Chemoenzymic synthesis of oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

Article abstract of DOI:10.1039/b204933c

The chemoenzymic synthesis is described of β-D-Galp-(1→4)-β-D-Glcp-(1→6)-[(β-D-Galp)- (1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→O(CH ₂)₆NH₂) 20 and β-D-Galp-(1→4)-β-D-Glcp-(1→6)-[(β-D-Galp)- (1→4)]-β-D-GlcpNAc-(1→3)-β-D-Galp-(1

Full text of DOI:10.1039/b204933c

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Chemoenzymic synthesis of oligosaccharide fragments of the
capsular polysaccharide of Streptococcus pneumoniae type 14
Dirk Michalik,† Johannes F. G. Vliegenthart and Johannis P. Kamerling*
1
Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and
Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands
Received (in Cambridge, UK) 22nd May 2002, Accepted 16th July 2002
First published as an Advance Article on the web 13th August 2002
The chemoenzymic synthesis is described of β--Galp-(1 4)-β--Glcp-(1 6)-[(β--Galp)-(1 4)]-β--GlcpNAc-
(1 3)-β--Galp-(1 O(CH2)6NH2) 20 and β--Galp-(1 4)-β--Glcp-(1 6)-[(β--Galp)-(1 4)]-β--GlcpNAc-
(1 3)-β--Galp-(1 4)-β--Glcp-(1 O(CH2)6NH2) 21, representing penta- and hexasaccharide fragments of the
Streptococcus pneumoniae type 14 capsular polysaccharide. In a chemical approach the intermediate tetra- and
pentasaccharide fragments 18 and 19, respectively, were synthesised, wherein the non-terminal N-acetyl-β--
glucosamine residues were not yet galactosylated. Both intermediates were found to be good acceptor substrates for
bovine milk β-1,4-galactosyltransferase. The title oligosaccharides form suitable compounds for conjugation with
carrier proteins, to be tested as potential vaccines in animal models.
Based on these findings, it was decided to synthesise longer
intact oligosaccharide fragments of the CPS of S. pneumoniae
Introduction
Streptococcus pneumoniae is a pathogenic bacterium causing
type 14.
infections of the lung (pneumonia), middle ear (otitis media)
Here, we report on the chemoenzymic synthesis of spacered
and meninges (meningitis). Pneumococcal infections are still
oligosaccharides, representing penta- and hexasaccharide
significant causes of morbidity and mortality all over the world.
fragments of the CPS of S. pneumoniae type 14 (Fig.1).
The initial success of antibiotic treatment is now negatively
influenced by a growing resistance against antibiotics.1 Immuno-
competentpeoplecanbeprotectedefficientlybyvaccinationwith
Fig. 1 Repeating unit of the capsular polysaccharide of Streptococcus
pneumoniae type 14.
the available 23-valent capsular polysaccharide (CPS) vaccines.2
The highest incidence of pneumococcal infections is, however,
in young children, the elderly and immunocompromised
Results and discussion
For the preparation of the 6-aminohexyl glycosides of the tetra-
patients. People in these groups do not respond adequately to
the T-cell independent polysaccharides as antigens.3 Conjuga-
tion of carbohydrate antigens to protein carriers represents an
approach to overcome these problems by converting T-cell
independent antigens into more immunogenic T-cell dependent
antigens.4
(18) and penta- (19) saccharide, the acceptors for the enzymic
galactosylation to obtain the corresponding spacered title
penta- (20) and hexa- (21) saccharides, three building blocks
were designed: the trisaccharide donor 13, the monosaccharide
acceptor 6, and the disaccharide acceptor 11. In the case of the
acceptors, benzyl groups were introduced to increase the solu-
bility of the generated protected tetra- and pentasaccharides in
the generally applied organic solvents, which is of importance
in the deprotection protocols.
Currently, neoglycoconjugate vaccines have been introduced,
amongst others against S. pneumoniae.5 These neoglycoproteins
are prepared by conjugation of isolated capsular polysacchar-
ides or a mixture of polysaccharide-derived oligosaccharides to
a protein carrier.6 Recent studies with S. pneumoniae type 6B
neoglycoproteins showed that a synthetic tetrasaccharide
fragment, i.e. one repeating unit of the 6B CPS, coupled to
keyhole limpet hemocyanin (KLH) was sufficient to generate a
protective antibody response in mice.7 Similar results were
shown for S. pneumoniae type 3 neoglycoproteins, consisting of
di-, tri- and tetrasaccharides coupled to the cross-reacting
material (CRM197) of modified diphtheria toxin in different
molar carbohydrate–protein ratios.8
In earlier reports we have described the chemoenzymic
synthesis of a spacered branched tetrasaccharide fragment
of the CPS of S. pneumoniae type 14, corresponding with
one repeating unit,9 as well as of alkyl-bridged hexa- and
octasaccharide mimics of fragments of the CPS.10 Especially,
the tetrasaccharide-containing CRM197 conjugate showed
promising immunological data when tested in mice models.11
The synthesis of monosaccharide acceptor 6 involved as the
first step the coupling of 2,3,4,6-tetra-O-acetyl-α--galacto-
pyranosyl trichloroacetimidate (1)12 to the spacer 6-azido-
hexan-1-ol using boron trifluoride–diethyl ether as a catalyst
(Scheme 1). After deacetylation ( 2, 37%), the formed product
was isopropylidenated at O-3 and O-4 with 2,2-dimethoxy-
propane in the presence of a catalytic amount of camphor-
sulfonic acid to give 3 (42%).13,14 Subsequent benzylation of the
remaining two hydroxy groups in 3 with benzyl bromide in
N,N-dimethylformamide using sodium hydride as base gave the
fully protected derivative 4 (95%). Deisopropylidenation of 4
with aqueous trifluoroacetic acid in dichloromethane yielded 5
(99%), which was selectively acetylated at O-4 via orthoester
formation using trimethyl orthoacetate and toluene-p-sulfonic
acid, and subsequent ring opening with aqueous acetic acid
(
6, 98%).15
For the synthesis of disaccharide acceptor 11, (2,6-di-O-
acetyl-3,4-O-isopropylidene-β--galactopyranosyl)-(1 4)-
2,3,6-tri-O-acetyl-α--glucopyranosyl trichloroacetimidate
(7)16 was coupled to the spacer 6-azidohexan-1-ol (Scheme 2)
† Present address: Institut für Organische Katalyseforschung an der
Universität Rostock e.V., Friedrich-Barnewitz-Strasse 4, D-18119
Warnemünde, Germany
DOI: 10.1039/b204933c
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981
This journal is © The Royal Society of Chemistry 2002
1973

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Scheme 1
Scheme 2
using boron trifluoride–diethyl ether as a catalyst ( 8, 36%).
Deacetylation of 8 and subsequent benzylation with benzyl
bromide in N,N-dimethylformamide using sodium hydride as
base gave the fully protected derivative 9 (64%). Deisopropyl-
idenation of 9 with aqueous trifluoroacetic acid in dichloro-
methane ( 10, 90%), followed by selective acetylation at O-4
of the galactose residue as described above for 6, gave 11 (95%).
Both the synthesis of tetrasaccharide 14 (leading to 18) and
pentasaccharide 15 (leading to 19) required the trisaccharide
donor 13. The latter compound was obtained from allyl
(2,3,4,6-tetra-O-acetyl-β--galactopyranosyl)-(1 4)-2,3,6-tri-
O-acetyl-β--glucopyranosyl)-(1 6)-2-deoxy-3,4-bis(O-p-
methylbenzoyl)-2-phthalimido-β--glucopyranoside (12)9 by
subsequent deallylation with PdCl2 and imidation with trichloro-
acetonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-
In the deprotection sequence the fully protected oligo-
saccharides 14 and 15 were deacylated and dephthaloylated
(33% ethanolic methylamine) followed by re-N,O-acetylation
to yield the N-acetyl protected derivatives 16 (65%) and 17
(57%), respectively. Complete conventional de-O-acetylation
of both products required long reaction times (>24 h), as
indicated by NMR spectroscopy. Finally, debenzylation (Pd/
C, H2, alkaline conditions, ammonia19) followed by hydrogen-
ation of the azido group (Pd/C, H2, weak acid conditions,
acetic acid), and subsequent purification on Toyopearl HW-
40S afforded 18 (96%) and 19 (82%), respectively. Verification
of the formed products was carried out by high-resolution
1
MS and NMR (2D H COSY, TOCSY and ROESY) (Tables
1 and 2).
The β-1,4-galactosyltransferase-catalysed syntheses of the
branched-chain oligosaccharides 20 and 21 was achieved by
galactosyl transfer from UDP-galactose to O-4 of the N-acetyl-
β--glucosamine residues of 18 and 19 (Scheme 4).20–23 Alkaline
phosphatase was added to the incubation mixtures to prevent
feedback inhibition by released UDP24,25 and to promote a high
ene as a catalyst17,18
(
13, 52%) (Scheme 3). Compound 14 was
prepared by coupling of 13 with 6 in dichloromethane using
trimethylsilyl trifluoromethanesulfonate as a catalyst (49%).
