Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors

Article abstract of DOI:10.1093/glycob/cwq056

Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugate

Full text of DOI:10.1093/glycob/cwq056

Glycobiology vol. 20 no. 8 pp. 982–990, 2010
doi:10.1093/glycob/cwq056
Advance Access publication on April 7, 2010
Continuous nonradioactive method for screening
trypanosomal trans-sialidase activity and its inhibitors
Paula A Sartor2, Rosalía Agusti3, Maria S Leguizamón2,
Introduction
Oscar Campetella4, and Rosa M de Lederkremer1,3
Trypanosomatids are flagellate protozoa, some of them being
causative agents of diseases such as Trypanosoma cruzi and
2Departamento de Microbiología, Facultad de Medicina, Universidad de
Buenos Aires, 1121 Buenos Aires, Argentina, 3CIHIDECAR, Departamento de
Trypanosoma brucei producing the American (Chagas disease)
Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de
and African (sleeping sickness and nagana) trypanosomiasis,
respectively. Although different in many aspects at the biolog-
ical and cellular levels, they are unable to synthesize sialic
acids (SAs) even when this sugar is highly required to com-
plete their life cycles. Parasites solve this gap by expressing
enzymes known as trans-sialidases (TS) that are able to trans-
fer sialyl residues among glycoconjugates. In the case of T.
brucei, the SA incorporation through the TS into the glycosyl-
phosphatidylinositol anchor of the procyclin surface protein
cover is essential for parasite survival in the tsetse fly (Naga-
mune et al. 2004). T. cruzi trans-sialidase (TcTS) transfers
α(2,3)-linked sialyl residues present in host sialoglycoconju-
gates to terminal β-galactopyranosyl units of mucins
(Schenkman et al. 1991; Ferrero-Garcia et al. 1993; Frasch
2000; de Lederkremer and Agusti 2009) that are widely distrib-
uted on the parasite surface (Tomlinson et al. 1994; Pereira-
Chioccola et al. 2000). Sialylated mucins are in turn involved
in the invasion of mammalian host cells and in the protection
against lysis by serum factors (Tomlinson et al. 1994; Pereira-
Chioccola et al. 2000). The TcTS is anchored to the membrane
by glycosylphosphatidylinositol (Agusti et al. 1997, 1998) and
is shed to the medium, then it can be detected in the blood dur-
ing the acute phase of the infection (Leguizamón, Campetella,
González Cappa et al. 1994; Buscaglia et al. 1999; Alvarez et
al. 2004) and may produce several abnormalities in the im-
mune system (Leguizamón et al. 1999; Mucci et al. 2002,
2005, 2006; Risso et al. 2007). Animals that survive infection
and chronic Chagas disease patients both elicit TcTS-neutraliz-
ing antibodies that inhibit the enzymatic activity and have been
used on the detection of T. cruzi infections (Leguizamón, Cam-
petella, González Cappa et al. 1994, Leguizamón, Campetella,
Russomando et al. 1994; Blejer et al. 2008). The essential role
of TcTS in the infection and Chagas disease pathogenesis to-
gether with the absence of a similar activity in mammals makes
this enzyme an attractive target for the development of new
chemotherapies.
Buenos Aires, Ciudad Universitaria, Pabellón II, 1428 Buenos Aires,
Argentina, and 4Instituto de Investigaciones Biotecnológicas, Universidad
Nacional de General San Martin, 1650 San Martín, Argentina
Received on December 7, 2009; revised on March 31, 2010; accepted on
April 1, 2010
Trypanosoma cruzi, the agent of American trypanosomiasis
is unable to synthesize sialic acid (SA). Instead of using the
corresponding nucleotide sugar as donor of the monosac-
charide, the transfer occurs from α-2,3-linked SA in the
host sialoglycoconjugates to terminal β-galactopyranosyl
units of the parasite mucins. For that purpose, T. cruzi ex-
presses a glycosylphosphatidylinositol-anchored trans-
sialidase (TcTS) that is shed into the milieu, being detected
in the blood during the acute phase of the infection. The
essential role of TcTS in infection and the absence of a sim-
ilar activity in mammals make this enzyme an attractive
target for the development of alternative chemotherapies.
However, there is no effective inhibitor toward this enzyme.
