Synthesis of some amino and carboxy analogs of galabiose; evaluation as inhibitors of the pilus protein PapG(J)96 from Escherichia coli

Article abstract of DOI:10.1016/S0008-6215(98)00020-2

The 2'-amino-2'-deoxy, 6-amino-6-deoxy, and 6-carboxy analogs of the reference inhibitor 2(trimethylsilyl)ethyl (α-D-galactopyranosyl)-(1→4)- β-d-galactopyranoside were synthesized and evaluated as inhibitors of the binding of the Escherichia coli-derived pilus protein PapG(J96), using an ELISA assay. The inhibitory efficiencies (K(rel); relative to the reference inhibitor) were: 157, 13, and < 8, respectively. The results support the previously proposed combining site model, where the protein carries a negatively charged amino acid residue near HO-2' and HO-6 of the galabioside.

Full text of DOI:10.1016/S0008-6215(98)00020-2

Carbohydrate Research 307 (1998) 233±242
Synthesis of some amino and carboxy analogs
of galabiose; evaluation as inhibitors of the pilus
protein PapGJ96 from Escherichia coli
Henrik C. Hansen, Goran Magnusson *
Organic Chemistry 2, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124,
S-221 00 Lund, Sweden
Received 2 October 1997; accepted 20 December 1997
Abstract
The 20-amino-20-deoxy, 6-amino-6-deoxy, and 6-carboxy analogs of the reference inhibitor 2-
(trimethylsilyl)ethyl (ꢀ-d-galactopyranosyl)-(1!4)-ꢁ-d-galactopyranoside were synthesized and
evaluated as inhibitors of the binding of the Escherichia coli-derived pilus protein PapGJ96, using
an ELISA assay. The inhibitory eciencies (Krel; relative to the reference inhibitor) were: 157, 13,
and <8, respectively. The results support the previously proposed combining site model, where
the protein carries a negatively charged amino acid residue near HO-20 and HO-6 of the galabio-
side. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: Galabiose analogs; Escherichia coli inhibitors; PapGJ96 adhesin; Combining site model
1. Introduction
HO-20 and HO-6 of galabiose are situated in close
proximity to charged amino acid residues in the
respective protein binding sites [4,5,8]. Therefore,
changing these hydroxyl groups for amino or car-
boxyl groups might improve the binding strength
between the galabiose analogs and the protein, due
to the possible formation of salt bridges.
We now report the synthesis of the three gala-
biose analogs 1±3 (Fig. 1) as well as an investiga-
tion of their inhibitory eciency against adhesion
of puri®ed E. coli-derived PapGJ96 adhesin [9].
We are currently interested in the development
of improved inhibitors of the pathogens that utilize
the galabiose moiety [Gal(ꢀ1±4)Gal; present in cell
surface glycolipids of the globo series] as an
anchoring point in the early stages of an infective
process. The work is based on our previous inves-
tigations of the galabiose epitopes employed by
uropathogenic Escherichia coli [1±5] (a Gram-
negative species), the piglet pathogen Streptococcus
suis [6,7] (a Gram-positive species), and E. coli-
derived Verotoxin [8], for their adhesion to glyco-
lipids on the surface of cells.
2. Results and discussion
The epitope mappings of the E. coli-derived
proteins PapGJ96 and Verotoxin indicated that
The strategy used for the synthesis of com-
pounds 1±3 (Fig. 1) was based on functionalization
of monosaccharides, since such compounds would
* Corresponding author. Fax: 46 46 222 8209.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00020-2

234
H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
The glycosyl acceptor 9 [12] was prepared in
53% yield by partial benzoylation of the known
[13] 2-(trimethylsilyl)ethyl (TMSEt) glycoside 8.
Although the yield is modest, the single step trans-
formation is, in our hands, the most ecient route
to compound 9. Silver tri¯ate-promoted glycosyla-
tion of 9 with the donor 7 gave the disaccharide 10
(63%). Compound 10 was de-O-benzoylated with
sodium methoxide and the product was hydro-
genated to remove the benzyl groups and reduce
the azide group to an amino group, thus providing
the galabioside analog 1 (60%).
De-O-benzylidenation of 11 [13] (Scheme 2) gave
the partially protected TMSEt galactoside 12 [14]
(84%). Selective tosylation in the 6-position gave
the monotosylate 13 (70%). Treatment of 13 with
sodium azide in the presence of 15-crown-5 at
90 ꢀC gave the desired 6-azido-6-deoxy derivative
Fig. 1. Galabiose analogs 1±3, tested as inhibitors of PapGJ96
adhesin
be more generally useful as building blocks than
functionalized disaccharides.
Treatment of the known [10] glycosyl bromide 4
(Scheme 1) with thiocresol and potassium hydroxide
gave the corresponding thioglycoside [11], which
was benzylated, using benzyl bromide and sodium
hydride, to yield the thioglycoside 5 (46% overall).
Removal of the thiocresyl group by treatment with
N-iodosuccinimide gave the hemiacetal 6 (94%).
Treatment of 6 with oxalyl bromide furnished the
glycosyl bromide donor 7, which was used for gly-
cosylation without further puri®cation.
Scheme 2. (a) aq HOAc, 90 ꢀC, 1.5 h; (b) TsCl, pyridine,
45!22 ꢀC, 22 h; (c) NaN3, 15-crown-5, 90 ꢀC, 5 h; (d)
AgOTf, TMU, 45!22 ꢀC, 12 h; (e) MeONa±MeOH, 20 h,
then H2, Pd-C, EtOH±aq HCl, 12 h; (f) NaN3, 15-crown-5,
110 ꢀC, 3 d, then MeONa±MeOH.
Scheme 1. (a) HSPhMe, KOH, 4 h; (b) BnBr, NaH, 12 h; (c)
NIS, 10:1 MeCN±H2O, 5 min; (d) (COBr)2, DMF, 200 min; (e)
BzCl, 1:6 pyridine±Me2CO; (f) AgOTf, TMU, 45!22 ꢀC,
12 h; (g) MeONa±MeOH, 12 h, then H2, Pd-C, EtOH±aq HCl,
12 h.

H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
235
14 (48%) and the isomer 15 (26%; formed by
benzoate migration in 13 and/or 14). An analog of
13, carrying a tri¯ate instead of the tosylate group,
was investigated in an attempt to raise the yield of
14. Treatment with sodium azide was performed at
room temperature, which gave 14 in approximately
the same yield as above.
Silver tri¯ate-promoted glycosylation of 14 with
the known [15] galactosyl chloride 16 gave the di-
saccharide 17 (86%). De-O-benzoylation of 17 gave
18, and hydrogenation of the crude product furn-
ished the galabioside analog 2 (67% overall). An
alternative route to 18 was also investigated. The
tosylate 13 was glycosylated with 16, using silver
tri¯ate as promoter, to provide the disaccharide
tosylate 19 (72%). Substitution of the tosyl group
of 19 with sodium azide required longer reaction
time and higher temperature (3 days, 110 ꢀC),
which also caused some de-O-benzoylation to
occur. Treatment of the crude product with sodium
methoxide yielded 18 (83% overall).
methyl ester 23 was obtained in 81% yield by
treatment of 22 with trimethylsilyldiazomethane
[18]. N-Iodosuccinimide/tri¯ic acid-induced glyco-
sylation of 23 with the known [19] galactosyl donor
24 gave the disaccharide 25 (80%). Glycosylation
of uronic acid acceptors with thioglycoside donors,
giving high ꢀ-selectivity, was recently published
[20]. De-O-benzylation of 25, followed by ester
hydrolysis, furnished the galabioside analog 3
(98%).
