Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

Article abstract of DOI:10.1016/j.carres.2006.03.033

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infecti

Full text of DOI:10.1016/j.carres.2006.03.033

Carbohydrate Research 341 (2006) 1488–1497
Synthesis of the O-linked pentasaccharide in glycoproteins
of Trypanosoma cruzi and selective sialylation by
recombinant trans-sialidase
*
´
´
Veronica M. Mendoza, Rosalıa Agusti, Carola Gallo-Rodriguez and
Rosa M. de Lederkremer*
CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pabello´n II, 1428 Buenos Aires, Argentina
Received 9 February 2006; received in revised form 14 March 2006; accepted 24 March 2006
Available online 21 April 2006
Abstract—The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid
in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection
and pathogenesis. The synthesis of the pentasaccharide, b-D-Galp-(1!2)-[b-D-Galp-(1!3)]-b-D-Galp-(1!6)-[b-D-Galf-(1!4)]-D-
GlcpNAc and the corresponding alditol, previously isolated by reductive b-elimination of the mucins, is described. The key step
was the 6-O-glycosylation of a easily accessible derivative of b-D-Galf-(1!4)-D-GlcpNAc with a b-D-Galp-(1!2)-[b-D-Galp-
(1!3)]-D-Galp donor using the trichloroacetimidate method. The b-linkage was diastereoselectively obtained by the nitrile effect.
The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal b-D-Galp residues
for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated prod-
uct was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal
(1!3) linked galactopyranose.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Trypanosoma cruzi; Pentasaccharide; Trichloroacetimidate glycosylation; trans-Sialidase; Sialic acid; Galactofuranose
1. Introduction
anchored to the membrane by a glycosylphosphatidyl-
inositol moiety.6,7 The O-linked chains in these mucin-
like, sialic acid acceptors are linked to the protein by
a-GlcpNAc and may be derived from two cores, b-D-
Galp-(1!4)-GlcpNAc or b-D-Galf-(1!4)-GlcpNAc.
The cores are further branched with various units of
Galf and/or Galp. Thus far, the galactofuranose form
was found in the mucins of strains belonging to lineage
1 (Fig. 1),8–11 whereas in the more infective Y12 and
CL13–15 strains, galactose in the mucins is only present
in the pyranose form. The mechanism by which the pres-
ence of Galf correlates with the parasite inactivation has
not been fully elucidated. Galf is an antigenic epitope16
and an immunological reaction could influence the
infection. On the other hand, as the Galp units are the
acceptors of the sialic acid in the trans-sialidase reaction
Trypanosoma cruzi is the agent of Chagas’ disease,
which currently affects approximately 20 million people
in Central and South America.1 On the basis of bio-
chemical and molecular studies, T. cruzi strains may
be grouped into two divergent genetic divisions desig-
nated as lineages 1 and 2. Lineage 1 is related with the
sylvatic cycle and lineage 2 is associated with the domes-
tic cycle, involved in human infection.2,3
The surface of T. cruzi is dominated by glycoinositol-
phospholipids (GIPLs)4,5 and mucin-like glycoproteins
*
Corresponding authors. Tel./fax: +54 11 4576 3352; e-mail
addresses: cgallo@qo.fcen.uba.ar; lederk@qo.fcen.uba.ar
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.033

V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
1489
Galfβ(1→4)-GlcNAc
Galfβ(1→2)-Galfβ(1→4)
Galpβ(1→2)-Galfβ(1→4)
GlcNAc
GlcNAc
Galfβ(1→4)
GlcNAc
GlcNAc
Galpβ(1→6)
Galfβ(1→4)
Galfβ(1→2)-Galfβ(1→4)
GlcNAc
GlcNAc
Galpβ(1→3)
Galpβ(1→6)
Galpβ(1→3)
Galpβ(1 6)
→
Galpβ(1→2)-Galfβ(1→4)
Galfβ(1→4)
GlcNAc
Galpβ(1→3)
Galpβ(1→2)
Galpβ(1→3)
Galpβ(1→6)
Galpβ(1→2)
Galpβ(1→6)
1
Galfβ(1→2)-Galfβ(1→4)
GlcNAc
Galpβ(1→3)
Galpβ(1→6)
Galpβ(1→2)
Figure 1. Oligosaccharides in mucins of Trypanosoma cruzi, lineage 1 (Refs. 8–11); pentasaccharide 1 is shown in blue.
that involves the host glycoproteins, it is interesting to
study the influence of the coexistence of Galf on these
properties.
sis was on the primary 6-hydroxyl group of the
GlcpNAc moiety.
For the synthesis of target pentasaccharide 1, a similar
convergent route could be employed by glycosylation of
the acceptor derivative of b-D-Galf-(1!4)-D-GlcpNAc 4
with a derivative of the trisaccharide 2,3-di-O-(b-D-
Galp)-D-Galp. In this case, the diastereoselectivity of
the glycosylation would be affected by the absence of
neighbouring group assistance at C-2.
