Synthesis of PEGylated lactose analogs for inhibition studies on T.cruzi trans-sialidase

Article abstract of DOI:10.1007/s10719-010-9300-7

Trypanosoma cruzi, the agent of Chagas disease, expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The enzyme is shed to the medium and may affect the immune system of the host. We have previously described that lactose derivatives effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Lactitol also prevented the apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. In this paper we report covalent conjugation of polyethylene glycol (PEG) with lactose, lactobionolactone and benzyl β-D-galactopyranosyl-(1→6)-2-amino-2- deoxy-α-D-glucopyranoside (1) with the hope to improve the bioavailability, though retaining their inhibitory properties. Different conjugation methods have been used and the behavior of the PEGylated products in the TcTS reaction was studied.

Full text of DOI:10.1007/s10719-010-9300-7

Glycoconj J (2010) 27:549–559
DOI 10.1007/s10719-010-9300-7
Synthesis of PEGylated lactose analogs for inhibition studies
on T.cruzi trans-sialidase
M. Eugenia Giorgi & Laura Ratier & Rosalía Agusti &
Alberto C. C. Frasch & Rosa M. de Lederkremer
Received: 4 May 2010 /Revised: 29 June 2010 /Accepted: 30 June 2010 /Published online: 20 July 2010
#
Springer Science+Business Media, LLC 2010
Abstract Trypanosoma cruzi, the agent of Chagas disease,
expresses a unique enzyme, the trans-sialidase (TcTS)
involved in the transfer of sialic acid from host glyco-
conjugates to mucins of the parasite. The enzyme is shed to
the medium and may affect the immune system of the host.
We have previously described that lactose derivatives
effectively inhibited the transfer of sialic acid to N-
acetyllactosamine. Lactitol also prevented the apoptosis
caused by the TcTS, although it is rapidly eliminated from
the circulatory system. In this paper we report covalent
conjugation of polyethylene glycol (PEG) with lactose,
lactobionolactone and benzyl β-D-galactopyranosyl-
(1→6)-2-amino-2-deoxy-α-D-glucopyranoside (1) with
the hope to improve the bioavailability, though retaining
their inhibitory properties. Different conjugation methods
have been used and the behavior of the PEGylated products
in the TcTS reaction was studied.
Introduction
The need to maintain drugs in blood is one of the main
goals in therapeutic applications, which are often hampered
by the short in vivo half-life of administered compounds.
Many factors are involved in the removal of substances
from the circulation, being renal excretion and inactivation
reactions by blood enzymes the main causes for the brief
plasma residence time of low-molecular mass non-protein
drugs [1]. Modification of biological molecules by covalent
conjugation with polyethylene glycol (PEG) has been used
to improve the bioavailability of drugs [2]. PEG is a linear
polyether diol with many useful properties such as
biocompatibility, solubility in aqueous and organic media,
lack of toxicity and very low immunogenicity. Probably, the
most important feature of PEG modification is that it
greatly extends the half-life (t1/2) of most proteins, and
results in a greatly increased plasma presence. This can be
attributed, in part, to the increase in molecular weight of the
conjugate beyond the limits of renal filtration and reduced
proteolysis of the conjugate [3]. PEG has been coupled to
antibodies or antibody fragments to prolong the circulating
half-lives in vivo [4]. Selective alkylation and acylation of
amino groups in a somatostatin analogue using two
different PEG reagents has been described [5]. Also, low
molecular-weight drugs have been PEGylated in order to
prolong the in vivo action [6] or for targeting drug delivery
[7].
.
.
.
Keywords Trans-sialidase PEGylation Inhibitors
Trypanosoma cruzi
:
:
M. E. Giorgi R. Agusti R. M. de Lederkremer (*)
CIHIDECAR, Departamento de Química Orgánica, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pabellón II,
There are few reports on the PEGylation of carbohydrate
molecules and these have been focused on polysaccharides
or on carbohydrates linked to proteins. Thus, the linear
glycan chitosan was coupled by amide bond formation
between the aminogroups and a carboxylic acid function-
alized PEG [8]. N-hydroxysuccinimide (NHS) esters are
frequently used for derivatization of primary amino groups.
1428 Buenos Aires, Argentina
e-mail: lederk@qo.fcen.uba.ar
:
L. Ratier A. C. C. Frasch
Universidad Nacional de General San Martín y Consejo Nacional
de Investigaciones Científicas y Técnicas,
INTI, Av. General Paz 5445, Edificio 24, Casilla de correo 30,
1650 General San Martín, Argentina

550
Glycoconj J (2010) 27:549–559
A heterodifunctional PEG with a protected amino group at
one end and a NHS ester at the other was used to locate
active molecules at the distal end of the PEG chain linked
to chitosan. An amino functionalized α-mannoside was
introduced in this way [9]. Chitosan partially amidated with
lactobionic acid was PEGylated for DNA carrier studies
[10]. These PEG-chitosan copolymers have shown im-
proved biocompatibility. Specific PEGylation of the carbo-
hydrate of ricin A-chain was accomplished by periodate
oxidation followed by hydrazide formation with a hydra-
zine derivative of PEG [11]. The method was applied to
proteins previously glycosylated with oligosaccharides
[12]. Also, a protein was modified by sequential enzymatic
transfer of galactose and PEGylated sialic acid from the
respective nucleotides [13]. However, few reports describe
direct PEGylation of small oligosaccharides. Diels-Alder
chemistry was used to immobilize several different mono-
saccharides bearing a cyclopentadiene group, attached through
a polyethylene glycol (PEG) linker, to a monolayer presenting
benzoquinone groups on a gold surface [14]. A recent paper
[15] describes the preparation of monosaccharide-capped
PEGylated quantum dots (PEG-QDs) by reaction of the
PEG-QD with a glycosidic thiol.
not report studies on toxicity in animals [30, 31]. Inhibitors
of TcTS directed to the β-galactosyl acceptor site should be
more specific, as other sialidases lack this interaction.
Lactose derivatives effectively inhibited the transfer of
sialic acid to N-acetyllactosamine, lactitol being the best
[32]. The lactose analogs are only moderate inhibitors.
However, they are non-toxic to animals [33]. In fact, Mucci
et al. proved that intravenous administration of 10 mg of
lactitol inhibited TcTS in mice blood by 95–98%. Fast
clearance from blood was solved using lactitol releasing
pellets which prevented apoptosis of spleen cells.
Here, we report PEGylation of lactose derivatives and of
benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-
D-glucopyranoside (1) with the hope to improve the
bioavailability, though retaining their inhibitory properties.
Different conjugation methods have been used and the
behavior of the PEGylated products in the TcTS reaction
was studied.
