 9.1
Hz, H-3IV), 5.45 (d, 1 H, J2,NH 8.7 Hz, NHCOCHI3);
/
ꢁ
/
/
/
/
1.49 (m, 2 H, CH2), 1.96, 2.01, 2.03, 2.05, 2.06, 2.07,
2.12, 2.15, and 2.16 (9 s, 9,3,6,3,3,6,3,3,3 H, 13 COCH3),
2.25, 2.27, 2.33, and 2.34 (4 s, each 3 H, 4 CH3C6H4CO),
3.02 (t, 2 H, CH2N3), 3.75 (dd, 1 H, J5,6a 7.9, J6a,6b 11.8
Hz, H-6aI), 3.80 (t, 1 H, H-4V), 4.45 (dd, 1 H, J1,2 8.5,
J2,3 10.7 Hz, H-2I), 4.52 (d, 1 H, J1,2 7.9 Hz, H-1VI), 4.57
(d, 1 H, J1,2 7.9 Hz, H-1II), 4.63 (d, 1 H, J1,2 7.7 Hz, H-
1V), 4.95 (dd, 1 H, J2,3 10.5, J3,4 3.3 Hz, H-3VI), 5.00 (t, 1
H, J2,3 9.6, J3,4 9.2 Hz, H-3II), 5.41 (d, 1 H, J3,4 3.7,
HRMS of C80H113N5O48 (M, 1911.655): [Mꢀ
/
H]ꢀ
found 1912.644, calcd 1912.663.
J4,5
8.1 Hz, H-1VI); 13C NMR (75.5 MHz, CDCl3): d 20.4Á
B
1 Hz, H-4III), 5.43 (d, 1 H, H-1I), 5.44 (d, 1 H, J1,2
/
/
3.25. 6-Azidohexyl b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranosyl-(103)-b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranoside (33)
D
-galactopyranosyl-(10
/
4)-b-
D
-
20.6 (COCH3), 21.4 and 21.5 (CH3C6H4CO), 25.3, 26.1,
28.4, and 28.9 (4 CH2), 51.0 (CH2N3), 54.8 (2 C) (C-2I,
C-2IV), 60.4 (C-6III), 61.6 (C-6VI), 62.2 (C-6V), 62.4 (C-
6II), 68.3 (C-6I), 68.7 (C-6IV), 69.6 (OCH2), 66.4 (C-4VI),
68.9 (2 C) (C-4III, C-2VI), 69.8 (2 C) (C-4I, C-4IV), 69.9
(C-3IV), 70.5 (C-5VI), 70.7 (C-3I), 70.8 (2 C) (C-3III, C-
3VI), 71.0 (C-2III), 71.3 (C-2II), 71.8 (C-2V), 72.3 (C-3II),
72.7 (C-5V), 72.8 (C-5II), 72.9 (C-3V), 74.1 (2 C) and 74.9
(C-5I, C-5III, C-5IV), 75.7 (C-4II), 76.1 (C-4V), 97.6 and
98.0 (C-1I, C-1IV), 100.3 (C-1III), 100.5 (C-1II), 100.8 (C-
1V), 100.9 (C-1VI), 165.1, 165.2, 165.3, and 165.4 (4
/
D-
/
D-galactopyranosyl-(10
/4)-b-D-
/
D-
To a soln of 32 (17 mg, 8.88 mmol) in MeOH (0.7 mL)
was added NaOMe (pH 10). The mixture was stirred for
4 h, then neutralized with Dowex 50ꢂ
8 (Hꢀ), filtered,
/
and concentrated. Gel-filtration of the residue on a Bio
Gel P-2 column, eluted with aq 0.1 M NH4HCO3 at a
flow rate of 40 mL/h, gave 33 after freeze-drying as a
CH3C6H4CO), 168.6Á
116H129N5O50 (M, 2391.770): [Mꢀ
2392.742, calcd 2392.778.
