Total synthesis of the Glc3Man N-glycan tetrasaccharide

Article abstract of DOI:10.1016/S0040-4020(02)01221-8

The total synthesis of the tetrasaccharide Glcα(1→2)Glcα(1→3)Glcα(1→3)ManαOMe, which corresponds to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved by the use of differentially protected selenoglycosides and thioglycosides as glycosyl donors, all of which possessed non-participating protection of the 2-hydroxyl group. Favourable anomeric stereoselectivity was achieved for the glycosylation reactions by the use of ether as solvent, or co-solvent. Global deprotection by catalytic hydrogenation with palladium acetate in a mixture of ethanol and acetic acid yielded the target tetrasaccharide.

Full text of DOI:10.1016/S0040-4020(02)01221-8

Tetrahedron 58 (2002) 9403–9411
Total synthesis of the Glc3Man N-glycan tetrasaccharide
b
b
b
S. C. Ennis,a I. Cumpstey,a A. J. Fairbanks,a T. D. Butters, M. Mackeen and M. R. Wormald
,
*
aDyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OX1 3QY, UK
bDepartment of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Received 20 June 2002; revised 5 September 2002; accepted 26 September 2002
Abstract—The total synthesis of the tetrasaccharide Glca(1!2)Glca(1!3)Glca(1!3)ManaOMe, which corresponds to the terminal
tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved by the use of differentially protected
selenoglycosides and thioglycosides as glycosyl donors, all of which possessed non-participating protection of the 2-hydroxyl group.
Favourable anomeric stereoselectivity was achieved for the glycosylation reactions by the use of ether as solvent, or co-solvent. Global
deprotection by catalytic hydrogenation with palladium acetate in a mixture of ethanol and acetic acid yielded the target
tetrasaccharide. q 2002 Published by Elsevier Science Ltd.
1. Introduction
of the Glc3Man tetrasaccharide [Glca(1!2)Glca(1!3)
Glca(1!3)ManaOMe 1] was undertaken. Following suc-
cessful synthesis, the aim is to study the conformation of the
tetrasaccharide in a variety of solvent systems and compare
the results to those obtained for the full-length glycan.
The construction of the oligosaccharide portion of N-linked
glycoproteins, and the process by which the oligosaccharide
is linked to the protein are now well understood.1 Transfer
of a dolichol bound tetradecasaccharide (Glc3Man9-
GlcNAc2) to certain asparagine residues of the nascent
glycoprotein is mediated by the enzyme oligosaccharyl
transferase (OST) during protein synthesis in the endo-
plasmic reticulum. Subsequent trimming of this tetradeca-
saccharide is initially mediated by a series of glycosidases,
which remove all the glucose and some of the mannose
residues sequentially. Further elaboration to one of the three
standard types of N-glycan (high mannose, complex or
hybrid) is then controlled by a series of glycosyltransferases
in the Golgi apparatus.
This paper details one of the synthetic routes undertaken to
allow access to this biologically important tetrasaccharide.5
The key synthetic consideration was control of anomeric
stereochemistry during the respective glycosylation reac-
tions, which necessitated that all glycosyl donors possessed
non-participating protecting groups at the 2-position. It was
therefore envisaged that all hydroxyl groups would either be
protected in the vast majority of cases as benzyl ethers, or in
the case of two hydroxyls on the mannose unit as 4,6-
benzylidene. This strategy had the advantage that a final
global deprotection step by catalytic hydrogenation would
allow access to the deprotected tetrasaccharide. Such
considerations revealed four potential building blocks
which were needed to complete the synthesis of 1 (Fig. 1).
Detailed herein are the synthetic routes taken to access these
building blocks, the subsequent glycosylation reactions
undertaken to construct the tetrasaccharide, and finally the
global deprotection step.
The glucose residues of the Glc3Man9GlcNAc2 tetradeca-
saccharide have been shown to be part of the recognition
motif for OST,2 as well as forming the substrate for
glucosidase I. The solution conformation of the glucose
portion of the Glc3Man7GlcNAc2 oligosaccharide in water
has been determined by NMR analysis.3 In addition the
conformation of the tetrasaccharide Glc3ManOPr in DMSO
has also been determined by NMR and molecular model-
ing.4 However, these two experimentally derived confor-
mations are significantly different. Therefore in order to
determine whether this difference is (i) due to an incorrect
analysis of one of the compounds, (ii) a result of the
different solvents used or (iii) a result of the different
primary sequences of the saccharides studied, the synthesis
2. Results and discussion
2.1. Synthesis of glycosyl donors
Firstly the required manno glycosyl acceptor 2 was accessed
from methyl mannopyranoside 6, via a two step reaction
sequence by way of the endo dibenzylidene derivative 7,
following a modification of the original literature pro-
cedure.6 Thus treatment of 6 with benzaldehyde dimethyl
Keywords: carbohydrates; glycosidation; thioglycosides; glycoproteins.
*
Corresponding author. Tel.: þ44-1865-275-647; fax: þ44-1865-275-674;
e-mail: antony.fairbanks@chem.ox.ac.uk
0040–4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020(02)01221-8

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S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
Figure 1.
Scheme 1. (i) Benzaldehyde dimethyl acetal, CSA, DMF, 608C, 7 53%, 8 35%, (ii) DIBAL, PhCH3, 2408C, 2 85%, 9 10%.
acetal gave a mixture of endo and exo dibenzylidene
derivatives 7 and 8 which were readily separable on a large
scale by crystallization. Attempted treatment of the endo
isomer 7 with LiAlH4 and AlCl3 did indeed give the desired
2-O-benzyl product 2 but only in moderate yield, since
competitive reduction of the 4,6-benzylidene was also
observed. However, treatment of 7 with di-iso-butyl-
aluminium hydride (DIBAL)7 in toluene at 2408C resulted
in the formation of the desired product 2 in an excellent
yield of 85%, together with a small amount of the undesired
regioisomer 9 (10%, Scheme 1).
acid, which effected removal of both acetonide protecting
groups and reversion to the pyranose form. Finally complete
acetylation by treatment with acetic anhydride and sodium
acetate gave the 3-O-allyl tetraacetate 11 (45% yield over
three steps). Introduction of selenium at the anomeric centre
was achieved by treatment with selenophenol and BF3
etherate to yield selenoglycoside 12 (81% yield). A
protecting group swap of acetates for non-participating
benzyls, necessary to avoid formation of b-linkages during
subsequent glycosylation, was achieved by Zemplen
deacetylation followed by complete benzylation with
benzyl bromide and sodium hydride in DMF (76% yield
over two steps) to yield the desired allyl protected
selenoglycoside 3. In addition, as later glycosylation
reactions using 3 as a donor proved to be only moderately
successful and displayed poor stereoselectivity (vide infra)
the allyl protecting group at OH-3 was also exchanged for
The required gluco glycosyl donor 3 in which the
3-hydroxyl was selectively protected was synthesized
from diacetone glucose 10 as follows. Allylation of 10 by
treatment with sodium hydride and allyl bromide in DMF
was subsequently followed by heating in aqueous sulfuric
Scheme 2. (i) Allyl bromide, NaH, DMF, rt, (ii) aq. H2SO4, D, (iii) Ac2O, NaOAc, 45% over three steps, (iv) PhSeH, BF3·OEt2, DCM, rt, 81%,
(v) NaOMe/MeOH, (vi) BnBr, NaH, DMF, rt, 76%, (vii) Ti(O-iso-Pr)4, C6H11MgCl, THF, rt, 85%, (viii) Ac2O, pyridine, rt, 91%.

