The p-methoxybenzyl ether as an in situ-removable carbohydrate-protecting group: A simple one-pot synthesis of the globotetraose tetrasaccharide

Article abstract of DOI:10.1039/b009448j

A one-pot synthesis of the globotetraose tetrasaccharide is reported. The synthetic method relies on the use of a p-methoxybenzyl ether as an in situ-removable protecting group. N-Iodosuccinimide/trifluoromethanesulfonic acid-promoted glycosylation of 2-b

Full text of DOI:10.1039/b009448j

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The p-methoxybenzyl ether as an in situ-removable carbohydrate-
protecting group: a simple one-pot synthesis of the globotetraose
tetrasaccharide
1
Sumita Bhattacharyya,a Bengt G. Magnusson,b Ulf Wellmarb and Ulf J. Nilsson*a
a Organic Chemistry 2, Lund University, POB 124, SE-221 00 Lund, Sweden
b Lundonia Biotech AB, IDEON, Sölvegatan 41, SE-223 70 Lund, Sweden
Received (in Cambridge, UK) 23rd November 2000, Accepted 17th January 2001
First published as an Advance Article on the web 16th February 2001
A one-pot synthesis of the globotetraose tetrasaccharide is reported. The synthetic method relies on the use of a
p-methoxybenzyl ether as an in situ-removable protecting group. N-Iodosuccinimide/trifluoromethanesulfonic acid-
promoted glycosylation of 2-bromoethyl-2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-β--galactopyranosyl)-β--
glucopyranoside 5 at Ϫ45 ЊC with phenyl 2,4,6-tri-O-benzyl-3-O-(4-methoxybenzyl)-1-thio-β--galactopyranoside
6 gives an intermediate trisaccharide from which the p-methoxybenzyl ether is removed by allowing the reaction
temperature to rise to 0 ЊC for 40 min. Again lowering the temperature to Ϫ45 ЊC, followed by addition of methyl
3,4,6-tri-O-acetyl-2-trichloroethoxycarbonylamino-2-deoxy-1-thio-β--galactopyranoside 8, affords the
globotetraose tetrasaccharide 11 in one-pot in an excellent yield of 76%.
orthoesters and glycosides show different selectivity towards
Introduction
axial and equatorial inositol hydroxy groups has also been
A growing appreciation of the importance of glycobiological
phenomena1,2 in life processes has sparked the development of
numerous novel powerful methods for glycoside synthesis3,4
over the last decade. Although a rich plethora of highly efficient
glycosylation methods today allows the chemical synthesis
of most oligosaccharide structures, many glycosylations are
more or less unpredictable with respect to yields and regio/
stereoselectivities. Thus, carbohydrate chemists are still often
forced to spend many hours in the purification and character-
ization of glycosylation products (which is in contrast to pep-
tide and nucleotide chemists who often are served by automated
equipment for rapid solid-phase synthesis). Various approaches
have been pursued in order to simplify glycoside synthesis, such
as enzymatic glycoside synthesis,5,6 solid-phase glycoside syn-
thesis7,8 and one-pot glycosylations creating two or more glyco-
sidic bonds without the need for intermediate work-up and
purification. One-pot glycosylations are attractive, since they
minimize the number of both individual reaction steps and,
more important, tedious purification steps. (Synthetic oligo-
saccharides are rarely crystalline and purification is frequently
performed using time-consuming silica chromatography.)
