A facile synthesis of the GalNAcβ1→4Gal target sequence of respiratory pathogens

Article abstract of DOI:10.1016/j.carres.2005.08.001

The carbohydrate sequence, GalNAcβ1→4Gal, is the target for the adhesion of several respiratory pathogens. The sequence was prepared in an optimized synthesis in forms that allow conjugation to scaffolds or surfaces.

Full text of DOI:10.1016/j.carres.2005.08.001

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2436–2442
Note
A facile synthesis of the GalNAcb1!4Gal target sequence
of respiratory pathogens
Reshma Autar, Rob M. J. Liskamp and Roland J. Pieters*
Utrecht Institute for Pharmaceutical Sciences, Department of Medicinal Chemistry, Utrecht University,
PO Box 80082, 3508 TB Utrecht, The Netherlands
Received 16 December 2004; received in revised form 2 August 2005; accepted 2 August 2005
Available online 19 August 2005
Abstract—The carbohydrate sequence, GalNAcb1!4Gal, is the target for the adhesion of several respiratory pathogens. The
sequence was prepared in an optimized synthesis in forms that allow conjugation to scaffolds or surfaces.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Bacterial adhesion; GalNAcb1!4Gal synthesis
Pulmonary pathogens such as those infecting cystic
fibrosis patients, Pseudomonas aeruginosa, Haemophilus
influenzae, and Staphylococcus aureus, were previously
shown to bind to the disaccharide sequence Gal-
NAcb1!4Gal present as part of glycolipids on the sur-
face of lung tissue.1 Conjugates of this sequence have
potential as antibacterial agents by preventing adhesion
and thus infection, as alternatives to conventional anti-
biotics. While we previously showed the synthesis and
evaluation of multivalent versions of the sequence,2
which resulted in a multivalent inhibition enhancement,
the synthesis of the disaccharide in a form ready for
conjugation had to be improved. The disaccharide syn-
thesis has been achieved in 1984 by Lemieux et al.3 but
required many steps and consequently had a low overall
yield. Since then, the synthesis of the GalNAcb1!4Gal
sequence was reported several times.4 However, in many
of these studies the sequence was part of a larger struc-
ture,4c–h necessitating the introduction of various pro-
tecting groups to allow attachment of additional sugar
residues, which again resulted in a low overall yield.
Furthermore, the obtained products were not always
in a form allowing further conjugation4a and often the
experimental data were incomplete or absent. Although
over time procedures have improved since the first
attempt, further improvement is needed for practical
applications. However, the sequence is challenging, since
it requires the attack of the relatively non-reactive axial
galactose C-4 hydroxyl. We here describe a facile syn-
thesis that has a 22% overall yield, requires few purifica-
tion steps, and the products are compatible with
conjugation to carboxylic acid or amine functionalities
or via copper-catalyzed cycloaddition with acetylenes,
the so-called ꢀclickꢁ chemistry.5 Besides application to
prevent microbial adhesion, for which the availability
of significant amounts of material has been a limiting
factor, further applications involving the association to
the bacterial surface include the use of carbohydrate
arrays,5c,6 for example, for bacterial typing for diagnos-
tic purposes.
