Synthesis of heteroanalogues of disaccharides as potential inhibitors of the processing mannosidase Class I enzymes

Article abstract of DOI:10.1016/S0008-6215(98)00162-1

Three disaccharide analogues of α-D-Man-(1→2)-α-D-Man-OMe in which the ring and/or glycosidic oxygen atoms have been replaced by sulfur have been synthesized as potential inhibitors of Class I mannosidase enzymes. Glycosylation of appropriately protected

Full text of DOI:10.1016/S0008-6215(98)00162-1

Carbohydrate Research 310 (1998) 17±25
Synthesis of heteroanalogues of disaccharides as
potential inhibitors of the processing
mannosidase Class I enzymes
Blair D. Johnston, B. Mario Pinto *
Department of Chemistry and Protein Engineering Network of Centres of Excellence, Simon Fraser
University, Burnaby, BC, Canada V5A 1S6
Received 1 February 1998; accepted 1 May 1998
Abstract
Three disaccharide analogues of ꢀ-d-Man-(1!2)-ꢀ-d-Man-OMe in which the ring and/or gly-
cosidic oxygen atoms have been replaced by sulfur have been synthesized as potential inhibitors of
Class I mannosidase enzymes. Glycosylation of appropriately protected methyl ꢀ-d-mannopyr-
anosides, having free alcohol or thiol functional groups at the 2-position, with 2,3,4,6-tetra-O-
acetyl-5-thio-ꢀ-d-mannopyranosyl trichloroacetimidate was selective for the synthesis of ꢀ-linked
disaccharides in the case of the 2-alcohols, but yielded an ꢀ/ꢁ mixture in the case of the 2-thiol
glycosyl acceptors. A disaccharide containing a single sulfur in the linkage was synthesized by
nucleophilic substitution of the 2-tri¯ate of a methyl ꢀ-d-glucopyranoside derivative by the thio-
late anion of 2,3,4,6-tetra-O-acetyl-1-thio-ꢀ-d-mannopyranose. Protecting groups were removed
by standard methods to give the free heterosubstituted disaccharides. # 1998 Elsevier Science Ltd.
All rights reserved
Keywords: 5-Thio-ꢀ-d-mannopyranosyl trichloroacetimidate; Thio sugars; Disaccharides, heteroatom ana-
logues; Mannosidase inhibitors
1. Introduction
collection of processing mannosidases then
removes the four ꢀ-1,2-mannose residues to yield
Man5(GlcNAc)2. The GlcNAc transferase I, which
initiates branching of complex oligosaccharides,
then adds GlcNAc, and mannosidase II then
removes one ꢀ-1,3 and one ꢀ-1,6 linked mannose
residue to yield GlcNAcMan3(GlcNAc)2. This oli-
gosaccharide product is extended by branching
GlcNAc transferases to give bi-, tri-, and tetra-
antennary structures [2]. Distal sugars which
include Gal, GalNAc, GlcNAc, Fuc, and sialic acid
residues, are ®nally added by the corresponding
The major biosynthetic pathway of complex N-
linked oligosaccharides in higher eukaryotes occurs
by processing of oligosaccharides via the trimming
pathway [1,2]. The trimming of the high mannose
structure, Glc3Man9(GlcNAc)2 by ꢀ-glucosidases I
and II removes the three glucose residues, and a
* Corresponding author. Fax: +1-604-291-3765; e-mail:
bpinto@sfu.ca
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00162-1

18
B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
glycosyltransferases to yield the wide variety of
structures found in mature glycoproteins [1,3]. Inter-
ference with the trimming process by glucosidase or
mannosidase inhibitors blocks maturation of oli-
gosaccharides to complex structures [4]. The dis-
play of immature or ``aberrant'' structures can
inhibit tumor growth and metastasis, and the
infectivity of viruses such as HIV [5]. Inhibition of
oligosaccharide processing by glycosidase inhibi-
tors might constitute, therefore, an e€ective ther-
apeutic strategy.
Thus far, we have reported the synthesis of het-
eroanalogues of disaccharides, ꢀ-Glc-(1!2)-Glc-
OR, in which the ring and/or glycosidic oxygen
atoms have been O/S [6], S/O [7], S/N [8] and S/S
[9], as potential inhibitors of Glucosidase I. We
now describe the synthesis of heteroanalogues of
disaccharides, ꢀ-d-Man-(1!2)-(ꢀ-d-Man-OMe (1±
3), in which the ring and/or glycosidic oxygen
atoms have been replaced by sulfur for evaluation
as inhibitors of the Class I mannosidases.
trichloroacetimidate glycosyl donor 5 that was of
sucient purity for direct use in glycosylation
reactions. A suitably blocked 2-thio-mannopyrano-
side acceptor was prepared by selective S-acetate
hydrazinolysis of methyl 3-O-acetyl-2-S-acetyl-
4,6-O-benzylidene-2-thio-ꢀ-d-mannopyranoside (7)
[11] to give the free thiol (8). Although this com-
pound appeared to be stable toward air oxidation
or acetate migration reactions at room tempera-
ture, it was generally prepared immediately prior to
use. Glycosylation of 8 with 5 was performed at
low temperature in CH2Cl2 using triethylsilyl tri-
¯ate catalysis, conditions that we have previously
used [6,8,12] to prepare heteroatom-linked dis-
accharides having 5-thio-ꢀ-d-glucopyranose as the
non-reducing monosaccharide unit. In the present
case, two products were formed that were sepa-
rated by column chromatography. The less polar
product (11) and the more polar product (12) were
isolated as crystalline solids, in a ratio of 1:1.2,
respectively, with an overall yield of 83%. The
assignment of ꢀ-5-thiomannopyranosyl stereo-
chemistry to compound 11 and ꢁ-5-thiomanno-
pyranosyl stereochemistry to compound 12 rests on
1
evidence from both 13C and H NMR chemical
shifts and coupling constants. The equatorial H-10
of the ꢀ-isomer 11 (ꢂ 4.24) is deshielded with
respect to the axial H-10 of the ꢁ-isomer 12 (ꢂ 4.06),
in accord with the general trends observed in glu-
copyranosides [13]. The observation of a larger
C10±H10 coupling constant (153 Hz) for isomer 11
compared to that for 12 (146 Hz) is also diagnostic
[14]. The typical values of ꢀ170 and ꢀ160 Hz
observed for C10±H10 coupling in ꢀ- and ꢁ-5-O-
glycopyranosides, respectively, are somewhat
smaller for 5-S-glycopyranosides [15] and, in cer-
tain cases, the di€erences are too small to provide
reliable evidence for assignment of anomeric con-
®guration. In the present case, however, the di€er-
ence of 7 Hz may be considered signi®cant. Further
corroboration is provided by the 13C chemical
shifts observed for C-30 and C-50, which are 70.09
and 40.59 ppm, respectively, for 11 and 73.30 and
44.91 ppm, respectively, for 12. These results are in
accord with the expected ꢃ-gauche up®eld shift [16]
for C-3 and C-5 of ꢀ-thioglycosides relative to ꢁ-
thioglycosides for which the ꢃ-anti e€ect at C-3
and C-5 is expected to be negligible [15]. Com-
pound 11 was converted to the heptaacetate 13 by
aqueous acetic acid hydrolysis of the 4,6-O-benzy-
lidene acetal, followed by acetylation with acetic
anhydride in pyridine. Zemplen deacetylation
2. Results and discussion
5-Thio-ꢀ-d-mannopyranose pentaacetate [10]
was converted to the hemiacetal derivative 4 by
selective hydrazinolysis of the anomeric acetate.