S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
9405
Scheme 3. (i) NaOMe, MeOH, rt, 75%, (ii) allyl bromide, NaH, DMF, 08C to rt, 95%, (iii) PhSeH, BF3·OEt2, DCM, 74%, (iv) NaOMe, MeOH, (v) BnBr, NaH,
DMF, 62% over two steps.
an acetate; removal by treatment of 3 with titanium tetra-
iso-propoxide and cyclohexyl magnesium chloride8 pro-
duced alcohol 13 (85% yield) and subsequent acetylation
with acetic anhydride in pyridine yielded the desired acetate
14 (91% yield, Scheme 2).
16. Finally the perbenzylated selenoglycoside 5 was
accessed from glucose pentaacetate in two steps following
standard procedures (Scheme 3).10
2.2. Glycosylation reactions
The known gluco 2-O-acetyl thiophenyl glycoside 15
(synthesized from glucose pentacetate via a 1,2-orthoester
in five steps as previously described)9 was deacetylated by
treatment with sodium methoxide in methanol to yield the
thiophenyl glycosyl acceptor 4. In addition 4 was sub-
sequently allylated to yield the allyl protected thioglycoside
With all building blocks in hand construction of the
glycosidic linkages was undertaken. The initial convergent
strategy involved glycosylation of allyl protected donor 3
with the manno glycosyl acceptor 2. Attempted glycosyl-
ation of 2 mediated by N-iodosuccinimide (NIS) with
catalytic triflic acid in a mixture of dichloroethane (DCE)
˚
Scheme 4. (i) NIS, AgOTf, DCE: diethyl ether, 1:1, 4 A molecular sieves, rt, 88%, 4:1, a/b, (ii) NaOMe, MeOH, rt, 83%, (iii) 16, MeOTf, 4 A molecular
˚
˚
sieves, diethyl ether, rt, 80%, 3:1, a/b, (iv) Ti(O-iso-Pr)4, C6H11MgCl, THF, rt, 83%, (v) 5, NIS, AgOTf, 4 A molecular sieves, DCE/diethyl ether, 1:1, rt, 73%,
3:1, a/b, (vi) Pd(OAc)2, H2, EtOH/acetic acid, 9:1, rt, 86%.

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S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
Table 1. 1H Assignment of Glca(1!2)Glca(1!3)Glca(1!3)ManaOMe 1 in CD3OD
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6/H-60
OMe
3.373
d
c
b
a
5.027 (3.7 Hz)
5.391 (3.7 Hz)
5.093 (4.4 Hz)
4.617
3.436
3.582
3.488
4.039
3.713
3.794
3.812
3.758
3.313
3.365
{3.343}
{3.521}
3.981
4.066
{3.637}
{3.839}
3.667
3.698
3.728
3.808
3.831
{3.866}
{3.816}
{3.769}
The peaks for Glc3ManOMe were assigned via COSY unless stated otherwise. Italics indicate peaks assigned via RELAY spectra (unresolved in COSY) and
braces ({ }) indicate peaks assigned via TOCSY spectra.
Table 2. 1H Assignment of Glca(1!2)Glca(1!3)Glca(1!3)ManaOMe 1 in D2O
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6/H-60
OMe
3.410
d
c
b
a
5.175 (3.7 Hz)
5.524 (3.7 Hz)
5.237 (4.4 Hz)
4.735
3.602
3.688
3.643
4.086
3.782
3.835
3.908
3.852
3.449
3.503
3.620
3.965
4.058
3.810
3.781a
3.801a
3.749a
{3.832}, {3.907}
{3.656}b
{3.775}
The peaks for Glc3ManOMe were assigned via COSY unless stated otherwise. Italics indicate peaks assigned via RELAY spectra (unresolved in COSY) and
braces ({ }) indicate peaks assigned via TOCSY spectra.
a
H-6 and H-60 indistinguishable.
{ } from H-2.
b
and ether as solvent only produced a moderate (53%) yield
of disaccharide product, and in addition unfortunately
proceeded with no stereoselectivity (a/b mixture, 1:1).
However, glycosylation of the acetate protected seleno-
glycoside 14 with 2 not only produced the desired
disaccharide 17 in a much improved 87% yield, but also
with the desired anomeric selectivity (a/b mixture, 4:1,
Scheme 4).
yield, again as a mixture of anomers but with favourable
stereoselectivity (a/b ratio, 3:1). Finally complete depro-
tection of tetrasaccharide 21 was achieved by catalytic
hydrogenation in the presence of palladium acetate in a
mixture of ethanol/acetic acid (9:1) to yield the desired
deprotected tetrasaccharide 1 (Scheme 4). This completely
deprotected material could satisfactorily be purified to
homogeneity by high performance anion exchange
chromatography (HPAEC PAD). Detailed two-dimensional
1H NMR studies revealed the following assignments
(Tables 1 and 2).
The convergent synthetic strategy next required glycosyl-
ation of the fully armed11 selenoglycoside 5 by the armed
thioglycoside 4, and therefore relied upon the selective
activation of one of the two anomeric leaving groups.
Although selective activation of selenoglycosides in the
presence of thioglycosides is well precedented,12 several
attempted glycosylation reactions of 4 with 5 under a range
of conditions (IDCP, silver triflate/K2CO3 and NIS/TfOH)
all met with failure. Thin layer chromatography (tlc)
indicated the formation of many reaction products,
presumably due to competitive activation of 4 and self-
condensation. In the face of this disappointment
several alternative strategies were considered,13 and in
the light of several possibilities a linear approach was
adopted.
3. Summary and conclusion
The total synthesis of the deprotected tetrasaccharide
Glca(1!2)Glca(1!3)Glca(1!3)ManaOMe 1 has been
successfully achieved. Although a convergent strategy was
precluded by difficulties encountered during the selective
activation of a selenoglycoside in the presence of a
thioglycoside, reversion to a linear approach allowed access
to the required tetrasaccharide in good yield. Although
favourable ratios of the desired a products could be
obtained by the use of ether a solvent or as a co-solvent
for the glycosylation reaction, some b products were
invariably produced, which were difficult to separate.
However, the completely deprotected tetrasaccharide
could be purified by high performance anion exchange
chromatography.
Deprotection of the acetate protected disaccharide 17 with
sodium methoxide in methanol yielded the disaccharide
glycosyl acceptor 18 (83% yield). This was followed by
methyl triflate mediated glycosylation of 18 with the allyl
protected thioglycoside 16 in ether14 as the reaction solvent
to yield the desired trisaccharide 19 in 80% yield (a/b ratio,
4:1; unfortunately this anomeric mixture was inseparable
and therefore the mixture was carried on as such for
subsequent transformations). Removal of the allyl protect-
ing group was again achieved by treatment of 19 with
titanium tetra-iso-propoxide and cyclohexyl magnesium
chloride, to yield the trisaccharide acceptor 20 (83% yield).
Glycosylation of 20 with the selenoglycoside 5 was
achieved by activation with NIS and silver triflate in
DCE/ether to yield the protected tetrasaccharide 21 in 73%
The solution NMR conformation of the tetrasaccharide is
currently being investigated in a number of different
solvents and these findings will be compared with those
already published for the Glc3Man7GlcNAc2 and Glc3-
ManOPr oligosaccharides. In addition, an alternative and
completely stereoselective route to this tetrasaccharide
using an iterative intramolecular glycosylation strategy is
currently under investigation. All of these results will be
published in due course.

S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
9407
4. Experimental
4.1. General methods
performed. The duration of each run was 40 min. Eluates
of each run were collected in fractions of 200 ml per micro
eppendorf tube. Combined fractions of the product of
interest were passed through a desalting column of Dowex
AG50W-X12 (Hþ) resin and subsequently, concentrated in
vacuo.