In the synthesis, first peracetylated galactose 1 was
brominated at C-1 (Scheme 1).7 In order to make the
thiophenyl derivative 2, the bromide was exposed to a
mixture of NBu4HSO4 and thiophenol in aq sodium car-
bonate and EtOAc. The acetate groups of 2 were subse-
quently removed using sodium methoxide, which was
followed by treatment with a,a-dimethoxytoluene and
a catalytic amount of p-TsOH in CH3CN. This yielded
the corresponding benzylidene compound, that could
be purified by crystallization and resulted in 4 after acet-
ylation of the remaining hydroxyl groups. The prepara-
tion thus far required no chromatography, gave a 70%
*
Corresponding author. E-mail: R.J.Pieters@pharm.uu.nl
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.08.001

R. Autar et al. / Carbohydrate Research 340 (2005) 2436–2442
2437
Ph
O
OAc
AcO
AcO
OAc
AcO
AcO
O
O
O
O
c-e
g
a or b
R
R
AcO
OAc
OAc
OAc
OAc
1
2 : R = SPh
3 : R = OBn
4 : R = SPh
f
5: R = OCH2CO2Bn
6: R = OBn
OBn
HO
O
R
AcO
OAc
O
OAc
AcO
AcO
OAc
AcO
AcO
7: R = OCH2CO2Bn
8: R = OBn
O
h
i
OAc
O
OBn
O
HNAc
AcO
O
HNTroc
O
+
R
R
AcO
OAc
OAc
OAc
AcO
10: R = OCH2CO2Bn
11: R = OBn
12: R = OCH2CO2Bn
13: R = OBn
O
SPh
AcO
j
14 : R = OH
HNTroc
k
l
9
15 : R = OC(=N)CCl3
16 : R = O(CH2)6N3
Scheme 1. Reagents and conditions: (a) (i) HBr, Ref. 7, quant; (ii) NBu4HSO4, PhSH Na2CO3, EtOAc, 15 min prod. 2; (b) Ref. 11, 81% of 3; (c)
NaOMe, MeOH, 2 h; (d) PhCH(OMe)2, p-TsOH, CH3CN, 1.5 h; (e) Ac2O, py 18 h, 70% for 6 from 3; 70% for 4 from 1; (f) HOCH2CO2Bn, NIS
˚
(1.3 equiv), TfOH, CH2Cl2, ꢀ70 ꢁC, 1 h, 76%; (g) NaCNBH3, HCl–Et2O, THF, 3 h, 75–80%; (h) NIS, TfOH, CH2Cl2, MS 4 A, ꢀ70 ꢁC to rt, 2 h,
73% (10), 90% (11); (i) (i) Zn powder, Ac2O, 6 h; (ii) ZnCl2, Ac2O–AcOH, 48 h. 70% (12), 81% (13); (j) Pd–C, THF, 18 h; (k) CCl3CN, DBU, CH2Cl2,
1 h; (l) HOC6H12N3, TMSOTf, CH2Cl2, ꢀ70 ꢁC to 0 ꢁC, 2 h. 71% from 13.
yield from the starting galactoside 1 and was performed
on a multigram scale. Glycosylation with benzyl glyco-
late and NIS–TfOH8 in dichloromethane, furnished 5
in 76% yield (Table 1). Liberation of the 4-OH by reduc-
tive opening of the benzylidene ring by NaCNBH3 was
followed by NIS promoted glycosylation with donor 9,
which was prepared in three steps from D-(+)-galactos-
amine hydrochloride in 66% overall yield.2 This donor,
which was also readily made in multigram amounts,
contained a Troc protecting group reported to signifi-
cantly increase donor reactivity in coupling reactions
while effecting good b-selectivity as well.9 The coupling
resulted solely in the b-linked-disaccharide 10. The Troc
group was replaced by an acetyl group by the action of
Zn in Ac2O. In order to facilitate deprotection at the
multivalent or otherwise conjugated stage, it was
deemed preferable to have only a single type of protect-
ing group present on the sugar, that is, acetyl groups.
While this was partially achieved under the Troc con-
verting conditions, a report of Yang et al.10 proved use-
ful for the selective conversion of the 6-OBn by ZnCl2 in
AcOH–Ac2O to an acetate, yielding 12. This represents
a protected GalNAcb1!4Gal derivative, ready for con-
jugation to amine functionalized scaffolds or surfaces.