The trichloroacetimidate method was used for the gly-
cosylation step. Reaction of 4,6-di-O-acetyl-2,3-di-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-D-galacto-
pyranose (2)22 with trichloroacetonitrile and DBU at
0 ꢁC gave the a-trichloroacetimidate 3 in 92% yield
(Scheme 1). The configuration at the anomeric centre
was confirmed from the 1H NMR spectrum (d 6.55
ppm J1,2 = 3.8 Hz for H-1) and 13C NMR spectrum (d
95.6 ppm for C-1).
In our laboratory, we have undertaken the chemical
synthesis of the parasite mucin oligosaccharides, in par-
ticular of those containing galactofuranose.17–19 The
metabolic pathways involved in the transference of Galf
in T. cruzi have not been elucidated, thus, these oligosac-
charides could be useful tools for biosynthetic studies.
Here, we report the first synthesis of free b-D-Galp-
(1!2)-[b-D-Galp-(1!3)]-b-D-Galp-(1!6)-[b-D-Galf-(1!
4)]-D-GlcpNAc (1, Scheme 1) and the corresponding
alditol. Pentasaccharide 1 is the major oligosaccharide
in the mucins of the G strain9 and presents two terminal
b-D-Galp residues for possible sialylation in the TcTS
reaction. We have recently demonstrated selective mono
sialylation of 2,3-di-O-(b-D-Galp)-D-Galp, the external
unit in the pentasaccharide 1.20 The acceptor substrate
selectivity was now studied for compound 1, its benzyl
glycoside and the corresponding alditol.
Due to the lack of a participating group at C-2 of do-
nor 3, to obtain the b-(1!6) linkage, the glycosylation
was performed in a participating solvent such as aceto-
nitrile taking advantage of the nitrile effect.23,24 The
condensation of benzyl 2,3,5,6-tetra-O-benzoyl-b-D-
galactofuranosyl-(1!4)-2-acetamido-3-O-benzoyl-2-de-
oxy-a-D-glucopyranoside (4)19 with imidate 3 in the
presence of TMSOTf as catalyst, and employing aceto-
nitrile as solvent at ꢀ40 ꢁC, gave pentasaccharide 5 dia-
stereoselectively in 57% yield. The a-glycosylation
product was not detected, and unreacted acceptor 4
was also recovered.
2. Results and discussion
We have previously synthesized the tetrasaccharide b-D-
Galp-(1!3)-b-D-Galp-(1!6)-[b-D-Galf-(1!4)]-D-Glcp-
NAc via a convergent route, by condensing a b-D-Galf-
(1!4)-D-GlcpNAc acceptor with a b-D-Galp-(1!3)-D-
Galp donor.19 This strategy was designed taking into ac-
count that the 4-hydroxyl group of N-acetylglucosamine
derivatives is a poor nucleophile in glycosylation reac-
tions.21 Thus, the last glycosylation step of that synthe-
1
13C and H NMR assignments for pentasaccharide
derivative 5 were performed on the basis of 1H–1H

1490
V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
OH
O
O
O
OBz
BzO
AcNH
OBn
OBz
Cl3CCN
DBU
OAc
O
OAc AcO
O
AcO
AcO
AcO
O
OAc
O
AcO OAc
OBz
OBz
4
O
O
AcO
AcO
OH
AcO
O
AcO
O
(92%)
TMSOTf, CH3CN
(57%)
NH
CCl3
OAc
AcO
OAc
O
O
O
AcO
AcO
AcO
AcO
2
3
HO
OH
HO
HO
OH
AcO OAc
AcO OAc
O
O
O
O
O
O
O
O
O
AcO
AcO
HO
H2/Pd(C)
O
O
O
AcO
O
HO
HO
OH
O
O
O
BzO
2 atm
O
OAc
O
NaOMe, MeOH
(92%)
OH
OBz
AcNH
AcNH
HO
(92%)
OBn
OBn
AcO
HO
OH
AcO
OBz
OH
OH
OBz
OBz
5
6
HO OH
HO OH
HO OH
HO OH
O
O
O
O
O
O
O
HO
O
HO
O
HO
HO OH
O
HO
HO OH
OH
AcNH
O
NaBH4
O
O
O
O
O
OH
O
HO
HO
OH
OH
OH
(80%)
AcNH
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
1
7
Scheme 1.
1
COSY and HETCOR experiments (Tables 1 and 2). The
13C NMR spectrum of 5 (Table 2) showed the reso-
nances for the anomeric carbons at 96.1 (GlcpNAc),
107.1 (Galf), 102.5 (Galp-b-(1!6)), 99.9 and 99.4 ppm
(Galp-b-(1!3) and Galp-b-(1!2)). In the 1H NMR
spectrum, the resonance for the newly constructed
b-(1!6) glycosidic linkage, appeared at 4.68 ppm with
a coupling constant of 7.5 Hz, indicating the b-pyranos-
idic configuration. Based on the COSY experiment, H-2
and H-3 of this internal b-Galp unit appeared at 3.89
and 3.98 ppm, respectively, shielded compared to the
H-2 and H-3 signals in the terminal b-Galp units. The
assignment of each spin system of Galp-b-(1!2) and
Galp-b-(1!3) could be interchanged.
of the mixture (37%) by H NMR spectroscopy showed
the signals for compound 5 together with the signals for
the a-(1!6) product in a 1:2.5 b:a ratio as indicated by
integration. In the 13C NMR spectrum, the anomeric
carbons of the a-(1!6) product appeared at 96.1
(GlcpNAc), 106.7 (Galf), 101.9 and 100.5 (Galp-b-
(1!3) and Galp-b-(1!2)); and 98.2 ppm (Galp-a-
(1!6)), the latter indicating the a-configuration. More-
over, the anomeric proton of the new glycosidic bond
appeared at 5.26 ppm as a doublet with a coupling
constant of 3.7 Hz. Deacylation of the mixture followed
by NMR analysis of the benzyl glycosides confirmed the
ratio obtained in the glycosylation reaction.