In vitro, 3′-sialyllactose (SL) as donor and radioactive lac-
tose as acceptor substrate are widely used to measure TcTS
activity. The radioactive sialylated product is then isolated
by anion exchange chromatography and measured. Here
we describe a new nonradioactive assay using SL or fetuin
as donor and benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as
acceptor. Disaccharide 1 was easily synthesized by regiose-
lective glycosylation of benzyl α-D-GlcNAc with tetra-O-
benzoyl-D-fucose followed by debenzoylation. Compound
1 lacks the hydroxyl group at C-6 of the acceptor galactose
and therefore is not a substrate for galactose oxidase. Our
method relies on the specific quantification of terminal ga-
lactose produced by trans-sialylation from the donor to the
6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotomet-
ric galactose oxidase assay. This method may also
discriminate sialidase and trans-sialylation activities by
running the assay in the absence of acceptor 1.
A high throughput-screening test is required to search for
these putative inhibitors. A radioactive method has been fre-
quently used to assay TS activity and to detect TcTS-
neutralizing antibodies (Schenkman et al. 1991; Leguizamón,
Campetella, González Cappa et al. 1994; Leguizamón, Campe-
tella, Russomando et al. 1994; Blejer et al. 2008). In this
method, α-(2,3)sialyllactose (SL) is used as SA donor and ra-
dioactive lactose as acceptor. After SA transference, the
sialylated radioactive product is captured with anionic resins
Keywords: galactose oxidase/inhibitors/trans-sialidase/
Trypanosoma cruzi
1To whom correspondence should be addressed: Tel. +54-11-4576-3352;
e-mail: lederk@qo.fcen.uba.ar
982 © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Nonradioactive assay for trans-sialidase screening
Scheme 1. Transfer of SA by TcTS from a sialyl donor to a D-Fucp unit in the acceptor substrate. Quantification of the terminal galactose residues generated in the
donor after the removal of the SA was performed by a coupled colorimetric/fluorimetric galactose oxidase test.
and quantified. Although very sensitive, this method generates
radioactive waste, demands hands-on time and is difficult to
escalate. On the other hand, several nonradioactive methods
have been proposed for TS activity determination (Lee and
Kim 2000; Agusti et al. 2004; Neres et al. 2006; Schrader
and Schauer 2007). The available nonradioactive methods
have different limitations. They continue being hands-on de-
manding techniques, and the continuous monitoring of the
trans-sialylated product is not yet possible given that most of
them require a stopping step. This, together with the fact that
most of the products are also substrates of the enzyme and al-
low the reversible reaction, the accurate determination of the
enzymatic activity and some kinetic properties is hampered.
A major drawback is that the available nonradioactive methods
are limited in terms of sensitivity. Therefore, the development
of sensitive one-step and easy to escalate assays for monitoring
TS activity is required to perform the screening of TS inhibi-
tors and also for diagnostic purpose. Here we describe a new
nonradioactive assay for TS activity that uses a SA donor, such
as fetuin or SL, and an acceptor resistant to galactose oxidase
activity such as a D-fucosylated sugar. Then, the trans-sialyla-
tion reaction is determined by a coupled colorimetric/
fluorimetric galactose oxidase test, which allows the continu-
ous quantification of the terminal galactosyl residues generated
after the removal of the SA. The reaction used is shown in
Scheme 1. Because the exposed galactose is oxidized by the
galactose oxidase, the TS reaction proceeds only in one direc-
tion. The galactose oxidase assay is currently proposed as a
tool for detection of galactose and galactosamine residues on
biological samples (Kinoshita et al. 2000) and as a test for de-
termination of neuraminidase from influenza virus and bacteria
(Nayak and Reich 2004; Williamson et al. 2008). The method
is based on the oxidation of the C-6 of galactose that generates
H2O2. The amount of H2O2 is then measured by a highly sen-
sitive method involving the use of horseradish peroxidase
(HRP) and Amplex Red Reagent (10-acetyl-3,7-dihydroxyphe-
noxazine), rendering resorufin that is in turn quantified by
absorbance or fluorescence emission determination (Mohanty
et al. 1997).
