Synthesis of the Glc3Man N-glycan tetrasaccharide by iterative allyl IAD

Article abstract of DOI:10.1016/j.carres.2006.02.022

The synthesis of the tetrasaccharide α-d-Glcp-(1→2)-α-d-Glcp-(1→3)-α-d-Glcp-(1→3)-α-d-Manp-OMe, corresponding to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved with complete stereocontr

Full text of DOI:10.1016/j.carres.2006.02.022

Carbohydrate Research 341 (2006) 1609–1618
Synthesis of the Glc3Man N-glycan tetrasaccharide by
iterative allyl IAD
Emanuele Attolino, Ian Cumpstey and Antony J. Fairbanks*
Chemistry Research Laboratory, Oxford University, Mansfield Road, Oxford OX1 3TA, UK
Received 31 January 2006; received in revised form 15 February 2006; accepted 22 February 2006
Available online 10 March 2006
Abstract—The synthesis of the tetrasaccharide a-D-Glcp-(1!2)-a-D-Glcp-(1!3)-a-D-Glcp-(1!3)-a-D-Manp-OMe, corresponding
to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved with com-
plete stereocontrol by the use of iterative allyl protecting group mediated intramolecular aglycon delivery (allyl IAD) demonstrating
the utility of intramolecular glycosylation for the stereocontrolled construction of multiple glycosidic linkages during the synthesis
of an oligosaccharide.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Carbohydrates; Intramolecular; Glycosylation; Allyl; Intramolecular aglycon delivery; Thioglycosides; Glycoproteins
1. Introduction
bearing the familiar variety of mature N-glycan struc-
tures (high mannose, complex, hybrid type).
Post-translational modification of proteins by glycosyl-
ation1 is well known to have an important role in protein
folding,2 is able to modulate protein stability and enzy-
matic activity,3 and in addition can also affect other
important protein properties, such as circulatory life-
time.4 The most common form of protein glycosylation
is N-linked glycosylation, in which the oligosaccharides
(N-glycans) are attached to the side chains of asparagine
residues. The attachment of N-glycans to a protein is
initiated by the enzyme oligosaccharyl transferase
(OST), which mediates the transfer of a 14-membered
dolichol phosphate bound oligosaccharide (Glc3Man9-
GlcNAc2) to particular asparagine residues (consensus
sequence Asn-X-Ser/The, where X 5 Pro).5 Following
this transfer sequential trimming of the oligosaccharide
chain occurs, which is initiated by two glycoprotein-pro-
cessing enzymes, glucosidases I and II, in the endo-
plasmic reticulum (ER). Diversification of N-glycan
structures then occurs following the action of a whole
variety of other glycosidases and glycosyl transferases,
leading to the eventual production of glycoproteins
At first glance it may appear that the terminal three
glucose residues are somewhat redundant as these resi-
dues are invariably cleaved during glycoprotein process-
ing. However, it is now known that the terminal glucose
trisaccharide part of the Glc3Man9GlcNAc2 oligosaccha-
ride is important for OST recognition,6 and it has even
been suggested that the Glc3 trisaccharide portion has a
well-defined conformation which functions as a recogni-
tion epitope. Moreover, it has also been demonstrated
that correct folding of the glycoprotein is dependent on
the binding of the lectin chaperones calnexin and calreti-
culin to a monoglucosylated GlcMan9GlcNAc2 moiety7
produced by sequential removal of two of the three termi-
nal glucose units from the tetradecasaccharide: the first
glucose being removed by glucosidase I8 and the second
by glucosidase II.9 These chaperones do not bind to the
nonglucosylated Man9GlcNAc2 glycan, so cleavage of
the third glucose residue by glucosidase II actually stops
protein folding. Indeed, if a protein has not achieved its
native conformation at this point, it is recognised and
re-glucosylated by a glucosyltransferase10 as part of a
quality control mechanism to complete the folding pro-
cess.11 Conversely if correct folding has occurred prior
to cleavage of the last glucose residue by glucosidase II,
*
Corresponding author. Fax: +44 1865 275 674; e-mail: antony.
fairbanks@chem.ox.ac.uk
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.02.022

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E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
the protein is not re-glucosylated and it then leaves the
ER to continue maturation.
groups to allow linking of glycosyl donor and acceptor
via the 2-hydroxyl group of the donor and the hydroxyl
of the acceptor which is to be glycosylated. The basic
reaction sequence involves isomerisation22 of the double
bond of the allyl group to produce a vinyl ether, which
then undergoes mixed acetal formation with an alcohol,
usually mediated by the addition of a source of iodo-
nium ions. Subsequent activation of the glycosyl donor
is then followed by a completely stereoselective intramo-
lecular glycosylation reaction, in which the acceptor
alcohol is delivered syn to the 2-hydroxyl of the donor
enforcing the formation of a 1,2-cis linkage.
In principle, mixed acetal formation may be per-
formed either way around, and so either 2-O-allyl pro-
tected glycosyl donors and deprotected acceptors (the
‘forward’ approach), or donors which possess a free
hydroxyl at the 2-position and acceptors in which the
hydroxyl to be glycosylated is allyl protected (the
‘reverse’ approach), may be used as the partners for this
reaction sequence (Fig. 1).
A notable feature of the IAD approach is that the
product formed after the intramolecular glycosylation
step possesses a free hydroxyl group at the 2-position
of the nonreducing terminus. Because the terminal link-
age of the target Glc3Man tetrasaccharide is an a-(1!2)
linkage, it was therefore envisaged that both of the two
terminal glucose residues could be added sequentially
using the same basic glycosyl donor in an iterative
IAD process. The strategy chosen involved the use of
a first IAD reaction between an allyl protected disaccha-
ride acceptor and a thioglycoside donor with OH-2 free,
and a second IAD reaction between the newly formed
trisaccharide as acceptor and a 2-O-allyl protected ver-
sion of the same thioglycoside donor. Access was there-
fore required to both an allyl protected disaccharide
glycosyl acceptor for the first IAD reaction, and a glyco-
syl donor with a free 2-hydroxyl group, which would act
The interesting and diverse roles played by residues
present in the glucose terminated arm have therefore ini-
tiated significant interest from the synthetic community.
The synthesis of the complete a-D-Glcp-(1!2)-a-D-Glcp-
(1!3)-a-D-Glcp-(1!3)-a-D-Manp-OMe tetrasaccharide
has been reported both by Ogawa12 and ourselves,13
and in addition we have previously published14 the
synthesis of fluorescence labeled disaccharides corre-
sponding to portions of the tetrasaccharide as substrates
for glucosidase II. Moreover, Ito and co-workers15 have
most recently completed the total synthesis of the
complete Glc3Man9GlcNAc2 tetradecasaccharide itself,
whilst other research groups have synthesised other
truncated portions of the glucose terminated arm.16
Our previous efforts were directed at accessing signifi-
cant pure quantities of the Glc3Man tetrasaccharide to
allow extensive conformational studies. However, our
previous synthesis was not completely stereoselective,
and inseparable mixtures of anomers were formed during
the attempted formation of the 1,2-cis-a-gluco linkages,
necessitating laborious final product purification by high
performance anion exchange chromatography (HPAEC).
This paper details a completely stereoselective approach
to the Glc3Man tetrasaccharide in which two of the termi-
nal a-gluco linkages are formed by successive intramole-
cular glycosylation reactions using the allyl IAD
approach.17,18 The complete stereocontrol achieved dur-
ing these two reactions greatly simplifies the whole synthe-
sis and facilitates product isolation and purification.
