Synthesis of allyl 2-O-(α-L-arabinofuranosyl)-6-O-(α-D-mannopyranosyl)-β-D-mannop yranoside, a unique plant N-glycan motif containing arabinose

Article abstract of DOI:10.1016/S0008-6215(00)00185-3

The synthesis of the trisaccharide allyl 2-O-(α-L-arabinofuranosyl)-6-O-(α-D-mannopyranosyl)-β-D-mannop yranoside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl β-mannoside with the acetylated α-D-mannosyl bromide gave the α-(1 → 6)-disaccharide as the main product and the crystalline 3,6-branched trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1 → 6) disaccharide with L-arabinofuranosyl bromide furnished a mixture of 3-O- and 2-O-α-L-Ara-trisaccharides from which the title compound was isolated. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00185-3

www.elsevier.nl/locate/carres
Carbohydrate Research 329 (2000) 431–439
Note
Synthesis of allyl
2-O-(a-
L
-arabinofuranosyl)-6-O-(a-
D
-mannopyranosyl)-
b-
D
-mannopyranoside, a unique plant N-glycan motif
containing arabinose
Jean-Pierre Utille *, Bernard Priem
Centre de Recherches sur les Macromole´cules Ve´ge´tales,
UPR CNRS 5301 associated with the Uni6ersity Joseph Fourier, BP53X, F-38041 Grenoble, France
Received 7 March 2000; accepted 14 June 2000
Abstract
The synthesis of the trisaccharide allyl 2-O-(a-
noside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl b-mannoside with the acetylated
a- -mannosyl bromide gave the a-(1“6)-disaccharide as the main product and the crystalline 3,6-branched
trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1“6) disaccharide with -arabinofuranosyl
-Ara-trisaccharides from which the title compound was isolated.
L
-arabinofuranosyl)-6-O-(a-
D
-mannopyranosyl)-b-D-mannopyra-
D
L
bromide furnished a mixture of 3-O- and 2-O-a-
L
© 2000 Elsevier Science Ltd. All rights reserved.
Keywords: L-Arabinose; Plant N-glycan; b-D-Allyl mannoside
Plant N-glycosylation exhibits a certain di-
versity, sharing both common and different
features by comparison to animal N-glycosy-
For instance, in a study on the analysis of
secreted glycoproteins of carrot, the presence
of
L
-rhamnose and -arabinose in N-glycosy-
L
lation. In addition to
which are found in all N-glycans, extra sugars
-GlcNAc, -Xyl, -Fuc, and -Gal can sub-
D
-Man and
D-GlcNAc
lation was suspected on the basis of radioiso-
tope labeling and enzymatic treatments [1].
D
D
L
D
Another report mentioned the presence of
L
-
stitute the inner core, resulting in the diversity
of all described plant N-glycoproteins.
arabinose in equal amount as -fucose in a
L
glycoprotein of red kidney seedling [2]. A few
years latter, while studying the structural di-
versity of free N-glycans in tomato fruit peri-
carp, Priem et al. [3] eventually described a
Despite the fact that an important number
of plant N-glycans have been fully character-
ized and shown to only contain the sugar
residues listed above, a few authors reported
the occurrence of ‘exotic’ sugars in the compo-
sition of some plant N-glycoprotein extracts.
structure containing a-
L
-arabinofuranose with
the following sequence:
* Corresponding author. Tel.: +33-47-6037668; fax: +33-
47-6517203.
E-mail address: utille@cermav.cnrs.fr (J.-P. Utille).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00185-3

432
J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
Scheme 1.
Here the
often found in the N-glycan core of plant
-Xylp
exhibit very similar conformational features,
and some bacterial hydrolases can either act as
L
-arabinose replaces the
D
-xylose
xylopyranoseanda-(1“3)-linked -fucopyran-
L
ose, often as part of N-glycoproteins and both
absent in mammals [6,7]. Hence, it is reasonable
to think that putative arabinose-containing
plant glycoproteins would be allergenic.
glycoproteins. In fact, a-
L-Araf and b-
D
a-
L
-arabinofuranosidases or b-
D
-xylopyranosi-
Here, we describe the synthesis of allyl 2-O-
dases (see for example [4], Swiss-Prot accession
c P49943). These enzymes all belong to the
family 43 of glycosylhydrolases based on their
(a -
pyranosyl)-b-
common motif with
L
- arabinofuranosyl) - 6 - O - (a -
D
- manno-
-mannopyranoside sharing a
-arabinose-containing N-
D
L
HCA plot similarity [5]. Thus,
D
-xylose- and
glycan of tomato. The b-allyl aglycon was
chosen to produce a neoglycoprotein which
may be used to raise a specific antibody for
screening of arabinosylated N-glycoproteins in
L
-arabinose-containing N-glycans might share
common building or hydrolytic enzymes, and
hence similar biosynthetic and/or hydrolytic
pathways.
1
plants. A full H and 13C NMR 1D and 2D
A practical consequence of the presence of
particular motifs in plant glycoproteins is that
they are often potent allergens for mammal
organisms. The best-known plant carbohydrate
spectroscopy analysis was done for all newly
described compounds (Scheme 1).
The key compound involved in the synthesis
of allyl (2,3,4,6-tetra-O-acetyl-a-
D
-manno-
allergenic determinants are b-(1“2)-linked
D-
pyranosyl)-(1“6)-b-
D
-mannopyranoside (4)

J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
433
Scheme 2. (a) CH3CN, Hg(CN)2–HgBr2 (50%). (b) Acetone, 2,2-dimethoxypropane, H+ (70%). (c) DCM, Ac2O, pyridine (97%).