The conformations of galabiose and its analogs
have been determined by NMR [21,22]. In addition
to coupling constants and NOE's, the chemical
shift of H-50 is a sensitive conformational probe. In
ordinary galabiosides, H-50 is situated close in
space to HO-3 (H-50±O-3 distance: <3 A), which
causes a strong (0.4±0.5 ppm relative to H-50 of
methyl ꢀ-d-galactopyranoside) deshielding of H-50.
In galabiose derivatives where O-3 is absent (as in
the deoxy analog [21]) or where a distortion
increases the H-5±O-3 distance [4], the deshielding
of H-50 is eliminated or reduced. In compounds 1±
3, the deshielding of H-50 amounts to 0.46, 0.40,
and 0.40 ppm, respectively, which clearly shows
that the overall conformations are similar to those
of other galabiosides.
Treatment of the known [15] acetal 20 (Scheme 3)
with iodine in methanol [16] gave the partially
benzylated galactoside 21 (96%). The hydro-
xymethyl group of 21 was oxidized by 2,2,6,6,-tet-
ramethylpiperidinyloxy radical (TEMPO) and
sodium hypochlorite [17] to give the galacturonic
acid derivative 22 (54%). The corresponding
The inhibitory eciencies of compounds 1±3
were determined essentially as described in earlier
investigations [2,4]. In short, a linker galabioside
was covalently bound to a microtiter plate to give
the glycoplate 26 (Fig. 2), and the inhibitors 1±3, as
well as the reference inhibitor 27, were serially
diluted in the wells of the plate. The puri®ed
PapGJ96 adhesin [9] (in complex with its chaperone
Scheme 3. (a) I2/MeOH, 4 d; (b) TEMPO, KBr, Bu4NBr,
NaOCl, NaHCO3, NaCl, 0 ꢀC, 100 min; (c) Me3SiCHN2,
22 ꢀC, 30 min; (d) NIS, F3CSO2OH, 1:2 CH2Cl2±Et2O,
65! 60 ꢀC, 4 h; (e) H2, Pd-C, 12 h, then aq NaOH, 4 h.
Fig. 2. Galabiose covalently linked to a microtiter plate (Gly-
coplate 26) and the refererence inhibitor 2-(trimethylsilyl)ethyl
galabioside 27.

236
H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
protein PapDJ96) was dissolved in PBS bu€er and
added to the wells. After incubation and washing
of the plate, the amount of bound protein was
determined by ELISA, using an anti-PapGJ96
antibody.
The inhibition data are depicted in Fig. 3. The
20-amino-20-deoxy compound 1 was ꢁ50% more
ecient than the reference compound 27, whereas
the 6-amino-6-deoxy compound 2 only retained
13% of the inhibitory eciency. The uronic acid
compound 3 was inecient as inhibitor.
The inhibition data support the binding pattern
suggested previously [1±5], where HO-20 and HO-6
are involved in a cooperative hydrogen bonding to
a carboxylic acid group in the PapGJ96 adhesin
(Fig. 4a). It should be noted that experimental evi-
dence for an intramolecular hydrogen bond
between HO-20 and HO-6 has been obtained by
(Krel=157) is consistent with a salt bridge between
the carboxylic acid group and protonated H2N-20
while maintaining the intramolecular hydrogen
bond, as depicted in Fig. 4b. The geometrical
requirements for intermolecular interactions are
obviously strict, since compound 2 with its amino
group in the 6-position has lost most of its binding
eciency (Krel=13). The lack of inhibitory e-
ciency of the carboxylic acid compound 3
strengthens the arguments for a carboxylic acid
group in the protein, since both groups would be
charged under the conditions used (pH 7.4) and
would therefore repel each other.
3. Experimental
General methods.ÐMelting points are uncor-
rected. NMR spectra were recorded on a 300 or a
NMR [4,21]. The increased binding of
1
Fig. 3. Eciency of compounds 1±3 and 27 as inhibitors of the binding of PapG/PapDJ96 complex to glycoplate 26. IC50, Krel and
ÁÁG values: 1 (1.4 mM, 157, 1.1 kJ mol 1), 2 (17.2 mM, 13, +5.0 kJ mol 1), 3 (>24.9 mM, <8, >+21.0 kJ mol 1), 27 (2.2 mM,
0.0 kJ mol 1).
Fig. 4. (a) The hydrogen bonding pattern of galabiosideÐPapGJ96 recognition, based on previous investigations [1±5]. (b) Suggested
hydrogen bonding and salt bridge formation in the recognition of compound 1 by PapGJ96
.

H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
237
400 MHz instrument. 1H NMR spectral assign-
ments were made by the double resonance techni-
was ®ltered through Celite and concentrated. The
residue was chromatographed (Varian Mega Bond
Elut Column C18; 9:1!8:2!7:3!6:4!5:5 H2O±
1
que (COSY) and chemical shifts for H resonances
are reported as though they were ®rst order. Con-
centrations were made using rotary evaporation
with a bath temperature at or below 40 ꢀC. Anhy-
drous Na2SO4 was used as drying agent for the
organic extracts in the workup procedures. Col-
umn chromatography was performed on SiO2
(Matrex LC-gel: 60A, 35-70 MY, Grace), and TLC
was performed on Kieselgel 60 F254 plates
(Merck). Compounds 4 [10], 9 [12], 11 [13], 16 [15],
20 [15], 24 [19], 26 [4], and 27 [13] were prepared as
described in the literature.
MeOH, 6 mꢀL of each) to give 2 (15.6 mg, 67%);
1
[ꢀ]25 +142 (c 0.2, H2O); H NMR (D2O): ꢂ 4.85
d
(d, 1 H, J 3.7 Hz, H-10), 4.36 (d, 1 H, J 7.8 Hz, H-
1), 4.18 (bt, 1 H, J 6.5 Hz, H-50), 3.93 (m, 1 H,
OCH2CH2Si), 3.90 (m, 2 H, H-40,4), 3.78 (dd, 1 H,
J 3.1, 10.6 Hz, H-30), 3.73 (dd, 1 H, J 3.6, 10.6 Hz,
H-20), 3.53±3.70 (m, 5 H, OCH2CH2Si, H-3,5,60),
3.39 (dd, 1 H, J 7.9, 10.1 Hz, H-2), 3.09 (dd, 1 H, J
7.9, 13.1 Hz, H-6a), 2.94 (dd, 1 H, J 3.9, 13.3 Hz,
H-6b), 1.81 (s, NH2), 0.90 (m, 2 H, CH2Si), 0.10
(s, 9 H, SiMe3); 13C NMR (D2O): ꢂ 102.5, 101.1,
79.3, 74.0, 72.9, 71.5, 71.3, 69.5, 69.3, 68.9, 68.7,
60.9, 40.8, 18.0, 2.2; HRMS calcd for
C17H35O10NSiNa (M+Na): 464.1928, found:
464.1942.
