The synthesis of a series of modified mannotrisaccharides as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation

Article abstract of DOI:10.1016/j.carres.2004.08.007

A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation. Methyl (α-d-mannopyranosyl)-(1→3)- [(α-d-mannopyranosyl)-(1→6)]-β-d-mannopyra

Full text of DOI:10.1016/j.carres.2004.08.007

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2487–2497
The synthesis of a series of modified mannotrisaccharides as probes
of the enzymes involved in the early stages of mammalian complex
N-glycan formation
Chris A. Tarling and Stephen G. Withers*
Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z1
Received 12 July 2004; accepted 17 August 2004
Available online 21 September 2004
Abstract—A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in
the early stages of mammalian complex N-glycan formation. Methyl (a-D-mannopyranosyl)-(1!3)-[(a-D-mannopyranosyl)-(1!6)]-
b-D-mannopyranoside (6) and methyl (2-deoxy-2-fluoro-a-D-mannopyranosyl)-(1!3)-[(2-deoxy-2-fluoro-a-D-mannopyranosyl)-
(1!6)]-b-D-mannopyranoside (8) were rapidly synthesized from unprotected methyl b-D-mannopyranoside (12). Methyl
(2-deoxy-2-fluoro-a-D-mannopyranosyl)-(1!3)-[(a-D-mannopyranosyl)-(1!6)]-b-D-mannopyranoside (7) and methyl (a-D-manno-
pyranosyl)-(1!3)-[(2-deoxy-2-fluoro-a-D-mannopyranosyl)-(1!6)]-b-D-mannopyranoside (9) were synthesized from the common
orthogonally protected precursor methyl 2-O-acetyl-4,6-O-benzylidene-b-D-mannopyranoside (15). The 2-deoxy-2-fluoro substitu-
tion common to trisaccharides 7–9 renders these analogues resistant to enzyme action in two distinct ways. Firstly the fluorine serves
as a non-nucleophilic isostere for the acceptor hydroxyl in studies with glycosyl transferases GnT-I and GnT-II (7 and 9, respec-
tively). Secondly it should render trisaccharide 8 stable to hydrolysis by the mannosidases Man-II and Man-III by inductive desta-
bilization of their oxocarbenium ion-like transition states. These analogues should be useful for structural studies on these enzymes.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: N-Acetylglucosaminyl transferase; Mannosyl hydrolase; Mannotrisaccharide; Fluorine; Inhibitors
1. Introduction
come involved without prior action of GnT-1. Absence
of this enzyme, and therefore a complete deficiency of
complex N-glycans, has been shown to be embryonic
lethal in mice.6,7
Glycoprotein glycans are protein bound oligosacchar-
ides arising from post-translational modifications of
proteins. These glycans, which can be either O- (Ser/
Thr) or N-linked (Asn), are vital for many cellular or
intercellular processes. Among the roles implicated for
these glycoconjugates are cell differentiation, cancer
metastasis, and bacterial and viral infection.1 The
assembly of complex N-glycans follows a series of
well-defined steps involving many sugar-processing en-
zymes (Chart 1).2 Entry into the biosynthetic pathway
of complex N-glycans is gained solely through the addi-
tion of an N-acetyl-glucosamine residue to an oligo-
mannose core, a process performed by the glycosyl
transferase GnT-I.3–5 No downstream enzymes can be-
Subsequent to the action of GnT-I, the next enzyme in
the pathway is Golgi a-mannosidase II (Man-II).8 This
enzyme is responsible for the hydrolysis of two terminal
mannose units to give the oligosaccharide 3 (Chart 1). In
the absence of Man-II a rescue pathway involving a dif-
ferent mannosidase (Golgi a-mannosidase III, Man-III)
prior to the action of GnT-I has been observed.9 In this
alternate route, the Man5 core 1 is first trimmed of its
two terminal mannose units to give 5, followed by
GnT-I mediated glycosylation to arrive at the same
oligosaccharide 3. It is upon this substrate that another
N-acetyl-glucosamine transferase, GnT-II, is responsible
for the introduction of a second branching GlcNAc
residue.10 Upon formation of this doubly N-acetyl-
glucosaminylated oligosaccharide 4, the pathway
*
Corresponding author. Tel.: +1 604 822 3402; fax: +1 604 822 2847;
e-mail: withers@chem.ubc.ca
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.08.007

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C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
Gn3
Gn3
Gn3
Gn4
β1,2
M4
M5
M4
M5
β1,2
β1,2
β1,2
α1,6
α1,6
α1,3
α1,3
M2
M3
M2
M3
M2
M3
M2
M3
α1,3
α1,6
α1,3
α1,6
α1,3
α1,6
α1,3
α1,6
M1
M1
M1
M1
β1,4
β1,4
β1,4
α1,4
GnT-I
Man-II
GnT-II
Gn2
β1,4
Gn2
β1,4
Gn2
β1,4
Gn2
β1,4
Gn1
β
Gn1
β
Gn1
β
Gn1
β
Asn
Asn
Asn
Asn
1
2
3
4
M2
M3
Man-III
α1,3
α1,6
GnT-I
M1
β1,4
Gn2
β1,4
Gn1
β
Asn
5
Chart 1. The early stages of the mammalian complex N-glycan pathway. Gn = N-acetyl-glucosamine; M = mannose.
diverges, with many different enzymes being responsible
for the complex structures that eventually arise from this
common precursor.
6 would be processed by the a-mannosyl hydrolases
Man-II and Man-III. Therefore, in the second ap-
proach, the 2-deoxy-2-fluoro substitution is used to
inductively destabilize the transition states involved in
the enzyme catalyzed reaction and thereby render the
trisaccharide 8 stable to these enzymes (Chart 2).
The aim of this work is to produce a series of modified
mannotrisaccharides, utilizing a divergent strategy, as
mechanistic or structural probes for the investigation
of the first four enzymes involved in the elaboration of
oligomannose 1 into complex N-glycans. Three dimen-
sional structures of both GnT-1 and Man-II have
recently been solved by X-ray crystallography.11,12
However, in neither case has a structure with all relevant
substrates present been determined. Missing compo-
nents of complexes are the mannotrisaccharide acceptor
for GnT-I and the trisaccharide substrate for Man-II.
The absence of structures including these key oligosac-
charides leaves much unknown regarding the details of
the binding pocket for these residues. One of the key
problems in attaining such binary (Man-II) or ternary
(GnT-I) complexes for crystallographic studies is pre-
vention of the natural enzyme-catalyzed process so that
a substrate complex, rather than a product complex,
may be observed. A commonly used strategy to combat
this problem involves the use of incompetent substrates;
compounds that structurally resemble the natural sub-
strate but which have been modified in such a way that
they are unable to participate in the enzyme catalyzed
reaction. In this study we describe the synthesis of fluo-
rinated substrate analogues that should block the action
of several enzymes in this pathway by two quite distinct
processes. In the first instance, fluorine is utilized as an
isostere for the hydroxyl group onto which the N-acetyl-
glucosamine residue is transferred by GnT-I or GnT-II
(7 and 9, respectively). It is expected, although not
known, that truncated mannotrisaccharides of the type
Previous work has shown that trisaccharide 6 is the
minimal optimal acceptor for GnT-I.13–15 The three
mannose units present are equivalent to the M1–M2–
M3 trisaccharide moiety present in oligosaccharide
1. In a previous study of acceptor specificity, the branched
mannotrisaccharides 2-deoxy-Man(a1!3)[Man(a1!6)]-
Manb-OMe and 2-O-methyl-Man(a1!3)[Man-(a1!6)]-
Manb-OMe were shown to be neither substrates nor
inhibitors of GnT-I.14 The removal of the key 2-
hydroxyl group presumably eliminates one or more
important hydrogen bonding interactions. That the 2-O-
methyl analogue did not bind in the GnT-I active site is
likely due to the increased steric bulk at this crucial posi-
tion (an event also observed upon an equivalent modifica-
tion to the acceptor for GnT-II).16 A more conservative
modification is to replace the 20-hydroxyl with a fluorine,
and indeed fluorinated analogues have been shown to
bind with similar affinities to those of the parent sugar.16
Removal of the 20-hydroxyl group will render the trisac-
charide 7 unable to accept the N-acetylglucosamine resi-
due offered by GnT-I. Such an incompetent substrate
analogue may well bind tightly and allow for structural
studies to be performed.