Similarly, a derivative was prepared that could be
conjugated to carboxylic acid functionalized scaffolds or
surfaces. While the previous synthesis necessitated the
early incorporation of the aglycon part, in this method
the use of an anomeric benzyl group allows for con-
jugation at the very end. As before, the synthesis started
with acetylated galactose which was now converted to
its C-1 benzyl functionalized derivative 3 by BF3ÆOEt2.11
The subsequent steps leading to the benzylidene deriva-
tive 6 and its ring opened counterpart 8 proceeded as
before, setting the stage for glycosylation with donor
9. The NIS-promoted glycosylation gave the desired b-
linked-product 11 in a high yield of 90%. Again the
two-step protecting group conversion by first Zn and
then ZnCl2 yielded 13. The anomeric benzyl group was
unaffected by the latter step, which selectively removed
the benzyl group at the sixth position, to our knowledge
a first observation of this type of selectivity. After
hydrogenation of 13, any desired spacer can be linked
to C-1 using trichloroimidate chemistry. To this end,
the hydrogenation product 14 was first converted to
the trichloroacetamidate 15 and subsequently coupled
in good yield to 6-azido-1-hexanol using TMSOTf as a
catalyst. This protected GalNAcb1!4Gal derivative
can be conjugated to carboxylic acid bearing scaffolds
or surfaces after hydrogenation or via copper-catalyzed
cycloaddition with acetylenes, the so-called ꢀclickꢁ
chemistry.5
In conclusion, we report a facile and efficient synthesis
of two linkable versions of the GalNAcb1!4Gal
sequence, an important sequence to which various bac-
terial pathogens bind.1,12 Of the two compounds, one

2438
R. Autar et al. / Carbohydrate Research 340 (2005) 2436–2442
Table 1. 1H NMR chemical shifts and coupling constants of the prepared compounds (CDCl3)
5
7
10
6
8
11
13
16
H-1
H-2
H-3
H-4
4.68
5.42
4.98
4.38
2.85
4.03
4.28
4.60
5.33
4.94
4.14
3.71
3.71
3.71
4.62
5.22
5.02
3.35
3.92
3.92
3.92
4.75
3.70
4.75
5.34
3.92
3.92
3.92
7.7
4.55
5.47
4.93
4.36
3.49
4.07
4.36
4.50
5.35
4.90
4.11
3.74
3.74
3.74
4.50
5.26
4.98
4.16
3.75
3.75
3.75
4.81
3.75
5.26
5.39
3.85
3.98
4.10
8.0
4.49
5.32
4.81
5.39
3.73
4.32
4.32
5.13
3.38
5.92
5.39
3.93
4.09
4.09
8.0
4.43
5.25
4.94
4.13
3.73
4.29
4.29
5.12
3.36
5.93
5.38
3.90
4.05
4.05
8.0
H-5
H-6a
H-6b
H-10
H-20
H-30
H-40
H-50
H-6a0
H-6b0
J1,2
8.0
8.0
8.0
8.0
J2,3
J3,4
10.4
3.6
10.1
3.0
10.2
3.0
10.2
10.2
10.2
2.7
10.4
10.4
2.5
J4,5
0
J1 ,2
0
0
0
8.2
11.5
3.3
8.2
11.3
3.3
0
J2 ,3
0
J3 ,4
CHPh
5.49
4.38
5.17
5.51
CH2C(O) (Linker)
CH2C6H5 (Linker)
CH2C6H5 (6-OBn)
CH2C6H5 (1-OBn)
4.34
5.16
4.54
4.35
5.17
4.53
4.57
4.62
4.90
4.59
4.63
4.81
4.81
4.65
4.93
4.64
4.81
CH2CCl3
4.75
OC6H12N3
1.37
1.59
3.27
3.48
3.90
contained a protected carboxylic acid while the other
featured a masked amino group, that is, an azide. They
were both synthesized in 22% overall yield or 15% when
considering the donor synthesis.
spectrometer (300 MHz) in CDCl3 and chemical shifts
are given in ppm relative to Me4Si (0 ppm). 13C NMR
spectra were recorded on a Varian G-300 spectrometer
at 75.4 MHz in CDCl3 (referenced to CDCl3 at
77.0 ppm) using the attached proton test (APT) se-
quence. Electrospray ionization (ESI) mass spectrome-
try was carried out using a Shimadzu LCMS QP-8000
single quadrupole benchtop mass spectrometer (m/z
range < 2000), coupled with a QP-8000 data system.
