Synthesis of the O-linked hexasaccharide containing β-d-Galf- (1→2)-β-d-Galf in Trypanosoma cruzi mucins

Article abstract of DOI:10.1039/c2ob25741f

The hexasaccharide β-d-Galp-(1→2)-[β-d-Galp-(1→3)]- β-d-Galp-(1→6)-[β-d-Galf(1→2)-β-d-Galf(1→4)] -d-GlcNAc (1) is the largest carbohydrate structure released as alditol by reductive β-elimination from mucins of some strains of T. cruzi. The terminal β-d-Galp units are sites of sialylation by trans-sialidase which transfers sialic acid from the host to the parasite. Hexasaccharide 1 was synthesized by a [3 + 3]-convergent strategy based on a nitrile assisted glycosylation, using the trichloroacetimidate method. The β-d-Galf- (1→2)-β-d-Galf-d-GlcNAc synthon was sequentially constructed from the reducing end to the non-reducing end employing benzyl α-d- galactofuranoside as starting material for the internal Galf unit. The choice of this novel precursor, obtained in one-reaction step from galactose, allowed the introduction of an orthogonal and participating levulinoyl group at O-2. Thus, the diastereoselective construction of the Galf-β(1→4)-GlcNAc linkage by the trichloroacetimidate method of glycosylation was achieved. The ¹H NMR spectrum of alditol 2 was identical to the product released by β-elimination from the parasite mucin. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25741f

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PAPER
Synthesis of the O-linked hexasaccharide containing β-D-Galf-(1→2)-β-D-Galf
in Trypanosoma cruzi mucins†
Gustavo A. Kashiwagi, Verónica M. Mendoza, Rosa M. de Lederkremer and Carola Gallo-Rodriguez*
Received 17th April 2012, Accepted 11th June 2012
DOI: 10.1039/c2ob25741f
The hexasaccharide β-D-Galp-(1→2)-[β-D-Galp-(1→3)]-β-D-Galp-(1→6)-[β-D-Galf(1→2)-β-D-Galf(1→4)]-
D-GlcNAc (1) is the largest carbohydrate structure released as alditol by reductive β-elimination from
mucins of some strains of T. cruzi. The terminal β-D-Galp units are sites of sialylation by trans-sialidase
which transfers sialic acid from the host to the parasite. Hexasaccharide 1 was synthesized by a [3 + 3]-
convergent strategy based on a nitrile assisted glycosylation, using the trichloroacetimidate method. The
β-D-Galf-(1→2)-β-D-Galf-D-GlcNAc synthon was sequentially constructed from the reducing end to the
non-reducing end employing benzyl α-D-galactofuranoside as starting material for the internal Galf unit.
The choice of this novel precursor, obtained in one-reaction step from galactose, allowed the introduction
of an orthogonal and participating levulinoyl group at O-2. Thus, the diastereoselective construction of
the Galf-β(1→4)-GlcNAc linkage by the trichloroacetimidate method of glycosylation was achieved. The
1H NMR spectrum of alditol 2 was identical to the product released by β-elimination from the parasite
mucin.
stages of the parasite.5 The striking feature of the O-linked
chains is that they are linked to the protein by α-GlcNAc instead
Introduction
The protozoan Trypanosoma cruzi is the etiologic agent of
Chagas disease, the American trypanosomiasis which is trans-
mitted to animals, including humans, by insect vectors of the
family Reduviidae.1–3 This neglected disease is a major public
health issue in Latin America, and is also becoming an important
health issue in the United States and Europe due to immigration
of GalNAc common in vertebrate mucins. By comparing struc-
tural data on the mucins from different strains it followed that the
O-linked oligosaccharides may be derived from two cores,
β-Galp-(1→4)-GlcNAc or β-Galf-(1→4)-GlcNAc. The cores are
further branched with various units of Galf and/or Galp. A puz-
zling fact is that, depending on the strain, galactose may be
found in the furanose configuration together with the more
common galactopyranose. Galactofuranose was first described in
mucins of the G strain, classified as T. cruzi I, with a sylvatic
origin.11,12 It was later found in other strains belonging to the
same group,13 more recently from drug-resistant Colombiana
strain, isolated from a chronic human case in Colombia.14 In the
strain Y of group II, galactose is only present in the pyranose
form,15 as well as in the mucins of the CL14 and CL Brener
strains,16–18 previously included in group II and now reclassified
as DTU VI.19 It is interesting that the Tulahuen strain, now
included in the new hybrid type VI expresses mucins with sub-
stitution of GlcNAc by either Galp or Galf.20 The biosynthetic
steps for the incorporation of Galf in some mucins deserves
further investigation since Galf is an antigenic epitope,21 which
is not present in the mammalian host. In our laboratory we have
undertaken the chemical synthesis of the mucin oligosacchar-
ides, in particular of those containing Galf (Fig. 1),22–26 which
should be useful tools for biosynthetic studies. Trans-sialidase
and infection by blood transfusion or organ transplants.4
A
dense glycocalyx covers the surface of the parasite and its com-
position is characteristic of each differentiation stage of the
complex living cycle.5,6 The interplay between the enzymatically
active trans-sialidase (TcTS) and the mucins in the surface of
T. cruzi is crucial for pathogenesis.7–10 The O-linked oligosac-
charides in the mucins account for up to 60% of the molecular
mass.6 They play the function of being acceptors of sialic acid,
which is not biosynthesized by the parasite but is needed to
build a negatively charged coat. Stage variations and inter-strain
differences in the epimastigote stage have been observed in the
carbohydrates of mucins.5 The structures of the O-linked oligo-
saccharides have been elucidated for the mucins of the insect
CIHIDECAR, Departamento de Química Orgánica, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, Pabellón II, 1428 Buenos Aires, Argentina.
E-mail: cgallo@qo.fcen.uba.ar; Fax: +54-11-4576-3352;
Tel: +54-11-4576-3346
studies have been performed on the substrates 3, 4 and 6.25,27
A
1
†Electronic supplementary information: Experimental for 8 and 24. H
and 13C NMR spectra for 1, 2, 10, 11, 13–26 and bidimensional spectra
for 1 and 26. See DOI: 10.1039/c1ce05336a
tetrasaccharide from mucin of the Y strain which only contains
galactose in the pyranose form, has been also synthesized.28 In
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Fig. 1 (a) Representative oligosaccharides present in mucins of T. cruzi that have already been synthesized ; (b) retrosynthesis of the target hexasac-
charide 1.
this work we describe the first synthesis of the ramified hexasac-
charide β-D-Galp-(1→2)-[β-D-Galp-(1→3)]-β-D-Galp-(1→6)-[β-D-
Galf-(1→2)-β-D-Galf-(1→4)]-D-GlcNAc (1, Fig. 1) and the corre-
sponding alditol 2. The NMR spectra of 2 could be compared with
the data for the alditol obtained by reductive β-elimination from the
mucins of the Tulahuen strain. Hexasaccharide 1 and the related
considered. Tetrasaccharide 3 and pentasaccharide 4 (Fig. 1)
share a ramified GlcNAc at the reducing end substituted at the
4-O and 6-O positions. Due to the recognized low nucleophili-
city of OH-4 in GlcNAc,29 the Galf moiety was first introduced
followed by glycosylation at O-6.24,25 Pentasaccharide 4 shares a
2,3-di-O-(β-D-Galp)-β-D-Galp unit with the target hexasaccharide
1 (Fig. 1). In the synthesis of 4,25 selective 1,2-trans β-glycosy-
lation on O-6 of the GlcNAc derivative was achieved by the use
of acetonitrile as solvent, taking advantage of the nitrile
effect,30–34 and overcoming the lack of neighboring group par-
ticipation in the Galp trisaccharide donor.
For that reason, retrosynthetic analysis for hexasaccharide
1 indicated a [3 + 3] nitrilium-based glycosylation between
already synthesized Galp-containing trisaccharide imidate 725
and the trisaccharide acceptor 8 (Fig. 1) which contains an
internal Galf.
hexasaccharide
(1→6)-[β-D-Galp-(1→2)-β-D-Galf-(1→4)]-D-GlcNAc
β-D-Galp-(1→2)-[β-D-Galp-(1→3)]-β-D-Galp-
constitute
the largest O-linked oligosaccharides found in mucins of T. cruzi.
Results and discussion
Synthetic strategies
Based on our experience in the synthesis of several oligosacchar-
ides present in mucins of T. cruzi, some aspects were initially
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Scheme 1
Several methods have been employed for β-galactofuranosyla-
tion which led to the development of new Galf precursors.35–41
As Galf is thermodynamically less stable compared to galacto-
pyranose, the synthesis of internal galactofuranosyl-containing
oligosaccharides requires the choice of a convenient galactofura-
nosyl precursor in accordance with a proper glycosylation
method. Examples of the synthesis of internal Galf-containing
oligosaccharides have been reported and each synthesis has its
advantages and drawbacks.26,35–37,42–49 The thioglycoside
method was employed for the first time in the challenging syn-
thesis of internal Galf-containing oligosaccharides from Myco-
bacterium tuberculosis as well as in the synthesis of the
repeating unit of varianose.44 More recently, a biotinylated tetra
β(1–5)galactofuranoside was also synthesized by this method.45
The precursor thiogalactofuranoside allowed the oligosaccharide
construction from the reducing end to the non-reducing end;
however, it required several reaction steps from galactose.
