Synthesis of monodeoxy analogues of the trisaccharide α-d-Glcp-(1→3)-α-d-Manp-(1→2)-α-d-ManpOMe recognised by Calreticulin/Calnexin

Article abstract of DOI:10.1016/j.carres.2006.03.015

Six (3,4,4′,6′,3″ or 6″)-monodeoxy analogues of the title trisaccharide (1-6) have been prepared utilising monodeoxy monosaccharide precursors. The reducing end deoxy derivatives were synthesised by N-iodosuccinimide/silver trifluoromethanesulfonate (NIS/

Full text of DOI:10.1016/j.carres.2006.03.015

Carbohydrate Research 341 (2006) 1533–1542
Synthesis of monodeoxy analogues of the trisaccharide
a-D-Glcp-(1!3)-a-D-Manp-(1!2)-a-D-ManpOMe
recognised by Calreticulin/Calnexin
Emiliano Gemma,a Martina Lahmanna and Stefan Oscarsonb,*
aDepartment of Chemistry, Go¨teborg University, S-412 96 Gothenburg, Sweden
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 1 February 2006; received in revised form 7 March 2006; accepted 12 March 2006
Available online 17 April 2006
Abstract—Six (3,4,40,60,300 or 600)-monodeoxy analogues of the title trisaccharide (1–6) have been prepared utilising monodeoxy
monosaccharide precursors. The reducing end deoxy derivatives were synthesised by N-iodosuccinimide/silver trifluoromethanesulfon-
ate (NIS/AgOTf)-promoted couplings of a common disaccharide thioglycoside donor 10 to suitably protected monodeoxy acceptors
9 and 12, affording trisaccharides, which after deprotection yielded target structures 1 and 2. The non-reducing end deoxy deriva-
tives could similarly be produced by halide-assisted glycosylations of a common disaccharide acceptor 17 with monodeoxy glycosyl
bromide donors (obtained from thioglycosides 18 and 20) to yield, after removal of protecting groups, target trisaccharides 3 and 4.
The analogues with the deoxy function in the middle mannose residue, were obtained through orthogonal halide-assisted coupling of
tetrabenzyl-glucopyranosyl bromide to monodeoxy thioglycoside acceptors to give thioglycoside disaccharides, which subsequently
were used as donors in NIS/AgOTf-promoted couplings to a common 2-hydroxy-mannose acceptor 15 to afford trisaccharides;
deprotection yielded the final target compounds 5 and 6.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Calreticulin substrates; Monodeoxy derivatives; Oligosaccharide synthesis
1. Introduction
OMe).5,6 Deoxy and deoxyfluoro analogues have fre-
quently been shown to be valuable and complementary
The control of the folding process of all proteins glycos-
ylated with N-glycan chains is highly dependent on
the recognition of the high-mannose-type glycan
Glc1Man9(GlcNAc)2 by the chaperone proteins Calreti-
culin/Calnexin.1–4 The binding to these lectins is medi-
ated mainly through the glucose-containing branch,
that is, the tetrasaccharide a-D-Glcp-(1!3)-a-D-Manp-
(1!2)-a-D-Manp-(1!2)-a-D-Manp. Isothermal titration
calorimetry studies of the interaction between Calreticu-
lin and synthesised truncated oligosaccharides of the
glucosylated branch showed a 25-fold stronger binding
for the trisaccharide unit, that is, a-D-Glcp-(1!3)-a-D-
Manp-(1!2)-a-D-Manp-(1!2)-a-OMe, compared to the
disaccharide unit (a-D-Glcp-(1!3)-a-D-Manp-(1!2)-a-
R4
O
R6
HO
OH
R3
O
R5
HO
O
HO
O
O
R1
R2
OMe
1 R1 = H, R2 = R3 = R4 = R5 = R6 = OH;
2
1
3
4
5
6
;
2 R = H, R = R = R = R = R = OH
3 R3 = H, R1 = R2 = R4 = R5 = R6 = OH;
4 R4 = H, R1 = R2 = R3 = R5 = R6 = OH;
5 R5 = H, R1 = R2 = R3 = R4 = R6 = OH;
6 R6 = H, R1 = R2 = R3 = R4 = R5 = OH
*
Figure 1. The target structures.
Corresponding author. E-mail: s.oscarson@organ.su.se
0008-6215/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.015

1534
E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
tools to determine the role of each hydroxyl group in
binding and 2-deoxy-2-fluoro analogues of the title tri-
saccharide have already been synthesised and investi-
gated.7 To investigate in further detail binding
interactions to Calreticulin, a series of deoxy analogues
of the glucose-containing trisaccharide were desired.
Herein we present the synthesis of six monodeoxy deriv-
atives of this trisaccharide (1–6, Fig. 1). Similar ana-
logues of the branched trimannoside core structure of
N-glycans have earlier been synthesised by us and pro-
ven to be most valuable tools in binding studies with
various lectins.8–11
BzO
O
BnO
HO
BnO
BnO
O
OH
O
OH
O
O
i, ii, iii
92%
OMe
iv
27%
OMe
OMe
8
9
7
Scheme 1. Synthesis of the 4-monodeoxy acceptor 9. Key: (i)
NaOCH3, CH3OH; (ii) NaH, BnBr, DMF; (iii) 80% aq TFA; (iv)
1. Bu2SnO, CH3OH; 2. BnBr, DMF.
verted into 9 by regioselective tin-mediated benzylation
of the equatorial 3-hydroxyl group.
Coupling of acceptor 9 with the common donor ethyl
2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1!3)-2,4,6-
tri-O-benzyl-1-thio-a-D-mannopyranoside (10), in
a
2. Results and discussion
NIS/AgOTf-promoted reaction gave the fully protected
trisaccharide 11 (87%). One-step deprotection by cata-
lytic hydrogenolysis afforded trisaccharide 1 in 86%
yield. The 3-deoxy derivative 2 was obtained from a
NIS/AgOTf-mediated glycosylation of the known 4,6-
O-benzylidene 3-deoxy derivative 1215 with donor 10,
affording trisaccharide 13 in high yield (86%) followed
by catalytic hydrogenolysis (87%).
For the synthesis of the non-reducing end deoxy
derivatives 3 and 4, a common disaccharide precursor
was also used (Scheme 3). This disaccharide, 17, pre-
pared from the known donor 1416 and acceptor 1517 in
a NIS/AgOTf-promoted coupling followed by removal
of the temporary 30-benzoate protecting group, was cho-
sen because it avoids the use of a deactivating 40,60-O-
benzylidene acetal and contains a 20-O-benzyl group
thus maximising the acceptor efficiency of the 30-hydr-
oxyl group. Coupling of the 3-monodeoxy glucosyl bro-
mide, prepared in situ from the corresponding ethyl
thioglycoside 18,18 under halide-assisted conditions
afforded the pure a-anomer 19 in acceptable yields
(46%). Target compound 3 was obtained after removal
of the benzyl ethers by catalytic hydrogenolysis (89%).
A similar sequence, starting with the 6-deoxy glucosyl
Because all the target structures are monodeoxy ana-
logues of the same trisaccharide, a possible pathway
would be to synthesise a precursor trisaccharide struc-
ture protected with a plethora of orthogonal protecting
groups, which then could be removed selectively to ex-
pose a single hydroxyl group for deoxygenation. How-
ever, our experiences with this approach have been
disappointing.12 Not only is the synthesis of such vari-
ably protected derivatives difficult, but selective deoxy-
genation at the oligosaccharide level is also frequently
problematic. Hence, all the target structures were even-
tually synthesised starting from monodeoxy monosac-
charide intermediates (9, 12, 18, 20, 23 and 29).