Results
Synthesis of benzyl β-D-fucopyranosyl-(1→6)-2-acetamido-2-
deoxy-α-D-glucopyranoside (1, Scheme 2)
In order to avoid interference from the acceptor substrate for
the galactose oxidase reaction, a 6-deoxy-galactose (D-Fuc)
analog of the common disaccharides (lactose or N-acetyllacto-
samine) used for the TS reaction was necessary. Benzyl β-D-
Fuc-(1→6)-α-D-GlcNAc (1) was chosen because the synthesis
was easier than that for the lactosamine analog, and it was pre-
viously shown that β-Galp-(1→6)GlcNAc, in particular the
benzyl glycoside, was a good acceptor in the TcTS reaction
(Agusti et al. 2007).
The starting synthons, tetra-O-benzoyl-α-D-fucopyranose (2)
and benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (3),
were easily prepared from the free commercial sugars. Glyco-
sylation of 3 with a limited amount of the fucose derivative 2,
promoted by tin(IV) chloride, afforded, selectively, the β-
1
(1→6) linked disaccharide (Scheme 2). Analysis of the H
and 13C NMR spectra confirmed the structure. As expected,
983

PA Sartor et al.
Scheme 2. Synthesis of benzyl β-D-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-α-D-glucopyranoside (1). Reagents and yield: (i) benzoyl chloride, pyridine,
yield 75%; (ii) SnCl4, CH2Cl2, yield 42%; (iii) NaOMe, MeOH, yield 94%.
the primary OH-6 of 3 was regioselectively glycosylated as
shown by the downfield shift (6 ppm) of the C-6 resonance with
respect to the same signal in benzyl α-D-GlcNAc. The signal at
δ 102.1 ppm in the anomeric region of the 13C NMR and at δ
4.85 ppm with J 7.8 Hz in the 1H NMR spectrum indicated the
β-configuration for the new glycosidic linkage.
Although the yield was only moderate (42%), the selective
formation of 4 by glycosidation of the nonprotected benzyl
glycoside 3, followed by the quantitative deprotection reaction
of the galactose unit are points in favor for this approach for
the preparation of 1.
same principle was used in our work to detect the galactose
residues exposed by the TS catalytic activity. In order to adapt
this method for TS detection, we used fetuin or SL as SA do-
nor and the novel disaccharide 1 as the acceptor considering
that this disaccharide cannot be oxidized by galactose oxidase.
The optimal conditions for detection of TcTS activity were
empirically settled (data not shown). The sensitivity of the as-
say was determined by measuring either the optical density or
fluorescence emission (Figure 2A, B). The activity of less than
10 ng of TcTS could be determined. Taking into account the
optimal temperatures for the enzymes involved, the colorimet-
ric reaction (Figure 2A) was performed incubating first, TcTS
with SL and 1 at room temperature (Riberao et al. 1997) and
increasing the temperature to 37°C after the addition of galac-
tose oxidase and HRP. The fluorometric method, more
sensitive, allowed the determination in one step at room tem-
perature (Figure 2B). On the other hand, the optical density
remained constant when the amount of TcTS was higher than
0.3 μg. As expected, the radioactive method allowed the detec-
tion of even smaller amounts of TcTS than the novel
colorimetric assay now described (Figure 2C). Nevertheless,
the small amount of enzyme required together with the possi-
bility to easily and fast assay multiple inhibitors in a multiwell
array render this new method of high interest.
Testing of the synthetic disaccharide as SA acceptor in the TS
reaction
By HPAEC-PAD Analysis. Evaluation of disaccharide 1 as ac-
ceptor in the TS reaction was performed using SL as donor and
recombinant TcTS (Buschiazzo et al. 1996). The reaction was
followed by high pH anion exchange chromatography with
pulse amperometric detection (HPAEC-PAD) (Figure 1). Trans-
fer of SA was fast and reached equilibrium in about 15 min.
Sialylation was very effective, with 57% transfer of SA from
SL to 1 (Figure 1B). Since lactose generated from sialyllactose
is also present, this value indicates that both acceptors display
similar efficiency. A KM value of 0.13 mM was obtained
for 1. The minor peak at 8.0 min corresponds to SA and is
already present in the sample of SL (Figure 1A). The activity
as neuraminidase of the TcTS is low in the presence of an
appropriate acceptor (Agusti et al. 2004). Transfer of SA to
compound 1 was almost completely inhibited (Figure 1C) by
previous incubation of the enzyme with a neutralizing antibody
(Risso et al. 2007).