2-(Trimethylsilyl)ethyl (2-amino-2-deoxy-a-d-
galactopyranosyl)-(1!4) - b -d - galactopyranoside
(1).ÐCompound 10 was dissolved in MeOH
(10 mL) and the mixture was treated with a cataly-
tic amount of MeONa for 12 h, neutralized with
Duolite C436 (H+) resin, and concentrated. The
resulting residue was chromatographed (3:1
EtOAc±heptane). The puri®ed material was dis-
solved in a mixture of EtOH (10 mL) and 0.1 M aq
HCl (0.780 mL, 0.078 mmol), and hydrogenated
(H2, Pd-C, 1 atm). After 12 h the mixture was ®l-
tered through Celite and concentrated. The residue
was chromatographed (10:4:1 CH2Cl2±MeOH±
2-(Trimethylsilyl)ethyl (a-d-galactopyranosyl)-
(1!4)-b-d-galactopyranosiduronic acid (3).Ð
Compound 25 (99 mg, 0.098 mmol) was dissolved
in HOAc (10 mL) and hydrogenated (H2, Pd-C, 1
atm). After 12 h the mixture was concentrated. The
residue was dissolved in water (4 mL) and 2 M aq
NaOH (0.14 mL, 0.28 mmol) was added at ambient
temperature. After 4 h the mixture was neutralized
with Duolite C436 (H+) resin, ®ltered, and con-
centrated. The residue was chromatographed
(10:5:1 CH2Cl2±MeOH±H2O) to give 3 (44 mg,
Et3N) to give 1 (20.7 mg, 60%); [ꢀ]24 +87ꢀ (c
d
1
0.6, H2O); H NMR (D2O): ꢂ 4.92 (d, 1 H, J
3.8 Hz, H-10), 4.34 (d, 1 H, J 7.8 Hz, H-1), 4.24 (bt,
1 H, J 6.4 Hz, H-50), 3.95 (bd, 1 H, J 2.7 Hz, H-4),
3.91 (m, 1 H, OCH2CH2Si), 3.87 (bd, 1 H, J
2.7 Hz, H-40), 3.75 (dd, 1 H, J 3.1, 10.8 Hz, H-30),
3.54±3.74 (m, 7 H, OCH2CH2Si, H-3,5,6,60), 3.40
(dd, 1 H, J 7.8, 10.1 Hz, H-2), 3.04 (dd, 1 H, J 3.6,
10.9 Hz, H-20), 1.78 (s, NH2, partial exchange by
D2O), 0.90 (m, 2 H, CH2Si), 0.11 (s, 9 H, SiMe3);
13C NMR (D2O): ꢂ 102.6, 99.3, 76.9, 75.2, 73.0,
71.6, 71.3, 69.3, 68.7, 68.6, 61.0, 60.7, 51.4, 18.0,
2.2; HRMS calcd for C17H35O10NSiNa (M+Na):
464.1928, found: 464.1927.
98%); [ꢀ]22 +50ꢀ (c 1.0, H2O); H NMR data
1
d
(D2O): ꢂ 4.92 (d, 1 H, J 3.3 Hz, H-10), 4.34 (d, 1 H,
J 7.8 Hz, H-1), 4.24 (d, 1 H, J 2.7 Hz, H-4), 4.18
(bt, 1 H, J 6.2 Hz, H-50), 3.92±4.07 (m, 2 H, H-5
and OCH2CH2Si), 3.88 (bd, 1 H, J 3.0 Hz, H-40),
3.76 (dd, 1 H, J 3.2, 10.4 Hz, H-30), 3.53±3.69 (m, 4
H, H-20,60,3 and OCH2CH2Si), 3.41 (dd, 1 H, J 7.8,
9.9 Hz, H-2), 0.81±1.02 (m, 2 H, CH2Si), 0.1 (s, 9
H, SiMe3); 13C NMR (D2O): ꢂ 179.0, 102.1, 100.0,
78.2, 72.8, 71.3, 70.9, 69.6, 69.4, 69.0, 68.7, 61.0,
17.9, 2.1; HRMS calcd for C17H31O12SiNa2 (M-
H+2Na): 501.1380, found: 501.1383.
2-(Trimethylsilyl)ethyl (a-d-galactopyranosyl)-
(1!4)-6-amino-6-deoxy-b-d-galactopyranoside
(2).ÐCompound 17 (55 mg, 0.053 mmol) was dis-
solved in MeOH (8 mL) and the mixture was trea-
ted with a catalytic amount of MeONa for 20 h,
neutralized with Duolite C436 (H+) resin, and
concentrated. The resulting residue was chromato-
graphed (1:1 EtOAc±heptane). The puri®ed mate-
rial was dissolved in a mixture of EtOH (6 mL) and
0.1 M aq HCl (0.530 mL, 0.053 mmol), and hydro-
genated (H2, Pd-C, 1 atm). After 12 h the mixture
4-Methylphenyl 2-azido-3,4,6-tri-O-benzyl-2-
deoxy-1-thio-b-d-galactopyranoside (5).Ð3,4,6-Tri-
O-acetyl-2-azido-2-deoxy-ꢀ-d-galactopyranosyl
bromide [10] (4; 500 mg, 1.27 mmol) was dissolved
in CHCl3 (3 mL) and the mixture was added drop-
wise to a mixture of KOH (120 mg, 2.14 mmol),
thiocresol (190 mg, 1.53 mmol), and EtOH (3 mL)
over 1 h, followed by stirring at 22 ꢀC for 4 h.