Under similar conditions 15 was obtained by coupling of 13
with 11 (38%).
1974
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981

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Scheme 3
conversion of the acceptors. Final products were isolated after
purification on Toyopearl HW-40S ( 20, 80%; 21, 96%).
Verification of the formed products was carried out by high-
resolution MS and NMR (2D 1H COSY, TOCSY and ROESY)
(Tables 3 and 4).
Experimental
General procedures
Reactions were monitored by TLC on Silica Gel 60 F254
(Merck) with detection either by UV light or charring with
either 10% H2SO4 in EtOH or 0.2% orcinol in 20% methanolic
H2SO4. Solutions were concentrated under reduced pressure at
Conjugation of the oligosaccharides to CRM197 (cross-
reactive material) and immunological studies are in progress.
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981
1975

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Scheme 4
Table 3 1H NMR data (COSY, TOCSY, ROESY) of compound 20
Table 1 1H NMR data (COSY, TOCSY, ROESY) of compound 18
δH
δH
Proton
Gal aa
GlcNAc b
Glc c
Gal db
Proton
Gal aa
GlcNAc b
Glc c
Gal db
Gal ec
H-1
H-2
H-3
H-4
H-5
H-6
H-6Ј
4.37
3.55
3.70
4.14
n.d.c
n.d.
n.d.
4.70
3.75
3.56
3.56
3.61
4.21
3.89
4.54
3.36
3.66
3.66
3.56
3.99
3.81
4.45
3.55
3.67
3.93
n.d.
n.d.
n.d.
H-1
H-2
H-3
H-4
H-5
H-6
H-6Ј
4.38
3.55
3.70
4.16
n.d.d
n.d.
n.d.
4.73
3.75
3.73
3.86
3.73
4.28
3.96
4.56
3.37
3.67
3.67
3.61
3.99
3.82
4.46
3.55
3.67
3.94
n.d.
n.d.
n.d.
4.54
3.54
3.67
3.93
n.d.
n.d.
n.d.
OCH2(CH2)5NH2
CH2NH2
OCH2CH2(CH2)2CH2CH2NH2
O(CH2)2(CH2)2(CH2)2NH2
NHCOCH3
3.92, 3.67
2.99
1.70–1.60 (4 H)
1.45–1.35 (4 H)
2.04
OCH2(CH2)5NH2
CH2NH2
OCH2CH2(CH2)2CH2CH2NH2
O(CH2)2(CH2)2(CH2)2NH2
NHCOCH3
3.92, 3.67
2.99
1.70–1.60 (4 H)
1.43–1.37 (4 H)
2.03
a Gal a: Gal(β1-O(CH2)6NH2). b Gal d: Gal(β1–4)Glc. c n.d., not
determined.
a Gal a: Gal(β1-O(CH2)6NH2). b Gal d: Gal(β1–4)Glc. c Gal e: Gal-
(β1–4)GlcNAc. d n.d., not determined.
Table 2 1H NMR data (COSY, TOCSY, ROESY) of compound 19
Table 4 1H NMR data (COSY, TOCSY, ROESY) of compound 21
δH
δH
Proton
Glc aa
Gal bb
GlcNAc c
Glc dc
Gal ed
Proton
Glc aa
Gal bb
GlcNAc c
Glc dc
Gal ed
Gal fe
H-1
H-2
H-3
H-4
H-5
H-6
H-6Ј
4.48
3.29
3.63
n.d.e
n.d.
3.97
3.79
4.43
3.60
3.76
4.16
n.d.
n.d.
n.d.
4.69
3.76
3.56
3.56
3.61
4.21
3.88
4.53
3.36
3.66
n.d.
n.d.
3.99
3.81
4.45
3.55
3.67
3.93
n.d.
n.d.
n.d.
H-1
H-2
H-3
H-4
H-5
H-6
H-6Ј
4.48
3.29
3.63
3.62
n.d.f
3.98
3.79
4.43
3.58
3.72
4.12
n.d.
n.d.
n.d.
4.70
3.72
3.81
3.88
n.d.
4.28
3.96
4.56
3.37
3.67
n.d.
3.61
3.99
3.82
4.45
3.54
3.67
3.92
n.d.
n.d.
n.d.
4.54
3.54
3.66
3.92
n.d.
n.d.
n.d.
OCH2(CH2)5NH2
CH2NH2
OCH2CH2(CH2)2CH2CH2NH2
O(CH2)2(CH2)2(CH2)2NH2
NHCOCH3
3.92, 3.68
2.99
1.71–1.60 (4 H)
1.46–1.36 (4 H)
2.04
OCH2(CH2)5NH2
CH2NH2
OCH2CH2(CH2)2CH2CH2NH2
O(CH2)2(CH2)2(CH2)2NH2
NHCOCH3
3.93, 3.68
2.99
1.71–1.60 (4 H)
1.45–1.37 (4 H)
2.03
a Glc a: Glc(β1-O(CH2)6NH2). b Gal b: Gal(β1–4)Glc(β1-O(CH2)6NH2).
c Glc d: Glc(β1–6)GlcNAc. d Gal e: Gal(β1–4)Glc(β1–6)GlcNAc. e n.d.,
not determined.
a Glc a: Glc(β1-O(CH2)6NH2). b Gal b: Gal(β1–4)Glc(β1-O-
(CH2)6NH2). c Glc d: Glc(β1–6)GlcNAc. d Gal e: Gal(β1–4)Glc(β1–6)-
GlcNAc. e Gal f: Gal(β1–4)GlcNAc. f n.d., not determined.
40 ЊC. Column chromatography was performed on Silica Gel 60
(0.063–0.200 mm, Merck). Gel-permeation chromatography
was performed on Toyopearl® HW-40S (Supelco) (2.0 × 100
cm). Optical rotations were measured with a Perkin-Elmer 241
polarimeter. [α]-Values are given in 10Ϫ1 deg cm2 gϪ1. 1H NMR
spectra (300 MHz) were recorded with a Bruker AC 300
1976
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981

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spectrometer; only selected NMR data are reported. 2D DQF
1H–1H COSY spectra, 2D TOCSY spectra with 7 ms and 100
ms mixing times, and 2D H ROESY spectra (300 ms mixing
6-Azidohexyl 2,6-di-O-benzyl-3,4-O-isopropylidene-ꢀ-D-galacto-
pyranoside 4
1
To a solution of 3 (195 mg, 0.56 mmol) in DMF (10 ml) was
added sodium hydride (80 mg, 80% suspension in oil). The
mixture was stirred for 10 min, benzyl bromide (0.2 ml, 1.65
mmol) was added, and the stirring was continued for 2.5 h at
room temperature. Then, the mixture was poured onto ice–
water (300 ml). After extraction with CH2Cl2, the combined
organic layers were washed with water, dried (Na2SO4), filtered,
and concentrated. Column chromatography (toluene–EtOAc,
10 : 1) gave 4, isolated as a colourless syrup (281 mg, 95%); TLC
(toluene–EtOAc, 2 : 1) Rf 0.79; [α]2D2 ϩ9 (c 1.1, CHCl3) (Found:
C, 66.52; H, 7.49; N, 7.72. C29H39N3O6 requires C, 66.27; H,
7.48; N, 7.99%); δH(300 MHz; CDCl3) 7.40–7.20 (10 H, m, 2
Ph), 4.82 and 4.79 (each 1 H, 2 d, PhCH2O), 4.63 and 4.56 (each
1 H, 2 d, PhCH2O), 4.29 (1 H, d, J1,2 8.1, H-1), 3.94 (1 H, dt,
OCHHCH2), 3.89 (1 H, dd, J3,4 6.7, J4,5 1.4, H-4), 3.51 (1 H,
dt, OCHHCH2), 3.38 (1 H, m, H-5), 3.23 (2 H, t, CH2N3),
1.68–1.53 (4 H, m, OCH2CH2(CH2)2CH2), 1.47–1.32 (4 H, m,
OCH2CH2(CH2)2CH2), 1.36 and 1.32 (each 3 H, 2 s, 2 Me);
δC(75.5 MHz; CDCl3) 138.3 and 138.1 (2 i-Ph), 128.3–127.3
(C-ar), 109.8 [C(CH3)2], 102.8 (C-1), 79.6, 79.0, 73.8 and 72.1
(C-2,-3,-4,-5), 73.5 (2 C) (2 PhCH2O), 69.5 (2 C) (C-6,
OCH2CH2), 51.3 (CH2N3), 29.5, 28.7, 26.4 and 25.6
[CH2(CH2)4CH2N3], 27.7 and 26.3 [C(CH3)2].
times) were recorded at 300 K using a Bruker AMX 500
spectrometer. δH(ppm)-Values are given relative to the signal for
internal Me4Si (δ 0, CDCl3) or acetone (δ 2.225, D2O). J-Values
are given in Hz. 13C NMR spectra (75.5 or 125.7 MHz) were
recorded with a Bruker AC 300 or AMX 500 spectrometer.