Materials and methods
Materials and general methods
Glycans have been recognized as candidates for chemo-
therapy [16]. In this respect, Trypanosoma cruzi, the agent
of American trypanosomiasis [17], expresses a unique
enzyme, the trans-sialidase (TcTS) involved in the transfer
of sialic acid from host glycoconjugates to mucins of the
parasite [18, 19]. This is the only way trypanosomes may
incorporate sialic acid, instead of using the corresponding
nucleotide sugar as donor. Oligosaccharides of the mucins
have been synthesized and their acceptor and inhibitory
properties were studied [20, 21]. Recombinant TcTS has
also been used for the preparation of sialylated oligosac-
charides [22]. Sialic acid on the surface of the parasite is
involved in host cell invasion and in protection of the
parasite against complement [23] and from killing by anti
α-galactosyl antibodies [24]. Also, in Trypanosoma brucei,
the agent of sleeping sickness, incorporation of sialic acid
through a trans-sialidase is essential for the parasite to
survive in the insect vector [25].Trans-sialidase, being a
virulence factor in the mammal and/or insect stages of
trypanosomes and being specific for the parasite, with no
equivalent in the human host, is an interesting target for
drug design. The 3D structure of TcTS shows two (sub)sites
in the active center: the sialic acid-binding site and the
galactose-binding site [26, 27]. Potential inhibitors are
usually classified depending on the active site region they
target. In the last years several laboratories have been active
in seeking inhibitors for TcTS, mainly directed to the sialic
acid binding site [28, 29]. In particular, some natural
products or synthetically modified natural products proved
to be good inhibitors in vitro of the TcTS but the authors do
Methoxypolyethylene glycol amine 750 (MW: 750)(CH3O-
PEG750-NH2), Methoxypolyethylene glycol amine 5000
(MW: 5000)(CH3O-PEG5000-NH2) and Methoxypolyethy-
lene glycol 5000 acetic acid N-hydroxysuccinimidyl ester
(MW: 5000)(CH3O-PEG5000-NHS ester) were purchased
from Fluka (Steinheim, Germany), Methoxypolyethylene
glycol N-hydroxysuccinimidyl ester (MW: 685.71) (CH3
O-PEG12-NHS ester) was purchased from Pierce Biotech-
nology (Rockford, USA). Lactobionic acid, lactose and 3’-
sialyllactose were purchased from Sigma Chemical Co.
[D-glucose-1-14C] lactose was purchased from Amersham
Biosciences. NMR spectra were recorded with a Bruker
AM 500 spectrometer at 500 MHz (1H) and 125 MHz (13C)
at 30°C. Assignments were supported by 2D HSQC
experiments. High resolution electrospray ionization mass
spectra (ESI-TOF) were recorded on BRUKER microTOF-
Q II ESI-Qq-TOF spectrometer. MALDI-TOF spectra were
collected on a PE BIOSYSTEMS DE-STR MALDI-TOF
spectrometer. Optical rotations were measured with Perkin-
Elmer 343 polarimeter. Analytical TLC was performed on
0.2 mm Silica Gel 60 F254 (Merck) aluminium supported
plates. Detection was effected by spraying with 10% (v/v)
sulfuric acid in ethanol containing 0.5% p-anisaldehyde and
charring. Column chromatography was performed on Silica
Gel 60 (230–400 mesh, Merck). Melting points were
determined with a Fisher-Johns apparatus. FT-IR spectra
were determined with a 510 P Nicolet FT-IR spectropho-
tometer (Thermo Fisher Scientific, Inc., Waltham, MA,
USA) using the KBr disk method, at 4,000−250 cm-1; 32–

Glycoconj J (2010) 27:549–559
551
64 scans were taken with a resolution of 2–4 cm-1.
Radioactivity was measured on WinSpectral 1414 liquid
scintillation counter (Wallac).
Synthesis of benzyl β-D-galactopyranosyl-(1→6)-2-
amino-2-deoxy-α-D-glucopyranoside (1).
102.4 (C-1’), 96.2 (C-1), 74.3 (C-3), 71.5-71.4 (C-4, C-3´,
C-5´), 70.5 (C-2), 69.8 (C-2´), 69.6 (C-6), 69.2 (PhCH2),
68.1 (C-4´), 61.9 (C-6’), 53.6 (C-5), 23.1 (CH3CO). Anal.
Calcd for C49H47NO15: C, 66.13; H, 5.32; N, 1.57. Found:
C, 65.74; H, 5.31; N, 1.68. ESI-TOF MS m/z: calcd for
[M + H]+: 890.3018, found: 890.3036. Calcd for [M +
Na]+: 912.2838, found: 912.2847.
Synthesis of benzyl β-D-galactopyranosyl-(1→6)-2-
acetamido-2-deoxy-α-D-glucopyranoside (4). To a solution
of compound 3 (731 mg, 0.82 mmol) in anhydrous MeOH
(10 mL) at 0°C, 0.5 M NaOMe in MeOH (10 mL) was
added. After stirring for 1 h at 0°C and 2 h at room
temperature, TLC examination showed only one compound
more polar than 3. The solution was passed through a
column of Amberlite IR 120 (plus) H+ resin eluting the
product with methanol. The solvent was evaporated and the
remaining methyl benzoate was removed by successive co-
evaporations with water, to afford 291 mg of 4 as a white
solid (75 % yield). Crystallization from EtOH gave mp
234-235°C. The physical constants and spectra were the
same as previously reported [35].
Synthesis of benzyl 2-acetamido-2-deoxy-α-D-glucopyr-
anoside (2). To a solution of N-acetyl-D-glucosamine (3 g,
13,56 mmol) in benzyl alcohol (21 mL), BF3 diethyl ether
complex (320 μl, 2.7 mmol) was added and the mixture
was heated with stirring at 80°C for 4 h. After this time,
TLC showed disappearance of the starting compound and
the product was precipitated by addition of diethyl ether
(100 mL). The benzyl glycosides were obtained in 83%
yield as a mixture of α and β anomers (8:2 ratio) (Rf 0.66
1
and 0.60 respectively, nPrOH-EtOH-H2O, 7:1:2). H NMR
(D2O, 500 MHz):δ anomeric region 4.83 (d, 0.8 H, J=
3.5 Hz, H-1α), 4.42(d, 0.2 H, J=8.5 Hz, H-1β) Pure benzyl
2-acetamido-2-deoxy-α-D-glucopyranoside was obtained
by fractional crystallization from ethanol. The melting
point and optical rotation were in agreement with those
previously reported [34].