Very recently, regioselective one pot syntheses of trisacchar-
ides with internal Galf were developed using partially protected
thiogalactofuranosides.46 A drawback is that the required stereo-
selective protection of Galf thioglycosides seems rather difficult.
For instance, selective benzoylation at low temperature of p-tolyl
5,6-isopropylidene-1-thio-β-D-galactofuranoside afforded the
2-O- and 3-O-benzoyl derivatives in a 1.7 : 1 ratio.46
Also, the carbobenzoxy method has been successfully
employed in the challenging synthesis of agelagalastatin, which
contains an internal β-D-Galf.47 This method was also employed
for the synthesis of cyclic galactooligofuranosides as well as in
the synthesis of a tetrasaccharide constituent of the mycobacter-
ial cell wall. However, this method failed in constructions of
some internal-Galf linkages and was overcome by a fluoride
replacement of the carbobenzoxy group in a later step.48 On the
other hand, the precursor carboxybenzyl galactofuranoside donor
can be synthesized via the galactofuranosyl chloride in four reac-
tion steps from pentaacetylgalactofuranose, involving the use of
mercuric salts.47
trisaccharide was constructed from the non-reducing end to the
reducing end. By this approach, only moderate yields were
achieved in the glycosylation of the GlcNAc unit due to the
absence of a participating group at O-2 of the β-D-Galf-(1→2)-
Galf donor. In view of the low anomeric effect in furanoses,35,36
we had considered that the steric effect of the substituent in O-2
would prevail in the control of glycosylation. However, it was
not as important as expected, and moderate diastereoselectivities
were obtained, depending on the nature of the glycosylation
solvent.26
For that reason, in the present study, a sequential strategy from
the reducing end to the non-reducing end, was designed for the
synthesis of trisaccharide 8. The terminal β-D-Galf of 8 would be
installed in O-2 by glycosylation of partially protected β-Galf
(1→4)GlcNAc 10 with known tetra-O-benzoyl-β-D-galactofura-
nosyl trichloroacetimidate (9, Fig. 1).50 The internal Galf precur-
sor 11 should possess a participating group at O-2 to ensure
glycosylation diastereoselectivity and should be orthogonal to
the benzoyl and silyl groups of the known GlcNAc acceptor
1224 successfully employed in the synthesis of the oligosacchar-
ides of Fig. 1.24–26 A levulinoyl group was chosen in this case,
and donor 11 could be prepared from benzyl α-D-galactofurano-
side (13). The anomeric benzyl group, orthogonal to the benzoyl
and the levulinoyl protecting-groups, could be easily removed
by hydrogenolysis for further activation as trichloroacetimidate.
Synthesis of 11, the precursor of the internal Galf
Based in our experience in the one-step anomeric O-alkylation
of galactose,51,52 starting material benzyl α-D-galactofuranoside
(13) was obtained crystalline in 67% yield by treatment of galac-
tose with 1.2 equiv. of benzyl bromide and NaH, in DMPU as
solvent (Scheme 1). The 2,3,5,6-tetra-O-acetyl derivative has
been previously synthesized also using O-anomeric alkylation.53
In the 1H NMR spectrum of 13, the anomeric hydrogen appeared
at δ 5.09 as a doublet with J1,2 = 4.6 Hz, indicating the H-1–H-2
cis disposition of α-D-Galf. Also, the H-2 appeared as a doublet
of doublets with J = 8.0 and 4.6 Hz. The 13C NMR spectrum
showed the resonance of the C-1 at δ 100.9 confirming the
α-configuration, and the downfield shifted signal of C-4 at δ
82.0 which is characteristic of the furanose form. In order to
selectively introduce a levulinoyl group in O-2, the 5- and 6-O
positions of 13 were temporarily protected. Thus reaction of 13
with dimethoxypropane in acetone and catalytic amounts of
p-toluenesulfonic acid gave the 5,6-O-isopropylidene derivative
14 which was obtained crystalline from the crude mixture in
The trichloroacetimidate method34 of glycosylation has been
extensively applied for the synthesis of internal Galf-containing
oligosaccharides,26,35–37,42,49 being pivotal in the aldonolactone
approach,49 where D-galactono-1,4-lactone, which can be selec-
tively substituted by protecting groups, acts as precursor of the
Galf. By this methodology, oligosaccharides of Leishmania, and
Mycobacterium tuberculosis were synthesized in high yields. Tri-
saccharide 8 (Fig. 1) was recently synthesized by the aldonolac-
tone approach as an intermediate in the synthesis of trisaccharide
526 (Fig. 1). In this case, a derivative of D-galactono-1,4-lactone
was employed for the introduction of the internal Galf, and the
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94% yield. Selective 2-O-benzoyl and 2-O-pivaloyl protection
has been previously described for allyl52b and pentenyl α-D-
galactofuranoside54 derivatives, respectively, probably as a con-
sequence of the increment of the 2-O-nucleophilicity by the
intramolecular hydrogen bond with the α-anomeric oxygen.54
For that reason, and in order to introduce a levulinoyl group at
that position, different conditions of levulinoylation were
assayed. The best results were obtained by reaction of 14 with
0.8 equiv of levulinic acid, dicyclohexylcarbodiimide (0.8 equiv)
and DMAP at −18 °C in order to reduce the 2,3-di-O-levulinoyl
byproduct. In this case, the reaction afforded the 2-O-Lev deriva-
tive 15 in 38% yield, the 2,3-di-O-Lev product 16 in 6% yield
and the starting material 14 was recovered in 31% yield as
NMR spectrum of 20, the signal for the anomeric hydrogen
appeared 0.71 ppm downfield shifted compared to the same
signal in 19, whereas the signal for the H-2 remained almost at
the same δ. The reaction of 20 with levulinic acid, DMAP and
dicyclohexylcarbodiimide gave the 2-O-Lev derivative 21
(97%). Substitution at O-2 was confirmed by the signal for H-2
(δ 5.29) shifted 0.80 ppm downfield in comparison with the
1
same signal in 20, in the H NMR spectrum. Hydrogenolysis of
the anomeric benzyl group of 21 with H2/10% Pd(C) afforded
22 in 95% yield as a 3 : 7 α : β anomeric mixture established by
integration of the anomeric protons at δ 5.67 (J1,2 4 Hz, α
1
anomer) and 5.56 (bs, β anomer) in the H NMR spectrum. The
anomeric center was further activated by treatment with
trichloroacetonitrile and DBU to yield the precursor of the
internal Galf, β-trichloroacetimidate 11, in 90% yield.
1
described in the ESI† (Scheme 1). In the H NMR spectrum of
15, the H-2 signal appeared 0.82 ppm shifted downfield com-
pared to the same signal of its precursor 14, confirming the 2-O-
acyl substitution.
However, the moderate yield obtained for the 2-O-levulinoyl
derivative 15 which included a tedious purification, prompted us
to introduce this protecting group later. For that purpose, a bulky
TBDPS group was chosen due to its resistance to acid hydroly-
sis. Thus, treatment of 14 with 1.25 equiv of tert-butyldiphenyl-
chlorosilane and imidazole gave the silyl derivative 17
selectively (84%, Scheme 2). Interestingly, in the 1H NMR spec-
trum, the signal of H-1 (δ 4.49) appeared shifted 0.5 ppm upfield
compared to the same signal of 14. Hydrolysis of the isopropyli-
dene group of 17 with acetic acid–water at 80 °C gave benzyl 2-
O-tert-butyldiphenylsilyl-α-D-galactofuranoside (18). An analyti-
cal sample of 18 was purified for characterization. The crude
product was treated with benzoyl chloride in pyridine to afford
Synthesis of β-D-Galf-(1→2)-β-D-Galf-(1→4)-D-GlcNAc fragment 8
With trichloroacetimidate 11 in hand, the construction of the
β-Galf(1→4)GlcNAc linkage was attempted (Scheme 3). Reac-
tion of benzyl 2-acetamido-3-O-benzoyl-6-O-t-butyldiphenylsi-
lyl-2-deoxy-α-D-glucopyranoside 1224 with donor 11 in the
presence of TMSOTf as catalyst gave, diastereoselectively, disac-
charide 23 which crystallized from the crude reaction mixture in
66% yield. The signal at δ 105.5 for C-1′ in the 13C NMR spec-
trum indicated a β-configuration for the new anomeric center,
which was in agreement with the H-1′ and H-2′ resonances that
1
appeared as singlets at 5.60 and 5.03 ppm in the H NMR spec-
trum. Delevulinoylation of 23 with hydrazine in pyridine–acetic
acid55 gave acceptor 10 in 91% yield, ready for glycosylation.
Construction of the β-Galf(1→2)Galf linkage was achieved dia-
stereoselectively by glycosylation of 10 with trichloroacetimidate
950 and TMSOTf as catalyst to yield known trisaccharide 24 in
81% yield with the same physical properties compared to that
obtained by the aldonolactone approach.26 Compound 24 was
synthesized in a total yield of 21% from benzyl α-D-galactofura-
noside (13) in 11 steps diastereoselectively, while the shorter
aldonolactone approach took 9 steps in 10% total yield from
D-galactono-1,4-lactone requiring tedious purification of mix-
tures of anomers.