Hitherto disclosed reports concerning one-pot glycosylations
typically involve multiple donors and acceptors possessing dif-
ferent reactivities. Sequential activation of glycosyl donors
carrying anomeric leaving groups displaying orthogonal
reactivities9–13 have been used in one-pot oligosaccharide syn-
theses. Furthermore, the most common approaches towards
one-pot multiple glycosylation reactions have involved elegant
fine-tuning of glycosyl donor reactivities14–21 by modifying the
leaving group substitution pattern and/or the donor protecting
groups, which has allowed the use of a single promotor to selec-
tively activate a more reactive glycosyl donor, followed by a less
reactive donor. Such fine-tuning of donor reactivities have also
been combined with use of glycosyl donors of orthogonal
reactivities.22–24 Reactivity differences between glycosyl accep-
tors have also allowed one-pot syntheses of di- to hexa-
saccharides25 and reaction-rate differences between intra- and
intermolecular glycosylations have been exploited in one-pot
trisaccharide syntheses.26 The observation that n-pentenyl
utilized in one-pot trisaccharide synthesis.27
Quantification of glycosyl donor reactivities28–30 has recently
been undertaken in order to simplify the planning of one-pot
oligosaccharide syntheses. Such quantitative measurements and
fine-tuning of glycosyl donor reactivities allowed the construc-
tion of a computer program/database as an aid in designing
synthetic strategies towards oligosaccharides29 and an oligo-
saccharide library.30
In the methods described above involving reactivity fine-
tuning and the use of only one promotor, the most reactive
donor (or acceptor) reacts first, followed by the less reactive
donor (or acceptor). Thus, an inherent limitation of these
methods is that reactive glycosyl donors cannot carry reactive
hydroxy groups that are to be glycosylated by less reactive
glycosyl donors.
In this paper we address this problem by introducing a novel
approach in which a p-methoxybenzyl ether is used as an in situ-
removable temporary protecting group for a reactive hydroxy
group in a one-pot reaction involving two sequential glycosyla-
tions. A p-methoxybenzyl ether is stable towards N-iodo-
succinimide–trifluoromethanesulfonic acid (NIS–TfOH) at
Ϫ45 ЊC and can thus block a highly reactive hydroxy group
during glycosylation of a less reactive alcohol with a reactive
glycosyl donor. When the first glycosylation reaction is com-
plete, the p-methoxybenzyl ether is cleaved by simply elevating
the temperature to 0 ЊC to expose a second, more reactive,
hydroxy group for glycosylation. A second glycosyl donor
can subsequently be added at Ϫ45 ЊC to complete the second
glycosylation in one pot.
The usefulness of the present method is demonstrated with
the first one-pot synthesis of the globotetraose (Gb4) tetra-
saccharide. Apart from being interesting from a biological
point of view,31–33 the Gb4 tetrasaccharide is
a rather
challenging target for one-pot glycosylations involving the
construction of a 1,2-cis glycosidic (α--galacto), as well as
a
2-amino-2-deoxy-β--galactopyranosidic linkage. Several
reports describe step wise34 or block syntheses35,36 of the Gb4
tetrasaccharide, as well as of elongated Gb4-containing
oligosaccharides.37–46
886
J. Chem. Soc., Perkin Trans. 1, 2001, 886–890
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b009448j

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Scheme 1 Reagents and conditions: i, PhCH(OMe)2, PTSA, MeCN (89%); ii, BzBr, pyridine, CH2Cl2 (71%); iii, AcOH, water, 80 ЊC (70%); iv, BzCl,
pyridine, CH2Cl2, Ϫ78 ЊC (82%); v, MeSSiMe2, Me3SiOTf, MS AW-300, (CH2Cl)2 (96%); vi, NIS, TfOH, CH2Cl2, Et2O, MS AW-300, Ϫ45 ЊC;
vii, TfOH, 0 ЊC, 40 min; viii, NIS, 8, Ϫ45 ЊC (76% for vi–viii one pot).
β-directing group removable under mild condtions.29,51,52 How-
Results and discussion
ever, earlier reports on the preparation of N-Troc-protected
The design of building blocks for one-pot multiple glyco-
thioglycosides of galactosamine have involved laborious multi-
sylation reactions deserves some special attention. Protective-
step syntheses from glucosamine, including inversion of con-
group patterns have to be chosen to ensure complete
stereoselectivity in each individual glycosylation reaction in
order to avoid the formation of complex product mixtures.
figuration at C-4.51 During the course of this work, we
have found that compound 8 could be obtained in only
three high-yielding steps from galactosamine hydrochloride;
N-Troc protection and acetylation45 (99% over two steps),
The lactoside acceptor
followed by treatment with trimethyl(methylthio)silane and
trimethylsilyl trifluoromethanesulfonate (96%) (Scheme 1).