Synthesis of benzyl (2,3,4,6-tetra-O-benzoyl-β-D-
galactopyranosyl)-(1→6)-2-acetamido-2-deoxy-α-D-gluco-
pyranoside (3).To an externally cooled (0°C) solution of
1,2,3,4,6-penta-O-benzoyl-β-D-galactopyranose [35]
(1.67 g, 2.36 mmol) in dry Cl2CH2 (6 mL) under argon,
tin(IV) chloride (0.27 mL, 2.31 mmol) was added. After
15 min of stirring at 0°C, benzyl 2-acetamido-2-deoxy-α-
D-glucopyranoside (0.60 g, 1.42 mmol) and dry CH3CN
(0.6 mL) were added, and stirring was continued for 16 h at
room temperature. TLC showed a main compound of Rf
0.69, minor amounts of less polar compounds and remain-
ing starting material (15% CH3OH in CH2Cl2). The mixture
was diluted with Cl2CH2 (40 mL) and poured into cold
saturated aqueous NaHCO3 with vigorous stirring. The
aqueous layer was extracted with Cl2CH2 (2×50 mL), and
the combined organic solutions were washed with water
until pH 7, dried (MgSO4), filtered, and concentrated under
reduced pressure. The product was purified by column
chromatography (toluene-EtOAc,1:15) to give compound 3
(731 mg, 42.4%), mp 118–120°C; [α]D:+86,8º (c1,
CHCl3). 1H NMR (CDCl3): δ 8.05-7.2 (m, 25 H, aromatic),
6.0 (dd,1H, J3´,4´=3.5 Hz, J4´,5´=1 Hz, H-4’), 5.91 (d, 1H,
Synthesis of benzyl β-D-galactopyranosyl-(1→6)-2-
amino-2-deoxy-α-D-glucopyranoside (1). Compound 4
(203 mg, 0.43 mmol) was dissolved in 1.2 mL of 1 M
KOH and heated at 100°C for 16 h. After this time, TLC
showed that the reaction was complete, (Rf 0.36, n-PrOH-
EtOH-H2O, 7:1:2). The solution was passed through a
column (1.5 cm×6 cm) containing Amberlite IR 120 (plus)
H+ resin and eluted with 0.1 N HCl to afford 110 mg of 1
1
(0.29 mmol, 67% yield).[α]D:+45,4º (c1, H2O). H NMR
(D2O): δ 7.47 (m, 5H, aromatic), 5.24 (d, 1H, J1,2=3.7 Hz,
H-1), 4.81, 4.66 (2d, 2H, J=11.6 Hz, PhCH2), 4.43 (d, 1H,
J
1´,2´=7.9 Hz, H-1’), 4.10 (dd, 1H, J6a,6b=11.2 Hz, J5,6a=
1.7 Hz, H-6a), 3.93 (dd, 1H, J4´,5´=0.7 Hz, J3´,4´=3.5 Hz,
H-4´), 3.89 (dd, 1H, J2,3=10.5 Hz, J3,4=9.1, H-3), 3.89 (dd,
1H, J5,6b=4.3 Hz, H-6b), 3.87 (ddd, 1H, J4,5=9.5 Hz, H-5),
3.79 (dd, 1H, J5´,6´a=7.9 Hz, J6´a,6´b=11.7 Hz, H-6´a), 3.75
(dd, 1H, J5´,6´b=4.3 Hz, H-6´b), 3.7 (ddd, 1H, H-5´), 3.66
(dd, 1H, J2´,3´=10.0 Hz, H-3´), 3.64 (dd, 1H, H-4), 3.56
(dd, 1H, H-2´), 3.37 (dd, 1H, H-2). 13C NMR (D2O): δ
136.5, 128.8, 128.6, 128.5 (aromatic), 103.4 (C-1’), 94.5 (C-
1), 75.1 (C-5’), 72.7 (C-3´), 71.2 (C-5), 70.7 (C-2´), 70.0
(PhCH2), 69.9 (C-3), 69.1 (C-4), 68.6 (C-4´), 67.8 (C-6),
60.9 (C-6’), 53.8 (C-2). ESI-TOF MS m/z: calcd for [M +
H]+:464.1684 found:432.1863. Calcd for [M + Na]+: 454.
1684 found: 454.1677.
J=8.5 Hz, NH), 5.85 (dd, 1H, J2´,3´=10.5 Hz, J1´,2´
7.8 Hz, H-2’), 5.64 (dd, 1H, H-3’), 4.93 (d, 1H, H-1’), 4.7
(d, 1H, J1,2=3.5 Hz, H-1), 4.67, 4.42 (2dd, 2H, J6´a,6´b
=
=
11.5 Hz, J6´a,5´=6.5 Hz, J6´b,5´=6.5 Hz, H-6´a, H-6´b), 4.57,
4.19 (2d, 2H, J=11.7 Hz, PhCH2), 4.35 (ddd, 1H, H-5´),
4.27 (dd, 1H, J5,6a=8.5 Hz, J6a,6b=11.5 Hz, H-6a), 3.88
(ddd, 1H, J4,5=9 Hz, J5,6b=4 Hz, H-5), 3.83-3.77 (2dd, 2H,
H-2, H-6), 3.58 (dd, 1H, J2,3=10.2, J3,4=9 Hz, H-3), 3.39
(dd, 1H, H-4), 1.90 (s, 3H, CH3). 13C NMR (CDCl3): δ
172.1(CONH), 166.1-165.3 (PhCO), 136-128 (aromatic),
PEGylation of compound 1
Preparation of benzyl β-D-galactopyranosyl-(1→6)-2-
CH3O-PEG12amido-2-deoxy-α-D-glucopyranoside (5). A
solution of benzyl β-D-galactopyranosyl-(1→6)-2-amino-
2-deoxy-α-D-glucopyranoside (1) (38 mg, 0.09 mmol) and

552
Glycoconj J (2010) 27:549–559
CH3O-PEG12-NHS ester (50 mg, 0.07 mmol) in 1 mL
phosphate buffer (50 mM, pH 8) was left at room
temperature for 24 h . The reaction was monitored by
TLC. The PEGylated compound 5 (Rf 0.33, EtOH-H2O-
NH4OH(c), 9:1: 0.15) appeared as a black spot by spraying
with the sulfuric acid reagent and heat charring. Unreacted
PEG is not detected with this reagent and compound 1 had
Rf 0.51. The product was purified by passing through a RP-
18 column (2 g, Strata, Phenomenex). Some non-reacted
compound 1 and salts were eluted with water and
compound 5 was eluted with methanol. After evaporation
of methanol under vacuum followed by lyophilization,
compound 5 was obtained as a white powder (59 mg, 67%).
1H NMR (D2O): δ 7.44 (m, 5 H, aromatic), 4.95 (d, 1H, J=
3.6 Hz, H-1), 4.54 (d, 1H, J=12 Hz, PhCH2), 4.43 (d, 1H,
J=7.8 Hz, H-1’), 4.15 (d, 1H, J=9.6 Hz, NH), 3.96-3.88
(m, 5H, sugar protons), 3.72-3.67 (m, 44H, OCH2CH2O),
3.66-3.56 (m, sugar protons), 3.37 (s, 3H, OCH3), 2.52 (dt,
2H, J=1,7 Hz, J=6 Hz, CH2CO). 13C NMR (D2O): δ,174.2
(CO), 137.0, 128.7, 128.4 (aromatic), 103.4 (C-1´), 96.1
(C-1), 75.1, 72.7, 71.1, 70.9, 70.8, 70.7, 68.6, 68.3, 61.0,
(sugar carbons), 69.6 (OCH2CH2O), 58.0 (OCH3), 53.6 (C-
2), 35.8 (CH2CO). MALDI-TOF MS m/z: calcd for [M (12
oxyethylene) + Na]+: 1,024.49; found: 1,024.4953.
ninhydrine and the appearance of the PEGylated sugar
amide 8 with the sulfuric acid reagent (Rf 0.35, EtOH-H2O-
HAcO, 80:5:15). The reaction was completed after 6 h. The
excess of CH3O-PEG750-NH2 was removed by passing
through a cationic exchange resin. The product was eluted
with water and chromatographed on a Sephadex G10
column (1.5 cm×7 cm). Conjugate 8 was recovered in the
first 40 mL of water. After evaporation a white solid was
1
obtained (209 mg, 66% yield). IR (C=O) 1,654 cm-1. H
NMR (D2O): δ 4.55 (d, 1H, J=7.8 Hz, H-1’), 4.42 (d, 1H,
J=2.75 Hz, H-2), 4.2 (m, 1H), 3.98 (dd, 1H, J=4.15 Hz, J=
6.45 Hz), 3.95-3.74 (m, sugar protons), 3.73-3.6 (m, 60H,
OCH2CH2O), 3.56 (dd, 2H, J=8 Hz, J=9.7 Hz,
NHCH2CH2), 3.46 (m, 2H, NHCH2), 3.38 (s, 3H, OCH3).
