Synthesis of triazole-linked pseudo-starch fragments

Article abstract of DOI:10.1016/j.carres.2006.09.026

Rapid assembly of starch fragment analogues was achieved using 'click chemistry'. Specifically, a pentadecasaccharide and two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to glucose or maltose core w

Full text of DOI:10.1016/j.carres.2006.09.026

Carbohydrate Research 342 (2007) 529–540
Synthesis of triazole-linked pseudo-starch fragments
Sergey A. Nepogodiev,* Simone Dedola, Laurence Marmuse,
Marcelo T. de Oliveira and Robert A. Field*
Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia,
Norwich NR4 7TJ, UK
Received 10 August 2006; accepted 16 September 2006
Available online 5 October 2006
Dedicated to the memory of Professor Nikolay K. Kochetkov
Abstract—Rapid assembly of starch fragment analogues was achieved using ‘click chemistry’. Specifically, a pentadecasaccharide
and two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to glucose or maltose
core were synthesised using Cu(I)-catalyzed [3+2] dipolar cycloaddition of azidosaccharides and 4,6-di-O-propargylated methyl a-D-
glucopyranoside and 6,60- and 40,60-di-O-propargylated p-methoxyphenyl b-maltoside.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Dipolar cycloaddition; Amylopectin mimics
1. Introduction
modelling9–11 and NMR spectroscopic studies12 have
been undertaken on amylopectin fragments. However,
isolation of well-characterised amylopectin fragments
incorporating branching points from natural starches
is very difficult since they represent only a small fraction
of the total polysaccharide material. Synthetic a-(1!4)-
glucans incorporating a-(1!6)-branch points have been
suggested as models of starch, which are useful tools for
physicochemical and biochemical studies. Synthesis of
oligosaccharides related to starch have been reported
in the literature13–19 but construction of challenging
1,2-cis-glucosidic linkages is known20 to be a serious
obstacle in the assembly of the large branched frag-
ments. The synthesis may be considerably simplified if
target structures are represented by pseudo-oligosaccha-
rides or oligosaccharide mimics in which certain glyco-
sidic bonds are substituted with non-glycosidic motifs.
There are several approaches to synthetic pseudo-oligo-
saccharides including the construction of C-,21 S-,22
amide,23,24 ureido and thioureido25,26 linkages between
monosaccharide residues. Recently, 1,2,3-triazole links
have emerged as a popular bridging unit in carbo-
hydrate chemistry because of the facile and efficient
method of their introduction, which is referred to as
Starch granules possess a well-organised physical struc-
ture that is characterised by repeating altered crystalline
and amorphous regions.1–3 It is believed that the semi-
crystallinity of starch is a result of the specific arrange-
ment of polysaccharide molecules of amylopectin, which
comprises 70–80% of most starches. Highly branched
amylopectin macromolecules consist of relatively short
a-(1!4)-D-glucan chains forming clusters attached
through a-(1!6)-linkages to a-(1!4)-D-glucan back-
bone. Theoretical models proposed for starch granule
structure suggest that short glucan chains within clusters
exist as double helices that are closely packed forming
crystalline lamellae, whereas regions of the granule
enriched with branch points are amorphous.4–8 The
branch points apparently play an important role in
propagation of the double helices, thus influencing the
whole granule structure. To gain insight into the struc-
tural features associated with branch points, computer
*
Corresponding authors. Tel.: +44 1603593985 (S.A.N.); e-mail
addresses: s.nepogodiev@uea.ac.uk; r.a.field@uea.ac.uk
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.09.026

530
S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
‘click chemistry.’27 The latter method is based on Cu(I)-
catalyzed version28,29 of Huisgen’s 1,3-dipolar cycload-
dition30 of azides and terminal alkynes and it has been
successfully applied for the synthesis of various glyco-
conjugates including multivalent glycosides,31–34 cyclo-
dextrin analogues,35,36 glycopeptide mimetics37,38 and
glycosidase inhibitors.39,40
OAc
O
AcO
AcO
OAc
O
AcO
O
R1
AcO
AcO
R2
R1,R2 = H, OAc
R1,R2 = PMP
1
2
a
Continuing our efforts on generating synthetic amylo-
pectin fragments,20 we describe herein the application of
‘click chemistry’ to the construction of amylopectin ana-
logues composed of two linear maltooligosaccharide
chains attached to a maltose or glucose unit through
heterocyclic bridges.41 This type of oligosaccharide
architecture is particularly interesting, given a potential
for the formation of double helices, which are assumed
to be crucial to starch granule organisation. The concept
of enforced interaction of two parallel glucan chains
through their attachment to a template was applied by
Vasella and co-workers42 in the construction of a cellu-
lose II mimic. The branching templates chosen in our
work were dipropargylated glucose and maltose deriva-
tive, whereas linear chains containing an azido group in
the reducing terminal anomeric positions comprised the
cycloaddition partner.
b
f
OR2
O
OR2
R1O
R1O
R2O
R1O
O
OR2
O
OR1
O
R1O
R1O
O
O
OPMP
OPMP
R1O
R1O
R1O
R1O
R1 = H R2 = Tr
R1 = Bn R2 = Tr
R1 = Bn R2 = H
R1 = H R2 = PhCH<
R1 = Bn R2 = PhCH<
R1 = Bn R2 = H
3
4
5
6
7
c
d
e
c
g
e
8
9
R1 = Bn R2 = CH2C CH
R1 = Bn R2 = CH2C CH
10
Scheme 1. Reagents and conditions: (a) p-methoxyphenol, CH2Cl2,
BF3ÆOEt2, 41%; (b) 1. MeOH, NaOMe, 2. TrCl, pyridine, 42%; (c)
BnBr, NaH, 94% (for 3), 92% (for 8); (d) TsOH, MeOH, CH2Cl2, 90%;
(e) CH„CCH2Br, NaH, THF, DMPU, 88% (for 6), 93% (for 10); (f)
1. NaOMe, MeOH, 2. PhCH(OMe)2, TsOH, DMF, 65%; (g) 90%
AcOH, 77%.
OAc
O
2. Results and discussion
OAc
O
OAc
O
AcO
AcO
AcO
OAc
O
AcO
AcO
Regioselectively propargylated maltose derivatives were
synthesised in the form of their p-methoxyphenyl b
maltosides, which were prepared starting from readily
available maltose peracetate 1.43 Glycosidation of 1 with
p-methoxyphenol in the presence of BF3ÆOEt2 gave an
a,b mixture of acetylated aryl glycosides from which
pure b-anomer 2 was isolated by crystallisation in 41%
yield (Scheme 1). Deacetylation of 2 followed by selec-
tive protection of primary positions with triphenylm-
ethyl groups using excess TrCl in pyridine afforded
6,60-di-O-trityl derivative 3 in 42% overall yield. For
the introduction of temporary 40,60 protecting groups,
p-methoxyphenyl maltoside was benzylidenated to pro-
duce acetal 7 in 65% yield starting from acetate 2. The
remaining hydroxy groups in 3 and 7 were benzylated
to give derivatives 4 and 8 in high yield. Acid-labile tri-
phenylmethyl and benzylidene groups were removed to
give 6,60-diol 5 and 40,60-diol 9 in 90% and 77% yield,
respectively. The reactions of dialkoxides prepared
in situ from diols 5 and 9 with propargyl bromide led
to target di-O-propargyl maltosides 6 and 10 in 88%
and 93% yield, respectively.
O
AcO
AcO
AcO
O
Br
AcO
n
AcO
Br
11
12 n = 1
13 n = 5
a
a
OAc
O
OR
O
OR
O
RO
RO
AcO
AcO
N3
AcO
RO
OR
O
RO
O
RO
O
N3
RO
n
14 n = 0 (83%)
RO
15 R = Ac n = 1 (65%)
16 R = Ac n = 5 (77%)
17 R = H n = 5 (95%)
b
Scheme 2. Reagents and conditions: (a) Me3SiN3, Bu4NF, THF; (b)
NaOMe, MeOH then NaOH, water, 20 ꢁC, 2 h.
lyzed reaction46 of 11–13 with NaN3–Bu4NHSO4 in
CH2Cl2–water mixture gave similar results.
Since Cu(I)-catalyzed cycloaddition of azides to alky-
nes was first developed,28,29 a number of different Cu
salts and a variation of conditions for this ‘click reac-
tion’ have been suggested.31,35,47–49 In the case of pro-
Peracetylated b-glycosyl azides of glucopyranose 14,44
maltotriose 15 and maltoheptaose 16 were synthesised in
65–83% yield by the reaction of the corresponding gly-
cosyl bromides 11, 1245 and 1345 with Me3SiN3 in the
presence of Bu4NF44 (Scheme 2). Phase transfer cata-
tected di-O-propargyl derivatives
acetylated glycosyl azides 14–16, we used (Ph3P)3ÆCuBr
6 and 10 and

S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
531
as a catalyst in the presence of DIPEA as a base31,32 but
instead of microwave irradiation a longer reaction time
(12 h) was applied. The cycloaddition products were
purified on silica gel where they were the slowest moving
carbohydrate components of reaction mixtures. Thus,
compounds 18, 19, 21 and 22 (Scheme 3) were obtained
in yields ranging between 65% and 27%, with yields
decreasing when increasing the length of the azidooligo-
saccharide chain.