/
170.9 (COCH3); HRMS of
white powder (9.4 mg, 89%); Rf 0.19 (1:3 CH2Cl2Á
MeOH); [a]2D0
18 (c 0.5, water); HRMS of
C46H79N5O31 (M, 1197.475): [Mꢀ
H]ꢀ found
1198.508, calcd 1198.483. For H NMR data, see Table
4.
/
C
/
H]ꢀ found
ꢁ
/
/
1
3.24. 6-Azidohexyl (2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-(2-acetamido-3,4-di-O-acetyl-2-
deoxy-b- 3)-(2,4,6-tri-O-acetyl-b-
-glucopyranosyl)-(10
-galactopyranosyl)-(104)-(2,3,6-tri-O-acetyl-b-
glucopyranosyl)-(106)-2-acetamido-3,4-di-O-acetyl-2-
deoxy-b- -glucopyranoside (32)
D-
/
D-
/
3.26. 6-Aminohexyl b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranosyl-(103)-b-
glucopyranosyl-(106)-2-acetamido-2-deoxy-b-
glucopyranoside (4)
D
-galactopyranosyl-(10
/
4)-b-D-
-
D
/
/
D
D
/
D-
/
D-galactopyranosyl-(10
/4)-b-D-
/
/
D
-
D
To a soln of 31 (30 mg, 12.5 mmol) in MeOH (2 mL) and
CH2Cl2 (0.3 mL) was added NaOMe (pH 10), and the
mixture was stirred for 24 h. After neutralization with
To a soln of 33 (8 mg, 6.67 mmol) in t-BuOH (3 mL) and
water (2 mL) were added 10% PdÁC (30 mg) and 2
/
drops of aq 25% NH3, and the mixture was stirred for 24
h under H2, then filtered over cotton, and concentrated.
Gel-filtration of the residue on a Bio Gel P-2 column,
eluted with aq 0.1 M NH4HCO3 at a flow rate of 40 mL/
h, gave 4 after freeze-drying as a white powder (5.3 mg,
Dowex 50ꢂ
8 (Hꢀ) and filtration, the mixture was
/
concentrated. To a solution of the residue in 1-BuOH (5
mL) was added 1,2-diaminoethane (1 mL), and the
mixture was stirred overnight at 80 8C, then, co-con-
centrated with toluene. A solution of the residue in Py
(10 mL) and Ac2O (10 mL) was stirred overnight, then
co-concentrated with toluene. Column chromatography
67%); Rf 0.16 (2:1:1 AcOHÁ
(c 0.3, H2O); HRMS of C46H81N3O31 (M, 1171.485):
[Mꢀ
H]ꢀ found 1172.486, calcd 1172.492. For 1H
NMR data, see Table 5.
/
1-BuOHÁ
/
water); [a]2D0
ꢁ18
/
/
(9:1 CH2Cl2ÁMeOH) of the residue afforded 32, iso-
/

J.A.F. Joosten et al. / Carbohydrate Research 338 (2003) 2611Á
/2627
2627
6. Klugman, K. P. Clin. Microbiol. Rev. 1990, 3, 171Á
196.
7. Robbins, J. B.; Austrian, R.; Lee, C. J.; Rastogi, S. C.;
/
3.27. 6-Aminohexyl b-
glucopyranosyl-(106)-[b-
acetamido-2-deoxy-b-
-glucopyranosyl-(10
galactopyranosyl-(104)-b- -glucopyranosyl-(10
-galactopyranosyl-(104)]-2-acetamido-2-deoxy-b-
glucopyranoside (2)
D
-galactopyranosyl-(10
-galactopyranosyl-(10
3)-b-
/
4)-b-D-
/
D
/4)]-2-
D
/
D-
Schiffman, G.; Henrichsen, J.; Makala, P. H.; Broome, C.
¨
V.; Facklam, R. R.; Tiesjema, R. H.; Parke, J. C. J., Jr. J.
¨
/
D
/
6)-[b-
D
/
D-
Infect. Dis. 1983, 148, 1136Á1159.