13C NMR: 174.4 (C-1), 103.5 (C-1´), 81.0, 75.3, 72.5, 72.4,
71.4, 70.4, 68.8, 68.6, 61.9, 61.0 (sugar carbons), 71.0,
70.9, 69.6, 69.4 (OCH2CH2O), 58.0 (OCH3), 38.6
(NHCH2). MALDI-TOF MS m/z: calcd. for [M (16
oxyethylene) + Na]+: 1,098.55; found: Mn 1,130.9, Mw
1,143.0, Mp 1,098.55.
Preparation of N-(CH3O-PEG 5000)-lactobionamide
(9). Lactobionolactone 7 (40.8 mg, 0.12 mmol) and
CH3O-PEG5000-NH2 (200 mg, 0.04 mmol) were dissolved
in anhydrous dimethylformamide (2 mL) under argon and
the reaction mixture was heated at 100°C for 15 h. After
this time CH3O-PEG5000-NH2 was not longer detected by
ninhydrine on a TLC plate. The solution was extensively
dialyzed (cut-off 3500) and lyophilized yielding a white
powder (136 mg, 65.5%). IR (C = O) 1,672 cm-1. 1H NMR
(D2O): δ 4.55 (d, J=7.8 Hz, H-1’); 4.41 (d, J=2.75 Hz, H-
2), 4.36 (m, 1H), 4.2-3.88 (m, sugar protons), 4.18 (m, 1H,
NH), 3.88-3.5 (m, OCH2CH2O), 3.43 (m, NHCH2), 3.37 (s,
OCH3). ESI-TOF MS m/z: calcd. for [M (95 oxyethylene) +
NH4]+: 4,569; found Mn 4,586.6, Mw 4611.5, Mp 4,569.
Preparation of benzyl β-D-galactopyranosyl-(1→6)-2-
CH3O-PEG5000amido-2-deoxy-α-D-glucopyranoside (6).
Compound 1 (26 mg, 0.04 mmol) was conjugated with
CH3O-PEG5000-NHS ester (150 mg, 0.024 mmol) as
described for 5. After 24 h, analysis by TLC showed a
product close to the origin of the plate. Excess of
disaccharide and salts were removed by dialysis against
water (cut off 3500). After lyophilization compound 6
1
(148 mg, 97%) was obtained as a white powder. H NMR
(D2O): δ 7.44 (m, aromatic), 4.98 (d, 3.7 Hz, H-1), 4.55 (d,
J=11.8 Hz, PhCH2), 4.44 (d, J=7.8 Hz, H-1’), 4.18 (d, J=
9.5, NH), 4.09-3.9 (m, sugar protons); 3.87-3.56 (m,
OCH2CH2O); 3.37 (s, OCH3). ESI-TOF MS m/z: calcd
for [M (104 oxyethylene) + NH4 ]+: 5,099; found Mn
4992.95, Mw 5007.41, Mp 5097.
PEGylation of lactose by reductive amination
Preparation of 1-amino-(CH3O-PEG750)-lactitol (10).
Lactose (100 mg, 0.3 mmol) and CH3O-PEG750-NH2
(75 mg, 0.1 mmol) were dissolved in 3 mL of water, the
pH was adjusted to 8 with 10% acetic acid and NaBH3CN
(30 mg, 0.5 mmol) was added. The reaction was kept at
50°C for 96 h and monitored by TLC which showed
disappearance of CH3O-PEG750-NH2 with the ninhydrine
reagent whereas conjugate 10 was detected with the sulfuric
acid reagent (Rf 0.16, EtOH-H2O, 8:2). The solution was
evaporated under vacuum, boric acid was removed by
successive co-evaporations with methanol and finally the
product was purified by passage through a cationic resin.
The excess of lactose was eluted with water and compound
10 was eluted with HCl 1 N. Lyophilization afforded 78 mg
(71.3% yield) of a white powder. 1H NMR (D2O) δ 4.54 (d,
1H, J=8 Hz, H-1’), 4.24 (m,1H, NH), 3.97-3.77 (m, 12H,
PEGylation of lactobionolactone
Lactobionolactone (7) was prepared from lactobionic acid
as previously described [36]. Analysis by TLC showed
disappearance of lactobionic acid, Rf 0.38, and formation of
the lactone, Rf 0.78 (EtOH-H2O-HAcO, 60:25:15). IR
(CO): 1,741 cm-1
Preparation of N-(CH3O-PEG750)-lactobionamide (8).
Lactobionolactone 7 (100 mg, 0.29 mmol) and CH3O-
PEG750-NH2 (300 mg, 0.4 mmol) were dissolved in
anhydrous dimethylformamide (1.5 mL) under argon and
the reaction mixture was heated at 120°C. The reaction was
monitored by TLC, detecting the PEGamine reagent with

Glycoconj J (2010) 27:549–559
553
sugar protons), 3.76-3.64 (m, 48 H, OCH2CH2O), 3.59-
3.56 (dd, 2H, NH-CH2CH2O), 3.44-3.31 (m, 3H, NH-
CH2CH2O and H-6), 3.39 (s, 3H, OCH3), 3.22 (m, 1H,
CH2(a)NH), 3.02 (m, 1H, CH2(b)NH). 13C NMR: 102.8 (C-
1´), 78.6, 75.3, 72.4, 70.9, 70.4, 68.7, 67.7, 65.3, 61.9, 61.2
(sugar carbons), 71.0, 69.6, 69.4 (OCH2CH2O), 58.1
(OCH3), 49.5 (C-1), 47.1 (NHCH2). ESI-TOF MS m/z:
calcd. for [M (13 oxyethylene) + Na]+: 952.49; found Mn
980.46, Mw 985.91, Mp 952.49.
Results and Discussion
Synthesis of PEG-conjugates
Three different chemical approaches were used for PEGy-
lation of disaccharide derivatives containing β-D-Galp as
non-reducing unit. 1. Amide formation between the amino
group of benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-
deoxy-α-D-glucopyranoside (1) and a succinimidyl activat-
ed ester of PEG (PEG-NHS ester). 2. Amide formation
between the carboxyl group of lactobionic acid and an
amino-derivatized PEG (PEGamino). 3. Reductive amina-
tion of lactose with a PEGamino in the presence of
NaBH3CN. The conjugates were characterized by NMR
and mass spectrometry, MALDI-TOF or ESI-TOF were
used. Except for the monodisperse CH3O-PEG12-NHS
ester, all PEGylations were performed with polydisperse
PEGs and the values for Mn (number average molecular
weight), Mw (weight average molecular weight) and Mp
(molecular weight at peak top) were obtained. In all cases
the PDI value (polydispersity index) was close to 1. The
match between the experimental and calculated values
confirmed the identity of the conjugates. Our purpose was
to study the influence of PEGylation on the inhibitory
properties of the above mentioned disaccharides in the
TcTS reaction.