The bis(2-deoxy-2-fluoro)-compound 8, may prove to
be an interesting mechanistic probe for both Golgi a-
mannosidase II and III since this trisaccharide strongly
resembles the terminal M3–M4–M5 portion of the nat-
ural substrate for these enzymes. The presence of the

C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
2489
OH
O
OH
O
F
F
HO
HO
HO
HO
HO
HO
HO
HO
O
O
HO
HO
HO
HO
O
O
O
O
O
O
O
O
OH
O
OH
O
OH
O
OH
O
HO
O
HO
O
HO
O
HO
O
OMe
OMe
OMe
OMe
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
OH
F
F
OH
6
7
8
9
Chart 2. Modified mannotrisaccharides for probing enzymes involved in complex N-glycan biosynthesis.
2-fluorine substitution without a chemically activated
leaving group should render these sugars stable to
hydrolysis by a glycosidase, a strategy, which has been
employed with much success in the investigation of
b-glycosidases.17
The final enzyme in this pathway, GnT-II, is respon-
sible for the addition of an N-acetyl glucosamine residue
to the 2-position of M3 in the M1–M2(Gn3)–M3 tetra-
saccharide. We envisage that a non-competent analogue,
with a 2-deoxy-2-fluoro sugar in place of M3, should be
readily accessible from GnT-I enzyme-mediated glycos-
ylation of trisaccharide 9.15
synthesis of target compounds 6 and 8, however, a
more traditional approach must be employed for targets
7 and 9.
The differentially protected mannose unit 15 contain-
ing a synthetically challenging b-mannosyl linkage was
required as the key building block for access to target
trisaccharides 7 and 9. The installation of this b-methyl
glycosidic linkage was previously reported via a stannyl-
ene acetal mediated alkylation of 3,4,6-tri-O-benzyl
mannopyranose.22 This approach was particularly
appealing to our synthetic route since after methylation
the 2-hydroxyl group remains free and can be orthogo-
nally protected. Briefly, regio- and stereoselective glycos-
ylation of 10 is accomplished via formation of a cyclic
1,2-stannylene acetal with dibutyltin oxide under
Dean–Stark conditions, followed by treatment with
methyl iodide to give the required b-methyl glycoside
11.22 For the synthesis of targets 6 and 8 via the Hinds-
gaul route, fully deprotected b-methyl mannoside 12 was
required. Catalytic hydrogenolysis of the benzyl ethers
gave the tetrol starting material 12 for this route. Target
compounds 7 and 9 require a more complex precursor,
so mannoside 11 was further elaborated. After protec-
tion of the remaining free hydroxyl at the 2-position as
its acetyl derivative, the benzyl ethers were removed by
catalytic hydrogenolysis to give the triol 14. Regioselec-
tive protection of the 4- and 6-hydroxyls with benzalde-
hyde dimethyl acetal afforded the required key building
block methyl 2-O-acetyl-4,6-O-benzylidene-b-D-manno-
pyranoside 15 (Scheme 1).
Target mannosides 6 and 8 were readily amenable to
synthesis via the Hindsgaul methodology (Scheme 2).21
Glycosylation of the unprotected b-methyl mannoside
12 with 2.5equiv of either mannosyl bromide 16 or
2-deoxy-2-fluoro-mannosyl bromide 17 donor gave a
complex mixture of products from which a mixture of
isomeric trisaccharides was isolated by column chroma-
tography. Reaction of these two mixtures with sodium
periodate followed by global deprotection of the acetate
groups with sodium methoxide allowed, in both cases,
isolation of the desired mannotrisaccharide 6 or 8.
Although the obtained yields were low (14% for 6 and
These compounds should prove to be useful tools for
detailed kinetic and structural studies of this important
group of enzymes.
2. Results and discussion
Whilst several synthetic strategies to 2,4-di-O-protected
mannosides are reported in the literature,18–20 Kaur
and Hindsgaul have developed a rapid synthesis of a
structurally related analogue of the branched mannotri-
saccharide 6 requiring no prior protection steps.21 This
strategy involved mannosylation of unprotected octyl
b-D-mannopyranoside with 2.5equiv of donor sugar,
giving a complex mixture of products. However, by
making ingenious use of the fact that the desired 3,6-
disubstituted mannoside contain no vicinal diols about
the core bisecting sugar residue, the unwanted regio-
isomers could be selectively oxidized by treatment with
sodium periodate. Subsequent to Zemplen deprotection
of the acetyl protecting groups on the terminal sugar res-
idues, the desired product was readily isolated by col-
umn chromatography. Although the reported yield is
low (17% over these three key steps), this synthesis rep-
resents an extremely efficient route into this class of
compounds due to the significant number of protecting
group manipulation steps that are avoided. A limitation
of this approach for our purposes is that it is applicable
only to trisaccharide targets arising from the same
donor sugar. This method is therefore suitable for the

2490
C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
Ph
OR1
O
R2O
R2O
OAc
O
OH
O
BnO
BnO
O
a
e
O
R2O
OMe
BnO
OH
HO
OMe
15
10
R1 R2
b
11
12
13
14
H
H
Ac
Ac
Bn
H
Bn
H
c
d
Scheme 1. Reagents and conditions: (a) Bu2SnO, MeI, toluene/benzene (3:1), 73%; (b) H2, Pd/C, MeOH, 88%; (c) Ac2O, pyridine, 96%; (d) H2, Pd/C,
MeOH, 88%; (e) PhCH(OMe)2, TsOH, DMF, 56%.
OAc
O
AcO
AcO
R
HO
HO
O
AcO
HO
OH
O
HO
HO
a, b, c
Br
O
O
16
+
OH
O
HO
OMe
HO
O
OMe
or
12
F
AcO
AcO
HO
HO
O
HO
AcO
R
R
Br
17
6
8
OH
F
Scheme 2. Reagents and conditions: (a) HgBr2, Hg(CN)2, MeCN, 16 or 17; (b) NaIO4, MeOH, H2O; (c) MeONa, MeOH, 14% for 6, 5% for 8.
5% for 8), they still represent a more efficient route into
this class of compounds than traditional synthetic
routes involving multiple protection and deprotection
steps.
amine–borane complex. In contrast to previous reports
using this reagent,24,25 only the 6-O-benzyl ether regio-
isomer was observed. Confirmation of this assignment
was established by the observation (1H NMR) of an in-
duced downfield chemical shift (1.3ppm) of the H-4 pro-
ton upon acetylation to provide 20. Deprotection of the
benzyl ether of 20 by catalytic hydrogenolysis gave the
6-hydroxy disaccharide 21 ready for the final
glycosylation.
The target trisaccharide 7 was realized by addition of
the final mannosyl moiety using the bromide donor 16
(Scheme 4). Global deprotection was accomplished
employing Zemplen conditions. It is worth noting that
this deprotection was slow to proceed to completion, a
phenomenon previously reported for the Zemplen
deprotection of structurally similar acylated branched
mannotrisaccharides.26
The regioisomeric trisaccharide 9 was synthesized
from the common, selectively protected mannoside 15.