[1,2,3]-triazole rings in 18, 19, 21 and 22 followed from
two separate triazole resonances of H-5 (dH ꢀ 7.7) in
1H NMR spectra and pairs of signals corresponding to
C-4 (dC ꢀ 121.4 and 121.8) and C-5 (dC ꢀ 145.5, 145.9)
in 13C NMR spectra. From the rest of the considerably
overlapping resonances of acetylated glucopyranose res-
idues only clusters corresponding to signals of a-ano-
meric carbons of the core maltoside unit (dC ꢀ 95–96)
and C-6 (dC ꢀ 61.5–62) carbon atoms were reliably dis-
tinguishable. Therefore, NMR data clearly indicated the
formation of a single isomer in each case, which for the
copper(I)-catalyzed cycloaddition reaction is known to
be the 1,4-substituted [1,2,3]-triazole.28 Regioselectivity
of cycloaddition in the synthesis of 6,60-di-substituted
derivatives 18–20 also followed from the observation
of only one pair of doublets of aromatic protons
(dH ꢀ 6.9 and dH ꢀ 7.0) belonging to the anomeric p-
methoxyphenyl group in the 1H NMR spectra. We
noted previously20 that the chemical shifts of these sig-
nals are highly sensitive to the stereochemistry of a
substituent at the 6 position of a p-methoxyphenyl
b-maltoside unit.
The formation of cycloaddition products was con-
firmed by MALDI-TOFMS, which clearly showed the
presence of the expected sodium adducts of molecular
ions: 1744.7 (18), 2897.1 (19), 5202.7 (21) and 5207.6
(22). The structure of these compounds was also con-
1
firmed by NMR spectroscopy. Both H and 13C NMR
spectra of 18, 19, 21 and 22 revealed very close but dis-
tinguishable resonances (dH ꢀ 5.7–5.9 and dC ꢀ 85–86)
corresponding to the anomeric centre of the glucopyra-
nose residues attached to N-1 of the triazole unit. Char-
acteristic signals of anomeric carbon atoms of the core
0
maltoside unit (dC-1 ꢀ 102 and dCꢁ1 ꢀ 97) as well as res-
onances corresponding to p-methoxyphenyl group
(dOMe ꢀ 55.5, dC ꢀ 115 and 118 for aromatics) were
observed in the 13C NMR spectra. The presence of two
It has been previously demonstrated35 that protected
pseudo-oligosaccharides incorporating [1,2,3]-triazole
6
+
14
6
+
15 or 16
a
a
RO
RO
N
N
f
c O
O
RO
RO
RO
RO
RO
N
OPMP
OBn
e
RO
RO
O
OR
a O
O
RO
O
RO
RO
N
N
c O
N
N
f
RO
d O
O
O
N
RO
OPMP
OBn
RO
RO
RO
RO
RO
n
N
b O
OBn
O
OR
OR
a O
e O
OBn
O
RO
RO
O
O
RO
N
N
d O
RO
BnO
OBn
O
N
b O
OBn
RO
n
O
OBn
OR
O
BnO OBn
18 R = Ac (63%)
19 R = Ac n = 1 (59%)
20 R = H n = 1 (51%)
21 R = Ac n = 5 (38%)
b
RO
f
O
RO
RO
RO
OPMP
e O
RO
RO
O
a O OBn
OBn
a
RO
N
N
BnO
d O
10
+
16
RO
RO
O
N
RO
5
f
O
RO
RO
RO
OR
O
O
b O
OBn
e O
RO
RO
O
RO
N
N
c O
O OBn
RO
O
N
RO
5
OR
22 R = Ac (27%)
Scheme 3. Reagents and conditions: (a) (Ph3P)3CuBr, i-Pr2NEt, toluene, 20 ꢁC, 12 h, (b) 1. H2, Pd/C, 1:1 EtOAc–EtOH, 35 ꢁC, 24 h, 2. NaOMe,
MeOH then NaOH, water, 20 ꢁC, 17 h.

532
S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
bridges can be safely deprotected using conventional
techniques. As an example, we performed a complete
deprotection of pseudo-octasaccharide 19 using catalytic
hydrogenolysis followed by two-step deacetylation, first
with NaOMe in MeOH and then NaOH in water. After
purification by gel-permeation chromatography, depro-
tected compound 20 was obtained in modest 51% yield.
The protecting groups are currently necessary during the
preparation of sugar components by cycloaddition
‘click’ reactions and are useful during the purification
of protected pseudo-oligosaccharides. However, the pro-
tected carbohydrate hydroxyl groups are not essential
for the cycloaddition reaction itself, which may be car-
ried out in aqueous media and is not affected by the
presence of alcohols. To investigate the possibility of di-
rect synthesis of unprotected pseudo-oligosaccharides,
we attempted ‘click’ reactions between unprotected
maltoheptaosyl azide 17 and methyl 4,6-di-O-propar-
gyl-a-D-glucopyranoside 28 (Scheme 4). Glycosyl azide
17 was readily available by deacetylation of compound
16 (Scheme 2). For the regioselective introduction of
propargyl groups into positions 4 and 6 of methyl gluco-
pyranoside 23, the 2-OH and 3-OH groups were first
protected as a temporary butanediacetal (BDA).50 It is
known that acetalation of 23 proceeds with the forma-
tion of an inseparable mixture of 2,3-diacetal 24 and
3,4-diacetal 25.51 In order to obtain pure 4,6-diol 24,
the mixture of products prepared by the reaction of 23
with butane-2,3-dione–CH(OMe)3 in the presence of
CSA was converted into a mixture of 6-O-trityl ethers
by reaction with Ph3CCl in pyridine and isomer 26
was isolated by chromatography in 38% overall yield.
Removal of triphenylmethyl group in 26 with MeOH–
C5H5NÆTsOH led to diol 24 in 76% yield. Alkylation
of 24 with propargyl bromide followed by hydrolysis
of diacetal 27 with 90% CF3CO2H gave target 4,6-di-
O-propargyl ether 28 in 68% yield over two steps. The
position of propargyl groups was confirmed by the
downfield shift of resonances of H-2 and H-3 in the
1H NMR spectrum of an acetylated sample of 28 (dH-2
4.85, dH-3 5.46) compared to the corresponding
resonances in the spectrum of diol 28 (dH-2 3.55,
dH-3 ꢀ 3.83).
Cycloaddition (‘click’) reaction between maltoheptao-
syl azide 17 and dialkyne 28 was carried out at 70 ꢁC in
water in the presence of cat. CuSO4 and 0.1 equiv of
sodium ascorbate.28 Without any prior workup, the
product was purified by aqueous gel-permeation
chromatography to afford 29 in 89% yield. The elution
volume of 29 on a GPC column was noticeably lower
than that of one of the precursors, 17, indicating the for-
mation of a compound with much higher molecular
mass. A peak at m/z 2648.8, present in the MALDI-
TOF mass spectrum of the ‘click’ reaction product ob-
tained from 17 and 28, corresponded to an [M+Na]+ ad-
duct of 29. In the 13C NMR spectrum of 29, intensive
signals corresponding to C-1–C-6 of a-D-glucopyranose
residues of maltoheptaosyl chains can be observed along
with weaker resonances, which can be assigned to the
core glucopyranose residue (d 54.5, OMe), C-1 of gluco-
pyranoses attached to triazole (d 86.8, OMe) and triazole
units (d 123.9 and 124.0, C-5; d 143.4 and 143.5, C-4).
To summarize, azide-alkyne 1,3-dipolar cycloaddition
of protected saccharides proceeded in moderate to good
yields. However, subsequent deprotection, which proved
technically demanding, gave only modest yields of target
pseudo-oligosaccharides. In contrast, saccharide depro-
tection prior to the key coupling ‘click’ reaction proved
facile access to the ultimate deprotected target
compound.
HO
HO
R1O
R2O
HO
OMe
OMe
O
O
a
O
O
Me
+
O
O
HO
Me
Me
O
Me
HO
HO
OMe
OMe
OMe
OMe
OMe
24 R1 = R2 = H
26 R1 = CPh3, R2 = H
23
25
b
c
d
27 R1 = R2= CH2C CH
e
O
O
O
HO
HO
OMe
28
HO
17, f
O
HO
HO
HO
HO
HO
O
HO
HO
29
O
HO
HO
N
N
N
HO
O
HO
O
N
HO
O
OH
5
HO
HO
O
O
OMe
OH
HO
O
O
N
Detailed investigation of conformational properties of
branch points of amylopectin by molecular modelling
has shown9,10 that strands of a double helix formed by
two parallel a-(1!4)-glucan chains can be connected
through an a-(1!6)-glucosidic linkage with minimal
distortion of the original polysaccharide conformation.