It is worthwhile to mention that this simple and high-yielding
synthesis of the powerful β--GalNAc donor 8 is particu-
larly attractive in view of the high cost of galactosamine
hydrochloride.
In the present project a lactoside acceptor had to fulfil two
requirements; it should be equipped with an aglycon primed
for straightforward conjugation chemistry once the Gb4 tet-
rasaccharide assembly was completed and it should display
low reactivity of the HO-4Ј to ensure high stereoselectivity in
the α-galactosylation reaction. The first requirement was met
by using the 2-bromoethyl aglycon, since it is readily con-
verted into various functionalities via bromide substitution
reactions.47 The second requirement was fulfilled by benzoyl-
ation of 2-, 3-, 6-, 2Ј-, 3Ј- and 6Ј-OH of the 2-bromoethyl
β-lactoside 1 via 4Ј,6Ј-benzylidenation, benzoylation, acetal
hydrolysis and finally regioselective 6Ј-benzoylation to give
the desired lactoside acceptor 5 (Scheme 1). The electron-
withdrawing benzoyl groups significantly decrease the nucleo-
philicity of 4Ј-OH and such acceptors have proven to yield
excellent stereoselectivity in syntheses of the α--gal(1→4)--
gal glycosidic linkage.48
One-pot glycosylations
Initial attempts to glycosylate the lactoside 5 with the galactosyl
donor 6 yielded mixtures of the desired product 9 together with
the corresponding trisaccharide 10. The amount of 10 formed
was dependent on the reaction temperature, which prompted us
to examine the possibilities of performing a one-pot reaction
between the lactoside acceptor 5, the galactosyl donor 6 and the
β--GalNAc donor 8 towards the protected Gb4-derivative 11.
Indeed, NIS–TfOH (0.2 eq.)-promoted glycosylation53,54 of 5
with 6 proceeded smoothly in dichloromethane–diethyl ether at
Ϫ45 ЊC to consume all of the donor 6 and to give the inter-
mediate trisaccharide 9. Once all donor 6 was consumed,
simply raising the temperature of the reaction mixture to
0 ЊC, as well as adding more TfOH (0.2 eq.) to speed up
p-methoxybenzyl ether cleavage, allowed the safe and clean
conversion of 9 into 10 in 40 min according to TLC. The tem-
perature was again lowered to Ϫ45 ЊC and the galactosamine
donor 8 was added to give the tetrasaccharide 11 in an excellent
yield of 76% in one pot (Scheme 1).
Important advantages of our one-pot glycosylation strategy
are that only one class of glycosyl donors (simple and stable
thioglycosides) and one promotor system (NIS/TfOH) are
needed. Yet, our method does not depend on the fine-tuning of
donor and/or acceptor reactivities (i.e., fine-tuning leaving
group reactivities and/or alcohol nucleophilicities) by intro-
ducing special anomeric leaving-groups or hydroxy-protecting
groups, since the second hydroxy group to be glycosylated is
protected during the first glycosylation reaction. In our example
The ꢀ-D-Gal donor
The known phenyl 2,4,6-tri-O-benzyl-3-O-(4-methoxy-
benzyl)-1-thio-β--galactopyranoside49 6 was chosen as the
α--galactosyl donor, because it carries a stable, yet readily
activated, anomeric leaving group, a non-participating 2-O-
benzyl group and a p-methoxybenzyl protective group, which
can be selectively removed under mildly acidic conditions50
(Scheme 1).