δC(ppm)-Values are given relative to the signal for CDCl3
(δC76.9) or internal acetone (δC 31.08). Elemental analyses
were carried out with a CHNS-932 (Fa. Leco). Exact masses of
final products were measured by nano ES-TOF MS using a
Micromass LCTOF mass spectrometer at a resolution of 5000
FWHM. Gold-coated capillaries were loaded with 1 µl of
sample (conc. 20 µM) dissolved in 1 : 1 acetonitrile–water with
0.1% formic acid. Pentaphenylalanine was added as internal
standard. The capillary voltage was set at 1500 V and the cone
voltage was set at 30 V.
6-Azidohexyl ꢀ-D-galactopyranoside 2
To a solution of 2,3,4,6-tetra-O-acetyl-α--galactopyranosyl
trichloroacetimidate 112 (1.9 g, 3.86 mmol) and 6-azidohexan-
1-ol (1.08 g, 7.56 mmol) in dry CH2Cl2 (40 ml) was added
powdered 4 Å molecular sieves (1 g), and the suspension was
stirred for 2 h at room temperature. Then, at 0 ЊC BF3ؒEt2O (0.3
ml) was added. After stirring for 2 h at room temperature,
the mixture was neutralised with Et3N, diluted with CH2Cl2,
filtered through Celite, and the filtrate was concentrated.
Column chromatography (CH2Cl2–MeOH, 50 : 1) of the
residue gave a mixture of the intermediate product (CH2Cl2–
MeOH, 20 : 1, Rf 0.78) and 6-azidohexan-1-ol (Rf 0.44), which
was dissolved in MeOH (60 ml). A solution of NaOMe (100
mg) in MeOH (10 ml) was added, and the mixture was stirred
for 2.5 h at room temperature, then neutralised with Dowex
50W-X8 (Hϩ-form), filtered, and concentrated. Column
chromatography (CH2Cl2–MeOH, 20 : 1) of the residue gave 2,
isolated as an amorphous solid (433 mg, 37%); TLC (CH2Cl2–
MeOH, 5 : 1) Rf 0.37; [α]2D4 Ϫ23 (c 0.3, CHCl3) (Found: C, 47.02;
H, 7.38; N, 13.40. C12H23N3O6 requires C, 47.21; H, 7.59; N,
13.76%); δH(300 MHz; CD3OD) 4.18 (1 H, d, J1,2 7.2, H-1), 3.87
(1 H, dt, OCHHCH2), 3.80 (1 H, dd, J3,4 3.0, J4,5 0.8, H-4), 3.52
(1 H, dt, OCHHCH2), 3.25 (2 H, t, CH2N3), 1.66–1.52 (4 H,
m, OCH2CH2(CH2)2CH2), 1.43–1.32 (4 H, m, OCH2CH2-
(CH2)2CH2); δC(75.5 MHz; CD3OD) 105.0 (C-1), 76.5, 75.0,
72.6 and 70.3 (C-2,-3,-4,-5), 70.5 (OCH2CH2), 62.4 (C-6), 52.4
(CH2N3), 30.6, 29.8, 27.6 and 26.6 [CH2(CH2)4CH2N3].
6-Azidohexyl 2,6-di-O-benzyl-ꢀ-D-galactopyranoside 5
To a cooled (0 ЊC) solution of 4 (281 mg, 0.53 mmol) in CH2Cl2
(15 ml) was added 90% aq. trifluoroacetic acid (1 ml). The
mixture was stirred for 1.5 h at 0 ЊC, then diluted with CH2Cl2,
and washed with cold water, saturated aq. Na2CO3, and water.
The organic layer was dried (Na2SO4), filtered, and concen-
trated. Column chromatography (toluene–EtOAc, 2 : 1) of the
residue gave 5, isolated as a colourless syrup (257 mg, 99%);
TLC (toluene–EtOAc, 2 : 1) Rf 0.29; [α]2D4 ϩ3 (c 0.5, CHCl3)
(Found: C, 64.53; H, 7.24; N, 8.44. C26H35N3O6 requires C,
64.31; H, 7.27; N, 8.65%); δH(300 MHz; CDCl3) 7.37–7.25
(10 H, m, 2 Ph), 4.96 and 4.68 (each 1 H, 2 d, PhCH2O), 4.59
(2 H, s, PhCH2O), 4.36 (1 H, d, J1,2 7.5, H-1), 3.99 (1 H, m, H-
4), 3.96 (1 H, dt, OCHHCH2), 3.23 (2 H, t, CH2N3), 2.58 and
2.52 (each 1 H, 2 d, 2 OH), 1.69–1.53 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.46–1.35 (4 H, m, OCH2CH2(CH2)2CH2);
δC(75.5 MHz; CDCl3) 138.4, 137.9, 128.5 (4 C), 128.0 (2 C),
127.9, 127.8 and 127.7 (2 C) (2 Ph), 103.7 (C-1), 79.1, 74.6, 73.7,
73.3, 73.2, 69.7, 69.4 and 69.0 (C-2,-3,-4,-5,-6, OCH2CH2, 2
PhCH2O), 51.4 (CH2N3), 29.6, 28.8, 26.5 and 25.8
[CH2(CH2)4CH2N3].
6-Azidohexyl 3,4-O-isopropylidene-ꢀ-D-galactopyranoside 3
To a solution of 2 (433 mg, 1.25 mmol) in 2,2-dimethoxy-
propane (15 ml) and acetonitrile (15 ml) was added camphor-
sulfonic acid (20 mg), and the mixture was stirred for 48 h
at room temperature. After neutralisation with Et3N and co-
concentration with toluene, a mixture of MeOH–H2O (10 : 1)
(50 ml) was added to the residue, and the solution was boiled
under reflux for 3 h, then concentrated. Column chroma-
tography (toluene–EtOAc, 1 : 1) of the residue gave 3, isolated
as a colourless syrup (206 mg, 42%); TLC (EtOAc) Rf 0.53; [α]2D2
ϩ9 (c 1.1, CHCl3) (Found: C, 52.36; H, 7.69; N, 11.98.
C15H27N3O6 requires C, 52.16; H, 7.88; N, 12.17%); δH(300
MHz; CDCl3) 4.18 (1 H, d, J1,2 8.2, H-1), 4.16 (1 H, dd, J3,4 5.5,
J4,5 2.0, H-4), 4.10 (1 H, dd, J2,3 7.3, H-3), 3.93 (1 H, dt, OCH-
HCH2), 3.56 (1 H, dd, H-2), 3.51 (1 H, dt, OCHHCH2), 3.27 (2
H, t, CH2N3), 2.48 and 2.18 (each 1 H, 2 br s, 2 OH), 1.68–1.55
(4 H, m, OCH2CH2(CH2)2CH2), 1.47–1.34 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.52 and 1.35 (each 3 H, 2 s, 2 Me); δC(75.5 MHz;
CDCl3) 110.3 [C(CH3)2], 102.2 (C-1), 78.8, 73.8, 73.6 and 73.4
(C-2,-3,-4,-5), 69.7 (OCH2CH2), 62.3 (C-6), 51.2 (CH2N3),
29.3, 28.6, 26.3 and 25.4 [CH2(CH2)4CH2N3], 28.0 and 26.2
[C(CH3)2].
6-Azidohexyl 4-O-acetyl-2,6-di-O-benzyl-ꢀ-D-galactopyranoside
6
To a solution of 5 (190 mg, 0.39 mmol) in acetonitrile (5 ml)
were added trimethyl orthoacetate (0.2 ml) and toluene-p-
sulfonic acid monohydrate in catalytic amounts, and the
mixture was stirred for 30 min. Then, 80% aq. HOAc (6.0 ml)
was added and stirring was continued for another 30 min. The
mixture was diluted with CH2Cl2 and washed with cold water,
saturated aq. Na2CO3, and water. The organic layer was dried
(Na2SO4), filtered, and concentrated. Column chromatography
(toluene–EtOAc, 2 : 1) gave 6, isolated as a colourless syrup
(202 mg, 98%); TLC (toluene–EtOAc, 2 : 1) Rf 0.50; [α]2D2 Ϫ4
(c 1.2, CHCl3) (Found: C, 63.94; H, 7.04; N, 7.79. C28H37N3O7
requires C, 63.74; H, 7.07; N, 7.96%); δH(300 MHz; CDCl3)
7.37–7.25 (10 H, m, 2 Ph), 5.40 (1 H, dd, J3,4 3.5, J4,5 1.0, H-4),
4.96 and 4.67 (2 H, 2 d, PhCH2O), 4.54 and 4.47 (2 H, 2 d,
PhCH2O), 4.39 (1 H, d, J1,2 7.7, H-1), 3.97 (1 H, dt, OCH-
HCH2), 3.22 (2 H, t, CH2N3), 2.32 (1 H, d, OH), 2.06 (3 H, s,
Ac), 1.69–1.52 (4 H, m, OCH2CH2(CH2)2CH2), 1.48–1.35 (4 H,
m, OCH2CH2(CH2)2CH2); δC(75.5 MHz; CDCl3) 170.9
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981
1977

View Article Online
(COCH3), 138.3 and 137.8 (2 i-Ph), 128.5–127.7 (C-ar), 103.7
(C-1), 79.2, 72.5, 72.0 and 69.6 (C-2,-3,-4,-5), 74.7 and 73.6 (2
PhCH2O), 70.1 (OCH2CH2), 68.3 (C-6), 51.3 (CH2N3), 29.5,
28.7, 26.5 and 25.7 [CH2(CH2)4CH2N3], 20.8 (COCH3).