Glycosylation of 15 with the mannosyl bromide donor
16 was observed to proceed only in low yield. A more
successful glycosylation was accomplished when the
corresponding trichloroacetimidate donor 23 was used
(Scheme 5). Regioselective ring opening of the benzylid-
ene moiety again afforded only the 6-O-benzyl regio-
isomer 25. Simple protecting group manipulations
With the two symmetric mannoside targets 6 and 8 in
hand, attention turned to the synthetically more com-
plex targets 7 and 9. The routes to these two trisacchar-
ides both utilize the same orthogonally protected
precursor 15. Elaboration of this core structure to target
7 began with glycosylation of the 3-hydroxyl with 2-
deoxy-2-fluoro-mannose (Scheme 3). Glycosylation of
15 was achieved in good yield using 3,4,6-tri-O-acetyl-
2-deoxy-2-fluoro-a-D-mannosyl bromide 17 as the
donor. The disaccharide product 18 was difficult to purify
from unreacted monosaccharide acceptor 15. Therefore,
the reaction mixture was subjected to acetylation condi-
tions in order to facilitate purification of 18. Although
the glycosyl donor 17 has no C-2 acetoxy group to pro-
vide neighboring group participation, only formation of
the a-linked anomer 18 was observed, as determined by
1H and 19F NMR.23 This stereochemical outcome is
likely due to a combination of the anomeric effect
exerted by the endocyclic oxygen and the dipolar inter-
action from the axially oriented C-2 fluorine. Regio-
selective reductive ring opening of the benzylidene acetal
to the benzyl ether 19 was accomplished with trimethyl-

C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
2491
Ph
R1O
R2O
OAc
O
O
O
OAc
O
F
AcO
AcO
O
O
O
15
a
b
+
OMe
O
OMe
AcO
AcO
AcO
AcO
AcO
17
Br
R1 R2
O
18
AcO
AcO
F
F
19
20
21
Bn
Bn
H
H
Ac
Ac
c
d
Scheme 3. Reagents and conditions: (a) i. HgBr2, Hg(CN)2, MeCN; ii. Ac2O, pyridine, 64%; (b) Me3NÆBH3, CH2Cl2, 52%; (c) Ac2O, pyridine, 90%;
(d) H2, Pd/C, MeOH, 78%.
OR
O
RO
RO
RO
OAc
O
HO
AcO
a
O
O
+
16
O
OMe
OR
O
RO
O
AcO
AcO
OMe
O
AcO
F
21
RO
RO
RO
F
R
22
7
Ac
H
b
Scheme 4. Reagents and conditions: (a) HgBr2, Hg(CN)2, MeCN, 85%; (b) NaOMe, MeOH, 71%.
Ph
OAc
O
R1O
O
O
OAc
O
OAc
O
AcO
AcO
R2O
O
O
15
a
b
+
OMe
O
OMe
AcO
AcO
AcO
AcO
AcO
23
24
O
R1 R2
NH
O
OAc
AcO
OAc
AcO
25
26
27
Bn
Bn
H
H
Ac
Ac
c
Cl3C
d
Scheme 5. Reagents and conditions: (a) TMSOTf, CH2Cl2, 73%; (b) Me3NÆBH3, CH2Cl2, 57%; (c) Ac2O, pyridine, 88%; (d) H2, Pd/C, MeOH, 87%.
gave disaccharide 27 ready for addition of the final sugar
moiety.
a prolonged reaction time under Zemplen conditions to
give the target compound 9.
An appropriate 2-deoxy-2-fluoro-mannosyl donor
was readily synthesized from 3,4,6-tri-O-acetyl glucal
using the Selectfluor procedure described by Dax and
co-workers.27 The hemi-acetal was then converted to
the required trichloroacetimidate donor 28 upon treat-
ment with trichloroacetonitrile. Glycosylation of the
6-position of disaccharide 27 with donor 28 gave the
protected mannotrisaccharide 29 (Scheme 6). As was
previously observed in the synthesis of 24, the trichloro-
acetimidate donor 28 was found to give superior yields
over the equivalent glycosyl bromide. Global deprotec-
tion of the acetate groups was again accomplished with
Structural studies and kinetic analysis of these four
key compounds (6–9) with GnT-I and Man-II are cur-
rently in progress.
3. Experimental
3.1. General methods
All reagents were obtained from commercial suppliers
and were used without further purification. Col-
umn chromatography was performed with silica gel

2492
C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
F
RO
RO
O
RO
OAc
O
HO
AcO
F
AcO
AcO
O
a
+
O
O
O
OMe
AcO
OR
O
RO
O
AcO
AcO
OMe
O
28
NH
O
OAc
AcO
27
RO
RO
Cl3C
RO
OR
R
29
9
Ac
H
b
Scheme 6. Reagents and conditions: (a) TMSOTf, CH2Cl2, 57%; (b) NaOMe, MeOH, 73%.
D-mannoside 12 (1.0g, 1.97mmol) in MeOH (20mL)
was added 10% Pd/C (100mg) and catalytic AcOH and
the mixture stirred under an atmosphere of hydrogen
for 12h. The reaction mixture was then filtered through
Celite and the filtrate concentrated under diminished
pressure. The crude material was purified by flash col-
umn chromatography over silica gel (5 ! 20% MeOH–
CHCl3) to yield the title compound 14 a colorless oil
(470mg, 1.97mmol, 100%). 1H NMR (400MHz,
CDCl3): dH 5.27 (1H, d, J 3.4Hz, H-2), 4.65 (1H, s,
H-1), 3.86 (1H, dd, J 1.7 and 12.4Hz, H-6a), 3.74
(1H, dd, J 3.4 and 9.7Hz, H-3), 3.65 (1H, dd, J 6.6
and 12.4Hz, H-6b), 3.48 (1H, t, J 9.7Hz, H-4), 3.42
(3H, s, OCH3), 3.36 (1H, ddd, J 1.7, 6.6 and 9.7Hz,
H-5), 2.07 (3H, s, OAc). 13C NMR (100MHz, CDCl3):
dC 173.6, 99.6, 76.5, 72.0, 71.4, 67.1, 61.0, 57.0 and
20.3.
(230–400mesh). TLC analysis was performed on pre-
coated E. Merck 60 F-254 silica plates and visualized
by staining with 10% ammonium molybdate in 2M
H2SO4 followed by heating. Methylene chloride was dis-
tilled over calcium hydride. Methanol was distilled over
˚
magnesium and iodine. DMF was dried over 4A molecu-
lar sieves. All 1H NMR assignments were based on COSY
experiments.
3.1.1. Methyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranoside (13). Known methyl 3,4,6-tri-O-benzyl-b-
D-mannoside 1119 (2g, 4.3mmol) in 2:1 pyridine–Ac2O
(30mL) was stirred overnight at room temperature.