2. Results and discussion
Allyl IAD19,20 is a particular intramolecular glycosyl-
ation strategy21 based on the use of allyl protecting
O
O
O
O
O
X
HO
X
RO
RO
+
RO
acceptor
'forward' approach
allyl protected
donor
I+
O
X
O
RO
RO
Intramolecular
glycosylation
O
O
I
O
HO
O
O
RO
RO
I+
'reverse' approach
O
O
O
O
O
X
+
RO
RO
RO
OH
allyl protected
acceptor
donor
Figure 1. Allyl IAD: ‘Forward’ and ‘Reverse’ approaches.

E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
1611
as a divergent donor; OH-2 free for the first IAD
sequence, OH-2 allyl protected for the second.
With the allyl protected disaccharide acceptor in
hand, attention then turned to the required donors.
Thus the thioglycoside acceptor 724 was accessed as pre-
viously described as the donor for the first IAD
sequence. With the second IAD reaction in mind, a
portion of the thioglycoside 7 was then allylated by
treatment with sodium hydride and allyl bromide in
DMF to yield the allyl protected donor 9, and subse-
quent Wilkinson’s catalyst mediated double bond iso-
merisation then produced enol ethers 10. With all key
building blocks in hand, the two IAD reactions were
investigated in turn (Scheme 2). Thus, isomerisation of
the double bond of allyl protected disaccharide acceptor
5 yielded enol ethers 6. Previous reports on allyl IAD
have mainly focussed on a two-step approach, in which
tethering (i.e., mixed acetal formation) and glycosyl-
ation are performed as separate steps. However, it has
also been demonstrated that both operations may be
performed sequentially in a single reaction vessel (the
one-pot approach)18a,20m provided that an excess of gly-
cosyl donor is present; the caveat being that any excess
alcohol remaining after tethering can lead to competitive
and nonstereoselective intermolecular glycosylation.
In this instance it was decided to investigate both
approaches for the first IAD reaction and apply the
most efficient and practical for the second.
The synthesis of the allyl protected disaccharide
acceptor was undertaken first. The 3-O-allyl protected
gluco tetraacetate 1 was accessed as described previ-
ously,13 and was then converted into the b-thioglycoside
acceptor 2 by treatment with p-thiocresol and boron
trifluoride etherate in dichloromethane. Subsequent
deacetylation and immediate benzylation gave the re-
quired thioglycoside donor 3, possessing nonparticipat-
ing protection at the 2-position. The known manno
glycosyl acceptor 4 was accessed as previously de-
scribed,23 and glycosylation of this acceptor 4 with
donor 3 gave the desired disaccharide 5 as expected.
However, it was necessary to investigate a range of reac-
tion conditions to maximise both product yield and a-
stereoselectivity for this step. Thioglycoside activation
in ether, using either NIS with catalytic triflic acid or
methyl triflate, only produced the desired disaccharide
in low to moderate yield (45% and 34% yields, respec-
tively), though in both cases reasonably good stereo-
selectivity was observed (a:b ratio >3:1). However,
glycosylation in ether mediated with iodine, silver tri-
flate and 2,6-di-tert-butyl-4-methyl pyridine (DTBMP),
produced the desired disaccharide 5 in excellent yield
and with a high level of stereocontrol (87% yield, a:b
ratio, 23:1, Scheme 1).
Mixed acetal formation was achieved by treatment of
a mixture of 6 and 7 with iodine, silver triflate and
DTBMP (78% yield). Intramolecular glycosylation of
the purified tethered intermediates was then undertaken.
Several methods of thioglycoside activation were inves-
tigated, including NIS/TfOH, NIS/TMSOTf, NIS/
TMSOTf/DTBMP and I2/AgOTf/DTBMP. However,
none proved particularly satisfactory and all either led
to the formation of multiple products as observed by
TLC, or to very slow reaction. Attention then turned
to the use of a variant on DMTST activation recently
OAc
O
AcO
OAc
O
OAc
1
(i)
OR
O
reported by Fugedi and Tatai,25 involving thioglycoside
RO
¨
STol
O
activation by treatment with a mixture of Me2S2 and tri-
flic anhydride. In concurrence with Fugedi, this reagent
¨
OR
2 R=Ac
3 R=Bn
combination proved to be much more potent than the
use of DMTST, but unfortunately substantial decompo-
sition was also observed. The reported conditions were
therefore modified by adding the base DTBMP to the
activation mixture and pleasingly this produced a highly
efficient and clean reaction yielding trisaccharide 8 in
65% yield, and, as expected, with complete control of
anomeric stereochemistry. Attention then turned to the
one-pot approach as a possibly more efficient alternative
to the two-step procedure above (yield 51% over the two
steps). A mixture of donor alcohol 7 and acceptor 6
(1.1 equiv) was stirred with iodine, silver triflate and
DTBMP and once mixed acetal formation was complete
(as monitored by TLC) the activation mixture of Me2S2/
Tf2O/DTBMP was added. However, this approach
proved less satisfactory; following work-up, trisaccharide
(ii),(iii)
OBn
O
Ph
O
O
(iv)
HO
4
OMe
Ph
BnO
OBn
O
O
O
O
OMe
O
BnO
BnO
O
5
Scheme 1. Reagents and conditions: (i) TolSH, BF3ÆOEt2, CH2Cl2,
0 ꢁC, 78%; 2b:2a, 35:1; (ii) Na, MeOH, rt; (iii) NaH, BnBr, DMF, 0 ꢁC,
89% over two steps; (iv) I2, AgOTf, DTBMP, CH2Cl2, ꢀ78 ꢁC, 87%,
5a:5b, 23:1.

1612
E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
OBn
O
OBn
O
O
Ph
Ph
O
O
O
(i)
O
O
BnO
BnO
OMe
O
OMe
O
6
5
BnO
BnO
BnO
BnO
O
O
OBn
O
(ii),(iii)
O
BnO
BnO
BnO
Ph
+
O
O
O
BnO
BnO
BnO
O
HO
OMe
O
STol
STol
BnO
BnO
BnO
O
OH
(iv)
7
8
BnO
BnO
BnO
BnO
O
O
O
(v)
BnO
BnO
STol
(vi),(vii)
O
10
9
OH
O
OBn
O
BnO
HO
HO
HO
O
O
O
Ph
BnO
O
HO
(viii)
BnO
HO
HO
HO
O
O
O
HO
BnO
O
OMe
O
OMe
O
BnO
BnO
BnO
HO
HO
BnO
O
O
O
HO
HO
BnO
O
HO
12
11
Scheme 2. Reagents and conditions: (i) Wilkinson’s catalyst, n-BuLi, THF, 70 ꢁC, 97%; (ii) 7, I2, AgOTf, DTBMP, CH2Cl2, 0 ꢁC!rt, 78%; (iii)
Me2S2, Tf2O, DTBMP, CH2Cl2, 0 ꢁC!rt, 65%; (iv) allyl bromide, NaH, DMF, 0 ꢁC!rt, 98%; (v) Wilkinson’s catalyst, n-BuLi, THF, 70 ꢁC, 95%;
(vi) 10, I2, AgOTf, DTBMP, CH2Cl2, 0 ꢁC!rt, 77%; (vii) Me2S2, Tf2O, DTBMP, CH2Cl2, 0 ꢁC!rt, 58%; (viii) H2, Pd(OAc)2, AcOH, EtOH, 98%.
8 was isolated in only 35% yield, although again
glycosylation had occurred with complete control of
anomeric stereochemistry. In light of this, the two-step
approach was therefore applied to the second IAD reac-
best of our knowledge, this represents the first synthesis
in which IAD has been used in an iterative manner for
the completely stereocontrolled formation of two suc-
cessive glycosidic linkages in the same oligosaccharide.