1. Experimental
1.1. General remarks
Chemicals were obtained from commercial sources and
used without further purification unless stated other-
wise. The solvents CH2Cl2, MeOH and dioxane were
purchased from Biosolve, the Netherlands. The solvents
1.2. Synthesis
1.2.1. Multi-step protocol for synthesis of phenyl 2,3-di-
O-acetyl-4,6-O-benzylidene-1-thio-b-D-galactopyranoside
(4)—optimized conditions. Acetylated galactose 1 was
obtained from galactose (2.5 g, 13.9 mmol) and con-
verted to the corresponding bromide.7 It was dissolved
in EtOAc (100 mL) and added to a mixture of Bu4NH-
SO4 (4.7 g, 13.9 mmol), thiophenol (2 mL, 19.5 mmol) in
1 M Na2CO3 (100 mL) and vigorously stirred at rt for
15 min. After the addition of EtOAc (5 mL), the organic
phase was separated and washed with 1 M NaOH
(50 mL), water (2 · 50 mL) and brine (50 mL), dried
(Na2SO4), concentrated, and dissolved in MeOH
(100 mL). In order to deprotect the hydroxyl groups,
˚
CH2Cl2 and MeOH were stored on molecular sieves, 4 A
˚
and 3 A, respectively. Activated molecular sieves were
prepared by flame-drying under diminished pressure fol-
lowed by a nitrogen flush. Column chromatography was
performed on E. Merck Kieselgel 60 (40–63 lm). TLC
analysis was performed using E. Merck pre-coated silica
gel 60 F-254 plates; spots were visualized by UV light
(254 nm) and/or by charring after treatment with 10%
H2SO4–MeOH. For neutralization, Dowex 50 · 8 (H+-
form; 20–50 mesh) purchased from Fluka was used.
1H NMR spectra were recorded on a Varian G-300

R. Autar et al. / Carbohydrate Research 340 (2005) 2436–2442
2439
NaOMe (33% v/v in MeOH, 2 mL) was added and the
mixture was stirred for 2 h, after which it was neutral-
ized with Dowex/H+ ion-exchange resin, filtered, and
concentrated under diminished pressure. Prior to benz-
ylidene formation the residue was subjected to high
vacuum, thereby removing all traces of MeOH and facil-
itating the attachment of a,a-dimethoxytoluene. The
product was suspended in CH3CN (10 mL), and after
the addition of a catalytic amount of p-TsOH (80 mg,
The reaction was monitored with TLC analysis
(2% MeOH–CH2Cl2). When completed, solid NaHCO3,
(ꢁ2 g), CH2Cl2 (40 mL) and satd aq NaHCO3 (25 mL)
were added, and the mixture was filtered over hyflo.
After the addition of additional CH2Cl2 (40 mL), the or-
ganic phase was washed with aq NaHCO3 (satd
2 · 50 mL), water (50 mL) and dried with Na2SO4. After
concentration, product 7 was purified with silica gel
column chromatography with 2% MeOH–CH2Cl2 as
an eluent. Yield: 0.96 g (80%, clear oil): 1H NMR
(300 MHz, CDCl3): d = 2.03, 2.08 (2 · s, 6H,
2 · C(O)CH3), 7.30 (m, 10H, 10 · Harom); 13C NMR
(75.4 MHz, CDCl3): d = 20.7, 20.8 (2 · OC(O)CH3),
64.5, 66.6, 69.0, 73.6 (C-6, OCH2CO, 2 · CH2C6H5),
67.6, 68.7, 73.0, 73.1 (C-2, C-3, C-4, C-5), 100.4 (C-1),
127.7–128.5 (10 · CHarom), 135.1, 137.4 (2 · Carom),
169.2, 169.9, 170.2 (3 · C@O); HRMS m/z calcd for
C26H30O10 [M+Na]+ 525.1737, found 525.1418.
˚
0.4 mmol), 3 A molecular sieves and a,a-dimethoxy-
toluene (3.11 mL, 20.9 mmol), the mixture was stirred
for 90 min at rt, yielding phenyl 4,6-O-benzylidene-1-
thio-b-D-galactopyranoside. After crystallization from
EtOH, this compound was obtained pure in 50% yield.
More material was harvested (up to 20%) after filtration
over a silica plug to remove excess a,a-dimethoxytolu-
ene, followed by another crystallization from EtOH.