Competitive radioactive assay for TS activity
Trans-sialidase activity was measured as the transfer of
sialic acid from 1 mM of 3’-sialyllactose to 12 μM [D-
glucose-1-14C] lactose (55 mCi/mmol) by 0.5 ng of purified
TcTS enzyme in 30 μl of 20 mM Hepes-Na (pH 7.5), 0.2%
BSA, 30 mM NaCl. After 60 min at room temperature, the
reaction was stopped by dilution with 1 ml of water. QAE-
Sephadex (Amersham Pharmacia Biotech) was added and
the resin was washed twice with water. Negatively charged
compounds were eluted with 800 μl of 1 M NaCl and
quantified in a WinSpectral 1414 liquid scintillation
counter. When required, the purified enzyme was diluted
in the reaction buffer before use (0.5 ng of TcTS611/2
rendered about 4,000 cpm per hour).
Increasing concentrations (0.2 to 2.0 mM) of the
PEGylated sugars were preincubated for 20 min with the
enzyme at room temperature prior to the addition of 1 mM
of 3’-sialyllactose and [D-glucose-1-14C] lactose.
PEGylation of benzyl β-D-galactopyranosyl-(1→6)-2-
amino-2-deoxy-α-D-glucopyranoside
Results are expressed in percentage of inhibition of
trans-sialidase activity. Radioactivity measured without
addition of any inhibitor was considered as 0% of inhibition
(100% of enzymatic activity).
We have previously shown that the disaccharide β-D-
galactopyranosyl-(1→6)-2-acetamido-2-deoxy-α-D-
glucose as well as its benzyl glycoside 4, are good
acceptors for sialic acid in the reaction catalyzed by TcTS
[20]. They are also competitive inhibitors when 3’-
sialyllactose was used as the sialic acid donor and N-
acetyl-lactosamine as the acceptor. A simplified preparation
of compound 4, that relies on the selective glycosylation at
6-O of benzyl α-D-GlcNAc (2) is now presented. Although
the total yield is only moderate (42%), the present synthesis
of 4 avoids the cumbersome protection and deprotection
steps. Deacylation to afford the reactive amino group was
accomplished by alkaline hydrolysis (Scheme 1).
Assay of PEG-conjugates in blood
Adult mice (C3H strain) were intravenously injected with
200 μl of sterile PBS containing 30 mg (29.5 mM) of
compound 6, 28 mg of CH3O-PEG5000-NHS ester
(29.5 mM,), 44 mg (50 mM) of compound 9 or 40 mg of
CH3O-PEG5000-NH2 (50 mM). Blood was extracted from
the tail at different times and serum was used to assay free
or combined PEG.
The colorimetric test described by Nag et al [37] was
adapted to assay the PEG-conjugates in blood. Briefly,
10 μl of sera, 0.5 mL of CHCl3 and 0.5 mL of the
ferrothiocyanate reagent (prepared by dissolving 16.2 g
anhydrous ferric chloride (FeCl3) and 30.4 g ammonium
ferrothiocyanate (NH4SCN) in 1 L of water) were vigor-
ously shaken for 30 min and later centrifuged at 5,000 rpm
for 2 min. Absorbances from chloroformic layers were
recorded at 510 nm. A calibration curve using CH3O-
PEG5000-NHS ester was performed.
Monomethoxy polyethyleneglycol activated as the N-
hydroxysuccinimide ester (CH3O-PEG-NHS ester) was
used for amidation of the 2-amino group in the sugar with
the PEG chain (Scheme 2). Conjugation was performed
using two different molecular weight PEG derivatives, a
low molecular weight PEG, which introduces 12 oxy-
ethylene groups (CH3O-PEG12-NHS ester) and a high
molecular weight, polydisperse PEG (CH3O-PEG5000-
NHS ester). The reaction was mild. Amidation took place
in buffer pH 8 at room temperature and was monitored by

554
Glycoconj J (2010) 27:549–559
Scheme 1 Synthesis of benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside (1). Conditions: a 16 h, r.t.; b 1 h, 0°C→2 h
r.t.; (c) 1 M KOH, 16 h, 100°C
TLC being detected as a black spot after heating with the
sulfuric acid reagent. The starting CH3O-PEG-NHS ester
did not stain with this reagent. After purification by passing
through a RP-18 cartridge or by dialysis against water and
lyophilization, in the case of the high molecular weight
product, compounds 5 and 6 were obtained as white
powders in 61% and 97% yield, respectively. The 1H
NMR spectrum of 5 (Fig. 1a) indicated PEGylation with
one equivalent of PEG, by integration of the only OCH3
signal, which appeared as a singlet at δ 3.37 ppm and the
two anomeric signals at δ 4.95 ppm (J1,2=3.6 Hz) for the
α-GlcN and at δ 4.43 ppm (J1’,2’=7.8 Hz) due to the β-
Galp. One of the protons of the PhCH2, substituent in the
sugar, was shown at δ 4.54 ppm with the characteristic high
coupling constant J=12 Hz, the other proton was sup-
pressed together with the DHO signal. The PEG oxy-
methylene protons appeared as a strong signal at δ
3.70 ppm, which integrated to about 11 oxyethylene
groups. The methylene linked to the amide carbonyl
appeared at higher field (δ 2.52 ppm). The 13C NMR
spectrum (Fig. 2a) showed among others the anomeric
signals at δ 103.4 ppm (β-Galp) and δ 96.1 ppm (α-GlcN),
and the C-2 signal of the amino sugar at δ 53.6 ppm. For
the PEG moiety the OCH3 signal appeared at 58.0 ppm, the
methylene next to the carbonyl sugar amide appeared at
35.8 ppm and the oxymethylene groups of the PEG chain
appeared as a strong peak at 69.6 ppm. MALDI-TOF mass
spectral analysis showed a main peak of [M + Na]+ at 1024
corresponding to the conjugate 5 with 12 oxyethylene
groups.
Accordingly, the higher molecular weight PEG-
conjugate 6 showed signals with similar chemical shifts
(not shown). ESI-TOF mass spectral analysis of 6 revealed
a series of 44 Da (OCH2CH2) spaced peaks corresponding
+
to the NH4 adducts. In this case, ammonium acetate was
added to help ionization. A main peak of [M + NH4]+ at
5,097 was observed. This value corresponded to conjugate
6 with 104 oxyethylene groups.