This suggests that the branch points might actually serve
to initiate the organisation of glucan chains into double
helices, leading to a crystalline arrangement. Clearly, the
O
HO
O OH
O
N
HO
5
OH
Scheme 4. Reagents and conditions: (a) 1,4-butanedione, (MeO)3CH,
CSA, reflux, 18 h; (b) TrCl, C5H5N, 40 ꢁC, 18 h, 38% (steps a and b);
(c) C5H5NÆHOTs, MeOH, 37 ꢁC, 72 h, 76%; (d) HCCCH2Br, NaH,
DMF; (e) 90% CF3CO2H, 20 ꢁC, 1 h, 68% (steps d and c); (f) 0.2 equiv
sodium ascorbate, 0.01 equiv CuSO4, water, 70 ꢁC, 2 h, 89%.

S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
533
4. Experimental
4.1. General methods
All solvents were dried and freshly distilled using stan-
dard procedures. The reactions were carried out at room
temperature (22–24 ꢁC) unless otherwise stated. Cation-
exchange resin (Dowex 50WX8-200 (H+) or Amberlite
IR-120 (H+) was pre-washed with water and dry MeOH
before use. Solns of reaction products were dried with
MgSO4 or Na2SO4 before the solvents were removed
by evaporation under was added slowly pressure at
25–40 ꢁC. Thin-layer chromatography (TLC) was per-
formed on aluminium-backed, pre-coated silica gel
plates (Silica Gel 60 F254, E. Merck). Spots were de-
tected by immersion in a 5% ethanolic soln of H2SO4,
followed by heating to 200 ꢁC. Column chromatography
was performed on a Biotage Horizon purification system
using pre-packed silica gel cartridges. Evaporation of
solvents was performed under was added slowly pres-
sure at 25–40 ꢁC. Reagents and dry solvents were added
via syringes through septa. FT-IR spectra were recorded
on a Perkin–Elmer 1720X spectrometer. Optical rota-
tions were measured at 18 ꢁC using a Perkin–Elmer
141 polarimeter. NMR spectra were recorded at 24 ꢁC
with a Varian Unity Plus spectrometer at 400 MHz
(1H) or 100.6 MHz (13C) and Varian Gemini 2000 spec-
trometer at 75 MHz (13C), respectively, using Me4Si (for
solns in CDCl3) or MeOH (d 49.9, for solns in D2O) as
internal standards. Resonance allocations were made
with the aid of COSY and HSQC experiments when nec-
essary. Signals corresponding to resonances of nuclei of
aromatic fragments are generally not listed because of
the complexity of the corresponding regions of NMR
spectra. High resolution electrospray ionisation mass
spectra (ESIMS) were obtained on Finnigan MAT 900
XLT mass spectrometer. For compounds with Mw
>1000 mass spectra were recorded with an Applied Bio-
systems Voyager-DE-STR (MALDI-TOF) mass spec-
trometer or with a Waters ZQ4000 (ESIMS) mass
spectrometer and experimental data were matched to
theoretical isotope patterns.
Figure 1. Molecular model of pseudo-pentadecasaccharide 29.
introduction of an unnatural branch point may lead to
more extensive disruption of the original double helix.
In order to evaluate the potential or otherwise stability
of the double helix in the ‘click’ product 29, we carried
out molecular mechanics optimisation of its structure
using OPLS-AA force field.52 An initial model of 29
was generated using crystallographic data53 for A-amy-
lose, the crystal lattice of which has a-(1!4)-linked glu-
can chains arranged in left-handed double helices.
Crystallographic coordinates were taken for two hepta-
saccharide fragments of each parallel strand of the dou-
ble helix of A-amylose and joined through the reducing
ends of these heptasaccharides to a core bis(triazole)-
glucopyranoside unit. It was found that the double
helical conformation similar to that of the A-form of
double helical amylose was preserved in an optimised
structure of pseudo-starch fragment 29. At least superfi-
cially, the bis(triazole)glycopyranoside unit appears to
be suitable as a surrogate for branch points in amylopec-
tin structure (Fig. 1).
3. Conclusion
In summary, we have described the first application of
‘click chemistry’ based on cycloaddition of substituted
azide and alkynes to the synthesis of well-defined
branched oligosaccharide mimics. Starting from diprop-
argylated maltosides and azido maltooligosaccharides,
this modular approach allowed the construction of a
number of [1,2,3]-triazole-based analogues of amylopec-
tin fragments in one simple coupling step. As synthesis
of starting glycosyl azides and propargyl ethers of malt-
ose required the presence of protecting groups, the cru-
cial cycloaddition step can also be carried out with
protected carbohydrate components. The protecting
groups can be removed after the isolation of a ‘click’
product or, alternatively, unprotected glycosyl azides
and sugar propargyl ethers can be applied. The latter
concept was demonstrated by the efficient synthesis of
pseudo-pentadecasaccharide 29. The analogues of starch
fragments incorporating triazole bridges between two
parallel maltoheptaosyl chains and branching maltose
or glucose unit have potential for templating formation
of double helices observed in crystalline regions of nat-
ural starches. Studies to investigate such assembly pro-
cesses are ongoing.
4.2. p-Methoxyphenyl 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-a-D-glucopyranosyl)-b-D-glucopyranoside
(2)
Maltose octaacetate (1) (13.0 g, 19.1 mmol) was pre-
pared by the reaction of maltose monohydrate (6.9 g,
19 mmol) with Ac2O (70 mL) in the presence of iodine
(500 mg) as described in lit.43 To a stirred soln of 1
and p-methoxyphenol (10.85 g, 87 mmol) in dry CH2Cl2
(100 mL) BF3ÆOEt2 (13.0 mL, 54.6 mmol) was added
slowly at 0 ꢁC. After stirring for 12 h at room tempera-
ture, the mixture was diluted with CH2Cl2 (150 mL),
washed successively with water (2 · 80 mL), satd aq

534
S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
NaHCO3 (2 · 80 mL), water (2 · 80 mL) and dried. The
solvent was evaporated and the residue was crystallised
from Et2O (250 mL) to give glycoside 2 (5.8 g, 41%); mp
130–132 ꢁC; [a]D +49 (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): d 2.01, 2.03, 2.04, 2.05, 2.06, 2.11
(·2) (21H, 6 s, Ac), 3.78 (4H, m, OMe, H-5a), 3.97
(1H, m, H-5b), 4.01 (2H, m, H-4b, H-6), 4.26 (2H, m,
950.4103 [M+NH4]+. Calcd for C57H56O12+NH4
950.4110.
4.4. p-Methoxyphenyl 2,3-di-O-benzyl-6-O-triphenylm-
ethyl-4-O-(2,3-di-O-benzyl-6-O-triphenylmethyl-a-D-
glucopyranosyl)-b-D-glucopyranoside (4)
0
2 · H-6), 4.49 (1H, dd, J5,6 2.9 Hz, J6;6 12.2 Hz, H-6),
Benzylation of maltoside (3) (3.01 g, 3.22 mmol) was
achieved by treatment of its soln in dry DMF (40 mL)
with 60% suspension of NaH in mineral oil (1.28 g,
32.2 mmol) followed by addition of BnBr (3.80 mL,
32.2 mmol) at 0 ꢁC and stirring the mixture at room
temperature for 17 h. After careful addition of MeOH
(10 mL) at 0–10 ꢁC and concentration, the mixture was
diluted with CH2Cl2 (200 mL), washed with water
(3 · 80 mL), dried and concentrated again. The product
was purified by chromatography (toluene/EtOAc,
10:0!9:1) to 4 as a white foam (3.21 g, 94%); [a]D
4.87 (1H, dd, J1,2 3.9 Hz, J2,3 10.4 Hz, H-2b), 4.99
(1H, d, J1,2 7.6 Hz, H-1a), 5.06 (2H, m, H-2a, H-4a),
5.32 (1H, t, J2,3 ꢂ J3,4 8.8 Hz, H-3a), 5.37 (1H, t,
J2,3 ꢂ J3,4 10.2 Hz, H-3b), 5.44 (1H, d, J1,2 3.9 Hz, H-
1b), 6.82 (2H, JAB 9.0 Hz, C6H4–OMe), 6.94 (2H, JAB
9.0 Hz, C6H4–OMe); 13C NMR (100 MHz, CDCl3): d
20.7 (·2), 20.8 (·2), 20.9, 21.0, 21.1 (·7, CH3C@O),
55.8 (OMe), 61.7, 63.0 (C-6a, C-6b), 68.2, 68.7, 69.5,
70.2, 72.2, 72.4, 72.8, 75.5 (C-2a, C-2b, C-3a, C-3b, C-
4a, C-4b, C-5a, C-5b), 95.7 (C-1a), 99.8 (C-1b), 114.7,
114.9, 116.1, 118.9 (C6H4–OMe), 150.9, 155.9 (quat. C
of C6H4–OMe), 169.7, 169.9, 170.2, 170.5, 170.6,
170.5, 170.8 (CH3C@O); HRESIMS: found m/z
760.2659 [M+NH4]+. Calcd for C33H42O19+NH4
760.2659.