The collected crystals were acetylated using Ac2O
(40 mL) and pyridine (60 mL) and after completion
the mixture was concentrated and coevaporated with
toluene, EtOH and CH2Cl2, giving 4 in 70% overall yield
(4.32 g, white solid). Spectroscopic data were consistent
with those in Ref. 13.
1.2.4. Benzyloxycarbonylmethyl O-(3,4,6-tri-O-acetyl-
2-deoxy-2-(20,20,20-trichloroethoxycarbonyl-amino))-(b-D-
galactopyranosyl)-(1!4)-2,3-di-O-acetyl-6-O-benzyl-b-
D-galactopyranoside (10). Compound 7 (0.96 g, 1.9
mmol) and 9 (0.87 g, 1.82 mmol) were dissolved in dry
CH2Cl2 (2.5 mL) and stirred under argon in the presence
1.2.2. Benzyloxycarbonylmethyl 2,3-di-O-acetyl-4,6-O-
benzylidene-b-D-galactopyranoside (5). To a soln of 4
(444 mg, 1 mmol) in dry CH2Cl2 (10 mL) were added
˚
of activated crushed 4 A molecular sieves. After 30 min,
the mixture was cooled to ꢀ70 ꢁC and NIS (580 mg,
2.4 mmol) and a cat. amount of TfOH were added.
The reaction mixture was stirred while the temperature
was allowed to rise to 20 ꢁC. Progression of the reaction
was checked with TLC analysis 1:1 EtOAc–hexane.
After 2 h, the reaction mixture was filtered over hyflo,
CH2Cl2 (to a volume of 150 mL) was added after which
the mixture was washed with aq NaHCO3 (satd,
100 mL), Na2SO3 (satd, 100 mL) and water (100 mL).
The organic phase was dried with Na2SO4 and concen-
trated under diminished pressure. Purified product
was obtained after silica gel column chromatography
with 2:3 EtOAc–hexane as eluent. Yield: 1.28 g (73%,
˚
4 A molecular sieves (crushed and activated) and benzyl
glycolate (117 lL, 1.2 mmol). The mixture was first
stirred at rt for 20 min under N2, after which it was
cooled to ꢀ70 ꢁC. Then, NIS (318 mg, 1.3 mmol) and
a cat. amount of TfOH (17 lL) was added and stirring
was continued for approx. 1 h (monitored by TLC,
1:1 EtOAc–hexane). When complete conversion was
reached, the mixture was filtered over Celite, taken up
in CH2Cl2 (75 mL) and subsequently washed with aq
NaHCO3 (sat., 75 mL), water (75 mL) and brine
(75 mL). The organic layer was dried using NaSO4,
concentrated under diminished pressure and subjected
to silica gel column chromatography with 1:2 EtOAc–
hexane as eluent. Yield: 380 mg (76%, white solid):
1H NMR (300 MHz, CDCl3): d = 2.05, 2.07 (2 · s,
6H, 2 · C(O)CH3), 7.34–7.51 (m, 10H, 10 · Harom);
13C NMR (75.4 MHz, CDCl3): d = 20.7, 20.9
(2 · OC(O)CH3), 64.0, 66.5, 68.6 (C-6, OCH2CO,
CH2C6H5), 66.3, 67.9, 71.6, 73.0 (C-2, C-3, C-4, C-5),
100.0, 100.9 (C-1, PhCH), 126.2–129.0 (10 · CHarom),
135.2, 137.3 (2 · Carom), 169.4, 169.7, 170.6 (3 · C@O);
HRMS m/z calcd for C26H28O10 [M+Na]+ 523.1580,
found 523.1535.
23
white solid): ½aꢂD ꢀ23.8 (c 1.0 CHCl3); 1H NMR
(300 MHz, CDCl3): d = 1.99, 2.00, 2.01, 2.12, 2.13
0
(5 · s, 15H, 5 · C(O)CH3), 5.51 (d, JNH,2 = 7.1 Hz,
1H, NH), 7.27–7.43 (m, 10H, 10 · Harom); 13C NMR
(75.4 MHz, CDCl3): d = 20.6 (5 · C(O)CH3), 52.8
(C-20), 61.3, 64.4, 66.6, 69.1, 73.4, 74.2 (6 · CH2),
66.5, 68.7, 68.9, 70.4, 72.9, 73.6, 73.7 (C-2, C-3, C-30,
C-4, C-40, C-5, C-50), 100.2, 101.0 (C-1, C-10), 127.5–
128.6 (10 · CHarom), 135.2, 137.9 (2 · Carom), 153.9
(C@O Troc), 169.3–170.3 (6 · C@O); HRMS m/z
calcd for C41H48Cl3NO19 [M+Na]+ 986.1784, found
986.1278.