Compound 4 was reacted with trichloroacetonitrile
and potassium carbonate in CH2Cl2 to give the

B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
19
yielded methyl 2-S-(5-thio-ꢀ-d-mannopyranosyl)-
1
2-thio-ꢀ-d-mannopyranoside (1). The H and 13C
NMR spectra of 1 showed the up®eld chemical
shifts (H-10, ꢂ 4.30 and C-10, ꢂ 57.70) typical of S,S
acetals compared to O,O acetals.
With dithiodisaccharide 1 in hand, we were
encouraged to apply the same approach to the
synthesis of the analogous 5-thio-ꢀ-d-mannopyr-
anosyl disaccharide with oxygen in the linkage.
Reaction of 5 with methyl 3-O-benzoyl-4,6-O-ben-
zylidene-ꢀ-d-mannopyranoside (9) [17] using con-
ditions similar to those previously successful for
the preparation of 11, did not result in the expected
disaccharide 14 as a major product. Instead, 14
was produced as a minor product and the orthoe-
ster derivative 17 was produced as the major pro-
duct (ratio of 17:14=5:2) We have previously
observed that, in related glycosylations using
the trichloroacetimidate of 5-thio-ꢀ-d-glucose,
orthoesters may predominate if the reactions are
quenched at low temperature [7,12]. Suitable con-
ditions for the separation of 14 and 17 by column
chromatography could not be found, but a sample
of pure 17 was obtained by fractional crystal-
Our approach to the mannose disaccharide 3
containing sulfur in the glycosidic linkage was
based on the use of 1-thiolates in nucleophilic dis-
placement reactions. Such an approach has already
been used with some success in the synthesis of S-
linked glucose disaccharides [19]. For the present
purposes, the direct displacement method, leading
to a mannose disaccharide, required a nucleophilic
displacement reaction at the unreactive 2-position
of a glucose derivative. Fortunately, compound 18
[20], which has a tri¯ate at the 2-position of a
methyl ꢀ-d-glucopyranoside derivative, is readily
available and was found to react with the thiolate
anion of 2,3,4,6-tetra-O-acetyl-1-thio-ꢀ-d-manno-
pyranoside (6) [21] in N,N-dimethylformamide, to
give the disaccharide 19, albeit in modest yield.
Deprotection by sequential acid-catalyzed hydro-
lysis of the benzylidene acetal and base-catalyzed
methanolysis of the esters yielded methyl 2-S- (ꢀ-d-
mannopyranosyl)-2-thio-ꢀ-d-mannopyranoside (3),
which was further characterized as the heptaacetate
20.
1
lization. H NMR analysis indicated that the con-
®guration of the orthoester moiety of 17 was exo
(orthoester CH3, ꢂ 1.65), with the non-reducing
monosaccharide portion adopting a boat con-
formation (3JH1 ,H2 =8.1 Hz). The remaining mix-
ture of 14 and 17 (ratio ꢀ1:1) was subjected to
treatment with catalytic amounts of triethysilyl
tri¯ate in dichloromethane at room temperature in
order to ascertain whether rearrangement of 17
to 14 was feasible. Although 17 was consumed,
the low yield of 14 obtained by this procedure
prompted us to consider methyl 3,4,6-tri-O-benzyl-
a-d-mannopyranoside (10) [18] as an alternative
glycosyl acceptor. Reaction of 5 with 10 using
triethylsilyl tri¯ate catalysis as before, except that
the reaction mixture was allowed to warm to room
temperature prior to quenching with triethylamine,
yielded the disaccharide 15 in moderate yield.
Because of the resistance of sulfur-containing
molecules to palladium-catalyzed hydrogenolysis
due to catalyst poisoning, deprotection of 15 was
e€ected by a dissolving metal reduction using
sodium in a mixed solvent system consisting of
tetrahydrofuran and liquid ammonia. Acetylation
of the crude product yielded the crystalline
heptaacetate 16. Zemplen deacetylation then
gave methyl 2-O-(5-thio-ꢀ-d-mannopyranosyl)-ꢀ-
d-mannopyranoside (2).
0
0
3. Experimental
General methods.ÐTLC was performed using
aluminum plates, precoated with Merck silica gel
60-F254, using appropriate mixtures of hexanes±
EtOAc or toluene±EtOAc for development. Visua-
lization was achieved by exposure of the dried

20
B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
plates to UV light or by spraying with a solution of
1% ceric sulfate and 1.5% molybdic acid in 10%
aqueous H2SO4 and heating. Column chromato-
graphy was performed using silica gel 60 (Merck,
230±400 mesh).
Methyl 3-O-Acetyl-4,6-O-benzylidene-2-thio-a-d-
mannopyranoside (8).ÐThe thiolacetate 7 [11]
(382 mg, 1.00 mmol) was dissolved in dry pyridine
(2 mL) and placed under a nitrogen atmosphere.
Hydrazine hydrate (0.052 mL, 1.07 mmol) was
added via syringe and the mixture stirred at room
temperature for 2 h. Volatile material was removed
in vacuo to give a cloudy syrup, which was applied
to a silica gel column with a minimum amount of
EtOAc and eluted with 3:1 hexanes±EtOAc to yield
the thiol 8 as a colorless glass (281 mg, 82%); [ꢀ]d
+7.0 (c 2.3, CHCl3); H ꢂ 7.5±7.3 (m, 5H, Ph),
5.57 (s, 1H, PhCH), 5.38 (dd, 1H, J2,3 4.9, J3,4
10.1 Hz, H-3), 4.82 (d, 1H, J1,2 0.9 Hz, H-1), 4.27
(dd, 1H, J5,6a 4.6, J6a,6b 10.1 Hz, H-6a), 4.10 (dd,
J4,5 9.8 Hz, H-4), 3.97 (ddd, 1H, J5,6b 10.1 Hz, H-
5), 3.84 (dd, 1H, H-6b), 3.76 (ddd, 1H, J2,SH
8.7 Hz, H-2), 3.39 (s, 3H, OCH3), 2.11 (s, 3H,
COCH3), 1.74 (d, 1H, SH); 13C NMR: ꢂ 169.92
(COCH3), 137.27, 129.06, 128.23, 126.26 (Ph),
103.40 (C-1), 102.06 (PhCH), 76.13 (C-4), 69.12 (C-
3), 68.87 (C-6), 64.26 (C-5), 55.16 (OCH3), 42.98
(C-2), 20.94 (COCH3).