(C-2a,-2b,-3a,-3b,-4a,-4b,-5a,-5b), 75.3, 74.8, 73.3 and 73.1
(2 C) (5 PhCH2O), 69.6 (OCH2CH2), 68.8 and 68.2 (C-6a,-6b),
51.2 (CH2N3), 29.5, 28.7, 26.4 and 25.6 [CH2(CH2)4CH2N3],
27.8 and 26.3 [C(CH3)2].
6-Azidohexyl (2,6-di-O-acetyl-3,4-O-isopropylidene-ꢀ-D-
galactopyranosyl)-(1 4)-2,3,6-tri-O-acetyl-ꢀ-D-gluco-
pyranoside 8
6-Azidohexyl (2,6-di-O-benzyl-ꢀ-D-galactopyranosyl)-(1 4)-
2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside 10
To a cooled (0 ЊC) solution of 9 (576 mg, 0.6 mmol) in CH2Cl2
(15 ml) was added 90% aq. trifluoroacetic acid (1 ml). The
mixture was stirred for 1.5 h at 0 ЊC, then diluted with CH2Cl2
and washed with cold water, saturated aq. Na2CO3, and water.
The organic layer was dried (Na2SO4), filtered, and concen-
trated. Column chromatography (toluene–EtOAc, 3 : 1) of the
residue gave 10, isolated as a colourless syrup (497 mg, 90%);
TLC (toluene–EtOAc, 2 : 1) Rf 0.40; [α]2D0 ϩ15 (c 1, CHCl3)
(Found: C, 69.47; H, 6.70; N, 4.69. C53H63N3O11 requires C,
69.34; H, 6.92; N, 4.58%); δH(300 MHz; CDCl3) 7.40–7.19
(25 H, m, 5 Ph), 4.99–4.57 (7 H, m, 3 PhCH2O, PhCHHO), 3.20
(2 H, t, CH2N3), 2.49 and 2.43 (each 1 H, 2 d, 2 OH), 1.70–1.50
(4 H, m, OCH2CH2(CH2)2CH2), 1.46–1.32 (4 H, m, OCH2-
CH2(CH2)2CH2); δC(75.5 MHz; CDCl3) 139.1, 138.6, 138.3,
138.1 and 137.9 (5 i-Ph), 128.4–127.1 (C-ar), 103.5 and 102.4
(C-1a,-1b), 82.7, 81.7, 79.9, 76.5, 75.0, 73.4, 72.8 and 68.6
(C-2a,-2b,-3a,-3b,-4a,-4b,-5a,-5b), 75.1, 74.7 (2 C), 73.3 and
73.1 (5 PhCH2O), 69.6 (OCH2CH2), 68.7 and 68.2 (C-6a,-6b),
51.2 (CH2N3), 29.5, 28.6, 26.4 and 25.6 [CH2(CH2)4CH2N3].
To a solution of (2,6-di-O-acetyl-3,4-O-isopropylidene-β--
galactopyranosyl)-(1 4)-2,3,6-tri-O-acetyl-α--glucopyrano-
syl trichloroacetimidate 716 (2.5 g, 3.4 mmol) and 6-azido-
hexan-1-ol (1.02 g, 7.1 mmol) in dry CH2Cl2 (50 ml) was added
powdered 4 Å molecular sieves (2 g). After stirring for 2 h
at room temperature, BF3ؒEt2O (0.25 ml) was added to the
suspension at 0 ЊC, and stirring was continued for another 2 h at
room temperature. Then, the mixture was neutralised with
Et3N, diluted with CH2Cl2, filtered through Celite, and concen-
trated. Column chromatography (toluene–EtOAc, 3 : 1) of the
residue gave 8, isolated as a colourless syrup (872 mg, 36%);
TLC (toluene–EtOAc, 2 : 1) Rf 0.20; [α]2D0 ϩ8 (c 1, CHCl3)
(Found: C, 52.06; H, 6.53; N, 6.06. C31H47N3O16 requires C,
51.88; H, 6.60; N, 5.85%); δH(300 MHz; CDCl3) 5.18 (1 H, t,
J2a,3a 9.6, J3a,4a 9.6, H-3a), 4.89 (1 H, dd, J1a,2a 8.0, H-2a), 4.85
(1 H, dd, J1b,2b 7.4, J2b,3b 6.3, H-2b), 4.44 (1 H, d, H-1a), 4.35
(1 H, d, H-1b), 3.93 (1 H, m, H-5b), 3.83 (1 H, dt, OCHHCH2),
3.74 (1 H, t, H-4a), 3.60 (1 H, ddd, J4a,5a 9.9, J5a,6a 1.9, J5a,6aЈ 5.0,
H-5a), 3.46 (1 H, dt, OCHHCH2), 3.25 (2 H, t, CH2N3), 2.12,
2.10, 2.06, 2.04 and 2.03 (each 3 H, 5 s, 5 Ac), 1.67–1.53 (4 H,
m, OCH2CH2(CH2)2CH2), 1.43–1.32 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.53 and 1.31 (each 3 H, 2 s, 2 Me); δC(75.5 MHz;
CDCl3) 170.6, 170.3, 169.9, 169.4 and 169.0 (COCH3), 110.7
[C(CH3)2], 100.5 and 100.4 (C-1a,-1b), 76.7, 76.0, 72.9, 72.7
(2 C), 72.3, 71.6 and 70.8 (C-2a,-2b,-3a,-3b,-4a,-4b,-5a,-5b),
69.7 (OCH2CH2), 63.0 and 62.1 (C-6a,-6b), 51.2 (CH2N3), 29.1,
28.6, 26.2 and 25.3 [CH2(CH2)4CH2N3], 27.2 and 26.0
[C(CH3)2].
6-Azidohexyl (4-O-acetyl-2,6-di-O-benzyl-ꢀ-D-galacto-
pyranosyl)-(1 4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside 11
To a solution of 10 (390 mg, 0.42 mmol) in acetonitrile (5 ml)
were added trimethyl orthoacetate (0.2 ml) and toluene-p-
sulfonic acid monohydrate (catalytic amounts), and the mixture
was stirred for 30 min. Then, 80% aq. HOAc (6.6 ml) was
added, and stirring was continued for 30 min. After dilution
with CH2Cl2, the mixture was washed with cold water, saturated
aq. Na2CO3, and water. The organic layer was dried (Na2SO4),
filtered, and concentrated. Column chromatography (toluene–
EtOAc, 2 : 1) of the residue gave 11, isolated as a colourless
syrup (389 mg, 95%); TLC (toluene–EtOAc, 2 : 1) Rf 0.51; [α]2D0
Ϫ4 (c 1, CHCl3) (Found: C, 68.74; H, 6.68; N, 4.39.
C55H65N3O12 requires C, 68.80; H, 6.82; N, 4.38%); δH(300
MHz; CDCl3) 7.40–7.15 (25 H, m, 5 Ph), 5.33 (1 H, d, J3b,4b 3.3,
J4b,5b 0, H-4b), 4.98 and 4.76 (each 1 H, 2 d, PhCH2O), 4.85 and
4.73 (each 1 H, 2 d, PhCH2O), 4.80 and 4.67 (each 1 H, 2 d,
PhCH2O), 4.60 and 4.42 (each 1 H, 2 d, PhCH2O), 4.45 and
4.24 (each 1 H, 2 d, PhCH2O), 4.47 and 4.36 (each 1 H, 2 d,
J1a,2a/1b,2b 7.7/8.0, H-1a,-1b), 3.21 (2 H, t, CH2N3), 2.29 (1 H, d,
OH), 2.02 (3 H, s, Ac), 1.70–1.51 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.48–1.30 (4 H, m, OCH2CH2(CH2)2CH2);
δC(75.5 MHz; CDCl3) 170.8 (COCH3), 139.0, 138.6, 138.2,
138.1 and 137.8 (5 i-Ph), 128.2–127.2 (C-ar), 103.5 and 102.2
(C-1a,-1b), 82.6, 81.6, 80.0, 76.3, 75.0, 72.3, 71.9 and 69.5
(C-2a,-2b,-3a,-3b,-4a,-4b,-5a,-5b), 75.1, 74.9, 74.7, 73.3 and
73.1 (5 PhCH2O), 69.6 (OCH2CH2), 68.1 and 67.1 (C-6a,-6b),
51.2 (CH2N3), 29.5, 28.6, 26.4 and 25.6 [CH2(CH2)4CH2N3],
20.6 (COCH3).
6-Azidohexyl (2,6-di-O-benzyl-3,4-O-isopropylidene-ꢀ-D-
galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl-ꢀ-D-gluco-
pyranoside 9
A solution of 8 (770 mg, 1.07 mmol) in MeOH (70 ml) was
stirred with NaOMe at pH 8–9 for 24 h at room temperature.