1.2.3. Benzyloxycarbonylmethyl 2,3-di-O-acetyl-6-O-benz-
yl-b-D-galactopyranoside (7). Ethereal HCl was added
dropwise to a stirred mixture of 5 (1.19 g, 2.38 mmol),
NaCNBH3 (1.38 g, 21.9 mmol) and molecular sieves
1.2.5. Benzyloxycarbonylmethyl O-(3,4,6-tri-O-acetyl-2-
deoxy-2-acetamido-b-D-galactopyranosyl)-(1!4)-2,3,6-
tri-O-acetyl-b-D-galactopyranoside (12). Compound 10
(0.52 g, 0.54 mmol) was dissolved in Ac2O (50 mL),
˚
(4 A) in dry THF (20 mL) until gas evolution ceased.

2440
R. Autar et al. / Carbohydrate Research 340 (2005) 2436–2442
˚
excess Zn (2.5 g) was added and the mixture was vigor-
ously stirred for 6 h. When TLC analysis (2% MeOH in
CH2Cl2) showed that complete conversion had taken
place, the mixture was filtered over hyflo, the filter cake
was washed with CH2Cl2 (20 mL) and the filtrates were
concentrated under diminished pressure at 50 ꢁC, fol-
lowed by coevaporation with toluene (2 · 50 mL). The
crude Troc-deprotected residue was dissolved in a mix-
ture of Ac2O (8 mL) and AcOH (4 mL), ZnCl2 (0.55 g,
4.1 mmol) was added and the mixture was stirred for
48 h. After filtration over hyflo, the reaction mixture
was concentrated. The residue was taken up in CH2Cl2
(150 mL), washed with water (100 mL), NaHCO3 (satd,
100 mL), water (100 mL) and brine (100 mL). Finally,
compound 12 was obtained in pure form after column
chromatography with 4:1 EtOAc–hexane as eluent.
4.41 mmol) and 4 A molecular sieves were added to this
mixture, after which ethereal HCl was added dropwise
until TLC analysis (2% MeOH in CH2Cl2) revealed
complete conversion. The reaction mixture was
quenched with solid NaHCO3, (ꢁ1 g), CH2Cl2 (20 mL)
and satd NaHCO3 (5 mL) were added and the mixture
was filtered over hyflo. The organic phase was washed
with satd NaHCO3 (2 · 50 mL), water (50 mL) and
dried with Na2SO4. After concentration, the product
was purified by silica gel column chromatography with
2% MeOH–CH2Cl2 as an eluent. Yield: 160 mg (75%,
1
clear oil): H NMR (300 MHz, CDCl3): d = 1.99, 2.06
(2 · s, 6H, 2 · C(O)CH3), 7.25–7.37 (m, 10H, 10 ·
H
arom); 13C NMR (75.4 MHz, CDCl3): d = 20.6, 20.7
(2 · C(O)CH3), 67.7, 69.2, 73.1, 73.3 (C-2, C-3, C-4,
C-5), 69.0, 70.3, 73.6 (C-6, 2 · CH2C6H5), 99.7 (C-1),
127.5–128.4 (10 · CHarom), 136.9, 137.5 (2 · Carom),
169.5, 170.3 (2 · C@O); HRMS m/z calcd for
C24H28O8 [M+Na]+ 467.1682, found 467.1620.
23
Overall yield: 0.34 g (70%, white solid): ½aꢂD ꢀ27.2 (c
1.0 CHCl3); spectroscopic data were consistent with
those reported in Ref. 2; HRMS m/z calcd for
C35H45NO19 [M+Na]+ 806.2484, found 806.2555.