The ꢁ-D-GalNAc donor
The N-trichloroethoxycarbonyl (N-Troc)-protected methyl
thioglycoside donor 8 was chosen for the synthesis of the
β--GalNAc linkage, because it is activated by the same promo-
tor as the α--galactosyl donor 6 discussed above and because
the N-Troc group has proven to be a reactive and reliable
J. Chem. Soc., Perkin Trans. 1, 2001, 886–890
887

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above, the most reactive benzylated galactosyl donor 6 reacts
first with the least reactive alcohol 5, followed by cleavage of the
p-methoxybenzyl ether to expose the more reactive alcohol 10
for glycosylation with the less reactive Troc-protected galacto-
samine donor 8. In comparison, the design of a one-pot proto-
col for the synthesis of the relatively demanding globotetraose
tetrasaccharide, based on donor/acceptor reactivity fine-tuning,
is not straightforward.
The N-Troc group of 11 was transformed into the corre-
sponding N-acetyl derivative 12, which subsequently was
deprotected to give the deblocked 2-bromoethyl spacer-
glycoside 13 of globotetraose in high yield (Scheme 2). Finally,
compound 13 was per-O-acetylated (→ 14) in order to simplify
1H NMR analyses of the tetrasaccharide structure.
sulfonic acid (PTSA) (0.5 g) were added. Triethylamine (10 mL)
was added after 4 h, and the precipitate was filtered off,
washed with cold ethanol and left under vacuum overnight
to give 2 (42.6 g, 89%); mp 194–197 ЊC (from EtOH); [α]D25 Ϫ21
(c 0.3 in MeOH); δH (500 MHz; CD3OD) 7.54 (2H, m,
ArH), 7.35 (3H, m, ArH), 5.63 (1H, s, PhCH), 4.49 (1H, d,
J 6.8, H-1Ј), 4.38 (1H, d, J 7.8, H-1), 4.23 (1H, br d, J 2.9,
H-4Ј), 4.19 (1H, dd, J 1.5 and 8.6, H-6Ј), 4.10 (1H, ddd,
J 6.4, 6.9 and 9.9, OCH2CH2), 3.27 (1H, dd, J 8.0 and 8.9,
H-2); m/z 559.0781 (Mϩ ϩ Na. C21H29BrNaO11 requires m/z,
559.0791).
2-Bromoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-4,6-O-
benzylidene-ꢁ-D-galactopyranosyl)-ꢁ-D-glucopyranoside 3
To a solution of compound 2 (755 mg, 1.40 mmol) in pyridine
(30 mL) at 0 ЊC was added benzoyl bromide (1.16 mL, 9.84
mmol). After 160 min, the reaction mixture was poured onto a
mixture of dichloromethane and saturated aq. NaHCO3, and
the organic layer was dried (Na2SO4), filtered and concentrated.
Flash chromatography (SiO2: heptane–EtOAc 2 : 1) gave 3 (1.05
g, 71%); mp 253–254 ЊC (from heptane–EtOAc); [α]D25 ϩ114
(c 1.1 in CHCl3); δH (400 MHz; CDCl3) 5.85 (1H, t, J 9.2, H-3),
5.80 (1H, dd, J 8.0 and 10.5, H-2Ј), 5.34 (1H, dd, J 7.7 and 9.2,
H-2), 5.31 (1H, s, PhCH), 5.19 (1H, dd, J 3.6 and 10.5, H-3Ј),
4.85 (1H, d, J 7.9, H-1Ј), 4.78 (1H, d, J 7.6, H-1), 4.66 (1H, dd,
J 2.0 and 11.8, H-6), 4.37 (1H, dd, J 4.3 and 11.9, H-6), 4.31
(1H, d, J 3.5, H-4Ј), 4.24 (1H, t, J 9.3, H-4), 4.01 (1H, ddd,
J 5.6, 6.9 and 11.2, OCH2CH2), 3.89 (1H, ddd, J 2.3, 4.3 and
9.7, H-5), 3.79 (1H, br d, J 10.9 and H-6Ј), 3.77 (1H, dt, J 7.0
and 11.3, OCH2CH2), 3.59 (1H, dd, J 1.7 and 12.3, H-6Ј), 3.33
(2H, m, CH2Br); m/z 1079.2131 (Mϩ ϩ Na. C56H49BrNaO16
requires m/z, 1079.2101).