In the assembly of the reducing end deoxy derivatives
1 and 2, a known common disaccharide donor 1013 was
used for the preparation of both target compounds
(Scheme 2). For the synthesis of the 4-deoxy derivative
1, a suitable 2-hydroxy acceptor 9 was obtained in a
four-step sequence from the published 4-deoxy com-
pound 714 (Scheme 1). Exchange of the 6-O-benzoyl
group for a benzyl group followed by removal of the
2,3-O-isopropylidene acetal gave diol 8, which was con-
RO
O
BnO
OH
O
RO
RO
RO
RO
RO
BnO
O
RO
O
OMe
SEt
9
i
RO
BnO
BnO
BnO
11: R = Bn
1: R = H (86%)
O
ii
O
O
87%
BnO
BnO
OBn
RO
O
BnO
R1O
OMe
O
i
O
R1O
10
Ph
R1O
R1O
OR1
O
R1O
86%
R1O
OH
O
O
O
R2O
R2O
O
O
O
OMe
13: R1 = Bn, R2, R2 = PhCH
2: R1 = R2 = H (87%)
12
iii
OMe
Scheme 2. Synthesis of the 4 and 3-monodeoxy analogues 1 and 2. Key: (i) NIS/AgOTf, CH2Cl2; (ii) H2, Pd/C, CH3OH/EtOAc 5:1; (iii) H2, Pd/C,
CH3OH/1 M aq HCl.

E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
1535
RO
BnO
BnO
OBn
O
RO
BnO
BnO
RO
RO
OBn
O
O
OR
O
SEt
RO
BzO
O
BnO
BnO
BnO
18
iii, iv
RO
O
SEt
14
15
O
19 R = Bn
3 R = H
(89%)
v
O
RO
46 %
RO
i
BnO
BnO
BnO
RO
OH
O
O
81%
O
OMe
BnO
iii, iv
BnO
BnO
16 R = Bz
17 R = H (90 %)
BnO
BnO
O
RO
RO
OMe
OMe
RO
RO
ii
OR
73 %
O
RO
O
O
SEt
RO
O
21 R = Bn
4 R = H
BnO
20
v
O
RO
(quant.)
RO
OMe
Scheme 3. Synthesis of the 300- and 600-monodeoxy analogues 3 and 4. Key: (i) NIS/AgOTf, CH2Cl2; (ii) NaOCH3, CH3OH; (iii) Br2, CH2Cl2; (iv)
Et4NBr, CH2Cl2; (v) H2, Pd/C, CH3OH/THF 1:1.
donor 20,19 produced the 600-deoxy trisaccharide 21 in
good yield (73%), which afforded target compound 4
after deprotection.
TBDPSO
TBDPSO
i, ii, iii
58%
O
OH
O
O
HO
O
HO
Preparation of the 40-deoxy trisaccharide 5 and 60-
deoxy analogue 6, both having the deoxy function in
the non-reducing mannose moiety, turned out to be
more difficult.12 Attempts to stereoselectively introduce
an a-glucopyranoside residue at the 3 position of a 2,6
protected 4-deoxy mannoside under in situ anomerisa-
tion conditions afforded only low amounts of the desired
product, even after prolonged reaction times (1–2
weeks). Earlier experiments had shown that a free 2-hy-
droxyl increases the reactivity of the 3-hydroxyl group
and that regioselective 3-O-glycosylation of 2,3-unpro-
tected mannose acceptors is possible.8 Hence, the 4-
deoxy 2,3-diol 23 (Scheme 4) was prepared according
to standard procedures from the 2,3-O-isopropylidene
derivative 22.20 Deoxygenation under Barton–McCom-
bie conditions21 followed by hydrolysis of the isopropyl-
idene acetal with 90% aqueous TFA provided the
acceptor 23 (58%). Glycosylation with 2,3,4,6-tetra-O-
benzyl-D-glucopyranosyl bromide, prepared in situ from
ethyl 2,3,4,6-tetra-O-benzyl-1-thio-b-D-glucopyranoside
24,22 under halide-assisted conditions produced disac-
charide 25 in acceptable isolated yield (54%). The cou-
pling reaction occurred with good regioselectivity
(3.4:1 for 3-O-glycosylation) and with complete a-stereo-
selectivity. After 2-O-benzoylation (94%) yielding donor
26, the fully protected trisaccharide 27 was assembled in
good yield (76%) using acceptor 15 and NIS/AgOTf as
promoter. A three-step deprotection sequence then
afforded target compound 5 (76%).
SEt
SEt
22
23
BnO
iv
OTBDPS
O
BnO
BnO
OR
54%
O
BnO
O
BnO
O
BnO
BnO
SEt
25 R = H
26 R = Bz (94%)
SEt
v
BnO
24
vi
67%
HO
BnO
HO
O
OTBDPS
O
HO
HO
BnO
BnO
OH
OBz
O
O
HO
BnO
O
O
HO
vii, viii, ix
BnO
O
O
O
O
HO
HO
76%
BnO
BnO
5
OMe
27
OMe
Scheme 4. Synthesis of the 40-monodeoxy analogue 5. Key: (i) Im2CS,
DCE; (ii) n-Bu3SnH, AIBN, benzene; (iii) AcOH/H2O/TFA 10:2:1 (v/
v/v); (iv) 1. Br2, CH2Cl2; 2. Et4NBr, CH2Cl2; (v) BzCl, pyridine; (vi)
15, NIS, AgOTf, CH2Cl2; (vii) NaOCH3, CH3OH; (viii) TBAF, THF;
(ix) H2, Pd/C, CH3OH/THF 1:1.
glucopyranosyl bromide gave disaccharide 29 (54%)
with complete a-selectivity. Again, 2-O-benzoylation
(85%) furnished a donor 30, which was coupled in a
NIS/AgOTf-mediated reaction with the same acceptor
15 as above to produce trisaccharide 31 (63%). Removal
of the 20-O-benzoate with sodium methoxide, followed
by hydrogenolysis of benzyl groups then afforded the
60-deoxy analogue 6 (77%).
In conclusion, six monodeoxy trisaccharides ana-
logues, 1–6, of the Glc-containing branch of the high-
mannose-type glycan Glc1Man9(GlcNAc)2, needed for
detailed binding studies with the Calreticulin/Calnexin
The last target compound in this series, derivative 6,
was prepared similarly starting from the known ethyl
6-deoxy thiomannoside 2823 (Scheme 5). Regioselective
(2.6:1 for 3-O-glycosylation) halide-assisted coupling be-
tween diol acceptor 28 and 2,3,4,6-tetra-O-benzyl-D-

1536
E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
The organic phase was washed with H2O, dried with
BnO
BnO
BnO
OH
O
Na2SO4 and concentrated. The residue was dissolved in
80% aqueous TFA (10 mL) and stirred at rt for 2 h. Tol-
uene was added and the mixture subsequently concen-
trated and co-evaporated with toluene several times.