1.2.8. Benzyl (3,4,6-tri-O-acetyl-2-deoxy-2-(20,20,20-tri-
chloroethoxycarbonyl-amino))-(b-D-galactopyranosyl)-(1!
4)-2,3-di-O-acetyl-6-O-benzyl-b-D-galactopyranoside (11).
Donor 9 (213 mg, 0.44 mmol) and acceptor 8 (220 mg,
0.42 mmol) were dissolved in dry CH2Cl2 (10 mL), acti-
1.2.6. Benzyl 2,3-di-O-acetyl-4,6-O-benzylidene-b-D-
galactopyranoside (6). Compound 3, obtained from
galactose penta-acetate according to Ref. 11, (0.74 g,
81%, 1.62 mmol) was deacetylated after stirring for 2 h
in a solution of NaOMe (33% v/v in MeOH, 2 mL) in
MeOH (100 mL). After neutralization with Dowex/H+
ion-exchange resin, the resin was removed by filtration
and the filtrate was concentrated under diminished
pressure and coevaporated several times with CH2Cl2.
Thus obtained deacetylated intermediate was then sus-
pended in CH3CN (6 mL). Next, a,a-dimethoxytoluene
(363 lL, 2.43 mmol) and a cat. amount of p-TsOH
(9 mg) were added and the mixture was stirred. After a
few minutes, the product precipitated as a white solid.
More CH3CN was added (20 mL), and stirring was con-
tinued for 50 min. The solvent was evaporated under
diminished pressure and the benzylidene sugar was crys-
tallized from EtOH. Acetylation of the intermediate was
accomplished by stirring for 18 h in a mixture of Ac2O
(10 mL) and pyridine (15 mL). After completion of the
reaction, the mixture was concentrated and coevapo-
rated with toluene, EtOH and CH2Cl2, giving com-
pound 6.14 Overall yield: 0.50 g (70%, white solid): mp
˚
vated ground molecular sieves (4 A) were added and the
mixture was stirred under argon for 30 min. Then, the
mixture was cooled to ꢀ70 ꢁC and NIS (134 mg,
0.55 mmol) and a cat. amount of TfOH were added. Stir-
ring was continued for 2 h, while the mixture was allowed
to warm up to rt. Successful product formation was ob-
served by TLC analysis 1:1 EtOAc–hexane. The mixture
was filtered over hyflo, CH2Cl2 (40 mL) was added and
the mixture was washed with aq NaHCO3 (satd,
30 mL), Na2SO3 (satd, 30 mL) and water (30 mL).
The organic phase was then dried with NaSO4 and
concentrated under diminished pressure. The product
was obtained in pure form after silica gel column chroma-
tography with 1:2 EtOAc–hexane as eluent. Yield: 343 mg
23
(90%, white solid): mp 58 ꢁC; ½aꢂD ꢀ33.3 (c 1.0 CHCl3);
1H NMR (300 MHz, CDCl3): d = 1.97, 1.99, 2.00, 2.11,
2.14 (5 · s, 15H, 5 · C(O)CH3), 5.39 (m, 2H, 40-H,
NH), 7.33 (m, 10H, 10 · Harom); 13C NMR (75.4 MHz,
CDCl3): d = 20.5, 20.6 (5 · C(O)CH3), 52.7 (C-20), 61.2
(C-60), 66.4, 68.9, 69.4, 70.3, 73.1, 73.5, 73.9 (C-2, C-3,
C-30, C-4, C-40, C-5, C-50), 69.2, 70.1, 73.3, 74.1
(2 · CH2C6H5, C-6, CH2CCl3), 95.7 (CCl3), 99.4, 100.8
(C-1, C-10), 127.3–128.3 (10 · CHarom), 136.9, 138.0
(2 · Carom), 153.8, 168.9, 170.0, 170.2 (6 · C@O); HRMS
m/z calcd for C39H46Cl3NO17 [M+Na]+ 928.1729, found
928.0938.