CH2Cl2 and sat aq NaHCO3 were added and the
organic layer was dried and concentrated. The
residue was dissolved in DMF (2 mL) and benzyl

238
H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
bromide (0.59 mL, 4.96 mmol) and NaH (80% in
oil, 150 mg, 4.96 mmol) were added. After 12 h at
22 ꢀC, MeOH (5 mL) was added. After 30 min,
H2O (15 mL) and toluene (40 mL) were added. The
organic layer was washed with H2O, dried, and
concentrated. The residue was chromatographed
residue was chromatographed (1:50 EtOAc-
toluene) to give 9 (129 mg, 53%); mp 88±89 ꢀC
(from EtOAc±heptane); [ꢀ]25 +49ꢀ (c 1, CHCl3);
d
1H NMR (CDCl3): ꢂ 7.96±8.07 (6 H, Ph), 7.32±7.62
(9 H, Ph), 5.78 (dd, 1 H, J 7.9, 10.3 Hz, H-2), 5.37
(dd, 1 H, J 3.2, 10.3 Hz, H-3), 4.77 (d, 1 H, J
7.9 Hz, H-1), 4.71 (dd, 1 H, J 6.3, 11.3 Hz, H-6a),
4.63 (dd, 1 H, J 6.7, 11.4 Hz, H-6b), 4.38 (bs, 1 H,
H-4), 4.10 (bt, 1 H, J 6.7 Hz, H-5), 4.00±4.07 (m, 1
H, CH2CH2O), 3.64 (m, 1 H, CH2CH2O), 2.76 (bd,
1 H, J 4.9 Hz, HO-4), 0.82±1.02 (m, 2 H, CH2Si),
0.07 (s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 166.9,
166.4, 165.8, 128.7±133.0, 101.3, 74.8, 72.7, 70.2,
(1:5!1:3 EtOAc±heptane) to give 5 (339 mg, 46%);
[ꢀ]23
14ꢀ (c 0.9, CHCl3); H NMR (CDCl3): ꢂ
1
d
7.00±7.60 (19 H, Ph), 4.91 (d, 1 H, J 11.5 Hz,
CH2Ph), 4.76 (d, 1 H, J 11.7 Hz, CH2Ph), 4.69 (d, 1
H, J 11.5 Hz, CH2Ph), 4.56 (d, 1 H, J 11.5 Hz,
CH2Ph), 4.52 (d, 1 H, J 13.2 Hz, CH2Ph), 4.46 (d,
1 H, J 11.7 Hz, CH2Ph), 4.38 (d, 1 H, J 10.0 Hz, H-
1), 3.98 (d, 1 H, J 2.4 Hz, H-4), 3.84 (t, 1 H, J
9.9 Hz, H-2), 3.45 (dd, 1 H, J 2.7, 9.8 Hz, H-3),
2.33 (s, 3 H, PhCH3); 13C NMR (CDCl3): ꢂ 138.5,
138.1, 137.8, 137.5, 133.5, 129.7, 128.6, 128.5,
128.2, 128.04, 128.00, 127.9, 127.7, 127.5, 86.6,
82.5, 76.7, 74.4, 73.6, 72.4, 72.1, 68.5, 61.5, 21.3;
HRMS calcd for C34H35O4N3SNa (M+Na):
604.2246, found: 604.2246.
2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-galacto-
pyranosyl bromide (7).ÐCompound 5 (50 mg,
0.086 mmol) was dissolved in 10:1 MeCN±H2O
(1.3 mL), and N-iodosuccinimide (21.4 mg,
0.095 mmol) was added. The mixture was stirred
for 5 min at 22 ꢀC and Et3N (0.3 mL) and toluene
(5 mL) were added. The mixture was concentrated
and the residue was chromatographed (1:2 EtOAc±
heptane) to give 6 (38.5 mg, 94%); HRMS calcd
for C27H29O5N3Na (M+Na): 498.2005, found:
498.1991. Compound 6 (223 mg, 0.469 mmol) was
dissolved in CH2Cl2 (5 mL) and DMF (0.020 mL,
0.00026 mmol), and oxalyl bromide (0.050 mL,
0.534 mmol) was added. The mixture was stirred
under Ar at 22 ꢀC for 200 min. Cold toluene
(30 mL) and cold sat aq NaHCO3 (10 mL) were
added. The organic layer was dried and con-
centrated to give crude 7, which was used without
further puri®cation.
67.9, 67.8, 63.3, 18.4,
1.1 Anal. Calcd for
C32H36O9Si: C, 64.8; H, 6.1. Found: C, 64.5; H,
6.0.
2-(Trimethylsilyl)ethyl (2-azido-3,4,6-tri-O-benzyl-
2-deoxy-a-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-
benzoyl-b-d-galactopyranoside (10).ÐA mixture of
compound 9 [12] (152 mg, 0.256 mmol), tetra-
methylurea (0.090 mL, 0.75 mmol), silver tri¯ate
(145 mg, 0.564 mmol), molecular sieves (4A), and
toluene (4 mL) was cooled to 45 ꢀC, and a solu-
tion of 7 (253 mg, 0.469 mmol) in toluene (5 mL)
was added. The mixture was stirred for 12 h, while
the temperature was allowed to slowly reach
ꢁ22 ꢀC. The mixture was ®ltered through Celite
and concentrated, and the residue was chromato-
graphed (1:5 EtOAc±heptane) to give 10 (169 mg,
63%); [ꢀ]24 +72ꢀ (c 1.1, CHCl3); 1H NMR
d
(CDCl3): ꢂ 7.93±8.10 (6 H, Ph), 7.15±7.64 (24 H,
Ph), 5.76 (dd, 1 H, J 7.7, 10.6 Hz, H-2), 5.27 (dd, 1
H, J 2.9, 10.6 Hz, H-3), 5.00 (d, 1 H, J 3.5 Hz, H-
10), 4.77 (d, 1 H, J 7.7 Hz, H-l), 4.47 (d, 1 H, J
2.8 Hz, H-4), 4.44 (d, 1 H, J 11.0 Hz, CH2Ph), 4.34
(dd, 1 H, J 5.0, 9.3 Hz, H-5), 4.17 (dd, 1 H, J 2.5,
10.6 Hz, H-30), 4.04 (dd, 1 H, J 3.5, 10.6 Hz, H-20),
3.66 (dt, 1 H, J 6.6, 10.2 Hz, OCH2CH2Si), 3.39 (t,
1 H, J 8.5 Hz, H-60a), 2.91 (dd, 1 H, J 5.1, 8.4 Hz,
H-60b), 0.94 (m, 2 H, CH2Si), 0.05 (s, 9 H,
SiMe3); 13C NMR (CDCl3): ꢂ 166.4, 166.1, 165.4,
138.5, 138.1, 137.7, 133.4, 133.3, 133.1, 127.5±129.9
(Ar), 100.9, 99.9, 75.0, 74.9, 74.2, 73.2, 72.9, 72.3,
72.1, 69.73, 69.66, 67.5, 67.2, 62.0, 60.5, 18.0, 1.5;
HRMS calcd for C59H63O13N3SiNa (M+Na):
1072.4028, found: 1072.4022.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-b-d-
galactopyranoside (12).Ð2-(Trimethylsilyl)ethyl 2,3-
di-O-benzoyl-4,6-O-benzylidene-ꢁ-d-galactopyr-
anoside [13] (11; 900 mg, 1.56 mmol) was treated
with aq 80% HOAc (10 mL) at 90 ꢀC for 90 min.
The mixture was cooled to ꢁ22 ꢀC and CH2Cl2
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzoyl-b-d-
galactopyranoside (9) [12].ÐTo a solution of 2-
(trimethylsilyl)ethyl-b-d-galactopyranoside [13] (8;
116 mg, 0.414 mg) in dry acetone (0.8 mL) and dry
pyridine (0.132 mL) was added benzoyl chloride
(0.193 mL, 1.65 mmol) dropwise at 78 ꢀC. The
reaction was carefully monitored by TLC (1:2
EtOAc±heptane). When the amount of 9 (Rf 0.34)
was at its optimum, MeOH (0.1 mL) was added
and the temperature was allowed to reach ꢁ22 ꢀC.