Time course of TS reaction and sialidase activity
The TS colorimetric reaction was allowed to proceed by add-
ing all components, from time zero incubating the mixture at
37°C and evaluating the optical densities at different time
points. The sialidase activity was also evaluated by performing
the assay without any acceptor. As expected, the color gener-
ated by the sialidase activity was significantly lower than that
measured for the transferase activity (Figure 3A). The kinetic
curve for both activities showed good linearity along the time
points tested (r=0.99 in both cases). A control assay by the
radioactive method showed no inhibition of TS activity in
the presence of Amplex Red Reagent, resorufin, H2O2 and
TS Activity Detection by a Colorimetric/Fluorometric As-
say. Kinoshita et al. (2000) have introduced a test for the
determination of galactose/N-acetylgalactosamine in biological
samples. They employed a galactose oxidase method in com-
bination with the detection of H2O2 by a highly sensitive
fluorometric/colorimetric assay (Mohanty et al. 1997). The
984

Nonradioactive assay for trans-sialidase screening
Fig. 1. Analysis of benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as acceptor substrate in the TcTS reaction. (A) Compound 1 and SL, without enzyme; (B) 1 was
incubated with SL and TcTS for 15 min at 25°C and the reaction mixture analyzed by HPAEC-PAD; (C) the same as above but TcTS was preincubated with a
neutralizing antibody. A CarboPac PA-100 ion exchange analytical column was eluted with 50 mM NaAcO in 100 mM NaOH at 1 mL/min. L, lactose; SA, sialic
acid; SL, sialyllactose; S-1, sialylated disaccharide 1.
HRP (data not shown). According to the previous results, the
fluorometric determination was performed at room temperature
(Figure 3B). In order to evaluate the sialidase activity in the
same experiment, the enzyme was incubated with all the re-
agents except for the acceptor 1 which was then added and
incubation continued to evaluate the sialyl transferase activity.
In the absence of a suitable sialyl unit acceptor such as 1, the
TcTS displayed a poor sialidase activity.
It is important to point out that these assays rendered mea-
surable colorimetric/fluorometric data that allow continuously
measuring the TS activity through time because no stopping
step is required and the exposed galactose residues are oxi-
dized by galactose oxidase being therefore not suitable for
resialylation.
Detection of TS activity inhibition
To assay whether the test might be useful to determine TS inhi-
bition, purified neutralizing antibodies from mouse (mAb 13G9)
or from human patients (purified AbsNt) or high concentrations
of DANA (2,3-dehydro-2-deoxy-N-acetylneuraminic acid)
were included in the test. Inhibitors were allowed to react with
TS for 15 min and then the substrates fetuin and 1 were incor-
porated to each well. The activity was measured by the adapted
galactose oxidase method after incubation at 37°C. A significant
decrease (P < 0.01) in the optical density was observed in com-
parison with the activity for TS in the absence of inhibitors
(Figure 4). To evaluate if the decrease of the optical density
could be due to interference in color development, two positive
controls were included adding asialofetuin to each well contain-
985

PA Sartor et al.
ing TS inhibitors. Galactose residues from asialofetuin were
quickly oxidized by galactose oxidase, increasing the optical
density without any interference from the inhibitors tested. In
addition, Clostridium perfringes neuraminidase was also includ-
ed as a positive control (C+). Unfortunately, the straightforward
detection of neutralizing antibodies in human serum samples
was hampered by a violet interference developed during the
reaction that could not be prevented by preincubation of sera at
56°C for 30 min (data not shown).
Discussion
Trans-sialidases are crucial in the biology of African and
American pathogenic trypanosomes. Their notable transferase
activity is of interest also for biotechnology applications. Be-
cause it is very effective and specific for the sialylation of
oligosaccharides, the TS from T. cruzi has been used with pre-
parative purposes (de Lederkremer and Agusti 2009).
Therefore, the development of a one-step assay for TS activity
screening/measurement is of great interest to perform high
throughput screening of substrates and inhibitors. Due to its
relevance in Chagas disease diagnosis, the possible use of
the TS inhibition assay (TIA) (Leguizamón et al. 1997; Blejer
et al. 2008), where neutralizing antibodies present in mamma-
lian sera are detected, also requires a user-friendly and
nonradioactive detection system.