(d) CH3CN, 35% aq soln HBF4 (96%). (e) Toluene, Hg(CN)2–HgBr2, 2,3,5-tri-O-benzoyl-L-Araf bromide (32%). (f) Ac2O,
pyridine, rt (95%). (g) MeO−Na+, MeOH, resin H+ (99%).
was the b-allyl mannoside 3 prepared by reac-
tion of tetraacetate 1 [8] with an excess of allyl
bromide in toluene at room temperature in the
presence of silver oxide [9], which furnished in
high yield (98%) a mixture of acetylated man-
noside 2a,b [10] in a 7:13 ratio. Crystallization
from diethyl ether–cyclohexane led to the iso-
lation of a major amount (]60%) of the pure
2b isomer. Chromatography on silica gel al-
lowed the isolation of both pure isomers from
the mother liquor. Subsequently, glycosylation
of 3 in acetonitrile (obtained by Zemple´n
method [11] from 2b) with 2,3,4,6-tetra-O-ace-
matography furnished pure 4 [21] which eluted
first and crystallized from diethyl ether–hex-
anes after a few weeks at −18 °C. The disac-
charide 5 eluted next and was an amorphous
solid containing minor contaminants1 as
evidenced by 13C NMR spectroscopy. In-
terestingly, the yield of 5 was enhanced
when conventional washings with saturated
aqueous phases were omitted and the mixture
directly chromatographed after evaporation
in vacuo of the reaction solvent. However,
the procedure suffered from the traces of mer-
cury salts easily eliminated in the next step.
Thus, treatment of 5 in dry acetone with
tyl-a- -mannopyranosyl bromide [12] in the
D
presence of Hg(CN)2–HgBr2 gave in 16%
yield the allyl 3,6-di-O-(2,3,4,6-tetra-O-acetyl-
1 Among the minor compounds expected to be formed
during the glycosylation, the 1,3-linked disaccharide showed a
faster moving rate than 5 and was isolated pure; the presence
of 1,2 and 1,4 linked disaccharides (not isolated) with 5 were
supposed to be eliminated during step purification’s of 6 and
7.
a-
D
-mannopyranosyl)-b- -mannopyranoside
D
(4) and the target disaccharide allyl 6-O-
(2,3,4,6-tetra-O-acetyl-a- -mannopyranosyl)-
b- -mannopyranoside (5) (50%) as major
products (Scheme 2). Purification by chro-
D
D

434
J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
dimethoxypropane in large excess (10 fold)
catalyzed by dry camphorsulfonic acid af-
forded in 2 h the 2,3-O-acetonide 6 in 70%
yield2. 6 was isolated pure by chromatography
on silica gel with EtOAc–hexanes–Et3N
(1:1:traces) or acetylated with DCM–Ac2O–
pyridine (10:1:1) to give in good yield (68%
yield, 2 steps) 7 as a more stable product. The
compound 7 was further hydrolyzed at 0 °C
(ice–water bath) by aqueous tetrafluoroboric
acid in acetonitrile according to the method of
Albert et al. [13] affording the 2,3-diol 8 in a
quantitative yield. At this stage, attempts to-
wards selective OH-3 acetylation with DCM–
Ac2O–pyridine failed (ꢀ1:1 ratio of
OAc-3:OAc-2), so the direct glycosylation of
NMR spectroscopy in D2O at 500 MHz for
1H and 125 MHz for 13C. Comparison of the
earlier H NMR data from sample labeled
1
NR5b in Ref. [3] with those of 12 showed an
excellent accord for the a- -arabinofuranosyl
L
moiety (other data were not available from [3]
due to close overlap).
In conclusion, we achieved a stereocon-
trolled synthesis of this new type of oligosac-
charide constituting a unique plant N-glycan
motif containing a-
the previously reported syntheses of b-(1“2)-
-xylopyranosyl containing oligosaccharides
[17,18]. The b- -configuration of the allyl
aglycon was assigned by comparison with the
b- -mannosyl unit (1“4)-linked to a chito-
L
-arabinose, completing
D
D
D
diol 8 with 2,3,5-tri-O-benzoyl- -arabinofuran-
L
biosyl moiety observed in the core structure of
N-glycans. Furthermore, the presence of the
allyl group may serve as nascent reactive
group for coupling to carriers by a spacer
arms in order to initiate antibody production.
osyl bromide [14] was performed in dry tolu-
ene with Hg(CN)2–HgBr2 as promoters [15].
From the reaction mixture, the 3-O- (9) and
2-O-a- -arabinofuranosyl (10) trisaccharides
L
were identified (total yield 60%, 5:6 ratio) and
isolated by chromatography. Subsequent
acetylation of 10 with a 10:1:1 mixture of
DCM–Ac2O–pyridine gave 11, the structure
of which was unambiguously determined by
1D and 2D NMR spectroscopy at 400 MHz
(100 MHz for 13C NMR) showing the ex-
pected (1H NMR) deshielding effects for a
protons belonging to secondary esterified alco-
hol positions and high field shifts for protons
attached to 2-O- and 6-O-glycosylated centers
(H-2, H-6a,b, 4.11, 3.28, 3.35 ppm compared
with 5.35, 4.03, 4.18 ppm for the acetylated
b-mannoside 2). Furthermore, a characteristic
feature (13C NMR) was the deshielding effect
on the C-2 signal of the b-allyl mannosyl unit
confirming the arabinofuranosyl glycosylation
at C-2 (C-2, 71.90 ppm for 11 compared with
68.73 for 2). In addition, small scalar cou-
plings between H-1¦, H-2¦ and H-3¦ belonging
to the arabinofuranosyl unit unequivocally
1. Experimental
General methods.—Melting points were de-
termined in capillary tubes with a Bu¨chi 535
melting point apparatus and are uncorrected.