Deacylation of pentasaccharide 5 with sodium meth-
oxide gave crystalline benzyl glycoside 6 in 92% yield.
Taking into account that 6 was used as substrate for a
preparative trans-sialylation reaction, NMR assign-
ments were needed for comparison with the signals for
the sialylated product (Tables 1 and 2), and were per-
formed based on 1H–1H COSY, HETCOR and NOESY
experiments.
It is important to point out the influence of the solvent
in the glycosylation reaction. When the reaction was
performed in CH2Cl2 as solvent, at ꢀ12 ꢁC for 90 min
and then at 5 ꢁC for 16 h, low diastereoselectivity in
the glycosidic bond formation was obtained, as two
products could be distinguished by TLC, but could
not be separated by column chromatography. Analysis

V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
Table 1. 1H NMR data for 5 and 6 (500 MHz, CDCl3 and D2O, respectively)
1491
Compound
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
Other signals
0
(J1,2
)
(J2,3
)
(J3,4
)
(J4,5
)
(J5,6a
)
(J6b,6a
)
(J5,6b
)
ðJH;H Þ
5
GlcpNAc-OBn
5.02
(3.7)
5.43
(b.s.)
4.68
(7.5)
4.72
(7.7)
4.96
(8.0)
4.43
(11.0)
5.31
(1.4)
3.89
(9.3)
5.08
(n.d.)
5.21
5.64
(9.0)
5.66
(4.7)
3.98
(3.8)
5.08
(3.0)
5.00
(3.7)
4.12
(10.0)
4.40
(4.3)
5.37
(1.2)
5.35
(1.2)
5.36
4.21
(4.3)
5.53
(7.8)
4.00
(5.5)
3.90
(6.8)
3.81
(6.5)
4.36
(11.0)
4.30
(12.1)
4.35
(10.9)
4.19
(11.2)
4.13
3.96
(6.2)
4.24
(3.2)
4.08
(8.3)
4.16
(6.8)
4.00
(6.7)
5.82 (NH)
(9.5)
CH2Ph
4.85, 4.57
(11.6)
CH3CONH
1.77 (s)
Galf-b-(1!4)
Galp-b-(1!6)
Galp-b-(1!2)a
Galp-b-(1!3)a
(10.5)
(1.0)
(11.2)
6
GlcpNAc-OBn
4.90
(3.5)
5.28
(2.5)
4.49
(7.5)
4.78
(7.5)
4.60
(7.1)
3.85
(n.d.)
3.79b,e
(n.d.)
4.07–4.00
(n.d.)
3.89
(n.d.)
3.88b,e
4.13
(10.4)
3.82e
(n.d.)
CH2Ph
4.69, 4.51
(12.0)
CH3CONH
1.89 (s)
(<1)
3.78e
(n.d.)
3.67e
(n.d.)
3.60e
(n.d.)
3.62e
(n.d.)
Galf-b-(1!4)
Galp-b-(1!6)
Galp-b-(1!2)
Galp-b-(1!3)
3.64–3.59e
(n.d.)
3.88–3.65e
(n.d.)
3.91
(9.7)
3.95
(3.2)
4.15
(n.d.)
3.83d,e
(n.d.)
3.87d,e
(n.d.)
3.57c
(9.9)
3.62–3.55e
(n.d.)
3.88–3.65e
(n.d.)
3.56c
(10.0)
3.62–3.55e
(n.d.)
3.88–3.65e
(n.d.)
a The assignments for the residues could be interchanged.
b –d Assignment could be interchanged.
e Assigned from two-dimensional spectra.
Table 2. 13C NMR data for compounds 5 and 6 (125 MHz, CDCl3 and D2O, respectively)
Compound
C-1
C-2
C-3
C-4
C-5
C-6
Other signals
5
GlcpNAc-OBn
Galf-b-(1!4)
Galp-b-(1!6)
Galp-b-(1!2)a
Galp-b-(1!3)a
96.1
52.4
82.4
78.8
70.4g
69.8
72.5
76.9
75.8
69.8g
71.1
76.2
81.4
68.8
67.0
66.8
70.9
69.8
70.6
70.7
70.8
69.1
63.2
60.5
61.0
60.3
69.8 (CH2Ph)
23.0 (CH3CO)
107.1
102.5
99.4
99.9
6
GlcpNAc-OBn
Galf-b-(1!4)
Galp-b-(1!6)
Galp-b-(1!2)
Galp-b-(1!3)
96.4
108.3
102.4
103.4
104.5
54.2
82.6c
76.3
70.7
72.0
69.9b
76.3
78.5
70.3b
70.9
75.2
76.0
75.7
68.3
63.2
61.8f
61.5f
61.4f
70.5 (CH2Ph)
22.4 (CH3CO)
81.8c
69.4
83.5
73.5d
73.4d
69.7e
69.2e
a The assignments for the residues could be interchanged.
b–gAssignment could be interchanged.