Then, the solution was neutralised with Dowex 50W-X8 (Hϩ
form), filtered, and concentrated. Column chromatography
(CH2Cl2–MeOH, 5 : 1) of the residue gave an amorphous solid
(530 mg) that was dissolved in DMF (25 ml). Sodium hydride
(171 mg, 80% suspension in oil) was added, and the mixture was
stirred for 30 min at room temperature. After adding benzyl
bromide (0.8 ml, 6.6 mmol), the mixture was stirred overnight,
then poured onto ice–water. The organic layer was separated
and the water phase was extracted (3×) with CH2Cl2. The com-
bined organic layers were collected, washed with water, dried
(Na2SO4), filtered, and concentrated. Column chromatography
(toluene–EtOAc, 10 : 1) of the residue gave 9, isolated as a
colourless syrup (657 mg, 64%); TLC (toluene–EtOAc, 10 : 1)
Rf 0.40; [α]2D0 ϩ15 (c 1, CHCl3) (Found: C, 70.49; H, 7.04; N,
4.39. C56H67N3O11 requires C, 70.20; H, 7.05; N, 4.39%); δH(300
MHz; CDCl3) 7.40–7.19 (25 H, m, 5 Ph), 4.93 and 4.74 (each 1
H, 2 d, PhCH2O), 4.87 and 4.71 (each 1 H, 2 d, PhCH2O), 4.79
and 4.66 (each 1 H, 2 d, PhCH2O), 4.57 and 4.42 (each 1 H, 2 d,
PhCH2O), 4.50 and 4.31 (each 1 H, 2 d, PhCH2O), 4.41 and
4.37 (each 1 H, 2 d, J1a,2a/1b,2b 8.5/7.7, H-1a,-1b), 4.10 (1 H, dd,
J3b,4b 5.6, J4b,5b 1.5, H-4b), 4.03 (1 H, t, J2b,3b 5.9, H-3b), 3.92
and 3.51 (each 1 H, 2 dt, OCH2CH2), 3.21 (2 H, t, CH2N3),
1.68–1.52 (4 H, m, OCH2CH2(CH2)2CH2), 1.47–1.36 (4 H, m,
OCH2CH2(CH2)2CH2), 1.40 and 1.34 (each 3 H, 2 s, 2 Me);
δC(75.5 MHz; CDCl3) 138.9, 138.6, 138.5, 138.4 and 138.2 (5 i-
Ph), 128.2–127.1 (C-ar), 109.6 [C(CH3)2], 103.5 and 101.8
(C-1a,-1b), 82.9, 81.8, 80.6, 79.3, 76.3, 75.0, 73.5 and 71.9
(2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl)-(1 4)-(2,3,6-tri-
O-acetyl-ꢀ-D-glucopyranosyl)-(1 6)-2-deoxy-3,4-bis-
(O-p-methylbenzoyl)-2-phthalimido-ꢀ-D-glucopyranosyl
trichloroacetimidate 13
To
a solution of allyl (2,3,4,6-tetra-O-acetyl-β--galacto-
pyranosyl)-(1 4)-(2,3,6-tri-O-acetyl-β--glucopyranosyl)-
(1 6)-2-deoxy-3,4-bis(O-p-methylbenzoyl)-2-phthalimido-β-
-glucopyranoside (12)9 (3.47 g, 2.9 mmol) in 96% aq. HOAc
(75 ml) were added PdCl2 (2.3 g) and NaOAc (2.0 g), and the
mixture was sonicated in an ultrasonic bath for 24 h at room
temperature. After filtration through Celite, CH2Cl2 was added
1978
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981

View Article Online
to the filtrate, and the organic layer was washed with cold
water, dried (Na2SO4), filtered, and concentrated. Column
chromatography (toluene–EtOAc, 2 : 1) of the residue gave a
colourless syrup (2.93 g) that was dissolved in CH2Cl2 (40 ml),
and trichloroacetonitrile (0.92 ml, 9.15 mmol) was added.
Then, at 0 ЊC 1,8-diazabicyclo[5.4.0]undec-7-ene (100 µl) was
added, and the mixture was stirred for 5 h at room tempera-
ture. After concentration, column chromatography (toluene–
EtOAc, 2 : 1) of the residue gave 13, isolated as an amorphous
solid (1.70 g, 52%); TLC (toluene–EtOAc, 1 : 1) Rf 0.51; [α]2D0
ϩ11 (c 1, CHCl3) (Found: C, 53.38; H, 4.79. C58H61Cl3N2O26
requires C, 53.24; H, 4.70%); δH(300 MHz; CDCl3) 8.78 (1 H,
s, OCNHCCl3), 7.81–7.05 (12 H, m, 2 COC6H4CH3 and Phth),
6.78 (1 H, d, J1a,2a 8.8, H-1a), 6.28 (1 H, dd, J2a,3a 9.3, J3a,4a 9.3,
H-3a), 5.49 (1 H, t, J4a,5a 9.3, H-4a), 5.35 (1 H, d, J3c,4c 3.1,
J4c,5c 0, H-4c), 4.69 (1 H, d, J1b,2b 7.6, H-1b), 4.47 (1 H, d, J1c,2c
7.9, H-1c), 2.36 and 2.29 (each 3 H, 2 s, 2 COC6H4CH3),
2.17–1.63 (21 H, m, 7 Ac); δC(75.5 MHz; CDCl3) 170.3–168.9
(COCH3), 165.4 and 165.1 (2 COC6H4CH3), 160.3 (OCNH-
CCl3), 101.0 and 99.9 (C-1b,-1c), 93.5 (C-1a), 68.2, 61.2, 60.7
(C-6a,-6b,-6c), 55.3 (C-2a), 21.5–20.5 (COC6H4CH3 and
COCH3).
6-Azidohexyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-
(1 4)-(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1 6)-
(2-deoxy-3,4-bis(O-p-methylbenzoyl)-2-phthalimido-
ꢀ-D-glucopyranosyl)-(1 3)-(4-O-acetyl-2,6-di-
O-benzyl-ꢀ-D-galactopyranosyl)-(1 4)-2,3,6-tri-
O-benzyl-ꢀ-D-glucopyranoside 15
To a solution of 13 (327 mg, 0.25 mmol) and 11 (302 mg, 0.31
mmol) in dry CH2Cl2 (10 ml) was added powdered 4 Å molec-
ular sieves (500 mg), and the suspension was stirred for 1 h at
room temperature. Then, at 0 ЊC trimethylsilyl trifluoro-
methanesulfonate (63 µl, 0.35 mmol) was added, and the mix-
ture was stirred for 2 h at room temperature, neutralised with
Et3N, diluted with CH2Cl2, filtered through Celite, and the
filtrate was concentrated. Column chromatography (toluene–
EtOAc, 5 : 1) of the residue gave 15, isolated as a white crystal-
line powder (202 mg, 38%); Mp 93–94 ЊC; TLC (toluene–
EtOAc, 2 : 1) Rf 0.47; [α]2D2 Ϫ0.4 (c 1, CHCl3) (Found: C, 63.14;
H, 5.92; N, 2.73. C111H124N4O37 requires C, 63.30; H, 5.93; N,
2.66%); δH(300 MHz; CDCl3) 7.79 and 7.60 (each 2 H, 2 m,
COC6H4CH3), 7.47 (4 H, br s, Phth), 7.37–6.96 (29 H, m, 5 Ph,
COC6H4CH3), 6.25 (1 H, dd, J2c,3c 10.8, J3c,4c 9.1, H-3c), 5.66 (1
H, d, J1c,2c 8.2, H-1c), 5.56 (1 H, dd, J3b,4b 3.6, J4b,5b 0.8, H-4b),
5.40 (1 H, dd, J4c,5c 10.2, H-4c), 5.32 (1 H, dd, J3e,4e 3.5, J4e,5e 1.2,
H-4e), 5.23 (1 H, dd, J2d,3d 9.5, J3d,4d 9.0, H-3d), 5.07 (1 H, dd,
J2e,3e 10.7, J1e,2e 7.8, H-2e), 4.91 (1 H, dd, J3e,4e 3.5, H-3e), 4.88
(1 H, dd, J1d,2d 7.9, H-2d), 4.87 and 4.62 (each 1 H, 2 d,
PhCH2O), 4.83 and 4.68 (each 1 H, 2 d, PhCH2O), 4.65 (d, 1 H,
H-1d), 4.52–4.40 (6 H, m, 3 PhCH2O), 4.43 (1 H, dd, H-2c),
4.42 (2 H, m, H-1a,-1e), 4.25 (1 H, d, J1b,2b 7.5, H-1b), 4.02 (1
H, H-5c), 3.92 (1 H, H-3b), 3.87 (1 H, H-5b), 3.85 and 3.47
(each 1 H, OCH2CH2), 3.84 (1 H, H-5e), 3.82 (1 H, H-4a), 3.69
(1 H, dd, J4d,5d 9.8, H-4d), 3.55 (1 H, H-5d), 3.41 (1 H, H-3a),
3.34 (1 H, H-2a), 3.31 (1 H, H-2b), 3.19 (2 H, t, CH2N3), 3.15
(1 H, H-5a), 2.34 and 2.26 (each 3 H, 2 s, 2 COC6H4CH3), 2.13,
2.04, 2.03, 2.01, 1.94 and 1.90 (3,6,3,3,6,3 H, 6 s, 8 Ac), 1.61–
1.47 (4 H, m, OCH2CH2(CH2)2CH2), 1.40–1.30 (4 H, m,
OCH2CH2(CH2)2CH2); δC(75.5 MHz; CDCl3) 170.3, 170.2,
170.1, 169.9 (2 C), 169.6, 169.4 and 168.9 (8 COCH3), 167.2
(br s, COPhth), 165.5 and 165.2 (2 COC6H4CH3), 103.5 (C-1b),
102.2 (C-1a), 101.1 (C-1e), 101.0 (C-1d), 98.5 (C-1c), 82.6
(C-3a), 81.7 (C-2b), 79.1 (C-2a), 78.0 (C-3b), 76.4 (C-4d), 76.3
(C-4a), 75.2 (PhCH2O), 75.1 (C-5a), 74.9 (PhCH2O), 74.5
(C-5c), 74.5, 73.1 and 73.0 (3 PhCH2O), 72.7 (C-3d), 72.3
(C-5d), 72.0 (C-2d), 71.8 (C-5b), 71.1 (C-3e), 70.6 (C-5e), 70.4
(C-3c), 69.9 (C-4c), 69.8 (C-4b), 69.6 (OCH2CH2), 69.1 (C-2e),
68.4 (C-6c), 68.0 (C-6a), 67.2 (C-6b), 66.6 (C-4e), 62.1 (C-6d),
60.7 (C-6e), 55.3 (C-2c), 51.3 (CH2N3), 29.6 [CH2CH2-
(CH2)4N3], 28.7 [CH2(CH2)3CH2CH2N3], 26.5 and 25.7
[CH2CH2(CH2)2CH2CH2N3], 21.6 and 21.5 (2 COC6H4CH3),
20.8, 20.7 (3 C), 20.6, 20.5 and 20.4 (2 C) (8 COCH3).