Optical rotations were determined with a
Perkin–Elmer Type 241 polarimeter at 25 °C
using a 10 cm cell. All reactions were moni-
tored by thin layer chromatography (TLC)
run on aluminum plates precoated with Silica
Gel 60 F254 (E. Merck, Darmstadt, Germany);
detection was effected by charring with a
1:15:15 mixture of H2SO4–MeOH–water.
Flash column chromatography was performed
using Silica Gel 60 (0.0063-0.200, E. Merck).
1D and 2D NMR spectra for compounds
1–10 were recorded at 30 °C with a Bruker
AM300 spectrometer operating at 300 MHz
ruled out the a- -configuration (1,2-trans)
L
1
for H and 75 MHz for 13C using CDCl3
[16]. Finally, deprotection of 11 by Zemple´n
trans-esterification [11] procured the title com-
or D2O as solvents. Assignments of NMR
signals were made by first-order analysis of
spectra and were supported by DEPT ex-
periments, homo- and heteronuclear two-
dimensional correlation spectroscopy (COSY,
COSYRCT2, XHCORRD.AU). Spectra for
the trisaccharide 11 were recorded on a
Bruker AMX 400 spectrometer using CDCl3
pound namely allyl 2-O-(a-
L
-arabinofuran-
-manno-
osyl)-6-O-(a-
D
-mannopyranosyl)-b-
D
pyranoside (12), fully analyzed by 1D and 2D
2 Partial hydrolysis of the b-allyl aglycon was observed in
non perfect dry conditions.

J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
435
(m, 1 H, ꢀCHꢁCH2), 5.22 (dd, 1 H, J2,3 3.30
Hz, H-2), 5.25 (t, 1 H, J3,4=J4,5 9.90 Hz,
H-4), 5.27 (m, 1 H, ꢀCHꢁCH2), 5.34 (dd, 1 H,
as solvent. Spectra for the target product 12
were obtained at 45 °C with a VARIAN 500
1
Unity+ apparatus; total assignments of H
13
signals (500 MHz) were made using 1D and
2D spectroscopy supported by 1D TOCSY
experiments and 13C signals (125 MHz) were
assigned from HMQC 2D spectroscopy. Atom
numbering was labeled as prime for the a-
(1“6)-mannosyl unit and double prime for
H-3), 5.86 (m, 1 H, ꢀCHꢁCH2); C NMR (75
MHz, CDCl3): l 20.67–20.80 (4CH3COO),
62.46 (C-6), 66.07, 68.42, 68.94, 69.48 (C-2,
C-3, C-4, C-5), 68.47 (OCH2CHꢁCH2), 96.43
(C-1),
118.17
(ꢀCHꢁCH2),
132.80
(ꢀCHꢁCH2), 169.66–170.55 (4CH3COO).
Anomer 2b [10]: 884 mg (53%), crystallized
from cyclohexane–diethyl ether; mp 106–
107 °C; Rf 0.16 (2:1, cyclohexane–EtOAc);
the a- -arabinosyl unit. Mass spectra in the
L
FAB(+) mode were recorded with a Nermag
R 10 10C spectrometer.
1
2,3,4,6-Tetra-O-acetyl- -mannopyranose
D
[h]D −46° (c 1, CHCl3); H NMR (300 MHz,
(1). Per-O-acetyl mannose a,b (5.65 g, 14.49
mmol) obtained from mannose by conven-
tional acetylation (Ac2O, C5H5N, rt) was dis-
solved in 1:4 DMF–CH3CN (60 mL) and
treated with NH2NH2, CH3CO2H [19] (1.47 g,
15.97 mmol). After 15 h at rt the soln was
concd in vacuo. The residue was extracted
with CH2Cl2, washed with water, then dried
(Na2SO4) and the organic phase concd. The
solid was purified by column chromatography
(1:1, EtOAc–hexanes) to yield amorphous 1
[8,19] (4.3 g, 85%) as a quite pure a anomer
(a,b, 99:1); [h]D +19.8° (c 1, CHCl3); Rf 0.24
CDCl3): l 1.96, 2.01, 2.06, 2.16 (4 s,
CH3COO), 3.55 (m, 1 H, H-5), 3.90–4.24 (2
m, 2 H, OCH2ꢀCHꢁCH2), 4.03 (dd, 1 H, J5,6b
2.49, J6a,6b 12.30 Hz, H-6b), 4.18 (dd, 1 H, J5,6a
5.54 Hz, H-6a), 4.59 (broad d, 1 H, J1,2 B1
Hz, H-1), 4.94 (dd, 1 H, J2,3 3.32, J3,4 10.00
Hz, H-3), 5.13 (t, 1 H, J4,5=J3,4 10.00 Hz,
H-4), 5.17–5.28 (2 m, 2 H, ꢀCHꢁCH2), 5.35
(broad dd, 1 H, H-2), 5.68–5.81 (m, 1 H,
ꢀCHꢁCH2); 13C NMR (75 MHz, CDCl3): l
20.45–20.76 (4CH3COO), 62.40 (C-6), 65.99
(C-4), 68.73 (C-2), 70.05 (ꢀOCH2CHꢁCH2),
70.97 (C-3), 72.21 (C-5), 97.19 (C-1), 117.97
(ꢀCHꢁCH2), 132.95 (–CHꢁCH2), 169.48,
169.91, 170.25, 170.56 (4CH3COO).