MeOH (5mL) was added and the mixture stirred for a
further 0.5h. The reaction mixture was then concen-
trated under diminished pressure and poured onto
EtOAc. The organic layer was washed with satd NaH-
CO3 (· 2) and water and dried over MgSO4. The solvent
was evaporated under diminished pressure and the crude
material purified by flash column chromatography over
silica gel (3:1 hexane–EtOAc) to give the title compound
3.1.3. Methyl 2-O-acetyl-4,6-O-benzylidene-b-D-manno-
pyranoside (15). To a soln of methyl 2-O-acetyl-b-D-
mannoside 14 (460mg, 1.95mmol) in DMF (3mL)
containing benzaldehyde dimethyl acetal (3.0g, 19.5
mmol) was added catalytic p-TsOH (10mg). The reac-
tion mixture was stirred at room temperature for 12h
then poured onto EtOAc. The organic layer was washed
with satd NaHCO3, water (· 5), and brine. The organic
layer was then dried over MgSO4 and the solvent evap-
orated under diminished pressure. The crude material
was purified by flash column chromatography over silica
gel (3:2 EtOAc–hexane) to give the product 15 as a col-
orless crystalline solid (357mg, 1.10mmol, 56%). Mp
149–150ꢁC (EtOAc/hexane). 1H NMR (400MHz,
CDCl3): dH 7.49–7.45 (2H, m, Ar), 7.38–7.34 (3H, m,
Ar), 5.57 (1H, s, ArCH), 5.46 (1H, dd, J 1.2 and
3.6Hz, H-2), 4.50 (1H, d, J 1.2Hz, H-1), 4.34 (1H, dd,
J 5.0 and 10.5Hz, H-6a), 3.95 (1H, dd, J 3.6 and
9.8Hz, H-3), 3.87 (1H, t, J 10.5Hz, H-6b), 3.84 (1H, t,
J 9.8Hz, H-4), 3.51 (3H, s, OCH3), 3.39 (1H, ddd, J
5.0, 9.8, and 10.5Hz, H-5), 2.18 (3H, s, OAc). 13C
NMR (100MHz, CDCl3): dC 170.8, 137.0, 129.3,
1
13 as a colorless oil (2.1g, 4.1mmol, 96%). H and 13C
NMR data were found to be in accordance with the lim-
ited data previously reported in the literature.28 1H
NMR (400MHz, CDCl3): dH 7.33–7.24 (13H, m, Ar),
7.18–7.15 (2H, m, Ar), 5.61 (1H, d, J 3.3Hz, H-2),
4.86 (1H, d, J 10.8Hz, OCH2Ara), 4.74 (1H, d, J
11.2Hz, OCH2Arb), 4.65 (1H, d, J 12.0Hz, OCH2Arc),
4.55 (1H, d, J 12.0Hz, OCH2Arc), 4.50 (1H, d, J
10.8Hz, OCH2Ara), 4.48 (1H, d, J 11.2Hz, OCH2Arb),
4.90 (1H, s, H-1), 3.80–3.74 (3H, m, H-4, H-6a and H-
6b), 3.65 (1H, dd, J 3.3 and 9.3Hz, H-3), 3.51 (3H, s,
OCH3), 3.47 (1H, ddd, J 2.3, 4.8, and 9.7Hz, H-5),
2.19 (3H, s, OAc). 13C NMR (100MHz, CDCl3): dC
170.7, 138.3, 138.3, 137.6, 128.5, 128.4, 128.4, 128.2,
128.0, 127.9, 127.9, 127.7, 127.6, 100.0, 80.4, 75.6,
75.2, 74.4, 73.6, 71.5, 69.3, 67.9, 57.2, and 21.1.
3.1.2. Methyl 2-O-acetyl-b-D-mannopyranoside (14). To
a solution of methyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-

C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
2493
128.4, 126.3, 102.2, 100.6, 78.7, 70.9, 69.8, 68.5, 66.9,
57.5, and 20.9. HRMS-ESI: calcd for C16H20O7Na:
347.1107; found: 347.1094. Anal. Calcd for C16H20O7:
C, 59.25; H, 6.22. Found: C, 58.89; H, 6.04.
those used for compound 6. The crude mixture was puri-
fied by flash column chromatography over silica (7:3:1
CH2Cl2–MeOH–H2O) to give the title compound 8 as
1
a colorless powder (15mg, 0.03mmol, 5%). H NMR
(400MHz, D2O): dH 5.17 (1H, d, J 7Hz, H-10), 4.97
(1H, d, J 7Hz, H-100), 4.79–4.60 (2H, m partially ob-
scured by water peak, H-20 and H-200), 4.43 (1H, s, H-
1), 4.00 (1H, d, J 2.6Hz, H-2), 3.89–3.53 (14H, m),
3.42–3.34 (1H, m), 3.36 (3H, s, OCH3). 13C NMR
(100MHz, D2O): dC 100.8, 99.2 (d, J 30Hz), 96.5 (d, J
30Hz), 89.4 (d, J 173Hz), 89.3 (d, J 172Hz), 81.0,
73.8, 73.1, 72.5, 69.8, 69.6 (d, J 17Hz), 69.4 (d, J
17Hz), 66.5, 66.5, 65.5, 65.4, 60.3, 60.3, 56.7. 19F
NMR (376MHz, D2O): dF ꢀ128.1 (ddd, J 7, 31, and
50Hz, F-20), ꢀ129.5 (ddd, J 7, 31, and 49Hz, F-200).
HRMS-ESI: calcd for C19H32F2O16Na: 545.1658;
found: 545.1655.
3.1.4.
Methyl
(a-D-mannopyranosyl)-(1!3)-[(a-D-
mannopyranosyl)-(1!6)]-b-D-mannopyranoside (6). To
a soln of methyl b-D-mannoside 12 (165mg, 0.85mmol)
˚
in MeCN (100mL) containing 4A molecular sieves,
mercuric bromide (760mg, 2.12mmol) and mercuric
cyanide (540mg, 2.12mmol) was added donor 16
(870mg, 2.12mmol). The mixture was stirred at 35ꢁC
for 16h then filtered through Celite. The filtrate was
concentrated under diminished pressure then redissolved
in CH2Cl2 and extracted with water. The organic layer
was washed sequentially with water, NaHCO3 soln,
and brine then dried over MgSO4 and the solvent evap-
orated under diminished pressure. The crude material
was fractionated by flash column chromatography over
silica (2:1 EtOAc–hexane ! EtOAc) and the trisaccha-
ride-containing fractions (Rf 0.19–0.38, 2:1 EtOAc–hex-
ane) pooled and used in the next step without further
purification. The crude trisaccharide mixture (280mg)
was dissolved in MeOH–water (1:2, 15mL) containing
sodium periodate (300mg) and stirred at room tempera-
ture overnight. The reaction mixture was poured onto
water, extracted with CH2Cl2, the organic layer was
dried over MgSO4, and the solvent was evaporated
under diminished pressure. Without further purification
the crude material was dissolved in MeOH (10mL) and
catalytic sodium methoxide was added. The mixture was
stirred at room temperature for 4h. The sodium meth-
oxide was neutralized with Amberlite IR-120 H+ resin
and the solvent evaporated under diminished pressure.
The crude mixture was purified by flash column chroma-
tography over silica (7:4:1 CH2Cl2–MeOH–H2O) to give
the title compound 6 as a colorless powder (60mg,
3.1.6. Methyl 2-O-acetyl-(3,4,6-tri-O-acetyl-2-deoxy-2-
fluoro-a-D-mannopyranosyl)-(1!3)-4,6-O-benzylidene-b-
D-mannopyranoside (18). To a soln of acceptor 15
(590mg, 1.82mmol) and donor 17 (1.2g, 3.23mmol) in
˚
MeCN (20mL) containing 4A molecular sieves was
added mercuric cyanide (816mg, 3.23mmol) and merc-
uric bromide (1.16g, 3.23mmol). The reaction mixture
was allowed to stir at room temperature for 12h. TLC
analysis showed the formation of product, but the prod-
uct and starting material were observed to have very
similar mobility under all solvent systems investigated.
The reaction mixture was filtered through Celite, con-
centrated under diminished pressure then concentrate
was redissolved in EtOAc and the organic layer washed
with satd NaHCO3 solution and brine, dried over
MgSO4, and concentrated under diminished pressure.
To resolve the separation problem, the crude reaction
mixture was acetylated with 2:1 pyridine–Ac2O
(30mL) overnight at room temperature. MeOH (5mL)
was added and the mixture stirred for a further 0.5h.