The mixture was ®ltered, diluted with CH2Cl2,
washed with H2O, dried, and concentrated. The

H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
239
(30 mL) was added. The organic layer was washed
with sat aq NaHCO3, dried, and concentrated. The
residue was chromatographed (1:1 EtOAc±hep-
tane) to give crystalline 12 (641 mg, 84%); mp 117±
15 (36 mg, 26%) and 14 (67 mg, 48%). Compound
14 had: [ꢀ]22 +43ꢀ (c 0.5, CHCl3); H NMR
(CDCl3): ꢂ 7.25±8.05 (10 H, Ph), 5.73 (dd, 1 H, J
1
d
7.9, 10.3 Hz, H-2), 5.32 (dd, 1 H, J 3.2, 10.3 Hz, H-
3), 4.76 (d, 1 H, J 7.9 Hz, H-1), 4.26 (m, 1 H, H-4),
4.08 (m, 1 H, OCH2CH2Si), 3.90 (ddd, 1 H, J 1.0,
4.2, 8.2 Hz, H-5), 3.81 (dd, 1 H, J 8.0, 12.8 Hz, H-
6a), 3.64 (dt, 1 H, J 6.6, 10.2 Hz, OCH2CH2Si),
3.36 (dd, 1 H, J 4.3, 12.8 Hz, H-6b), 2.40 (d, 1 H, J
5.5 Hz, HO-4), 0.82±1.02 (m, 2 H, CH2Si), 0.06
(s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 166.3, 165.8,
134.0, 133.6, 130.3, 130.2, 130.0, 129.3, 128.9,
128.7, 101.2, 74.8, 74.5, 70.0, 68.4, 68.0, 51.4, 18.3,
1.1; HRMS calcd for C25H31O7N3SiNa
(M+Na): 536.1829, found: 536.1843. Compound
15 had: 1H NMR (CDCl3): ꢂ 7.41±8.22 (10 H, Ph),
5.56 (d, 1 H, J 3.6 Hz, H-4), 5.34 (dd, 1 H, J 7.8,
10.0 Hz, H-2), 4.74 (d,1 H, J 7.9 Hz, H-1), 3.93 (dd,
1 H, J 3.3, 8.6 Hz, H-5), 3.24 (dd, 1 H, J 3.4,
13.2 Hz, H-6), 2.78 (bd, 1 H, J 6.6 Hz, HO-3), 0.80±
1.08 (m, 2 H, CH2Si), 0.05 (s, 9 H, SiMe3).
120 ꢀC; [ꢀ]23 +77ꢀ (c 0.9, CHCl3); 1H NMR
d
(CDCl3): ꢂ 7.90±8.00 (m, 4 H, Ph), 7.22±7.54 (m, 6
H, Ph), 5.76 (dd, 1 H, J 8.0, 10.3 Hz, H-2), 5.29
(dd, 1 H, J 3.2, 10.3 Hz, H-3), 4.74 (d, 1 H, J
8.1 Hz, H-1), 4.42 (bd, 1 H, J 3.2 Hz, H-4), 3.78 (t,
1 H, J 5.4 Hz, H-5), 3.59 (dt, 1 H, J 6.6, 10.0 Hz,
OCH2CH2Si), 3.31 (bs, 1 H, OH), 2.82 (bs, 1 H,
OH), 0.90 (m, 2 H, CH2Si), 1.0 (s, 9 H, SiMe3);
13C NMR (CDCl3): ꢂ 166.1, 165.5, 133.4, 133.1,
129.9, 129.8, 129.7, 129.1, 128.4, 128.3, 101.0, 74.7,
74.2, 69.9, 68.0, 67.7, 62.2, 18.0, 1.5; HRMS
calcd for C25H32O8SiNa (M+Na): 511.1764,
found: 511.1774. The data were in good agreement
with those published [14].
2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-6-O-
tosyl-b-d-galactopyranoside (13).ÐCompound 12
(640 mg, 1.31 mmol) was dissolved in dry pyridine
(12 mL), and the mixture was cooled to 45 ꢀC
under Ar. p-Toluenesulfonyl chloride (tosyl chlor-
ide; 315 mg, 1.65 mmol) was added, and the mix-
ture was stirred for 22 h, while slowly raising the
temperature to ꢁ22ꢀC. CH2Cl2 (30 mL) and sat aq
NaHCO3 (10 mL) were added, and the organic
layer was dried and concentrated. The residue was
chromatographed (1:2 EtOAc±heptane) to give 13
2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzyl-
a-d-galactopyranosyl)-(1!4)-6-azido-2,3-di-O-
benzoyl-6-deoxy-b-d-galactopyranoside (17).ÐTo
a mixture of compound 14 (109 mg, 0.212 mmol),
N,N,N0,N0-tetramethylurea (0.039mL, 0.325 mmol),
molecular sieves (4A), and dry toluene (4 mL) was
added a solution of 2,3,4,6-tetra-O-benzyl-ꢀ-d-
galactopyranosyl
chloride
[15]
(16;160 mg,
(589 mg, 70%); [ꢀ]23 +50ꢀ (c 1.5, CHCl3); H
0.296 mmol) in dry toluene (7 mL). The mixture
was cooled to 45 ꢀC and silver tri¯ate (82 mg,
0.319 mmol) was added. The mixture was stirred
under Ar for 12 h, while slowly raising the tem-
perature to ꢁ22 ꢀC. The mixture was ®ltered
through Celite and concentrated, and the residue
was chromatographed (1:4 EtOAc±heptane) to give
1
d
NMR (CDCl3): ꢂ 7.32±8.00 (14 H, Ph), 5.66 (dd, 1
H, J 8.0, 10.3 Hz, H-2), 5.27 (dd, 1 H, J 3.2,
10.3 Hz, H-3), 4.68 (d, 1 H, J 7.9 Hz, H-1), 3.56 (dt,
1 H, J 6.8, 10.0 Hz, OCH2CH2Si), 2.42 (s, 3 H,
PhCH3), 2.24 (d, 1 H, J 5.9 Hz, HO-4), 0.87 (m, 2
H, CH2Si), 0.08 (s, 9 H, SiMe3); 13C NMR
(CDCl3): ꢂ 166.3, 165.8, 145.6, 133.9, 133.5, 132.8,
130.4, 130.3, 130.2, 130.0, 129.3, 128.9, 128.7,
128.5, 101.1, 74.6, 72.5, 70.0, 68.3, 68.0, 67.3,
22.1, 18.4, 1.0; HRMS calcd for C32H38O10SSiNa
(M+Na): 665.1853, found: 665.1866.
2-(Trimethylsilyl)ethyl 6-azido-2,3-di-O-benzoyl-
6-deoxy-b-d-galactopyranoside (14) and 2-(tri-
methylsilyl)ethyl 6-azido-2,4-di-O-benzoyl-6-deoxy-
b-d-galactopyranoside (15).ÐTo a solution of
compound 13 (174 mg, 0.271 mmol) in DMF
(4 mL) and 15-crown-5 (0.055 mL, 0.278 mmol)
was added NaN3 (110 mg, 1.69 mmol). The mixture
was stirred at 90 ꢀC for 5 h, and cooled to 22 ꢀC.