Moreover, it has been demonstrated that allyl IAD
may be efficiently achieved either in the forward (allyl
protected donor) or reverse (allyl protected acceptor)
senses. Finally, it has also been demonstrated that the
tion. Thus tethering of trisaccharide acceptor
8
(1.0 equiv) with enol ethers 10 (1.1 equiv) by treatment
with iodine, silver triflate and DTBMP gave intermediate
mixed acetals (77% yield), which then underwent intra-
molecular glycosylation, again using the modified Fugedi
¨
addition of the base DTBMP to the Fugedi thioglyco-
¨
conditions (Me2S2/Tf2O/DTBMP), to yield desired tetra-
saccharide 11 (58% yield), again with complete control of
anomeric stereochemistry. Finally, global deprotection of
benzyl ethers and benzylidene acetal by catalytic hydro-
genation in the presence of a palladium acetate catalyst
in a mixture of acetic acid and ethanol gave the deprotec-
ted tetrasaccharide 12 (98% yield, Scheme 2). In contrast
to our previous synthesis, tetrasaccharide was easily puri-
fied as no other stereoisomeric products had been formed
during either of the two intramolecular glycosylation
reactions.
side activation conditions (Me2S2, Tf2O) produces a
highly reactive activation system for thioglycosides,
which in particular leads to high yielding clean intra-
molecular glycosylation reactions; a process that none
of the other commonly used thioglycoside activators
that were investigated was capable of achieving. Further
investigations into the use of these conditions in combi-
nation with the IAD approach for the synthesis of more
complex biologically important oligosaccharides are
currently in progress, and the results will be reported
in due course.
3. Summary and conclusion
4. Experimental
4.1. General methods
The stereoselective synthesis of the tetrasaccharide
a-D-Glcp-(1!2)-a-D-Glcp-(1!3)-a-D-Glcp-(1!3)-a-D-
Manp-OMe 12 has been successfully achieved using two
successive intramolecuar glycosylation reactions. To the
Melting points were recorded on a Kofler hot block and
1
are uncorrected. H NMR spectra were recorded on a

E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
1613
1
Bruker DPX 400 (400 MHz) or on a Bruker AMX 500
(500 MHz) spectrometer. 13C NMR spectra were re-
corded on a Bruker AC 200 (50.3 MHz), or on a Bruker
DPX 400 (100.6 MHz) spectrometer. Multiplicities were
assigned using the DEPT sequence. All chemical shifts
are quoted on the d-scale in parts per million (ppm).
The abbreviations at, dat, etc, refer to apparent triplet,
double apparent triplet, etc. All NMR experiments were
performed at a probe temperature of 30 ꢁC. Infrared
spectra were recorded on a Perkin–Elmer 150 Fourier
Transform spectrophotometer. Low resolution mass
spectra were recorded on a Micromass Platform 1 APCI
using atmospheric pressure chemical ionisation (APCI).
High resolution mass spectra (electrospray) were per-
formed on a Waters 2790-Micromass LCT electrospray
ionisation mass spectrometer. Optical rotations were
measured on a Perkin–Elmer 241 polarimeter with a path
length of 1 dm. Concentrations are given in g/100 mL.
Microanalyses were performed by the microanaly-
tical services of the Inorganic Chemistry Laboratory,
Oxford. Thin layer chromatography (TLC) was carried
out on Merck Kieselgfel 0.22–0.25 mm thickness glass
backed sheets, pre-coated with 60F254 silica. Plates were
developed using 5% w/v ammonium molybdate in 2 M
sulfuric acid. Flash column chromatography was carried
out using Sorbsil C60 40/60 silica. Solvents and reagents
were dried and purified before use according to standard
procedures; CH3OH was distilled from sodium hydride,
CH2Cl2 was distilled from calcium hydride, pyridine was
distilled from calcium hydride and stored over potas-
sium hydroxide and THF was distilled from a solution
of sodium benzophenone ketyl immediately before use.
Petrol was distilled between 40 and 60 ꢁC before use to
remove nonvolatile fractions.
1744 (s, C@O) cmꢀ1; H NMR (400 MHz, CDCl3): d
2.08, 2.08, 2.15 (9H, 3 · s, 3 · COCH3), 2.34 (3H, s,
ArCH3), 3.57–3.63 (1H, m, H-5), 3.59 (1H, at,
J = 9.2 Hz, H-3), 4.02–4.11 (2H, m, OCH2CH@CH2),
4.13–4.20 (2H, m, H-6, H-60), 4.56 (1H, d, J1,2
=
10.1 Hz, H-1), 4.96 (1H, at, J = 9.5 Hz, H-2), 5.00
(1H, at, J = 9.8 Hz, H-4), 5.13 (1H, d, JZ = 10.4 Hz,
CH@CHEHZ), 5.19 (1H, dd, Jgem = 1.6 Hz, JE =
17.2 Hz, CH@CHEHZ), 5.75 (1H, ddat, J = 5.5 Hz,
CH@CH2), 7.11, 7.40 (4H, 2 · d, J = 8.1 Hz, Ar–H);
13C NMR (100.6 MHz, CDCl3): d 21.2, 21.3, 21.5,
21.6 (4 · q, 3 · COCH3, ArCH3), 63.0, 73.5 (2 · t, C-6,
CH2CH@CH2), 69.9, 71.7, 76.4, 81.7 (4 · d, C-2, C-3,
C-4, C-5), 86.8 (d, C-1), 117.5 (t, CH@CH2), 129.0,
138.8 (2 · s, Ar–C) 130.0, 133.7 (2 · d, Ar–CH), 134.6
(d, CH@CH2), 169.6, 169.7, 171.2 (3 · s, 3 · C@O);
m/z (ES+) 475 (M+Na+, 100), 470 (M+NH4þ, 77%);
ESIMS m/z calcd for C22H32NO8S [M+NH4]+:
470.1849. Found 470.1850. Anal Calcd for C22H28O8S:
C, 58.39; H, 6.24. Found: C, 58.50; H, 6.27. In addition
a small quantity of the corresponding a-thioglycoside 2a
was also produced (50 mg, 2%) as a colourless oil;
21
½aꢁD +163.3 (c 1.8, CHCl3); IR: 1748 (s, C@O) cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 2.03, 2.11, 2.16, 2.32
(12H, 4 · s, 3 · COCH3, ArCH3), 3.80 (1H, at,
J = 9.6 Hz, H-3), 4.04 (1H, dd, J5,6 = 2.2 Hz,
0
J6;6 ¼ 12:3 Hz, H-6), 4.11 (1H, dd, Jgem = 13.1 Hz,
Jvic = 5.7 Hz, OCHH0CH@ CH2), 4.20–4.26 (2H, m,
H-60, OCHH0CH@CH2), 4.48 (1H, ddd, J4,5
=
0
10.1 Hz, J5;6 ¼ 5:6 Hz, H-5), 5.02 (1H, dd,
J1,2 = 5.7 Hz, J2,3 = 10.0 Hz, H-2), 5.04 (1H, at,
J = 9.8 Hz, H-4), 5.17 (1H, d, JZ = 10.4 Hz, CH@
CHEHZ), 5.25 (1H, d, JE = 17.2 Hz, CH@CHEHZ),
5.78–5.88 (1H, m, CH@CH2), 5.82 (1H, d, H-1), 7.11,
7.33 (4H, 2 · d, J = 8.0 Hz, Ar–H); 13C NMR
(100.6 MHz, CDCl3): d 20.7, 20.8, 20.9, 21.0 (4 · q,
3 · COCH3, ArCH3), 62.2, 73.8 (2 · t, C-6, CH2CH@
CH2), 68.4, 69.7, 73.1, 77.3 (4 · d, C-2, C-3, C-4, C-5),
85.6 (d, C-1), 116.9 (t, CH@CH2), 129.8, 132.4, 134.3
(3 · d, Ar–CH, CH@CH2), 128.8, 138.0 (2 · s, Ar–C),
169.4, 169.8, 170.7 (3 · s, 3 · C@O); m/z (ES+) 475
(M+Na+, 43), 470 (M+NH4þ, 64%). ESIMS m/z calcd
for C22H32NO8S [M+NH4]+: 470.1849. Found
470.1854.