Melting points were recorded on a Kofler hot block and are
uncorrected. Proton nuclear magnetic resonance (dH)
spectra were recorded on a Bruker DPX 400 (400 MHz)
or on a Bruker AMX 500 (500 MHz) spectrometer.
Multiplicities are quoted as singlet (s), doublet (d), doublet
of doublets (dd), doublet of doublet of doublets (ddd), triplet
(t), apparent triplet (at) or doublet of doublet of triplets
(ddt). Carbon nuclear magnetic resonance (dC) spectra were
recorded on a Bruker AC 200 (50.3 MHz), or on a Bruker
DPX 400 (100.6 MHz) spectrometer. Multiplicities were
assigned using DEPT sequence. All chemical shifts are
quoted on the d-scale in parts per million (ppm). All NMR
experiments were performed at a probe temperature of
308C. Two-dimensional phase-sensitive COSY, RELAY
and TOCSY spectra, referenced to internal acetone at
4.1.1. Methyl (S),(R)-2,3:4,6-di-O-benzylidene-a-D-man-
nopyranoside 7 and methyl(R),(R)-2,3:4,6-di-O-benzyl-
idene-a-D-mannopyranoside 8. Benzaldehyde dimethyl
acetal (40 ml, 266 mmol) was added to a solution of methyl-
a-D-mannopyranoside 6 (20 g, 103 mmol) and camphor
sulfonic acid (300 mg, 1.29 mmol) in DMF (220 ml). The
resulting solution was heated to 608C on a rotary evaporator
under a pressure of 250 mbar. After 3 h, TLC (petrol/ethyl
acetate, 3:1) indicated complete conversion of starting
material (Rf 0.0) to two products (Rf 0.50 and 0.80). Further
benzaldehyde dimethyl acetal (20 ml, 133 mmol) and
camphor sulfonic acid (150 mg, 0.65 mmol) was added to
the reaction mixture. After 2 h, TLC (petrol/ethyl acetate,
3:1) indicated the formation of a single product (Rf 0.80).
The solvent was removed in vacuo, the residue co-
evaporated with toluene (50 ml), then dissolved in DCM
(300 ml), and washed with saturated sodium bicarbonate
solution (150 ml) and brine (150 ml). The organic phase was
then dried (MgSO4), filtered and concentrated in vacuo. The
resulting residue was purified by recrystallization (ethyl
acetate/methanol) to give the exo-dibenzylidene compound
8 (13.2 g, 35%) as a white crystalline solid; mp 171–1738C;
[a]D24¼27.3 (c, 1.1, CHCl3) [lit.15 mp 176–1808C;
[a]2D0¼21.9 (c, 0.76, CHCl3)]; dH (400 MHz, CDCl3) 3.42
1
2.217 ppm, were used to assign the H resonances of 1.
Sequence-specific assignments were made by comparison
with reported assignments for glucosylated oligomannose-
type oligosaccharides.3 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 (electro-
spray) were performed on a Waters 2790-Micromass LCT
electrospray ionisation mass spectrometer, or by the EPSRC
Mass Spectrometry Service Centre, Department of
Chemistry, University of Wales, Swansea on a MAT900
XLT electrospray ionisation mass spectrometer. Optical
rotations were measured on a Perkin–Elmer 241 polari-
meter with a path length of 1 dm. Concentrations are given
in g/100 ml. Microanalyses were performed by the micro-
analytical services of the Inorganic Chemistry Laboratory,
Oxford. TLC was carried out on Merck Kieselgel 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; methanol was
distilled from sodium hydride, dichloromethane (DCM)
was distilled from calcium hydride, pyridine was distilled
from calcium hydride and stored over potassium hydroxide
and tetrahydrofuran was distilled from a solution of sodium
benzophenone ketyl immediately before use. Petrol was
distilled between 40 and 608C before use to remove
involatile fractions. HPAEC was carried out on a Dionex
BioLC system (Dionex, USA) with pulsed amperometric
detection (PAD) on a CarboPac PA-10 column (4£250 mm)
connected to a guard column (4£50 mm). The detector
settings used were E1¼þ0.05 V, t1¼0–400 ms,
E2¼þ0.75 V, t2¼410–600 ms and E3¼20.15 V,
t3¼610–1000 ms. Elution was performed isocritically
using 70 mM NaOH as the solvent at a flow rate of
1 ml/min at 308C. Before sample injection, the column was
eluted with the solvent until detector readings stabilized.
The solution of crude Glc3ManOMe sample was centrifuged
for 10 min at 13000 rpm to remove particulate matter which
settled to the bottom of the centrifuge tube. Multiple
injections (25 ml each) of crude Glc3ManOMe were
(3H, s, OMe), 3.83–3.94 (3H, m, H-4, H-5, H-6), 4.16 (1H,
0
0
d, J2,3¼5.4 Hz, H-2), 4.37 (1H, d, J5,6 ¼5.4 Hz, H-6 ), 4.64
(1H, dd, J2,3¼5.4 Hz, J3,4¼7.8 Hz, H-3), 5.03 (1H, s, H-1),
5.66 (1H, s, O2CHPh), 6.31 (1H, s, O2CHPh), 7.26–7.57
(10H, m, Ar-H); the mother liquor was concentrated in
vacuo and the resulting residue was purified by recrystal-
lization (ethanol) to give the desired endo-dibenzylidene
derivative 7 (19.5 g, 53%) as a white crystalline solid; mp
94–958C; [a]2D4¼258 (c, 1, CHCl3) [lit.15 mp 95–978C;
[a]2D0¼263 (c, 1, CHCl3)]; dH (400 MHz, CDCl3) 3.44 (3H,
s, OMe), 3.73–3.86 (3H, m, H-4, H-5, H-6), 4.31 (1H, d,
0
J2,3¼6.2 Hz, H-2), 4.33 (1H, dd, J5,6¼4.2 Hz, J6,6 ¼9.8 Hz,
H-60), 4.49 (1H, at, J¼6.6 Hz, H-3), 5.10 (1H, s, H-1), 5.54
(1H, s, O2CHPh), 5.99 (1H, s, O2CHPh), 7.27–7.57 (10H,
m, Ar-H).
4.1.2. Methyl 2-O-benzyl-(R)-4,6-O-benzylidene-a-D-
mannopyranoside 2 and methyl 3-O-benzyl-(R)-4,6-O-
benzylidene-a-D-mannopyranoside 9. The endo-dibenzyl-
idene derivative 7 (100 mg, 0.27 mmol) was dissolved in
freshly distilled toluene (5 ml). The solution was stirred
under an atmosphere of argon and cooled to 2408C. Di-iso-
butyl aluminium hydride (0.20 ml, 0.30 mmol of a 1.5 M
solution in toluene) was added and the reaction was allowed
to reach room temperature. After 20 min TLC (petrol/ethyl
acetate, 3:1) indicated partial conversion of starting material
(Rf 0.8) to a product (Rf 0.4). Additional di-iso-butyl
aluminium hydride (0.20 ml, 0.30 mmol of a 1.5 M solution
in toluene) was added to the reaction mixture. After 30 min
TLC (petrol/ethyl acetate, 3:1) indicated complete conver-
sion of starting material (Rf 0.8) to two products (Rf 0.4 and
0.3). Methanol (2 ml) was added dropwise to the reaction

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S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
mixture, followed by DCM (50 ml). The organic phase was
washed with water (25 ml), brine (25 ml), dried (MgSO4),
filtered and concentrated in vacuo. The resulting residue
was purified by column chromatography (petrol/ethyl
acetate, 3:1) to give the 2-O-benzyl alcohol 2 (87 mg,
85%) as a white crystalline solid; mp 44–468C; [a]2D4¼þ1.1
(c, 1.0, CHCl3) [lit.15 mp 42–448C; [a]2D0¼þ2 (c, 1,
CHCl3)]; dH (400 MHz, CDCl3) 2.40 (1H, d,
J3,OH¼7.7 Hz, OH), 3.37 (3H, s, OMe), 3.79–3.87 (3H,
m, H-2, H-5, H-6), 3.92 (1H, at, J¼9.7 Hz, H-4), 4.06–4.11
1 M HCl (20 ml) was added to the reaction mixture, the
product was then extracted with DCM (2£200 ml), organic
phase dried (MgSO4), filtered and concentrated in vacuo.