The resulting residue was purified by silica gel chroma-
tography (30:1, CH2Cl2/CH3OH) to give 8 (230 mg) in
92% yield. [a]D +56 (c 1.0, CHCl3), Lit.24 +18; 1H
NMR (CDCl3): d 1.63 (q, 1H, H-4b, J = 12 Hz), 1.75
(m, 1H, H-4a), 2.11 (d, 1H, OH, J = 8.5 Hz), 2.15 (d,
1H, OH, J = 7 Hz), 3.38 (s, 3H, OCH3), 3.51 (dd, 1H,
H-6b, J6b,6a = 10 Hz, J6b,5 = 4 Hz), 3.55 (dd, 1H, H-6a,
J6a,5 = 6 Hz), 3.73 (m, 1H, H-2), 3.88–4.01 (m, 2H, H-
3, H-5), 4.58 (2d, 2H, CH2Ph, J = 12 Hz), 4.78 (d, 1H,
H-1, J1,2 = 1.1 Hz), 7.25–7.34 (m, 5H, H-Aromatic).
O
OR
i
BnO
O
BnO
HO
BnO
54%
O
SEt
HO
28
29 R = H
30 R = Bz (85%)
iii
SEt
ii
BnO
BnO
63%
HO
HO
HO
O
O
OBz
O
BnO
OH
BnO
BnO
O
O
HO
O
BnO
BnO
iv, v
O
HO
O
O
77 %
O
HO
HO
BnO
MeO
31
OMe
6
Scheme 5. Preparation of the 60-monodeoxy analogue 6. Key: (i) 1. 24,
Br2, CH2Cl2; 2. Et4NBr, CH2Cl2; (ii) BzCl, pyridine; (iii) 15, NIS,
AgOTf, CH2Cl2; (iv) NaOCH3, CH3OH; (v) H2, Pd/C, CH3OH/THF
1:1.
3.2.2. Methyl 3,6-di-O-benzyl-4-deoxy-a-D-lyxo-hexopyr-
anoside (9). The obtained diol 8 (225 mg, 0.84 mmol)
was dissolved in dry CH3OH (5 mL) containing dibutyl-
tin oxide (245 mg, 0.98 mmol) and heated at 50 °C for
3 h. The solvent was removed under reduced pressure
and the residue dissolved in DMF (5 mL). Benzyl bro-
mide (235 lL, 1.96 mmol) was added and the solution
heated at 110 °C for 12 h. Satd aq NaHCO3 was poured
into the mixture, which was extracted with EtOAc. The
organic phase was washed with 1 N HCl and H2O and
dried over MgSO4. After concentration, silica gel chro-
matography (6:1, toluene/EtOAc) afforded 9 (80 mg,
chaperone proteins, have been efficiently synthesised.
The approach involved the use of monodeoxy monosac-
charide building blocks and a number of common di-
and monosaccharide acceptors and donors.
3. Experimental
1
3.1. General methods
27%). [a]D +31 (c 1.0, CHCl3); H NMR (CDCl3): d
1.77 (m, 2H, H-4b, H-4a), 2.40 (br s, 1H, OH), 3.37 (s,
3H, OCH3), 3.50 (dd, 1H, H-6b, J6b,6a = 10 Hz,
J6b,5 = 4 Hz), 3.60 (dd, 1H, H-6a, J6a,5 = 6 Hz), 3.83
(m, 1H, H-5), 3.91 (m, 2H, H-2, H-3), 4.44–4.69 (m,
4H, CH2Ph), 4.81 (d, 1H, H-1, J1,2 = 1.5 Hz), 7.28–
7.35 (m, 10H, H-Aromatic); 13C NMR (CDCl3): d 28.5
(C-4), 54.9 (OCH3), 66.7, 67.4, 70.1, 72.9, 73.0, 73.5
(C-2, C-3, C-5, C-6, CH2Ph), 101.1 (C-1), 127–129,
138.1, 138.3 (C-Aromatic). Anal. Calcd for C21H26O5:
C, 70.37; H, 7.31. Found: C, 70.27; H, 7.26.
CH2Cl2 was distilled from calcium hydride. Organic
solutions were concentrated under reduced pressure at
<45 °C (bath temperature). NMR spectra were recorded
1
at 400 MHz for H and at 100 MHz for 13C. Chemical
shifts are reported relative to chloroform [dH 7.26, dC
(central of triplet) 77.0] or to HOD (dH 4.80). TLC
was performed on Silica Gel 60 F254 with detection
by charring with 8% sulfuric acid. Silica gel (0.040–
0.063 mm) was used for column chromatography.
3.2. Methyl 3,6-di-O-benzyl-4-deoxy-a-D-lyxo-
hexopyranoside (9)
3.3. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-benzyl-a-D-mannopyranosyl-(1!2)-
3,6-di-O-benzyl-4-deoxy-a-D-lyxo-hexopyranoside (11)
3.2.1. Methyl 6-O-benzyl-4-deoxy-a-D-lyxo-hexopyran-
oside (8). Methyl 6-O-benzoyl-2,3-O-isopropylidene-4-
deoxy-a-D-lyxo-hexopyranoside 7 (300 mg, 0.93 mmol)
was dissolved in CH3OH (10 mL) and a catalytic amount
of 1 M NaOCH3 in CH3OH was added. After being
stirred overnight, the reaction was neutralised with
Dowex 50 (H+) ion exchange resin. The resin was filtered
off and the filtrate concentrated and dried in vacuo. The
crude residue was dissolved in DMF (4 mL), and NaH
(60%, 74 mg, 1.86 mmol) was added at 0 °C. After
10 min, benzyl bromide (167 lL, 1.39 mmol) was added
and the ice-bath removed. After 2 h, ice was added to
the reaction, and the mixture extracted with EtOAc.
Acceptor 9 (60 mg, 0.17 mmol) and ethyl 2,3,4,6-tetra-
O-benzyl-a-D-glucopyranosyl-(1!3)-2,4,6-tri-O-benzyl-
1-thio-a-D-mannopyranoside (10, 145 mg, 0.14 mmol)
were dissolved in CH2Cl2 (2 mL) and the solution stirred
˚
under N2 for 10 min in the presence of powdered 4 A
molecular sieves. N-iodosuccinimide (44 mg, 0.2 mmol)
was added and thereafter a catalytic amount of silver tri-
fluoromethanesulfonate. After 45 min the reaction was
neutralised with Et3N, and the mixture filtered through
a Celite pad and concentrated. Silica gel chromatogra-
phy of the residue (15:1, toluene/EtOAc) gave 11
(160 mg, 87%). [a]D +30 (c 1.0, CHCl3); 1H NMR

E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
1537
(CDCl3): d 1.81 (m, 2H, H-4b, H-4a), 3.32 (s, 3H,
OCH3), 3.52 (m, 3H), 3.71 (m, 4H), 3.99 (m, 8H), 4.21
(m, 1H), 4.35 (m, 2H), 4.44–4.68 (m, 13H), 4.78–4.97
(m, 4H), 5.15 (2br s, 2H), 5.34 (d, 1H, J = 2 Hz),
7.05–7.40 (m, 45H, H-Aromatic); 13C NMR (CDCl3):
d 29.3 (C-4), 54.8 (OCH3), 67.9, 68.3, 69.7, 70.3, 70.9,
72.2, 72.8, 73.3, 73.5, 73.9, 74.6, 74.8, 75.6, 77.1, 77.7,
79.7, 79.8, 81.9 (C-2-3, C-5-6, C-20-60, C-200-600, CH2Ph),
98.4, 99.1, 101.1 (C-1, C-10, C-100), 127-129, 138.0, 138.4,
138.5, 138.6, 138.7, 138.9 (C-Aromatic). Anal. Calcd for
C82H88O15: C, 74.98; H, 6.75. Found: C, 75.10; H, 6.71.