Hydrogenolysis of the anomeric benzyl group of 6
either with hydrogen and palladium-on-carbon or
ammonium formate, 10% palladium-on-carbon in
CH3OH at 65 ꢁC gave the pure free pentasaccharide
b-D-Galp-(1!2)-[b-D-Galp-(1!3)]-b-D-Galp-(1!6)-[b-
D-Galf-(1!4)]-D-GlcpNAc (1) in high yield as a a:b
mixture in a 4:1 ratio. Further reduction of 1 with
NaBH4 gave the corresponding alditol 7. The chemical
cule when incubated with native trans-sialidase and 30-
sialyllactose as donor; however, the site of sialylation
was not determined. We have previously shown20 that
selective sialylation of benzyl 2,3-di-O-(b-D-Galp)-b-D-
Galp, the terminal constituent of pentasaccharide 1,
occurred at the less hindered (1!3) linked Galp unit.
Compounds 1, 6 and 7 were assayed as acceptor sub-
strates for TcTS, using conditions previously described
for incubation with the donor 30-sialyllactose25
(Fig. 2). In all cases, the reaction was fast and reached
equilibrium after about 30 min. The progress of the
transference of sialic acid was followed by HPAEC.
As expected, sialylated pentasaccharide S-1 (Fig. 2A)
was eluted later (CarboPac PA-100 column) than
1
shifts in the H and 13C NMR spectra matched with
those reported for the alditol released by reductive
b-elimination from mucins of T. cruzi (G strain).8
Acosta-Serrano et al.9 described that the pentasaccha-
ride alditol 7, obtained by reductive b-elimination from
mucins of T. cruzi, accepted only one sialic acid mole-

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V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
Figure 2. Analysis of compounds 1, 6 and 7 as substrates of the trans-sialidase of Trypanosoma cruzi. Compounds 1 (A), 6 (B), 7 (C) at 1 mM
concentrations were incubated for 15 min at 25 ꢁC with 1 mM 30-sialyllactose (SL) and 40 ng trans-sialidase in 20 mM Tris buffer (pH 7) and
analyzed by HPAEC-PAD. For all panels, the upper chromatogram represents incubation in the absence of enzyme. A CarboPac PA-100 column
was used and eluted with 70 mM NaOAc in 100 mM NaOH. Compounds S1, S6 and S7 are the sialylated derivatives of 1, 6 and 7, respectively.
sialyllactose. The nonreducing sialylated benzyl glyco-
side S-6 (Fig. 2B) and sialylated pentasaccharide alditol
S-7 (Fig. 2C) were less retained on the column than sial-
yllactose. The three compounds were shown to be good
acceptors of sialic acid, with transference values of 60–
70%. Similar values were previously obtained for the
2,3-di-O-(b-D-Galp)-D-Galp trisaccharide and homolo-
gous derivatives. These results indicated that the pres-
ence of the Galf branch does not affect the acceptor
properties of the pentasaccharide.
With the aim of confirming the site of sialylation in
the pentasaccharide, a preparative sialylation reaction
was performed using benzyl glycoside 6 as the substrate
(Scheme 2). The benzyl glycoside 6 was selected because
the hydrophobic aglycon allowed easier purification, by
exchange chromatography, of the product from the
remaining sialyllactose that was used as donor. More-
over, the absence of anomeric mixtures facilitated anal-
ysis of the results by NMR spectroscopy. The reaction
was monitored by HPAEC and, under the used condi-
tions, 68% sialylation of compound 6 was obtained.
No evidence of the presence of the other monosialylated
or disialylated compounds was found. The reaction
product was purified using an AG1X2 (acetate form)
resin column. After elution of neutral sugars with water,
the acidic components were eluted with 200 mM pyridi-
nium acetate buffer, pH 5.0.
The sialyl derivative S-6 was characterized on the
basis of mass spectrometry and NMR spectroscopy.
The expected molecular mass of the trans-sialylation
product was confirmed by Q-TOF positive ion spray
mass spectrometry. Peaks at 1251.46, 1273.43 and
1289.42, assigned to [M+H]+, [M+Na]+ and [M+K]+
established that monosialylation had occurred. The
peak corresponding to the loss of sialic acid at 960.34
was observed.
1
The H NMR spectrum (Fig. 3) of the purified sialyl-
ated oligosaccharide S-6 showed, inter alia, the presence
HO
OH
HOOC
O
HO
O
OH
HO
O
OH
HO
O
OH
HO
HO
OH
O
O
O
O
O
O
HO
O
O
AcNH
trans-sialidase
Sialyllactose
HO
HO
O
O
HO
HO
HO
OH
O
HO
OH
O
HO
O
O
O
O
OH
OH
Lactose
AcNH
AcNH
HO
HO
OBn
OBn
HO
HO
OH
OH
OH
OH
OH
OH
S-6
6
Scheme 2.