The reaction mixture was then concentrated under di-
minished pressure and poured onto EtOAc. The organic
layer was washed with 0.1M HCl, satd NaHCO3 (· 2)
and brine, dried over MgSO4, and the solvent was evap-
orated under diminished pressure. The crude material
was purified by flash column chromatography over silica
gel (2:1!3:1 EtOAc–hexane) to give the title compound
18 as a colorless oil (720mg, 1.17mmol, 64%). Mp 88–
90ꢁC (EtOAc/hexane). 1H NMR (400MHz, CDCl3):
dH 7.38–7.32 (5H, m, Ar), 5.56 (1H, s, PhCH), 5.51
(1H, dd, J 1.1 and 3.6Hz, H-2), 5.34 (1H, dd, J 10.3
and 9.3Hz, H-40), 5.32 (1H, d, J 6.8Hz, H-10), 5.05
(1H, ddd, J 2.3, 10.3, and 28.6Hz, H-30), 4.76 (1H, dt,
J 2.3 and 49.8Hz, H-20), 4.56 (1H, d, J 1.1Hz, H-1),
4.34 (1H, dd, J 4.9 and 10.5Hz, H-6a), 4.26 (1H, dd, J
4.2 and 12.6Hz, H-6a0 ), 4.15–4.10 (2H, m, H-50 and
H-60b), 4.08 (1H, dd, J 3.6 and 9.6Hz, H-3), 3.98 (1H,
t, J 9.6Hz, H-4), 3.88 (1H, t, J 10.5Hz, H-6b), 3.53
1
0.12mmol, 14%). H NMR (400MHz, D2O): dH 4.95
(1H, d, J 1.6Hz, H-10), 4.76 (1H, d, J 1.5Hz, H-100),
4.44 (1H, s, H-1), 3.99 (1H, d, J 3.0Hz, H-2), 3.92
(1H, dd, J 1.6 and 3.2Hz, H-20), 3.85 (1H, dd, J 1.5
and 3.3Hz, H-200), 3.84–3.48 (14H, m), 3.43–3.35 (1H,
m), 3.37 (3H, s, OAc). 13C NMR (100MHz, D2O): dC
102.2, 100.9, 80.5, 74.6, 73.9, 73.1, 70.4, 70.2, 70.0,
69.9, 69.8, 69.7, 66.6, 66.6, 65.6, 65.2, 65.2, 60.8, 56.7.
HRMS-ESI: calcd for C19H34O16Na: 541.1745; found:
541.1736.
3.1.5. Methyl (2-deoxy-2-fluoro-a-D-mannopyranosyl)-
(1!3)-[(2-deoxy-2-fluoro-a-D-mannopyranosyl)-(1!6)]-
b-D-mannopyranoside (8). To
a
soln of methyl
b-D-mannoside 12 (120mg, 0.62mmol) in acetonitrile
˚
(25mL) containing 4A molecular sieves, mercuric bro-
mide (490mg, 1.36mmol) and mercuric cyanide
(340mg, 1.36mmol) was added donor 17 (470mg,
1.27mmol). The reaction conditions were similar to

2494
C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
(3H, s, OCH3), 3.41 (1H, ddd, J 4.9, 9.6, and 10.5Hz, H-
5), 2.22 (3H, s, OAc), 2.07 (3H, s, OAc), 2.04 (3H, s,
OAc), 2.04 (3H, s, OAc). 13C NMR (100MHz, CDCl3):
dC 170.7, 170.7, 170.1, 169.6, 136.8, 129.2, 128.4, 125.9,
101.7, 100.5, 98.0 (d, J 30Hz), 88.6 (d, J 179Hz), 78.5,
77.4, 73.6, 70.2, 69.7 (d, J 17Hz), 69.4, 68.4, 66.8,
65.1, 62.0, 57.7, and 20.8 (4 · OAc). 19F NMR
(376MHz, CDCl3): dF ꢀ127.7 (ddd, J 7, 29, and
50Hz). HRMS-ESI: calcd for C28H35FO14Na:
637.1909; found: 637.1919. Anal. Calcd for
C28H35FO14: C, 54.72; H, 5.74. Found: C, 54.87; H,
5.77; and methyl 2,3-di-O-acetyl-4,6-O-benzylidene-b-D-
mannopyranoside (100mg, 0.27mmol, 15%). Mp 105–
dd, J 6.6 and 9.7Hz, H-6b), 3.47 (3H, s, OCH3), 3.42
(1H, ddd, J 4.7, 6.6, and 9.4Hz, H-5), 2.16 (3H, s,
OAc), 2.06 (3H, s, OAc), 2.05 (3H, s, OAc), 2.03 (3H,
s, OAc). 13C NMR (100MHz, CDCl3): dC 170.7,
170.7, 170.2, 169.7, 137.2, 128.7, 128.2, 128.0, 99.9,
98.6 (d, J 30Hz), 86.8 (d, J 179Hz), 77.5, 74.0, 73.1,
71.2, 71.0, 70.0, 69.8 (d, J 17Hz), 69.2, 65.3, 62.1,
57.3, and 20.8 (4 · OAc). 19F NMR (376MHz, CDCl3):
dF ꢀ127.7 (ddd, J 7, 28 and 50Hz). HRMS-ESI: calcd
for C28H37FO14Na: 639.2065; found: 639.2034.
3.1.8.
Methyl
2,4-di-O-acetyl-(3,4,6-tri-O-acetyl-2-
deoxy-2-fluoro-a-D-mannopyranosyl)-(1!3)-6-O-benzyl-
b-D-mannopyranoside (20). Disaccharide 19 (230mg,
0.37mmol) in 2:1 pyridine–Ac2O (15mL) was stirred
overnight at room temperature. MeOH (2mL) was
added and the mixture stirred for a further 0.5h. The
reaction mixture was then concentrated under dimin-
ished pressure, redissolved in EtOAc, then washed with
1M HCl, satd NaHCO3 (· 2) and water, and dried over
MgSO4. The solvent was evaporated under diminished
pressure and the crude material purified by flash column
chromatography over silica gel (1:1 hexane–EtOAc) to
give the title compound 20 as a colorless oil (220mg,
0.33mmol, 90%). 1H NMR (400MHz, CDCl3): dH
7.33–7.24 (5H, m, Ar), 5.43 (1H, d, J 3.4Hz, H-2),
5.32 (1H, t, J 10.1Hz, H-40), 5.18 (1H, t, J 9.7Hz, H-
4), 5.05 (1H, d, J 7Hz, H-10), 5.03 (1H, ddd, J 2.3,
10.1, and 29Hz, H-30), 4.59 (1H, dt, J 2.3 and 50Hz,
H-20), 4.50 (2H, s, PhCH2), 4.47 (1H, s, H-1), 4.23
(1H, dd, J 4.3 and 12.7Hz, H-60a), 4.17–4.10 (2H, m,
H-50 and H-60b), 3.92 (1H, dd, J 3.4 and 9.7Hz, H-3),
3.64–3.51 (3H, m, H-5, H-6a, and H-6b), 3.50 (3H, s,
OCH3), 2.19 (3H, s, OAc), 2.06 (3H, s, OAc), 2.03
(3H, s, OAc), 2.02 (3H, s, OAc), 1.91 (3H, s, OAc).
13C NMR (100MHz, CDCl3): dC 170.7, 170.6, 169.9,
169.6, 169.6, 137.7, 128.4, 127.9, 127.8, 99.8, 98.7 (d, J
31Hz), 86.9 (d, J 180Hz), 76.2, 73.7, 73.6, 70.0, 69.7,
69.6, 69.6, 69.3 (d, J 17Hz), 65.2, 61.9, 57.4, 20.9, and
20.7 (4 · OAc). 19F NMR (376MHz, CDCl3): dF
ꢀ126.8 (ddd, J 7, 29, and 50Hz). HRMS-ESI: calcd
for C30H39FO15Na: 681.2171; found: 681.2148.
1
106ꢁC (EtOAc/hexane). H NMR (400MHz, CDCl3):
dH 7.44–7.43 (2H, m, Ar), 7.34–7.33 (3H, m, Ar), 5.56
(1H, dd, J 1.3 and 3.4Hz, H-2), 5.55 (1H, d, J 1.2Hz,
PhCH), 5.12 (1H, ddd, J 1.3, 3.4, and 10.4Hz, H-3),
4.60 (1H, t, J 1.3Hz, H-1), 4.35 (1H, ddd, J 1.2, 4.9,
and 10.5Hz, H-6a), 4.01 (1H, td, J 1.2 and 10.5Hz, H-
4), 3.90 (1H, td, J 1.2 and 10.5Hz, H-6b), 3.52 (3H, d,
J 1.3Hz, OCH3), 3.52–3.47 (1H, m, H-5), 2.18 (3H, d,
J 1.3Hz, OAc), 2.00 (3H, d, J 1.3Hz, OAc). 13C NMR
(100MHz, CDCl3): dC 170.3, 170.0, 137.0, 129.2,
128.3, 126.2, 101.9, 100.3, 75.7, 70.3, 69.3, 68.5, 67.3,
57.7, 20.8, and 20.8. HRMS-ESI: calcd for C18H22O8Na:
389.1212; found: 389.1202. Anal. Calcd for C18H22O8:
C, 59.01; H, 6.05. Found: C, 59.17; H, 6.24.