(d, 1H, J = 1.8 Hz), 5.12 (m, 2H), 5.56 (s, 1H, CHPh),
7.12–7.52 (m, 40H, H-Aromatic); 13C NMR (CDCl3):
d 29.8 (C-3), 54.8 (OCH3), 68.8, 69.5, 69.6, 71.1, 71.2,
72.4, 73.1, 73.4, 73.6, 74.1, 74.6, 74.8, 74.9, 75.7, 77.9,
78.2, 79.9, 81.9 (C-2, C-4-6, C-20-60, C-200-600, CH2Ph),
99–100 (C-1, C-10, C-100), 102.1 (CHPh), 126–129,
137.7, 137.8, 138.3, 138.4, 138.4, 138.6, 138.8, 138.8
(C-Aromatic). Anal. Calcd for C75H80O15: C, 73.75;
H, 6.60. Found: C, 73.64; H, 6.53.
3.6. Methyl a-D-glucopyranosyl-(1!3)-a-D-mannopyran-
osyl-(1!2)-3-deoxy-a-D-arabino-hexopyranoside (2)
3.4. Methyl a-D-glucopyranosyl-(1!3)-a-D-mannopyran-
osyl-(1!2)-4-deoxy-a-D-lyxo-hexopyranoside (1)
Compound 13 (140 mg, 0.11 mmol) was dissolved in
CH3OH (5 mL), a catalytic amount of Pd/C and 1 N
HCl (20 lL) were added, and the mixture was stirred
under H2 (1 atm). After 3 h the catalyst was removed
by filtration and the solvent evaporated. Purification of
the residue on a 600 mg C18 MAXI-CLEAN cartridge
(purchased from Alltech) afforded 2 (50 mg, 87%). [a]D
Compound 11 (70 mg, 0.053 mmol) was dissolved in
CH3OH/EtOAc (5:1 v/v, 10 mL). A catalytic amount
of Pd/C was then added and the reaction stirred under
1 atm H2. After two days, the catalyst was filtered off
and the solution concentrated. The residue, dissolved
in distilled H2O, was applied to a 600 mg C18 MAXI-
CLEAN cartridge (purchased from Alltech) and eluted
with H2O, to give pure 1 (23 mg, 86%). [a]D +117 (c
1
+113 (c 1.0, H2O); H NMR (D2O): d 1.74 (m, 1H, H-
3b), 2.21 (dt, 1H, H-3a, J = 13 Hz, J = 4 Hz), 3.39 (t,
1H, J = 9 Hz), 3.42 (s, 3H, OCH3), 3.55 (dd, 1H,
J = 10 Hz, J = 4 Hz), 3.63 (m, 1H), 3.69–3.98 (m,
13H), 4.11 (br s, 1H), 4.78 (br s, 1H), 4.99 (d, 1H,
J = 1.4 Hz), 5.24 (d, 1H, J = 4 Hz); 13C NMR (D2O): d
30.1 (C-3), 54.7 (OCH3), 60.9, 61.1, 61.3, 61.8, 66.3,
69.9, 70.4, 71.9, 72.5, 73.0, 73.1, 73.4, 73.7, 78.4 (C-2,
C-4-6, C-20-60, C-200-600), 98.4, 98.5, 100.5 (C-1, C-10, C-
100); MALDI-TOFMS calcd for C19H34O15 [M+Na]+:
525.1795, [M+K]+: 541.1535. Found 525.49, 541.52.
1
0.5, H2O); H NMR (D2O): d 1.67 (m, 2H, H-4b, H-
4a), 3.36 (s, 3H, OCH3), 3.53 (dd, 1H, J = 10 Hz,
J = 4 Hz), 3.59–3.89 (m, 13H), 3.95 (m, 1H), 4.07 (m,
1H), 4.20 (br s, 1H), 4.98 (d, 1H, J = 1.4 Hz), 5.00 (d,
1H, J = 1.5 Hz), 5.23 (d, 1H, J = 4 Hz); 13C NMR
(D2O): d 29.6 (C-4), 54.8 (OCH3), 60.8, 61.1, 64.2,
65.2, 66.4, 69.3, 69.8, 70.0, 71.9, 72.5, 73.0, 73.5, 76.4,
78.4 (C-2-3, C-5-6, C-20-60, C-200-600), 100.2, 100.5,
102.1 (C-1, C-10, C-100); MALDI-TOFMS calcd for
C19H34O15 [M+Na]+: 525.1795, [M+K]+: 541.1535.
Found 525.49, 541.48.
3.7. Methyl 3-O-benzoyl-2,4,6-tri-O-benzyl-a-D-manno-
pyranosyl-(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyran-
oside (16)
3.5. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-benzyl-a-D-mannopyranosyl-(1!2)-
4,6-O-benzylidene-3-deoxy-a-D-arabino-hexopyranoside
(13)
Ethyl 3-O-benzoyl-2,4,6-tri-O-benzyl-1-thio-a-D-manno-
pyranoside (14, 255 mg, 0.43 mmol) and acceptor 15
(180 mg, 0.39 mmol) were dissolved in CH2Cl2 (2 mL)
˚
and stirred for 10 min in the presence of 4 A powdered
Donor 10 (145 mg, 0.14 mmol) and methyl 4,6-O-benz-
ylidene-3-deoxy-a-D-arabino-hexopyranoside 12 (50 mg,
0.19 mmol) were dissolved in CH2Cl2 (1 mL) and the
solution stirred for 15 min in the presence of powdered
molecular sieves under N2. N-Iodosuccinimide
(144 mg, 0.64 mmol) and a catalytic amount of silver tri-
fluoromethanesulfonate were added. The reaction was
stirred for 30 min and then neutralised by dropwise
addition of Et3N. The mixture was directly applied onto
a silica gel column, eluted with toluene/EtOAc (15:1).
Disaccharide 16 (315 mg, 81%) was thus obtained. [a]D
˚
4 A molecular sieves under N2. N-iodosuccinimide
(48 mg, 0.21 mmol) was added and thereafter a catalytic
amount of silver trifluormethanesulfonate. The reaction
was neutralised with Et3N after 30 min and filtered
through a Celite pad. The filtrate was washed with
aqueous Na2S2O3 and H2O and concentrated. The crude
material was purified on a silica gel column (30:1!15:1,
toluene–EtOAc) affording trisaccharide 13 (150 mg,
1
ꢀ6 (c 1.0, CHCl3); H NMR (CDCl3): d 3.28 (s, 3H,
OCH3), 3.73–3.94 (m, 7H), 4.09–4.14 (m, 4H), 4.35 (d,
1H, CH2Ph, J = 12 Hz), 4.47–4.72 (m, 10H), 4.83 (br
s, 1H, H-1), 4.86 (d, 1H, CH2Ph, J = 12 Hz), 5.24 (br
s, 1H, H-10), 5.56 (dd, 1H, H-30, J3 ,4 = 8.5 Hz,
0
0
1
0
0
86%). [a]D +41 (c 0.5, CHCl3); H NMR (CDCl3): d
J3 ,2 = 3.5 Hz), 7.05–7.40 (m, 30H, H-Aromatic), 7.45
(t, 2H, H-Aromatic), 7.58 (t, 1H, H-Aromatic), 8.05
(d, 2H, H-Aromatic); 13C NMR (CDCl3): d 54.7
(OCH3), 69.3, 69.6, 71.8, 72.0, 72.6, 72.7, 73.4, 73.5,
2.05 (m, 2H, H-3b, H-3a), 3.32 (s, 3H, OCH3), 3.56
(m, 3H), 3.64–3.96 (m, 9H), 4.12 (m, 3H), 4.24
(m, 1H), 4.42–4.68 (m, 11H), 4.78–4.97 (m, 3H), 5.04

1538
E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
73.9, 74.2, 74.6, 75.0, 75.3, 76.1, 80.3 (C-2-6, C-20-60,
CH2Ph), 99.7 (C-1, C-10), 127.5–128.8, 129.9, 130.1,
132.9, 138.0, 138.1, 138.3, 138.4, 138.5, 138.7 (C-Aro-
matic), 165.6 (PhC@O). Anal. Calcd for C62H64O12: C,
74.38; H, 6.44. Found: C, 74.49; H, 6.35.