The resulting residue was dissolved in DMF (80 ml), benzyl
bromide (4.8 ml, 40.5 mmol) and sodium hydride (60% in
mineral oil, 2.16 g, 54 mmol) were then added. An air
condenser was attached to the flask and the reaction mixture
was stirred at room temperature. After 18 h, TLC (petrol/
ethyl acetate, 4:1) indicated complete conversion of starting
material (Rf 0.0) to a major product (Rf 0.6). Methanol
(50 ml) was added portionwise in order to quench the
reaction. DCM (200 ml) was added, the organic phase was
washed with water (150 ml), dried (MgSO4), filtered and
concentrated in vacuo. The resulting residue was purified by
flash column chromatography (petrol/ethyl acetate, 6:1) to
give the benzylated selenoglycoside 3 (4.3 g, 76%) as a
white crystalline solid; (found C, 68.70; H, 6.07.
C36H38O5Se requires C, 68.55; H, 6.08%); mp 62–638C
(petrol/ether); [a]2D0¼216.5 (c, 1.2, CHCl3); nmax (KBr
Disc) 1641 (CvC) cm21; dH (500 MHz, CDCl3) 3.49–3.52
(1H, m, H-5), 3.52 (1H, at, J¼9.5 Hz, H-2), 3.59 (1H, at,
J¼8.8 Hz, H-4), 3.66 (1H, at, J¼9.2 Hz, H-3), 3.79 (1H, dd,
0
0
(1H, m, H-3), 4.27 (1H, dd, J5,6 ¼4.1 Hz, J6,6 ¼9.5 Hz,
H-60), 4.70 (1H, d, J¼11.7 Hz, OCH2Ph), 4.76 (1H, d,
J1,2¼1.1 Hz, H-1), 4.77 (1H, d, J¼11.7 Hz, OCH2Ph), 5.59
(1H, s, O2CHPh), 7.27–7.51 (10H, m, Ar-H); and the
undesired 3-O-benzyl alcohol 9 (10 mg, 10%) as a colour-
less oil; [a]2D4¼þ30 (c, 0.4, EtOH) [lit.15 [a]2D0¼þ33 (c, 2,
EtOH)]; dH (400 MHz, CDCl3) 2.67 (1H, s, OH), 3.39 (3H,
s, OMe), 3.79–3.93 (3H, m, H-3, H-5, H-6), 4.06 (1H, d,
J2,3¼3.0 Hz, H-2), 4.10 (1H, at, J¼9.2 Hz, H-4), 4.29 (1H,
dd, J5,6 ¼4.1 Hz, J6,6 ¼9.5 Hz, H-60), 4.72 (1H, d,
J¼11.6 Hz, OCH2Ph), 4.78 (1H, s, H-1), 4.86 (1H, d,
J¼11.6 Hz, OCH2Ph), 5.62 (1H, s, O2CHPh), 7.27–7.53
(10H, m, Ar-H).
0
0
0
0
J5,6¼4.4 Hz, J6,6 ¼10.9 Hz,0 H-6), 3.83 (1H, dd, J5,6
¼
0
1.9 Hz, J6,6 ¼10.9 Hz, H-6 ), 4.37–4.44 (2H, m, CH2-
CHvCH2), 4.60 (1H, d, J¼12.5 Hz, OCH2Ph), 4.64 (1H,
d, J¼11.4 Hz, OCH2Ph), 4.67 (1H, d, J¼12.5 Hz,
OCH2Ph), 4.78 (1H, d, J¼10.2 Hz, OCH2Ph), 4.87 (1H, d,
J¼11.4 Hz, OCH2Ph), 4.89 (1H, d, J¼10.2 Hz, OCH2Ph),
4.88 (1H, d, J1,2¼9.7 Hz, H-1), 5.23 (1H, dd, JZ¼10.4 Hz,
Jgem¼1.3 Hz, CHvCHEHZ), 5.36 (1H, dd, JE¼17.2 Hz,
Jgem¼1.5 Hz, CHvCHEHZ), 6.02 (1H, ddt, J¼5.7 Hz,
JZ¼10.8 Hz, JE¼17.2 Hz, CHvCH2), 7.24–7.76 (20H, m,
Ar-H); dC (50.3 MHz, CDCl3) 69.0, 73.5, 74.6, 75.2, 75.4
(5£t, C-6, OCH2CHvCH2, 3£OCH2Ph), 77.7, 80.2, 81.3,
83.1, 86.6 (5£d, C-1, C-2, C-3, C-4, C-5), 117.2 (t,
OCH2CHvCH2), 127.8, 127.9, 128.1, 128.3, 128.7,
129.3, 134.8, 135.2 (8£d, Ar-CH), 138.3, 138.4, 138.6
(3£s, Ar-C); m/z (APCIþ) 654 (MþNaþ, 100%).
4.1.3. Phenyl 2,4,6-tri-O-acetyl-3-O-allyl-1-seleno-b-D-
glucopyranoside 12. Selenophenol (4.1 ml, 38.7 mmol)
and boron trifluoride etherate (2.5 ml, 19.4 mmol) were
added to a stirred solution of peracetate 11 (5.0 g,
12.9 mmol) in DCM (30 ml), under argon at room
temperature. After 3 h, TLC (petrol/ethyl acetate, 1:1)
indicated complete conversion of starting material (Rf 0.5)
to a major product (Rf 0.6). DCM (100 ml) was added to the
reaction mixture, which was then washed with water
(100 ml), brine (100 ml). The organic phase was dried
(MgSO4), filtered and concentrated in vacuo. The resulting
residue was purified by recrystallization from ether/petrol to
give the selenoglycoside 12 (5.1 g, 81%) as a white
crystalline solid; (found C, 51.90; H, 5.28. C21H26O8Se
requires C, 51.84; H, 5.39%); mp 113–1158C; [a]2D0¼228.3
(c, 1.0, CHCl3); nmax (KBr Disc) 1740 (CO2CH3), 1641
(CvC) cm21; dH (500 MHz, CDCl3) 2.12, 2.18 (9H, 2£s,
3£CH3CO2), 3.61–3.66 (2H, m, H-3, H-5), 4.10–4.13 (2H,
m, CH2CHvCH2), 4.20–4.21 (2H, m, H-6, H-60), 4.87 (1H,
d, J1,2¼10.2 Hz, H-1), 5.04 (1H, dd, J2,3¼8.4 Hz, H-4), 5.08
(1H, dd, J1,2¼10.2 Hz, J2,3¼8.4 Hz, H-2), 5.18 (1H, dd,
Jgem¼2.7 Hz, JZ¼10.3 Hz, CHvCHEHZ), 5.24 (1H, dd,
Jgem¼2.9 Hz, JE¼17.2 Hz, CHvCHEHZ), 5.80 (1H, ddt,
J¼4.8 Hz, JZ¼10.3 Hz, JE¼17.2 Hz, CHvCH2), 7.31–
7.67 (5H, m, Ar-H); dC (50.3 MHz, CDCl3) 20.6, 20.7, 21.0
(3£q, 3£CH3CO2), 69.5, 72.2, 77.4, 81.2, 81.7 (5£d, C-1,
C-2, C-3, C-4, C-5), 62.5, 73.3 (2£t, C-6, OCH2CHvCH2),
117.2 (t, OCH2CHvCH2), 128.1, 128.5, 129.4, 134.4, 134.9
(1£s, 4£d, Ar-C), 169.6 (s, 2£MeCO2), 171.0 (s, MeCO2);
m/z (APCIþ) 509 (MþNaþ, 80%), 293 (50), 169 (50), 109
(100).