1
13
(1:1, EtOAc–hexanes); H and C NMR (see
Ref. [20]).
Allyl 2,3,4,6-tetra-O-acetyl-h,i- -manno-
D
Allyl i- -mannopyranoside (3). The acety-
D
pyranoside (2a,b). To a soln of 1 (1.5 g, 4.31
mmol) and Ag2O (3 g, 12.93 mmol) in toluene
(30 mL) was added allyl bromide (1 mL, 11.82
mmol) over 2 h. The mixture was stirred for 4
days at rt, then filtered on a pad of silica gel
(10 g); the gel was washed with EtOAc and
the organic phases concd in vacuo. The solid
(yield 98%) was purified by column chro-
matography (2:1, hexanes–EtOAc); the a
anomer was eluted first followed by the b-allyl
mannoside 2b (the b anomer was also ob-
tained pure by crystallization (diethyl ether–
hexanes) from the reaction mixture with a
]32% yield).
lated product 2b (800 mg, 2.06 mmol) depro-
tected under classical conditions by using
Zemple´n reagent [11] (MeO−Na+, MeOH, rt)
afforded 3 (453 mg) in a quantitative yield.
The solid obtained after stirring the soln with
Amberlite H+ resin for 5 min and concn
under reduced pressure was a colorless syrup:
[h]D −55° (c 1, water); Rf 0.31 (9:1, CH3CN–
1
water); H NMR (300 MHz, D2O): l 3.22 (m,
1 H, H-5), 3.43 (t, 1 H, J4,5=J3,4 940 Hz,
H-4), 3.50 (dd, 1 H, J2,3 3.12 Hz, H-3), 3.60
(dd, 1 H, J5,6b 6.40, J6a,6b 12.21 Hz, H-6b),
3.79 (dd, 1 H, J5,6a 2.32 Hz, H-6a), 3.86 (broad
dd, 1 H, J1,2B1 Hz, H-2), 4.04–4.10 and
4.20–4.27 (2 m, 2 H, OCH2ꢀCHꢁCH2), 4.56
(broad d, 1 H, H-1), 5.12–5.26 (2 m, 2 H,
ꢀCHꢁCH2), 5.77–5.91 (m, 1 H, ꢀCHꢁCH2);
13C NMR (75 MHz, D2O): l 61.46 (C-6),
67.20 (C-4), 70.43 (OCH2CHꢁCH2), 70.90 (C-
2), 73.35 (C-3), 76.58 (C-5), 99.21 (C-1),
118.74 (ꢀCHꢁCH2), 133.86 (ꢀCHꢁCH2).
Pure product 2a [10]: yield 701 mg (42%);
1
Rf 0.22 (2:1, cyclohexane–EtOAc); H NMR
(300 MHz, CDCl3): l 1.95, 2.00, 2.07, 2.11 (4
s, CH3COO), 3.98 (m, 1 H, H-5), 4.00 (m, 1
H, OCH2ꢀCHꢁCH2), 4.07 (dd, 1 H, J5,6b 2.59,
J6a,6b 12.25 Hz, H-6b), 4.15 (m, 1 H,
OCH2ꢀCHꢁCH2), 4.23 (dd, 1 H, J5,6a 5.18 Hz,
H-6a), 4.83 (d, 1 H, J1,2 1.65 Hz, H-1), 5.20

436
J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
Allyl 6 - O - (2,3,4,6 - tetra - O - acetyl - h -
D
-
Allyl 2,3 - O - isopropylidene - 6 - O - (2,3,4,6-
tetra - O - acetyl - h - - mannopyranosyl) - i - -
mannopyranosyl)-i- -mannopyranoside (5). A
D
D
D
soln of 3 (99 mg, 0.45 mmol) in dry CH3CN
mannopyranoside (6). To a soln of disaccha-
ride 5 (100 mg, 0.18 mmol) in dry acetone (5
mL) was added 2,2 dimethoxypropane (0.22
mL, 1.80 mmol) and a catalytic amount of
camphorsulfonic acid. The reaction was moni-
tored by TLC; after 2 h the initial product (Rf
0.10, EtOAc) disappeared totally and a major
reaction product was present (Rf 0.63,
EtOAc); the soln was neutralized with Et3N.
Conventional work up was performed; the
organic phase was evaporated and gave a
solid 6 which failed to crystallize. The product
was purified by column chromatography
(EtOAc–hexanes–Et3N traces); yield 70% (75
mg); [h]D −3° (c 1, CHCl3); FABMS: [M+
,
(10 mL) was stirred for 2 h with 4 A molecu-
lar sieves. To the mixture were added succes-
sively Hg(CN)2 (190 mg, 0.75 mmol), a
catalytic amount of HgBr2 and 2,3,4,6-tetra-
O-acetyl-a- -mannopyranosyl bromide [12]
D
(267 mg, 0.65 mmol); the latter, added in two
fractions, was poured into the flask with a 2 h
interval between additions. The soln was
stirred at rt for 15 h, then deposited at the top
of a column containing 5 g of silica gel and
filtered; the column was washed successively
with EtOAc (100 mL) and CH3CN (50 mL).