1
19 (81% from 17). The H NMR spectrum of 19 showed the
H-5 and H-3 signals shifted downfield at 5.71 and 6.07 ppm due
to benzoylation, compared to the same signals in 17 and 18,
confirming also the 2-OH substitution by TBDPS in 17 and 18.
The TBDPS group resisted the hydrolysis as demonstrated by
the absence of benzyl tetra-O-benzoyl-α-D-galactofuranoside as
byproduct.
In order to introduce the levulinoyl group, removal of the silyl
group was performed by treatment of 19 with acetic acid-buf-
fered tetrabutylammonium fluoride solution in THF to give the
1
2-OH free derivative 20 in 83% yield. Interestingly, in the H
Scheme 2 Synthesis of internal galactofuranosyl building block 11.
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Scheme 3 Synthesis of hexasaccharide 1 and its alditol 2.
Synthesis of hexasaccharide 1 and alditol 2
centers of the two terminal β-D-Galp (δ 99.3 and 99.1), showing
similar δ values for these centers when compared to pentasac-
charide 4.25 The signals for the other three anomeric centers of
25 appeared at 106.6 (internal GalfB), 105.9 (terminal Galf C)
and 95.9 (GlcNAc), as expected. The anomeric β-configuration
of the new glycosidic linkage was demonstrated by the coupling
constant of J1,2 7.5 Hz for the Galp(1→6) H-1D signal at δ 4.70
Desilylation of 24 with 5% HF (48 wt% in H2O) in acetonitrile
as already described26 gave acceptor 8 with the 6-OH free in
88% yield (as shown in the ESI†), ready for glycosylation. As
mentioned above, pentasaccharide 4 was synthesized by reaction
with O-[2,3-di-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
4,6-di-O-acetyl-α-D-galactopyranosyl] trichloroacetimidate25 (7)
taking advantage of the nitrile effect. In that case, only the
desired β-isomer was obtained in acetonitrile despite the absence
of anchimeric participation, whereas a mixture of β : α anomers
in 1 : 2.5 ratio was obtained when the glycosylation was carried
in CH2Cl2 as solvent.25 Reaction of acceptor 8 with ramified
Galp trichloroacetimidate 7 in acetonitrile as solvent in the pres-
ence of catalytic amounts of TMSOTf (0.16 equiv) gave hexa-
saccharide 25 diastereoselectively in 56% yield. No
α-glycosylation product was detected and unreacted acceptor 8
was also recovered, raising the yield of 25 up to 66%. On the
basis of the HSQC and COSY experiments, the 1H and 13C
NMR spectra assignments for 25 were performed. In the 13C
NMR spectrum, the signal for the new β-D-Galp(1→6) linkage
appeared at 101.8 ppm, more deshielded than for the anomeric
1
in the H NMR spectrum. On the other hand, H-2D and H-3D
resonances for the internal Galp unit appeared shielded due to
glycosylation, compared to the same signals of the benzoylated
terminal Galp. The next step was the deacylation of 25 with
sodium methoxide to give benzyl glycoside 26 in 99% yield.
Studies on 26 as substrate in the trans-sialidase reaction are cur-
rently being performed to establish if the reaction is selective for
one of the two terminal Galp as in the case of the pentasacchar-
ide 4. For that reason full NMR assignments for compound 26
1
were necessary. H and 13C NMR assignment were performed
based on HSQC, COSY and ROESY experiments. Due to signal
overlapping, the δ values of H-2D and H-3D of the internal Galp
in the 1H NMR spectrum were determined from the HSQC spec-
trum shown in the ESI.† Each terminal β-D-Galp was identified
based on a ROESY experiment. The signal of Galp(β1→2) H-1E
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at δ 4.82 correlated with the Galp(β1→6) H-2D at δ 3.95. More-
over, the Galp(β1→3) H-1F at δ 4.66 correlated with Galp
(β1→6) H-3D at δ 3.98.
reaction flask. After cooling to 0° C, benzyl bromide (4.25 mL,
36.2 mmol) was added and the suspension was stirred at 25 °C
for 16 h until TLC showed no D-galactose left. The resulting
solution was cooled to 0° C and CH3OH (0.5 mL) was slowly
added. The reaction mixture was extracted with hexane (12 ×
130 mL) and the remaining syrup was dissolved in distilled
water (70 mL) and extracted with EtOAc until TLC analysis
showed no more product in the aqueous phase. The organic
layers were combined, dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromato-
graphy (25 : 1 CH2Cl2–MeOH) to give a syrup that crystallized
from EtOAc (20 mL) to yield 5.53 g of crystalline 13 (67%): Rf
0.45 (6 : 1 CH2Cl2–MeOH), mp 124–125 °C (needles), [α]D
The hexasaccharide alditol 2 was also prepared with the aim
of comparing it with the alditol isolated from the natural
source.20 Hydrogenolysis of the benzyl glycoside of 26 was per-
formed by treatment with H2 and Pd(C). The free hexasaccharide
1 (99%) was obtained as a mixture of anomers in α : β ratio of
3 : 2, estimated by the integration of terminal Galf H-1C of the
α-anomer at δ 5.24 and GlcNAc H-1A of the β-anomer at δ 4.70,
1
in the H NMR spectrum. Further reduction of 1 with NaBH4
1
gave alditol 2 (90%), with the same H NMR spectrum as pre-
viously depicted.20
1
+94.9° (c 1, H2O). H NMR (D2O, 500 MHz): δ 7.45–7.38 (m,
5H, aromatic), 5.09 (d, 1H, J = 4.6 Hz, H-1); 4.82, 4.63 (2d, 2H,
J = 12.0 Hz, CH2Ph), 4.17 (t, 1H, J = 7.0 Hz, H-3), 4.12 (dd,
1H, J = 8.0, 4.6 Hz, H-2), 3.80 (dd, 1H, J = 7.0, 6.0 Hz, H-4),
3.73 (ddd, 1H, J = 7.0, 6.5, 4.0 Hz, H-5), 3.68 (dd, 1H, J = 11.5,
4.0 Hz, H-6a), 3.56 (dd, 1H, J = 11.5, 6.5 Hz, H-6b). 13C NMR
(D2O, 125.8 MHz): δ 137.8, 129.4, 129.1, 128.9 (C-arom.),
100.9 (C-1), 82.0 (C-4), 77.0 (C-2), 75.1 (C-3), 73.5 (C-5), 70.6
(CH2Ph), 62.8 (C-6).
Conclusion
The novel ramified hexasaccharide β-D-Galp-(1→2)-[β-D-Galp-
(1→3)]-β-D-Galp-(1→6)-[β-D-Galf-(1→2)-β-D-Galf-(1→4)]-D-GlcNAc
(1) was synthesized and its alditol confirmed the structure of the
natural oligosaccharide released from mucins of the Tulahuen
strain. The nitrilium effect mediated glycosylation allowed the
introduction of a ramified Galp trisaccharide donor that lacks
anchimeric assistance demonstrating the convenient choice of
the convergent [3 + 3] strategy. Benzyl α-D-galactofuranoside,
synthesized in one step from galactose, proved to be a suitable
precursor for the construction of internal Galf-containing oligo-
saccharides. This precursor together with the robust trichloroace-
timidate method of glycosylation allowed the synthesis of the
benzyl hexasaccharide 26, useful also for studies on the
unknown Galf transferases of T. cruzi.
Anal. calcd for C13H18O6: C 57.77, H 6.71. Found: C 57.67,
H 6.52.
Benzyl 5,6-O-isopropylidene-α-D-galactofuranoside (14). To a
cooled solution (0 °C) of 13 (2.05 g, 7.58 mmol) in a mixture of
2,2-dimethoxypropane (3.4 mL, 27.2 mmol) and acetone
(17 mL), p-toluenesulfonic acid (30 mg, 0.17 mmol) was added
with stirring. After 45 min, 28% NH4OH (0.2 mL) was added
until pH = 10 and the mixture was concentrated under vacuum.
Crystallization of the residue from hexane–EtOAc (13 : 10) gave
2.22 g of 14 (94%): Rf 0.30 (20 : 1 CH2Cl2–MeOH); mp
100–101 °C, [α]D +65.7° (c 1, CHCl3); 1H NMR (CDCl3,
500 MHz): δ 7.35–7.26 (m, 5H, arom.), 4.98 (d, 1H, J = 4.2 Hz,
H-1), 4.85, 4.52 (2d, 2H, J = 11.6 Hz, CH2Ph), 4.17 (apparent q,
1H, J = 6.7 Hz, H-5), 4.05–4.00 (m, 2H, H-2, H-3), 3.99 (dd,
1H, J = 8.6, 6.6 Hz, H-6a), 3.90 (dd, 1H, J = 7.0, 8.6 Hz, H-6b),
3.80 (t, 1H, J = 7.0 Hz, H-4), 3.68 (bs, 1H, –OH), 3.10 (d, 1H,
J = 9.4 Hz, –OH), 1.43, 1.37 (2s, 6H, (CH3)2C). 13C NMR
Experimental
General
TLC was performed on 0.2 mm silica gel 60 F254 aluminium
supported plates. Detection was effected by exposure to UV
light or by spraying with 10% (v/v) H2SO4 in EtOH and char-
ring. Column chromatography was performed on silica gel 60
(230–400 mesh). NMR spectra were recorded with a Bruker
AVANCE II 500 spectrometer at 500 MHz (1H) and 125.8 MHz
(13C) or with a Bruker AC 200 at 200 MHz (1H) and 50.3 MHz
(13C). Chemical shifts are given relative to the signal of internal
(CDCl3, 125.8 MHz):
δ 136.9–128.0 (C-arom.), 109.7
((CH3)2C), 99.8 (C-1), 81.5 (C-4), 78.0 (C-2), 77.5 (C-5), 76.3
(C-3), 69.7 (CH2Ph), 64.9 (C-6), 26.5, 25.3 ((CH3)2C).