4.1.5. Phenyl 2,4,6-tri-O-benzyl-1-seleno-b-D-glucopyra-
nose 13. Titanium tetra-iso-propoxide (1.2 ml, 4.0 mmol)
was added to a stirred solution of the allyl protected
selenoglycoside 3 (2.5 g, 4.0 mmol) in THF (50 ml) at room
temperature under argon. Cyclohexylmagnesium chloride
(8.0 ml, 16.0 mmol) was added to the reaction mixture over
a period of 1 h (via a syringe pump). After a further 1 h,
TLC (petrol/ethyl acetate, 3:1) indicated complete conver-
sion of starting material (Rf 0.5), to a major product (Rf 0.4).
Water (50 ml) was added to the reaction mixture, the
product was then extracted with ether (3£150 ml), the
organic phase was dried (MgSO4), filtered and concentrated
in vacuo. The resulting residue was purified by flash column
chromatography (petrol/ethyl acetate, 4:1) to give the
alcohol 13 (2.0 g, 85%) as a colourless oil; (found C,
66.72; H, 6.02. C33H34O5Se requires C, 67.10; H, 5.81%);
[a]2D6¼213.6 (c, 0.96, CHCl3); nmax (film, CHCl3) 3435
(OH) cm21; dH (400 MHz, CDCl3) 3.41 (1H, at, J¼9.7 Hz,
H-2), 3.47 (1H, ddd, J4,5¼9.7 Hz, J5,6¼4.2 Hz,
4.1.4. Phenyl 3-O-allyl-2,4,6-tri-O-benzyl-1-seleno-b-D-
glucopyranoside 3. Sodium (430 mg) was dissolved in
methanol (20 ml) and the mixture was added to a stirred
solution of acetylated selenoglycoside 12 (4.4 g, 9.0 mmol)
in methanol (80 ml) at 08C under argon. After 1 h 45 min,
TLC (petrol/ethyl acetate, 1:2) indicated complete conver-
sion of starting material (Rf 0.8) to a major product (Rf 0.4).
0
J5,6 ¼1.9 Hz, H-5), 3.56 (1H, at, J¼9.6 Hz, H-4), 3.74–
0
3.78 (2H, m, H-3, H-6), 3.82 (1H, dd, J5,6 ¼1.8 Hz,
0
0
J6,6 ¼11.0 Hz, H-6 ), 4.55 (1H, d, J¼12.0 Hz, OCH2Ph),
4.63 (1H, d, J¼11.2 Hz, OCH2Ph), 4.65 (1H, d, J¼12.0 Hz,
OCH2Ph), 4.69 (1H, d, J¼10.8 Hz, OCH2Ph), 4.79 (1H, d,

S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
9409
J¼11.2 Hz, OCH2Ph), 4.85 (1H, d, J1,2¼9.8 Hz, H-1), 4.93
(1H, d, J¼10.8 Hz, OCH2Ph), 7.20–7.76 (20H, m, Ar-H);
dC (100.6 MHz, CDCl3) 69.0, 73.4, 74.7, 75.0 (4£t, C-6,
3£OCH2Ph), 77.4, 78.7, 79.9, 81.2, 82.6 (5£d, C-1, C-2,
C-3, C-4, C-5), 127.6, 127.7, 127.8, 127.9, 128.1, 128.3,
128.4, 128.5, 128.6, 129.0 (10£d, Ar-CH), 128.7, 138.1,
138.2 (3£s, Ar-C); m/z (ESþ) 608 (MþNH4þ, 100%).
4.1.8. Phenyl 2-O-allyl-3,4,6-tri-O-benzyl-1-thio-b-D-
glucopyranoside 16. A solution of the alcohol 4 (2.6 g,
4.8 mmol) and allyl bromide (0.83 ml, 9.6 mmol) in DMF
(40 ml) was stirred at 08C under argon. Sodium hydride
(383 mg, 9.58 mmol, 60% in mineral oil) was added and the
reaction was allowed to reach room temperature. After 2 h,
TLC (petrol/ethyl acetate, 3:1) indicated complete conver-
sion of starting material (Rf 0.7) to a major product (Rf 0.8).
Methanol (5 ml) was added portionwise and the solution
was co-evaporated with toluene in vacuo. The resulting
residue was dissolved in DCM (200 ml), washed with water
(50 ml) and brine (50 ml). The organic phase dried
(MgSO4), filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (petrol/ethyl
acetate, 4:1) to give the allyl protected thioglycoside 16
(2.7 g, 95%) as a white crystalline solid; (found C, 74.59; H,
6.55. C36H38O5S requires C, 74.20; H, 6.57%; HRMSþ
NHþ4 : 600.2784. C36H38O5S requires: 600.2793); mp
51–528C (petrol/ethyl acetate); [a]2D5¼217.3 (c, 1.0,
CHCl3); dH (400 MHz, CDCl3) 3.39 (1H, at, J¼9.5 Hz,
H-2), 3.50–3.53 (1H, m, H-5), 3.62 (1H, at, J¼9.2 Hz, H-4),
3.67 (1H, at, J¼8.6 Hz, H-3), 3.72 (1H, dd, J5,6¼4.7 Hz,
10.9 Hz, H-60), 4.26 (1H, dd, J¼6.0 Hz, Jgem¼11.9 Hz,
CHH0CHvCH2), 4.39 (1H, dd, J¼5.6 Hz, Jgem¼11.8 Hz,
CHH0CHvCH2), 4.54 (1H, d, J¼11.9 Hz, OCH2Ph), 4.59
(1H, d, J¼11.7 Hz, OCH2Ph), 4.61 (1H, d, J¼11.9 Hz,
OCH2Ph), 4.62 (1H, d, J1,2¼10.0 Hz, H-1), 4.83 (1H, d,
J¼11.7 Hz, OCH2Ph), 4.84 (1H, d, J¼10.8 Hz, OCH2Ph),
4.92 (1H, d, J¼10.8 Hz, OCH2Ph), 5.20 (1H, dd, Jgem¼1.2
Hz, JZ¼10.5 Hz, CHvCHEHZ), 5.30 (1H, dd, Jgem¼1.6 Hz,
JE¼17.2 Hz, CHvCHEHZ), 5.99 (1H, ddt, J¼5.8 Hz,
JZ¼10.5 Hz, JE¼17.1 Hz, CHvCH2), 7.20–7.60 (20H, m,
Ar-H); dC (50 MHz, CDCl3) 69.5, 71.6, 73.9, 74.7, 75.0
(5£t, C-6, 3£OCH2Ph, OCH2CHvCH2), 75.3, 78.1, 79.5,
87.2, 87.9 (5£d, C-1, C-2, C-3, C-4, C-5), 117.9 (t,
OCH2CHvCH2), 127.9, 128.0, 128.1, 128.2, 128.3,
128.4, 128.7, 128.8, 128.9, 129.4, 132.5, 135.1 (12£d,
Ar-CH, OCH2CHvCH2), 136.6, 138.5, 138.7, 138.9 (4£s,
Ar-C); m/z (APCIþ) 606 (MþNaþ, 80%), 181 (45), 132
(100), 121 (80).