V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
1493
Figure 3. 1H NMR spectrum of sialylated compound 6 (D2O, 500 MHz).
of the diagnostic signals for the sialyl residue at 1.75 ppm
(H-3a) and 2.70 ppm (H-3e). Integration of these signals
confirmed that only one sialic acid unit has been intro-
duced. Also, one new singlet appeared at d 1.97 corre-
sponding to the NAc group of neuraminic acid.
The selectivity of the transfer reaction with respect to
the two terminal b-D-Galp units was studied by two-
dimensional NMR experiments (COSY, HETCOR
and NOESY). The signal of H-3 at the site of sialylation
was now at d 4.04 ppm appearing as the characteristic
doublet of doublets (J = 10, 3.2 Hz). The signal for the
H-2 (COSY spectrum) that correlates with this signal
was also shifted downfield (d 3.59) and correlates with
H-1 at d 4.65 ppm assigned to the Galp-b-(1!3) linked
unit (E). NOESY correlation (Fig. 4) between this H-1
(unit E) and H-3 of the internal Galp-b-(1!6) (unit C)
confirmed selective sialylation of the terminal Galp-b-
(1!3) linked unit. On the other hand, the chemical
Figure 4. NOESY spectrum (500 MHz) of a-D-NeupAc-(2!3)-b-D-Galp-(1!3)-[b-D-Galp-(1!2)]-b-D-Galp-(1!6)-[b-D-Galf-(1!4)]-a-D-GlcpNAc-
OBn (S-6).

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V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
shifts for the Galp-b-(1!2) unit were not affected by
sialylation.
pyranose22 (2, 342 mg, 0.37 mmol) in anhydrous CH2Cl2
(3 mL) cooled to 0 ꢁC, trichloroacetonitrile (1.71 mmol,
0.17 mL) and DBU (0.17 mmol, 26 lL) were added.
After 1 h at rt, the solution was carefully concentrated
under reduced pressure and the residue was immediately
purified by column chromatography (7:3:0.03, toluene/
EtOAc/TEA) to give 368 mg of foamy trichloroacetimi-
date 3 (92%, 0.34 mmol) as the only product: Rf = 0.49
(1:2, toluene/EtOAc), [a]D +32.4 (c 1, CHCl3); 1H NMR
(CDCl3): d 8.59 (br s, 1H, NH), 6.55 (d, 1H, J = 3.8 Hz,
H-1), 5.49 (dd, 1H, J = 3.6, 1.2 Hz, H-4), 5.35 (dd, 1H,
J = 3.5, 1.1 0Hz, H-40 or H-400), 5.33 (dd, 1H, J = 3.4,
1.0 Hz, H-4 or H-400), 5.13 (dd, 1H, J = 7.7, 10.5 Hz,
H-20 or H-200), 5.08 (dd, 1H, J = 7.7, 10.6 Hz, H-20 or
H-200), 4.99 (dd, 1H, J = 10.6, 3.6 Hz, H-30 or H-300),
4.97 (dd, 1H, J = 10.5, 3.4 Hz, H-30 or H-300), 4.64 (2d,
2H, J = 7.7 Hz, H-10 and H-100), 4.26 (m, 1H, H-5),
4.23 (dd, 1H, J = 10.0, 3.6 Hz, H-3), 4.20–4.13 (m,
3H), 4.10 (dd, 1H, J = 3.8, 10.0 Hz, H-2), 4.10 (dd,
1H, J = 11.3, 6.5 Hz), 4.01 (dd, 1H, J = 11.1, 6.6 Hz);
3.99 (dd, 1H, J = 11.4, 7.3 Hz), 3.91 (dt, 1H, J = 6.7,
1.2 Hz, H-50 or H-500), 3.89 (dt, 1H, J = 6.6, 1.2 Hz,
H-50 or H-500), 2.17, 2.15, 2.11, 2.10, 2.08 (5s, 15H,
CH3CO), 2.07 (s, 6H, CH3CO), 1.97 (s, 3H, CH3CO);
13C NMR (CDCl3): d 170.5, 170.3, 170.0, 169.9, 169.6,
169.4, 168.9 (CH3CO), 160.7 (CCl3CNH), 101.3, 100.4
(C-10, C-100), 95.6 (C-1); 75.7, 72.1 (C-2, C-3), 70.9
(·2), 70.6, 70.2, 69.8 (·2), 69.7, 69.5, 69.1, 67.0, 66.7
(C-40, C-400), 62.1 (C-6), 61.3 (C-60, C-600), 21.0, 20.9,
20.7 (·3), 20.6 (·3), 20.5 (·2) (CH3CO).
In conclusion, pentasaccharide 1 was chemically syn-
thesized, for the first time, by a convergent approach
employing the ‘nitrile effect’ concept. Analysis by
NMR spectroscopy of alditol 7 confirmed the structure
of the oligosaccharide present in the natural sources.