128.4, 138.2, 138.3, 138.4, 138.5, 138.5, 138.6, 138.9
(C-Aromatic). Anal. Calcd for C82H88O15: C, 74.98;
H, 6.75. Found: C, 75.06; H, 6.67.
3.10. Methyl 3-deoxy-a-D-ribo-hexopyranosyl-(1!3)-a-
D-mannopyranosyl-(1!2)-a-D-mannopyranoside (3)
3.8. Methyl 2,4,6-tri-O-benzyl-a-D-mannopyranosyl-
(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (17)
Compound 19 (50 mg, 0.038 mmol) was dissolved in
CH3OH/THF (1:1 v/v, 3 mL) and a catalytic amount
of Pd/C (5 mg, 10%) was added. The reaction mixture
was set under hydrogen atmosphere at atmospheric pres-
sure and stirred at room temperature. After 5 h, the slur-
ry was filtered through a sandwich of filters (10 lm on
top of a 5 lm filter pellet) and concentrated. The residue
was dissolved in distilled H2O, applied on a C18 column
(10 g) and eluted with H2O to give 3 (17 mg, 0.034 mmol,
89%). [a]D +126 (c 1.4, H2O); 1H NMR (D2O): d 1.82 (q,
1H, H-3b00, J = 12 Hz), 2.16 (m, 1H, H-3a00), 3.39 (s, 3H,
OCH3), 3.56–3.98 (m, 16H), 4.26 (m, 1H), 4.99 and 5.02
Compound 16 (310 mg, 0.31 mmol) was dissolved in
CH3OH (10 mL) and a catalytic amount of 1 M sodium
methoxide in CH3OH was added. After 3 days, the reac-
tion was neutralised with Dowex 50 (H+) ion exchange
resin and the solvent removed under reduced pressure.
Silica gel column chromatography (6:1, toluene/EtOAc)
of the residue afforded 17 (250 mg, 90%). [a]D +9 (c 1.0,
CHCl3); 1H NMR (CDCl3): d 2.29 (d, 1H, OH,
J = 9 Hz), 3.26 (s, 3H, OCH3), 3.65–3.85 (m, 8H),
3.89–3.94 (m, 2H), 4.03 (dt, 1H, J = 9 Hz, J = 3.5 Hz),
4.09 (br s, 1H), 4.27 (d, 1H, CH2Ph, J = 12 Hz), 4.47–
4.57 (m, 5H, CH2Ph), 4.62–4.73 (m, 4H, CH2Ph), 4.78
(d, 1H, J = 1.8 Hz), 4.86 (d, 2H, CH2Ph, J = 12 Hz),
5.24 (br s, 1H), 7.19–7.38 (m, 30H, H-Aromatic); 13C
NMR (CDCl3): d 54.7 (OCH3), 69.3, 69.5, 71.4, 71.6,
71.8, 72.4, 72.7, 73.4, 74.7, 75.1, 75.2, 78.2, 80.2 (C-2-
6, C-20-60, CH2Ph), 98.7, 99.9 (C-1, C-10), 127.5-128.5,
137.9, 138.4, 138.4, 138.5, 138.6, 138.6 (C-Aromatic).
Anal. Calcd for C55H60O11: C, 73.64; H, 6.74. Found:
C, 73.72; H, 6.84.
(br s, 1H, H-10 and br s, 1H, H-1), 5.14 (d, 1H, H-100,
13
J1 ;2 ¼ 3:6 Hz); C NMR (D2O): d 34.5 (C-300), 55.0
00 00
(OCH3), 60.9, 61.1, 61.2, 64.5, 66.4, 67.0, 67.1, 70.0,
70.4, 72.7, 73.3, 73.5, 78.1, 78.7 (C-2-6, C-20-60, C-200,
C-400-600), 99.3, 99.5, 102.4 (C-1, C-10, C-100); MALDI-
TOFMS calcd for C19H34O15 [M+Na]+: 525.1795,
[M+K]+: 541.1535. Found 525.49, 541.47.
3.11. Methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyran-
osyl-(1!3)-2,4,6-tri-O-benzyl-a-D-mannopyranosyl-(1!2)-
3,4,6-tri-O-benzyl-a-D-mannopyranoside (21)
3.9. Methyl 2,4,6-tri-O-benzyl-3-deoxy-a-D-ribo-hexo-
pyranosyl-(1!3)-2,4,6-tri-O-benzyl-a-D-mannopyran-
osyl-(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (19)
Ethyl 2,3,4-tri-O-benzyl-6-deoxy-1-thio-D-glucopyran-
oside (20, 133 mg, 0.28 mmol) was dissolved in CH2Cl2
and bromine (0.42 mmol, 22 lL) added at 0 °C. The reac-
tion mixture was stirred under N2 for 1 h and then con-
centrated. The crude glucosyl bromide was added to a
solution of acceptor 17 (130 mg, 0.15 mmol) and Et4NBr
(65 mg, 0.41 mmol) in CH2Cl2 (1 mL) and DMF
(100 lL). After two weeks of stirring at rt in the presence
Ethyl 2,4,6-tri-O-benzyl-3-deoxy-1-thio-a-D-ribo-hexo-
pyranoside (18, 120 mg, 0.25 mmol) was dissolved in
CH2Cl2, and bromine (0.38 mmol, 20 lL) added at
0 °C. The reaction mixture was stirred under N2 for
1 h and then concentrated. The obtained crude glucosyl
bromide was dissolved in CH2Cl2 and added to a solu-
tion of acceptor 17 (120 mg, 0.13 mmol) and Et4NBr
(60 mg, 0.38 mmol) in CH2Cl2 (1 mL) and DMF
(100 lL). After two weeks of stirring at rt in the presence
˚
of powdered 4 A molecular sieves, the reaction mixture
was filtered through a Celite pad and concentrated. Silica
gel column chromatography (20:1, toluene/EtOAc) of
the residue afforded 21 (140 mg, 73%). [a]D +51 (c 0.6,
1
of powdered 4 A molecular sieves, the reaction mixture
CHCl3); H NMR (CDCl3): d 1.17 (d, 3H, H-600,
˚
was filtered through a Celite pad and concentrated. Sil-
ica gel column chromatography (20:1, toluene/EtOAc)
of the residue afforded 19 (80 mg, 46%). [a]D ꢀ25
J = 6 Hz), 3.13 (t, 1H, J = 9 Hz), 3.28 (s, 3H, OCH3),
3.49 (dd, 1H, J = 10 Hz, J = 3.5 Hz), 3.71–3.77 (m,
5H), 3.81–4.06 (m, 6H), 4.12 (br s, 1H), 4.19 (m, 1H),
4.44–4.70 (m, 14H), 4.76–4.93 (m, 5H), 5.08 (m, 2H),
5.23 (s, 1H), 7.11–7.39 (m, 45H, H-Aromatic); 13C
NMR (CDCl3): d 18.2 (C-600), 54.8 (OCH3), 67.2, 67.4,
69.4, 69.7, 71.7, 72.0, 72.5, 72.7, 73.3, 73.4, 73.9, 74.5,
74.9, 75.1, 75.2, 75.6, 78.1, 80.2, 80.2, 81.7, 84.2 (C-2-6,
C-20-60, C-200-500, CH2Ph), 98.6, 99.1, 100.1 (C-1, C-10,
C-100), 127.1–129.1, 138.3, 138.4, 138.5, 138.5, 138.7,
138.8, 138.9 (C-Aromatic). Anal. Calcd for C88H82O15:
C, 74.98; H, 6.75. Found: C, 74.90; H, 6.79.