H2O and CH2Cl2 were added. The organic layer
was dried and concentrated, and the residue was
chromatographed (1:5!1:3 EtOAc±heptane) to give
17 (189 mg, 86%); [ꢀ]22 +89ꢀ (c 0.7, CHCl3); H
1
d
NMR (CDCl3): ꢂ 5.73 (dd, 1 H, J 7.8, 10.6 Hz, H-
2), 5.19 (dd, 1 H, J 2.9, 10.6 Hz, H-3), 4.92 (d, 1 H,
J 11.4 Hz, CH2Ph), 4.90 (d, 1 H, J 11.1 Hz,
CH2Ph), 4.84 (d, 1 H, J 11.9 Hz, CH2Ph), 4.81 (d, 1
H, J 3.6 Hz, H-10), 4.80 (d, 1 H, J 11.8 Hz, CH2Ph),
4.73 (d, 1 H, J 7.8 Hz, H-1), 4.62 (d, 1 H, J 11.4 Hz,
CH2Ph), 4.55 (d, 1 H, J 11.2 Hz, CH2Ph), 4.36
(bdd, 1 H, J 5.2, 9.4 Hz, H-50), 4.25 (d, 1 H, J
11.9 Hz, CH2Ph), 4.24 (d, 1 H, J 2.9 Hz, H-4), 4.21
(d, 1 H, J 11.9 Hz, CH2Ph), 4.16 (bs, 1 H, H-40),
4.12 (m, 1 H, OCH2CH2Si), 4.09 (dd, 1 H, J 5.5,
9.7 Hz, H-30), 4.04 (dd, 1 H, J 3.4, 10.1 Hz, H-20),
3.76±3.85 (m, 2 H, H-5,6a), 3.64 (dt, 1 H, J 6.7,
10.2 Hz, OCH2CH2Si), 3.49 (t, 1 H, J 8.7 Hz, H-
60a), 3.21 (dd, 1 H, J 8.9, 18.2 Hz, H-6b), 3.13 (dd,

240
H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
1 H, J 5.0, 8.5 Hz, H-60b), 0.96 (m, 2 H, CH2Si),
0.03 (s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 167.0,
165.7, 127.8±139.3, 101.6, 101.2, 79.0, 75.5, 75.4,
74.8, 74.75, 73.5, 72.5, 70.2, 70.1, 68.0, 67.9, 51.6,
18.3, 1.0; HRMS calcd for C59H65O12N3SiNa
(M+Na): 1058.4235, found: 1058.4243.
H-2), 4.93 (d, 1 H, J 11.2 Hz, CH2Ph), 4.90 (d, 1 H,
J 11.5 Hz, CH2Ph), 4.82 (dd, 1 H, J 3.0, 10.6 Hz,
H-3), 4.81 (d, 1 H, J 11.7 Hz, CH2Ph), 4.77 (d, 1 H,
J 11.7 Hz, CH2Ph), 4.70 (d, 1 H, J 2.9 Hz, H-10),
4.62 (d, 1 H, J 11.5 Hz, CH2Ph), 4.60 (d, 1 H, J
11.2 Hz, CH2Ph), 4.48 (s, 2 H, CH2Ph), 4.46 (d, 1
H, J 7.8 Hz, H-1), 4.30 (bdd, 1 H, J 5.2, 8.5 Hz, H-
50), 4.15 (bs, 1 H, H-40), 3.99±4.09 (m, 3 H, H-20,30
and OCH2CH2Si), 3.95 (d, 1 H, J 2.6 Hz, H-4),
3.47-3.77 (m, 5 H, H-60,6a,5 and OCH2CH2Si),
3.13 (dd, 1 H, J 4.1, 12.9 Hz, H-6b), 2.06 (s, 3 H,
Ac), 1.92 (s, 3 H, Ac), 0.85±1.05 (m, 2 H, CH2Si),
0.02 (s, 9 H, SiMe3).
2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzyl-
a-d-galactopyranosyl)-(1!4)-6-azido-6-deoxy-b-d-
galactopyranoside (18).ÐCompound 19 (191 mg,
0.164 mmol) was dissolved in a mixture of dry
DMF (8 mL) and 15-crown-5 (0.035 mL,
0.164 mmol). NaN3 (75 mg, 1.15 mmol) was added
and the mixture was stirred at 110 ꢀC for 3 days,
then cooled to ꢁ22 ꢀC. H2O (10 mL) and CH2Cl2
(25 mL) were added and the organic layer was iso-
lated, dried, and concentrated. The crude material
was dissolved in MeOH and a catalytic amount of
MeONa was added. After 12 h, the mixture was
neutralized with Duolite 436 (H+) resin and con-
centrated. The residue was chromatographed (1:2
2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzyl-
a-d-galactopyranosyl)-(1!4)-2,3-di-O-benzoyl-6-
O-tosyl-b-d-galactopyranoside (19).ÐTo a mixture
of compound 13 (300 mg, 0.467 mmol), compound
16 [15] (313 mg, 0.56 mmol), N,N,N0,N0-tetra-
methylurea (0.069 mL, 0.575 mmol), molecular
sieves (4A), and dry toluene (14 mL), was added
silver tri¯ate (145 mg, 0.564 mmol) at 45 ꢀC under
Ar. The temperature was slowly raised to ꢁ22 ꢀC.
After 12 h, the mixture was ®ltered through Celite
and concentrated, and the residue was chromato-
graphed (1:3 EtOAc±heptane) to give 19 (390 mg,
EtOAc±heptane) to give 18 (113 mg, 83%); [ꢀ]22
d
+8ꢀ (c 1.1, CHCl3); 1H NMR (CDCl3): ꢂ 7.15±7.45
(20 H, Ph), 4.94 (d, 1 H, J 11.5 Hz, CH2Ph), 4.89
(d, 1 H, J 4.2 Hz, H-l0), 4.87 (d, 1 H, J 11.8 Hz,
CH2Ph), 4.83 (d, 1 H, J 11.8 Hz, CH2Ph), 4.79 (d, 1
H, J 11.8 Hz, CH2Ph), 4.63 (d, 1 H, J 11.7 Hz,
CH2Ph), 4.56 (d, 1 H, J 11.6 Hz, CH2Ph), 4.47 (d, 1
H, J 11.5 Hz, CH2Ph), 4.39 (d, 1 H, J 11.4 Hz,
CH2Ph), 4.27 (d, 1 H, J 7.5 Hz, H-1), 4.16 (bdd, 1
H, J 3.5, 8.6 Hz, H-50), 3.93±4.12 (m, 3 H, H-20,30
and OCH2CH2Si), 3.90 (bd, 1 H, J 1.9 Hz, H-40),
3.78 (dd, 1 H, J 8.2, 13.1 Hz, H-6a), 3.68 (bd, 1 H,
J 1.7 Hz, H-4), 3.54±3.69 (m, 3 H, H-5, 60a and
OCH2CH2Si), 3.48 (dd, 1 H, J 7.6, 10.1 Hz, H-2),
3.37 (bt, 1 H, J 10.6 Hz, H-3), 3.30 (dd, 1 H, J 3.9,
9.6 Hz, H-60b), 3.15 (dd, 1 H, J 3.8, 13.2 Hz, H-6b),
2.38 (s, 1 H, OH), 2.12 (bs, 1 H, OH), 0.97±1.16
(m, 2 H, CH2Si), 0.03 (s, 9 H, SiMe3); 13C NMR
(CDCl3): ꢂ 138.80, 138.78, 138.6, 138.0, 128.93,
128.9, 128.8, 128.7, 128.6, 128.4, 128.3, 128.25,
128.1, 127.9, 103.2, 101.3, 82.0, 79.2, 76.8, 75.2,
75.1, 75.0, 74.54, 74.5, 74.2, 73.3, 72.4, 71.7, 70.2,
68.0, 51.9, 18.6, 1.0; HRMS calcd for C45H57
O10N3SiNa (M+Na): 850.3711, found: 850.3723.