6-Azidohexyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-
(1 4)-(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1 6)-(2-
deoxy-3,4-bis(O-p-methylbenzoyl)-2-phthalimido-ꢀ-D-
glucopyranosyl)-(1 3)-4-O-acetyl-2,6-di-O-benzyl-
ꢀ-D-galactopyranoside 14
To a solution of 13 (325 mg, 0.25 mmol) and 6 (128 mg, 0.24
mmol) in dry CH2Cl2 (10 ml) was added powdered 4 Å
molecular sieves (500 mg), and the suspension was stirred for
2 h at room temperature. Then, at 0 ЊC trimethylsilyl trifluoro-
methanesulfonate (50 µl, 0.27 mmol) was added, and the
mixture was stirred for 2 h at room temperature, neutralised
with Et3N (40 µl, 0.29 mmol), diluted with CH2Cl2, filtered
through Celite, and the filtrate was concentrated. Column
chromatography (toluene–EtOAc, 3 : 1) of the residue gave 14,
isolated as an amorphous solid (201 mg, 49%); TLC (toluene–
EtOAc, 2 : 1) Rf 0.42; [α]2D3 Ϫ2 (c 1, CHCl3) (Found: C, 60.05;
H, 5.69; N, 3.27. C84H96N4O32 requires C, 60.28; H, 5.78; N,
3.35%); δH(300 MHz; CDCl3) 7.78 and 7.61 (each 2 H, 2 m,
COC6H4CH3), 7.52 (4 H, br s, Phth), 7.35–7.22 and 7.08 (8
and 2 H, 2 m, 2 Ph), 7.15 and 7.04 (each 2 H, 2 m,
COC6H4CH3), 6.24 (1 H, dd, J2b,3b 10.8, J3b,4b 9.1, H-3b), 5.67
(1 H, d, J1b,2b 8.2, H-1b), 5.54 (1 H, dd, J3a,4a 3.8, J4a,5a 1.0, H-
4a), 5.36 (1 H, dd, J4b,5b 10.2, H-4b), 5.33 (1 H, dd, J3d,4d 3.4,
J4d,5d 1.2, H-4d), 5.24 (1 H, dd, J2c,3c 9.5, J3c,4c 9.0, H-3c), 5.07
(1 H, dd, J2d,3d 10.4, J1d,2d 7.7, H-2d), 4.92 (1 H, dd, J3d,4d 3.4,
H-3d), 4.89 (1 H, dd, J1c,2c 7.8, H-2c), 4.69 (1 H, d, H-1c), 4.54
and 4.37 (each 1 H, 2 d, PhCH2O), 4.53 and 4.05 (each 1 H, 2
d, PhCH2O), 4.46 (1 H, d, H-1d), 4.35 (1 H, d, J1a,2a 7.8, H-
1a), 4.42 (1 H, dd, J2b,3b 10.8, H-2b), 3.98 (1 H, dd, J2a,3a 9.7,
H-3a), 3.74 (1 H, dd, J4c,5c 9.9, H-4c), 3.35 (1 H, dd, J2a,3a 9.7,
H-2a), 3.10 (2 H, t, CH2N3), 2.33 and 2.26 (each 3 H, 2 s, 2
COC6H4CH3), 2.11, 2.06, 2.05, 2.04, 2.03, 2.02, 1.93 and 1.91
(each 3 H, 8 s, 8 Ac), 1.55–1.35 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.30–1.20 (4 H, m, OCH2CH2(CH2)2CH2); δC(75.5
MHz; CDCl3) 170.3 (2 C), 170.1, 170.0 (2 C), 169.8, 169.5 and
169.0 (8 COCH3), 167.7 (br s, COPhth), 165.5 and 165.2 (2
COC6H4CH3), 103.6 (C-1a), 101.2 (C-1d), 100.9 (C-1c), 98.4
(C-1b), 78.6 (C-2a), 77.4 (C-3a), 76.4 (C-4c), 74.6 (C-5b), 74.0
and 73.3 (2 PhCH2O), 72.8 (C-3c), 72.3 (C-5c), 72.1 (C-2c),
71.8 (C-5a), 71.1 (C-3d), 70.6 (C-5d), 70.4 (C-3b), 69.9 (2 C)
(C-4b, OCH2CH2), 69.7 (C-4a), 69.0 (C-2d), 68.2 (C-6b), 67.6
(C-6a), 66.7 (C-4d), 62.1 (C-6c), 60.7 (C-6d), 55.3 (C-2b),
51.2 (CH2N3), 29.4 [CH2CH2(CH2)4N3], 28.6 [CH2(CH2)3-
CH2CH2N3], 26.4 and 25.6 [CH2CH2(CH2)2CH2CH2N3], 21.6
and 21.5 (2 COC6H4CH3), 20.9, 20.8, 20.7 (2 C), 20.6 (2 C) and
20.4 (2 C) (8 COCH3).
6-Azidohexyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-
(1 4)-(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1 6)-(2-
acetamido-3,4-di-O-acetyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1 3)-
4-O-acetyl-2,6-di-O-benzyl-ꢀ-D-galactopyranoside 16
A solution of 14 (201 mg, 0.12 mmol) in MeOH (50 ml) was
stirred with NaOMe at pH 8–9 for 24 h at room temperature.
Then, the solution was neutralised with Dowex 50W-X8 (Hϩ
form), filtered, and concentrated. Column chromatography
(CH2Cl2–MeOH, 5 : 1) of the residue gave an amorphous solid
(112 mg); TLC (CH2Cl2–MeOH, 5 : 1) Rf 0.14. A solution of
the residue in 33% ethanolic methylamine (10 ml) was stirred
for 24 h at room temperature, then concentrated and co-
concentrated with toluene; TLC (CH2Cl2–MeOH, 2 : 1) Rf 0.41.