Anal. calcd for C16H22O6: C 61.92, H 7.15. Found: C 61.54,
H 6.98.
acetone standard at 2.22 and 30.89 ppm for H NMR and 13C
1
NMR spectra, respectively when recorded in D2O. H and 13C
1
assignments were supported by 2D COSY, and HSQC exper-
iments. ROESY experiment was performed when indicated.
High resolution mass spectra (HRMS) were recorded on a
BRUKER micrOTOF-Q II spectrometer. Melting points were
determined with a Fisher–Johns apparatus and are uncorrected.
Optical rotations were measured with a Perkin-Elmer 343 polari-
meter with a path length of 1 dm at 25 °C.
Benzyl 2-O-tert-butyldiphenylsilyl-5,6-O-isopropylidene-α-D-
galactofuranoside (17). To a cooled solution (0 °C) of 14 (1.9 g,
6.16 mmol) and imidazole (0.5 g, 7.4 mmol) in anhydrous DMF
(15 mL), t-butyldiphenylsilylchloride (1.8 mL, 7.68 mmol, 1.25
equiv) was slowly added with stirring. After stirring for 20 h at
25 °C, the mixture was diluted with CH2Cl2 (250 mL), washed
with H2O (3 × 200 mL), dried (Na2SO4), filtered and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (6 : 1 hexane–EtOAc) to afford
syrupy 17 (2.89 g, 84%): Rf 0.38 (4 : 1 hexane–EtOAc), [α]D
Benzyl α-D-galactofuranoside (13). Dried ground D-galactose
(5.5 g, 30.5 mmol) was partially dissolved in DMPU (50 mL)
under argon atmosphere and was slowly cannula-added to 60%
oil suspension of sodium hydride (1.82 g, 47.7 mmol) pre-
viously washed with hexane (2 × 40 mL). The remaining
D-galactose with additional DMPU (12 mL) was added to the
1
+60.0° (c 1, CHCl3); H NMR (CDCl3, 200 MHz): δ 7.74–7.62
(m, 4H, arom.), 7.44–7.24 (m, 11H, arom.), 4.76, 4.33 (2d, 2H,
J = 11.9 Hz, CH2Ph), 4.49 (d, 1H, J = 4.2 Hz, H-1), 4.28–4.16
This journal is © The Royal Society of Chemistry 2012
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(m, 2H, H-5, H-3), 4.12 (dd, 1H, J = 7.7, 4.2 Hz, H-2), 3.95 (dd,
1H, J = 8.5, 6.4 Hz, H-6a), 3.84 (dd, 1H, J = 8.5, 7.0 Hz, H-6b),
3.70 (t, 1H, J = 7.2 Hz, H-4), 1.71 (d, 1H, J = 4.2 Hz, OH),
1.40, 1.35 (2s, 6H, (CH3)2C), 1.09 (s, 9H, (CH3)3CSi). 13C
NMR (CDCl3, 50.3 MHz): δ 137.7–127.5 (C-arom.), 109.6
((CH3)2C), 100.4 (C-1), 81.4 (C-4), 79.0, 78.1, 76.2 (C-2, C-3,
C-5), 69.0 (CH2Ph), 64.9 (C-6), 26.8 ((CH3)3CSi), 26.6, 25.3
((CH3)2C), 19.2 (CH3)3CSi); HRMS (ESI/APCI) m/z calcd for
C32H40NaO6Si (M + Na)+: 571.2486. Found: 571.2508.
2 equiv), and the stirring continued at rt for 6 h. The mixture
was diluted with CH2Cl2 (200 mL), washed with H2O (5 ×
150 mL), dried (Na2SO4), filtered and concentrated under
reduced pressure. Column chromatography of the residue
(15 : 4 hexane–EtOAc) gave 20 (1.3 g, 83%) as a foamy solid: Rf
0.49 (3 : 2 hexane–EtOAc), [α]D +49.7° (c 0.5, CHCl3); 1H
NMR (CDCl3, 500 MHz): δ 8.05–7.93 (m, 6H, arom.),
7.53–7.23 (m, 14H, arom.), 5.78 (dt, 1H, J = 5.5, 4.0 Hz, H-5),
5.71 (t, 1H, J = 7.0 Hz, H-3), 5.20 (d, 1H, J = 4.5 Hz, H-1),
4.98, 4.63 (2d, 2H, J = 11.5 Hz, CH2Ph), 4.76 (dd, 1H, J = 12.0,
4.0 Hz, H-6a), 4.66 (dd, 1H, J = 12.0, 6.0 Hz, H-6b), 4.55 (dd,
1H, J = 6.5, 5.5 Hz, H-4), 4.49 (dd, 1H, J = 7.0, 4.5 Hz, H-2),
3.00 (bs, 1H, OH). 13C NMR (CDCl3, 125.8 MHz): δ 166.0,
165.9, 165.7 (PhCO), 136.6–128.0 (C-arom.), 100.2 (C-1), 79.1
(C-4), 78.0 (C-3), 76.5 (C-2), 71.5 (C-5), 70.2 (CH2Ph), 63.0
(C-6); HRMS (ESI/APCI) m/z calcd for C34H30NaO9 (M +
Na)+: 605.1782, found: 605.1779.
Benzyl
3,5,6-tri-O-benzoyl-2-O-tert-butyldiphenylsilyl-α-D-
galactofuranoside (19). A suspension of 17 (1.86 g, 3.39 mmol)
in a mixture of 3 : 2 HOAc–H2O (11.6 mL) was warmed at
80 °C with stirring. After 10 min, the solution was cooled to rt
and pyridine (11.5 mL) was added to avoid decomposition of the
product during concentration. The residue was co-evaporated
with toluene (8 × 4 mL) to give 1.72 g of crude benzyl 2-O-tert-
butyldiphenylsilyl-α-D-galactofuranoside (18). Purification of an
analytical sample gave pure 18 as a syrup: Rf 0.64 (EtOAc); [α]D
Benzyl 3,5,6-tri-O-benzoyl-2-O-levulinoyl-α-D-galactofurano-
side (21). To a cooled solution (0 °C) of 20 (1.19 g, 2.04 mmol)
and DMAP (74.8 mg, 0.61 mmol) in CH2Cl2 (9 mL), levulinic
acid (229 μL, 2.24 mmol) was added with stirring followed by
DCC (462 mg, 2.24 mmol) and the mixture was allowed to
reach room temperature. After 14 h of stirring, the solution was
diluted with CH2Cl2 (100 mL), washed with 10% NaHCO3 (3 ×
80 mL), water (3 × 80 mL), dried and concentrated under
reduced pressure. The residue was purified by column chromato-
graphy (5 : 2 hexane–EtOAc) to give 1.35 g of 21 (97%; Rf 0.41,
1
+57.9° (c 1, CHCl3); H NMR (CDCl3, 500 MHz): δ 7.71–7.65
(m, 4H, arom.), 7.43–7.29 (m, 11H, arom.), 4.57, 4.35 (2d, 2H,
J = 11.5 Hz, CH2Ph), 4.42 (d, 1H, J = 4.0 Hz, H-1), 4.42 (t, 1H,
J = 7.5 Hz, H-3), 4.12 (dd, 1H, J = 8.0, 4.5 Hz, H-4), 3.71 (dd,
1H, J = 7.0, 4.5 Hz, H-2), 3.61 (apparent q, 1H, J = 5.0 Hz,
H-5), 3.58 (dd, 1H, J = 11.5, 5.5 Hz, H-6a), 3.55 (dd, 1H, J =
11.5, 5.0 Hz, H-6b), 2.74 (bs, 3H, –OH), 1.08 (s, 9H,
(CH3)3CSi). 13C NMR (CDCl3, 125.8 MHz): δ 137.2–127.7 (C-
arom.), 100.9 (C-1), 82.2 (C-2), 78.8 (C-4), 75.5 (C-3), 72.3
(C-5), 70.3 (CH2Ph), 63.9 (C-6), 26.8 (CH3)3CSi), 19.2
(CH3)3CSi); HRMS (ESI/APCI) m/z calcd for C29H36NaO6Si
(M + Na)+: 531.2173. Found: 531.2173.