Scheme 2 Reagents: i, aZn, AcOH; bAc2O, MeOH (86%); ii, aH2, Pd/C,
AcOH; bNaOMe, MeOH (79%); iii, Ac2O, pyridine, DMAP (88%).
Conclusions
The present report shows that a p-methoxybenzyl ether can be
used as an in situ-removable protecting group during an
N-iodosuccinimide/trifluoromethanesulfonic
acid-promoted
glycosylation at Ϫ45 ЊC with a thioglycoside donor. Reaction-
temperature manipulation (0 ЊC) allows convenient removal of
the p-methoxybenzyl ether, thus setting the stage for glycosyl-
ation in one pot with a second thioglycoside donor. The
usefulness of this approach was demonstrated with an efficient
and high-yielding one-pot synthesis of the globotetraose
tetrasaccharide 11. We believe the strategy of using the
p-methoxybenzyl ether as a temporary protecting group in one-
pot glycosylations is an attractive approach that will become a
useful tool for the synthesis of complex oligosaccharides. The
strategy is an important complement to and may be used in
combination with existing methods based on donor/acceptor
reactivity fine-tuning. The scope and limitation of this strategy
(e.g., the stability of various carbohydrate p-methoxybenzyl
ethers at low temperature, the use of multiple p-methoxybenzyl
ethers for the synthesis of branched oligosaccharides, the use of
other acid-labile temporary protecting groups, etc.) need to
be addressed and are currently under investigation in our
laboratory.
2-Bromoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-ꢁ-D-
galactopyranosyl)-ꢁ-D-glucopyranoside 4
Compound 3 (1.05 g, 0.99 mmol) was heated to 80 ЊC in 80%
acetic acid (20 mL) for 6 h, then the solution was concentrated.
Flash chromatography (SiO2; CH2Cl2–acetone 9 : 1) gave 4 (668
mg, 70%); mp 214–216 ЊC (from heptane–EtOAc); [α]D25 ϩ78
(c 0.8 in CHCl3); δH (400 MHz; CDCl3) 5.76 (2H, br t, J 9.1,
H-3 and -2Ј), 5.43 (1H, dd, J 7.6 and 9.4, H-2), 5.11 (1H, dd,
J 3.2 and 10.4, H-3Ј), 4.81 (1H, d, J 8.0, H-1Ј), 4.79 (1H, d,
J 7.7, H-1), 4.64 (1H, dd, J 2.0 and 11.9, H-6), 4.42 (1H, dd,
J 4.9 and 11.9, H-6), 4.21 (1H, t, J 9.4, H-4), 4.20 (1H, br d,
J 4.2, H-4Ј), 4.04 (1H, ddd, J 5.3, 6.8 and 11.3, OCH2CH2), 3.88
(1H, ddd, J 2.0, 4.7 and 9.7, H-5), 3.79 (1H, dt, J 6.9 and 11.2,
OCH2CH2); m/z 991.1792 (Mϩ ϩ Na. C49H45BrNaO16 requires
m/z, 991.1789).
2-Bromoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-ꢁ-D-
galactopyranosyl)-ꢁ-D-glucopyranoside 5
To a solution of compound 4 (247.1 mg, 0.255 mmol) and
pyridine (4 mL) in dichloromethane (6 mL) at Ϫ78 ЊC was
added benzoyl chloride (32.5 µL, 0.28 mmol). More benzoyl
chloride (25 µL, 0.21 mmol) was added after 150 min. The
reaction was quenched with methanol (10 mL) after 6 h and
the mixture was concentrated. Flash chromatography (SiO2;
heptane–EtOAc 2 : 1) gave 5 (225.1 mg, 82%); mp 197–199 ЊC
(from heptane–EtOAc); [α]D25 ϩ54 (c 0.9 in CHCl3); δH (400
MHz; CDCl3) 5.78 (1H, t, J 9.2, H-3), 5.75 (1H, dd, J 8.0
and 9.4, H-2Ј), 5.44 (1H, dd, J 7.9 and 9.7, H-2), 5.16 (1H,
dd, J 3.2 and 10.4, H-3Ј), 4.80 (1H, d, J 7.9, H-1Ј),
4.77 (1H, d, J 7.8, H-1), 4.61 (1H, dd, J 2.0 and 12.1, H-6), 4.46
(1H, dd, J 4.4 and 12.0, H-6), 4.21 (1H, t, J 9.3, H-4), 4.04 (1H,
ddd, J 5.3, 6.8 and 11.3, OCH2CH2), 3.87 (1H, ddd, J 2.0, 4.7
and 9.7, H-5), 3.79 (1H, dt, J 7.1 and 11.3, OCH2CH2), 3.36
(2 H, m, CH2Br); m/z 1095.2070 (Mϩ ϩ Na. C56H49BrNaO17
requires m/z, 1095.2051).