ꢁ
Optical rotations were measured at 21 C with a
Rudolph Research Autopol II polarimeter. Melt-
ing points were determined with a Fisher±Johns
1
melting point apparatus and are uncorrected. H
and 13C NMR spectra were recorded in CDCl3
unless otherwise stated, using a Bruker AMX-400
spectrometer operating at 400.13 and 100.6 MHz
for proton and carbon, respectively. All assign-
ments were con®rmed with the aid of two-dimen-
ꢁ
1
1
1
sional H,1H (COSYDFTP) or H,13C (INVBTP)
correlation experiments using standard Bruker
pulse programs with inverse-detection. Chemical
shifts and coupling constants were obtained from a
®rst-order analysis of the spectra.
2,3,4,6-Tetra-O-acetyl-5-thio-a-d-mannopyranose
trichloroacetimidate (5).ÐA solution of 1,2,3,4,
6-penta-O-acetyl-5-thio-ꢀ-d-mannopyranose [10]
(410 mg, 1.01 mmol) in N,N-dimethylformamide
(6 mL) was stirred with hydrazine acetate (205 mg,
2.23 mmol) at room temperature for 1.5 h. The
mixture was diluted with EtOAc (100 mL) and
washed with half-satd aq NaCl solution
(2Â10 mL), dried over anhydrous Na2SO4 and
concentrated to give crude hemiacetal (4) as a col-
orless oil. Compound 4 was dissolved in CH2Cl2
(20 mL) and stirred with anhydrous K2CO3 (1.2 g)
and trichloroacetonitrile (1.1 mL, 11 mmol) for
20 h at room temperature. The mixture was diluted
with an equal volume of diethyl ether and ®ltered
through Celite. Solvent removal gave the trichloro-
acetimidate (5) quantitatively and of sucient
purity for use in glycosylation reactions. Chroma-
tography on silica gel (2:1 Et2O±hexanes) gave
pure 5 as a colorless foam (478 mg, 93%); [ꢀ]d
Methyl 3-O-acetyl-4,6-O-benzylidene-2-S-(2,3,4,6-
tetra-O-acetyl-5-thio-a-d-mannopyranosyl)-2-thio-
a-d-mannopyranoside (11) and Methyl 3-O-acetyl-
4,6-O-benzylidene-2-S-(2,3,4,6-tetra-O-acetyl-5-thio-
b-d-mannopyranosyl)-2-thio-a-d-mannopyranoside
(12). The trichloroacetimidate
5
(508 mg,
1.00 mmol) and thiol 8 (279 mg, 0.820 mmol) were
dissolved in CH2Cl2 (4 mL). The mixture was
ꢁ
cooled to 30 C and stirred under a nitrogen
atmosphere while triethylsilyl tri¯ate (0.020 mL,
0.088 mmol) was added via syringe. An immediate
reaction to produce a precipitate ensued. After
0.5 h, triethylamine (0.020 mL) was added to
quench the reaction and the mixture was warmed
to room temperature. Dilution with CH2Cl2
(50 mL), ®ltration, washing with water (2Â10 mL),
drying (MgSO4), and concentration gave a color-
less syrup. Analysis by TLC (2:1 hexanes±EtOAc)
indicated that two products had formed. Separa-
tion by chromatography on silica gel (3:2 hexanes±
EtOAc) gave the faster-eluting ꢀ-isomer (11)
(209 mg, 37%) as a colorless syꢁrup which crystal-
lized on standing: mp 111±113 C; [ꢀ]d +96.7ꢁ (c
ꢁ
1
+140 (c 1.00, CHCl3); H NMR: ꢂ 8.81 (bs, 1H,
NH), 6.05 (d, 1H, J1,2 4.1 Hz, H-1), 5.58 (dd, 1H,
J2,3 3.1Hz, H-2), 5.54 (t, 1H, J3,4=J4,5 10.5 Hz, H-4),
5.36 (dd, 1H, H-3), 4.31 (dd, 1H, J5,6a 5.7, J6a,6b
12.0 Hz, H-6a), 4.10 (dd, 1H, J5,6b 3.5 Hz, H-6b),
3.62 (ddd, 1H, H-5), 2.21, 2.07, 2.05, 2.00 (4s, each
3H, 4 COCH3); 13C NMR: ꢂ 170.46, 169.71,
169.67, 169.58 (4 COCH3), 159.58 (CNH), 90.51
(CCl3), 77.61 (C-1), 70.14 (C-2), 69.37 (C-4), 68.54
(C-3), 61.59 (C-6), 40.68 (C-5), 20.91, 20.63, 20.58,
20.55 (4 COCH3). Anal. Calcd for C16H20
Cl3NO9S: C, 37.77, H, 3.96; N, 2.75. Found: C,
37.67; H, 3.90; N, 2.74.
1
1.08, CHCl3); H NMR: ꢂ 7.5±7.3 (m, 5H, Ph),
5.57 (s, 1H, PhCH), 5.54 (dd, 1H, J2,3 4.6, J3,4
0
0
0
0
9.8 Hz, H-3), 5.45 (t, 1H, J3 ,4 =J4 ,5 =9.9 Hz, H-
0
40), 5.38 (dd, 1H, J1 2 4.1, J2 ,3 3.0 Hz, H-20), 5.25
(dd, 1H, H-30), 4.88 (d, 1H, J1,2 1.2 Hz, H-1), 4.33
0
0
0
(dd, 1H, J5 ,6a 6.0, J6a ,6b 12.0 Hz, H-6a0), 4.26 (dd,
0
0
0
0

B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
21
1H, J5,6 4.5, J6a,6b 9.9 Hz, H-6a), 4.24 (d, 1H, H-10),
9.5 Hz, H-40), 5.37 (dd, 1H, J1 ,2 4.3, J2 ,3 3.1 Hz,
H-20), 5.28 (dd, 1H, H-30), 5.25 (dd, 1H, J4,5 9.7 Hz,
H-4), 4.89 (d, 1H, J1,2 1.6 Hz, H-1), 4.33 (dd, 1H,
0
0
0
4.13 (dd, 1H, J5 ,6b 4.1 Hz, H-6b0), 3.99 (t, 1H, J4,5
9.6 Hz, H-4), 3.93 (ddd, 1H, J5,6b 9.9 Hz, H-5), 3.81
(t, 1H, H-6b), 3.75 (dd, 1H, H-2), 3.67 (ddd, 1H,
H-50), 3.41 (s, 3H, OCH3), 2.18, 2.09(2), 2.06, 2.02
(5 COCH3); 13C NMR: ꢂ 170.27, 170.16, 169.79,
169.51, 169.30 (5 COCH3), 137.2, 128.97, 128.15,
126.17 (Ph), 102.84 (C-1), 101.92 (PhCH), 77.70
(C-4), 71.73 (C-20), 70.09 (C-30), 69.54, 69.41 (C-3,
C-40), 68.74 (C-6), 64.00 (C-5), 61.86 (C-60), 55.12
0
0
J5 ,6a 6.0, J6a ,6b 11.9 Hz, H-6a0), 4.25 (d, 1H, H-
10), 4.20 (dd, 1H, J5,6a 4.7, J6a,6b 12.3 Hz, H-6a),
4.13 (dd, 1H, J5,6 2.5 Hz, H-6b), 4.12 (dd, 1H,
0
0
0
0
J5 ,6b 4.5 Hz, H-6b0), 3.92 (ddd, 1H, H-5), 3.63
(ddd, 1H, H-50), 3.62 (dd, 1H, H-2), 3.41 (s, 3H,
OCH3), 2.17, 2.12, 2.08, 2.07, 2.05, 2.04, 2.01 (7s,
each 3H, COCH3); 13C NMR: ꢂ 170.49, 170.31,
169.91, 169.71, 169.47, 169.31(2C), (7 COCH3),
101.57 (C-1), 71.53 (C-20), 70.41 (C-3), 69.84 (C-30),
69.42 (C-40), 68.51 (C-5), 67.17 (C-4), 62.26 (C-6),
61.84 (C-60), 55.35 (OCH3), 50.83 (C-10), 50.19 (C-
2), 40.51 (C-50), 20.96, 20.79(2C), 20.65(3C), 20.58,
(7 COCH3) Anal. Calcd for C27H38O16S2: C, 47.50;
H, 5.61. Found: C, 47.71 ; H, 5.68.