1
3 : 2 hexane–EtOAc) as a syrup: [α]D +58.4° (c 1, CHCl3); H
NMR (CDCl3, 500 MHz): δ 8.06–7.92 (m, 6H, arom.),
7.53–7.20 (m, 14H, arom.), 6.03 (t, 1H, J = 6.8 Hz, H-3), 5.80
(apparent q, 1H, J = 5.0 Hz, H-5), 5.44 (d, 1H, J = 4.5 Hz, H-1),
5.29 (dd, 1H, J = 7.0, 4.5 Hz, H-2), 4.92, 4.53 (2d, 2H, J = 11.5 Hz,
CH2Ph), 4.76 (dd, 1H, J = 12.0, 4.5 Hz, H-6a), 4.67 (dd, 1H,
J = 12.0, 6.0 Hz, H-6b), 4.56 (t, 1H, J = 5.7 Hz, H-4), 2.70–2.56
(m, 4H, (CH2)2, 2.09 (s, 3H, CH3CO). 13C NMR (CDCl3,
125.8 MHz): δ 206.0 (CH3CO), 171.9 (CH2CO), 165.9, 165.6,
165.4 (PhCO), 137.0–127.5 (C-arom.), 99.6 (C-1), 78.7 (C-4),
76.9 (C-2), 74.5 (C-3), 71.4 (C-5), 70.4 (CH2Ph), 62.9 (C-6),
37.6, 27.6 ((CH2)2), 29.6 (CH3CO); HRMS (ESI/APCI)
m/z calcd for C39H36NaO11 (M + Na)+: 703.2150. Found:
703.2143.
Crude 18 was dissolved in dry pyridine (9 mL), cooled to
0 °C and benzoyl chloride (1.45 mL, 12.5 mmol, 3.7 equiv) was
slowly added with stirring. After stirring for 30 min at 0 °C and
16 h at rt, the mixture was poured into ice-water (150 g). After
20 min, the mixture was extracted with CH2Cl2 (150 mL) and
the organic phase was washed with 10% HCl (3 × 100 mL),
H2O (100 mL), 10% NaHCO3 (3 × 80 mL), H2O (3 × 80 mL)
until pH 7, dried (Na2SO4), and concentrated. Silica gel column
chromatography (10 : 1 hexane–EtOAc) of the residue gave 19
(2.24 g, 80.5% from 17) as a foamy solid: Rf 0.50 (20 : 1
1
toluene–EtOAc), [α]D +43.8° (c 1, CHCl3); H NMR (CDCl3,
500 MHz): δ 8.05–7.52 (4m, 11H, arom.), 7.48–7.26 (m, 15H,
arom.), 7.21–7.11 (2m, 4H, arom.), 6.07 (dd, 1H, J = 7.8, 6.9 Hz,
H-3), 5.71 (dt, 1H, J = 5.4, 4.5 Hz, H-5), 4.87, 4.30 (2d, 2H, J =
11.8 Hz, CH2Ph), 4.74 (dd, 1H, J = 11.9, 4.3 Hz, H-6a), 4.62
(dd, 1H, J = 11.9, 5.7 Hz, H-6b), 4.55 (dd, 1H, J = 7.8, 4.6 Hz,
H-2), 4.49 (d, 1H, J = 4.6 Hz, H-1), 4.34 (dd, 1H, J = 6.9,
5.2 Hz, H-4), 1.00 (s, 9H, (CH3)3CSi); 13C NMR (CDCl3,
125.8 MHz): δ 165.9, 165.8, 165.6 (PhCO), 137.3–127.6
(C-arom.), 100.3 (C-1), 78.3 (C-4), 76.9, 76.85 (C-2, C-3), 71.9
(C-5), 69.7 (CH2Ph), 62.9 (C-6), 26.7 ((CH3)3CSi), 19.1
(CH3)3CSi); HRMS (ESI/APCI) m/z calcd for C50H48NaO9Si
(M + Na)+: 843.2960. Found: 843.2978.
3,5,6-Tri-O-benzoyl-2-O-levulinoyl-α,β-D-galactofuranose (22).
A mixture of 21 (1.45 g, 2.13 mmol) dissolved in EtOAc
(45 mL) and 10% Pd(C) Deguzza type (530 mg) was hydrogen-
ated for 72 h at 45 psi (3 atm) at rt. The catalyst was filtered and
the filtrate was concentrated under vacuum to give 22 (1.2 g,
95%) as an amorphous solid in a 3 : 7 α : β anomeric mixture: Rf
1
0.40 (1 : 1 hexane–EtOAc), [α]D −10.1° (c 1, CHCl3), H NMR
(CDCl3, 500 MHz): δ for the β-anomer 8.15–7.95 (m, 6H,
arom.), 7.60–7.35 (m, 9H, arom.), 6.03 (dt, 0.7H, J = 7.0, 4.3
Hz, H-5), 5.91 (dt, 0.3H, J = 6.5, 3.5 Hz, H-5 α anomer), 5.84
(t, 0.3H, J = 4.7 Hz, H-3 α anomer), 5.67 (d, 0.3H, J = 4.0 Hz
H-1 α anomer), 5.56 (bs, 0.7H, H-1), 5.44 (dd, 0.7H, J = 4.5,
1.0 Hz, H-3), 5.33 (dd, 0.3H, J = 4.7, 4.0 Hz, H-2 α anomer),
5.27 (d, 0.7H, J = 1.0 Hz, H-2), 4.81 (dd, 0.3H, J = 12.0, 3.5
Hz, H-6a α anomer), 4.78 (t, 0.7H, J = 4.3 Hz, H-4), 4.74 (dd,
0.7H, J = 12.0, 4.5 Hz, H-6a), 4.69 (dd, 0.3H, J = 12.0, 6.5 Hz,
Benzyl 3,5,6-tri-O-benzoyl-α-D-galactofuranoside (20). To a
stirred cold solution (0 °C) of 19 (2.22 g, 2.7 mmol) in anhy-
drous DMF (10 mL), was added glacial acetic acid (286 μl, 1.85
equiv) and a solution of 1 M TBAF in anhydrous THF (5.4 mL,
6328 | Org. Biomol. Chem., 2012, 10, 6322–6332
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H-6b α anomer), 4.68 (dd, 0.7H, J = 12.0, 7.0 Hz, H-6b), 4.47
(dd, 0.7H, J = 6.5, 4.5 Hz, H-4 α anomer), 3.31 (bs, 0.7H,
HO-1), 2.77–2.68 (m, 1.3H, (CH2)2 α and β anomers),
2.62–2.44 (m, 2H, (CH2)2 α and β anomers), 2.33–2.27 (m,
0.7H, (CH2)2 Lev α and β anomers), 2.14, 2.13 (2s, 3H, CH3CO
α and β anomers); 13C NMR (CDCl3, 125.8 MHz): δ for the
β-anomer 206.8, 206.3 (CH3CO α and β anomers), 171.8
(CH2CO), 171.7 (CH2CO α anomer), 166.1–165.4 (PhCO),
133.5–128.3 (C-arom.), 100.9 (C-1), 96.1 (C-1 α anomer), 82.0
(C-4), 81.9 (C-2), 79.3 (C-4 α anomer), 77.7 (C-3), 77.0 (C-2 α
anomer), 75.9 (C-3 α anomer), 71.9 (C-5 α anomer), 70.5 (C-5),
63.4 (C-6), 63.1 (C-6 α anomer), 37.9, 27.7 ((CH2)2 α anomer),
37.7, 27.5 ((CH2)2), 29.7 (CH3CO α and β anomers); HRMS
(ESI/APCI) m/z calcd for C32H30NaO11 (M + Na)+: 613.1680.
Found: 613.1687.
(m, 3H, arom.), 7.52–7.48 (m, 4H, arom.), 7.44–7.24 (m, 18H,
arom.), 5.74 (d, 1H, J = 9.7 Hz, NH), 5.66 (ddd, 1H, J = 8.3,
3.5, 2.8 Hz, H-5′), 5.60 (bs, 1H, H-1′), 5.57 (dd, 1H, J = 10.7,
9.7 Hz, H-3), 5.31 (d, 1H, J = 3.8 Hz, H-3′), 5.03 (s, 1H, H-2′),
4.91 (d, 1H, J = 3.8 Hz, H-1), 4.62, 4.44 (2d, 2H, J = 11.9 Hz,
CH2Ph), 4.49 (ddd, 1H, J = 10.7, 9.7, 3.8 Hz, H-2), 4.38 (t, 1H,
J = 9.5 Hz, H-4), 4.38 (dd, 1H, J = 12.3, 8.3 Hz, H-6a′), 4.29
(dd, 1H, J = 12.3, 2.8 Hz, H-6b′), 4.09 (t, 1H, J = 3.7 Hz, H-4′),
4.07 (dd, 1H, J = 12.3, 3.1 Hz, H-6a), 3.84 (m, 2H, H-6b, H-5),
2.36 (t, 2H, J = 7.2 Hz, (CH2)2), 2.02 (t, 2H, J = 7.2 Hz,
(CH2)2), 2.00 (s, 3H, CH3CO Lev), 1.76 (s, 3H, CH3CONH),
1.10 (s, 9H, (CH3)3CSi); 13C NMR (CDCl3, 125.8 MHz):
δ 205.6 (CH3CO Lev), 171.6 (CH2COO), 169.9 (CH3CONH),
166.7, 165.9, 165.4 (PhCO), 136.8–127.5 (C-arom.), 105.5
(C-1′), 96.4 (C-1), 83.1 (C-4′), 81.3 (C-2′), 77.5 (C-3′), 72.2
(C-3), 71.9 (C-4), 71.8 (C-5), 70.3 (C-5′), 69.7 (CH2Ph), 63.8
(C-6′), 62.4 (C-6), 52.4 (C-2), 37.6, 27.2 ((CH2)2), 29.5
(CH3CO Lev), 26.7 ((CH3)3CSi), 23.1 (CH3CONH), 19.5
((CH3)3CSi).