Experimental
General methods
1H NMR spectra were recorded with Bruker ARX 500, DRX
400 and ARX 300 MHz instruments (J-values are given in Hz).
FAB-HRMS spectra were recorded with a JEOL SX102
instrument. Optical rotations were measured with a Perkin-
Elmer 241 polarimeter ([α]D-values are given in units of 10Ϫ1 deg
cm2 gϪ1). Reactions were monitored by TLC on Silica Gel FG254
(E. Merck, Darmstadt, Germany).
2-Bromoethyl 4-O-(4,6-O-benzylidene-ꢁ-D-galactopyranosyl)-ꢁ-
D-glucopyranoside 2
2-Bromoethyl 4-O-(β--galactopyranosyl)-β--glucopyrano-
side55 1 (40 g, 89 mmol) was suspended in acetonitrile (2 L)
and α,α-dimethoxytoluene (18 mL, 120 mmol) and toluene-p-
888
J. Chem. Soc., Perkin Trans. 1, 2001, 886–890

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Methyl 3,4,6-tri-O-acetyl-2-(2,2,2-trichloroethoxycarbonyl-
amino)-2-deoxy-1-thio-ꢁ-D-galactopyranose 8
J 7.6 and 10.6, H-2Ј), 5.42 (1H, br d, J 3.2, H-4ٞ), 5.37 (1H, dd,
J 7.9 and 9.2, H-2), 5.17 (1H, t, J 3.1, H-3Ј), 5.14 (1H, t, J 3.3,
H-3ٞ), 5.04 (1H, d, J 7.7, H-1Ј), 4.91 (1H, d, J 8.4, H-1ٞ), 4.67
(1H, d, J 7.7, H-1), 4.64 (1H, d, J 3.4, H-1Љ), 4.55 (1H, dd, J 4.1,
6.3, H-2ٞ), 4.32 (1H, m, H-2Љ), 3.70 (2H, m, OCH2CH2), 3.33
(3H, m, CH2Br, H-5Ј), 2.20, 2.15, 2.00, 1.87 (3H each, 4 s, Ac);
m/z 1856.5060 (Mϩ ϩ Na. C97H96BrNNaNO30 requires m/z,
1856.5098).
To a mixture of 1,3,4,6-tetra-O-acetyl-2-(2,2,2-trichloroethoxy-
carbonylamino)-2-deoxy-α/β--galactopyranose45,56 7 (α/β 1 : 3;
47.7 g, 91.0 mmol), trimethyl(methylthio)silane (16.7 g, 140
mmol) and MS AW-300 (activated, 10 g) in 1,2-dichloroethane
(400 mL) was added trimethylsilyl trifluoromethanesulfonate
(20 g, 91.0 mmol). The mixture was filtered after two days,
diluted with dichloromethane, washed with saturated aq.