Methyl 2-S-(5-thio-a-d-mannopyranosyl)-2-thio-
a-d-mannopyranoside (1).ÐTo a solution of dis-
accharide 13 (122 mg, 0.179 mmol) in anhyd
MeOH was added a 1 M solution of NaOMe in
MeOH (1.0 mL), and the mixture was kept at room
temperature for 1.5 h. The solution was neutralized
with Rexyn 101 H+ ion-exchange resin, ®ltered
and concentrated to a syrup. Anhydrous toluene
(20 mL) was added and removed in vacuo to give 1
(65 mg, 94%) as a colorless glass: [ꢀ]d +218ꢁ (c
0
0
(OCH3), 51.66 (C-10, JC1 ,H1 153 Hz), 51.30 (C-2),
40.59 (C-50), 20.80, 20.74, 20.49 (COCH3). Anal.
Calcd for C30H38O14S2: C, 52.47; H, 5.58. Found:
C, 52.40; H, 5.55.
0
0
The slower-eluting ꢁ-isomer 12 (258 mg, 46%)
was isolated as a colorless syrup which slowly crys-
tallized on standing: mp 109±112 ꢁC; [ꢀ]d 34.8ꢁ (c
1.15, CHCl3); 1H NMR: ꢂ 7.5±7.3 (m, 5H, Ph), 5.70
(dd, 1H, J1 2 1.9, J2 ,3 2.9 Hz, H-20), 5.52 (s, 1H,
0
0
0
0
PhCH), 5.43 (dd, 1H, J2,3 4.7, J3,4 10.3 Hz, H-3),
0
0
0
0
0
5.39 (t, 1H, J3 ,4 =J4 ,5 =10.3 Hz, H-4 ), 4.89 (d,
1H, J1,2 1.2 Hz, H-1), 4.83 (dd, 1H, H-30), 4.22 (dd,
1H, J5,6a 4.6, J6a,6b 10.2 Hz, H-6a), 4.18 (dd, 1H,
J5 ,6a 4.4, J6a ,6b 11.8 Hz, H-6a0), 4.14 (dd, 1H,
0
0
0
0
J5 ,6b 6.3 Hz, H-6b0), 4.06 (d, 1H, H-10), 3.89 (ddd,
1H, J4,5 9.7, J5,6b 10.3 Hz, H-5), 3.76 (t, 1H, H-6b),
3.73 (dd, 1H, H-4), 3.67 (dd, 1H, H-2), 3.41 (s, 3H,
OCH3), 3.27 (ddd, 1H, H-50), 2.22, 2.17, 2.08, 2.03
1.98 (5 COCH3); 13C NMR: ꢂ 170.31(2C), 170.11,
169.78, 169.62, (5 COCH3), 137.19, 129.06, 128.21,
126.21 (Ph), 102.72 (C-1), 101.96 (PhCH), 77.11
(C-4), 73.30 (C-30), 71.85 (C-20), 68.91 (C-3), 68.73
(C-6), 68.55 (C-40), 64.04 (C-5), 62.04 (C-60), 55.14
0
0
1
0.87, H2O); H NMR (D2O): ꢂ 4.99 (d, 1H, J1,2
1.2 Hz, H-1), 4.29 (d, 1H, J1 ,2 3.6 Hz, H-10), 4.25
0
0
(dd, 1H, J2 ,3 2.9 Hz, H-20), 4.13 (dd, 1H, J2,3 4.7,
0
0
0
0
0
0
J3,4 9.7 Hz, H-3), 3.94 (dd, 1H, J5 ,6a 3.3, J6a ,6b
12.0 Hz, H-6a0), 3.82 (dd, 1H, J5,6a 2.2, J6a,6b
12.0 Hz, H-6a), 3.78 (dd, 1H, J5 ,6b 7.1 Hz, H-6b0),
0
0
(OCH3), 52.48 (C-2), 50.10 (C-10, JC1 ,H1 146 Hz),
44.91 (C-50), 21.29, 20.95, 20.71, 20.55, 20.44 (5
COCH3). Anal. Calcd for C30H38O14S2: C, 52.47;
H, 5.58. Found: C, 52.21; H, 5.53.
3.76 (dd, 1H, J2 ,3 2.8, J4 ,5 9.9 Hz, H-40), 3.69 (dd,
0
0
0
0
0
0
0
0
1H, J5,6 5.8 Hz, H-6b), 3.67 (dd, 1H, J3 ,4 9.5 Hz,
H-30), 3.58 (ddd, 1H, H-5), 3.49 (t, 1H, J4,5 9.7 Hz,
H-4), 3.46 (dd, 1H, H-2), 3.36 (s, 3H, OCH3), 3.25
(ddd, 1H, H-50); 13C NMR (D2O): ꢂ 104.53 (C-1),
75.41 (C-5), 74.97 (C-20), 74.73 (C-30), 72.59 (C-40),
72.21 (C-3), 70.68 (C-4), 63.39 (C-6), 62.94 (C-60),
57.70 (C-10), 57.52 (OCH3), 55.94 (C-2), 47.72 (C-
50) Anal. Calcd for C13H24O9S2: C, 40.20; H, 6.23.