1
+16.5 (c 1.05, CHCl3); H NMR (400 MHz, CDCl3): d
0
3.03 (1H, dd, J5,6 4.7 Hz, J6;6 9.7 Hz, H-6a), 3.21–3.30
(2H, m, H-5, H-60a), 3.36–3.41 (2H, m, H-3b, H-6b),
3.45–3.53 (3H, m, H-2b, H-4b, H-60b), 3.72 (3H, s,
OMe), 3.86–3.97 (3H, m, H-2a, H-3a, H-5a), 4.15–5.13
(10H, H-1a, H-4a, CH2Ph), 5.78 (1H, d, J1,2 3.5 Hz,
H-1b), 6.79 (2H, d, JAB 8.9 Hz, C6H4OMe), 6.98 (2H,
d, JAB 8.9 Hz, C6H4OMe); 13C NMR (100 MHz,
CDCl3): d 55.6 (OMe), 62.2 (C6a), 63.9 (C-6b), 70.6
(C-4a), 71.2 (C-5b), 73.1, 73.2 (2 · CH2Ph), 74.6 (C-
5a), 74.9, 75.1, 75.9 (3 · CH2Ph), 78.1, 79.7 (C-2b, C-
4b), 82.1 (C-3b), 82.3 (C-3a), 85.4 (C-3a), 86.3, 86.8
(2 · CPh3), 95.0 (C-1b), 102.9 (C-1a), 114.8, 118.6
(2 · C6H4–OMe), 151.9, 155.5 (2 · quat. C6H4–OMe);
ESIMS: m/z 1406.8 [M+Na]+.
4.3. p-Methoxyphenyl 6-O-triphenylmethyl-4-O-(6-O-tri-
phenylmethyl-a-D-glucopyranosyl)-b-D-glucopyranoside
(3)
A soln of maltoside 2 (4.00 g, 5.27 mmol) in dry MeOH
(40 mL) was treated with 0.2 M NaOMe in MeOH
(10 mL) and the mixture was stirred until the reaction
was complete (TLC). The mixture was neutralised with
Dowex 50WX8-200 (H+) resin, the resin was removed
by filtration and the filtrate was concentrated and dried.
The residue was dissolved in pyridine (30 mL), Ph3CCl
(5.80 g, 21.1 mmol) was added, the mixture was stirred
for 2 days and pyridine was removed by evaporation.
The residue was dissolved in CH2Cl2 (150 mL), succes-
sively washed with 1 M HCl (2 · 80 mL), NaHCO3
(2 · 80 mL), water (80 mL) and the soln was dried and
concentrated. The residue was purified by column chro-
matography (3:2 Me2CO–CH2Cl2) to afford compound
3 (3.10 g, 42%) as amorphous white solid; [a]D +21.5
4.5. p-Methoxyphenyl 2,3-di-O-benzyl-4-O-(2,3,4-tri-O-
benzyl-a-D-glucopyranosyl)-b-D-glucopyranoside (5)
Detritylation of 4 (2.55 g, 1.84 mmol) was carried out in
the presence of TsOH (2.0 g) in 1:1 MeOH–CHCl3
(40 mL) for 5 h. Then the mixture was neutralised
with a satd aq NaHCO3 soln, concentrated to a half
of the volume, diluted in CH2Cl2 (100 mL), washed with
water (2 · 40 mL) and the organic layer was dried. The
product was purified by chromatography (25:75!3:7
toluene–EtOAc) to give diol 5 as a white amorphous so-
1
(c 1.05, CHCl3); H NMR (400 MHz, CD3OD): d 2.86
0
0
(1H, dd, J5,6 4 Hz, J6;6 10.2 Hz, H-6), 2.97 (1H, dd,
1
0
J5,6 8.4 Hz, J6;6 , H-6), 3.21–3.30 (3H, m, H-6, H-5a,
lid (1.49 g, 90%); [a]D +27 (c 1.16, CHCl3); H NMR
H-5b), 3.43–3.71 (13H, m, H-2, H-3, H-4, H-5, H-6,
OMe), 4.80 (1H, d, J1,2 7.8 Hz, H-1a), 4.80 (1H, d, J1,2
2.9 Hz, H-1b), 6.83 (2H, d, JAB 9.0 Hz, C6H4OMe),
7.06 (2H, d, JAB 9.0 Hz, C6H4OMe); 13C NMR
(400 MHz, CDCl3): d 3.39–3.49 (2H, m, H-2b, H-4b),
3.57–3.65 (2H, m, H-6, H-5a), 3.69–3.96 (5H, m, H-2a,
H-5b, OMe), 3.86–3.96 (5H, m, 3 · H-6, H-4a, H-3b),
4.15 (1H, t, J3,4 ꢂ J4,5 8.8 Hz, H-4a), 4.49–5.02 (11H,
m, H-1a, CH2Ph), 5.75 (1H, d, J1,2 3.8 Hz, H-1b), 6.84
(2H, d, JAB 9.6 Hz, C6H4OMe), 6.98 (2H, d, JAB
9.6 Hz, C6H4OMe); 13C NMR (100 MHz, CDCl3): d
55.9 (OMe), 61.6, 62.3 (C-6a, C-6b), 71.5 (C-3a), 72.6
(C-2a), 73.7, 74.3 (CH2Ph), 75.0 (C-5a), 75.2, 75.5,
(100 MHz, CD3OD):
d
54.9 (OMe), 62.3, 63.8
(C-6a, C-6b), 69.9, 72.2, 72.8, 73.4, 73.9, 74.4, 77.4,
79.3, (C-2–C-5), 86.2, 86.8 (CPh3), 100.9 (C-1b), 102.1
(C-1a), 114.3, 118.3 (2 · C6H4–OMe), 152.0, 155.4
(2 · quat. C6H4–OMe); HRESIMS: found m/z

S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
535
75.8 (3 · CH2Ph), 78.4, 79.5 (C-2b, C-4b), 82.1, 82.3 (C-
3b, C-5b), 84.9 (C-4a), 97.1 (C-1b), 102.8 (C-1a), 115.0,
118.5 (2 · C6H4–OMe), 150.9, 155.9 (2 · quat. C6H4–
OMe); HRESIMS: found m/z 916.4269 [M+NH4]+.
Calcd for C54H58O12+NH4 916.4269.
H-6, OMe), 4.79 (1H, d, J1,2 7.7 Hz, H-1a), 4.86 (1H,
s, CHPh), 5.22 (1H, d, J1,2 3.7 Hz, H-1b), 6.83 (2H, d,
JAB 9.2 Hz, C6H4OMe), 7.05 (2H, d, JAB 9.2 Hz,
C6H4–OMe), 6.71 (2H, d, JAB 9.0 Hz, C6H4OMe), 6.96
(2H, d, JAB 9.0 Hz, C6H4–OMe); 13C NMR (75 MHz,
CDCl3): d 55.7 (OMe), 61.2, 63.8 (C-6a, C-6b), 68.7,
7.90, 73.1, 73.3, 74.8, 76.0, 79.8, 80.7 (C-2–C-5), 101.8,
102.0 (C-1a, C-1b), 102.2 (CHPh), 115.6, 119.4
(2 · C6H4–OMe), 153.3, 158.95 (2 · quat. C6H4OMe);
HRESIMS: found m/z 554.2230 [M+NH4]+. Calcd for
C26H32O12+NH4 554.2232.
4.6. p-Methoxyphenyl 2,3-di-O-benzyl-6-O-(prop-2-ynyl)-
4-O-(2,3,4-tri-O-benzyl-6-O-(prop-2-ynyl)-a-D-glucopyr-
anosyl)-b-D-glucopyranoside (6)
A mixture of maltoside 5 (500 mg, 0.556 mmol) and
propargyl bromide (2.22 mmol, 200 lL) was dried by
evaporation of its toluene soln (three times) and then
dissolved in 3:1 THF–DMPU (5 mL). The soln was
cooled to 0 ꢁC and 60% suspension of NaH in mineral
oil (90 mg, 2.22 mmol) was added slowly under nitrogen.