1
(c 2.9, CHCl3); H NMR (CDCl3): d 1.96 (m, 1H, H-
3b00), 2.34 (m, 1H, H-3a00), 3.25 (s, 3H, OCH3), 3.41–
3.64 (m, 3H), 3.71–3.81 (m, 6H), 3.89–4.10 (m, 6H),
4.26–4.64 (m, 19H), 4.84 (m, 2H), 5.21 (br s, 2H),
7.15–7.36 (m, 45H, H-Aromatic); 13C NMR (CDCl3):
d 31.0 (C-300), 54.7 (OCH3), 68.4, 69.4, 69.7, 70.5, 70.8,
71.3, 71.7, 71.8, 71.9, 72.3, 73.3, 73.3, 73.5, 73.7, 74.4,
75.1, 75.2, 75.3, 77.8, 80.1 (C-2-6, C-20-60, C-200, C-400-
600, CH2Ph), 97.9, 99.3, 100.0 (C-1, C-10, C-100), 127.0–

E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
1539
3.12. Methyl 6-deoxy-a-D-glucopyranosyl-(1!3)-a-D-
mannopyranosyl-(1!2)-a-D-mannopyranoside (4)
24.7 (SCH2CH3), 26.9 (C(CH3)3), 31.5 (C-4), 66.3,
66.4, 69.1, 70.7 (C-2-3, C-5-6), 84.1 (C-1), 127.8, 129.8,
133.4, 135.7 (C-Aromatic). Anal. Calcd for C24H34O4S-
Si: C, 64.53; H, 7.67. Found: C, 64.42; H, 7.61.
Compound 21 (109 mg, 0.089 mmol) was dissolved in
CH3OH/THF (1:1 v/v, 5 mL) and a catalytic amount
of Pd/C (5 mg, 10%) was added. The reaction mixture
was set under an hydrogen atmosphere at atmospheric
pressure and stirred at room temperature. After 5 h,
the slurry was filtered through a sandwich of filters
(10 lm on top of a 5 lm filter pellet) and concentrated.
The residue was dissolved in distilled H2O, applied on a
C18 column (10 g) and eluted with H2O, to give pure 4
(42 mg, 0.089 mmol, quant.). [a]D +116 (c 0.7, H2O);
1H NMR (D2O): d 1.26 (d, 3H, H-600, J = 6 Hz), 3.16
(t, 1H, J = 9 Hz), 3.40 (s, 3H, OCH3), 3.54–3.91 (m,
14H), 3.96 (br s, 1H), 4.17 (br s, 1H), 4.98 and 5.02
3.14. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-deoxy-6-O-tert-butyldiphenylsilyl-
a-D-lyxo-hexopyranosyl-(1!2)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (27)
3.14.1. Ethyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-deoxy-6-O-tert-butyldiphenylsilyl-
1-thio-a-D-lyxo-hexopyranoside (26). Ethyl 2,3,4,6-
tetra-O-benzyl-1-thio-b-D-glucopyranoside (24, 264 mg,
0.45 mmol) was dissolved in CH2Cl2 (1 mL), and bro-
mine (0.68 mmol, 35 lL) added at 0 °C. The reaction
mixture was stirred under N2 for 1 h and concentrated.
The crude glucosyl bromide was added to a solution
of acceptor 23 (90 mg, 0.20 mmol) and Et4NBr
(104 mg, 0.49 mmol) in CH2Cl2 (1 mL) and DMF
(100 lL). The reaction mixture was stirred at rt in the
(br s, 1H, H-1 and br s, 1H, H-10), 5.17 (d, 1H, H-100,
13
J1 ;2 ¼ 3 Hz); C NMR (D2O): d 16.7 (C-600), 55.0
00 00
(OCH3), 61.1, 61.2, 66.2, 67.1, 68.5, 70.1, 70.4, 72.2,
72.7, 72.8, 73.5, 75.1, 78.5, 78.9 (C-2-6, C-20-60, C-200-
500), 99.5, 100.7, 102.2 (C-1, C-10, C-100); MALDI-
TOFMS calcd for C19H34O15 [M+Na]+: 525.1795,
[M+K]+: 541.1535. Found 525.51, 541.50.
˚
presence of 4 A powdered molecular sieves for 36 h. This
was then directly applied on a silica gel column and
eluted with toluene/EtOAc (15:1!10:1) to yield 25
(105 mg, 54%). Compound 25 was dissolved in pyridine
(5 mL) and benzoyl chloride (18 lL, 0.15 mmol) added.
The reaction mixture was stirred overnight, concen-
trated and co-evaporated with toluene several times.