O-(3,5,6-tri-O-benzoyl-2-O-levulinoyl-β-D-galactofuranosyl)
trichloroacetimidate (11). To a stirred solution of 22 (1.79 g,
3.03 mmol) and trichloroacetonitrile (1.52 mL, 15.2 mmol) in
anhydrous CH2Cl2 (20 mL), cooled to 0 °C, DBU (128 μl,
0.91 mmol, 0.3 equiv) was slowly added and the mixture was
allowed to reach rt. After 25 min, the solution was concentrated
under reduced pressure at room temperature, and the residue was
purified by column chromatography (7 : 1 : 0.2 toluene–EtOAc–
TEA) to afford syrupy 11 (2.01 g, 90%): Rf 0.5 (5 : 1 toluene–
EtOAc); [α]D −24.5° (c 0.7, CHCl3); 1H NMR (CDCl3,
500 MHz): δ 8.70 (s, 1H, NH), 8.15–7.95 (m, 6H, arom.),
7.60–7.34 (m, 9H, arom.), 6.55 (bs, 1H, H-1), 6.10 (ddd, 1H,
J = 6.2, 5.4, 3.3 Hz, H-5), 5.55 (dd, 1H, J = 3.8, 0.6 Hz, H-3),
5.51 (d, 1H, J = 0.6 Hz, H-2), 4.78 (t, 1H, J = 3.5 Hz, H-4),
4.74 (m, 2H, H-6a, H-6b), 2.69–2.52 (m, 2H, (CH2)2),
2.41–2.35 (m, 1H, (CH2)2), 2.22–2.14 (m, 1H, (CH2)2), 2.12
(s, 3H, CH3CO); 13C NMR (CDCl3, 125.8 MHz): δ 205.8
(CH3CO), 171.4 (CH2CO); 165.6, 165.5, 164.1 (PhCO), 160.2
(CNH), 133.7–128.4 (C-arom.), 102.8 (C-1), 84.9 (C-4), 79.9
(C-2), 77.0 (C-3), 69.9 (C-5), 63.4 (C-6), 37.7, 27.4 ((CH2)2),
29.7 (CH3CO); HRMS (ESI/APCI) m/z calcd for
C34H30Cl3NNaO11 (M + Na)+: 756.0777. Found: 756.0798.
Anal. calcd for C70H71NO17Si: C 68.55, H 5.84. Found:
C 68.55, H 5.55.
Benzyl 3,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→4)-2-acet-
amido-3-O-benzoyl-6-O-tert-butyldiphenylsilyl-2-deoxy-α-D-glu-
copyranoside (10). To a solution of 23 (826 mg, 0.68 mmol) in
a mixture of pyridine–HOAc (4 : 1 v/v, 7 mL), cooled to 0 °C,
hydrazine monohydrate (212 μL, 4.4 mmol, 6 equiv) was added
with stirring. After 20 min at rt, TLC analysis showed complete
conversion of 23 (Rf 0.43, 3 : 2 hexane–EtOAc, twice developed)
into a new product (Rf 0.48), and the mixture was diluted with
CH2Cl2 (180 mL) and subsequently washed with 5% HCl (3 ×
70 mL), 10% NaHCO3 (2 × 70 mL) and H2O (70 mL), dried
with Na2SO4 and concentrated. The crude product was purified
by column chromatography (3 : 2 hexane–EtOAc) to give glassy
10 (689 mg, 91%): Rf 0.5 (3 : 2 hexane–EtOAc, twice devel-
1
oped), [α]D +15.5° (c 1, CHCl3); H NMR (CDCl3, 500 MHz):
δ 8.03–7.57 (m, 13H, arom.), 7.52–7.18 (m, 22H, arom.), 5.79
(d, 1H, J = 9.7 Hz, NH), 5.55 (dd, 1H, J = 10.8, 9.4 Hz, H-3),
5.48 (dt, 1H, J = 7.4, 3.9 Hz, H-5′), 5.38 (bs, 1H, H-1′), 4.98 (d,
1H, J = 3.7 Hz, H-1), 4.90 (dd, 1H, J = 5.6, 2.4 Hz, H-3′), 4.67,
4.48 (2d, 2H, J = 11.9 Hz, CH2Ph), 4.48 (ddd, 1H, J = 10.8, 9.7,
3.7 Hz, H-2), 4.24 (t, 1H, J = 10.0 Hz, H-4), 4.22 (dd, 1H, J =
12.0, 7.4 Hz, H-6a′), 4.22 (dd, 1H, J = 5.6, 3.8 Hz, H-4′), 4.19
(dd, 1H, J = 12.0, 4.0 Hz, H-6b′), 4.09 (ddd, 1H, J = 5.1, 2.4,
1.0 Hz, H-2′), 3.88 (dd, 1H, J = 11.8, 3.6 Hz, H-6a), 3.82 (dd,
1H, J = 11.8, 1.6 Hz, H-6a), 3.80 (m, 1H, H-5), 2.79 (d, 1H, J =
5.1 Hz, 2-OH), 1.79 (s, 3H, CH3CONH), 1.07 (s, 9H,
Benzyl 3,5,6-tri-O-benzoyl-2-O-levulinoyl-β-D-galactofurano-
syl-(1→4)-2-acetamido-3-O-benzoyl-6-O-tert-butyldiphenylsilyl-
2-deoxy-α-D-glucopyranoside (23). A solution of trichloroaceti-
midate 11 (2.28 g, 3.10 mmol, 1.1 equiv) in anhydrous CH2Cl2
(30 mL) was added to a mixture of benzyl 2-acetamido-3-O-
benzoyl-6-O-t-butyldiphenylsilyl-2-deoxy-α-D-glucopyranoside
(12)24 (1.85 g, 2.83 mmol) and activated 4 Å powdered molecu-
lar sieves (3.4 g) with stirring under argon atmosphere. After
10 min of vigorous stirring at rt, the mixture was cooled to
−20 °C and TMSOTf (110 μl, 0.61 mmol, 0.2 equiv) was added,
and stirring continued for 2 h at −20 °C. TLC monitoring
showed a small amount of unreacted 12 and the mixture was
stirred for additional 16 h (−20 °C), then neutralized with Et3N
(85 μl, 0.61 mmol, 0.2 equiv), filtered and concentrated under
reduced pressure to give an off-white residue (4.3 g). The crude
product was recrystallized twice from CH3OH (from 9.5 mL and
4 mL, respectively) yielding 2.3 g of pure 23 (66%): Rf 0.35
(3 : 2 : 0.2 hexane–EtOAc–TEA), mp 162–163 °C (CH3OH);
[α]D +12.0° (c 2, CHCl3); 1H NMR (CDCl3, 500 MHz): δ
7.99–7.93 (m, 6H, arom.), 7.83–7.77 (m, 4H, arom.), 7.68–7.61
(CH3)3CSi); 13C NMR (CDCl3, 125.8 MHz):
δ 170.0
(CH3CONH), 166.9, 165.8, 165.6 (PhCO), 135.9–127.5
(C-arom.), 108.6 (C-1′), 96.4 (C-1), 81.3 (C-3′), 81.0 (C-2′),
80.5 (C-4′), 74.1 (C-4), 72.5 (C-3), 71.8 (C-5), 70.3 (C-5′), 69.6
(CH2Ph), 63.4 (C-6′), 62.3 (C-6), 52.3 (C-2), 26.8 ((CH3)3CSi),
23.1 (CH3CONH), 19.3 ((CH3)3CSi).
Anal. calcd for C65H65NO15Si: C 69.19, H 5.81, N 1.24.
Found: C 69.08, H 5.55, N 1.37.
Benzyl
2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl-(1→2)-
3,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→4)-2-acetamido-3-
O-benzoyl-6-O-tert-butyldiphenylsilyl-2-deoxy-α-D-glucopy-
This journal is © The Royal Society of Chemistry 2012
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ranoside (24).26 To a solution of dried acceptor 10 (956 mg,
0.85 mmol) and O-(2,3,5,6-tetra-O-benzoyl-β-D-galactofurano-
syl) trichloroacetimidate50 (9, 780 mg, 1.05 mmol, 1.2 equiv) in
freshly distilled anh CH2Cl2 (22 mL), activated 4 Å powdered
molecular sieves (1.6 g) were added, and the suspension was
stirred under argon atmosphere for 10 min. The mixture was then
cooled to −20 °C and after 15 min of vigorous stirring, TMSOTf
(18.5 μl, 0.10 mmol, 0.12 equiv) was added and the stirring con-
tinued for 23 h at −20 °C until TLC examination showed total
consumption of acceptor 10 (Rf 0.41, 1.0 : 0.9 hexane–EtOAc).