4.2. para-Tolyl 2,4,6-tri-O-acetyl-3-O-allyl-1-thio-b-D-
glucopyranoside (2b) and para-tolyl 2,4,6-tri-O-acetyl-3-
O-allyl-1-thio-a-D-glucopyranoside (2a)
Tetraacetate 1 (2.00 g, 5.15 mmol) was suspended in
freshly distilled CH2Cl2 (10 mL) under Ar in a flame-
dried flask. p-Thiocresol (766 mg, 6.18 mmol) was added
and the mixture cooled to 0 ꢁC. BF3ÆOEt2 (0.95 mL,
7.73 mmol) was added and the reaction mixture was stir-
red under Ar. After 1.5 h, TLC (3:2, petrol–EtOAc)
indicated formation of a major product (Rf = 0.3) and
a minor product (Rf = 0.35). Triethylamine (2 mL) was
then added, the mixture was diluted with ether
(100 mL), and washed with water (100 mL). The aque-
ous layer was re-extracted with ether (50 mL), and the
combined organic extracts were washed with brine
(50 mL), dried (MgSO4), filtered and concentrated
in vacuo. The residue was purified by flash column chro-
matography (3:2, petrol–EtOAc) to afford the desired b-
thioglycoside 2b (1.81 g, 78%) as a white solid, mp 127–
4.3. para-Tolyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-b-D-
glucopyranoside (3)
Acetylated thioglycoside 2 (1.77 g, 3.92 mmol) was sus-
pended in CH3OH (10 mL). Sodium (5 mg, 0.20 mmol)
was dissolved in CH3OH (5 mL) and the solution then
added to the reaction mixture, which was then stirred
under Ar. After 2 h, TLC (EtOAc) indicated formation
of a single product (Rf = 0.6) and complete consump-
tion of the starting material (Rf = 0.7). The mixture
was concentrated in vacuo, and the resulting residue
21
128 ꢁC (ether/petrol); ½aꢁD ꢀ14.4 (c 0.9, CHCl3); IR:

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E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
was then dissolved in DMF (30 mL) and the solution
cooled to 0 ꢁC. Benzyl bromide (2.09 mL, 17.6 mmol)
and sodium hydride (940 mg, 23.5 mmol) were added
and the reaction mixture was stirred under Ar. After
2 h, TLC (5:1, petrol–EtOAc) indicated the formation
of two products (Rf = 0.5 and 0.7) and complete con-
sumption of starting material (Rf = 0.0). Further benzyl
bromide (0.35 mL, 2.94 mmol) and sodium hydride
(156 mg, 3.91 mmol) were added, and the reaction mix-
ture was stirred under Ar. After a further 14 h, TLC
(5:1, petrol–EtOAc) indicated the formation of a single
product (Rf = 0.7). CH3OH (8 mL) was added slowly,
and the mixture was then diluted with ether (200 mL),
and washed with water (200 mL). The organic extracts
were dried (MgSO4), filtered, concentrated in vacuo
and the residue was purified by flash column chromato-
graphy (4:1, petrol–ether) to afford benzylated thio-
glycoside 3 (2.08 g, 89%) as a white, crystalline solid,
via cannula. The reaction was then allowed to warm
to rt. After 24 h, TLC (3:1, petrol–EtOAc) indicated
the formation of a major product (Rf = 0.4) and con-
sumption of starting materials (Rf = 0.6 and 0.3). The
reaction was quenched by the addition of sodium thio-
sulfate (40 mL of a 10% aqueous solution), diluted with
ether (40 mL), and then filtered through Celiteꢂ. The
organic phase was washed with sodium thiosulfate
(2 · 100 mL of a 10% aqueous solution), dried (MgSO4),
filtered and concentrated in vacuo. The resulting residue
was purified by flash column chromatography (5:1, pet-
rol–EtOAc then 15:1, toluene–EtOAc) to afford pure
a-disaccharide 5 (118 mg, 83%) as a colourless oil;
21
½aꢁD +93.0 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d 3.33 (3H, s, OCH3), 3.46 (1H, dd, J1,2 = 3.6 Hz,
J2,3 = 9.6 Hz, H-2b), 3.54 (1H, at, J = 9.4 Hz, H-4b),
3.64–3.71 (2H, m, H-6b, H-60b), 3.78–4.15 (5H, m, H-
2a, H-5a, H-6a, H-3b, H-5b), 4.18–4.26 (2H, m, H-60a,
OCHH0CH@CH2), 4.31, 4.54 (2H, ABq, JAB = 12.5 Hz,
PhCH2), 4.31–4.35 (1H, m, H-4a), 4.39 (1H, dd,
J2,3 = 2.9 Hz, J3,4 = 10.0 Hz, H-3a), 4.42–4.49 (1H, m,
OCHH0CH@CH2), 4.45, 4.58 (2H, ABq, JAB = 12.2 Hz,
PhCH2), 4.47, 4.88 (2H, ABq, JAB = 11.0 Hz, PhCH2),
4.71 (1H, d, J1,2 = 1.3 Hz, H-1a), 4.78, 4.93 (2H, ABq,
JAB = 11.9 Hz, PhCH2), 5.09 (1H, dd, JZ = 10.4 Hz,
21
1
mp 75 ꢁC (EtOH); ½aꢁD ꢀ6.5 (c, 1.0, CHCl3); H NMR
(400 MHz, CDCl3): d 2.32 (3H, s, ArCH3), 3.41–3.51
(2H, m, H-2, H-5), 3.53–3.61 (2H, m, H-3, H-4), 3.73
0
(1H, dd, J5,6 = 4.8 Hz, J6;6 ¼ 10:9 Hz, H-6), 3.79 (1H,
dd, J5;6 ¼ 2:1 Hz, H-60), 4.33 (1H, ddat, Jgem = 12.3 Hz,
0
Jvic 5.7 Hz, J = 1.4 Hz, OCHH0CH@CH2), 4.38 (1H,
ddat, Jvic = 5.7 Hz, J = 1.5 Hz, OCHH0CH@CH2),
4.55, 4.62 (2H, ABq, JAB = 12.1 Hz, PhCH2), 4.58
(1H, d, J1,2 = 9.7 Hz, H-1), 4.59, 4.83 (2H, ABq,
Jgem = 1.0 Hz, CH@CHEHZ), 5.22 (1H, dd, Jgem
=
1.6 Hz, JE = 17.2 Hz, CH@CHEHZ), 5.47 (1H, s,
PhCH) 5.50 (1H, d, H-1b), 5.96 (1H, ddat, J = 5.5 Hz,
CH@CH2), 7.00–7.47 (25H, m, Ar–H); 13C NMR
(100.6 MHz, CDCl3): d 54.8 (q, OCH3), 63.9 (d, C-5a),
68.6 (t, C-6b), 68.9 (t, C-6a), 70.5 (t, PhCH2), 70.8 (d,
C-5b), 72.7 (d, C-3a), 73.4, 73.8, 74.3, 75.0 (4 · t,
OCH2CH@CH2, 3 · PhCH2), 77.2, 77.3 (2 · d, C-2a,
C-4b), 78.6 (d, C-2b), 79.6 (d, C-4a), 81.0 (d, C-3b),
96.9 (d, C-1b), 100.6 (d, C-1a), 102.3 (d, PhCH), 116.6
(t, CH@CH2), 126.4, 127.0, 127.1, 127.6, 127.6, 127.8,
127.8, 127.9, 128.1, 128.2, 128.2, 128.3, 128.4, 129.2
(14 · d, Ar–CH), 135.4 (d, CH@CH2), 137.4, 138.0,
138.0, 138.3, 138.5 (5 · s, Ar–C); m/z (ES+) 867
(M+Na+, 27), 862 (M+NH4þ, 100%); ESIMS m/z calcd
for C51H60NO11 [M+NH4]+: 862.4166. Found 862.4160.