The residue was purified by silica gel chromatography
(30:1, toluene/EtOAc), affording disaccharide 26
(110 mg, 94%). [a]D +35 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 1.07 (s, 9H, C(CH3)3), 1.29 (t, 3H,
SCH2CH3), 1.92 (d, 1H, H-4b, J = 12 Hz), 2.08 (q,
1H, H-4a, J = 12 Hz), 2.60–2.69 (m, 2H, SCH2CH3),
3.45–3.87 (m, 8H), 4.08 (m, 1H), 4.26 (m, 1H), 4.38
(t, 2H, CH2Ph, J = 12 Hz), 4.57 (t, 2H, CH2Ph,
J = 12 Hz), 4.65–4.82 (m, 4H, CH2Ph), 4.96 (d, 1H,
3.13. Ethyl 4-deoxy-6-O-tert-butyldiphenylsilyl-1-thio-
a-D-lyxo-hexopyranoside (23)
Compound 22 (270 mg, 0.54 mmol) was dissolved in
CH2Cl2 (10 mL) together with 1,10-thiocarbonyldiimid-
azole (191 mg, 1.07 mmol) and the reaction was heated
at reflux for 10 h. The solvent was removed under re-
duced pressure and the brown residue was passed
through a silica gel column (6:1, toluene/EtOAc). The
crude product was dissolved in benzene (5 mL) and n-
Bu3SnH (2.60 mmol, 700 lL) was added. The mixture
was degassed and put under N2 atmosphere. AIBN
(ꢁ20 mg) was added, the solution was heated at reflux
for 10 min and then concentrated. The residue was par-
titioned between pentane–CH3CN and the CH3CN
layer washed twice with pentane. After concentration
of the CH3CN layer, the crude product was dissolved
in AcOH/H2O/TFA (10:2:1 v/v, 13 mL) and the solu-
tion kept at 0 °C for 2 h. The reaction mixture was then
co-evaporated with toluene several times and the result-
ing residue purified by chromatography on a silica gel
column (5:1, toluene/EtOAc) affording 23 (140 mg,
H-10, J1 ;2 ¼ 4 Hz), 5.35 (br s, 1H, H-2), 5.52 (s, 1H,
H-1), 7.03 (m, 2H, H-Aromatic), 7.22–7.45 (m,
23H, H-Aromatic), 7.54 (t, 2H, H-Aromatic), 7.67–
7.71 (m, 4H, H-Aromatic), 8.06 (d, 3H, H-Aromatic),
8.18 (d, 1H, H-Aromatic); 13C NMR (CDCl3): d
15.0 (SCH2CH3), 19.4 (C(CH3)3), 25.4 (SCH2CH3),
26.9 (C(CH3)3), 29.1 (C-4), 66.6, 68.2, 69.6, 70.9,
72.3, 73.0, 73.5, 74.6, 75.6, 80.2, 81.6 (C-2-3, C-5-6, C-
20-60, CH2Ph), 82.4 (C-1), 96.4 (C-10), 127.5–128.9,
129.7, 129.9, 130.3, 130.7, 133.1, 133.4, 133.6, 134.6,
135.7, 135.8, 138.0, 138.5, 138.9 (C-Aromatic), 165.7
(C@O).
0
0
1
58%). [a]D +87 (c 1.0, CHCl3); H NMR (CDCl3): d
1.05 (s, 9H, C(CH3)3), 1.26 (t, 3H, SCH2CH3), 1.59 (q,
1H, H-4b, J = 12 Hz), 1.80 (m, 1H, H-4a), 2.06 (d,
1H, OH, J = 8.5 Hz), 2.24 (d, 1H, OH, J = 6.5 Hz),
2.52–2.67 (m, 2H, SCH2CH3), 3.63 (dd, 1H, H-6b,
J6b,6a = 10.6 Hz, J6b,5 = 5 Hz), 3.73 (dd, 1H, H-6a,
J6a,5 = 6 Hz), 3.83 (m, 1H), 3.96 (m, 1H), 4.23 (m,
1H), 5.33 (d, 1H, H-1, J1,2 = 1.1 Hz), 7.34–7.45 (m,
6H, H-Aromatic), 7.65–7.67 (m, 4H, H-Aromatic); 13C
NMR (CDCl3): d 14.8 (SCH2CH3), 19.3 (C(CH3)3),
3.14.2. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyran-
osyl-(1!3)-2-O-benzoyl-4-deoxy-6-O-tert-butyldiphenyl-
silyl-a-D-lyxo-hexopyranosyl-(1!2)-3,4,6-tri-O-benzyl-
a-D-mannopyranoside (27). Compounds 15 (100 mg,
0.21 mmol) and 26 (110 mg, 0.10 mmol) were dissolved
in CH2Cl2 (4 mL) and the solution was stirred for

1540
E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
17H), 4.97 and 5.04 (br s, 1H, H-10 and br s, 1H, H-
˚
30 min under N2 in the presence of 4 A powdered mole-
cular sieves. N-Iodosuccinimide (35 mg, 0.15 mmol) was
then added and thereafter a catalytic amount of silver
trifluormethanesulfonate. After 2 h, the reaction was
neutralised by dropwise addition of Et3N and the mix-
ture directly applied onto a silica gel column and eluted
with toluene/EtOAc (20:1) to afford 27 (101 mg, 67%).
1), 5.10 (d, 1H, H00-10, J1 ;2 ¼ 3:9 Hz), 5.50 (br s, 1H,
0
0
13
H-20); C NMR (D2O): d 26.1 (C-40), 55.0 (OCH3),
60.8, 61.1, 64.3, 67.1, 68.0, 69.8, 70.0, 70.4, 71.1,
71.5, 72.4, 72.7, 73.1, 78.5 (C-2-6, C-20-30, C-50-60, C-
200-600), 96.6, 99.6, 103.0 (C-1, C-10, C-100); MALDI-
TOF MS calcd for C19H34O15 [M+Na]+: 525.1795,
[M+K]+: 541.1535. Found 525.48, 541.50.
1
[a]D +49 (c 1.4, CHCl3); H NMR (CDCl3): d 1.08 (s,
9H, C(CH3)3), 1.78 (d, 1H, H-4b0, J = 12 Hz), 2.05 (q,
1H, H-4a0, J = 12 Hz), 3.29 (s, 3H, OCH3), 3.51 (m,
3H), 3.66–3.94 (m, 10H), 4.08 (m, 2H), 4.26 (m, 2H),
4.40 (d, 1H, CH2Ph, J = 11 Hz), 4.51–4.80 (m, 12H),
4.89 (d, 1H, CH2Ph, J = 11 Hz), 5.04 (d, 1H, H-100,
3.16. Ethyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-O-benzyl-1-thio-a-D-rhamnopyr-
anoside (30)
J1 ;2 ¼ 4 Hz), 5.38 (d, 1H, H-10, J1 ;2 ¼ 1:1 Hz), 5.50
(br s, 1H, H-20), 7.05 (m, 2H, H-Aromatic), 7.14–7.43
(m, 42H, H-Aromatic), 7.54 (t, 1H, H-Aromatic), 7.71
(m, 4H, H-Aromatic), 8.08 (d, 1H, H-Aromatic); 13C
NMR (CDCl3): d 19.4 (C(CH3)3), 26.9 (C(CH3)3), 28.7
(C-40), 54.7 (OCH3), 66.8, 68.1, 69.6, 69.7, 70.2, 70.9,
71.4, 71.6, 71.9, 72.7, 73.3, 73.4, 73.8, 74.6, 74.9, 75.2,
75.5, 79.9, 80.0, 81.6 (C-2-6, C-20-30, C-50-60, C-200-600,
CH2Ph), 96.1, 99.6, 100.0 (C-1, C-10, C-100), 127.4–
128.5, 129.7, 129.9, 130.4, 132.9, 133.5, 133.6, 135.7,
135.8, 138.1, 138.5, 138.5, 138.7, 139.0 (C-Aromatic),
165.8 (PhC@O). Anal. Calcd for C91H98O16Si: C,
74.06; H, 6.69. Found: C, 74.14; H, 6.60.
Compound 24 (396 mg, 0.68 mmol) was dissolved in
CH2Cl2 and bromine (1.03 mmol, 53 lL) was added
at 0 °C. The reaction mixture was stirred under N2
for 1 h and then concentrated. The crude glucosyl bro-
mide was added to a solution of acceptor 2823 (100 mg,
0.33 mmol) and Et4NBr (156 mg, 0.73 mmol) in
CH2Cl2 (1 mL) and DMF (100 lL). After 40 h of stir-
00 00
0
0
˚
ring at rt in the presence of powdered 4 A molecular
sieves, the reaction mixture was directly applied onto
a silica gel column and eluted (10:1, toluene/EtOAc).