The reaction mixture was stirred at room temperature
for 6 h, carefully treated with water (10 mL) and diluted
with EtOAc (100 mL). The organic phase was washed
with brine (3 · 30 mL), water (2 · 30 mL) and dried
(Na2SO4). The product was purified by chromatography
(4:1 hexane–EtOAc) affording 6 (478 mg, 88%) as a syr-
up; [a]D +23 (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 2.30–2.37 (2H, m, CH2C„CH), 3.52 (1H,
dd, J1,2 3.6 Hz, J2,3 9.8 Hz, H-2b), 3.62–3.95 (13H, m,
OMe, H-2a, H-3a, H-3b, H-4b, H-5a, H-5b, 4 · H-6),
4.10 (1H, t, J3,4 ꢂ J4,5 9.5 Hz, H-4a), 4.11–4.26 (4H,
m, CH2C„CH), 4.52 (1H, d, J 11.6 Hz, CH2Ph), 4.60
(1H, d, J 11.6 Hz, CH2Ph), 4.69 (2H, d, J 11.5 Hz,
CH2Ph), 4.76–5.01 (7H, m, H-1b, CH2Ph), 5.69 (1H,
d, J1,2 3.6 Hz, H-1b), 6.84 (2H, d, JAB 9.2 Hz,
C6H4OMe), 7.02 (2H, d, JAB 9.2 Hz, C6H4OMe); 13C
NMR (75 MHz, CDCl3): d 55.6 (OMe), 58.5, 58.8
(CH2C„CH), 68.0, 68.5 (2 · C-6), 70.8, 72.3, 73.3,
73.9, 74.3, 74.7, 74.8 (·2), 74.9, 75.0, 75.4, 79.2, 79.3,
79.5, 82.0 (·2), 84.6 (C-2a, C-2b, C-3a, C-3b, C-4a, C-
4b, C-5a, C-5b, 5 · CH2Ph, 2 · CH2C„CH), 96.9 (C-
1b), 102.7 (C-1a), 114.6, 118.5 (2 · C6H4–OMe), 151.5,
155.5 (2 · quat. C6H4–OMe); HRESIMS: found m/z
992.4583 [M+NH4]+. Calcd for C60H62O12+NH4
992.4580.
4.8. p-Methoxyphenyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
benzyl-4,6-O-benzylidene-a-D-glucopyranosyl)-b-D-gluco-
pyranoside (8)
To a soln of maltoside 7 (334 mg, 0.62 mmol) in dry
DMF (5 mL), a 60% suspension of NaH in mineral oil
(186 mg, 4.65 mmol) was added slowly followed by the
addition of BnBr (550 lL, 4.65 mmol) at 0 ꢁC. The mix-
ture was then stirred for 12 h at room temperature, care-
fully treated with MeOH (1 mL) and concentrated. The
residue was mixed with CH2Cl2 (100 mL), the organic
soln was washed with water (3 · 30 mL), dried and con-
centrated again. The product was purified by chroma-
tography (0:10!4:1 EtOAc–hexane) to give 8 (560 mg,
92%) as an amorphous solid; [a]D +3.8 (c 1.1, CHCl3);
1H NMR (400 MHz, CDCl3): d 3.60 (1H, dd, J1,2
3.8 Hz, J2,3 9.4 Hz, H-2b), 3.66–3.98 (10H, m, OMe,
H-3a, H-4b, H-5a, H-5b, 3 · H-6), 4.08 (1H, t,
J2,3 ꢂ J3,4 9.4 Hz, H-3b), 4.25 (2H, m, H-4a, H-6),
4.62–5.12 (11H, m, H-1a, CH2Ph), 5.62 (1H, s, CHPh),
5.80 (1H, d, J1,2, H-1b), 6.89 (2H, d, JAB 9.1 Hz,
C6H4OMe), 7.13 (2H, d, JAB 9.1 Hz, C6H4–OMe); 13C
NMR (75 MHz, CDCl3): d 55.6 (OMe), 63.3, 68.8,
68.9, 72.1, 74.0, 74.5, 78.7 (·2), 82.2, 82.3, 84.9 (C-2–
C-6), 73.5 (·2), 73.8, 74.9, 75.3, (5 · CH2–Ph), 97.5 (C-
1b), 101.2 (C-1a), 102.9 (CH–Ph), 114.7, 118.8
(2 · C6H4–OMe), 151.6, 155.65 (2 · quat. C6H4OMe);
HRESIMS: found m/z 1004.4588 [M+NH4]+. Calcd
for C61H64O12+NH4 1004.4580.
4.7. p-Methoxyphenyl 4-O-(4,6-O-benzylidene-a-D-
glucopyranosyl)-b-D-glucopyranoside (7)
4.9. p-Methoxyphenyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
benzyl-a-D-glucopyranosyl)-b-D-glucopyranoside (9)
Maltoside 2 (2.94 g, 3.88 mmol) was deacetylated with
0.04 M NaOMe in MeOH (38 mL) as described in the
preparation of 3. Deprotected maltoside was dissolved
in DMF (20 mL) and PhCH(OMe)2 (1.16 mL,
7.76 mmol) and TsOHÆH2O (50 mg) were added. The
mixture was stirred for 12 h, neutralised with Et3N
and concentrated. The resulting residue was purified
by column chromatography (7:3 Me2CO–CH2Cl2) to
give 7 (1.30 g, 65%); [a]D +27 (c 0.9, MeOH); 1H
NMR (400 MHz, CD3OD): d 3.91–4.22 (15H, m, H-2–
A soln of the benzylidene derivative 8 (548 mg,
0.55 mmol) in 1:10 AcOH–water (11 mL) was heated
at 80 ꢁC for 4 h. After concentration, the residue was
purified by chromatography (4:1 hexane–EtOAc) afford-
ing 9 (381 mg, 77%) as a syrup; [a]D +16 (c 0.9, CHCl3);
1H NMR (400 MHz, CDCl3): d 3.40 (1H, dd, J1,2
3.6 Hz, J2,3 9.5 Hz, H-2b), 3.50 (1H, t, J3,4 ꢂ J4,5
8.8 Hz, H-4b), 3.64–3.87 (10H, m, H-3b, H-5, H-6,
OMe), 3.86 (1H, t, J2,3 ꢂ J3,4 8.8 Hz, H-3a), 4.12 (1H,
t, J3,4 ꢂ J4,5 8.9 Hz, H-4a), 4.53–5.04 (11H, m, H-1a,

536
S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
CH2Ph), 5.66 (1H, d, J1,2, H-1b), 6.81 (2H, d, JAB
8.8 Hz, C6H4OMe), 7.20 (2H, d, JAB 8.8 Hz,
C6H4OMe); 13C NMR (75 MHz, CDCl3): d 55.5
(OMe), 62.2, 70.3, 71.9, 72.3, 73.0 (·2), 73.4, 73.9, 74.7
(·2), 75.1, 79.0, 81.0, 81.9, 84.7 (C-2–C-6, 5 · CH2Ph),
96.5 (C-1b), 102.7 (C-1a), 114.6, 118.6 (2 · C6H4–
OMe), 151.5, 155.5 (2 · quat. C6H4OMe); HRESIMS:
found m/z 916.4266 [M+NH4]+. Calcd for C54H58O12+
NH4 916.4267.
Na2SO4 and concentrated. The crude product was puri-
fied by chromatography (3:2!1:1 hexane–EtOAc)
affording azide 15 (630 mg, 65%) as a white amorphous
1
solid; mmax 2119 cmꢁ1 (N3); [a]D +65 (c 1.2, CHCl3); H
NMR (400 MHz, CDCl3): d 2.00 (·2), 2.01, 2.02, 2.04
(·2), 2.06 (·2), 2.11, 2.17 (10 · Ac), 3.83 (1H, m, H-5),
3.92–4.07 (5H, m, H-4a, H-4b, H-5b, H-5c, H-6),
4.17–4.53 (5H, m, 5 · H-6), 4.71–4.80 (3H, m, H-1a,
H-2a, H-2b), 4.86 (1H, dd, J1,2 4.0 Hz, J2,3 10.6 Hz,
H-2c), 5.08 (1H, t, J3,4 ꢂ J4,5 10.1 Hz, H-4c), 5.24–5.30
(2H, m, H-1b, H-1c, H-3c), 5.33–5.43 (3H, m, H-1b,
H-3b, H-3c); 13C NMR (75 MHz, CDCl3): d 20.2–20.5
(CH3C@O), 61.15, 62.1, 62.5 (3 · C-6), 67.6, 68.3,
69.8, 70.2, 71.2, 71.4, 72.3, 73.3, 73.9, 74.6 (C-2–C-5),
87.1 (C-1a), 95.5, 95.7 (C-1b, C-1c), 169.3–170.5
(CH3C@O); HRESIMS: found m/z 972.2704
[M+Na]+. Calcd for C38H51N3O25+Na 972.2693.
4.10. p-Methoxyphenyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
benzyl-4,6-di-O-(prop-2-ynyl)-a-D-glucopyranosyl)-b-D-
glucopyranoside (10)
Diol 8 (368 mg, 0.410 mmol) and propargyl bromide
(1.64 mmol, 146 lL) were dried by concentration (three
times) of their soln in dry toluene, then dissolved in 3:1
THF-DMPU (4 mL) and the soln was cooled to 0 ꢁC. A
60% suspension of NaH in mineral oil (66 mg, 1.64) was
added slowly to the mixture which was stirred for 6 h at
room temperature. After careful addition of water
(10 mL), the mixture was diluted with EtOAc
(100 mL) and the organic phase was washed with brine
(3 · 30 mL), water (2 · 30 mL) and dried with Na2SO4.