JAB = 11.1 Hz, PhCH2), 4.74, 4.88 (2H, ABq, JAB
=
10.1 Hz, PhCH2), 5.18 (1H, daq, JZ = 10.4 Hz, J =
1.4 Hz, CH@CHEHZ), 5.30 (1H, daq, J = 1.7 Hz,
JE = 17.1 Hz, CH@CHEHZ), 5.98 (1H, ddat,
CH@CH2), 7.04–7.52 (19H, m, Ar–H); 13C NMR
(100.6 MHz, CDCl3): d 21.1 (q, ArCH3), 69.0, 73.4,
74.5, 75.1, 75.5 (5 · t, C-6, CH2CH@CH2, 3 · PhCH2),
77.7, 79.0, 80.7, 86.4, 87.6 (5 · d, C-1, C-2, C-3, C-4,
C-5), 116.9 (t, CH@CH2), 127.5, 127.7, 127.8, 127.9,
128.1, 128.3, 128.4, 129.6, 132.7, 134.9 (11 · d, Ar–
CH, CH@CH2), 137.7, 138.1, 138.1, 138.4 (4 · s, Ar–
C); m/z (ES+) 619 (M+Na+, 32), 614 (M+NH4
,
þ
100%); ESIMS m/z calcd for C37H44NO5S [M+NH4]+:
614.2940. Found 614.2935. Anal Calcd C37H40O5S: C,
74.47; H, 6.76. Found: C, 74.45; H, 6.79.
4.5. Methyl 2,4,6-tri-O-benzyl-3-O-prop-10-enyl-a-D-
glucopyranosyl-(1!3)-2-O-benzyl-(R)-4,6-O-benzyl-
idene-a-D-mannopyranoside (6)
4.4. Methyl 3-O-allyl-2,4,6-tri-O-benzyl-a-D-gluco-
pyranosyl-(1!3)-2-O-benzyl-(R)-4,6-O-benzylidene-
a-D-mannopyranoside (5)
Wilkinson’s catalyst (86 mg, 0.093 mmol) was dissolved
in distilled THF (4 mL) and degassed. n-Butyl lithium
(0.09 mL, 0.14 mmol, 1.6 M solution in hexanes) was
added and the mixture stirred for 10 min under Ar. Allyl
protected disaccharide 5 (786 mg, 0.93 mmol) was dis-
solved in distilled THF (4 mL) and the mixture heated
to 70 ꢁC. The catalyst solution prepared above was then
added via cannula under Ar. After 2 h, 30 min, TLC
(4:1, petrol–EtOAc) indicated the presence of a single
compound (Rf = 0.3). The reaction mixture was allowed
Iodine (62 mg, 0.17 mmol), silver trifluoromethanesulfo-
nate (63 mg, 0.25 mmol), 2,6-di-tert-butyl-4-methylpyr-
˚
idine (70 mg, 0.34 mmol) and 4 A molecular sieves were
suspended in freshly distilled ether (2 mL) and cooled
to ꢀ78 ꢁC whilst stirring under Ar. Thioglycoside 3
(100 mg, 0.168 mmol) and methyl 2-O-benzyl-4,6-O-
benzylidene-a-D-mannopyranoside
4
(62 mg, 0.17
mmol) were dissolved in freshly distilled ether (3 mL)
and this solution was then added to the reaction vessel

E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
1615
to cool to rt, diluted with CH2Cl2 (20 mL) and then
concentrated in vacuo. The residue was purified by flash
column chromatography (4:1, petrol–EtOAc) to afford
vinyl ethers 6 (745 mg, 95%) as a pale yellow oil; partial
EtOAc) indicated the complete consumption of starting
material (Rf = 0.52), and the formation of three main
products (Rf = 0.42, 0.28 and 0.20). The reaction mix-
ture was cooled to 0 ꢁC, then dioxane (5 mL) and an
aqueous NaH2PO4/Na2HPO4 buffer (pH 6.0, 5 mL)
were added, and the mixture was stirred for a further
10 min. The mixture was filtered, diluted with CH2Cl2
(20 mL) and the two phases separated. The organic
phase was dried (MgSO4), filtered, and concentrated in
vacuo. The crude residue was dissolved in acetone
(5 mL) and water (0.5 mL). NIS (11 mg, 0.049 mmol)
was added and the mixture was stirred overnight at rt.
The reaction mixture was then treated with sodium thio-
sulfate (5 mL of a 10% aqueous solution), and diluted
with CH2Cl2 (20 mL). The aqueous phase was washed
with CH2Cl2 (10 mL) and the combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo.
The resulting residue was purified by flash column chro-
matography (40:1!2:1, CH2Cl2–ether) to afford trisac-
1
data: H NMR (400 MHz, CDCl3) (E:Z, 1:2.6): d 1.51
(3H, dd, J = 1.6, 6.8 Hz, OCH@CHCH3–E), 1.64 (3H,
dd, J = 1.6, 6.8 Hz, OCH@CHCH3–Z); m/z (ES+) 867
(M+Na+, 21), 862 (M+NH4þ, 100%); ESIMS m/z calcd
for C51H60NO11 [M+NH4]+: 862.4166. Found 862.4161.
4.6. Methyl 3,4,6-tri-O-benzyl-a-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-benzyl-a-D-glucopyranosyl-(1!3)-2-
O-benzyl-(R)-4,6-O-benzylidene-a-D-mannopyranoside
(8)
Iodine (138 mg, 0.54 mmol), silver trifluoromethanesulf-
onate (142 mg, 0.55 mmol), 2,6-di-tert-butyl-4-methyl-
˚
pyridine (284 mg, 1.38 mmol) and 4 A molecular sieves
were added to freshly distilled CH2Cl2 (5 mL) in a
flame-dried flask. The mixture was cooled to ꢀ78 ꢁC
and stirred for 20 min under an atmosphere of Ar. Enol
ethers 6 (390 mg, 0.46 mmol) and thioglycoside 724
(308 mg, 0.55 mmol) were dissolved in freshly distilled
CH2Cl2 (5 mL) and added to the reaction vessel via can-
nula under an atmosphere of Ar. The reaction was
allowed to warm to rt whilst stirring. After 15 h, TLC
(10:1, toluene–EtOAc) indicated complete conversion
of starting materials (Rf = 0.48 and 0.44) to a major
product (Rf = 0.52). The reaction mixture was diluted
with CH2Cl2 (20 mL) and quenched by addition of
sodium thiosulfate (20 mL of a 10% aqueous solution).
The mixture was filtered and the two phases separated.
The aqueous layer was extracted with CH2Cl2
(2 · 20 mL), and the combined organic extracts were
dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by flash column chromatogra-
phy (20:1, toluene–EtOAc) to afford intermediate mixed
acetals (548 mg, 77%) as a mixture of diastereomers as a
colourless oil, a portion of which was used directly for
the next step; m/z(ES+) 1549 (M+Na+, 38), 1544
(M+NH4þ, 100%); m/z (isotopic distribution) expected
mass 1547 (MNH4þ, 23%), 1546 (MNH4þ+2, 55%),
1545 (MNH4þ+1, 97%), 1544 (MNH4þ, 100%), found
1547 (MNH4þ+3, 14%), 1546 (MNH4þ+2, 48%), 1545
(MNH4þ+1, 94%), 1544 (MNH4þ, 100%).