A solution of the residue in pyridine (12 ml) and Ac2O (7 ml)
was stirred overnight at room temperature, then concentrated
and co-concentrated with toluene. Column chromatography
(CH2Cl2–MeOH, 50 : 1) of the residue gave 16, isolated as an
amorphous solid (112 mg, 65%); TLC (CH2Cl2–MeOH, 20 : 1)
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981
1979

View Article Online
Rf 0.23; [α]2D4 Ϫ17 (c 0.25, CHCl3) (Found: C, 55.13; H, 6.35; N,
3.93. C66H88N4O31 requires C, 55.30; H, 6.19; N, 3.91%); δH(300
MHz; CDCl3) 7.43–7.21 (10 H, m, 2 Ph), 5.43 (1 H, dd, J3a,4a
3.8, J4a,5a 1.0, H-4a), 5.34 (1 H, dd, J3d,4d 3.4, J4d,5d 1.3, H-4d),
5.23 (1 H, dd, J2c,3c 9.3, J3c,4c 9.0, H-3c), 5.08 (1 H, dd, J2d,3d 10.4,
J1d,2d 7.8, H-2d), 5.02 and 4.54 (each 1 H, 2 d, PhCH2O), 4.95
(1 H, dd, H-3d), 4.87 (1 H, H-3b), 4.86 (1 H, dd, J1c,2c 7.7,
H-2c), 4.79 (1 H, H-4b), 4.76 (1 H, d, NH), 4.72 (1 H, d, J1b,2b
8.4, H-1b), 4.62 (1 H, d, H-1c), 4.53 and 4.39 (each 1 H, 2 d,
PhCH2O), 4.50 (1 H, d, H-1d), 4.40 (1 H, d, J1a,2a 7.8, H-1a),
3.94 (1 H, OCHHCH2), 3.93 (1 H, H-5c), 3.86 (1 H, H-5d), 3.84
(1 H, H-2b), 3.84 (1 H, H-3a), 3.78 (1 H, H-4c), 3.61 (1 H, H-
5a), 3.54 (1 H, H-2a), 3.53 (1 H, H-5b), 3.52 (1 H, OCHHCH2),
3.18 (2 H, t, CH2N3), 2.12, 2.05, 2.04, 2.02, 1.98 and 1.94
(6,6,3,3,3,9 H, 6 s, 10 Ac), 1.65–1.46 (4 H, m, OCH2CH2-
(CH2)2CH2), 1.59 (3 H, s, NHAc), 1.39–1.31 (4 H, m, OCH2-
CH2(CH2)2CH2); δC(75.5 MHz; CDCl3) 170.8, 170.3 (2 C),
170.2, 170.1, 170.0, 169.7, 169.5 (2 C), 169.3 and 169.0
(11 COCH3), 103.6 (C-1a), 101.4 (C-1b), 101.2 (C-1d), 100.5
(C-1c), 80.0 (C-2a), 77.0 (C-3a), 76.3 (C-4c), 74.5 (PhCH2O),
73.8 (C-5b), 73.4 (PhCH2O), 72.8 (C-3c), 72.6 (C-3b), 72.6
(C-5a), 72.3 (C-5c), 72.1 (C-2c), 71.1 (C-3d), 70.7 (C-5d), 69.9
(OCH2CH2), 69.6 (C-4a), 69.1 (C-4b), 69.1 (C-2d), 68.1 (C-6a),
68.1 (C-6b), 66.7 (C-4d), 62.1 (C-6c), 60.8 (C-6d), 54.2 (C-2b),
51.3 (CH2N3), 29.5 [CH2CH2(CH2)4N3], 28.7 [CH2(CH2)3CH2-
CH2N3], 26.5 and 25.7 [CH2CH2(CH2)2CH2CH2N3], 22.9
(NHCOCH3), 20.9 (2 C), 20.8, 20.7, 20.6 (4 C) and 20.5 (2 C)
(10 COCH3).
74.7 (2 PhCH2O), 73.6 (C-5c), 73.4 (2 PhCH2O), 72.7 (3 C)
(C-3c,-3d,-5b), 72.3 (C-5d), 72.0 (C-2d), 71.1 (C-3e), 70.7
(C-5e), 69.9 (C-4b), 69.7 (OCH2CH2), 69.2 (C-4c), 69.2 (C-2e),
68.5 (C-6c), 68.2 (C-6a), 67.6 (C-6b), 66.7 (C-4e), 62.1 (C-6d),
60.8 (C-6e), 54.3 (C-2c), 51.4 (CH2N3), 29.6 [CH2CH2-
(CH2)4N3], 28.8 [CH2(CH2)3CH2CH2N3], 26.5 and 25.7
[CH2CH2(CH2)2CH2CH2N3], 22.9 (NHCOCH3), 20.8, 20.7
(3 C), 20.6 (3 C), 20.5, 20.4 (2 C) (10 COCH3).
6-Aminohexyl (ꢀ-D-galactopyranosyl)-(1 4)-(ꢀ-D-glucopyrano-
syl)-(1 6)-(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1 3)-
ꢀ-D-galactopyranoside 18
To a solution of 16 (56 mg, 39 µmol) in dry MeOH (5 ml) was
added NaOMe (45 mg), and the solution was stirred overnight
at room temperature. Then, water (2 ml) was added, and after
stirring overnight at room temperature, the mixture was neu-
tralised with Dowex 50W-X8 (Hϩ form), filtered, and concen-
trated. To a solution of the crude de-O-acetylated intermediate
(42 mg) in a mixture of 2-methylpropan-2-ol (8 ml) and water
(8 ml) were added 3 drops of ammonia and Pd/C (100 mg, 10%
Pd). The suspension was stirred for 3 h at room temperature in
a hydrogen atmosphere, after which ammonia was removed by
bubbling nitrogen through the solution. Then, two drops of
HOAc and fresh Pd/C (50 mg) were added. The suspension was
stirred for another 24 h at room temperature in a hydrogen
atmosphere, neutralised with ammonia, and filtered through
Celite. The filtrate was concentrated, and the residue was
applied to a Toyopearl HW-40S column, eluted with 0.1 M
NH4OAc at a flow rate of 40 ml hϪ1. The appropriate fractions
were lyophilised to give 18 (30 mg, 96%); TLC (HOAc–
butanol–H2O, 2 : 1 : 1) Rf 0.25; [α]2D5 Ϫ2.3 (c 1.2, H2O); δC(125.7
MHz; D2O) 175.8 (COCH3), 103.8, 103.6 (2 C) and 103.5
(C-1a,-1b,-1c,-1d), 83.2, 79.3, 76.2, 75.6 (2 C), 75.5, 75.2, 74.4,
73.6, 73.4, 71.8, 71.2, 70.7, 70.5, 69.5, 69.4 and 69.1 (C-2a,
-2c,-2d,-3a,-3b,-3c,-3d,-4a,-4b,-4c,-4d,-5a,-5b,-5c,-5d,-6b,OCH2-
CH2), 61.9, 61.6 and 61.0 (C-6a,-6c,-6d), 56.5 (C-2b), 40.3
(CH2NH2), 29.3, 27.6, 26.1 and 25.4 [CH2(CH2)4CH2NH2],
23.0 (COCH3). For 1H NMR data (500 MHz, D2O), see
Table 1. High-resolution MS data of C32H58N2O21 (M, 806.3532):
M ϩ H found 807.3596, calculated 807.3610.
6-Azidohexyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-
(1 4)-(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1 6)-(2-
acetamido-3,4-di-O-acetyl-2-deoxy-ꢀ-D-glucopyranosyl)-
(1 3)-(4-O-acetyl-2,6-di-O-benzyl-ꢀ-D-galactopyranosyl)-
(1 4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside 17
A solution of 15 (105 mg, 0.05 mmol) in MeOH (25 ml) was
stirred with NaOMe at pH 8–9 for 24 h at room temperature.
Then, the solution was neutralised with Dowex 50W-X8 (Hϩ
form), filtered, and concentrated. Column chromatography
(CH2Cl2–MeOH, 5 : 1) of the residue gave an amorphous solid
(64 mg); TLC (CH2Cl2-MeOH, 5 : 1) Rf 0.20. A solution of the
residue in 33% ethanolic methylamine (10 ml) was stirred for 24
h at room temperature, then concentrated and co-concentrated
with toluene; TLC (CH2Cl2–MeOH, 2 : 1) Rf 0.14. A solution
of the residue in pyridine (10 ml) and Ac2O (5 ml) was stirred
for 20 h at room temperature, then concentrated and co-
concentrated with toluene. Column chromatography (CH2Cl2–
MeOH, 40 : 1) of the residue gave 17, isolated as an amorphous
solid (53 mg, 57%); TLC (CH2Cl2–MeOH, 20 : 1) Rf 0.43; [α]2D2
Ϫ114 (c 1, CHCl3) (Found: C, 59.59; H, 6.22; N, 2.94.