The organic soln was concd, giving a solid
crude mixture (420 mg). Column chromatog-
raphy (EtOAc, silica gel 50 g) of the solid first
eluted the trisaccharide allyl 3,6-di-O-(2,3,4,6-
1
Na]+ 613; H NMR (300 MHz, CDCl3): l
1.37, 1.54 (2CH3, iso), 1.96, 1.99, 2.08, 2.12 (4
s, CH3COO), 3.43 (m, 1 H, H-5), 3.77 (m, 1
H, H-5%), 3.79 (2 dd, 2 H, H-6a, H-6b), 4.08
(m, 2 H, H-3, H-4), 4.10–4.22 (m, 2 H, H-6%a,
H-6%b), 4.19–4.44 (m, 2 H, OCH2 CHꢁCH2),
4.21 (dd, 1 H, H-2), 4.79 (d, J1,2 2.25 Hz, H-1),
4.84 (d, 1 H, J1%,2% 1 Hz, H-1%), 5.21 (dd, 1 H,
H-2%), 5.23 (t, 1 H, H-4%), 5.30 (dd, 1 H, H-3%),
5.90 (m, 1 H, ꢀCHꢁCH2); 13C NMR (75 MHz,
CDCl3): l 20.66–20.84 (4 s, CH3COO), 26.11,
27.69 (2CH3, iso) (4CH3COO), 62.37 (C-6%),
66.15 (C-4%), 67.12 (C-6), 68.42 (C-4), 68.98
(C-3%), 69.42 (C-2%), 69.54 (C-5%), 69.94
(ꢀOCH2 CHꢁCH2), 73.77 (C-5), 74.37 (C-2),
80.17 (C-3), 96.69 (C-1%), 97.20 (C-1), 110.93
(Cꢀ(CH3)2), 118.26 (ꢀCHꢁCH2), 133.53
(ꢀCHꢁCH2), 169.69–170.66 (4CH3COO).
Allyl 2,3-O-isopropylidene-4-O-acetyl-6-O-
tetra - O - acetyl - a -
D
- mannopyranosyl) - b - -
D
mannopyranoside (4) (60 mg, 0.07 mmol, 16%
yield) followed by the disaccharide 5 with
minor contaminants (130 mg, 0.24 mmol, 50%
yield). Trisaccharide 4 crystallized from di-
ethyl ether–hexanes after few weeks at −
18 °C; mp 175–176 °C; [h] +22° (c 1,
CHCl3); FABMS: (M+Na)+ 903; Rf 0.59
(EtOAc); 13C NMR (CDCl3, 75 MHz): l
20.63–20.83 (8CH3COO), 62.46, 62.81 (C-6¦,
C-6%), 65.35, 66.03, 66.34 (3 CH), 66.68 (C-6),
68.36, 68.95, 69.04, 69.24, 69.46, 69.62 (6
CH), 69.73 (OCH2 CHꢁCH2), 70.45, 74.68 (2
CH), 84.69 (C-3), 99.35, 98.04, 99.70 (C-1,
C-1%, C-1¦), 118.19 (ꢀCHꢁCH2), 133.40
(ꢀCHꢁCH2), 169.77–170.51 (8CH3COO).
Compound 5 failed to crystallize; Rf 0.30
(4:1, EtOAc–acetone); [h]D −33° (c 1,
1
CHCl3); FABMS: (M+Na)+ 573; H NMR
(2,3,4,6-tetra-O-acetyl-h-
D
-mannopyranosyl)-
(300 MHz, CDCl3): l 1.95, 2.00, 2.05, 2.10 (4
s, CH3COO), 3.30–4.35 (11 H, H-2, H-3, H-4,
H-5, H-5%, H-6a, H-6b, H-6%a, H-6%b,
OCH2ꢀCHꢁCH2), 4.56 (d, 1 H, J1,2 B1 Hz,
H-1), 4.91 (d, 1 H, J1%,2% 1.20 Hz, H-1%), 5.15–
5.34 (m, 5 H, H-2%, H-3%, H-4%, ꢀCHꢁCH2),
5.86 (m, 1 H, ꢀCHꢁCH2); 13C NMR (75 MHz,
CDCl3): l 20.82–20.93 (4CH3COO), 62.37 (C-
6%), 67.25 (C-6), 69.99 (OCH2ꢀCHꢁCH2),
65.80, 66.98, 68.45, 69.47, 69.67, 70.82, 73.54,
74.86 (C-2, C-2%, C-3, C-3%, C-4, C-4%, C-5,
C-5%), 98.05, 98.30 (C-1, C-1%), 118.15 (ꢀCH–
CH2), 133.54 (ꢀCHꢁCH2), 169.71–170.89
(4CH3COO).