Furthermore, the present study unequivocally estab-
lished the sialylation site of the pentasaccharide at the
less hindered (1!3)-linked galactopyranose.
3. Experimental
3.1. General methods
Melting points were determined with a Thomas-Hoover
apparatus and are uncorrected. Optical rotations were
measured with a Perkin–Elmer 343 polarimeter. TLC
was performed on 0.2 mm Silica Gel 60 F254 (Merck)
aluminium supported plates. Column chromatography
was performed on Silica Gel 60 (230–400 mesh, Merck).
Detection was effected by exposure to UV light or by
spraying with 10% (v/v) sulfuric acid in EtOH and char-
ring. NMR spectra were recorded with a Bruker AM 500
spectrometer at 500 MHz (1H) and 125 MHz (13C) at
30 ꢁC unless otherwise indicated. Chemical shifts are
given relative to the signal of internal acetone standard
at 2.16 and 30.8 ppm for 1H NMR and 13C NMR
spectra, respectively, when recorded in D2O. 1H and
13C assignments were supported by DEPT 135, homo-
nuclear COSY, HETCOR and HMQC experiments.
HMQC experiments were recorded in a Bruker Avance
DPX-400. High resolution electrospray ionization mass
spectra (ESIMS) were recorded on a Micromass
Q-TOF Ultima Tandem Quadrupole/Time-of-Flight
Instrument.
3.3. Benzyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1!2)-[2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1!3)]-4,6-di-O-acetyl-b-D-galactopyranosyl-(1!6)-
[2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-(1!4)]-2-
acetamido-3-O-benzoyl-2-deoxy-a-D-glucopyranoside (5)
For the sialylation experiments, a recombinant TcTS
expressed in Escherichia coli was kindly provided by
To a flask containing recently purified and dried benzyl
2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-(1!4)-2-
acetamido-3-O-benzoyl-2-deoxy-a-D-glucopyranoside19
´
A. C. C. Frasch (UNSAM, General San Martın, Buenos
Aires Argentina). 30-Sialyllactose was obtained from
bovine colostrum by an adaptation of a reported
method.26 Analysis by HPAEC-PAD was performed
using a Dionex DX-300 HPLC system with a pulse
amperometric detector (PAD) and a CarboPac PA-100
ion exchange analytical column (4 · 250 mm), equipped
with a guard column PA-100 (4 · 50 mm), with 70 mM
NaAcO in 100 mM NaOH at a flow rate of 1.0 mL/
min at rt.
(4, 285 mg, 0.29 mmol), and activated 4 A powdered
˚
molecular sieves, a solution of trichloroacetimidate (3,
368 mg, 0.34 mmol) in freshly distilled anhydrous
CH3CN (10 mL) was added, and the suspension was
cooled to ꢀ40 ꢁC under an argon atmosphere. After
15 min of vigorous stirring, TMSOTf (0.115 mmol,
21 lL) was added and the stirring continued for 4 h at
ꢀ40 ꢁC, and then for 16 h at ꢀ20 ꢁC. The reaction was
quenched by the addition of Et3N (0.12 mmol, 16 lL),
the mixture was allowed to reach rt, filtered and the
molecular sieves were washed with CH3CN. The filtrate
was concentrated under reduced pressure and the resi-
due was purified by column chromatography (1:1.7,
n-hexane/EtOAc). A first fraction gave 100 mg of unre-
acted acceptor 4 (Rf = 0.60; 1:2, toluene/EtOAc). The
next fraction afforded 314 mg of a foamy solid which
3.2. O-[2,3-Di-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyr-
anosyl)-4,6-di-O-acetyl-a-D-galactopyranosyl] trichloro-
acetimidate (3)
To a stirred solution of 2,3-di-O-(2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl)-4,6-di-O-acetyl-a,b-D-galacto-

V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
1495
crystallized from 1:1 hexane/ether and was characterized
Galf for a anomer), 5.27 (d, 0.2H, J = 2.4 Hz, Galf for
b anomer), 5.15 (d, 0.8H, J = 3.5 Hz, a-GlcpNAc),
4.64 (d, 0.2H, J = 8.3 Hz, Galp b-(1!2) or Galp b-
(1!3) for b anomer), 4.59 (d, 0.8H, J = 7.6 Hz, Galp
b-(1!2) or Galp b-(1!3) for a anomer), 4.50 (d,
0.2H, J = 7.4 Hz, Galp b-(1!6) for b anomer), 4.49
(d, 0.8H, J = 7.7 Hz, Galp b-(1!6) for a anomer).
b-GlcNAc, Galp b-(1!2) or Galp b-(1!3) for b
anomer and Galp b-(1!2) or Galp b-(1!3) for a
anomer signals are overlapped with DHO signal. 13C
NMR (D2O): d anomeric region 108.4 (Galf), 104.7
(Galp b-(1!3)), 103.6 (Galp b-(1!2)), 102.4 (Galp
b-(1!6)), 95.7 (b-GlcpNAc), 91.3 (a-GlcpNAc). ESIMS
m/z calcd for C32H55NO26Na [M+Na]+: 892.2910.