PEGylation of lactobionolactone
We had previously determined that lactobionic acid was a
good acceptor of sialic acid and inhibitor of TcTS [32]. In
this work we took advantage of the carboxyl group already
present in this disaccharide to form an amide linkage with
an amino functionalized PEG (Scheme 3). Again, we used
two different molecular weight derivatives, CH3O-PEG750-
NH2 and CH3O-PEG5000-NH2. In the case of the high
MW PEG, a molecular excess of the sugar was used since it
could be easily removed by dialysis after reaction. The
lactobionic acid was activated as lactobionolactone [36] and
then heated with the corresponding PEGamino in anhy-
drous dimethylformamide. The reaction could be monitored
by TLC. Whereas the PEGamino starting material was
detected with the ninhydrin reagent, the sugar conjugate
was only revealed with the sulfuric acid reagent. In the case
of the lower MW conjugate 8, purification from excess
PEGamino was accomplished by cation exchange chroma-
tography, which retained excess reagent, followed by
Sephadex G-10 column chromatography. After lyophiliza-
Scheme 2 Synthesis of N-
PEGylated derivatives of benzyl
β-D-galactopyranosyl-(1→6)-2-
amino-2-deoxy-α-D-glucopyra-
noside. Conditions: 24 h, r.t

Glycoconj J (2010) 27:549–559
555
-OCH2CH2O-
-OCH2CH2O-
a
a
-OCH3
-OCH2Ph H-1’
H-1
NH
5.0
4.8
4.6
44.
4.2
4.0
3.8
3.6
3.4
3.2
ppm
180
160
140
120
100
80
60
40
ppm
-OCH2CH2O-
-OCH2CH2O-
b
b
-OCH3
-NHCH2-
H-1’ H-2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
ppm
180
160
140
120
100
80
60
40
ppm
-OCH2CH2O-
-OCH2CH2O-
c
c
-OCH3
-CH2NH-
H-1’
NH
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
32.
ppm
180
160
140
120
100
80
60
40
ppm
Fig. 1 1H NMR spectra of PEGylated sugar derivatives in D2O
(500 MHz). a. benzyl β-D-galactopyranosyl-(1→6)-2-CH3O-PEG12a-
mido-2-deoxy-α-D-glucopyranoside. b. N-(CH3O-PEG750)-lactobio-
namide. c. N-(CH3O-PEG750)-lactitol
Fig. 2 13C NMR spectra of PEGylated sugar derivatives in D2O
(500 MHz). a. benzyl β-D-galactopyranosyl-(1→6)-2-CH3O-PEG12a-
mido-2-deoxy-α-D-glucopyranoside. b. N-(CH3O-PEG750)-lactobiona-
mide. c. N-(CH3O-PEG750)-lactitol
tion, compound 8 was obtained as a white powder in 66%
yield. The IR spectrum of the conjugate showed the amide
carbonyl at 1,654 cm-1 and disappearance of the lactone
carbonyl at 1,740 cm-1.
the OCH3 signal, which appeared as a singlet at δ 3.38 ppm
and the only anomeric signal at δ 4.55 ppm (J=7.8 Hz) due
to the β-Galp. A characteristic doublet at δ 4.42 ppm (J=
2.8 Hz) due to the H-2 of the derivatized lactobionamide
was also observed. The oxymethylene groups of the
repeating units gave a strong signal centered at δ
The 1H NMR spectrum of 8 (Fig. 1b) indicated
PEGylation with one equivalent of PEG, by integration of

556
Glycoconj J (2010) 27:549–559
Scheme 3 Lactobionolactone amidation with PEG-NH2. Conditions: Dimethylformamide (DMF), 6 h, 120°C for 8 and 15 h, 100°C for 9
3.70 ppm. The 13C NMR spectrum (Fig. 2b) showed among
others the carbonyl signal of the amide at δ 174.4 ppm and
the anomeric signal at δ 103.4 ppm (β-Galp). For the PEG
moiety the OCH3 signal appeared at 58.0 ppm, the
methylene next to the amide appeared at 38.6 ppm and
the oxymethylene groups of the chain appeared as strong
peaks at 69.6-69.4 ppm. MALDI-TOF mass spectral
analysis revealed a series of 44 Da spaced peaks
corresponding to the Na+ adducts. A main peak of [M +
Na]+ at 1,098 was observed corresponding to conjugate
8 with 16 oxyethylene groups.
pH 8, conditions under which the selective reduction of the
imine takes place. The reaction was monitored by TLC,
which showed disappearance of the starting PEGamino
compound when revealing with the ninhydrin reagent.
Purification of the reaction mixture was performed by
means of a cation exchange resin that retained the non-
reacted PEGamino. Boric acid was then eliminated by
co-evaporations with methanol. A white solid of CH3O-
PEG750-NH-lactitol (10) was obtained with 71% yield.
1
The H NMR spectrum of 10 (Fig. 1c) indicated PEGyla-
tion with one equivalent of PEG, by integration of the only
OCH3 signal which appeared as a singlet at δ 3.39 ppm and
the anomeric signal at δ 4.54 ppm (J=8.0 Hz) due to the β-
Galp. Characteristic signals at δ 4.24 ppm due to the NH of
the secondary amine and at δ 3.22 and δ 3.02 ppm due to the
methylene protons obtained by reduction of the lactose
anomeric carbon were also observed. The 13C NMR spectrum
(Fig. 2c) showed among others the only anomeric signal at δ
102.8 ppm (β-Galp) and the -CH2NHCH2- signals at δ
49.6 ppm and 47.2 ppm. For the PEG moiety the OCH3
signal appeared at 58.1 ppm and the oxymethylene groups of
the chain appeared as a strong peak at 69.5 ppm. ESI-TOF
mass spectral analysis revealed a series of 44 Da spaced
peaks corresponding to the Na+ adducts. A main peak of
[M + Na]+ at 952.49 was observed corresponding to the
conjugate 10 with 13 oxyethylene groups.
For the isolation of the higher MW conjugate 9 the
excess of lactobionolactone was removed by dialysis and,
after lyophilization, 9 was obtained as a white powder in
66% yield. IR spectroscopy confirmed the presence of the
amide group by showing its characteristic absortion band at
1,672 cm-1 and the disappearance of the lactone band above
1
1,700 cm-1. The H NMR spectrum of 9 (not shown) was
similar to the spectrum of 8. ESI-TOF mass spectral
analysis revealed a series of 44 Da spaced peaks
corresponding to the [NH4]+ adducts, accompanied by
lower intensity peaks from the [H]+ adducts. A main peak
of [M + NH4]+ at 4,569 was observed corresponding to
conjugate 9 with 95 oxyethylene groups.