/
8. Butler, J.; Breiman, R.; Campbell, J.; Lipman, H.;
Broome, C.; Facklam, R. J. Am. Med. Assoc. 1993, 270,
To a soln of 4 (5.3 mg, 4.52 mmol) in aq 50 mM sodium
cacodylate buffer pH 7.5 (500 mL), containing 5 mM
MnCl2, BSA (0.5 mg), and NaN3 (0.02%), were added
alkaline phosphatase (10 U), UDP-galactose (8.2 mg,
13.4 mmol), and b-1,4-galactosyltransferase (1 U). The
reaction mixture was incubated for 20 h at 37 8C, then,
water (100 mL) was added. UDP-Galactose was re-
1826Á1831.
/
˚ ˚
¨
9. Ortqist, A.; Hedlund, J.; Burman, L.-A.; Elbel, E.; Hofer,
¨
.
M.; Leinonen, M.; Lindblad, I.; Sundelof, B.; Kalin, M.
¨
Lancet 1998, 351, 399Á
10. Beuvery, E. C.; Van Rossem, F.; Nagel, J. Infect. Immun.
1982, 37, 15Á22.
/
403.
/
11. Rennels, M. B.; Edwards, K. M.; Keyserling, H. L.;
Reisinger, K. S.; Hogerman, D. A.; Madore, D. V.;
Chang, I.; Paradiso, P. R.; Malinoski, F. J.; Kimura, A.
moved on a Dowex 1ꢂ
8 (Clꢁ) column, eluted with
/
water. The eluate was concentrated, and the residue
applied on a Bio Gel P-2 column eluted with aq 0.1 M
NH4HCO3 at a flow rate of 40 mL/h. The appropriate
fractions were freeze-dried to give 2 (1.5 mg, 22%); Rf
Pediatrics 1998, 101, 604Á611.
/
12. Jansen, W. T. M.; Hogenboom, S.; Thijssen, M. J. L.;
Kamerling, J. P.; Vliegenthart, J. F. G.; Verhoef, J.;
Snippe, H.; Verheul, A. F. M. Infect. Immun. 2001, 69,
0.07 (2:1:1 AcOHÁ
C58H101N3O41 (M, 1495.591): [Mꢀ
1496.614, calcd 1496.598. For H NMR data, see Table
6.
/
1-BuOHÁ
/
water); HRMS of
H]ꢀ found
787Á793.
/
/
13. Benaissa-Trouw, B.; Lefeber, D. J.; Kamerling, J. P.;
1
Vliegenthart, J. F. G.; Kraaijeveld, K.; Snippe, H. Infect.
Immun. 2001, 69, 4698Á
14. Niggemann, J.; Kamerling, J. P.; Vliegenthart, J. F. G.
Bioorg. Med. Chem. 1998, 6, 1605Á1612.
15. Niggemann, J.; Kamerling, J. P.; Vliegenthart, J. F. G. J.
Chem. Soc. Perkin Trans. 1 1998, 3011Á3020.
/
4701.
/
Acknowledgements
/
We thank H.I.V. Amatdjais-Groenen (Nijmegen Uni-
versity) for measuring the elemental analysis data and C.
Versluis (Utrecht University) for recording the HRMS
spectra. This work was financially supported by the EU,
project BIO CT 95-0138.
16. Mawas, F.; Niggemann, J.; Jones, C.; Corbel, M. J.;
Kamerling, J. P.; Vliegenthart, J. F. G. Infect. Immun.
2002, 70, 5107Á
17. Michalik, D.; Vliegenthart, J. F. G.; Kamerling, J. P. J.
Chem. Soc. Perkin Trans. 1 2002, 1973Á1981.
18. Lindberg, B.; Lonngren, J.; Powell, D. A. Carbohydr. Res.
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¨
1977, 58, 177Á186.
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19. Koeman, F. A. W.; Meissner, J. W. G.; van Ritter, H. R.
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