The crude product was purified by chromatography
(9:1!4:1 hexane–EtOAc) affording 10 (370 mg, 93%)
as a gum; [a]D +25 (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): d 2.41–2.45 (2H, m, 2 · CH2C„
CH), 3.43 (1H, dd, J1,2 3.6 Hz, J2,3 9.8 Hz, H-2b),
3.46–3.53 (2H, m, H-4b, H-6), 3.67–3.89 (11H, m,
H-2a, H-3b, H-4a, H-5a, H-5b, 3 · H-6, OMe), 4.02
(1H, dd, J 2.2 Hz, J 16 Hz, CH2C„CH), 4.10 (1H, t,
J2,3 8.6 Hz, H-4a), 4.18 (1H, dd, J 2.2 Hz, J 16 Hz,
CH2C„CH), 4.38 (2H, m, CH2C„CH), 4.74–5.02
(11H, m, H-1a, CH2Ph), 5.66 (1H, d, J1,2 3.6 Hz,
H-1b), 6.80 (2H, d, JAB 9.0 Hz, C6H4OMe), 7.04 (2H,
d, JAB 9.0 Hz, C6H4OMe); 13C NMR (75 MHz, CDCl3):
d 55.5 (OMe), 58.4 (CH2C„CH), 60.0 (CH2C„CH),
67.7 (C-6), 68.9 (C-6), 70.4, 72.7, 73.2, 73.4, 73.9, 74.1
(·2), 74.7, 74.8, 75.4, 77.2, 78.9, 79.3, 80.0, 81.5, 82.0,
84.6 (C-2–C5, 5 · CH2–Ph, 2 · CH2C„CH), 96.8 (C-
1a), 102.7 (C-1b), 114.6, 118.5 (2 · C6H4–OMe), 151.5,
155.4 (2 · quat. C6H4OMe); HRESIMS: found m/z
992.4585 [M+NH4]+. Calcd for C60H66O12+NH4
992.4585.
4.12. 2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl-{(1!4)-
2,3,6-tri-O-acetyl-a-D-glucopyranosyl}5-(1!4)-2,3,6-tri-
O-acetyl-b-D-glucopyranosyl azide (16)
Peracetylated maltoheptaose 1345 (1.08 g, 0.505 mmol)
was dissolved in dry CH2Cl2 (6 mL), the soln was cooled
with an ice bath and 33% HBr in AcOH (2 mL) was
added. After 2 h at room temperature the soln was
diluted with CH2Cl2 (100 mL) and washed successively
with ice-water (2 · 30 mL), cold satd aq NaHCO3
(3 · 30 mL) and water (30 mL). The organic phase was
separated, concentrated and crystallised (Et2O–hexane)
affording a-maltoheptaosyl bromide 13 (1.00 g, 96%);
1H NMR (400 MHz, CDCl3): d 3.86–4.28 (22H, m),
4.43–4.60 (6H, m), 4.65–4.75 (5H, m), 4.83 (1H, J
3.8 Hz, J 10.2 Hz), 5.05 (1H, t, J 9.8 Hz), 5.24–5.42
(10H, m, H-1b-g), 5.58 (1H, t, J 9.5 Hz), 6.48 (1H, d,
J1,2 3.8 Hz, H-1a); 13C NMR (75 MHz, CDCl3): d 86.0
(C-1a), 95.6, 95.7 (5 · C-1); MALDI-TOFMS m/z
found: 2163.6 [M+Na]+. Calcd for C86H115O57Br+Na
2163.5. To a soln of 13 (1.00 g, 0.46 mmol), Bu4NHSO4
(340 mg, 0.50 mmol) and NaN3 (260 mg, 2.00 mmol) in
CH2Cl2 (10.0 mL) was added satd aq NaHCO3
(10.0 mL). The mixture was stirred for 2 h and diluted
with EtOAc (100 mL). The organic phase was separated,
washed successively with satd aq NaHCO3 (40 mL) and
water (2 · 40 mL), dried and concentrated. After purifi-
cation by chromatography (85:15!75:25 toluene–
Me2CO), the crude product was crystallised from EtOH
affording azide 16 (740 mg, 77%); mp 127–130 ꢁC; mmax
2119 cmꢁ1 (N3); [a]D +159 (c 1.0, CHCl3); 13C NMR
(75 MHz, CDCl3): d 20.3, 20.6 (CH3C@O), 61.3, 62.1,
62.3, 62.4, 62.5 (C-6), 67.9, 68.3, 68.8, 68.9, 69.0, 69.2,
69.9, 70.3, 70.4, 71.4, 71.5, 72.4, 73.3, 73.4, 73.5, 74.1,
74.8 (C-2–C-5), 87.3 (C-1a), 95.5, 95.6 (C-1), 169.4–
170.7 (CH3C@O). MALDI-TOFMS: m/z 2124.5
[M+Na]+.
4.11. (2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl)-
(1!4)-(2,3,6-tri-O-acetyl-a-D-glucopyranosyl)-(1!4)-
2,3,6-tri-O-acetyl-b-D-glucopyranosyl azide (15)
To a soln of bromide45 2 (1 g, 1.01 mmol) and Me3SiN3
(174 lL, 1.31 mmol) in dry THF (10 mL), a 1 M soln of
TBAF in THF (1.31 mL) previously dried over activated
˚
4 A molecular sieves was added. The soln was stirred for
4 h, filtered through a plug of silica gel, dried over

S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
537
4.13. a-D-Glucopyranosyl-{(1!4)-a-D-glucopyranosyl}5-
(1!4)-b-D-glucopyranosyl azide (17)
155.4 (C6H4–OMe); MALDI-TOFMS: m/z 1744.7
[M+Na]+.
To a soln of acetate 16 in THF/MeOH (4:1, 5 mL),
0.1 M NaOMe in MeOH (0.5 mL) was added and
the mixture was stirred at room temperature. After
10 min a white precipitate formed, the mixture was
stirred for 1 h and concentrated to dryness. The resi-
due was dissolved in water (5 mL), stirred for 2 h
and neutralised with Amberlite IRA-120 (H+). The re-
sin was filtered off and the filtrate was freeze-dried to
give 17 (95%) as a white powder, [a]D +160 (c 1.0,
H2O); 13C NMR (75 MHz, D2O, signal intensity is
not indicated): d 59.9 (C-6), 68.7, 70.6, 70.9, 71.0,
72.1, 72.3, 75.6, 75.8, 75.9, 76.2, 76.3 (C-2–C-5), 89.4
(C-1x), 99.0–99.2 (C-1); MALDI-TOFMS: m/z
1200.5 [M+Na]+.
4.16. p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-a-D-
glucopyranosyl)-(1!4)-(2,3,6-tri-O-acetyl-a-D-glucopyr-
anosyl)-(1!4)-(1-(2,3,6-tri-O-acetyl-a-D-glucopyrano-
syl)-1H-[1,2,3]triazol-4-ylmethyl)-(4!6)-(2,3,4-tri-O-
benzyl-a-D-glucopyranosyl)-(1!4)-[(2,3,4,6-tetra-O-ace-
tyl-a-D-glucopyranosyl)-(1!4)-(2,3,6-tri-O-acetyl-a-D-
glucopyranosyl)-(1!4)-(1-(2,3,6-tri-O-acetyl-a-D-gluco-
pyranosyl)-1H-[1,2,3]triazol-4-ylmethyl)-(4!6)]-2,3-di-
O-benzyl-b-D-glucopyranoside (19)
This was synthesised following the general procedure
using p-methoxyphenyl maltoside 6 (111 mg, 0.11 mmol)
and maltotriosyl azide 15 (235 mg, 0.31 mmol). The res-
idue was purified by chromatography (7:3 EtOAc–hex-
ane) to give 19 (207 mg, 65%) as a white solid; [a]D
+61 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
1.77–2.16 (60H, s, CH3–C@O), 3.50 (1H, dd, J1,2
3.6 Hz, J2,3 9.8 Hz, H-2b), 4.93 (1H, d, J1,2 9.0 Hz, H-
1a), 5.30–5.48 (8H, m, 4 · H-1e,f, H-2c, H-2d, H-3c,
H-3d), 5.61 (1H, d, J1,2 3.6 Hz, H-1b), 5.75 (1H, d, J1,2
8.8 Hz, H-1c or H-1d), 5.85 (1H, d, J1,2 9.1 Hz, H-1d
or H-1c), 6.87 (2H, JAB 9.1 Hz, C6H4OMe), 7.02 (2H,
JAB 9 Hz, C6H4OMe), 7.69 (1H, s, triazole), 7.72 (1H,
s, triazole); 13C NMR (75 MHz, CDCl3): d 55.6
(OMe), 85.0, 85.1 (C-1c, C-1d), 95.7 (·2), 96.1 (·2) (C-
1e–C-1h), 97.1 (C-1b), 102.3 (C-1a), 114.7, 118.2
(C6H4–OMe), 121.4, 121.8 (C-5 triazole), 145.5, 145.9
(C-4 triazole), 151.4, 155.4 (C6H4–OMe); MALDI-
TOFMS: m/z 2897.1 [M+Na]+.