22
charide 8 (26 mg, 65%) as a colourless oil; ½aꢁD +73.1
(c 1.1, CHCl3); IR: 3374 (br, OH) cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 2.36 (1H, br s, OH-2c), 3.19
(2H, br s, H-6, H-60), 3.36 (3H, s, OCH3), 3.42 (1H,
dd, J1,2 = 3.7 Hz, J2,3 = 9.8 Hz, H-2b), 3.65–3.79 (8H,
m), 3.82–3.89 (2H, m), 4.13 (1H, d, J = 12.3 Hz,
PhCHH0), 4.16, 4.47 (2H, ABq, JAB = 11.1 Hz, PhCH2),
4.21–4.25 (3H, m, H-3b, H-5, H-6), 4.34–4.44 (5H, m),
4.48, 4.67 (2H, ABq, JAB = 11.8 Hz, PhCH2), 4.72
(1H, d, J = 11.0 Hz, PhCHH0), 4.75 (1H, br s, H-1a),
4.82, 4.95 (2H, ABq, JAB = 12.0 Hz, PhCH2), 4.83
(2H, s, PhCH2), 4.91 (1H, d, J = 10.4 Hz, PhCHH0),
5.36 (1H, s, PhCH), 5.51 (1H, d, J1,2 = 2.9 Hz, H-1c),
5.62 (1H, d, H-1b), 7.07–7.44 (40H, m, Ar–H); 13C
NMR (100.6 MHz, CDCl3): d 54.8 (q, OCH3), 63.8,
70.1, 70.5, 72.1, 72.8, 76.1, 76.5, 76.8, 77.4, 78.6, 79.9,
83.3 (12 · d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b,
C-5b, C-2c, C-3c, C-4c, C-5c), 67.9, 68.3, 68.9 (3 · t, C-
6a, C-6b, C-6c), 70.3, 73.2, 73.6, 74.2, 74.3, 75.2 (6 · t,
7 · PhCH2), 96.1 (d, C-1b), 98.6 (d, C-1c), 100.7 (d, C-
1a), 102.4 (d, PhCH), 126.3, 127.3, 127.4, 127.4, 127.5,
127.7, 127.7, 127.8, 127.8, 127.8, 128.0, 128.1, 128.1,
128.1, 128.2, 128.3, 128.3, 128.4, 128.5, 129.2 (20 · d,
Ar–CH), 137.2, 137.6, 137.8, 137.9, 137.9, 138.0, 138.7,
138.8 (8 · s, Ar–C); m/z (ES+) 1275 (M+K+, 11), 1259
(M+Na+, 100), 1254 (M+NH4þ, 64%); ESIMS m/z
calcd for C75H84NO16 [M+NH4]+: 1254.5790. Found
1254.5782.
Mixed acetals prepared above (50 mg, 0.0327 mmol),
2,6-di-tert-butyl-4-methylpyridine (27 mg, 0.0655 mmol)
˚
and 4 A molecular sieves were added to freshly distilled
CH2Cl2 (2 mL) in a flame-dried flask under an atmo-
sphere of Ar and the mixture was cooled to 0 ꢁC whilst
stirring. Dimethyldisulfide (6.0 lL, 0.0655 mmol) and
triflic anhydride (11.0 lL, 0.0655 mmol) were dissolved
in freshly distilled CH2Cl2 (0.25 mL) and after 5 min
the mixture was added to the reaction vessel under an
atmosphere of Ar. The reaction mixture was allowed
to warm to rt and after 90 min, TLC (10:1, toluene–
4.7. para-Tolyl 2-O-allyl-3,4,6-O-benzyl-1-thio-b-D-
glucopyranose (9)
Alcohol 724 (0.393 g, 0.707 mmol) was dissolved in
anhydrous DMF (20 mL) in a flame-dried flask under
an atmosphere of Ar. The solution was cooled to 0 ꢁC
whilst stirring and sodium hydride (53 mg, 1.3 mmol,
60% dispersion in mineral oil) was added portion-wise

1616
E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
over a 10 min period. Allyl bromide (0.1 mL, 2 mmol)
was added and the reaction mixture was allowed to
warm to room temperature. After 38 h, TLC (3:1, pet-
rol–EtOAc) indicated the formation of a single product
(Rf = 0.6) and complete consumption of starting mate-
rial (Rf = 0.4). The reaction was quenched by the addi-
tion of CH3OH (10 mL), and then partitioned between
ether (50 mL) and brine (50 mL of a saturated aqueous
solution). The organic extracts were washed with brine
(2 · 50 mL of a saturated aqueous solution), dried
(MgSO4), filtered and concentrated in vacuo. The result-
ing residue was purified by flash column chromato-
graphy (5:1, petrol–EtOAc) to afford allyl protected
thioglycoside 9 (0.412 g, 98%) as a white crystalline
to rt and was then diluted with CH2Cl2 (50 mL) and
concentrated in vacuo. The resulting residue was puri-
fied by flash column chromatography (5:1, petrol–
EtOAc) to afford enol ethers 10 (144 mg, 95%) as an
off-white crystalline solid, mp 50–53 ꢁC (EtOAc/petrol);
1
IR: (KBr) 1456 (w, C@C) cmꢀ1; H NMR (400 MHz,
CDCl3) (E:Z, 1:2.9): d 1.58 (1H, dd, J = 1.5 Hz,
J = 6.8 Hz, OCH@CHCH3-E), 1.67 (3H, dd, J =
1.6 Hz, J = 6.8 Hz, OCH@CHCH3ꢀZ), 2.32 (3H, s,
ArCH3), 3.46–3.59 (2H, m, H-4, H-5), 3.59 (1H, at,
J = 9.6 Hz, H-3), 3.70 (1H, at, J = 8.7 Hz, H-2), 3.72–
3.82 (2H, m, H-6, H-60), 4.46 (1H, aquint, J = 6.7 Hz,
OCH@CHCH3–Z), 4.55, 4.62 (2H, ABq, JA,B
=
12.1 Hz, OCH2Ph), 4.58 (1H, d, J1,2 = 10.4 Hz, H-1),
4.58, 4.83 (2H, ABq, JA,B = 10.7 Hz, OCH2Ph), 4.72,
4.83 (2H, ABq, JA,B = 10.2 Hz, OCH2Ph), 5.03 (1H,
m, OCH@CHCH3–E), 6.04 (1H, dd, JZ 6.2 Hz,
OCH@CHCH3–Z), 6.10 (1H, dd, JE 12.1 Hz,
OCH@CHCH3–E), 7.04 (2H, d, J 8.5 Hz, Ar–H), 7.20-
7.38 (15H, m, Ar–H), 7.48 (2H, d, J 8.0 Hz); 13C
NMR (100.6 MHz, CDCl3) (Z isomer): d 9.4 (q,
CH@CHCH3), 21.1 (q, ArCH3), 69.0 (t, C-6), 73.4,
75.1, 75.6 (3 · t, 3 · PhCH2), 79.2, 79.2, 82.3 (3 · d,
C-3, C-4, C-5), 86.0, 86.3 (2 · d, C-1, C-2), 100.2 (d,
CH@CHCH3), 127.5, 127.6, 127.8, 127.9, 128.3, 128.3,
128.4, 129.6, 133.7, 133.7 (10 X d, Ar–CH), 138.0,
138.3 (2 · s, Ar–C), 146.5 (d, CH@CHCH3); m/z
(ES+) 619 (M+Na+, 100), 614 (M+NH4þ, 57%); ESIMS
m/z calcd for C37H44NO5S [M+NH4]+: 614.2940.
Found 614.2938. Anal Calcd for C37H40O5SÆ1/2H2O:
C, 73.36; H, 6.82. Found: C, 73.47; H, 6.76.