Found: C, 40.31; H, 6.21.
Methyl 3,4,6-tri-O-acetyl-2-S-(2,3,4,6-tetra-O-
acetyl-5-thio-a-d-mannopyranosyl)-2-thio-a-d-manno-
pyranoside (13).ÐA solution of the disaccharide 11
(167 mg, 0.243 mmol) in 80% aq HOAc (10 mL)
was stirred at room temperature for 2 h and at
ꢁ
45 C for 5 h. The solvent and benzaldehyde were
removed in vacuo and the residue was acetylated
by treatment with Ac2O (1.5 mL) and 4-dimethyla-
minopyridine (0.020 g) in pyridine (4 mL) at room
temperature for 4 h. Volatile material was removed
in vacuo and the residue was puri®ed by column
chromatography (2:3 hexanes±EtOAc,) to yield 13
(152 mg, 92%) as an amorphous solid: [ꢀ]d
+131.7ꢁ (c 1.04, CHCl3); 1H NMR: ꢂ 5.51 (dd, 1H,
Methyl 3-O-benzoyl-4,6-O-benzylidene-2-O-(2,3,
4,6-tetra-O-acetyl-5-thio-a-d-mannopyranosyl)-a-
d-mannopyranoside (14) and 3,4,6-Tri-O-Acetyl-
1,2-(methyl 3-O-benzoyl-4,6-O-benzylidene-a-d-
mannopyranos-2-yl)-5-thio-b-d-mannopyranose Orth-
oacetate (17).ÐCrude trichloroacetimidate
5
(prepared from 5-thio-ꢀ-d-mannopyranose pen-
taacetate (408 mg, 1.00 mmol)) and methyl 3-O-
benzoyl-4,6-O-benzylidene-ꢀ-d-mannopyranoside
0
0
0
J2,3 4.5, J3,4 9.5 Hz, H-3), 5.43 (t, 1H, J3 ,4 =J4 ,5 =
0

22
B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
(9) [17] (446 mg, 1.15 mmol) were dissolved in
CH2Cl2 and stirred with 4 A molecular sieves
(ꢀ1 g) for 20 min at room temperature under N2.
was monitored by TLC. After 0.5 h, the formation
of several minor decomposition products was
noted, but the major spot corresponding to the
mixture of 14 and 17 remained invariant. Triethy-
lamine (0.04 mL) was added and the mixture was
processed as before. Puri®cation by column chro-
matography (1:1 hexanes±EtOAc) yielded dis-
accharide 14 (105 mg, 14%) as an amorphous solid.
A similar reaction of 5 with 9, in which the mix-
ture was allowed to warm to room temperature for
1 h prior to quenching, gave 14 (57% yield) in
approximately 90% purity after column chroma-
tography. Pure 14 was obtained as colorless nee-
dles by crystallization and recrystallization (ether±
ꢁ
The solution was cooled in a 40 C bath and
triethylsilyl tri¯ate (0.020 mL, 0.088 mmol) was
added via syringe. After 0.5 h, during which the
ꢁ
temperature had risen to 35 C, the reaction was
quenched by the addition of triethylamine
(0.02 mL) and the mixture was allowed to warm to
room temperature. Dilution with CH2Cl2 (50 mL),
®ltration, washing with satd aqueous NaHCO3 and
water (10 mL), drying (MgSO4), and concentration
gave a colorless foam. Analysis by TLC (hexanes±
EtOAc or toluene±EtOAc mixtures) showed only a
single major product spot. This product was iso-
lated by column chromatography (1:1 hexanes±
EtOAc) as a colorless foam (516 mg). The 1H
NMR spectrum of this product, however, clearly
showed a mixture of two compounds in an
approximate ratio of 5:2. One of the major com-
ponents of the mixture, orthoester derivative (17)
(258 mg, 35%), was isolated by selective crystal-
lization fromꢁether±hexanes as colorless needles:
ꢁ
hexanes): mp 147±149 ꢁC; [ꢀ]d +38.6
(c 1.1,
1
CHCl3); H NMR: ꢂ 8.1±7.2 (m, 10H, Ph), 4.88
(dd, 1H, J1 ,2 3.9, J2 ,3 2.6 Hz, H-20), 5.70 (dd, 1H,
J2,3 3.3, J3,4 10.3 Hz, H-3), 5.64 (s, 1H, PhCH),
0
0
0
0
0
0
0
0
0
5.46 (t, 1H, J3 ,4 =J4 ,5 =10.1 Hz, H-4 ), 5.41 (dd,
1H, H-30), 4.78 (d, 1H, J1,2 1.7 Hz, H-1), 4.66 (d,
1H, H-10), 4.33±4.21 (m, 4H, H-2, H-4, H-6a, H-
0
6a0), 4.11 (dd, 1H, J5 ,6b 4.0, J6a ,6b 11.9 Hz, H-6b ),
4.00±3.90 (m, 2H, H-5, H-6b), 3.50 (ddd, 1H, J5,6a
5.8 Hz H-50), 3.45 (s, 3H, OCH3), 2.08, 2.07, 2.06,
2.03 (4s, each 3H, COCH3); 13C NMR: ꢂ 170.45,
169.78, 169.73, 169.51 (4 COCH3), 165.43 (COPh),
137.22, 133.28, 129.92, 129.29, 128.89, 128.51,
128.13, 126.17 (Ph), 101.77 (PhCH), 100.78 (C-1),
83.76 (C-10), 76.56 (C-4), 76.43 (C-2), 70.40 (C-20),
70.34(2C) (C-3, C-40), 69.37 (C-30), 68.76 (C-6),
63.95 (C-5), 62.00 (C-60), 55.11 (OCH3), 38.62 (C-
50), 20.99, 20.84, 20.65, 20.62 (4 COCH3) Anal.
Calcd for C35H40O15S: C, 57.37; H, 5.50. Found:
C, 57.30; H, 5.60.