Found: 892.2916.
as pentasaccharide 5 (57%), Rf = 0.40 (1:2, toluene/
1
EtOAc), mp 118–120 ꢁC, [a]D ꢀ19.2 (c 1, CHCl3); H
NMR (CDCl3, Table 1): only the protecting groups
are listed d 8.01–7.85 (m, 10H, arom.), 7.61–7.23 (m,
20 H, arom), 2.17, 2.07, 2.04, 2.02, 2.01, 2.00, 1.97,
1.96 (8s, 30H, CH3CO); 13C NMR (CDCl3, Table 2):
only the protecting groups are listed d 171.0, 170.3,
170.2, 169.9, 169.8, 169.7, 169.3, 168.9 (CH3CO), 167.1
(CH3CONH), 165.8, 165.6, 165.4, 165.3 (PhCO);
136.8, 133.5, 133.3, 133.0, 132.9, 129.9, 129.8, 129.6,
129.5, 129.4, 128.9, 128.7, 128.6, 128.5, 128.4, 128.3,
128.2, 128.1 (arom.), 20.9, 20.6, 20.5, 20.4, 20.3
(CH3CO). Anal. Calcd for C94H101NO41: C, 59.40; H,
5.36; N, 0.74. Found: C, 58.92; H, 5.29; N, 0.74.
3.4. Benzyl b-D-galactopyranosyl-(1!2)-[b-D-galacto-
pyranosyl-(1!3)]-b-D-galactopyranosyl-(1!6)-[b-D-
galactofuranosyl-(1!4)]-2-acetamido-2-deoxy-a-D-gluco-
pyranoside (6)
3.6. b-D-Galactopyranosyl-(1!2)-[b-D-galactopyranosyl-
(1!3)]-b-D-galactopyranosyl-(1!6)-[b-D-galactofuran-
osyl-(1!4)]-2-acetamido-2-deoxy-D-glucitol (7)
To a 0 ꢁC solution of pentasaccharide 1 (20 mg,
0.023 mmol) in CH3OH/H2O (12:1), NaBH4 (30 mg,
0.79 mmol, 34 equiv) was added in four portions with
stirring. The reaction was allowed to stand for 16 h at
5 ꢁC, until TLC analysis showed that the reaction was
complete. The resulting mixture was purified by elution
with CH3OH/H2O (9:1) through a column of Amberlite
IR 120 cationic resin. Evaporation of the solvent, and
co-evaporation with CH3OH (·3), gave 15.9 mg
(0.081 mmol, 80% yield) of the desired alditol 7, which
was recrystallized from absolute EtOH to give a very
hygroscopic crystalline product: 158–160 ꢁC; Rf = 0.18
(7:1:2, n-PrOH/EtOH/H2O); [a]D +1.1 (c 0.4, H2O);
1H NMR (D2O): d 5.26 (d, 1H, J = 2.2 Hz, H-1 Galf),
4.83 (d, 1H, J = 8.0 Hz, H-1 Galp b-(1!2) or b-
(1!3)), 4.66 (d, 1H, J = 7.6 Hz, H-1 Galp b-(1!2) or
b-(1!3)), 4.58 (d, 1H, J = 7.7 Hz, H-1 Galp b-(1!6)),
4.20 (d, 1H, J = 3.3 Hz, H-4 Galp b-(1!6)), 4.18–4.15
(m, 2H), 4.10 (dd, 1H, J = 6.8, 3.5 Hz), 4.07 (dd, 1H,
J = 6.8, 4.0 Hz), 4.08–4.03 (m, 1H), 4.00 (dd, 1H,
J = 9.7, 3.3 Hz, H-3 Galp b-(1!6)), 3.95 (dd, 1H, J =
9.7, 7.7 Hz, H-2 Galp b-(1!6)), 3.93–3.91 (m, 3H),
3.87–3.63 (m, 19H), 3.61 (dd, 1H, J = 9.9, 7.6 Hz, H-2
Galp b-(1!2) or b-(1!3)), 3.54 (dd, 1H, J = 10.0,
7.9 Hz, H-2 b-(1!2) or b-(1!3)), 2.05 (s, 3H, CH3CO);
13C NMR (D2O): d 175.7 (CH3CONH), 109.4 (C-1
Galf); 105.3, 104.2 (C-1 Galp b-(1!2) and b-(1!2)),
103.2 (C-1 Galp b-(1!6)), 83.9, 83.8, 82.7, 79.1, 77.8,
77.6, 76.6, 76.4, 75.9, 74.1, 74.0, 72.4, 72.1 (C-6
GlcpNAc), 71.9, 71.1, 70.1, 70.0, 69.9, 69.6, 64.1 (C-6
Galf), 62.3, 62.2, 62.1 (·2) (C-1 GlcpNAc, C-6 Galp b-
(1!6), b-(1!2) and b-(1!3)), 52.3 (C-2 GlcpNAc),
23.4 (CH3CO). ESIMS m/z calcd for C32H57NO26Na
[M+Na]+: 894.3067. Found: 894.3073. Calcd for
C32H58NO26 [M+H]+: 872.3247. Found: 872.3275.