In the last years other methods which avoid the use of radio-
active substrates have been described. For instance, HPAEC
has been used to define TS substrate specificity and for
screening of inhibitors (Agusti et al. 2004). Moreover, two
fluorimetric assays have been reported based on the use of
a 4-methylumbelliferyl glycoside. Schrader et al. (2003) used
4-methylumbelliferyl-β-D-galactoside as SA acceptor and SL as
donor and adapted this method to a 96-well-plate fluorescence
test. In this test, a further step of acid hydrolysis to release and
measure 4-methyl umbelliferone is necessary for better sensitiv-
ity. Neres et al. (2006) have used 4-methylumbelliferyl-α-D-N-
acetylneuraminic acid as SA donor; this method was described
for the screening of inhibitors of TS from T. cruzi (Neres et al.
2009); however, it does not discriminate sialidase from TS
activities. Also, a spectrophotometric assay has been de-
scribed by Lee et al. (2000) that used o-nitrophenyl-β-D-
galactopyranoside (ONPG) as SA acceptor and the remnant
nonsialylated ONPG is detected by a colorimetric assay.
The test described in the present work avoids radioactively
labeled compounds, requires small amounts of substrate and
enzyme, uses equipment available for routine test in all diag-
nosis laboratories, requires low manipulation without washing
or stopping steps and is easy to escalate since multiwell plates
can be used. From the biochemist point of view, it allows the
continuous quantification of the product, may discriminate be-
tween sialidase and TS activities and, because the reaction is
unidirectional, it allows the formal determination of enzymatic
parameters. The D-fucose disaccharide substrate is easily pre-
pared from the monosaccharide constituents. The benzyl
glycoside of the disaccharide is used instead of the free sugar
because it is as good as acceptor, is more stable and is obtained
in a previous step in the synthesis of the free sugar. The re-
placement of D-galactose by D-fucose as the acceptor unit
does not impair the TS reaction as shown by HPAEC-PAD
Fig. 2. Comparison of sensitivities for the nonradioactive and radioactive
assays. (A) Colorimetric assay: different amounts of TcTS were incubated with
fetuin and 1 at room temperature for 30 min. Then, galactose oxidase, HRP and
Amplex Red Reagent were added and incubation continued at 37°C. The
absorbance at 540 nm was measured for each TcTS concentration. An
expansion of the more diluted area is shown in the inset. (B) Fluorometric
assay: different amounts of TcTS were mixed with SL, disaccharide 1,
galactose oxidase, HRP and Amplex Red Reagent. Fluorescence emission at
590 nm was read after 30 min of incubation at room temperature. Fluorescence
readings are expressed as arbitrary units. Inset: expansion of values obtained at
the lower TcTS amounts tested. (C) Radioactive assay: different amounts of
TcTS were incubated with SL and [D-glucose-1-14C]-lactose. After 30 min of
incubation at room temperature, a slurry of quaternary aminoethyl-Sephadex
was added to bind the 14C-sialyllactose product, washed with water and the
counts per minute retained were quantified. An expansion of the more diluted
area is shown in the inset.
986

Nonradioactive assay for trans-sialidase screening
analysis (Figure 1). It proves that the hydrogen bond of the
OH-6 of the acceptor substrate with the Glu362 of the enzyme
through a water molecule, shown by the crystal structure of the
enzyme (Buschiazzo et al. 2002), is not crucial for the action of
the enzyme. Once established that the D-fucose disaccharide
was a good substrate in the TS reaction, it could be coupled
to the galactose oxidase assay for determination of TcTS activ-
ity and the study of inhibitors for this enzyme. A galactose
oxidase kit (Molecular Probes, Carlsbad, CA) developed for
the analysis of sialidases may be used.
By employing the assay proposed here, we may measure
both sialidase and TS activities simply by running a control
without the fucose disaccharide. The galactose oxidase assay
will then measure terminal galactosyl residues in the donor,
produced by SA hydrolysis. In this respect, enzymes with dif-
ferent TS and sialidase activities have been described
(Tiralongo et al. 2003; Ratier et al. 2008). The test can be used
for monitoring TS purification, elicitation of neutralizing anti-
bodies and for the high throughput screening of potential
inhibitors. Although throughout this work we used TcTS, the
enzyme from the African trypanosomes might be also deter-
mined following this assay.