0
0
0
0
mp 154±155 C; [ꢀ]d 63.7ꢁ (c 1.6, CHCl3); H
1
NMR: ꢂ 8.1±7.2 (m, 10H, Ph), 5.60 (s, 1H, PhCH),
5.52 (dd, 1H, J2,3 3.5, J3,4 10.5 Hz, H-3), 5.43 (d,
1H, J1 ,2 8.1 Hz, H-10), 5.30 (dd, 1H, J3 ,4 10.4,
0
0
0
0
J4 ,5 6.0 Hz, H-40), 5.17 (dd, 1H, J2 ,3 3.2 Hz, H-30),
4.88 (dd, 1H, H-20), 4.65 (d, 1H, J1,2 1.3 Hz, H-1),
0
0
0
0
4.36 (dd, 1H, J5 ,6a 8.0, J6a ,6b 11.3 Hz, H-6a0), 4.30
(dd, 1H, H-2), 4.27 (dd, 1H, J5,6a 4.2, J6a,6b 9.7 Hz,
H-6a), 4.21 (dd, 1H, J4,5 9.2 Hz, H-4), 4.08 (dd, 1H,
0
0
0
0
J5 ,6b 6.8 Hz, H-6b0), 3.93 (ddd, 1H, J5,6b 9.9 Hz,
H-5), 3.85 (t, 1H, H-6b), 3.23 (ddd, 1H, H-50), 2.06,
2.02 2.00 (3s, each 3H, COCH3), 1.65 (s, 3H,
orthoester CH3); 13C NMR: ꢂ 170.33, 169.78,
169.08, (3 COCH3), 165.93 (COPh), 137.35, 133.20,
129.92, 129.70, 128.88, 128.47, 128.14, 126.13 (Ph),
122.05 (orthoester CCH3), 101.75 (PhCH), 100.75
(C-1), 78.74 (C-10), 76.03 (C-4), 74.95 (C-20), 71.49
(C-2), 70.81 (C-30), 70.07 (C-3), 68.87 (C-6), 66.15,
66.11 (C-40, C-60), 64.04 (C-5), (55.10 (OCH3),
43.73 (C-50), 21.11, 20.71, 20.60(2C) (3 COCH3, 1
orthoester CCH3) Anal. Calcd for C35H40O15S: C,
57.37; H, 5.50. Found: C, 57.40; H, 5.55.
0
0
Methyl 3,4,6-tri-O-benzyl-2-O-(2,3,4,6-tetra-O-
acetyl-5-thio-a-d-mannopyranosyl)-a-d-mannopyr-
anoside (15).ÐA solution of the trichloroace-
timidate 5 (149 mg, 0.293 mmol) and methyl 3,4,6-
tri-O-benzyl-ꢀ-d-mannopyranoside (10) [18] (224 mg,
0.482 mmol) in CH2Cl2 (4 mL) was stirred at room
temperature with freshly activated, crushed 4A
molecular sieves (ꢀ1 g) for 15 min and then cooled
in a dry-ice/acetone bath. Triethylsilyl tri¯ate
(0.020 mL, 0.088 mmol) was added via syringe and,
after 10 min, the cooling bath was removed, thus
allowing the reaction mixture to warm to room
temperature. After 1.5 h, the mixture was re-cooled
The crystallization mother liquors were con-
centrated in vacuo to give a syrup (250 mg) con-
sisting of 14 and 17 in an approximately 1:1 ratio.
This syrup was dissolved in CH2Cl2 (10 mL) and
stirred with 4 A molecular sieves (ꢀ1 g) at room
temperature. Triethylsilyl tri¯ate (0.020 mL,
0.088 mmol) was added and the reaction mixture
ꢁ
to 30 C and quenched by the addition of excess
triethylamine (0.050 mL). Dilution with CH2Cl2
(100 mL), washing with 2 N aq HCl (10 mL), satd
aq NaHCO3 (10 mL) and water (10 mL) followed
by drying (anhyd MgSO4) and concentration gave

B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
23
a yellow oil which was puri®ed by chromatography
on silica gel (3:1 toluene±EtOAc) to yield 15 as a
colorless syrup (185 mg, 76%). This disaccharide
slowly oxidized and hydrolyzed upon storage with
exposure to moist air: Evidence for the formation
(2:3 hexanes±EtOAc,) to yield 16 (136 mg, 68%) as
ꢁ
a crystalline solid: mp 141±143 C; [ꢀ]d +67.3ꢁ (c
1.04, CHCl3); 1H NMR: ꢂ 5.47 (t, 1H, J3 ,4
0
0
=
0
0
0
0
0
J4 ,5 =9.9 Hz, H-4 ), 5.42 (dd, 1H, J2 ,3 2.8 Hz, H-
30), 5.38 (dd, 1H, J1 ,2 3.9 Hz, H-20), 5.34 (t, 1H,
J3,4=J4,5=9.8 Hz, H-4), 5.28 (dd, 1H, J2,3 2.9 Hz,
H-3), 4.79 (d, 1H, J1,2 1.8 Hz, H-1), 4.25 (d, 1H, H-
0
0
1
of benzꢁaldehyde was obtained by H NMR. [ꢀ]d
1
+116.7 (c 1.52, CHCl3); H NMR: ꢂ 7.4±7.1 (m,
15H, Ph), 5.65 (dd, 1H, J1 ,2 3.9, J2 ,3 3.0 Hz, H-
10), 4.30 (dd, 1H, J5 ,6a 5.5, J6a ,6b 11.9 Hz, H-6a0),
4.25 (dd, 1H, J5,6a 4.7, J6a,6b 12.3 Hz, H-6a), 4.17
0
0
0
0
0
0
0
0
20), 5.43 (t, 1H, J3 ,4 =J4 ,5 =10.3 Hz, H-4 ), 5.37
(dd, 1H, H-30), 4.85 (d, 1H, H-10), 4.80 and 4.50
(2d, 2H, JAB 10.8 Hz, PhCH2), 4.76 (d, 1H, J1,2
1.9 Hz, H-1), 4.72 and 4.64 (2d, 2H, JAB 11.9 Hz,
PhCH2), 4.62 and 4.59 (2d, 2H, JAB 12.2 Hz,
0
0
0
0
0
0
0
(dd, 1H, J5,6 2.4 Hz, H-6b), 4.08 (dd, 1H, J5 ,6b
3.8 Hz, H-6b0), 4.04 (dd, 1H, H-2), 3.91 (ddd, 1H,
H-5), 3.51 (ddd, 1H, H-50), 3.41 (s, 3H, OCH3),
2.16, 2.14, 2.09, 2.07, 2.03, 2.02, 2.01 (7s, each 3H,
COCH3); 13C NMR: ꢂ 170.91, 170.45, 170.36,
169.75, 169.67, 169.33, 168.98 (7 COCH3), 99.75
(C-1), 83.11 (C-10), 76.29 (C-2), 71.59 (C-20), 70.72
(C-3), 69.61 (C-40), 69.53 (C-30), 68.45 (C-5), 66.64
(C-4), 62.26 (C-6), 61.98 (C-60), 55.16 (OCH3),
39.58 (C-50), 20.92, 20.59(6C), (7 COCH3) Anal.
Calcd for C27H38O17S: C, 48.65; H, 5.75. Found:
C, 48.54; H, 5.68.