4.1.6. Phenyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-seleno-b-D-
glucopyranoside 14. Acetic anhydride (6 ml, 64 mmol)
was added to a stirred solution of the alcohol 13 (1.9 g,
3.2 mmol) in pyridine (50 ml) at room temperature, under
argon. After 20 h, TLC (toluene/acetone, 9:1) indicated
complete conversion of starting material (Rf 0.6) to a major
product (Rf 0.7). The reaction mixture was concentrated in
vacuo. The resulting residue was dissolved in DCM
(150 ml), washed with 1 M HCl (100 ml), the organic
phase dried (MgSO4), filtered and concentrated in vacuo.
The resulting residue was purified by flash column
chromatography (petrol/ethyl acetate, 4:1) to give the
acetate 14 (1.8 g, 91%) as a white crystalline solid; (found
C, 66.29; H, 5.65. C35H36O6Se requires C, 66.40; H,
5.74%); mp 78–798C (ethanol); [a]2D0¼221.2 (c, 1.3,
CHCl3); nmax (KBr disc) 1740 (CH3CO2) cm21; dH
(400 MHz, CDCl3) 1.83 (3H, s, CH3CO2), 3.48–3.54 (2H,
m, H-2, H-50), 3.69 (1H, at, J¼9.6 Hz, H-4), 3.72–3.81 (2H,
m, H-6, H-6 ), 4.52 (1H, d, J¼10.5 Hz, OCH2Ph), 4.54 (2H,
s, OCH2Ph), 4.55 (1H, d, J¼11.9 Hz, OCH2Ph), 4.65 (1H, d,
J¼11.9 Hz, OCH2Ph), 4.82 (1H, d, J¼10.5 Hz, OCH2Ph),
4.90 (1H, d, J1,2¼9.8 Hz, H-1), 5.27 (1H, at, J¼9.3 Hz,
H-3), 7.18–7.71 (20H, m, Ar-H); dC (50.3 MHz, CDCl3)
20.9 (q, CH3CO2), 68.6, 73.5, 74.5, 74.6 (4£t, C-6,
3£OCH2Ph), 75.9, 77.2, 79.5, 80.0, 82.8 (5£d, C-1, C-2,
C-3, C-4, C-5), 128.0, 128.1, 128.2, 128.4, 128.7,
129.3, 134.7 (7£d, Ar-CH), 138.0, 138.1, 138.4 (3£s,
Ar-C), 170.2 (s, CH3CO2); m/z (probe CIþ) 650 (MþNH4þ,
15%), 525 (15), 324 (15), 216 (20), 181 (25), 108 (55), 91
(100).
0
0
0
J6,6 ¼10.8 Hz, H-6), 3.79 (1H, dd, J5,6 ¼1.8 Hz, J6,6
¼
4.1.7. Phenyl 3,4,6-tri-O-benzyl-1-thio-b-D-glucopyrano-
side 4. Sodium (184 mg) was dissolved in methanol (20 ml)
and the mixture was added to a stirred solution of the acetate
15 (3.5 g, 6.0 mmol) in methanol (60 ml) at 08C under
argon. The reaction was allowed to reach room temperature.
After 1.5 h, TLC (petrol/ethyl acetate, 3:1) indicated
complete conversion of starting material (Rf 0.8) to a
major product (Rf 0.7). DCM (150 ml) was added to the
reaction mixture, which was then washed with 1 M HCl
(100 ml). The organic phase was dried (MgSO4), filtered
and concentrated in vacuo. The resulting residue was
purified by recrystallization (petrol/ether) to give the
alcohol 4 (2.4 g, 75%) as a white crystalline solid; mp
72–748C; [a]2D2¼29.8 (c, 1.12, CHCl3) [lit.16 mp 71–738C;
[a]2D5¼211.5 (c, 1.4, CHCl3)]; dH (500 MHz, CDCl3) 2.41
(1H, d, JOH,2¼2.1 Hz, OH), 3.48–3.56 (2H, m, H-2, H-5),
3.58–3.64 (2H, m, H-3, H-4), 3.75 (1H, dd, J5,6¼4.5 Hz,
4.1.9. Methyl (3-O-acetyl-2,4,6-tri-O-benzyl-a-D-gluco-
pyranosyl)-(1!3)-2-O-benzyl-(R)-4,6-O-benzylidine-a-
D-mannopyranoside 17. A solution of the selenoglycoside
14 (140 mg, 0.22 mmol), the alcohol 2 (60 mg, 0.16 mmol)
˚
and 4 A molecular sieves (powdered, 400 mg) in DCE/ether
(1:1, 1.5 ml) was stirred at room temperature, under argon,
for 2 h. NIS (107 mg, 0.44 mmol) and silver triflate (5 mg,
0.02 mmol) were then added to the reaction mixture. After
5 min, TLC (petrol/ethyl acetate, 3:1) indicated complete
conversion of starting materials (Rf 0.7 and 0.3) to a major
product (Rf 0.3). Collidine (1 ml) was then added in order to
quench the reaction. Ether (150 ml) was added, the reaction
mixture was then filtered through Celitew, and the filtrate
was washed with 10% aq. sodium thiosulfite (50 ml). The
organic phase was then dried (MgSO4), filtered and
concentrated in vacuo. The resulting residue was purified
by flash column chromatography (petrol/ethyl acetate, 4:1)
to give the disaccharide 17 (117 mg, 88%, 4:1 a/b mixture)
as a colourless oil; (HRMSþNHþ4 : 864.3959. C50H54O12
requires: 864.3965); a anomer (small quantity separated by
flash column chromatography (toluene/acetone, 20:1);
0
0
J6,6 ¼11.0 Hz, H-6), 3.80 (1H, dd, J5,6 ¼1.9 Hz,
0
0
J6,6 ¼11.0 Hz, H-6 ), 4.51 (1H, d, J1,2¼9.6 Hz, H-1), 4.56
(1H, d, J¼12.1 Hz, OCH2Ph), 4.60 (1H, d, J¼9.6 Hz,
OCH2Ph), 4.62 (1H, d, J¼12.1 Hz, OCH2Ph), 4.83 (1H, d,
J¼9.6 Hz, OCH2Ph), 4.85 (1H, d, J¼10.6 Hz, OCH2Ph),
4.91 (1H, d, J¼10.6 Hz, OCH2Ph), 7.12–7.60 (20H, m,
Ar-H).