i- -mannopyranoside (7). Compound 6 (59
D
mg, 0.1 mmol) in dry DCM (3 mL) was
acetylated by conventional method with Ac2O
(0.5 mL) and C5H5N (0.5 mL) for 24 h at rt;
then TLC (1:1, EtOAc–hexanes) showed that
the starting material had completely disap-
peared (Rf 0.18). The excess of reagent was
quenched by adding MeOH, then the soln was
concd under diminished pressure by co-evapo-
ration with toluene. The solid (Rf 0.32) was
purified by silica gel chromatography (1:1,
EtOAc–hexanes) and gave pure 7 which
failed to crystallize. [h]D −13° (c 1, CHCl3);

J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
437
FABMS: [M+Na]+ 655; 1H NMR (300
MHz, CDCl3): l 1.30, 1.49 (2 s, CH3, iso),
1.90–2.10 (5 s, CH3COO), 3.40 (dd, 1 H, J5,6b
2.78, J6a,6b 10.27 Hz, H-6b), 3.52 (m, 1 H,
H-5), 3.73 (dd, 1 H, J5,6a 7.92 Hz, H-6a),
3.99–4.08 (m, 2 H, H-5%, H-6b), 4.14 and 4.37
(m, 2 H, OCH2CHꢁCH2), 4.14–4.24 (m, 3 H,
H-2, H-3, H-6%a), 4.73 (d, 1 H, J1%,2%51 Hz,
H-1%), 4.76 (d, 1 H, J1,2 1.76 Hz, H-1), 4.91
(dd, 1 H, J3,4 6.75, J4,5 9.69 Hz, H-4), 5.13 (dd,
1 H, J2%,3% 3.25 Hz, H-2%), 5.18 and 5.25 (m, 2
H, ꢀCHꢁCH2), 5.20 (t, 1 H, J2%,3 =J3%,4% 9.30
Hz, H-4%), 5.27 (dd, 1 H, H-3%), 5.87 (m, 1 H,
ꢀCHꢁCH2); 13C NMR (75 MHz, CDCl3): l
20.51–20.83 (5CH3COO), 26.02, 27.30 (2CH3
iso), 60.19 (C-6%), 65.90 (C-4%), 67.12 (C-6),
68.45 (C-4), 68.86 (C-3%), 69.28 (C-2%), 70.19
(OCH2CHꢁCH2), 70.31 (C-5%), 72.59 (C-5),
74.28 (C-2), 76.92 (C-3), 96.58 (C-1%), 96.80
(C-1), 111.11 (C(CH3)2, iso), 118.66
(ꢀCHꢁCH2), 133.32 (ꢀCHꢁCH2), 169.46–
170.91 (5CH3COO).
69.86 (C-4), 69.91 (OCH2CHꢁCH2), 70.81 (C-
2), 72.44 (C-5), 72.69 (C-5%), 97.17 (C-1%),
98.24 (C-1), 118.25 (ꢀCHꢁCH2), 133.39
(ꢀCHꢁCH2), 169.66–170.76 (5CH3COO).
Allyl 2-O-(2,3,5-tri-O-benzoyl-h-
furanosyl)-4-O-acetyl-6-O-(2,3,4,6-tetra-O-
acetyl-h- -mannopyranosyl)-i- -mannopyran-
L
-arabino-
D
D
oside (10). To a soln of disaccharide 8 (35 mg,
0.06 mmol) in dry toluene (3 mL) and
Hg(CN)2 (30 mg, 0.13 mmol) was slowly
added 2 mL of a soln of 2,3,5-tri-O-benzoyl- -
L
arabinofuranosyl bromide [14,15] (240 mg,
0.43 mmol, 10 mL toluene). The mixture was
stirred at rt for 20 min, then MeOH (0.2 mL)
was added to quench the excess of bromide.
The soln was poured into a funnel and washed
with a satd aq soln of KBr and then distilled
water; the organic layers were isolated and
dried (Na2SO4). The soln was then concd in
vacuo and the solid chromatographed on sil-
ica gel with 1:1 EtOAc–hexanes as eluents;
the two trisaccharides 2-O- and 3-O-a- -Araf
L
Allyl 4-O-acetyl-6-O-(2,3,4,6-tetra-O-ace-
were isolated (60% yield), the first moving
tyl-h-
D
-mannopyranosyl)-i-
D
-mannopyran-
product (3-O-linked) 9 (17 mg, Rf 0.33, 1:1,
oside (8). To a soln of compound 7 (50 mg,
0.84 mmol) in CH3CN (5 mL) at 0 °C (ice–
water bath) was added tetrafluoroboric acid
(35% in water) according to the method of
Albert et al. [13]; after quantitative reaction
(TLC, 10 min) Et3N was added and the sol-
vent co-evaporated with toluene. Compound 8
was then purified on a short column of silica
gel (4:1, EtOAc–cyclohexane) and gave a
solid (45 mg, 96%); [h]D −4° (c 1, CHCl3); Rf
EtOAc–hexanes) followed by the 2-O-a- -
L
Araf isomer 10 (20 mg, Rf 0.27) as an amor-
phous compound.
9
13C NMR (75 MHz, CDCl3, selected
data): 97.15, 99.40, 105.80 (C-1, C-1%, C-1¦).
10 13C NMR (75 MHz, CDCl3): l 20.60–
20.80 (5CH3COO), 62.34, 63.42, 67.00 (C-6%,
C-5¦, C-6), 66.14, 67.27, 67.56, 68.49, 68.96,
69.45, 69.99, 73.02, 75.81, 77.41, 81.51, 82.93
(C-2, 2%, 2¦; C-3, 3%,3¦; C-4, 4%, 4¦; C-5, 5%,
OCH2CHꢁCH2), 96.24, 98.03 (C-1, C-1),
103.15 (C-1¦), 118.42 (ꢀCHꢁCH2), 128.32–
133.71 (CꢀAr, ꢀCHꢁCH2), 165.02–166.32 (3
C6H5COO),169.66–170.64 (5CH3COO).