Materials and methods
General procedures
Thin layer chromatography was performed using 0.2 mm silica
gel 60 F254 (Merck, Whitehouse Station, NJ) aluminum-
supported plates. Detection was done by ultraviolet light or
by spraying with 5% (v/v) sulfuric acid in EtOH and charring.
Column chromatography was performed on silica gel 60
(230–400 mesh, Merck). Melting points were determined with
a Fisher–Johns apparatus and are uncorrected. Optical rota-
tions were measured with a Perkin-Elmer 343 polarimeter.
NMR spectra were recorded with a Bruker AC 200 spectrome-
ter at 200 MHz (1H) and 50.3 MHz (13C) or with a Bruker AM
500 spectrometer at 500 MHz (1H) and 125 MHz (13C). N-acet-
yllactosamine, N-acetylglucosamine, D-fucose and chemical
Fig. 3. Assay of sialidase and trans-sialidase activities. TcTS was assayed in
the presence (dots) or absence (triangles) of disaccharide 1. (A) TcTS (0.1 µg)
was incubated at 37°C with SL as sialyl donor, galactose oxidase, HRP and
Amplex Red Reagent and OD540nm measured at different times. (B) The same
reaction as in A was performed at room temperature in the absence of 1 that
was incorporated after 20 min (arrow) and incubation continued. Fluorescence
was recorded at different incubation times and expressed as arbitrary units.
Fig. 4. Nonradioactive TcTS inhibition assay. Purified neutralizing antibodies from mouse (mAb 13G9) or from human patients (purified AbsNt, 2 µg) or 15 mM
DANA (2,3-dehydro-2-deoxy-N-acetylneuraminic acid) were allowed to react with TcTS (0.3 µg) for 15 min and then fetuin and disaccharide 1 substrates
were incorporated. Later, galactose oxidase, HRP and Amplex Red Reagent were added and incubated at 37°C. Two positive controls were included adding
asialofetuin (AF) to each well containing TcTS inhibitors (DANA + AF and AbsNt + AF). Clostridium perfringes neuraminidase was also included as another
positive control (C+). *P < 0.01.
987

PA Sartor et al.
reagents were purchased from Sigma (St Louis, MO). SL was
obtained from bovine colostrum by an adaptation of a reported
method (Veh et al. 1981). Galactose oxidase (EC 1.1.3.9 from
Dactylium dendroides), HRP (EC 1.11.1.7) and 10-acetyl-3,7-
dihydroxyphenoxazine (Amplex Red Reagent) were purchased
from Molecular Probes/Invitrogen (Invitrogen, Carlsbad,
CA). A recombinant TcTS expressed in Escherichia coli
was used for sialylation (Buschiazzo et al. 1996). Absorbances
were measured with a Multiscan EX-Labsystems Spectropho-
tometer. Analysis by HPAEC-PAD was performed using a
Dionex ICS-3000 HPLC system equipped with a pulse amper-
ometric detector. A CarboPac PA-100 ion exchange analytical
column (4 × 250 mm) equipped with a guard column PA-100
(4 × 50 mm) was eluted with 50 mM NaAcO in 100 mM
NaOH at a flow rate of 1.0 mL/min at room temperature.
Synthesis of benzyl β-D-fucopyranosyl-(1→6)-2-acetamido-2-
deoxy-α-D-glucopyranoside (1)
To a suspension of 4 (0.13 g, 0.17 mmol) in anhydrous MeOH at
0°C, 0.5 M NaOMe in MeOH (10 mL) was added. After stirring
for 1.5 h at room temperature, water (2 mL) was added, and the
solution passed through a column (1.5 cm × 2 cm) containing
BioRad AG 50 W-X12 (H+) resin (BioRad, Hercules, CA).
The solvent was evaporated and the remaining methyl benzoate
was removed by five successive coevaporations with water, to
afford 1 as a white solid (0.073 g, 94%), Rf 0.65 (7:1:2
nPrOH–EtOH–H2O). Crystallization from EtOH gave m.p.