3.1.7. Methyl 2-O-acetyl-(3,4,6-tri-O-acetyl-2-deoxy-2-
fluoro-a-D-mannopyranosyl)-(1!3)-6-O-benzyl-b-D-man-
nopyranoside (19). To a soln of acetal 18 (100mg,
˚
0.16mmol), 4A molecular sieves (100mg), and trimeth-
ylamine borane (60mg, 0.81mmol) in CH2Cl2 (6mL)
at 0ꢁC was added dropwise a suspension of aluminum
trichloride (87mg, 0.65mmol) in Et2O (3mL). The reac-
tion mixture was stirred at 0ꢁC for 1h then filtered
through Celite. CH2Cl2 (10mL) and 0.75M H2SO4
(1mL) were added and the mixture stirred for 1h at
room temperature. The mixture was transferred to a
separating funnel and the organic layer was washed with
water, satd NaHCO3 solution, and brine. The organic
layer was dried over MgSO4, the solvent evaporated
under diminished pressure, and the crude material then
purified by flash column chromatography over silica gel
(2:1 EtOAc–hexane) to give the title compound 19 as a
colorless solid (52mg, 0.084mmol, 52%). 1H NMR
(400MHz, CDCl3): dH 7.37–7.28 (5H, m, Ar), 5.39
(1H, d, J 7.3Hz, H-10), 5.39 (1H, dd, J 1.0 and 3.4Hz,
H-2), 5.09 (1H, t, J 10.3Hz, H-40), 5.07 (1H, ddd, J
2.4, 10.3, and 28Hz, H-30), 4.79 (1H, dt, J 2.4 and
50Hz, H-20), 4.61 (1H, d, J 11.7Hz, PhCH2), 4.53
(1H, d, J 11.7Hz, PhCH2), 4.44 (1H, d, J 1.0Hz, H-1),
4.24 (1H, dd, J 4.3 and 12.6Hz, H-60a), 4.14 (1H, dd, J
2.3 and 12.6Hz, H-60b), 4.13–4.10 (1H, m, H-50), 3.92
(1H, t, J 9.4Hz, H-4), 3.83 (1H, dd, J 4.7 and 9.7Hz,
H-6a), 3.78 (1H, dd, J 3.4 and 9.4Hz, H-3), 3.74 (1H,
3.1.9.
Methyl
2,4-di-O-acetyl-(3,4,6-tri-O-acetyl-2-
deoxy-2-fluoro-a-D-mannopyranosyl)-(1!3)-b-D-manno-
pyranoside (21). Benzyl ether 20 (34mg, 0.052mmol)
was dissolved in 2:1 MeOH–CH2Cl2 (1.5mL) containing
catalytic AcOH, and 10% Pd/C was added (5mg). The
reaction mixture was stirred under an atmosphere of
hydrogen for 12h then filtered through Celite. The sol-
vent was evaporated under diminished pressure and
the crude material purified by flash column chromatog-
raphy over silica gel (2:1 EtOAc–hexane) to give the title
compound 21 as a colorless solid (23mg, 0.04mmol,
78%). Mp 156–157ꢁC (EtOAc/hexane). 1H NMR
(300MHz, CDCl3): dH 5.45 (1H, dd, J 0.9 and 3.4Hz,

C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
2495
H-2), 5.31 (1H, t, J 10.1Hz, H-40), 5.14 (1H, t, J 9.8Hz,
H-4), 5.08 (1H, dd, J 1.8 and 8Hz, H-10), 5.02 (1H, ddd,
J 2.6, 10.1, and 28Hz, H-30), 4.61 (1H, dt, J 2.2 and
50Hz, H-20), 4.50 (1H, 0d, J 0.9Hz, H-1), 4.22 (1H, dd,
J 4.1 and 12.5Hz, H-6a), 4.17–4.06 (2H, m, H-50 and
H-60b), 3.97 (1H, dd, J 3.4 and 9.8Hz, H-3), 3.71 (1H,
dd, J 2.9 and 12.5Hz, H-6a), 3.63 (1H, dd, J 5.1 and
12.5Hz, H-6b), 3.50 (3H, s, OCH3), 3.38 (1H, ddd, J
2.9, 5.1, and 9.8Hz, H-5), 2.18 (3H, s, OAc), 2.07 (3H,
s, OAc), 2.06 (3H, s, OAc), 2.04 (3H, s, OAc), 2.03
(3H, s, OAc). 13C NMR (75MHz, CDCl3): dC 170.6,
170.6, 170.1, 169.8, 169.6, 99.8, 98.7 (d, J 30Hz), 86.8
(d, J 180Hz), 76.0, 74.5, 69.9, 69.6, 69.3 (d, J 17Hz),
68.8, 65.0, 61.8, 61.4, 57.4, 20.8, and 20.6 (4 · OAc).
19F NMR (282MHz, CDCl3): dF ꢀ126.9 (ddd, J 8, 28,
and 50Hz). HRMS-ESI: calcd for C23H33FO15Na:
591.1701; found: 591.1705. Anal. Calcd for C23H33-
FO15: C, 48.59; H, 5.85. Found: C, 48.98; H, 5.74.
32, and 49Hz). HRMS-ESI: calcd for C19H33FO15Na:
543.1701; found: 543.1705.
3.1.11. Methyl 2-O-acetyl-(2,3,4,6-tetra-O-acetyl-a-
D-mannopyranosyl)-(1!3)-4,6-O-benzylidene-b-D-manno-
pyranoside (24). To a cooled (ꢀ40ꢁC) soln of acceptor
15 (130mg, 0.40mmol) in CH2Cl2 (10mL) containing
the trichloroacetimidate donor 23 (490mg, 1.0mmol)
was added catalytic boron trifluoride diethyl etherate.
The reaction was allowed to warm to 0ꢁC over 4h then
Et3N (0.1mL) was added. The organic layer was washed
with satd NaHCO3 soln and brine, dried over MgSO4,
and concentrated under diminished pressure. The crude
material was purified by flash column chromatography
over silica gel (2:1 EtOAc–hexane) to give the title com-
pound 24 as a colorless crystalline solid (191mg,
1
0.29mmol, 73%). Mp 177–178ꢁC (EtOAc/hexane). H
NMR (400MHz, CDCl3): dH 7.40–7.37 (2H, m, Ar),
7.31–7.29 (3H, m, Ar), 5.78 (1H, s, PhCH), 5.52 (1H,
br s, H-2), 5.34 (1H, br s, H-20), 5.29 (1H, t, J 10.3Hz,
H-40), 5.19 (1H, s, H-10), 5.15 (1H, dd, J 3.4 and
10.3Hz, H-30), 4.56 (1H, s, H-1), 4.35 (1H, dd, J 4.9
and 10.4Hz, H-6a), 4.28 (1H, dd, J 4.8 and 13.0Hz,
H-60a), 4.11–4.08 (2H, m, H-50 and H-60b), 4.03–4.01
(2H, m, H-3 and H-4), 3.89 (1H, t, J 10.4Hz, H-6b),
3.53 (3H, s, OCH3), 3.42–3.36 (1H, m, H-5), 2.25 (3H,
s, OAc), 2.08 (3H, s, OAc), 2.06 (3H, s, OAc), 2.04
(3H, s, OAc), 1.94 (3H, s, OAc). 13C NMR (100MHz,
CDCl3): dC 170.8, 170.6, 169.9, 169.9, 169.7, 136.9,
129.0, 128.2, 125.9, 101.4, 100.5, 98.3, 78.7, 73.1, 70.1,
69.3, 69.1, 68.8, 68.3, 66.8, 65.5, 62.3, 57.6, and 20.8
(4 · OAc), 20.7 (OAc). HRMS-ESI: calcd for
C30H38O16Na: 677.2058; found: 677.2036. Anal. Calcd
for C30H38O16: C, 55.05; H, 5.85. Found: C, 55.04; H,
5.88.