An analytical sample was acetylated in 1:1 pyr-
idine±Ac2O (4 mL) overnight. The mixture was
concentrated and the residue was chromato-
graphed (1:3 EtOAc±heptane) to give 2-(trimethyl-
silyl)ethyl (2,3,4,6-tetra-O-benzyl-ꢀ-d-galactopyr-
anosyl)-(1!4)-2,3-di-O-acetyl-6-azido-6-deoxy-ꢁ-
72%); [ꢀ]24 +69ꢀ (c 0.9, CHCl3); 1H NMR
d
(CDCl3): ꢂ 7.19±7.99 (34 H, Ph), 5.70 (dd, 1 H, J
7.7, 10.5 Hz, H-2), 5.19 (dd, 1 H, J 2.9, 10.5 Hz, H-
3), 4.85 (d, 1 H, J 11.1 Hz, CH2Ph), 4.72±4.78 (m, 3
H, CH2Ph), 4.76 (d, 1 H, J 3.4 Hz, H-10), 4.69 (d, 1
H, J 7.8 Hz, H-1), 4.61 (d, 1 H, J 12.1 Hz, CH2Ph),
4.61 (dd, 1 H, J 5.4, 10.9 Hz, H-6a), 4.50 (d, 1 H, J
11.1 Hz, CH2Ph), 4.41 (dd, 1 H, J 6.9, 11.0 Hz, H-
6b), 4.29±4.36 (m, 2 H, H-4,50), 4.20 (d, 1 H, J
11.9 Hz, CH2Ph), 4.15 (d, 1 H, J 11.9 Hz, CH2Ph),
4.10 (bs, 1 H, H-40), 3.99±4.08 (m, 3 H, H-30,5 and
OCH2CH2Si), 3.97 (dd, 1 H, J 3.3, 10.2, H-20), 3.61
(dt, 1 H, J 6.8, 9.9 Hz, OCH2CH2Si), 3.44 (t, 1 H, J
8.8 Hz, H-60a), 3.06 (dd, 1 H, J 5.0, 8.5 Hz, H-60b),
2.4 (s, 3 H, PhCH3), 0.93 (m, 2 H, CH2Si), 0.04
(s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 166.8, 165.7,
145.3, 127.8±139.3, 101.4, 101.2, 79.4, 76.6, 76.2,
75.4, 75.1, 74.4, 74.1, 73.4, 73.0, 70.2, 70.0, 69.0,
68.0, 67.9, 22.0, 18.4, 1.0; HRMS calcd for
C66H72O15SSiNa (M+Na): 1187.4259, found:
1187.4250.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-b-d-galacto-
pyranoside (21).Ð2-(Trimethylsilyl)ethyl 2,3-di-O-
benzyl-4,6-O-benzylidene-b-d-galactopyranoside
[15] (20; 30 mg, 0.055 mmol) was dissolved in
CH2Cl2 (1 mL) and a solution of iodine (10 mg) in
MeOH (2 mL) was added. After 4 days, aq 10%
1
d-galactopyranoside; H NMR (CDCl3): ꢂ 7.24±
7.44 (m, 20 H, Ph), 5.23 (dd, 1 H, J 7.8, 10.6 Hz,

H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
Na2S2O7 was added. The organic layer was dried
give 23 (77 mg, 81%); [ꢀ]21
241
2ꢀ (c 1.8, CHCl3); 1H
d
and concentrated and the residue was chromato-
graped (2:1 EtOAc±heptane) to give 21 (24 mg,
NMR (CDCl3): ꢂ 7.28±7.41 (m, 10 H, Ph), 4.95 (d,
1 H, J 11.1 Hz, CH2Ph), 4.71±4.79 (m, 3 H,
CH2Ph), 4.39 (d, 1 H, J 7.7 Hz, H-1), 4.34 (bs, 1 H,
H-4), 4.07±4.16 (m, 1 H, OCH2CH2Si), 4.07 (bs, 1
H, H-5), 3.84 (s, 3 H, COOMe), 3.69 (dd, 1 H, J
7.7, 9.4 Hz, H-2), 3.58 (dd, 1 H, J 3.4, 9.3 Hz, H-3),
3.57±3.65 (m, 1 H, OCH2CH2Si), 2.57 (bs, 1 H,
HO-4), 1.00±1.13 (m, 2 H, CH2Si), 0.04 (s, 9 H,
SiMe3); 13C NMR (CDCl3): ꢂ 169.0, 139.0, 138.0,
129.0, 128.7, 128.5, 128.4, 128.3, 128.1, 103.3, 80.2,
78.8, 75.6, 74.1, 72.9, 68.4, 68.1, 53.0, 18.9, 1.0;
HRMS calcd for C26H36O7SiNa (M+Na):
511.2128, found: 511.2125.
96%); [ꢀ]21 +2ꢀ (c 1.0, CHCl3); 1H NMR
d
(CDCl3): ꢂ 7.25±7.42 (10 H, Ph), 4.95 (d, 1 H, J
11.1 Hz, CH2Ph), 4.70±4.79 (m, 3 H, CH2Ph), 4.40
(d, 1 H, J 7.7 Hz, H-1), 3.94±4.09 (m, 3 H, H-4,6
and OCH2CH2Si), 3.79±3.89 (m, 1 H, H-6), 3.66
(dd, 1 H, J 7.8, 9.3 Hz, H-2), 3.58±3.66 (m, 1 H,
OCH2CH2Si), 3.52 (dd, 1 H, J 3.5, 9.4 Hz, H-3),
3.47 (bt, 1 H, J 5.7 Hz, H-5), 2.65 (s, 1 H, HO-4),
2.20 (bs, 1 H, HO-6), 1.02±1.10 (m, 2 H, CH2Si),
0.04 (s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 139.1,
138.2, 128.9, 128.7, 128.5, 128.4, 128.3, 128.1,
103.8, 80.9, 79.4, 75.6, 74.3, 73.0, 68.0, 67.95,
63.0, 19.0, 1.0; HRMS calcd for C25H36O6SiNa
(M+Na): 483.2179, found: 483.2173.