sodium hydrogen carbonate, dried (Na2SO4), filtered and con-
centrated. Flash chromatography (SiO2; heptane–EtOAc 2 : 1 to
1 : 1 gradient) gave 8 (44.6 g, 96%); [α]D25 Ϫ18 (c 1 in CHCl3); δH
(400 MHz; CDCl3) 5.42 (1H, dd, J 0.7 and 3.2, H-4), 5.13 (1H,
dd, J 3.2 and 10.9, H-3), 5.03 (1H, d, J 9.6, NH), 4.82 (1H, d,
J 12.0, CH2CCl3), 4.69 (1H, d, J 12.0, CH2CCl3), 4.53 (1H, d,
J 10.3, H-1), 4.18 (1H, dd, J 6.8 and 11.3, H-6), 4.14 (1H, dd,
J 6.6 and 11.3, H-6), 4.04 (1H, br q, J 10.2, H-2), 3.96 (1H, dt,
J 0.9 and 6.7, H-5), 2.25 (3 H, s, SCH3), 2.18, 2.05, 2.01 (3 H
each, 3 s, Ac); m/z 531.9987 (Mϩ ϩ Na. C16H22Cl3NNaO9S
requires m/z, 531.9979).
2-Bromoethyl 4-O-{4-O-[3-O-(2-acetamido-2-deoxy-ꢁ-D-
galactopyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-D-galacto-
pyranosyl}-ꢁ-D-glucopyranoside 13
A solution of compound 12 (100 mg, 54 µmol) in AcOH (4 mL)
was hydrogenolysed over 10% Pd–C (40 mg) for 24 h, filtered
and concentrated. The residue was treated with methanolic
sodium methoxide (0.09 M; 2.5 mL) at room temperature for
16 h. The solution was neutralized with Duolite C436 (Hϩ)
resin, filtered and concentrated. Column chromatography
(SiO2, CH2Cl2–MeOH–H2O 10 : 5 : 1) gave 13 (35 mg, 79%); [α]D25
ϩ15 (c 0.2 in H2O); δH (400 MHz; D2O) 4.79 (1H, d, J 3.9,
H-1Љ), 4.51 (1H, d, J 8.4, H-1ٞ), 4.44 (1H, d, J 8.0, H-1), 4.39
(1H, d, J 7.7, H-1Ј), 4.25 (1H, br t, J 6.7, H-5Љ), 4.13 (1H, br d,
J 2.7, H-4Љ), 3.46 (1H, dd, J 7.8 and 10.3, H-2Ј), 3.23 (1H, dd,
J 8.1 and 9.0, H-2), 1.92 (3H, s, Ac); m/z 836.1831 (Mϩ ϩ Na.
C28H48BrNNaO21 requires m/z, 836.1800).
2-Bromoethyl 2,3,6-tri-O-benzoyl-4-O-{2,3,6-tri-O-benzoyl-4-O-
[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-acetyl-2-(2,2,2-trichloro-
ethoxycarbonylamino)-2-deoxy-ꢁ-D-galactopyranosyl)-ꢀ-D-
galactopyranosyl]-ꢁ-D-galactopyranosyl}-ꢁ-D-glucopyranoside 11
To a solution of 5 (2.27 g, 2.11 mmol) and 6 (2.10 g, 3.17 mmol)
in CH2Cl2–Et2O (1 : 2; 66 mL) was added MS AW-300 (acti-
vated, 1 g) and the mixture was stirred under N2 for 4 h. The
mixture was then cooled to Ϫ45 ЊC and NIS (922 mg, 4.13
mmol) and TfOH (50 µL, 0.2 eq.) were added. After 40 min,
TLC showed the complete disappearance of the donor 6 and
formation of the desired product 9. To the mixture was then
added TfOH (50 µL), the temperature was raised to 0 ЊC and
the mixture was stirred for 40 min. The temperature of the
reaction mixture was again lowered to Ϫ45 ЊC and the donor 8
(1.20 g, 2.34 mmol) and NIS (600 mg, 2.67 mmol) were added.