3.1.10. Methyl (2-deoxy-2-fluoro-a-D-mannopyranosyl)-
(1!3)-[(a-D-mannopyranosyl)-(1!6)]-b-D-mannopyrano-
side (7). To a soln of acceptor 21 (90mg, 0.158mmol)
and donor 16 (130mg, 0.316mmol) in MeCN (10mL)
˚
containing 4A molecular sieves was added mercuric
cyanide (75mg, 0.284mmol) and mercuric bromide
(108g, 0.284mmol). The reaction mixture was allowed
to stir at room temperature for 12h, then filtered
through Celite, and concentrated under diminished pres-
sure. The concentrate was redissolved in EtOAc and the
organic layer was washed with satd NaHCO3 soln and
brine, dried over MgSO4, and concentrated under
diminished pressure. The crude material was purified
by flash column chromatography over silica gel (2:1
EtOAc–hexane) to give the protected compound 22 as
a colorless oil (120mg, 0.134mmol, 85%). After column
chromatography the product was observed to contain a
co-eluting minor impurity. This material was therefore
deprotected and characterized as the final product de-
scribed below. To a soln of 22 (110mg, 0.122mmol) in
MeOH (10mL) was added catalytic sodium methoxide,
the mixture stirred at room temperature for 2days.
The sodium methoxide was then neutralized with
Amberlite IR-120 H+ resin and the solvent was evapo-
rated under diminished pressure. The crude material
was purified by flash column chromatography over silica
(7:4:1 CH2Cl2–MeOH–H2O) to give the title compound
3.1.12. Methyl 2-O-acetyl-(2,3,4,6-tetra-O-acetyl-a-D-
mannopyranosyl)-(1!3)-6-O-benzyl-b-D-mannopyranos-
ide (25). To a soln of acetal 24 (315mg, 0.48mmol),
˚
4A molecular sieves (100mg), and trimethylamine bor-
ane (176mg, 2.4mmol) in CH2Cl2 (20mL) at 0ꢁC was
added dropwise a suspension of aluminum trichloride
(257mg, 1.9mmol) in Et2O (10mL). The reaction mix-
ture was stirred at 0ꢁC for 1.5h then filtered through
Celite. CH2Cl2 (20mL) and 0.75M H2SO4 (5mL) was
added and the mixture was stirred for 1.5h at room tem-
perature. The mixture was transferred to a separating
funnel and the organic layer was washed with water,
satd NaHCO3 soln, and brine. The organic layer was
dried over MgSO4 and the solvent evaporated under
diminished pressure. The crude material was purified
by flash column chromatography over silica gel (2:1
EtOAc–hexane) to give the title compound 25 as a col-
orless oil (180mg, 0.27mmol, 57%). 1H NMR
(400MHz, CDCl3): dH 7.37–7.27 (5H, m, Ar), 5.40
(1H, dd, J 0.9 and 3.5Hz, H-2), 5.31 (1H, dd, J 1.7
1
7 as a colorless powder (45mg, 0.086mmol, 71%). H
NMR (400MHz, D2O): dH 5.23 (1H, d, J 7.9Hz, H-
10), 4.81 (1H, s, H-100), 4.78 (1H, d, J 49Hz, H-20),
4.49 (1H, s, H-1), 4.05 (1H, br s, H-2), 3.90–3.89 (1H,
m, H-200), 4.05–3.86 (1H, m, H-30), 3.81–3.56 (13 H,
m), 3.47–3.42 (1H, m), 3.42 (3H, s, OCH3). 13C NMR
(75MHz, D2O): dC 100.9, 99.4, 99.2 (d, J 30Hz), 89.5
(d, J 172Hz), 81.1, 74.0, 73.2, 72.6, 70.5, 69.9, 69.9,
69.5 (d, J 17Hz), 66.7, 66.7, 65.6, 65.3, 60.9, 60.4,
56.8. 19F NMR (376MHz, D2O): dF ꢀ128.1 (ddd, J 8,

2496
C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
and 3.4Hz, H-20), 5.28 (1H, t, J 10.2Hz, H-40), 5.23 (1H,
d, J 1.7Hz, H-10), 5.15 (1H, dd, J 3.4 and 10.2Hz, H-30),
4.61 (1H, d, J 11.8Hz, PhCH2), 4.53 (1H, d, J 11.8Hz,
PhCH2), 4.44 (1H, d, J 0.9Hz, H-1), 4.26 (1H, dd, J
4.6 and 12.3Hz, H-6a0 ), 4.16–4.09 (2H, m, H-50 and
H-60b), 3.95 (1H, dt, J 2.5 and 9.4Hz, H-4), 3.82 (1H,
dd, J 4.9 and 9.8Hz, H-6a), 3.77–3.73 (2H, m, H-3
and H-6b), 3.48 (3H, s, OCH3), 3.44–3.40 (1H, m, H-
5), 3.08 (1H, d, J 2.5Hz, C4-OH), 2.18 (3H, s, OAc),
2.11 (3H, s, OAc), 2.08 (3H, s, OAc), 2.04 (3H, s,
OAc), 1.96 (3H, s, OAc). 13C NMR (100MHz, CDCl3):
dC 170.7, 170.5, 170.0, 169.8, 169.8, 137.2, 128.5, 128.0,
127.8, 99.8, 98.8, 77.1, 73.8, 73.4, 70.8, 70.5, 69.9, 69.3,
69.1, 68.9, 65.5, 62.3, 57.2, 20.8, 20.7, 20.7, 20.6, 20.6.
HRMS-ESI: calcd for C30H40O16Na: 679.2214; found:
679.2192.
filtered through Celite. The solvent was evaporated
under diminished pressure and the crude material puri-
fied by flash column chromatography over silica gel
(EtOAc) to give the title compound 27 as a colorless
solid (87mg, 0.14mmol, 87%). Mp 191–193ꢁC
1
(EtOAc/hexane). H NMR (400MHz, CDCl3): dH 5.45
(1H, d, J 3.4Hz, H-2), 5.29 (1H, t, J 9.9Hz, H-40),
5.16–5.11 (2H, m, H-30 and H-4), 4.99 (2H, br s, H-10
and H-20), 4.50 (1H, d, J 0.8Hz, H-1), 4.24 (1H, dd, J
4.6 and 12.5Hz, H-6a0 ), 4.17–4.12 (2H, m, H-50 and
H-60b), 3.91 (1H, dd, J 3.4 and 9.9Hz, H-3), 3.69–3.65
(2H, m, H-6a and H-6b), 3.51 (3H, s, OCH3), 3.37–
3.33 (1H, m, H-5), 2.21 (3H, s, OAc), 2.12 (3H, s,
OAc), 2.12 (3H, s, OAc), 2.08 (3H, s, OAc), 2.03 (3H,
s, OAc), 1.97 (3H, s, OAc). 13C NMR (100MHz,
CDCl3): dC 170.7, 170.6, 170.5, 170.0, 169.8, 169.5,
99.8, 98.9, 76.3, 74.6, 70.6, 69.9, 69.4, 68.4, 68.1, 65.6,
62.1, 61.5, 57.3, 20.8, 20.8, 20.7, 20.6, 20.5, 20.5.