0
0
0
0
PhCH2), 4.31 (dd, 1H, J5 ,6a 5.3, J6a ,6b 11.9 Hz, H-
6a0), 4.04 (dd, 1H, J5 ,6b 3.7 Hz, H-6b0),.4.01 (dd,
1H, J2,3 2.7 Hz, H-2), 3.91 (dd, 1H, J3,4 9.1 Hz, H-
3), 3.85 (t, 1H, J4,5 9.1 Hz, H-4), 3.79±3.69 (m, 3H,
H-5, H-6a, H-6b), 3.50 (ddd, 1H, H-50), 3.37 (s,
3H, OCH3) 2.12, 2.08, 2.02, 1.98 (4s, each 3H,
COCH3); 13C NMR: ꢂ 170.41, 169.74, 169.55,
169.24, (4 COCH3), 138.59, 138.45, 138.33, (3 Cipso
Ph), 129.03, 128.42, 128.28, 128.04, 127.69, 127.58,
127.53, 127.45, 127.37 (Ph), 99.89 (C-1), 82.98 (C-
10), 80.12 (C-3), 75.51, 75.43 (C-2, C-4), 75.20,
73.29, 72.90 (3 CH2Ph), 71.90 (C-5), 70.93 (C-20),
70.48 (C-30), 69.59 (C-40), 69.52 (C-6), 62.14 (C-60),
54.74 (OCH3), 39.49 (C-50), 20.93, 20.56, (COCH3).
Methyl 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-
acetyl-5-thio-a-d-mannopyranosyl)-a-d-mannopyr-
anoside (16).ÐA solution of disaccharide 15
(242 mg, 0.298 mmol) in anhydrous tetrahydro-
furan (10 mL) was cooled in a 60 ꢁC bath.
Ammonia (ꢀ20 mL) was condensed into the ¯ask
and small pieces of sodium were added until a
dark-blue solution formed. When the blue color
had persisted for 15 min, it was discharged by the
addition of a few drops of methanol. The cooling
bath was removed and the ammonia evaporated by
gentle warming in a warm water bath. The tetra-
hydrofuran solvent was removed in vacuo and the
residue was acetylated by treatment with Ac2O
(7 mL) and 4-dimethylaminopyridine (0.050 g) in
pyridine (10 mL) at room temperature for 1.5 h.
Volatile material was removed in vacuo and the
residue was partitioned between CH2Cl2 (80 mL)
and satd aq NaHCO3 solution (15 mL). The
organic phase was washed with 2 N HCl solution
(10 mL), satd aq NaHCO3 solution (10 mL) and
water (10 mL). After drying (anhyd MgSO4), the
solvent was evaporated to give a pale yellow syrup
(181 mg) which slowly began to crystallize. The
product was puri®ed by column chromatography
0
0
Methyl 2-O-(5-thio-a-d-mannopyranosyl)-a-d-
mannopyranoside (2).ÐTo a solution of dis-
accharide 16 (136 mg, 0.204 mmol) in anhyd
MeOH was added a 1 M solution of NaOMe in
MeOH (1.0 mL), and the mixture was kept at room
temperature for 4 h. The solution was neutralized
with Rexyn 101 H+ ion-exchange resin, ®ltered
and concentrated to a syrup. Anhydrous toluene
(20 mL) was added and removed in vacuo to give 2
(72 mg, 95%) as a pale-yellow glass: [ꢀ]d +146ꢁ (c
1
0.63, H2O); H NMR (D2O): ꢂ 4.96 (d, 1H, J1,2
1.7 Hz, H-1), 4.84 (d, 1H, J1 ,2 3.7 Hz, H-10), 4.30
0
0
(dd, 1H, J2 ,3 2.1 Hz, H-20), 4.01 (dd, 1H, J2,3
0
0
0
0
0
0
3.4 Hz, H-2), 3.91 (dd, 1H, J5 ,6a 3.2, J6a ,6b 11.9
Hz, H-6a0), 3.86 (dd, 1H, J5,6a 2.2, J6a,6b 11.7 Hz,
H-6a), 3.85 (dd, 1H, J3,4 9.4 H-3), 3.77 (dd, 1H,
J5 ,6b 6.9 Hz, H-6b0), 3.76±3.71 (2H, m, H-30 and
H-40), 3.72 (dd, 1H, J5,6 6.0 Hz, H-6b), 3.64 (t, 1H,
J4,5 9.8 Hz, H-4), 3.56 (ddd, 1H, H-5), 3.37 (s, 3H,
OCH3), 3.05 (ddd, 1H, H-50); 13C NMR (D2O): ꢂ
102.33 (C-1), 89.06 (C-10), 79.91 (C-2), 75.32 (C-5),
74.39, 74.36 (C-20 and C-40), 73.21 (C-3), 72.24 (C-
30), 69.70 (C-4), 63.53 (C-6), 63.17 (C-60), 57.44
(OCH3), 46.88 (C-50) Anal. Calcd for C13H24O10S:
C, 41.93; H, 6.50. Found: C, 42.05; H, 6.61.
0
0
Methyl 4,6-O-benzylidene-2-S-(2,3,4,6-tetra-O-
acetyl-a-d-mannopyranosyl)-2-thio-a-d-mannopyr-
anoside (19).ÐA solution of 2,3,4,6-tetra-O-acetyl-
1-thio-ꢀ-d-mannopyranose
10.1 mmol) in dry N,N-dimethylformamide (20mL)
(6)
[21]
(3.66 g,
was cooled in an ice bath and stirred with 4 A

24
B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
1H, J1,2 1.3 Hz, H-1), 4.10 (dd, 1H, J2,3 4.7, J3,4 9.4
molecular sieves (2 g). Sodium hydride, 60% in oil
(0.40 g, 10.0 mmol) and, after 10 min, methyl 4,6-
O-benzylidene-2-O-tri¯uoromethanesulfonyl-ꢀ-d-
mannopyranoside (18) [20] (4.14 g, 10.0 mmol)
were added. The cooling bath was removed and the
mixture stirred at room temperature for 2 h.
Methylene chloride (200 mL) was used to transfer
the solution, with ®ltration, into a separatory fun-
nel for washing with satd. brine (50 ml). After dry-
ing (anhydr Na2SO4), the solvents were removed,
®rst with aspirator vacuum and then with high
vacuum. The residue was puri®ed by column chro-
matography (1:1 hexanes±EtOAc) to yield 19
(2.89 g, 46%) as a pale yellow syrup which slowly
crystalꢁlized on standing: mp 116±117 ꢁC; [ꢀ]d
H-3), 4.05 (dd, 1H, J2 ,3 3.4 Hz, H-20), 3.94 (ddd,
0
0
1H, J4 ,5 9.4, J5 ,6a 2.2, J5 ,6b 7.1 Hz, H-50), 3.85
0
0
0
0
0
0
(dd, 1H, J6a ,6b 12.3 Hz, H-6a0), 3.81 (dd, 1H, J5,6a
0
0
0
0
2.2, J6a,6b 12.3 Hz, H-6a), 3.70 (dd, 1H, J3 ,4
9.6 Hz, H-30), 3.68 (dd, 1H, H-6b0), 3.67 (dd, 1H,
J5,6b 5.8 Hz, H-6b), 3.58 (dd, 1H, H-40), 3.56 (ddd,
1H, J4,5 9.6 Hz, H-5), 3.46 (dd, 1H, H-4), 3.38 (dd,
1H, H-2), 3.32(s, 3H, OCH3); 13C NMR (D2O): ꢂ
104.52 (C-1), 89.73 (C-10), 76.41 (C-50), 75.59 (C-5),
74.25 (C-20), 73.81 (C-30), 72.26 (C-3), 70.64 (C-4),
70.05 (C-40), 63.76, 63.63 (C-6 and C-60), 57.64
(OCH3), 54.98 (C-2), Anal. Calcd for C13H24 O10S:
C, 41.93; H, 6.50. Found: C, 41.65; H, 6.68.