PEGylation of lactose
Previous results [32] indicated that lactitol was the
preferred acceptor in comparison with lactose and N-
acetyllactosamine, suggesting that better binding with the
enzyme takes place with the open chain alditol. Lactitol
also prevented parasite sialylation and partially inhibited
cell infection. PEGylation of lactose with a PEGamino
derivative by reductive amination retains the open chain
structure of lactitol, while providing a stable amino-bond
between the PEG derivative and the disaccharide
(Scheme 4). The reaction was performed incubating lactose
with CH3O-PEG750-NH2 in the presence of NaBH3CN at
Inhibition of sialylation of N-acetyllactosamine
by PEG-conjugates
To find out whether the PEG derivatives used in this study
retain the ability of inhibiting sialylation of lactose,
different concentrations of compounds 5,6,8,9 and 10 were
tested in transfer reactions containing 1 mM of 3’-
sialyllactose as a donor, 12 μM [D-glucose-1-14C]-lactose
as an acceptor, and TcTS (Table 1). Inhibition of sialylation
was quantified by measuring the radioactivity bound to an
anion exchange resin after incubation and comparing with
Scheme 4 Lactose modification by reductive amination with PEG-NH2 Conditions: pH 8, 96 h, 50°C

Glycoconj J (2010) 27:549–559
557
Table 1 TcTS inhibition by lactose analogs and their PEGylated
derivatives using the radioactive inhibition assay
rapid clearance from blood takes place by kidney filtration,
probably due to the low MW (5,000 Da) of the polymer
used for conjugation to the sugar. The results suggest the
use of homobifunctional or multi-arm PEGs for coupling
with lactose analogs. These PEG derivatives, having higher
molecular weights and carrying several active groups,
might prolong blood circulation though maintaining the
inhibitory properties. Multiarm (star) PEG derivatization
has been successfully used to improve the efficacy of small
drugs like N-acetyl cysteine [38].
Inhibitora
% Inhibition
IC50 (mM)
Lactitol
90
86
60
82
61
21
56
3.5
0.21
0.33
0.70
0.34
0.73
2.70
0.61
4.52
Latobionic Acid
1
5
6
8
9
The methods described in the present work for the
preparation, purification and characterization of the PEG-
conjugates provide useful data for conjugation of sugar
derivatives to the higher MW PEGs.
10
a 1 mM Concentration of each compound and the donor 3´-sialyllactose
were used
the radioactivity bound in the absence of inhibitor. The
PEGylated amides (5 and 6) of the aminodisaccharide 1
showed inhibition values similar to those of the original
disaccharide, even better for the shorter PEG conjugate.
These results indicate that the presence of a polyoxy-
ethylene chain attached to the glucoside unit do not impair
their ability to interact with the enzyme. However, the PEG
derivatives of the open chain sugars (8-10) gave lower
inhibition values for the TcTS reaction than the
corresponding precursors, being insignificant for the PEGy-
lated amine 10.
Stability in blood
To determine if the insertion of the long chain of PEG in
the disaccharides increased the permanence in blood of the
conjugates, the PEG derivatives 6 or 9 were injected in
mice and a sample of blood was taken at different times
after injection (0-120 min). Controls were injected with
equivalent amounts of PEG. Compounds 6 and 9 were
chosen because of the larger molecular weight of the PEG.
Quantification was performed using a colorimetric method
based on partitioning a chromophore present in the
ammonium ferrothiocyanate reagent from an aqueous to a
chloroform phase in the presence of PEG. This method was
previously reported for evaluation of PEG-protein conju-
gates [37]. The concentration of compound 6 in a blood
sample taken immediately after injection was 4 mg/mL and
diminished to 1 mg /ml after 30 min. This value remained
constant for 2 h (Fig. 3a). A similar behavior was observed
for compound 9 (Fig. 3b) and for controls injected with
CH3O-PEG5000-NHS ester or CH3O-PEG5000-NH2, re-
spectively. The recovery of PEGylated conjugate in urine
was estimated for 9. A value of 3.5 mg of compound 9 in
100 μL of urine was found after 30 min of injection,
decreasing to 1 mg /100 μL after 60 min. Although total
recovery values were not determined it was shown that
Fig. 3 Plasma clearance of PEG derivatives. Adult mice were
intravenously injected with 200 μl of sterile PBS containing: a.
30 mg of compound 6 (♦─♦) or 28 mg of CH3O-PEG5000-NHS ester
(o─o); b, 44 mg of compound 9 (♦─♦) or 40 mg of CH3O-PEG5000-
NH2 (o─o). Blood was extracted from the tail at different times and
10 μL serum was used to assay free or combined PEG by a
colorimetric assay [37]. Time 0 refers to samples taken immediately
after injection

558
Glycoconj J (2010) 27:549–559
14. Houseman, B.T., Mrksich, M.: Carbohydrate arrays for the
evaluation of protein binding and enzymatic modification. Chem
Biol 9, 443–454 (2002)
15. Kikkeri, R., Lepenies, B., Adibekian, A., Laurino, P., Seeberger, P.
H.: In vitro imaging and in vivo liver targeting with carbohydrate
capped quantum dots. J. Am. Chem. Soc. 131, 2110–2112 (2009)
16. Kawasaki, N., Itoh, S., Hashii, N., Takakura, D., Qin, Y., Huang,
X., Yamaguchi, T.: The significance of glycosylation analysis in
development of biopharmaceuticals. Biol. Pharm. Bull. 32, 796–
800 (2009)
17. Moncayo, A.: Chagas disease: current epidemiological trends alter the
interruption of vectorial and transfusional transmission in the Southern
Cone countries. Mem. Inst. Oswaldo. Cruz. 98, 577–591 (2003)
18. Schenkman, S., Jiang, M.S., Hart, G.W., Nussenzweig, V.: A
novel cell surface trans-sialidase of Trypanosoma cruzi generates
a stage-specific epitope required for invasion of mammalian cells.
Cell 65, 1117–1125 (1991)
The PEG-conjugates are potentially useful for studies on
inhibition of other galactose binding proteins. In this respect,
amino derivatives of lactulose have been synthesized and their
function as galectin inhibitors has been described [39].
Acknowledgments This work was supported by grants from
Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT),
National Research Council (CONICET) and Universidad de Buenos
Aires. A. C. C. Frasch, R. M. de Lederkremer and R. Agustí are
research members of CONICET and M. E. Giorgi is a fellow from
CONICET. The work of A. C. C. Frasch was partially supported by
International Research Scholar grants from the Howard Hughes
Medical Institute.
19. Frasch, A.C.C.: Functional diversity in the tans-sialidase and
mucin families in Trypanosoma cruzi. Parasitol. Today 16, 282–
286 (2000)
References
1. Mongardini, C., Veronese, F.M.: Stabilization of substances in
circulation. Bioconjugate Chem. 9, 418–450 (1998)
2. Veronese, F.M., Mero, A.: The impact of PEGylation on
biological therapies. BioDrugs. 22, 315–329 (2008)
3. Greenwald, R.B.: PEG drugs: an overview. J. Control. Release.
74, 159–171 (2001)
4. Chapman, A.P.: PEGylated antibodies and antibody fragments for
improved therapy: a review. Adv. Drug. Deliv. Res. 54, 531–545
(2002)
5. Morpurgo, M., Monfardini, C., Hofland, L.J., Sergi, M., Orsolini,
P., Dumont, J.M., Veronese, F.M.: Selective alkylation and
acylation of α and ∈ amino group with PEG in a somatostatin
analogue: tailored chemistry for optimized bioconjugates. Bio-
conjugate Chem. 13, 1238–1243 (2002)
20. Agustí, R., Giorgi, M.E., Mendoza, V.M., Gallo-Rodriguez, C.,
Lederkremer, R.M.: Comparative rate of sialylation by recombinant
trans-sialidase and inhibitor properties of synthetic oligosaccharides
from Trypanosoma cruzi mucins-containing galactofuranose and
galactopyranose. Bioorg. Med. Chem. 15, 2611–2616 (2007)
21. Lederkremer, R.M., Agustí, R.: Glycobiology of Trypanosoma
cruzi. Adv. Carbohydr. Chem. Biochem. 62, 311–366 (2009)
22. Kröger, L., Scudlo, A., Thiem, J.: Subsequent enzymatic galactosy-
lation and sialylation towards sialylated Thomsen-Friedenreich
antigen components. Adv. Synth. Catal. 348, 1217–1227 (2006)
23. Tomlinson, S., de Carvalho LC, Pontes, Vandekerckhove, F.,
Nussenzweig, V.: Role of sialic acid in the resistance of
Trypanosoma cruzi trypomastigotes to complement. J. Immunol.