To a flask containing benzyl derivative 5 (176 mg,
0.093 mmol) was added 2 mL of a cooled 0.5 M sodium
methoxide solution. After 1 h of stirring at 0 ꢁC, TLC
examination showed that the reaction was completed
giving only one more polar compound. H2O (0.2 mL)
was added and the resulting solution was decationized
by elution with CH3OH/H2O (9:1) through a column
of Amberlite IR 120 cationic resin. Evaporation of the
solvent, and co-evaporation with H2O three times, gave
82.2 mg (0.086 mmol) of 6 as a glassy solid (92% yield)
which was crystallized from EtOH/Et2O (1:2) to give
very hygroscopic crystals: mp 205–208 ꢁC; Rf = 0.43
(7:1:2, n-PrOH/EtOH/H2O); [a]D +43.2 (c 0.8, H2O);
1H NMR (D2O, Table 1): only the protecting groups
are listed d 7.41–7.33 (m, 5H, arom.); 13C NMR (D2O,
Table 2): only the protecting groups are listed d 174.8
(CH3CONH), 137.5, 129.3, 129.1, 128.9 (arom). Anal.
Calcd for C39H61NO26ÆH2O: C, 47.60; H, 6.49; N,
1.43. Found: C, 47.71; H, 6.45; N, 1.34.
3.5. b-D-Galactopyranosyl-(1!2)-[b-D-galactopyranosyl-
(1!3)]-b-D-galactopyranosyl-(1!6)-[b-D-galactofuran-
osyl-(1!4)]-2-acetamido-2-deoxy-a,b-D-glucopyranose (1)
To a solution of benzyl glycoside 6 (50 mg, 0.052 mmol)
in 9:1 CH3OH/H2O (4 mL), was added a catalytic
amount of 10% Pd/C Deguzza type E101 NE/W and
the suspension was hydrogenated for 5 h at 2 atm. The
catalyst was filtered and the solution was passed through
a C-8 reverse phase cartridge and eluted with H2O.
Evaporation of the solvent afforded compound 1
(41.7 mg, 92%) as a glassy solid: Rf = 0.25 (7:1:2, n-
1
PrOH/EtOH/H2O); [a]D ꢀ11.1 (c 1.0, H2O); H NMR
(D2O): d anomeric region 5.29 (d, 0.8H, J = 2.8 Hz,

1496
V. M. Mendoza et al. / Carbohydrate Research 341 (2006) 1488–1497
3.7. Synthesis of benzyl 5-N-acetyl-a-D-neuraminyl-
(2!3)-b-D-galactopyranosyl-(1!3)-[b-D-galactopyran-
osyl-(1!2)]-b-D-galactopyranosyl-(1!6)-[b-D-galacto-
furanosyl-(1!4)]-2-acetamido-2-deoxy-a-D-glucopyran-
oside (S-6)
Acknowledgements
We thank A. C. C. Frasch from Universidad Nacional
´
General San Martın, Argentina, for the kind gift of
trans-sialidase from T. cruzi. This work was supported
´
´
by Grants from Agencia Nacional de Promocion Cientı-
Benzyl pentasaccharide 6 (3.5 mg) and 30-sialyllactose
(3.0 mg) were incubated with 22.5 lg of recombinant
TcTS in 1 mL of 20 mM Tris buffer pH 7.6 containing
30 mM NaCl for 14 h at 25 ꢁC. The synthesis was per-
formed in triplicate and each reaction analyzed by
HPAEC. A typical chromatogram is shown in Figure
2. The three incubation mixtures were combined, and
sialylated benzyl b-D-galactopyranosyl-(1!2)-[b-D-
galactopyranosyl-(1!3)]-b-D-galactopyranosyl-(1!6)-
[b-D-galactofuranosyl-(1!4)]-2-acetamido-2-deoxy-a-
D-glucopyranoside (S-6) was purified by passing
through an anion exchange resin (AG1X2, acetate form,
BioRad, 1.2 · 15 cm). Neutral compounds, namely ben-
zyl b-D-galactopyranosyl-(1!2)-[b-D-galactopyranosyl-
(1!3)]-b-D-galactopyranosyl-(1!6)-[b-D-galactofuran-
osyl-(1!4)]-2-acetamido-2-deoxy-a-D-glucopyranoside (6)
and lactose were eluted with H2O and sialylated com-
pounds with 200 mM pyridinium acetate buffer pH 5.
Fractions (1.5 mL) were collected and analyzed by
HPAEC. The remaining sialyllactose was eluted with
the first 30 mL of 200 mM pyridinium acetate buffer,
further elution with the same buffer afforded sialylated
compound 6 (S-6). After neutralization with pyridine,
the pooled fractions were concentrated under vacuum
at rt. Compound S-6 was further purified by passing
through a SepPack C8 cartridge (Alltech) eluting with
H2O to obtain 3.6 mg of a colourless syrup: [a]D +9.0
fica y Tecnologica, Universidad de Buenos Aires and
Fundacion Antorchas. C. Gallo-Rodriguez and R. M.
de Lederkremer are research members of CONICET.
V. M. Mendoza is a fellow from CONICET.
´
´
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.carres.
2006.03.033.
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