Methyl [(2,3,4,6-tetra-O-benzyl-a-d-galactopyrano-
syl)-(1!4)-2,3-di-O-benzyl-b-d-galactopyranosid]
uronate (25).ÐA mixture of phenyl 2,3,4,6-tetra-O-
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-b-d-galacto-
pyranosiduronic acid (22).ÐTo a solution of com-
pound 21 (235 mg, 0.51 mmol) in CH2Cl2 (1.5 mL)
was added 2,2,6,6,-tetramethylpiperidinyloxy radi-
cal (TEMPO; 2 mg, 0.013 mmol), followed by a
solution of KBr (5.6 mg) and tetrabutylammonium
bromide (8.6 mg) in sat aq NaHCO3 (1.1 mL). The
mixture was cooled to 0 ꢀC and 7:3:6 aq NaOCl
(tech.)±sat aq NaHCO3±sat aq NaCl (4.5 mL) was
added dropwise until the reaction was completed
(100 min). The organic layer was dried and con-
centrated and the residue was chromatographed
(15:5:1 EtOAc±heptane±HOAc) to give 22 (130 mg,
benzyl-1-thio-ꢁ-d-galactopyranoside
(24)
[19]
(124 mg, 0.197 mmol), compound 23 (60 mg,
0.123 mmol), N-iodosuccinimide (48 mg, 0.213
mmol), molecular sieves (4A, 1 g), CH2Cl2 (3 mL)
and Et2O (6 mL) was cooled to 78 ꢀC under N2,
and tri¯ic acid (0.005 mL, 0.00006 mmol) was
added. The temperature was kept between 60 and
65 ꢀC. After 4 h, Et3N (1 mL) was added and the
mixture was allowed to reach ꢁ22 ꢀC. The mixture
was ®ltered through Celite, and Et2O (20 mL) was
added. The mixture was washed with sat aq
NaHCO3, the organic layer was dried and con-
centrated, and the residue was chromatographed
54%); [ꢀ]21
7ꢀ (c 0.8, CHCl3); 1H NMR
(1:7 EtOAꢀc±heptane) to give 25 (99 mg, 80%);
d
1
(CDCl3): ꢂ 7.27±7.41 (m, 10 H, Ph), 4.93 (d, 1 H, J
11.1 Hz, CH2Ph), 4.69±4.79 (m, 3 H, CH2Ph), 4.45
(d, 1 H, J 7.7 Hz, H-1), 4.36 (dd, 1 H, J 1.3, 3.3 Hz,
H-4), 4.02±4.13 (m, 2 H, H-5 and OCH2CH2Si),
3.60±3.77 (m, 2 H, H-2 and OCH2CH2Si), 3.58 (dd,
1 H, J 3.3, 9.3 Hz, H-3), 1.01±1.10 (m, 2 H, CH2Si),
0.04 (s, 9 H, SiMe3); 13C NMR (CDCl3): ꢂ 170.6,
138.8, 137.8, 129.0, 128.8, 128.5, 128.4, 128.2,
103.3, 80.0, 78.6, 75.7, 74.0, 73.0, 68.7, 68.0, 18.9,
1.0; HRMS calcd for C25H34O7SiNa (M+Na):
497.1972, found: 497.1962.
Methyl [2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-
b-d-galactopyranosid]uronate (23).ÐCompound
22 (91 mg, 0.192 mmol) was dissolved in a mixture
of MeOH (1 mL) and benzene (3.5 mL). Tri-
methylsilyldiazomethane [18] (2 M in hexane;
0.125 mL, 0.250 mmol) was added and the mixture
was stirred for 30 min. HOAc was added until the
yellow color disappeared. Toluene (10 mL) was
added and the mixture was concentrated. The resi-
due was chromatographed (1:3 EtOAc±heptane) to
[ꢀ]22 +30 (c 3.1, CHCl3); H NMR (CDCl3): ꢂ
d
7.20±7.45 (m, 30 H, Ph), 5.14 (d, 1 H, J 2.3 Hz, H-
10), 4.92 (d, 2 H, J 11.2 Hz, CH2Ph), 4.69±4.83 (m,
6 H, CH2Ph), 4.64 (d, 1 H, J 12.5 Hz, CH2Ph), 4.56
(d, 1 H, J 11.3 Hz, CH2Ph), 4.47 (d, 1 H, J 2.6 Hz,
H-4), 4.39 (d, 1 H, J 7.6 Hz, H-1), 4.35±4.42 (m, 1
H, H-50), 4.32 (d, 1 H, J 11.9 Hz, CH2Ph), 4.28 (d,
1 H, J 11.8 Hz, CH2Ph), 4.12±4.21 (m, 1 H,
OCH2CH2Si), 3.98±4.08 (m, 4 H, H-5,20,30,40), 3.77
(dd, 1 H, J 7.7, 9.8 Hz, H-2), 3.59±3.69 (m, 1 H,
OCH2CH2Si), 3.55 (s, 3 H, COOMe), 3.46±3.57
(m, 2 H, H-3,60a), 3.39 (dd, 1 H, J 5.4, 8.8 Hz, H-
60b), 1.03±1.19 (m, 2 H, CH2Si), 0.06 (s, 9 H,
SiMe3); 13C NMR (CDCl3): ꢂ 169.0, 139.4, 127.7,
104.0, 99.6, 80.9, 79.2, 79.0, 76.6, 75.7, 75.6, 75.4,
75.3, 74.0, 73.7, 73.3, 73.1, 73.0, 70.2, 68.9, 68.4,
52.6, 19.0, 0.9; HRMS calcd for C60H70O12SiNa
(M+Na): 1033.4534, found: 1033.4530.
Covalent coupling of galabioside to microtiter
plates.ÐMicrotiter plates functionalized with sec-
ondary amino groups (CovaLink1) microtiter

242
H.C. Hansen. G. Magnusson/Carbohydrate Research 307 (1998) 233±242
plates, A/S Nunc, P.O. Box 280, Kamstrup, DK-
4000 Roskilde, Denmark) were used for covalent
coupling of a carboxylic acid-functionalized gala-
bioside, as previously described [4,5], furnishing
the glycoplate 26 (Fig. 2).
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ELISA assay. Competitive inhibition of the PapG/
PapDJ96 complex by 1, 2, and 3.ÐThe PapG/
1
PapDJ96 complex [9] (0.1 mg mL in 2% BSA/
1
PBS, 0.050 mL microtiter well ) was added to the
glycoplate, containing 0.050 mL of the inhibitors
1±3 and the reference inhibitor 27, serially diluted
three times between each well. Every inhibitor was
investigated two or three times in order to obtain
mean values. After 45 min incubation, the plates
were washed three times with PBS and then
blocked for nonspeci®c binding by a 1 h incubation
with 0.4 mL 2% BSA in PBS. The PapG/PapDJ96
complex was detected by incubation with primary
anti-PapG/PapDJ96 rabbit antiserum (diluted 1/500
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and J. Kihlberg, J. Carbohydr. Chem., 13 (1994)
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1
in 2% BSA in PBS, 100 ꢃL well ) for 1 h, fol-
lowed by washing (three times) with Covabu€er
and addition of alkaline phosphatase-conjugated
antirabbit IgG (Sigma A7539, diluted 1/5000) and
phosphatase substrate 104 (Sigma 104). The optical
density was read at 405 nm. Incubations were at
24 ꢀC unless otherwise stated. The results are
shown in Fig. 3.
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Acknowledgements
We are grateful to Professor Scott Hultgren for a
generous gift of PapG/PapDJ96 protein. This work
was supported by grants from the Swedish Natural
Science Research Council and the National Insti-
tutes of Health (NIH, USA).
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