4.14. General procedure for the synthesis of 18, 19, 21 and
22 by 1,3-dipolar cycloaddition using protected
carbohydrates
A mixture of a di-O-propargylated maltoside (1.0
equiv), a glycosyl azide (2.2 equiv), i-Pr2NEt (3.0 equiv)
and (Ph3P)3CuBr (0.11 equiv) in dry toluene
(5.0 mL) was stirred for 12 h. The mixture was
concentrated and the product was purified by
chromatography.
4.15. p-Methoxyphenyl (1-(2,3,4,6-tetra-O-acetyl-a-D-
glucopyranosyl)-1H-[1,2,3]triazol-4-ylmethyl)-(4!6)-
(2,3,4-tri-O-benzyl-a-D-glucopyranosyl)-(1!4)-[(1-
(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-1H-
[1,2,3]triazol-4-ylmethyl)-(4!6)]-2,3-di-O-benzyl-b-D-
glucopyranoside (18)
4.17. Deprotection of 19
Compound 19 (90 mg, 0.031 mmol) was dissolved in 1:1
EtOAc–EtOH (12 mL) and hydrogenated in the pres-
ence of cat. 10% Pd/C for 24 h at 35 ꢁC. The catalyst
was removed by filtration, the filtrate was concentrated
and the residue was dissolved in CH2Cl2/MeOH mixture
(1:4, 5 mL). NaOMe (1 M) in MeOH (0.5 mL) was
added to the mixture, which was then stirred for 2 h
and concentrated. The residue was dissolved in water
(5 mL) and the soln was stirred for 17 h. The soln was
diluted with water, neutralised with Amberlite IRA-
120 (H+), the resin was filtered off, washed with water
and combined aqueous filtrates were concentrated. The
product was purified by gel chromatography (TSK gel
Toyopearl HW 40S, 1.5 · 85 cm) in 0.1% CF3CO2H.
The target product was collected in fractions eluted with
85–110 mL with a peak maximum at 100 mL; maltotri-
ose was eluted with a peak maximum at 111 mL. Depro-
tected compound 20 (25 mg, 51%), ESIMS: m/z 1583.1
[M+H]+.
This was synthesised from 6 (138 mg, 0.14 mmol) and 14
(116 mg, 0.31 mmol) and purified by chromatography
(6:4 EtOAc–hexane) to give 8 (153 mg, 63%) as a white
foam; [a]D ꢁ5.4 (c 1.06, CHCl3); H NMR (400 MHz,
1
CDCl3): d 1.79, 1.62, 1.95, 2.02 (·2), 2.03, 2.07 (24H,
s, Ac), 3.50 (1H, dd, J1,2 3.5 Hz, J2,3 9.7, H-2b), 3.59
(1H, t, J3,4 ꢂ J4,5 9.5 Hz, H-4b), 3.64–4.32 (16H, m, H-
3a, H-3b, H-4a, 4 · H-5, 8 · H-6, OMe), 4.50–5.00
(15H, m, H-1a, 10 · CH2Ph, 2 · CH2 triazole), 5.27–
5.58 (7H, m, H-1b, H-2c, H-2d, H-3c, H-3d, H-4c, H-
4d), 5.87 (1H, d, J1,2 3.3 Hz, H-1c or H-1d), 5.91 (1H,
d, J1,2 3.1 Hz, H-1d or H-1c), 6.85 (2H, JAB 9.0 Hz,
C6H4OMe), 7.00 (2H, JAB 9.0 Hz, C6H4OMe), 7.7
(1H, s, triazole), 8.02 (1H, s, triazole), dC (75 MHz,
CDCl3): d 19.9, 20.4, 20.45 (8 · CH3C@O), 55.5
(OMe), 60.5 (·2, C-6c, C-6d), 63.9, 64.0 (CH2 triazole),
68.4 (·2, C-6a, C-6b), 85.5, 85.7 (C-1c, C-1d), 97.6
(C-1b), 102.2 (C-1a), 113.8, 117.3 (C6H4–OMe), 120.8
(C-5 triazole), 144.8, 144.9 (C-4 triazole), 151.4,

538
S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
4.18. p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-a-D-
glucopyranosyl)-(1!4)-{2,3,6-tri-O-acetyl-a-D-
glucopyranosyl)-(1!4)}5-(2,3,6-tri-O-acetyl-a-D-
glucopyranosyl-1H-[1,2,3]triazol-4-ylmethyl)-(4!6)-
(2,3,4-tri-O-benzyl-a-D-glucopyranosyl)-(1!4)-
[(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-(1!4)-
{(2,3,6-tri-O-acetyl-a-D-glucopyranosyl)-(1!4)}5-
(2,3,6-tri-O-acetyl-a-D-glucopyranosyl-1H-[1,2,3]triazol-
4-ylmethyl)-(4!6)]-2,3-di-O-benzyl-b-D-glucopyranoside
(21)
4.20. Methyl [(20R,30R)-2,3-dimethoxybutane-20,30-diyl]-
6-O-triphenylmethyl-a-D-glucopyranoside (26)
Camphorsulfonic acid (230 mg, 1.00 mmol) was added
to a soln of 23 (2.00 g, 10.31 mmol), (MeO)3CH
(12.4 mL, 11.4 mmol) and 1,4-butanedione (2.0 mL,
23 mmol) in dry MeOH (40 mL). After refluxing for
18 h, the mixture was neutralised by the addition of
Et3N and concentrated to give a dark brown syrup.
The syrup was dissolved in 2:1 CH2Cl2–hexane
(200 mL) and the products were extracted with water
(6 · 50 mL). The combined aqueous extracts were con-
centrated and the residual water was removed by re-
peated evaporation of the solvent from a toluene soln
of the products. After drying the residue, a crude mix-
ture of 24 and 25 (3.1 g, 97%) was obtained as a yellow
syrup. 13C NMR (75 MHz, CDCl3): d 17.4, 17.5
(4 · acetal CMe), 47.6, 47.7 (·2), 47.8 (4 · acetal
OMe), 54.9, 55.1 (2 · anomeric OMe), 60.8, 61.6
(2 · C-6), 65.8, 67.6, 68.1, 69.3, 69.7 (·2), 69.9, 71.8
(C-2–C-5), 98.0, 99.4, 99.5, 99.6 (·2), 99.8 (2 · C-1,
4 · acetal Cquat.). The crude mixture was dissolved in
pyridine, Ph3CCl (2.79 g, 10.0 mmol) was added and
the mixture was stirred for 18 h at 40 ꢁC. CH2Cl2
(150 mL) was added, the mixture was washed with water
(50 mL), dried and concentrated. The residue was puri-
fied by chromatography (pet. ether/EtOAc, 8:2!1:1) to
afford trityl ether 26 (2.15 g, 38%), which was eluted
first. Compound 26, [a]D ꢁ48 (c 1.8, CHCl3); d 1H
NMR (400 MHz, CDCl3): d 1.33 (3H, s, acetal CMe),
1.36 (3H, s, acetal CMe), 3.28, 3.29 (6H, 2 · s, acetal
OMe), 3.39 (2H, m, H-6, H-60), 3.42 (3H, s, anomeric
OMe), 3.70–3.82 (3H, H-2, H-4, H-5), 4.20 (1H, t,
J2,3 ꢂ J3,4 9.0 Hz, H-3), 4.67 (1H, d, J 3.5 Hz, H-1),
7.22–7.33 (9H, m, Ph), 7.46–7.48 (6H, m, Ph); 13C
NMR (75 MHz, CDCl3): d 17.7, 17.6 (2 · acetal CMe),
47.7, 47.9 (2 · acetal OMe), 54.8 (anomeric OMe), 63.8
(C-6), 68.1, 69.2, 69.6, 70.5 (C-2–C-5), 87.0 (CPh3),
97.9 (C-1), 99.4, 99.9 (acetal Cquat.), 127.1, 127.9,
128.7, 143.8 (Ph); HRESIMS: found m/z 573.2465.
Calcd for C32H38O8+Na 573.2459. Acetylation of a
small sample of 26 gave its 4-acetate, 1H NMR
(400 MHz, CDCl3): d 1.25 (3H, s, acetal CMe), 1.34
(3H, s, acetal CMe), 1.73 (Ac), 3.08–3.18 (2H, m, H-6,
H-60), 3.20, (3H, s, acetal OMe), 3.26 (3H, s, acetal
OMe), 3.52 (3H, s, anomeric OMe), 3.86 (1H, dd, J1,2
3.5, J2,3 10.2 Hz, H-2), 4.08 (1H, t, J2,3 ꢂ J3,4 10.2 Hz,
H-3), 4.67 (1H, d, H-1), 4.99 (1H, t, J3,4 ꢂ J4,5
10.2 Hz, H-4), 7.14–7.33 (9H, m, Ph), 7.40–7.48 (6H,
m, Ph).