The reaction was quenched by addition of triethylamine (15 μl,
0.11 mmol) and the mixture was allowed to reach room tempera-
ture and then filtered over Celite. The filtrate was concentrated
under vacuum and the residue was purified by column chromato-
graphy (1.9 : 1 hexane–EtOAc) to give 1.18 g of 24 (81%) as an
amorphous solid (Rf 0.32 (1 : 0.9 hexane–EtOAc)). Physical
properties and 1H and 13C NMR spectra matched lit.26 mp
87–89 °C (hexane–toluene);26 [α]D +11° (c 1, CHCl3);26
1H NMR (CDCl3, 500 MHz): δ 8.06–7.59 (m, 21H), 7.53–7.23
(m, 34H), 5.90 (m, 1H), 5.81 (d, 1H, J = 9.7 Hz), 5.63 (ddd, 1H,
J = 8.0, 5.7, 2.9 Hz), 5.58 (bs, 1H), 5.58–5.56 (m, 1H), 5.54
(dd, 1H, J = 10.9, 9.6 Hz), 5.43 (bs, 1H), 5.37 (d, 1H, J = 3.4
Hz), 5.31 (d, 1H, J = 1.7 Hz), 4.92 (d, 1H, J = 3.8 Hz), 4.67,
4.44 (2d, 2H, J = 11.9 Hz), 4.53 (dd, 1H, J = 12.0, 7.0 Hz), 4.49
(dd, 1H, J = 5.6, 3.2 Hz), 4.44 (dd, 1H, J = 12.3, 2.9 Hz), 4.42
(ddd, 1H, J = 10.9, 9.7, 3.8 Hz), 4.37 (dd, 1H, J = 12.0, 4.0 Hz),
4.35 (dd, 1H, J = 12.3, 8.0 Hz), 4.23 (dd, 1H, J = 5.7, 3.4 Hz),
4.20 (bs, 1H), 4.16 (t, 1H, J = 9.6 Hz), 3.92 (dd, 1H, J = 11.7,
4.1 Hz), 3.87–3.82 (m, 2H), 1.78 (s, 3H), 0.99 (s, 9H); 13C
NMR (CDCl3, 50.3 MHz): δ 169.9, 167.0, 166.0, 165.8, 165.73,
165.65, 165.6, 165.3, 136.8–127.5 (C-arom.), 106.1, 105.4,
96.2, 84.7, 82.6, 82.3, 81.9, 77.6, 77.2, 72.3, 72.1, 70.5, 70.2,
69.4, 63.8, 63.4, 62.5, 52.5, 26.8, 23.1, 19.2.
unreacted acceptor 8 (60 mg, 15%, Rf 0.60; 1 : 2 toluene–
EtOAc). The next fraction afforded 360 mg of hexasaccharide 25
as a glassy solid (56%): Rf 0.37 (1 : 2 toluene–EtOAc), [α]D
+6.9° (c 1.5, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 8.07–7.82
(m, 16H, arom.), 7.59–7. 32 (m, 29H, arom.), 6.05 (dt, 1H, J =
7.5, 4.0 Hz, H-5C), 5.81 (d, 1H, J = 9.5 Hz, NH), 5.70 (dd, 1H,
J = 5.5, 1.0 Hz, H-3C), 5.65 (ddd, 1H, J = 8.5, 6.0, 2.5 Hz,
H-5B), 5.63 (bs, 1H, H-1C), 5.57 (dd, 1H, J = 10.5, 9.5 Hz,
H-3A), 5.47 (d, 1H, J = 3.5 Hz, H-3B), 5.45 (bs, 1H, H-1B), 5.41
(d, 1H, J = 1.0 Hz, H-2C), 5.36 (d, 1H, J = 3.0 Hz H-4F), 5.34
(d, 1H, J = 3.5 Hz, H-4E), 5.26 (d, 1H, J = 3.5 Hz, H-4D), 5.18
(dd, 1H, J = 10.0, 8.0 Hz, H-2F), 5.16 (dd, 1H, J = 10.5, 3.5 Hz,
H-3E), 5.08 (d, 1H, J = 8.0 Hz, H-1F), 5.05 (dd, 1H, J = 10.5,
8.0 Hz, H-2E), 5.01 (dd, 1H, J = 10.0, 3.5 Hz, H-3F), 4.91 (d,
1H, J = 3.5 Hz, H-1A), 4.85, 4.52 (2d, 2H, J = 11.5 Hz, CH2Ph),
4.83 (d, 1H, J = 8.0 Hz, H-1E), 4.77 (dd, 1H, J = 5.5, 4.0 Hz,
H-4C), 4.74 (dd, 1H, J = 11.5, 7.5 Hz, H-6aC), 4.70 (d, 1H, J =
7.5 Hz, H-1D), 4.62 (dd, 1H, J = 11.5, 4.0 Hz, H-6bC), 4.47 (dd,
1H, J = 12.0, 2.5 Hz, H-6aB), 4.38 (bs, 1H, H-2B), 4.38–4.31
(m, 3H, H-2A; H-4B and H-6bB), 4.32–4.24 (m, 3H, H-5A,
H-6aA, H-6aF), 4.17–4.10 (m, 2H, H-6bF, H-6aE), 4.09–4.03 (m,
3H, H-6bE, H-3D, H-5F), 3.99 (dd, 1H, J = 12.0, 7.0 Hz, H-6aD),
3.96–3.90 (m, 2H, H-6bD, H-5E), 3.88 (dd, 1H, J = 9.0, 7.5 Hz,
H-2D), 3.80–3.74 (m, 2H, H-6bA, H-5D), 3.70 (t, 1H, J = 9.5 Hz,
H-4A), 2.15, 2.08, 2.03, 1.98, 1.97, 1.96, 1.84 (8s, 27H,
CH3CO), 1.75 (s, 3H, CH3CONH); 13C NMR (CDCl3,
125.8 MHz): δ 170.3, 170.2 (× 2), 170.1, 169.9 (× 2), 169.8,
169.7, 169.6, 168.8 (CH3CO), 166.8, 166.1, 165.8, 165.6 (× 2),
165.5, 165.3, 165.2 (PhCO), 136.6, 133.4–132.8 (C-arom.),
129.9–128.2 (C-arom.), 106.6 (C-1B), 105.9 (C-1C), 101.8
(C-1D), 99.3 (C-1F)), 99.1 (C-1E), 95.9 (C-1A), 85.3 (C-2B), 82.6
(C-2C), 82.2 (C-4B), 81.7 (C-4C), 79.5 (C-2D), 77.3 (C-3B), 76.9
(C-3C), 75.7 (C-4A), 74.7 (C-3D), 71.7 (C-3A), 71.1 (C-3F), 70.7
(C-5E), 70.6 (C-5A), 70.5 (C-5D, C-5F), 70.3 (C-5, C-5C), 70.0
(C-3E, C-2F), 69.8 (C-2E), 69.6 (CH2Ph), 69.0 (C-6A), 68.2
(C-4D), 67.0, 66.7 (C-4E, C-4F), 63.4 (C-6B), 63.3 (C-6C), 61.2
(C-6D), 60.8 (C-6E), 60.3 (C-6F), 52.1 (C-2A), 23.0
(CH3CONH), 20.9, 20.8, 20.7, 20.6, 20.5 (× 2), 20.4 (× 2)
(CH3CO). The assignments for the Galp-β(1→2) (E) and Galp-
β(1→3) (F) could be interchanged. HRMS (ESI/APCI) m/z calcd
for C121H123NNaO49 (M + Na)+: 2396.7056. Found: 2396.6966.
Benzyl
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→2)-
[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)]-4,6-di-O-
acetyl-β-D-galactopyranosyl-(1→6)-[2,3,5,6-tetra-O-benzoyl-β-D-
galactofuranosyl-(1→2)-3,5,6-tri-O-benzoyl-β-D-galactofuranosyl-
(1→4)]-2-acetamido-3-O-benzoyl-2-deoxy-α-D-glucopyranoside
(25). To a flask containing O-[2,3-di-O-(2,3,4,6-tetra-O-acetyl-
β-D-galactopyranosyl)-4,6-di-O-acetyl-α-D-galactopyranosyl] tri-
chloroacetimidate25 7 (320 mg, 0.30 mmol, 1.1 equiv) and acti-
vated 4 Å powdered molecular sieves (210 mg), a solution of
acceptor 8 (400 mg, 0.27 mmol) in freshly distilled anhydrous
CH3CN (15 mL) was added, and the suspension was cooled to
−20 °C under argon atmosphere. After 10 min of vigorous stir-
ring, TMSOTf (8 μL, 44 μmol, 0.16 equiv) was added and the
stirring continued for 24 h at −20 °C. TLC analysis showed the
presence of unreacted acceptor 8 (Rf 0.65; 2 : 3 : 0.2 toluene–
EtOAc–TEA), trisaccharide O-(2,3,4,6-tetra-O-acetyl-β-D-galac-
topyranosyl)-(1→2)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyrano-
syl-(1→3)]-4,6-di-O-acetyl-α,β-D-galactopyranose (Rf 0.3) due
to remaining trichloroacetimidate 7, and a new spot (Rf 0.38).