Methyl 3,4,6-tri-O-acetyl-2-S-(2,3,4,6-tetra-O-
acetyl-a-d-mannopyranosyl)-2-thio-a-d-mannopyr-
anoside (20).ÐA solution of disaccharide 3 (1.10 g,
2.95 mmol) and 4-dimethylaminopyridine (0.020 g)
in pyridine (15 mL) and Ac2O (7.5 mL) was kept at
1
+92.2 (c 1.22, CHCl3); H NMR: ꢂ 7.5±7.3 (m,
5H, Ph), 5.59 (d, 1H, J1 ,2 1.5 Hz, H-10), 5.58 (s,
0
0
1H, PhCH), 5.44 (dd, 1H, J2 ,3 2.08 Hz, H-20), 5.28
0
0
0
0
0
0
(t, 1H, J3 ,4 =J4 ,5 =10.0 Hz, H-4 ), 5.26 (dd, 1H,
H-30), 4.87 (d, 1H, J1,2 0.8 Hz, H-1), 4.41 (dt, 1H,
J3,4 9.4, J3,OH=J2,3=4.6 Hz, H-3), 4.37 (ddd, 1H,
ꢁ
40 C for 1 h. Volatile material was removed in
vacuo and the residue was dissolved in CH2Cl2
(100 mL) and washed with satd NaHCO3 solution
(2Â15 mL), 2 N aq HCl (2Â15 mL), satd NaHCO3
solution (10 mL) and satd brine (10 mL). The solu-
tion was dried over anhyd MgSO4 and con-
centrated to give 20 (1.84 g, 94%) as a crystalline
J5 ,6a 6.2, J5 ,6b 2.4 Hz, H-50), 4.27 (dd, 1H, J6a ,6b
0
0
0
0
0
0
12.1 Hz, H-6a0), 4.25 (m, 1H, H-6a), 4.13 (dd, 1H, ,
H-6b0), 3.87 (m, 1H, H-4), 3.79 (m, 2H, H-5, H-
6b), 3.55 (dd, 1H, H-2), 3.38 (s, 3H, OCH3), 2.47(d,
1H, OH), 2.15, 2.10, 2.06, 1.99 (4s, each 3H,
COCH3); 13C NMR: ꢂ 170.46, 169.64(3C), (4
COCH3), 137.24, 129.20, 128.30, 126.28 (Ph),
102.67 (C-1), 102.33 (PhCH), 83.54 (C-10), 80.29
(C-4), 70.65 (C-20), 69.50 (C-30), 69.34 (C-50), 68.80
(C-6), 68.22 (C-3), 66.73 (C-40), 63.75 (C-5), 62.73
(C-60), 55.03 (OCH3), 51.11 (C-2), 20.75, 20.60,
20.52(2C), (4 COCH3) Anal. Calcd for C28H36
O14S: C, 53.50; H, 5.77. Found: C, 53.44; H, 5.85.
Methyl 2-S-(a-d-mannopyranosyl)-2-thio-a-d-
mannopyranoside (3).ÐA solution of disaccharide
19 (2.87 g, 4.56 mmol) in 80% aq HOAc (50 mL)
ꢁ
solid: mp 194±195 C (EtOH); [ꢀ]d +92.1ꢁ (c 1.08,
1
CHCl3); H NMR: ꢂ 5.50 (dd, 1H, J2,3 4.6, J3,4
9.6 Hz, H-3), 5.36±5.23 (m, 4H, H-20, H-30, H-40,
H-4), 5.32 (d, 1H, J1 ,2 1.3 Hz, H-10), 4.88 (d, 1H,
0
0
0
0
0
0
J1,2 1.4 Hz, H-1), 4.36 (ddd, 1H, J4 ,5 9.5, J5 ,6a 6.2,
J5 ,6b 2.4 Hz, H-50), 4.26 (dd, 1H, J6a ,6b 12.1 Hz,
H-6a0), 4.20 (dd, 1H, J5,6a 4.7, J6a,6b 12.3 Hz, H-6a),
4.12 (dd, 1H, J5,6b 2.5 Hz, H-6b), 4.09 (dd, 1H, H-
6b0), 3.91 (ddd, 1H, J4,5 9.9 Hz, H-5), 3.56 (dd, 1H,
H-2), 3.38 (s, 3H, OCH3), 2.15, 2.12, 2.09, 2.06,
2.05, 2.03, 2.00 (7s, each 3H, COCH3); 13C NMR:
ꢂ 170.62, 170.42, 170.00, 169.64(2C), 169.54, 169.37
(7 COCH3), 101.33 (C-1), 82.74 (C-10), 70.85 (C-
20), 70.27 (C-3), 69.37 (C-5), 69.08 (C-50), 68.70 (C-
30), 66.87 (C-4), 66.56 (C-40), 62.50 (C-60), 62.35 (C-
6), 55.20 (OCH3), 48.56 (C-2), 20.75, 20.68(2C),
20.58(2C), 20.50(2C) (7 COCH3) Anal. Calcd for
C27H38O17S: C, 48.65; H, 5.75 . Found: C, 48.64;
H, 5.72.
0
0
0
0
ꢁ
was warmed to 50 C for 4.5 h. After cooling, the
solvents were removed in vacuo and the residue
was dried by evaporation of toluene (2Â25 mL)
and kept under high vacuum at 50 ꢁC until the last
traces of benzaldehyde had been removed. The
pale yellow foam was dissolved in anhyd MeOH
(45 mL) and a 1 M solution of NaOMe in MeOH
(5.0 mL) was added. The mixture was kept at room
temperature for 19 h, neutralized with Rexyn 101
H+ ion-exchange resin, ®ltered and concentrated
to a syrup. The crude product was puri®ed by
chromatography on silica gel (4:2:0.3 EtOAc±
MeOH±H2O) to give 3 (1.12 g, 66%) as a pale-
Acknowledgements
The authors are grateful to the Protein Engi-
neering Network of Centres of Excellence and
Glycodesign Inc. for ®nancial support.
ꢁ
1
yellow foam.: [ꢀ]d +133 (c 1.0, H2O); H NMR
(D2O): ꢂ 5.32 (d, 1H, J1 ,2 1.4 Hz, H-10), 4.92 (d,
0
0

B.D. Johnston, B.M. Pinto/Carbohydrate Research 310 (1998) 17±25
25
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