21
solid, mp 59–61 ꢁC (EtOAc/petrol); ½aꢁD ꢀ18.6 (c 1.0,
CHCl3); IR: (KBr) 1648 (w, C@C) cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 2.31 (3H, s, ArCH3), 3.35 (1H,
at, J = 9.2 Hz, H-2), 3.57–3.81 (5H, m, H-3, H-4, H-5,
H-6, H-60) 4.25 (1H, dat, Jgem = 12.5 Hz, Jvic = 5.8 Hz,
J = 1.1 Hz, OCHH0CH@CH2), 4.39 (1H, dat, J =
1.5 Hz, OCHH0CH@CH2), 4.52, 4.62 (2H, ABq,
JA,B = 11.6 Hz, OCH2Ph), 4.54 (1H, d, J1,2 = 9.9 Hz,
H-1), 4.77, 4.83 (2H, ABq, JA,B = 12.2 Hz, OCH2Ph),
4.58, 4.82 (2H, ABq, JA,B = 10.5 Hz, OCH2Ph), 5.19
(1H, daq, JZ = 10.4 Hz, J = 0.9 Hz, CH@CHEHZ),
5.30 (1H, daq, JE = 17.1 Hz, J = 1.3 Hz, CH@CHEHZ),
5.99 (1H, ddat, CH@CH2), 7.04 (2H, d, J = 7.5 Hz, Ar–
H), 7.27–7.38 (15H, m, Ar–H), 7.46 (2H, d, J = 1.9 Hz,
Ar–H); 13C NMR (100.6 MHz, CDCl3): d 21.1 (q,
ArCH3), 69.0 (t, C-6), 73.4, 74.2, 75.0, 75.8 (4 · t,
3 · PhCH2, CH2CH@CH2), 77.0 (d, C-2), 77.3, 79.1
(2 · d, C-4, C-5), 86.7 (d, C-3), 87.6 (d, C-1), 117.3 (t,
CH@CH2) 127.5, 127.6, 127.7, 127.8, 127.9, 128.3,
128.4, 128.4, 129.6, 132.6, 134.7 (11 · d, CH@CH2,
Ar–CH), 137.6,þ 138.3, 138.4 (3 · s, Ar–C); m/z (ES+)
4.9. Methyl 3,4,6-tri-O-benzyl-a-D-glucopyranosyl-
(1!2)-3,4,6-tri-O-benzyl-a-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-benzyl-a-D-glucopyranosyl-(1!3)-2-O-benz-
yl-(R)-4,6-O-benzylidene-a-D-mannopyranoside (11)
614 (M+NH4
,
100%); ESIMS m/z calcd for
C37H44NO5S [M+NH4]+: 614.2940. Found 614.2933.
Anal calcd for C37H40O5S: C, 74.47; H, 6.76. Found:
C, 74.49; H, 6.78.
Iodine (11.6 mg, 0.0457 mmol), silver trifluoromethane-
sulfonate (12 mg, 0.0485 mmol), 2,6-di-tert-butyl-4-
˚
methylpyridine (20 mg, 0.097 mmol) and 4 A molecular
4.8. para-Tolyl 3,4,6-O-benzyl-2-O-prop-10-enyl-1-thio-b-
D-glucopyranose (10)
sieves were added to freshly distilled CH2Cl2 (2 mL) in
a flame-dried flask. The mixture was cooled to ꢀ78 ꢁC
and stirred for 20 min under an atmosphere of Ar. Enol
ethers 10 (23.5 mg, 0.0388 mmol) and trisaccharide 8
(40 mg, 0.0323 mmol) were dissolved in freshly distilled
CH2Cl2 (1 mL) and added to the reaction vessel via
cannula under an atmosphere of Ar. The reaction was
allowed to warm to rt whilst stirring. After 17 h, TLC
(10:1, toluene–EtOAc) indicated the complete consump-
tion of the trisaccharide (Rf = 0.2) and enol ethers
(Rf = 0.64). The reaction mixture was diluted with
CH2Cl2 (5 mL) and quenched by addition of sodium
thiosulfate (5 mL of a 10% aqueous solution). The mix-
ture was filtered and the two phases separated. The
aqueous layer was extracted with CH2Cl2 (10 mL) and
the combined organic extracts were dried (MgSO4), fil-
Wilkinson’s catalyst (93 mg, 0.10 mmol) was dissolved
in freshly distilled THF (5 mL) in a flame-dried flask
and degassed. n-Butyl lithium (0.10 mL, 0.16 mmol,
1.6 M solution in hexanes) was added and the mixture
was stirred for 20 min under an atmosphere of Ar. Allyl
protected thioglycoside 9 (152 mg, 0.26 mmol) was dis-
solved in freshly distilled THF (5 mL) in a flame-dried
flask and heated to 70 ꢁC under an atmosphere of Ar.
The catalyst solution prepared above was then added
via cannula. After 5 h, TLC (3:1, petrol–EtOAc) indi-
cated the formation of a single product (Rf = 0.65)
and the complete consumption of the starting material
(Rf = 0.60). The reaction mixture was allowed to cool

E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
1617
tered and concentrated in vacuo. The residue was
purified by flash column chromatography (20:1!10:1,
toluene–EtOAc) to afford intermediate mixed acetals
(48 mg, 77%) as a pale yellow foam as a mixture of
diastereomers; m/z (ES+) 1983 (M+Na+, 17) 1978
(M+NH4þ, 7%); m/z (isotopic distribution) expected
mass 1985 (MNa++2, 36%), 1984 (MNa++1, 70%),
1983 (MNa+, 100%), 1982 (MNa+ꢀ1, 78%), found
1985 (MNa++2, 37%), 1984 (MNa++1, 62%), 1983
(MNa+, 100%), 1982 (MNa+ꢀ1, 88%).
PhCHH0), 4.70 (1H, d, J1,2 = 1.3 Hz, H-1a), 4.70 (1H,
d, J = 10.6 Hz, PhCHH0), 4.73 (1H, d, J = 11.3 Hz,
PhCHH0), 4.82 (1H, d, J = 10.8 Hz, PhCHH0), 4.85
(1H, d, J = 10.5 Hz, PhCHH0), 5.02 (1H, d,
J = 11.4 Hz, PhCHH0), 5.07 (1H, d, J = 11.8 Hz,
PhCHH0), 5.38 (1H, s, PhCH), 5.46 (1H, d,
J1,2 = 3.2 Hz, H-1y), 5.66 (1H, d, H-1x), 6.99–7.53
(55H, m, Ar–H); 13C NMR (125.7 MHz, CDCl3): d
54.7 (q, OCH3), 63.6 (d, C-5a), 68.0, 68.3, 68.5, 68.7
(4 · t, C-6a, C-6x, C-6y, C-6d), 69.8, 70.0 (2 · d, C-5x,
C-5d), 71.0, 73.0, 73.4, 73.4, 74.4, 74.6, 74.8, 74.8, 75.8
(9 · t, 9 · PhCH2), 71.0 (d, C-5y), 71.9 (d, C-3a), 75.5
(d, C-2y), 76.8 (d, C-4d), 77.1 (d, C-2a), 77.7, 78.0, 78.3
(3 · d, C-3x, C-4x, C-4y), 78.7 (d, C-2x), 80.0 (d, C-4a),
80.3 (d, C-3y), 83.8 (d, C-3d), 95.9 (d, C-1x, C-1y, C-
1d), 100.9 (d, C-1a), 102.0 (d, PhCH), 126.2, 127.1,
127.1, 127.2, 127.3, 127.5, 127.6, 127.6, 127.7, 127.7,
127.9, 127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.2,
128.2, 128.3, 128.3, 128.4, 129.0 (24 · d, Ar–CH),
137.2, 137.6, 137.8, 138.0, 138.1, 138.1, 138.3, 138.3,
138.9, 139.1 (9 · s, Ar–C); m/z (ES+) Isotope Distribu-
tion calcd for C102H108O21Na (MNa+) 1694.7 (26),
1693.7 (61), 1692.7 (100), 1691.7 (88%). Found 1694.9
(28), 1693.9 (61), 1692.9 (100), 1691.9 (95%).