The mixture was allowed to reach 0 °C and was stirred for an
additional 20 h. The reaction was stopped by the addition of
Et3N (6.3 μL, 45 μmol), filtered and the molecular sieves were
washed with CH3CN. The filtrate was concentrated under
reduced pressure and the residue was purified by column chrom-
atography (1 : 1 toluene–EtOAc). The first fraction gave
Benzyl
β-D-galactopyranosyl-(1→2)-[β-D-galactopyranosyl-
(1→3)]-β-D-galactopyranosyl-(1→6)-[β-D-galactofuranosyl-(1→2)-
β-D-galactofuranosyl-(1→4)]-2-acetamido-2-deoxy-α-D-glucopyra-
noside (26). Compound 25 (270 mg, 0.11 mmol) was treated
with 2.5 mL of cooled 0.6 M NaOCH3–CH3OH and the mixture
was stirred at rt. After 2.5 h the resulting solution was diluted
with water (0.25 mL), and purified by cationic interchange
column chromatography (Amberlite IR-120 plus resin 200 mesh,
H+ form) eluting with 9 : 1 CH3OH–H2O (2 × 8 mL). The eluate
was concentrated under reduced pressure and the methyl benzo-
ate was co-evaporated with water (3 × 2.5 mL). Further purifi-
cation through a C18 cartridge, eluting with 95 : 5 H2O–CH3OH
followed by concentration at 30 °C gave 126 mg of 26 (99%), as
an amorphous hygroscopic solid: Rf 0.34 (7 : 1 : 2 n-propanol–
EtOH–H2O); [α]D +9.6° (c 1, CHCl3); 1H NMR (D2O,
500 MHz): δ 7.54–7.38 (m, 5H, arom.), 5.47 (d, 1H, J = 1.0 Hz,
H-1B), 5.24 (d, 1H, J = 1.5 Hz, H-1C), 4.96 (d, 1H, J = 3.5 Hz,
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H-1A), 4.82 (d, 1H, J = 7.5 Hz, H-1E), 4.74, 4.57 (2d, 2H, J =
12.0 Hz, CH2Ph), 4.66 (d, 1H, J = 7.0 Hz, H-1F), 4.53 (d, 1H,
J = 7.5 Hz, H-1D), 4.26–4.23 (m, 2H, H-2B and H-3B), 4.20 (dd,
1H, J = 11.5, 1.5 Hz, H-6aA), 4.19 (d, 1H, J = 3.5 Hz, H-4D),
4.14 (dd, 1H, J = 3.5, 1.5 Hz, H-2C), 4.12–4.08 (m, 2H, H-3C,
H-4B), 4.02 (dd, 1H, J = 6.5, 4.0 Hz, H-4C), 4.00–3.59 (m,
28H), 1.95 (s, 3H, CH3CONH); 13C NMR (D2O, 125.8 MHz):
δ 174.9 (CH3CONH), 137.6, 129.4, 129.0 (C-arom.), 107.1
(C-1B), 106.5 (C-1C), 104.6 (C-1F), 103.6 (C-1E), 102.6 (C-1D),
96.5 (C-1A), 86.0 (C-2B), 84.1 (C-4C), 83.6 (C-3D), 83.2 (C-4B),
82.3 (C-2C), 78.4 (C-4A), 77.5 (C-3C), 76.1 (C-2D, and undeter-
mined Galp C-5), 75.8 (undetermined Galp C-5); 75.4, 75.3
(C-3B and undetermined Galp C-5), 73.6, 73.5 (C-3E, C-3F);
72.1, 71.8 (C-2E, C-2F), 70.7 (CH2Ph), 71.4, 70.9, 70.4,; 69.8
(C-5A, C-5B, C-5C, C-3A); 69.9, 69.3 (C-4E, C-4F), 69.5 (C-4D),
68.6 (C-6A); 63.32, 63.27 (C-6B, C-6C), 62.0, 61.7, 61.5 (C-6E,
C-6F, C-6D), 54.4 (C-2A), 22.4 (CH3CONH). HRMS (ESI/APCI)
m/z calcd for C45H71NNaO31 (M + Na)+: 1144.3902. Found:
1144.3878.
column of Amberlite IR-120 plus, the resin was washed with
2 : 1 CH3OH–H2O and the combined solutions were concen-
trated to dryness. The residue was co-evaporated with methanol
(4 × 1 mL), dissolved in deionized water and the solution was
filtered through a C18 cartridge. Evaporation of the filtrate under
reduced pressure gave 14.4 mg of 2 (90%) as a glassy solid: [α]D
1
+24.1° (c 1, H2O); H NMR (D2O, 500 MHz): δ 5.43 (bs, 1H,
H-1B), 5.21 (d, 1H, J = 1.7 Hz, H-1C), 4.84 (d, 1H, J = 7.8 Hz),
4.65 (d, 1H, J = 7.5 Hz), 4.57 (d, 1H, J = 7.7 Hz, H-1D), 4.25
(dd, 1H, J = 3.5, 1.5 Hz, H-2B), 4.22 (dd, 1H, J = 6.5, 3.5 Hz,
H-3B), 4.19 (d, 1H, J = 3.0 Hz, H-4D), 4.18 (dd, 1H, J = 8.0, 3.0
Hz, H-6aA), 4.15–4.12 (m, 1H, H-2A), 4.13 (dd, 1H, J = 3.5, 1.5
Hz, H-2C), 4.11 (t, 1H, J = 6.0 Hz, H-4B), 4.09 (dd, 1H, J = 6.5,
3.5 Hz, H-3C), 4.08–4.04 (m, 1H), 4.02 (dd, 1H, J = 6.5, 4.0 Hz,
H-4C), 4.00 (dd, 1H, J = 10.0, 3.0 Hz, H-3D), 3.94 (dd, 1H, J =
10.0, 7.7 Hz, H-2D), 3.93–3.90 (m, 2H), 3.86–3.82 (m, 3H),
3.87–3.62 (m, 21H), 3.56 (dd, 1H, J = 10.0, 7.8 Hz), 3.60 (dd,
1H, J = 10.0, 7.5 Hz), 2.05 (s, 3H, CH3CONH), δ values
matched lit.;20 13C NMR (D2O, 125.8 MHz):
δ 175.1
(CH3CONH), 107.4 (C-1B), 107.0 (C-1C), 104.7, 103.5; 102.5
(C-1D), 87.1 (C-2B), 83.9 (C-4C), 83.8 (C-4B), 83.4 (C-3D), 82.1
(C-2C), 77.9 (C-4A), 77.3 (C-3C), 76.7 (C-2D), 76.04 (C-3B);
75.99, 75.8, 75.4, 73.5, 72.2, 71.8, 71.4; 71.3 (C-6A); 71.2, 70.8,
70.4, 70.0, 69.6; 69.4 (C-4D); 69.3, 69.0, 63.9, 63.5, 63.4, 61.8,
61.6, 61.5; 53.2 (C-2A), 22.8 (CH3CONH). HRMS (ESI/APCI)
m/z calcd for C38H67NNaO31 (M + Na)+: 1056.3589. Found:
1056.3550.
β-D-Galactopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→3)]-β-D-
galactopyranosyl-(1→6)-[β-D-galactofuranosyl-(1→2)-β-D-galac-
tofuranosyl-(1→4)]-2-acetamido-2-deoxy-α,β-D-glucopyranose (1). A
solution of 26 (33 mg, 0.029 mmol) dissolved in 9 : 1 CH3OH–
H2O (2 mL) and 10% Pd(C) (12 mg), was hydrogenated for 20 h
at 40 psi (3 atm) and rt. The catalyst was filtered over Celite and
the filtrate was concentrated at 25 °C, affording 1 (30 mg, 99%)
as a glassy hygroscopic solid, in a 3 : 2 α : β anomeric mixture:
[α]D +12.7° (c 0.5, H2O), Rf 0.15 (7 : 1 : 2 n-propanol–EtOH–
H2O); 1H NMR (D2O, 500 MHz): δ anomeric zone and diagnos-
tic signals 5.47 (m, 1H, H-1B in α and β anomer), 5.24 (d, 0.6H,
J = 1.5 Hz, H-1C in α anomer), 5.22 (d, 0.4H, J = 1.5 Hz, H-1C
in β anomer), 5.21 (d, 0.6H, J = 3.5 Hz, H-1A α anomer), 4.82
(Galp H-1 superimposed with DHO signal), 4.70 (d, 0.4H, J =
8.0 Hz, H-1A β anomer), 4.65 (d, 1H, J = 7.0 Hz, Galp H-1),
4.55 (d, 0.4H, J = 7.5 Hz, Galp H-1), 4.53 (d, 0.6H, J = 7.5 Hz,
Galp H-1), 2.04 (s, 3H, α and β anomers CH3CONH); 13C NMR
(D2O, 50.3 MHz): δ 107.1 (α and β anomers C-1B), 106.6,
(C-1C in β anomer) 106.5 (C-1C in α anomer); 104.6, 103.5,
102.6; 95.8 (β anomer C-1A), 91.4 (α anomer C-1A); 86.2, 85.9
(C-2B in α and β anomers), 84.1, 83.5, 83.4, 83.3, 82.3, 81.9,
78.5, 78.0, 77.5, 77.3, 76.4, 76.2, 76.1, 75.8, 75.4, 75.3, 74.3,
73.6, 73.5, 72.9, 72.0, 71.8, 71.4, 71.0, 70.0, 69.9, 69.7, 69.5,
69.3, 68.8 (C-6A α and β anomers), 63.3, 63.2, 62.1, 61.9, 61.7,
61.5, 57.2 (β anomer C-2A), 54.7 (α anomer C-2A), 22.8, 22.5
(α and β anomers CH3CONH). HRMS (ESI/APCI) m/z calcd for
C38H65NNaO31 (M + Na)+: 1054.3433. Found: 1054.3402.
Acknowledgements
This work was supported by grants from Agencia Nacional de
Promoción Científica y Tecnológica, Universidad de Buenos
Aires and CONICET. R. M. de Lederkremer and C. Gallo-Rodri-
guez are research members of CONICET.
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