9410
S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
[a]2D0¼þ44.3 (c, 1.13, CHCl3); nmax (thin film) 1741
(CH3CO2) cm21; dH (400 MHz, CDCl3) 1.98 (3H, s,
O2CH3), 3.34 (1H, s, OMe), 3.36 (1H, dd, J1,2¼3.7 Hz,
J2,3¼10.2 Hz, H-2b), 3.58–3.89 (7H, m), 4.12 (1H, d,
J¼13.0 Hz, OCH2Ph), 4.22–4.28 (1H, m, H-5a), 4.34–4.35
(2H, m, H-3a, H-6a0 ), 4.38 (1H, d, J¼11.3 Hz, OCH2Ph),
4.43 (1H, d, J¼12.0 Hz, OCH2Ph), 4.49 (1H, d, J¼13.0 Hz,
OCH2Ph), 4.52 (1H, d, J¼11.3 Hz, OCH2Ph), 4.61 (1H, d,
J¼12.0 Hz, OCH2Ph), 4.73 (1H, d, J1,2¼1.5 Hz, H-1a), 4.87
(1H, d, J¼12.5 Hz, OCH2Ph), 4.92 (1H, d, J¼12.5 Hz,
OCH2Ph), 5.47 (1H, s, O2CHPh), 5.53 (1H, d, J1,2¼3.6 Hz,
H-1b), 5.62 (1H, at, J¼9.7 Hz, H-3b), 6.82–7.47 (25H, m,
Ar-H); dC (100.6 MHz, CDCl3) 21.1 (q, CH3CO2), 45.8 (q,
OCH3), 63.9, 70.4, 72.8, 73.1, 75.5, 75.8, 76.5, 76.9 (8£d,
C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b, C-5b), 68.2, 68.9,
69.9, 76.7, 77.0, 77.3 (6£t, C-6a, C-6b, 4£OCH2Ph), 96.6,
100.5, 102.4 (3£d, C-1a, C-1b, O2CHPh), 126.4, 127.0,
127.2, 127.6, 127.7, 127.8, 127.9, 128.0, 128.2, 128.3,
128.5, 129.3 (12£d, Ar-CH), 137.7, 137.9, 138.0 (3£s, Ar-C),
169.9 (s, CH3CO2); m/z (ESþ) 864 (MþNH4þ, 100%).
(175 mg, 0.30 mmol), the disaccharide 18 (118 mg,
˚
0.15 mmol) and 4 A molecular sieves (powdered, 200 mg)
in ether (4 ml) was stirred at room temperature, under argon.
After 30 min, methyl triflate (0.03 ml, 0.30 mmol) was
added to the reaction mixture. After 18 h, TLC (petrol/ethyl
acetate, 3:1) indicated complete conversion of starting
materials (Rf 0.8 and 0.3) to a major product (Rf 0.4).
Triethylamine (0.5 ml) was then added to the reaction and
the resulting mixture was stirred at room temperature for
30 min. The reaction mixture was then filtered through
Celitew and concentrated in vacuo. The resulting residue
was purified by flash column chromatography (petrol/ethyl
acetate, 3:1) to give the trisaccharide 19 (155 mg, 80%,
inseparable 3:1 a/b mixture) as a colourless oil; (HRMSþ
NHþ4 : 1294.6098. C78H84O16 requires: 1294.6103); (a
anomer) dH (400 MHz, CDCl3) 3.12–3.21 (3H, m), 3.34
(3H, s, OCH3), 3.43 (1H, dd, J1,2¼3.5 Hz, J2,3¼9.8 Hz,
H-2c), 3.48 (1H, dd, J1,2¼3.7 Hz, J2,3¼9.9 Hz, H-2b), 3.62–
5.05 (31H, m), 4.73 (1H, d, J1,2¼1.7 Hz, H-1a), 5.35 (1H, s,
O2CHPh), 5.53 (1H, d, J1,2¼3.6 Hz, H-1c), 5.62 (1H, d,
J1,2¼3.7 Hz, H-1b), 5.62–5.68 (1H, m, CHvCH2), 7.03–
7.48 (40H, m, Ar-H); dC (100 MHz, CDCl3) 54.8 (q, OCH3),
64.0, 69.8, 70.5, 71.9, 75.5, 76.4, 76.7, 77.9, 78.6, 79.2,
80.0, 82.4 (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), 68.9, 73.1, 73.4, 73.5, 73.6,
74.4, 77.2, 77.3 (8£t, C-6a, C-6b, C-6c, OCH2CHvCH2,
7£OCH2Ph), 96.0, 97.8, 100.8, 102.4 (4£d, C-1a, C-1b,
C-1c, O2CHPh), 117.6 (t, OCH2CHvCH2), 126.3, 126.6,
127.3, 127.4, 127.6, 127.8, 127.9, 128.0, 128.1, 128.2,
128.4, 128.6, 129.1 (13£d, Ar-CH), 134.5, 137.8, 138.0,
138.9 (4£s, Ar-C); m/z (ESþ) 1295 (MþNH4þ, 100%).
4.1.10. Methyl (2,4,6-tri-O-benzyl-a-D-glucopyranosyl)-
(1!3)-2-O-benzyl-(R)-4,6-O-benzylidine-a-D-manno-
pyranoside 18. Sodium (12 mg) was dissolved in methanol
(2 ml) and the mixture was added to a stirred solution of the
acetylated disaccharide 17 (235 mg, 0.3 mmol, a/b 4:1) in
methanol (3 ml) at 08C, under argon. The reaction was then
allowed to reach room temperature. After 4 h, TLC
(petrol/ethyl acetate, 3:1) indicated complete conversion
of starting material (Rf 0.5) to a major product (Rf 0.45).
DCM (100 ml) was then added. The organic phase was
washed with 1 M HCl (20 ml), dried (MgSO4), filtered and
concentrated in vacuo. The resulting residue was purified by
flash column chromatography (petrol/ethyl acetate, 3:1) to
give the alcohol 18 (201 mg, 83%, a/b 4:1) as a colourless
oil; (a anomer) (HRMSþNHþ4 : 822.3838. C48H52O11
requires: 822.3853); [a]2D6¼þ73.4 (c, 0.41, CHCl3); nmax
(thin film) 3492 (OH) cm21; dH (500 MHz, CDCl3) 3.32
(1H, dd, J1,2¼3.7 Hz, J2,3¼9.5 Hz, H-2b), 3.33 (3H, s,
OMe), 3.55 (1H, at, J¼9.5 Hz, H-4b), 3.67–3.79 (2H, m,
H-6b, H-6b0 ), 3.80–3.87 (4H, m, H-2a, H-5a, H-6a, H-5b),
4.13 (1H, at, J¼9.1 Hz, H-3b), 4.19 (1H, d, J¼12.0 Hz,
OCH2Ph), 4.22–4.24 (1H, m, H-6a0 ), 4.31 (1H, at, J¼9.7 Hz,
H-4a), 4.38 (1H, dd, J2,3¼3.0 Hz, J3,4¼10.0 Hz, H-3a), 4.46
(1H, d, J¼12.1 Hz, OCH2Ph), 4.51 (1H, d, J¼11.3 Hz,
OCH2Ph), 4.54 (1H, d, J¼12.0 Hz, OCH2Ph), 4.59 (1H, d,
J¼12.0 Hz, OCH2Ph), 4.70 (1H, s, H-1a), 4.75 (1H, d,
J¼12.0 Hz, OCH2Ph), 4.90 (1H, d, J¼11.3 Hz, OCH2Ph),
4.91 (1H, d, J¼11.9 Hz, OCH2Ph), 5.41 (1H, s, O2CHPh),
5.52 (1H, d, J1,2¼3.5 Hz, H-1b), 6.99–7.43 (25H, m, Ar-H);
dC (100.6 MHz, CDCl3) 55.8 (q, OMe), 63.9, 70.4, 73.0,
73.4, 77.2, 75.6, 79.5, 79.6 (8£d, C-2a, C-3a, C-4a, C-5a,
C-2b, C-3b, C-4b, C-5b), 68.7, 68.9, 70.3, 73.5, 73.8, 74.4
(5£t, C-6a, C-6b, 4£OCH2Ph), 96.4, 100.6, 102.5 (3£d,
C-1a, C-1b, O2CHPh), 126.3, 126.4, 127.4, 127.5, 127.6,
127.8, 127.9, 128.0, 128.1, 128.3, 128.5, 128.6, 129.3 (13 x
d, Ar-CH), 137.4, 137.8, 138.0, 138.1, 138.6 (5£s, Ar-C);
m/z (ESþ) 822 (MþNHþ4 , 100%).