After 30 min, the reaction mixture was diluted with dichloro-
methane, filtered through Celite, washed successively with 10%
aq. Na2S2O3 and 1 M aq. NaHCO3, dried (Na2SO4) and con-
centrated. Column chromatography (SiO2; toluene–EtOAc,
12 : 1 to 6:1 gradient) gave 11 (3.2 g, 76%); [α]D25 ϩ39 (c 1 in
CHCl3); δH (400 MHz; CDCl3) 5.88 (1H, t, J 9.3, H-3), 5.73
(2H, dd, J 7.7 and 10.5, H-2Ј and NH), 5.47 (1H, br d, J 2.8,
H-4ٞ), 5.37 (1H, dd, J 7.9 and 9.3, H-2), 5.16 (1H, dd, J 3.0 and
7.8, H-3ٞ), 5.13 (1H, dd, J 2.7 and 10.4, H-3Ј), 5.05 (1H, d,
J 7.7, H-1ٞ), 4.94 (1H, d, J 8.3, H-1Ј), 4.67 (1H, d, J 7.9, H-1),
4.32 (1H, t, J 9.6, H-4), 4.25 (3H, m, H-2ٞ and OCH2CH2), 3.95
(1H, dd, J 3.0 and 10.5, H-2Љ), 3.32 (2H, m, CH2Br), 2.90 (1H,
dd, J 5.8 and 8.2, H-6Љ), 2.15, 2.01, 2.00 (3H each, 3 s, Ac);
m/z 1988.4076 (Mϩ ϩ Na. C98H95BrCl3NaO31 requires m/z,
1988.4035).
2-Bromoethyl 2,3,6-tri-O-acetyl-4-O-{2,3,6-tri-O-acetyl-4-O-
[2,4,6-tri-O-acetyl-3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-ꢁ-D-galactopyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-D-
galactopyranosyl}-ꢁ-D-glucopyranoside 14
Compound 13 (35 mg, 43 µmol) was acetylated with Ac2O (0.2
mL), pyridine (0.4 mL) and 4-(dimethylamino)pyridine
(DMAP) (10 mg) for 16 h. Concentration and column chrom-
atography of the residue (SiO2; EtOAc) gave 14 (50 mg, 88%);
[α]D25 ϩ44 (c 0.5 in CHCl3); δH (400 MHz; CDCl3) 5.65 (1H, d,
J 8.7, NH), 5.60 (1H, br d, J 2.9, H-4Љ), 5.35 (1H, br d, J 3.2,
H-4ٞ), 5.27 (1H, dd, J 3.2 and 11.2, H-3ٞ), 5.23 (1H, t, J 9.2,
H-3), 5.20 (1H, dd, J 3.5 and 10.6, H-2Љ), 5.13 (1H, dd, J 7.6
and 10.8, H-2Ј), 4.96 (1H, d, J 3.5, H-1Љ), 4.92 (1H, dd, J 8.0
and 9.3, H-2), 4.85 (1H, d, J 8.2, H-1ٞ), 4.76 (1H, dd, J 2.4 and
10.9, H-3Ј), 4.56 (1H, d, J 7.6, H-1Ј), 4.55 (1H, d, J 7.9, H-1),
4.20 (1H, dd, J 3.3 and 10.9, H-3Љ), 3.91 (1H, dt, J 8.7 and 11.0,
H-2ٞ), 3.45 (2H, m, CH2Br), 2.14, 2.13, 2.12, 2.10, 2.09, 2.07,
2.06, 2.05, 1.99, 1.92 (39 H, 10 s, Ac); m/z 1340.3063 (Mϩ ϩ Na.
C52H72BrNNaO33 requires m/z, 1340.3068).
Acknowledgements
This work is dedicated to the memory of Professor Göran
Magnusson. We will remember Göran as a close friend and as a
supportive and enthusiastic mentor and colleague.
Financial support by the Swedish Natural Science Research
Council and Lundonia Biotech is acknowledged.
2-Bromoethyl 2,3,6-tri-O-benzoyl-4-O-{2,3,6-tri-O-benzoyl-4-O-
[2,4,6-tri-O-benzyl-3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-ꢁ-D-galactopyranosyl)-ꢀ-D-galactopyranosyl]-
ꢁ-D-galactopyranosyl}-ꢁ-D-glucopyranoside 12
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