1
0.42 (EtOAc); FABMS: [M+Na]+ 615; H
NMR (300 MHz, CDCl3): l 1.96, 1.99, 2.08,
2.09, 2.12 (5, CH3COO), 3.50 (m, 1 H, H-5%),
3.54 (m, 1 H, H-5), 3.60 (dd, 1 H, J6a,6b 10.80,
J5,6b 3.30 Hz, H-6b), 3.77 (dd, 1 H, J5,6a 7.10
Hz, H-6a), 3.99 (broad dd, 1 H, J2,3 3.25 Hz,
H-2), 4.06 (dd, 1 H, J3,4 9.65 Hz, H-3), 4.11
(dd, 1 H, J5%,6%b 2.51, J6%a,6%b 12.17 Hz, H-6%b),
4.16 (m, 1 H, OCH2CHꢁCH2), 4.23 (dd, 1 H,
J5%,6%a 5.60 Hz, H-6%a), 4.35 (m, 1 H,
OCH2CHꢁCH2), 4.55 (d, 1 H, J1,2 B1 Hz,
H-1), 4.83 (d, 1 H, J1%,2% 1.35 Hz, H-1%), 4.98 (t,
1 H, J3,4 =J4,5 9.65 Hz, H-4), 5.20–5.35 (m, 2
Allyl 2-O-(2,3,5-tri-O-benzoyl-h-
furanosyl)-3,4-di-O-acetyl-6-O-(2,3,4,6-tetra-
O-acetyl-h- -mannopyranosyl)-i- -manno-
L
-arabino-
D
D
pyranoside (11). Compound 10 was acetylated
with Ac2O and C5NH5 at rt (overnight) and 11
was isolated by conventional workup, then
purified on a short column of silica gel; Rf
1
0.40 (1:1, EtOAc–hexanes); yield 95%; H
NMR (400 MHz, CDCl3): l 1.66, 1.70, 1.77,
1.79, 1.85, 2.07 (6 s, CH3COO), 3.28 (dd, 1 H,
J5,6b 2.60, J6a,6b 10.50 Hz, H-6b), 3.35 (m, 1 H,
H-5), 3.59 (dd, 1 H, J5,6a 7.20 Hz, H-6a), 3.83
(m, 3 H, H-6%b, H-5%, ꢀCH2CHꢁCH2), 4.01
13
H, ꢀCHꢁCH2), 5.90 (m, 1 H, ꢀCHꢁCH2); C
NMR (75 MHz, CDCl3): l 20.75–20.87
(5CH3COO), 62.41 (C-6%), 66.18 (C-4%), 66.73
(C-6), 68.53 (C-3), 68.95 (C-3%), 69.52 (C-2%),

438
J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
5.38 (m, 2 H, ꢀCHꢁCH2), 5.96–6.04 (m, 1
H, ꢀCHꢁCH2); 13C NMR (125 MHz, D2O,
45 °C): l 61.28 (C-6%), 61.56 (C-5¦), 66.68
(C-6), 67.08 (C-4), 67.48 (C-3), 70.29 (C-2%),
70.65 (C-3%), 70.94 (OCH2CHꢁCH2), 72.65
(C-5%), 73.03 (C-4%), 74.83 (C-2, C-5), 76.94
(C-3¦), 81.14 (C-2¦), 84.69 (C-4¦), 99.86 (C-
1%), 100.08 (C-1), 108.03 (C-1¦), 118.28
(ꢀCHꢁCH2), 134.00 (ꢀCHꢁCH2).
(dd, 1 H, J5%,6%a 5.15, J6%a,6%b 12.30 Hz, H-6%a),
4.08 (m, 1 H, ꢀCH2CHꢁCH2), 4.11 (broad
dd, 1 H, J1,2 ꢁ1, J2,3 3.25 Hz, H-2), 4.30 (m,
1 H, H-4¦), 4.35 (dd, 1 H, J4¦,5¦ 5.37, J5¦a,5¦b
11.54 Hz, H-5¦b), 4.39 (broad d, 1 H, H-1),
4.49 (dd, 1 H, J4¦,5¦a 3.24 Hz, H-5¦a), 4.56
(d, 1 H, J1%,2% 1.60 Hz, H-1%), 4.75 (dd, 1 H,
J3,4 10.09 Hz, H-3), 4.88 (m,
1
H,
ꢀCHꢁCH2), 4.95 (dd, 1 H, J2%,3% 3.32 Hz, H-
2%), 5.01 (m, 2 H, H-4, H-4%), 5.03 (m, 1 H,
ꢀCHꢁCH2), 5.06 (dd, 1 H, J3%,4% 9.97 Hz, H-
3%), 5.27 (broad dd, 1 H, J3¦,4¦ 4.10 Hz, H-
3¦), 5.39 (broad d, 1 H, J1¦,2¦B1 Hz, H-2¦),
5.49 (broad d, 1 H, H-1¦), 5.58 (m, 1 H,
ꢀCHꢁCH2); 13C NMR (100 MHz, CDCl3): l
20.6–20.7 (6CH3COO), 62.27 (C-6%), 64.06
(C-5¦), 65.99 (C-4%), 67.20 (C-4, C-6), 68.72
(C-5%), 69.01 (C-3%), 69.40 (C-2%), 70.04
(OCH2CHꢁCH2), 71.90 (C-2, C-3), 73.27 (C-
5), 77.76 (C-3¦), 81.34 (C-2¦), 82.17 (C-4¦),
97.15 (C-1%), 99.03 (C-1), 105.97 (C-1¦),
118.07 (ꢀCHꢁCH2), 128.25–133.47 (ꢀCHꢁ
CH2, Ar.), 165.12–166.45 (3 C6H5COO),
169.63–170.01 (6CH3COO).
Acknowledgements
The authors would like to thank C. Gey
and M.C. Brochier for recording the 400
and 500 MHz NMR spectra, respectively.
References
[1] A. Sturm, Eur. J. Biochem., 199 (1991) 169–179.
[2] Y. Lu, F. Wolf, Arch. Biochem. Biophys., 286 (1991)
147–152.
[3] B. Priem, R.K. Gitti, C.A. Bush, K.C. Gross, Plant
Physiol., 102 (1993) 445–458.
[4] T.R. Whitehead, Biochim. Biophys. Acta, 1244 (1995)
239–241.