This was synthesised following the general procedure
from
6
(60 mg, 0.061 mmol) and 16 (280 mg,
0.13 mmol). The product was purified by chromatogra-
phy (6:3!9:1 EtOAc–hexane) to give the title com-
pound (121 mg, 38%) as a white foam; [a]D +108 (c
1
1.1, CHCl3); H NMR (400 MHz, CDCl3): d 5.62 (1H,
d, J1,2 3.6 Hz, H1-b), 5.73 (1H, d, J1,2 8.8 Hz, H1-d),
5.84 (1H, d, J1,2 9.2 Hz, H1-c), 6.88 (2H, d, JAB
9.2 Hz, C6H4OMe), 7.01 (2H, d, JAB 9.0 Hz,
C6H4OMe), 7.67 (1H, s, triazole), 7.71 (1H, s, triazole);
13C NMR (75 MHz, CDCl3): d 55.6 (OMe) 84.7, 85.0
(C-1c, C-1d), 95.6, 95.7, 96.1 (C-1e, C-1f), 97.1 (C-1b),
102.2 (C-1a), 114.8, 118.2 (C6H4–OMe), 121.4, 121.8
(C-5 triazole), 145.5, 145.9 (C-4 triazole), 151.4,
155.3 (C6H4–OMe); MALDI-TOFMS: m/z 5202.7
[M+Na]+.
4.19. p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-a-D-
glucopyranosyl)-(1!4)-{2,3,6-tri-O-acetyl-a-D-
glucopyranosyl)-(1!4)}5-(2,3,6-tri-O-acetyl-a-D-
glucopyranosyl-1H-[1,2,3]triazol-4-ylmethyl)-(4!4)-
[(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-(1!4)-
{(2,3,6-tri-O-acetyl-a-D-glucopyranosyl)-(1!4)}5-
(2,3,6-tri-O-acetyl-a-D-glucopyranosyl-1H-
[1,2,3]triazol-4-ylmethyl)-(4!6)]-2,3-di-O-benzyl-b-
D-glucopyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-gluco-
pyranoside (22)
This was synthesised from maltoside 10 (60 mg,
0.061 mmol) and maltoheptaosyl azide 16 (280 mg,
0.13 mmol) as described in the general procedure. Puri-
fication of the product by chromatography (3:2!9:1
EtOAc–hexane) gave the title compound as a white
foam (85.5 mg, 27%); [a]D +126 (c 1.8, CHCl3); 1H
NMR (400 MHz, CDCl3): d 5.64 (1H, d, J1,2 3.3 Hz,
H1-b), 5.82 (1H, d, J1,2 8.8 Hz, H1-c/d), 5.87 (1H, d,
J1,2 9.0 Hz, H1-c/d), 6.81 (2H, d, JAB 9.0 Hz,
C6H4OMe), 7.03 (2H, d, JAB 9.0 Hz, C6H4OMe), 7.60
(1H, s, triazole), 7.74 (1H, s, triazole);13C NMR
(100 MHz, CDCl3): 85.5 (C-1c, C-1d), 95.2–96.2
(C-1e, C-1f), 97.1 (C-1b), 103.0 (C-1a), 114.8,
118.2 (C6H4–OMe), 121.4, 121.8 (C-5 triazole), 151.4,
155.3 (C6H4–OMe); MALDI-TOFMS: m/z 5202.7
[M+Na]+.
4.21. Methyl [(20R,30R)-2,3-dimethoxybutane-20,30-diyl]-
a-D-glucopyranoside (24)
To a soln of trityl ether 26 (2.00 g, 3.64 mmol) in dry
MeOH (40 mL), pyridinium p-toluenesulfonate (1.00 g,

S. A. Nepogodiev et al. / Carbohydrate Research 342 (2007) 529–540
539
3.98 mmol) was added and the mixture was heated at
4.23. Methyl a-D-glucopyranosyl-(1!4)-{a-D-glucopyr-
37 ꢁC for 72 h. The mixture was concentrated, CH2Cl2
(100 mL) was added and the soln was washed with water
(2 · 50 mL). The organic phase was dried and concen-
trated. The product was purified by column chromato-
graphy (1:1!3:7 toluene–EtOAc) followed by
crystallisation from Et2O to afford diol 24 (850 mg,
anosyl)-(1!4)}5-(a-D-glucopyranosyl-1H-[1,2,3]triazole-
4-ylmethyl)-(4!4)-[a-D-glucopyranosyl-(1!4)-{(a-D-
glucopyranosyl)-(1!4)}5-(a-D-glucopyranosyl-1H-
[1,2,3]triazole-4-ylmethyl)-(4!6)]-a-D-glucopyranoside
(29)
1
76%), mp 136.5–137.5 ꢁC; [a]D ꢁ58 (c 1.1, CHCl3); H
A soln of azide 17 (140 mg, 0.120 mmol), di-O-propar-
gyl derivative 28 (16 mg, 0.060 mmol), sodium ascorbate
(2.4 mg, 0.012 mmol) and CuSO4Æ5H2O (0.6 mg) in
water (0.65 mL) was heated at 70 ꢁC for 2 h. The soln
was diluted with water (1.35 mL) and subjected to gel
permeation chromatography (TSK gel Toyopearl HW
40S, 1.5 · 85 cm) in 0.1% CF3CO2H in water in four
separate injections. The target product was collected in
fractions eluted with 70–84 mL (peak maximum at
76 mL), the maltoheptaosyl azide 17 had a maximum
of elution peak at 86 mL on the same column. The com-
bined fractions were concentrated and the residual soln
was freeze-dried to give pseudo-pentadecasaccharide 28
as a white powder, yield 140 mg (89%), [a]D +155 (c
1.3, H2O); 13C NMR (75 MHz, D2O): d 54.5, 59.8 (high
intensity), 62.4, 63.7, 67.5, 68.5, 70.6 (high intensity),
70.95 (high intensity), 71.2, 71,5, 72.1, 72.3, 72.7 (high
intensity), 75.7–79.9 (high intensity), 86.8, 98.6, 99.2
(high intensity), 124.0, 124.1, 143.4, 143.5; MALDI-
TOFMS: m/z 2648.8 [M + NH4]+.
NMR (400 MHz, CDCl3): d 1.33, 1.36 (6H, 2 · s, acetal
CMe), 2.82 (1H, br s, OH), 3.27, 3.30 (2H, 2 · s, acetal
OMe), 3.41 (3H, s, anomeric OMe), 3.52 (1H, br s, OH),
3.65 (1H, m, H-5), 3.75–3.90 (3H, m, H-2, H-6, H-60),
4.04 (1H, t, J3,4 ꢂ J4,5 10.3 Hz, H-3), 4.75 (1H, d, J
3.5 Hz, H-1); 13C NMR (75 MHz, CDCl3): d 17.7
(2 · acetal CMe), 47.9, 48.1 (2 · acetal OMe), 55.1 (ano-
meric OMe), 61.7 (C-6), 67.6 (C-4), 68.3 (C-2), 69.4 (C-
3), 71.9 (C-5), 98.1 (C-1), 99.5, 99.95 (acetal Cquat.);
HRESIMS: found m/z 326.1809 [M+NH4]+. Calcd for
C13H24O8+NH4 326.1809.
4.22. Methyl 4,6-di-O-(prop-2-ynyl)-a-D-glucopyranoside
(28)
To a soln of diacetal 24 (350 mg, 1.14 mmol) in dry
DMF (5 mL) was added a 60% suspension of NaH in
mineral oil (100 mg, 2.5 mmol) and the mixture was stir-
red for 1 h under nitrogen. Then it was cooled to 0 ꢁC,
HC„CCH2Br (0.31 mL, 3.44 mmol) was added and
stirring was continued for 2 h at this temperature before
the reaction was quenched with MeOH. The mixture
was diluted with EtOAc (50 mL), washed with water
(4 · 10 mL) and the solvent was evaporated. The prod-
uct was purified by chromatography (4:1 toluene–
EtOAc) to give a brown syrup, which was taken up in
CF3CO2H (0.9 mL) and water (0.1 mL). After stirring
the mixture for 1 h it was diluted with toluene and con-
centrated. The procedure was repeated several times un-
til complete removal of CF3CO2H. The product was
purified by reverse phase (C-18) column chromatogra-
phy (H2O/MeCN, 92:8!80:20) to afford compound 28
(68%), mp 68–71 ꢁC (Et2O/pet. ether), [a]D +146 (c
Acknowledgements
We are grateful to the EPSRC and the Weston Founda-
tion for financial support and we are indebted to the
EPSRC Mass Spectrometry Service Centre, Swansea,
for invaluable support.
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