The product disaccharide 29 (130 mg, 47%) was dis-
solved in pyridine (3 mL) and benzoyl chloride
(28 lL, 0.23 mmol) was added. The mixture was stirred
at rt for 1 day, then concentrated and the residue co-
evaporated with toluene before being purified on a sil-
ica gel column (40:1, toluene/EtOAc) affording 30
(125 mg, 85%). [a]D +38 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 1.26–1.35 (m, 6H, SCH2CH3, H-6), 2.65
(m, 2H, SCH2CH3), 3.28 (br s, 2H), 3.53 (dd, 1H,
J = 10 Hz, J = 4 Hz), 3.63–3.74 (m, 3H), 3.94 (t, 1H,
J = 10 Hz), 4.10 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz),
4.16 (m, 1H), 4.27–4.37 (2d, 2H, CH2Ph, J = 12 Hz),
4.51–4.59 (2d, 2H, CH2Ph, J = 12 Hz), 4.66–4.74 (m,
4H, CH2Ph), 4.87 (d, 1H, CH2Ph, J = 12 Hz), 4.99
3.15. Methyl a-D-glucopyranosyl-(1!3)-4-deoxy-a-D-
lyxo-hexopyranosyl-(1!2)-a-D-mannopyranoside (5)
Compound 27 (93 mg, 0.063 mmol) was dissolved in
CH3OH/THF (5:1 v/v, 6 mL) and NaOCH3 (1 M in
CH3OH) was added. The mixture was left stirring over-
night and then neutralised with H+ ion exchange resin.
After removal of the resin by filtration and concentra-
tion of the filtrate, the residue was dissolved in a solu-
tion of n-BuN4F in THF (5 mL, approx. 0.1 M) and
stirred until removal of the silyl ether was complete
(TLC; 1:1, toluene/EtOAc). The solution was concen-
trated and the residue was purified by silica gel chro-
matography (4:1!1:1, toluene/EtOAc) to give methyl
2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1!3)-4-
deoxy-a-D-lyxo-hexopyranosyl-(1!2)-3,4,6-tri-O-benzyl-
a-D-mannopyranoside (60 mg, 84%). This compound
(60 mg, 0.053 mmol) was dissolved in CH3OH/THF
(1:1 v/v, 5 mL) and a catalytic amount of Pd/C
(5 mg, 10%) added. The reaction mixture was set under
an hydrogen atmosphere at atmospheric pressure and
stirred at room temperature. After 3 h, the slurry was
filtered through a sandwich of filters (10 lm on top
of a 5 lm filter pellet) and concentrated. The obtained
residue was dissolved in distilled H2O, applied on a C18
column (10 g) and eluted with H2O to give 5 (24 mg,
0.048 mmol, 91%). [a]D +86 (c 0.7, H2O); 1H NMR
(D2O): d 1.62 (q, 1H, H-4b0, J = 12 Hz), 1.85 (d, 1H,
H-4a0, J = 12 Hz), 3.38 (s, 3H, OCH3), 3.51–4.20 (m,
(d, 1H, H-10, J1 ;2 ¼ 3:5 Hz), 5.23 (d, 1H, CH2Ph,
J = 12 Hz), 5.38 (br s, 1H, H-1), 5.48 (br s, 1H, H-
2), 6.95–8.05 (m, 30H, H-Aromatic); 13C NMR
0
0
(CDCl3):
d
15.2 (SCH2CH3), 18.1 (C-6), 26.0
(SCH2CH3), 67.8, 68.6, 71.4, 73.2, 73.5, 74.6, 75.0,
75.4, 75.6, 77.2, 79.6, 79.9, 80.8, 81.7, 81.8 (C-1-5, C-
20-60, CH2Ph), 99.6 (C-10), 127–130, 133.3, 137.9,
138.3, 138.6, 138.7, 138.8 (C-Aromatic), 166.0
(PhC@O). Anal. Calcd for C56H60O10S: C, 72.70; H,
6.54. Found: C, 72.61; H, 6.63.
3.17. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranosyl-
(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (31)
Methyl 3,4,6-tri-O-benzyl-a-D-mannopyranoside (27,
100 mg, 0.21 mmol) and donor 30 (110 mg, 0.10 mmol)
were dissolved in CH2Cl2 (4 mL) and the solution was
˚
stirred for 30 min under N2 in the presence of 4 A pow-

E. Gemma et al. / Carbohydrate Research 341 (2006) 1533–1542
1541
dered molecular sieves. N-iodosuccinimide (35 mg,
0.15 mmol) was added followed by a catalytic amount
of silver trifluoromethanesulfonate. After 2 h, the reac-
tion was neutralised by dropwise addition of Et3N and
the mixture directly applied onto a silica gel column
and eluted (20:1, toluene/EtOAc) to yield 31 (101 mg,
71.9, 72.5, 72.9, 73.0, 78.1, 78.4 (C-2-6, C-20-50, C-200-
600), 99.7, 100.5, 102.1 (C-1, C-10, C-100); MALDI-TOF
MS calcd for C19H34O15 [M+Na]+: 525.1795,
[M+K]+: 541.1535. Found 525.52, 541.51.
1
67%). [a]D +35 (c 1.5, CHCl3); H NMR (CDCl3): d
Acknowledgements
1.08 (s, 9H, C(CH3)3), 1.78 (d, 1H, H-4b0, J = 12 Hz),
2.05 (q, 1H, H-4a0, J = 12 Hz), 3.29 (s, 3H, OCH3),
3.51 (m, 3H), 3.66–3.94 (m, 10H), 4.08 (m, 2H), 4.26
(m, 2H), 4.40 (d, 1H, CH2Ph, J = 11 Hz), 4.51–4.80
(m, 12H), 4.89 (d, 1H, CH2Ph, J = 11 Hz), 5.04 (d,
We are thankful to the Swedish Research Council and
EU (Glycotrain, Contract no. HPRN-CT-2000-00001)
for financial support.
1H, H-100, J1 ;2 ¼ 4 Hz), 5.38 (d, 1H, H-10, J1 ;2
¼
00 00
0
0
1:1 Hz), 5.50 (br s, 1H, H-20), 7.05 (m, 2H,
H-Aromatic), 7.14–7.43 (m, 42H, H-Aromatic), 7.54 (t,
1H, H-Aromatic), 7.71 (m, 4H, H-Aromatic), 8.08 (d,
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.03.015.
1H, H-Aromatic); 13C NMR (CDCl3):
d
19.4
(C(CH3)3), 26.9 (C(CH3)3), 28.7 (C-40), 54.7 (OCH3),
66.8, 68.1, 69.6, 69.7, 70.2, 70.9, 71.4, 71.6, 71.9, 72.7,
73.3, 73.4, 73.8, 74.6, 74.9, 75.2, 75.5, 79.9, 80.0, 81.6
(C-2-6, C-20-30, C-50-60, C-200-600, CH2Ph), 96.1, 99.6,
100.0 (C-1, C-10, C-100), 127.4–128.5, 129.7, 129.9,
130.4, 132.9, 133.5, 133.6, 135.7, 135.8, 138.1, 138.5,
138.5, 138.7, 139.0 (C-Aromatic), 165.8 (PhC@O). Anal.
Calcd for C82H86O16: C, 74.19; H, 6.53. Found: C,
74.18; H, 6.46.
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