[5] B. Henrissat, A. Bairoch, Biochem. J., 316 (1996) 695–
696.
[6] L. Faye, V. Gomord, A.C. Fitchette-Laine´, M.J.
Chrispeels, Anal. Biochem., 209 (1993) 104–108.
[7] I.B.H. Wilson, J.E. Harthill, N.P. Mullin, D.A. Ashford,
F. Altmann, Glycobiology, 8 (1998) 651–661.
[8] J.M. Lacombe, N. Rakotomanomana, A.A. Pavia, J.
Carbohydr. Chem., 9 (1990) 85–92.
[9] K.J. Kaur, O. Hindsgaul, Glycoconjugate J., 8 (1991)
90–94.
[10] (a) T.V. Rajanbubu, T. Fukumoga, G.S. Reddy,
J. Am. Chem. Soc., 111 (1989) 1759–1769. (b) W.
Klotz, R.R. Schmidt, Liebigs Ann. Chem. (1993) 683–
690.
[11] G. Zemple´n, Ber. Dtsch. Chem. Ges., 60 (1927) 1555.
[12] (a) K. Higashi, K. Nukayama, E. Shioya, T. Kusama,
Chem. Pharm. Bull., 39 (1991) 2502–2504. (b) R.P.
Spencer, C.L. Cavallaro, J. Schwartz, J. Org. Chem., 64
(1999) 3987–3995.
[13] R. Albert, K. Dax, R. Pleschko, A.E. Stu¨tz, Carbohydr.
Res., 137 (1985) 282–290.
[14] H.G. Fletcher, Jr., Methods Carbohydr. Chem., 2 (1963)
228–230.
[15] J. Hirsch, E. Petra´kova´, J. Schraml, Carbohydr. Res., 131
(1984) 219–226.
[16] (a) J.D. Stevens, H.G. Fletcher Jr., J. Org. Chem., 33
(1968) 1799–1805. (b) K. Mizutani, R. Kasai, M.
Nakamina, O. Tanaka, H. Matsuura, Carbohydr. Res.,
185 (1989) 27–38.
[17] J.G.M. Van der Ven, J. Kere´kgya´rto´, J.P. Kamerling, A.
Lipta´k, J.F.G. Vliegenthart, Carbohydr. Res., 264 (1994)
45–62 and references cited herein.
Allyl 2-O-(h-
L
-arabinofuranosyl)-6-O-(h-
D
-
mannopyranosyl)-i-
D
-mannopyranoside (12).
The esterified derivative 11 was stirred
overnight with MeONa in MeOH (6 equiv),
the soln was neutralized with ion-exchange
resin H+ (Amberlite IR 120), filtered, diluted
with distilled water; the aq soln was ex-
tracted twice with CHCl3 then freeze-dried
to give 12 (10 mg) as an amorphous solid;
Rf 0.26 (8:2, CH3CN–water); [h]D +8° (c 1,
1
water); H NMR (500 MHz, D2O, 45 °C): l
3.52 (m, 1 H, H-5), 3.63 (t, 1 H, J3,4 =J4,5
10.05 Hz, H-4), 3.67 (dd, 1 H, J2,3 3.10 Hz,
H-3), 3.70 (m, 2 H, H-4%, H-5%), 3.72 (dd, 1
H, J4¦,5¦b 6.03, J5¦a,5¦b 12.25 Hz), 3.77 (dd, 1
H, J5%,6%b 5.48, J6%a,6%b 12.06 Hz, H-6%b), 3.81
(dd, 1 H, J4¦,5¦a 3.48 Hz, H-5¦a), 3.82 (dd, 1
H, J5,6b 2.01, J6a,6b 11.33 Hz, H-6b), 3.83
(dd, 1 H, H-3%), 3.90 (dd, 1 H, J5%,6a 2.02 Hz,
H-6%a), 3.93 (dd, 1 H, J5,6a 5.66 Hz, H-6a),
3.96 (dd, 1 H, J2¦,3¦ 3.11, J3¦,4¦ 5.48 Hz, H-
3¦), 401 (dd, 1 H, J1%,2% 1.82, J2%,3% 3.47 Hz,
H-2%), 4.19 (broad dd, 1 H, H-2), 4.20–4.23
(m, 2 H, H-2¦, H-4¦), 4.17–4.22 and 4.34–
4.39 (m, 2 H, OCH2CHꢁCH2), 4.76 (broad
d, 1 H, J1,2 51 Hz, H-1), 4.92 (d, 1 H, H-
1%), 5.35 (d, 1 H, J1,251 Hz, H-1¦), 5.26–

J.-P. Utille, B. Priem / Carbohydrate Research 329 (2000) 431–439
439
[18] F.W. Lichtenthaler, T. Schneider-Adams, S. Immel, J.
Org. Chem., 59 (1994) 6735–6738.
[19] G. Excoffier, D. Gagnaire, J.P. Utille, Carbohydr. Res.,
39 (1975) 368–373.
[20] H. Franzyk, M. Meldal, H. Paulsen, K. Bock, J. Chem.
Soc., Perkin Trans. I, (1995) 2883–2898.
[21] (a) See Ref. [9]. (b) T. Ogawa, K. Sasujima, Car-
bohydr. Res., 93 (1981) 53–66. (c) F.M. Winnik,
J.R. Brisson, J.P. Carver, J.J. Krepinsky, Carbohydr.
Res., 103 (1982) 15–28. (c) S. Figneroo, S. Pe´rez, V.
Verez-Bencomo, J. Carbohydr. Chem., 17 (1998) 851–
868.
.