C93H116N4O36 requires C, 59.86; H, 6.27; N, 3.00%); δH(300
MHz; CDCl3) 7.41–7.08 (25 H, m, 5 Ph), 5.43 (1 H, dd, J3b,4b
3.5, J4b,5b 0.8, H-4b), 5.33 (1 H, dd, J3e,4e 3.5, J4e,5e 1.2, H-4e),
5.21 (1 H, t, J2d,3d 9.1, J3d,4d 9.1, H-3d), 5.07 (1 H, dd, J2e,3e 10.4,
J1e,2e 7.8, H-2e), 4.94 (1 H, dd, H-3e), 4.93 (1 H, H-3c), 4.88
(1 H, H-4c), 4.87 (1 H, H-2d), 4.85 (1 H, NH), 4.68 (1 H, d, J1c,2c
8.2, H-1c), 4.57 (1 H, H-1d), 4.51 (1 H, H-1a), 4.47 (1 H, d, H-
1e), 4.46 and 4.21 (each 1 H, 2 d, PhCH2O), 4.32 (1 H, d, J1b,2b
7.7, H-1b), 3.94 (1 H, H-4a), 3.88 (1 H, OCHHCH2), 3.86 (1 H,
H-5e), 3.84 (1 H, H-2c), 3.80 (1 H, H-5d), 3.77 (1 H, H-3b), 3.72
(1 H, H-4d), 3.60 (1 H, H-5c), 3.51 (1 H, H-3a), 3.50 (1 H, H-
5b), 3.49 (1 H, OCHHCH2), 3.47 (1 H, H-2a), 3.31 (1 H, H-5a),
3.20 (2 H, t, CH2N3), 2.13, 2.08, 2.03, 2.02, 1.99, 1.98, 1.97, 1.94
and 1.93 (3,3,3,3,3,3,3,6,3 H, 9 s, 10 Ac), 1.65–1.50 (4 H, m,
OCH2CH2(CH2)2CH2), 1.55 (3 H, s, NHAc), 1.44–1.33 (4 H, m,
OCH2CH2(CH2)2CH2); δC(75.5 MHz; CDCl3) 170.9, 170.3 (2
C), 170.1, 170.0 (2 C), 169.6, 169.5, 169.4, 169.3 and 169.0 (11
COCH3), 103.6 (C-1b), 102.2 (C-1a), 101.5 (C-1c), 101.2 (C-1e),
100.6 (C-1d), 82.5 (C-3a), 81.8 (C-2b), 80.6 (C-2a), 77.7 (C-3b),
76.4 (C-4a), 76.2 (C-4d), 75.3 (PhCH2O), 75.2 (C-5a), 74.9 and
6-Aminohexyl (ꢀ-D-galactopyranosyl)-(1 4)-(ꢀ-D-gluco-
pyranosyl)-(1 6)-(2-acetamido-2-deoxy-ꢀ-D-gluco-
pyranosyl)-(1 3)-(ꢀ-D-galactopyranosyl)-(1 4)-
ꢀ-D-gluco-pyranoside 19
To a solution of 17 (49 mg, 26 µmol) in dry MeOH (5 ml) was
added NaOMe (55 mg), and the solution was stirred overnight
at room temperature. Then, water (1 ml) was added, and after
stirring for 20 h at room temperature, the mixture was neutral-
ised with Dowex 50W-X8 (Hϩ form), filtered, and concen-
trated. To a solution of de-O-acetylated intermediate (40 mg) in
a mixture of 2-methylpropan-2-ol (8 ml) and water (6 ml) were
added 3 drops of ammonia and Pd/C (90 mg, 10% Pd). The
suspension was stirred for 2 h at room temperature in a
hydrogen atmosphere, after which ammonia was removed by
bubbling nitrogen through the solution. Then, two drops of
HOAc and fresh Pd/C (90 mg) were added. The suspension was
stirred for another 24 h at room temperature in a hydrogen
atmosphere, neutralised with ammonia, and filtered through
Celite. The filtrate was concentrated, and the residue was
applied to a Toyopearl HW-40S column, eluted with 0.1 M
NH4OAc at a flow rate of 40 ml hϪ1. The appropriate fractions
were lyophilised to give 19 (21 mg, 82%); TLC (HOAc–
butanol–H2O, 2 : 1 : 1) Rf 0.18; [α]2D5 Ϫ0.5 (c 0.9, H2O); δC(125.7
MHz; D2O) 175.8 (COCH3), 103.8 (2 C), 103.7 (2 C) and 102.9
(C-1a,-1b,-1c,-1d,-1e), 82.9, 79.3 (2 C), 76.2, 75.8, 75.7, 75.6,
75.5, 75.3, 75.2, 74.4, 73.7 (2 C), 73.4, 71.8, 71.3, 70.9, 70.5,
69.6, 69.4, 69.2 (C-2a,-2b,-2d,-2e,-3a,-3b,-3c,-3d,-3e,-4a,-4b,
-4c,-4d,-4e,-5a,-5b,-5c,-5d,-5e,-6c, OCH2CH2), 61.9 (2 C) and
1980
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981

View Article Online
61.0 (2 C) (C-6a,-6b,-6d,-6e), 56.5 (C-2c), 40.3 (CH2NH2), 29.3,
27.5, 26.1 and 25.4 [CH2(CH2)4CH2NH2], 23.0 (COCH3). For
1H NMR data (500 MHz, D2O), see Table 2. High-resolution
MS data of C38H68N2O26 (M, 968.4060): M ϩ H found
969.4154, calculated 969.4138.
23.0 (COCH3). For 1H NMR data (500 MHz, D2O), see Table
4. High-resolution MS data of C44H78N2O31 (M, 1130.4588):
M ϩ H found 1131.4506, calculated 1131.4666.
Acknowledgements
6-Aminohexyl (ꢀ-D-galactopyranosyl)-(1 4)-(ꢀ-D-gluco-
pyranosyl)-(1 6)-[(ꢀ-D-galactopyranosyl)-(1 4)]-
(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-
(1 3)-ꢀ-D-galacto- pyranoside 20
We thank Mrs G. Sanzberro and Mrs U. Rezabal for synthesiz-
ing compound 13. This work was supported by the European
Union programs CARENET-1 (grant ERB CHRX CT940442)
and VACNET (grant ERB BIO 4CT960158).
To a solution of 18 (10 mg, 12.3 µmol) in 50 mM sodium
cacodylate buffer pH 7.5 (600 µl) containing MnCl2 (5 mmol),
bovine serum albumin (0.8 mg) and NaN3 (0.02%), were added
alkaline phosphatase (7 U), UDP-galactose (10 mg, 16.3 µmol)
and β-1,4-galactosyltransferase (2.5 U). The mixture was
incubated for 19 h at 37 ЊC. Then, water (100 µl) was added, and
UDP-galactose was removed using a Dowex 1X8 (ClϪ form)
column with water as eluent. The eluate was concentrated, and
the residue was applied to a Toyopearl HW-40S column, eluted
with 0.1 M NH4OAc at a flow rate of 40 ml hϪ1. The appro-
priate fractions were lyophilised to give 20 (9.6 mg, 80%); TLC
(HOAc–butanol–H2O, 2 : 1 : 1) Rf 0.16; [α]2D5 Ϫ2.7 (c 0.6, H2O);
δC (125.7 MHz; D2O) 175.8 (COCH3), 103.8, 103.7, 103.6 (2 C)
and 103.5 (C-1a,-1b,-1c,-1d,-1e), 83.4, 79.3, 78.6, 76.2, 76.1,
75.6, 75.5, 75.1, 74.2, 73.5, 73.4 (2 C), 73.0, 71.8 (2 C), 71.2,
70.6, 69.4 (2 C), 69.1 and 68.4 (C-2a,-2c,-2d,-2e,-3a,-3b,-3c,
-3d,-3e,-4a,-4b,-4c,-4d,-4e,-5a,-5b,-5c,-5d,-5e,-6b, OCH2CH2),
61.9 (2 C), 61.8 and 61.0 (C-6a,-6c,-6d,-6e), 56.1 (C-2b), 40.3
(CH2NH2), 29.3, 27.5, 26.1 and 25.4 [CH2(CH2)4CH2NH2],
23.1 (COCH3). For 1H NMR data (500 MHz, D2O), see Table
3. High-resolution MS data of C38H68N2O26 (M, 968.4060): M
ϩ H found 969.4164, calculated 969.4138.
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6-Aminohexyl (ꢀ-D-galactopyranosyl)-(1 4)-(ꢀ-D-gluco-
pyranosyl)-(1 6)-[(ꢀ-D-galactopyranosyl)-(1 4)]-(2-acetamido-
2-deoxy-ꢀ-D-glucopyranosyl)-(1 3)-(ꢀ-D-galactopyranosyl)-
(1 4)-ꢀ-D-glucopyranoside 21
To a solution of 19 (11.9 mg, 12.3 µmol) in 50 mM sodium
cacodylate buffer pH 7.5 (600 µl) containing MnCl2 (5 mmol),
bovine serum albumin (0.5 mg) and NaN3 (0.02%), were added
alkaline phosphatase (11.5 U), UDP-galactose (10 mg, 16.3
µmol) and β-1,4-galactosyltransferase (2.5 U). The mixture was
incubated for 20 h at 37 ЊC. Then, water (100 µl) was added, and
UDP-galactose was removed using a Dowex 1X8 (ClϪ form)
column with water as eluent. The eluate was concentrated, and
the residue was applied to a Toyopearl HW-40S column, eluted
with 0.1 M NH4OAc at a flow rate of 40 ml hϪ1. The appropri-
ate fractions were lyophilised to give 21 (13.4 mg, 96%); TLC
(HOAc–butanol–H2O, 2 : 1 : 1) Rf 0.14; [α]2D5 Ϫ3.2 (c 0.8, H2O);
δC(125.7 MHz; D2O) 175.8 (COCH3), 103.8 (3 C), 103.6, 103.5
and 102.8 (C-1a,-1b,-1c,-1d,-1e,-1f ), 82.0, 79.2, 76.2, 76.1, 75.8,
75.6, 75.3, 75.1, 73.7, 73.5, 73.4, 72.9, 72.4 (3 C), 71.8, 71.3 and
69.4
(C-2a,-2b,-2d,-2e,-2f,-3a,-3b,-3c,-3d,-3e,-3f,-4a,-4b,-4c,
-4d,-4e,-4f,-5a,-5b,-5c,-5d,-5e,-5f,-6c, OCH2CH2), 62.5, 61.9,
61.3 (2 C) and 60.9 (C-6a,-6b,-6d,-6e,-6f ), 56.0 (C-2c), 40.3
(CH2NH2), 29.3, 27.5, 26.1 and 25.4 [CH2(CH2)4CH2NH2],
J. Chem. Soc., Perkin Trans. 1, 2002, 1973–1981
1981