HRMS-ESI: calcd for C25H36O17Na: 631.1850; found:
631.1851. Anal. Calcd for C25H36O17: C, 49.34; H,
5.96. Found: C, 49.72; H, 6.03.
3.1.13. Methyl 2,4-di-O-acetyl-(2,3,4,6-tetra-O-acetyl-a-
D-mannopyranosyl)-(1!3)-6-O-benzyl-b-D-mannopyran-
oside (26). Disaccharide 25 (150mg, 0.23mmol) in 2:1
pyridine–Ac2O (15mL) was stirred overnight at room
temperature. MeOH (5mL) was added and the mixture
stirred for a further 0.5h. The reaction mixture was then
concentrated under diminished pressure, redissolved in
EtOAc, then washed with 1M HCl, satd NaHCO3
(· 2) and water, and dried over MgSO4. The solvent
was evaporated under diminished pressure and the crude
material purified by flash column chromatography over
silica gel (3:2 EtOAc–hexane) to give the title compound
26 as a colorless solid (140mg, 0.20mmol, 88%). Mp
149–150ꢁC (EtOAc/hexane). 1H NMR (400MHz,
CDCl3): dH 7.34–7.26 (5H, m, Ar), 5.44 (1H, dd, J 1.0
and 3.4Hz, H-2), 5.27 (1H, t, J 10.0Hz, H-40), 5.18
(1H, t, J 10.0Hz, H-4), 5.17 (1H, dd, J 3.4 and
10.0Hz, H-30), 4.98 (1H, dd, J 1.8 and 3.4Hz, H-20),
4.96 (1H, d, J 1.8Hz, H-10), 4.52 (2H, s, PhCH2), 4.47
(1H, d, J 1.0Hz, H-1), 4.24 (1H, dd, J 4.8 and
12.6Hz, H-60a), 4.16–4.11 (2H, m, H-50 and H-60b), 3.85
(1H, dd, J 3.4 and 10.0Hz, H-3), 3.66–3.52 (3H, m, H-
5, H-6a, and H-6b), 3.51 (3H, s, OCH3), 2.21 (3H, s,
OAc), 2.11 (3H, s, OAc), 2.08 (3H, s, OAc), 2.03 (3H,
s, OAc), 1.99 (3H, s, OAc), 1.97 (3H, s, OAc). 13C
NMR (100MHz, CDCl3): dC 170.7, 170.4, 169.9,
169.9, 169.8, 169.4, 137.7, 128.3, 127.8, 127.6, 99.6,
98.9, 76.5, 73.8, 73.6, 70.0, 69.9, 69.7, 69.3, 69.0, 68.1,
65.6, 62.1, 57.2, 20.8, 20.7, 20.7, 20.6, 20.6, 20.5.
HRMS-ESI: calcd for C32H42O17Na: 721.2320; found:
721.2332. Anal. Calcd for C32H42O17: C, 55.01; H,
6.06. Found: C, 55.40; H, 6.11.
3.1.15. Methyl 2,4-di-O-acetyl-(2,3,4,6-tetra-O-acetyl-a-
D-mannopyranosyl)-(1!3)-[(3,4,6-tri-O-acetyl-2-deoxy-
2-fluoro-a-D-mannopyranosyl)-(1!6)]-b-D-mannopyran-
oside (29). To a cooled (ꢀ40ꢁC) soln of acceptor 27
(50mg, 0.08mmol) in CH2Cl2 (10mL) containing the tri-
chloroacetimidate donor 2829 (150mg, 0.33mmol) was
added catalytic boron trifluoride diethyl etherate. The
reaction mixture was allowed to warm to ꢀ20ꢁC over
4h then Et3N (0.1mL) was added. The organic layer
was washed with satd NaHCO3 soln and brine, dried
over MgSO4, and concentrated under diminished pres-
sure. The crude material was purified by flash column
chromatography over silica gel (2:1!4:1 EtOAc–
hexane) to give the title compound 29 as a colorless
crystalline solid (42mg, 0.05mmol, 57%). Mp 194–
1
195ꢁC (EtOAc/hexane). H NMR (400MHz, CDCl3):
dH 5.43 (1H, d, J 3.2Hz, H-2), 5.33–5.10 (5H, m, H-30,
H-300, H-4, H-40, and H-400), 5.01–4.96 (3H, m, H-10,
H-100, and H-20), 4.74 (1H, br d, J 50Hz, H-200), 4.47
(1H, s, H-1), 4.26–4.03 (6H, m, H-50, H-500, H-6a0 ,
H-60a0, H-60b, and H-6b00), 3.88–3.81 (2H, m, H-3 and H-
6a), 3.57–3.49 (2H, m, H-5 and H-6b), 3.49 (3H, s,
OCH3), 2.19 (3H, s, OAc), 2.11 (3H, s, OAc), 2.10
(3H, s, OAc), 2.08 (3H, s, OAc), 2.07 (3H, s, OAc),
2.05 (3H, s, OAc), 2.02 (3H, s, OAc), 1.99 (3H, s,
OAc), 1.96 (3H, s, OAc). 13C NMR (100MHz, CDCl3):
dC 170.6, 170.5, 170.4, 170.0, 169.9, 169.9, 169.8, 169.4,
169.3, 99.6, 98.9, 99.8 (d, J 29Hz), 86.6 (d, J 180Hz),
76.4, 73.0, 69.9, 69.8 (d, J 17Hz), 69.4, 68.6, 68.5,
68.1, 66.8, 65.6, 65.6, 62.1, 61.9, 60.3, 57.3, 20.7, 20.7,
20.6, 20.6, 20.6, 20.6, 20.6, 20.5, 20.5. 19F NMR
(376MHz, CDCl3): dF ꢀ128.2 (ddd, J 7, 28, and
50Hz). HRMS-ESI: calcd for C37H51FO24Na:
921.2652; found: 921.2630. Anal. Calcd for
3.1.14. Methyl 2,4-di-O-acetyl-(2,3,4,6-tetra-O-acetyl-
a-D-mannopyranosyl)-(1!3)-b-D-mannopyranoside (27).
Benzyl ether 26 (115mg, 0.17mmol) was dissolved in
MeOH (20mL) containing catalytic AcOH and 10%
Pd/C was added (15mg). The reaction mixture was
stirred under an atmosphere of hydrogen for 12h then

C. A. Tarling, S. G. Withers / Carbohydrate Research 339 (2004) 2487–2497
2497
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C37H51FO24: C, 49.44; H, 5.72. Found: C, 49.46; H,
5.69.
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2-fluoro-a-D-mannopyranosyl)-(1!6)]-b-D-mannopyrano-
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1
0.015mmol, 73%). H NMR (400MHz, D2O): dH 4.99
(1H, d, J 8Hz, H-100), 4.96 (1H, s, H-10), 4.74–4.60
(1H, m, partially obscured by water peak), 4.45 (1H, s,
H-1), 4.01 (1H, d, J 3.1Hz, H-2), 3.92 (1H, br s, H-
20), 3.88 (1H, dd, J 4.5 and 11.3Hz), 3.83–3.49 (13H,
m), 3.43–3.36 (1H, m), 3.38 (3H, s, OCH3). 13C NMR
(100MHz, D2O): dC 102.2, 100.9, 96.6 (d, J 30Hz),
89.3 (d, J 172Hz), 80.5, 73.9, 73.2, 72.5, 70.1 (d, J
17Hz), 70.2, 69.9, 69.8, 66.6, 66.5, 65.6, 65.6, 60.8,
60.4, 56.7. 19F NMR (376MHz, D2O): dF ꢀ129.4
(ddd, J 7, 31, and 49Hz). HRMS-ESI: calcd for
C19H33FO15Na: 543.1701; found: 543.1708.
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Acknowledgements
We thank the Protein Engineering Network of Centres
of Excellence of Canada (PENCE) and GlycoDesign
Inc. for financial support. C.A.T. thanks the Royal Soci-
ety (UK) for a postdoctoral fellowship.
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