1
188 ꢁC, lit.14 188–189 ꢁC; H NMR (300 MHz, CDCl3):
d = 2.02, 2.06 (2 · s, 6H, 2 · C(O)CH3), 7.28–7.54 (m,
10H, 10 · Harom); 13C NMR (75.4 MHz, CDCl3):
d = 20.6, 20.7 (2 · C(O)CH3), 66.2, 68.4, 71.8, 73.2 (C-
2, C-3, C-4, C-5), 68.7, 69.9 (C-6, CH2C6H5), 99.5,
100.8 (CHC6H5, C-1), 126.2–128.9 (10 · CHarom),
137.1, 137.4 (2 · Carom), 169.2, 170.6 (2 · C@O); ESI-
MS: m/z = 465.4 [M+Na]+.
1.2.9. Benzyl (3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-b-
D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl-b-D-galacto-
pyranoside (13). Compound 11 (395 mg, 0.436 mmol)
was dissolved in Ac2O (10 mL) and Zn powder (4 g)
1.2.7. Benzyl 2,3-di-O-acetyl-6-O-benzyl-b-D-galacto-
pyranoside (8). Compound 6 (250 mg, 0.48 mmol)
was dissolved in THF (10 mL). NaCNBH3 (277 mg,

R. Autar et al. / Carbohydrate Research 340 (2005) 2436–2442
2441
was added. In the second step, the intermediate still
bearing the benzyl group at C-6 was dissolved in a mix-
ture of Ac2O (10 mL) and AcOH (5 mL), to which
ZnCl2 (446, 3.27 mmol) was added. The product 13
was successfully obtained in 81% yield (256 mg, white
solid). Work-up as described above for 12, mp 70 ꢁC,
170.1, 170.4, 170.5, 170.8, 171.3 (7 · C@O); HRMS
m/z calcd for C32H48N4O17 [M+Na]+ 783.2912, found
783.3254.
Acknowledgements
23
25
lit.4b 82–92 ꢁC; ½aꢂD ꢀ33.6 (c 1.0 CHCl3), lit.4b ½aꢂD
ꢀ37.5 (c 0.64 CHCl3); 1H NMR (300 MHz, CDCl3):
d = 1.99–2.14 (7 · s, 21H, 7 · C(O)CH3), 6.01 (d,
These investigations were supported by the council for
Chemical Sciences of The Netherlands—Organization
for Scientific Research (CW-NWO).
0
JNH,2 = 6.9 Hz, 1H, NH), 7.28–7.38 (m, 5H, 5 · Harom);
13C NMR (75.4 MHz, CDCl3): d = 20.5, 20.7, 23.2
(7 · C(O)CH3), 53.2 (C-20), 61.4, 63.0 (C-6, C-60), 66.8,
67.8, 69.0, 70.1, 72.0, 72.6, 73.1 (C-2, C-3, C-30, C-4,
C-40, C-5, C-50), 70.4 (CH2C6H5), 98.7, 99.7 (C-1,
C-10), 127.6, 127.8, 128.2 (5 · CHarom), 169.4, 169.6,
170.0, 170.3, 170.4, 170.7, 171.2 (7 · C@O); HRMS
m/z calcd for C33H43NO17 [M+Na]+ 748.2429, found
748.1372.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2005.08.001.
1.2.10. 6-Azidohexyl (3,4,6-tri-O-acetyl-2-deoxy-2-acet-
amido-b-D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl-b-
D-galactopyranoside (16). Compound 13 (0.50 g,
0.689 mmol) was dissolved in THF (15 mL) and hydro-
genated overnight in a Parr-apparatus, using Pd(OH)2
on carbon (75 mg) as a catalyst. After complete conver-
sion to 14, the mixture was filtered over hyflo and con-
centrated under diminished pressure. Crude 14 was
dissolved in dry CH2Cl2 (10 mL) and stirred under N2.
DBU (82 lL, 0.55 mmol) and trichloroacetonitrile
(2.2 mL, 21.7 mmol) were added and the mixture was
stirred at rt for 1 h to yield 15. The reaction mixture
was coevaporated twice with toluene, after which the
concentrate was dissolved in dry CH2Cl2 (6 mL). To this
soln, 6-azido-1-hexanol (296 mg, 2.07 mmol) and
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