4.1.12. 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-benzylidine-a-D-mannopyrano-
side 20. Titanium tetra-iso-propoxide (0.05 ml, 0.18 mmol)
was added to a stirred solution of allylated trisaccharide 19
(225 mg, 0.18 mmol) in THF (10 ml) at room temperature
under argon. Cyclohexylmagnesium chloride (0.4 ml,
0.81 mmol) was added to the reaction mixture over a period
of 1 h (via a syringe pump). After a further 1 h, TLC
(petrol/ethyl acetate, 3:1) indicated complete conversion of
starting material (Rf 0.4), to a major product (Rf 0.3). Water
(50 ml) was added to the reaction mixture, the product was
then extracted with ether (3£50 ml), the organic phase was
dried (MgSO4), filtered and concentrated in vacuo. The
resulting residue was purified by flash column chromato-
graphy (petrol/ethyl acetate, 3.5:1) to give the deprotected
trisaccharide 20 (189 mg, 83%, as 3:1 a/b mixture) as a
colourless oil; (HRMSþNHþ4 : 1254.5798. C75H80O16
requires: 1254.5790); [a]2D5¼þ73.1 (c, 1.1, CHCl3); nmax
(film) 3446 (OH) cm21; (a anomer) dH (400 MHz, CDCl3)
2.37 (1H, d, JOH,2¼7.1 Hz, OH), 3.13–3.23 (2H, m), 3.36
(3H, s, OCH3), 3.42 (1H, dd, J1,2¼3.7 Hz, J2,3¼9.7 Hz,
H-2b), 3.44–4.96 (30H, m), 5.36 (1H, s, O2CHPh), 5.51
(1H, d, J1,2¼2.9 Hz, H-1c), 5.61 (1H, d, J1,2¼3.7 Hz, H-1b),
7.06–7.47 (40H, m, Ar-H); (b anomer) (400 MHz, CDCl3)
5.10 (1H, d, J1,2¼10.2 Hz, H-1c); dC (125 MHz, CDCl3)
54.8 (q, OCH3), 63.8, 67.9, 68.3, 68.9, 70.1, 70.3, 70.5, 72.0,
72.8, 73.2, 73.6, 74.2, 74.3, 75.3, 76.1, 76.5, 77.5, 78.6,
79.9, 83.3 (12£CH, 10£CH2), 96.1, 98.6, 100.7, 102.4 (4£d,
C-1a, C-1b, C-1c, O2CHPh), 126.3, 126.4, 127.1, 127.2,
127.3, 127.4, 127.5, 127.7, 127.8, 127.9, 128.0, 128.1,
4.1.11. Methyl (2-O-allyl-3,4,6-tri-O-benzyl-a-D-gluco-
pyranosyl)-(1!3)-(2,4,6-tri-O-benzyl-a-D-glucopyrano-
syl)-(1!3)-2-O-benzyl-(R)-4,6-O-benzylidine-a-D-man-
nopyranoside 19. A solution of the thioglycoside 16

S. C. Ennis et al. / Tetrahedron 58 (2002) 9403–9411
9411
128.2, 128.3, 128.4, 128.5 (16£d, Ar-CH), 137.2, 137.6,
Acknowledgements
137.8, 137.9, 137.9, 138.0, 138.7, 138.8 (8£s, Ar-C); m/z
(ESþ) 1255 (MþNHþ4 , 100%).
We gratefully acknowledge the EPSRC (Quota Awards to
S. C. E. and I. C.) for financial support. We also gratefully
acknowledge the use of the EPSRC Mass Spectrometry
Service (Swansea, UK) and the Chemical Database Service
(CDS) at Daresbury, UK.
4.1.13. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(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-benzyl-(R)-4,6-O-benzylidine-a-D-mannopyrano-
side 21. A solution of the selenoglycoside 5 (129 mg,
0.19 mmol), the trisaccharide 20 (120 mg, 0.097 mmol) and
˚
4 A molecular sieves (powdered, 200 mg) in DCE/ether
References
(1:1, 2 ml) was stirred at room temperature, under argon, for
30 min. NIS (51 mg, 0.21 mmol) and silver triflate (5 mg,
0.02 mmol) was then added to the reaction mixture. After
5 min, TLC (petrol/ethyl acetate, 3:1) indicated complete
conversion of starting materials (Rf 0.8 and 0.3) to a major
product (Rf 0.4). Collidine (0.5 ml) was then added in order
to quench the reaction. DCM (50 ml) was added, the
reaction mixture filtered through Celitew and the filtrate
washed with 10% aq. sodium thiosulfite (30 ml). The
organic phase was then dried (MgSO4), filtered and
concentrated in vacuo. The resulting residue was purified
by column chromatography (petrol/ethyl) acetate, 4:1) to
give the protected tetrasaccharide 21 (127 mg, 73% as a 3:1
a/b inseparable mixture) as a colourless oil; (a anomer
selected data) (400 MHz, CDCl3) 3.32 (3H, s, OCH3), 5.15
(1H, d, J1,2¼3.6 Hz, H-1), 5.35 (1H, s, O2CHPh), 5.60 (1H,
d, J1,2¼3.8 Hz, H-1), 5.78 (1H, d, J1,2¼3.2 Hz, H-1), 7.00–
7.45 (60H, m, ArH); m/z (electrospray) Isotope distribution
MþNHþ4 : 1781.0 (18), 1779.9 (32), 1778.9 (72), 1777.9
(100), 1776.9 (98). C109H114O21 requires: 1780.8 (11),
1779.8 (29), 1778.8 (67), 1777.8 (100), 1776.8 (83%).
1. See: Roth, J. Chem. Rev. 2002, 102, 285–303, and references
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Carbohydr. Res. 1983, 123, C12–C15.
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´ ´
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4.2. Methyl (a-D-glucopyranosyl)-(1!2)-(a-D-
glucopyranosyl)-(1!3)-(a-D-glucopyranosyl)-(1!3)-a-
D-mannopyranoside 1
12. See for example: (a) Mehta, S.; Pinto, B. M. Tetrahedron Lett.
1991, 32, 4435–4438. (b) Zuurmond, H. M.; van der Klein,
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13. Alternative strategies such as the use of an orthogonal glycosyl
donor in place of 5 to allow a convergent approach, and the use
of an iterative IAD-based linear approach were also investi-
gated, and the results of these studies will be published in due
course.
Palladium acetate (20 mg) was added to a solution of the
protected tetrasaccharide 21 (40 mg, 0.022 mmol) in
ethanol (2.25 ml) and glacial acetic acid (0.25 ml). The
resulting solution was degassed and stirred at room
temperature under an atmosphere of hydrogen. After 18 h,
TLC (CMAW) indicated complete conversion of starting
material (Rf 0.9) to a major product (Rf 0.1). The reaction
mixture was filtered through Celitew and concentrated in
vacuo to give the deprotected tetrasaccharide 1 (13 mg,
86%) as a crude colourless oil; (HRMSþNHþ4 : 698.2716.
C25H44O21 requires: 698.2719); [a]2D5¼þ84 (c, 0.2, MeOH);
m/z (electrospray) 698 (MþNHþ4 , 100%). See Tables 1 and
2 for NMR data.
¨
14. Wulff, G.; Rohle, G. Angew. Chem., Int. Ed. Engl. 1974, 13,
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