1
229–230°C; [α]D +95° (c 1, H2O); H NMR (CDCl3): δ 7.27
(m, 5H, BnH), δ 4.76 (d, 1H, H-1, J=3.4 Hz), δ 4.62, 4.40
(dd, 2H, PhCH2, J=12 Hz), δ 4.24 (d, 1H, NH, J=5.6 Hz), δ
3.94 (d, 1H, H-1′, J=9.6 Hz), δ 1.79 (s, 3H, CH3CO), δ 1.10
(d, 3H, H-6′, J=6.6 Hz). 13C NMR (CDCl3): δ 175.1 (CH3CO),
δ 129.4 (PhCH2), δ 104.2 (C-1′), δ 96.6 (C-1), δ 69.2 (C-6), δ
54.4 (C-2), δ 22.6 (CH3CO), δ 16.2 (C-6′). Anal. calcd. for
C21H31NO10.H2O: C, 53.05; H, 7.00; N, 2.95. Found: C,
52.97; H, 6.76; N, 2.94.
1,2,3,4-tetra-O-benzoyl-α-D-fucopyranose (2)
Benzoyl chloride (3 mL, 26 mmol) was slowly added to an ice-
cold solution of D-fucose (514 mg, 3.13 mmol) in anhydrous
pyridine (7 mL). The reaction was stirred overnight at room
temperature and then quenched with MeOH and processed as
usual. The product was obtained in 75% yield after recrystalli-
Radioactive assay for TS activity
1
zation from ethanol. The H and 13C NMR spectroscopic data
The recombinant TcTS was incubated with 1 mM SL and
12 μM [D-glucose-1-14C]-lactose (GE Healthcare, Waukesha,
WI; 54.3 mCi/mmol) in a final volume of 30 µL of 20 mM
Tris buffer pH 7.6. After 30 min of incubation at room tem-
perature, 1 mL of bidistilled water was added to stop the
reaction. A dense slurry of quaternary aminoethyl-Sephadex
A-25 (Sigma) was added to bind the 14C-labeled sialyllactose
reaction product. After being vortexed briefly, beads were
washed three times with bidistilled water, the bound material
was eluted with 800 µL of 0.5 M NaCl and the counts per
minute were quantified.
of 2 were in agreement with those already available in the
literature (Ross et al. 2001; Piochon et al. 2009).
Synthesis of benzyl 2,3,4-tri-O-benzoyl-β-D-fucopyranosyl-
(1→6)-2-acetamido-2-deoxy-α-D-glucopyranoside (4)
To an externally cooled (0°C) solution of 1,2,3,4-tetra-O-ben-
zoyl-α-D-fucopyranose (2; 0.300 g, 0.46 mmol) in dry CH2Cl2
(5 mL), tin(IV) chloride (0.065 mL, 0.56 mmol) was added.
After 15 min of stirring at 0°C, benzyl 2-acetamido-2-deoxy-
α-D-glucopyranoside (Kuhn and Baer 1958) (3; 0.174 g,
0.56 mmol) and dry CH3CN (0.3 mL) were added, and stirring
was continued for 20 h at room temperature. The mixture was
diluted with CH2Cl2 (30 mL) and poured into saturated aque-
ous NaHCO3 with vigorous stirring. The aqueous layer was
extracted with CH2Cl2 (2 × 50 mL), and the combined organic
solutions were washed with water until pH 7, dried (MgSO4),
filtered and concentrated. The resulting syrup was purified by
column chromatography (1:30 toluene-EtOAc). A first fraction
gave unreacted 1,2,3,4-tetra-O-benzoyl-α-D-fucopyranose
(15 mg). The next fraction afforded 150 mg of 4 (42%) (Rf=
0.49; 1:9, methanol/CH2Cl2); m.p. (EtOH) 203–204°C; [α]D
Enzyme kinetics
Reaction mixtures of 20 µL containing 20 mM Tris buffer,
pH 7, 30 mM NaCl, 1 mM SL as donor and 1 mM disaccharide
1 as acceptor substrate were incubated with 300 ng purified
TcTS. After incubation, reaction mixtures were diluted with
40 µL deionized water and analyzed by HPAEC-PAD. Sialyla-
tion of 1 was calculated as the percentage of the sialylated
product obtained over the total amount of SA (free or linked
to a saccharide).