Mixed acetals prepared above (42 mg, 0.0216 mmol),
2,6-di-tert-butyl-4-methylpyridine (9 mg, 0.0438 mmol)
˚
and 4 A molecular sieves were added to freshly distilled
CH2Cl2 (1 mL) in a flame-dried flask under an atmo-
sphere of Ar, and the mixture was cooled to 0 ꢁC whilst
stirring. Dimethyldisulfide (4.0 lL, 0.044 mmol) and tri-
flic anhydride (7.0 lL, 0.041 mmol) were dissolved in
freshly distilled CH2Cl2 (0.25 mL) and after 5 min the
mixture was added to the reaction vessel under an atmo-
sphere of Ar. The reaction was allowed to reach rt and
stirred. After 90 min, TLC (10:1, toluene–EtOAc) indi-
cated the complete consumption of starting material
(Rf = 0.49) and the formation of several products
(Rf = 0.15–0.49). The reaction mixture was cooled to
0 ꢁC, then dioxane (5 mL) and an aqueous NaH2PO4/
Na2HPO4 buffer (pH 6.0, 3 mL) were added and the
mixture stirred for a further 10 min. The mixture was fil-
tered and diluted with CH2Cl2 (20 mL). The organic ex-
tracts were dried (MgSO4), filtered and concentrated in
vacuo. The crude residue was dissolved in acetone
(3 mL) and water (0.3 mL). NIS (7 mg, 0.032 mmol)
was added and the mixture stirred overnight at rt. The
reaction mixture was treated with sodium thiosulfate
(5 mL of a 10% aqueous solution) and diluted with
CH2Cl2 (10 mL). The aqueous extracts were washed
with CH2Cl2 (10 mL) and the combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by flash column chromatogra-
phy (10:1, toluene–EtOAc) to afford tetrasaccharide 11
4.10. Methyl a-D-glucopyranosyl-(1!2)-a-D-gluco-
pyranosyl-(1!3)-a-D-glucopyranosyl-(1!3)-a-D-manno-
pyranoside (12)
Protected tetrasaccharide 11 (10 mg, 5.9 lmol) was dis-
solved in ethanol (2.25 mL) and acetic acid (0.25 mL),
and palladium(II) acetate (5 mg 0.022 mmol) was added.
The mixture was degassed and stirred under an atmo-
sphere of H2. After 22 h, TLC (CMAW) indicated the
formation of a single major product (Rf = 0.1). The
reaction mixture was filtered through Celite and concen-
trated in vacuo. The residue purified by size-exclusion
chromatography on Sephadex G-25 (water) to afford
25
deprotected tetrasaccharide 12 (4 mg, 98%); ½aꢁD +84
25
(21 mg, 58%) as a colourless foam; ½aꢁD +94 (c 0.25,
(c 0.2, MeOH); 1H NMR (500 MHz, D2O)13: d 3.41
(3H, s, OCH3), 3.46 (1H, H-4d), 3.53 (1H, H-4c), 3.60
(1H, H-2d), 3.62 (1H, H-4b), 3.65 (1H, H-2b), 3.66
(1H, H-4a), 3.69 (1H, H-2c), 3.76 (2H, H-6b, H-60b),
3.78 (1H, H-5a), 3.79 (1H, H-3d), 3.79 (2H, H-6c, H-
60c), 3.82 (1H, H-5b), 3.82 (2H, H-6d, H-60d), 3.83 (1H,
H-6a), 3.84 (1H, H-3c), 3.86 (1H, H-3a), 3.91 (1H, H-
3b), 3.91 (1H, H-60a), 3.96 (1H, H-5d), 4.06 (1H, H-5c),
4.09 (1H, H-2a), 4.74 (1H, H-1a), 5.17 (1H, d,
J1,2 = 3.7 Hz, H-1d), 5.24 (1H, d, J1,2 = 4.4 Hz, H-1b),
5.52 (1H, d, J1,2 = 3.7 Hz, H-1c); 13C NMR
(100.6 MHz, D2O): d 54.6 (q, OCH3), 60.0 (t, C-6c),
60.1 (t, C-6d), 60.1 (t, C-6b), 60.7 (t, C-6a), 65.9 (d, C-
3c), 69.1 (d, C-4d), 69.1 (d, C-4c), 69.6 (d, C-2a), 69.7
(d, C-4b), 70.1 (d, C-2b), 71.0 (d, C-5b), 71.1 (d, C-2d),
71.3 (d, C-5c), 71.6 (d, C-5d), 71.7 (d, C-3d), 72.4 (d,
C-4a), 72.7 (d, C-5a), 75.2 (d, C-2c), 78.4 (d, C-3a),
CHCl3); 1H NMR (500 MHz, CDCl3):ꢀ d 3.15–3.16
(1H, br m, H-2d), 3.26 (1H, dd, J5,6 = 1.6 Hz,
0
J6;6 ¼ 11:6 Hz, H-6d), 3.30 (1H, dd, J5,6 = 3.1 Hz, H-
60d), 3.34 (3H, s, OCH3), 3.35 (1H, at, J = 9.6 Hz, H-
3d), 3.43 (1H, at, J = 9.5 Hz, H-4d), 3.48 (1H, dd,
J1,2 = 3.7 Hz, J2,3 = 9.5 Hz, H-2x), 3.52 (1H, d,
0
J6;6 ¼ 10:0 Hz, H-6y), 3.65–3.76 (6H, m, H-4x, H-6x,
H-60x, H-2y, H-4y, H-60y), 3.82–3.87 (4H, m, H-2a, H-
5a, H-6a, H-5d), 3.91 (1H, dat, J = 9.9 Hz, H-5x), 3.96
(1H, at, J = 9.4 Hz, H-3y), 4.18–4.24 (4H, m, H-60a,
H-5y, 2 · PhCHH0), 4.30–4.58 (15H, m, H-3a, H-4a, H-
3x, H-1d, 11 · PhCHH0), 4.68 (1H, d, J = 11.2 Hz,
ꢀ It was not possible to distinguish glucose residues b and c from the
NMR data and consequently, they have been arbitrarily denoted as x
and y.

1618
E. Attolino et al. / Carbohydrate Research 341 (2006) 1609–1618
16. (a) Scaman, C. H.; Hindsgaul, O.; Palcic, M. M.;
Srivastava, O. P. Carbohydr. Res. 1996, 296, 203–213;
(b) Gemma, E.; Lahmann, M.; Oscarson, S. Carbohydr.
Res. 2005, 340, 2558–2562.
79.6 (d, C-3b), 95.7 (d, C-1d), 96.4 (d, C-1c), 100.5 (d, C-
1b), 100.6 (d, C-1a); m/z (ES+) 703 (M+Na+, 100%).
ESIMS m/z calcd for C25H48NO21 [M+NH4]+:
698.2719. Found 698.2716.
17. Fairbanks, A. J. Synlett 2003, 1945–1958.
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Acknowledgements
We gratefully acknowledge the EPSRC (Quota Award
to I.C.) and the European Union (Marie Curie Fellow-
ship to E.A.) for financial support. We also gratefully
acknowledge the use of the Chemical Database Service
(CDS) at Daresbury, UK.
19. For the original developments of the IAD approach, see:
(a) Barresi, F.; Hindsgaul, O. J. Am. Chem. Soc. 1991, 113,
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