For Km calculations, 1 mM SL and different concentrations
of disaccharide 1 were incubated with 300 ng purified TcTS in
20 µL of 20 mM Tris buffer, pH 7, 30 mM NaCl for 15 min at
25°C as before. Samples were then diluted five times, and
20 µL of each was analyzed by HPAEC. The extent of sialyla-
tion of 1 was calculated from the decrease in the concentration
of the donor substrate using galacturonic acid as internal stan-
dard, in comparison with the corresponding control without
enzyme. The Km values were obtained graphically by the Line-
weaver–Burk method (Lineweaver and Burk 1934).
1
+150° (c 1, CHCl3); H NMR (CDCl3): δ 8.11–7.23 (m, 20
H, aromatic), δ 5.80 (dd, 1H, H-2′, J=10.3, 7.8 Hz), δ 5.71 (d,
1H, H-4′, J=3.0 Hz), δ 5.57 (dd, 1H, H-3′, J=10.3, 3.0 Hz), δ
4.85 (d, 1H, H-1′, J=7.8 Hz), δ 4.69 (d, 1H, H-1, J=4.0 Hz), δ
4.57, 4.16 (2d, 2H, PhCH2, J=11.8 Hz), δ 4.1–4.06 (1H), δ
3.91 (m, 1H, H-5′), δ 3.87–3.74 (m, 3H), δ 3.65–3.38 (m,
2H), δ 1.94 (s, 3H, CH3CO), δ 1.35 (d, 3H, H-6′, J=
6.4 Hz). 13C NMR (CDCl3): δ 172.0 (CH3CO), δ 166.02,
165.7, 165.43 (3xPhCO), δ 128–134 (aromatic), δ 102.1 (C-
1′), δ 96.3 (C-1), δ 74.28, 72.0, 71.68, 71.07, 70.49, 69.95,
69.79, 69.39, δ 53.6 (C-2), δ 23.18 (CH3CO), δ 16.3 (C6′).
Anal. calcd. for C42H43NO13.1/2H2O: C, 64.77; H, 5.69; N,
1.8. Found: C, 64.75; H, 5.50; N, 1.95. High-resolution elec-
trospray ionization mass spectrometry m/z 770.2810 [M+H]+
(calcd. for C42H44NO13, 770.2813); m/z 792.2622 [M+Na]+
(calcd. for C42H43NO13Na, 792.2632).
Colorimetric/fluorometric assay for TcTS activity
The indicated amounts of TcTS were incubated with 5 µL of
fetuin (10 mg/mL, Molecular Probes) or 10 µL SL (10 mg/mL,
Sigma) and 1 (2 mg/mL) at room temperature for 30 min in
30 µL of 20 mM Tris–HCl buffer, pH 7, 30 mM NaCl, con-
988

Nonradioactive assay for trans-sialidase screening
taining 0.1% of bovine albumin. Then, 70 µL of a solution
containing 3 µL of galactose oxidase (2 U/mL), 0.25 µL of
HRP (1 U/mL) and 1 µL of Amplex Red Reagent (1 mM) in
buffer Tris–HCl 50 mM pH 7.2 with 1 mM CaCl2 was added
and incubated at 37°C. OD540nm was measured at different in-
cubation times. In the fluorometric assays, reactions were
performed incubating all the reagents at room temperature in
buffer Tris–HCl 50 mM pH 7.2 with 1 mM CaCl2 containing
0.1% of bovine albumin in a final volume of 200 µL and
measuring fluorescence at excitation/emission 530/590 nm
in an Aminco-Bowman Series 2 Luminescence Spectrometer
(Thermo Spectronic, Walthman, MA).
Acknowledgements
We thank A.C.C. Frasch from Universidad Nacional General
San Martín, Argentina for a kind gift of TcTS.
Abbreviations
DANA, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid;
HPAEC-PAD, high pH anion exchange chromatography with
pulse amperometric detection; HRP, horseradish peroxidase;
ONPG, o-nitrophenyl-β-D-galactopyranoside; SA, sialic acid;
SL, α-(2,3)sialyllactose; TcTS, T. cruzi trans-sialidase; TIA,
TS inhibition assay; TS, trans-sialidase.
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