153, 3141–3147 (1994)
6. Marcus, Y., Sasson, K., Fridkin, M., Shechter, Y.: Turning low-
molecular-weight drugs into prolonged acting prodrugs by
reversible pegylation: a study with gentamicin. J. Med. Chem.
51, 4300–4305 (2008)
7. Dixit, V., Van den Bossche, J., Sherman, D.M., Thompson, D.H.,
Andres, R.P.: Synthesis and grafting of thioctic acid-PEG-folate
conjugates onto Au nanoparticles for selective targeting of folate
receptor-positive tumor cells. Bioconjugate Chem. 117, 603–609
(2006)
8. Prego, C., Torres, D., Fernandez-Megia, E., Novoa-Carballal, R.,
Quiñoá, E., Alonso, M.J.: Chitosan-PEG nanocapsules as new
carriers for oral peptide delivery. Effect of chitosan pegylation
degree. J. Controlled Release. 111, 299–308 (2006)
9. Fernandez-Megia, E., Novoa-Carballal, R., Quiñoá, E., Riguera,
R.: Conjugation of bioactive ligands to PEG-grafted chitosan at
the distal end of PEG. Biomacromolecules 8, 833–842 (2007)
10. Park, I.L., Kim, T.H., Park, Y.H., Shin, B.A., Choi, E.S.,
Chowdhury, E.H., Akaike, T., Cho, C.S.: Galactosylated
chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting
DNA carrier. J. Controlled Release 76, 349–362 (2001)
11. Youn, Y.S., Na, D.H., Yoo, S.D., Song, S.C., Lee, K.C.:
Carbohydrate-specifically polyethylene glycol-modified ricin A-
chain with improved therapeutic potential. Int. J. Biochem. Cell.
Biol. 37, 1525–1533 (2005)
24. Pereira-Chioccola, V.L., Acosta-Serrano, A., Correia de Almeida,
I., Ferguson, M.A., Souto-Padron, T., Rodrigues, M.M., Trav-
assos, L.R., Schenkman, S.: Mucin-like molecules form a
negatively charged coat that protects Trypanosoma cruzi trypo-
mastigotes from killing by human anti-alpha-galactosyl anti-
bodies. J Cell Sci 113, 1299–1307 (2000)
25. Nagamune, K., Acosta-Serrano, A., Uemura, H., Brun, R., Kunz-
Renggli, C., Maeda, Y., Ferguson, M.A., Kinoshita, T.: Surface
sialic acids taken from the host allow trypanosome survival in
tsetse fly vectors. J Exp Med. 199, 1445–1450 (2004)
26. Amaya, F.M., Buschiazzo, A., Nguyen, T., Alzari, P.M.: The high
resolution structures of free and inhibitor-bound Trypanosoma
rangeli sialidase and its camparison with T. cruzi trans-sialidase. J.
Mol. Biol. 325, 773–784 (2003)
27. Buschiazzo, A., Amaya, M.F., Cremona, M.L., Frasch, A.C.C.,
Alzari, P.M.: The crystal structure and mode of action of trans-
sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol.
Cell. 10, 757–768 (2002)
28. Neres, J., Bryce, R.J., Douglas, K.T.: Rational drug design in
parasitology: trans-sialidase as a case study for Chagas disease.
Drug Discov. Today 13, 110–117 (2008)
29. Buchini, S., Buschiazzo, A., Withers, S.G.: A new generation of
specific Trypanosoma cruzi trans-sialidase inhibitors. Angew.
Chem. Int. Ed. Engl. 47, 2700–2703 (2008)
12. Salmaso, S., Semenzato, A., Bersani, S., Mastrotto, F., Scomparin,
A., Caliceti, P.: Site-selective protein glycation and PEGylation.
Euro. Polym. J. 44, 1378–1389 (2008)
13. DeFrees, S., Wang, Z.G., Xing, R., Scott, A.E., Wang, J., Zopf,
D., Gouty, D.L., Sjoberg, E.R., Panneerselvam, K., Brinkman-Van
del Linden, E.C., Bayer, R.J., Tarp, M.A., Clausen, H.: Glyco-
PEGylation of recombinant therapeutic proteins produced in
Escherichia coli. Glycobiology 16, 833–843 (2006)
30. Kim, J.H., Ryu, H.W., Shim, J.H., Park, K.H., Withers, S.G.:
Development of new and selective Trypanosoma cruzi trans-
sialidase inhibitors from sulfonamide chalcones and their deriva-
tives. Chembiochem 10, 2475–2479 (2009)
31. Arioka, S., Sakagami, M., Uematsu, R., Yamaguchi, H., Togame,
H., Takemoto, H., Hinou, H., Nishimura, S.: Potent inhibitor
scaffold against Trypanosoma cruzi trans-sialidase. Bioorg. Med.
Chem. 18, 1633–1640 (2010)

Glycoconj J (2010) 27:549–559
559
32. Agustí, R., Páris, G., Ratier, L., Frasch, A.C.C., Lederkremer, R.
M.: Lactose derivatives are inhibitors of Trypanosoma cruzi trans-
sialidase activity toward conventional substrates in vitro and in
vivo. Glycobiology 14, 659–670 (2004)
33. Mucci, J., Risso, M.G., Leguizamón, M.S., Frasch, A.C.C.,
Campetella, O.: The trans-sialidase from Trypanosoma cruzi
triggers apoptosis by target cell sialylation. Cell. Microbiol. 8,
1086–1095 (2006)
34. Kuhn, R., Baer, H.H., Seelinger, A.: Zur Methylierung Von N-
Acetylglucosamin-Derivaten. Liebigs Ann. Chem. 611, 236–241
(1958)
35. Gallo-Rodriguez, C., Varela, O., Lederkremer, R.M.: One-pot
synthesis of β-D-Galf(1→4)[β-D-Galp(1→6)]-D-GlcNAc, a
‘core’ trisaccharide linked O-glycosidically in glycoproteins of
Trypanosoma cruzi. Carbohydr. Res. 305, 163–170 (1998)
36. Isbell, H.S., Frush, H.L.: Lactonization of aldonic acids. Meth.
Carbohydr. Chem. 2, 16–18 (1963)
37. Nag, A., Mitra, G., Ghosh, P.C.: A colorimetric assay for estimation
of polyethylene glycol and polyethylene glycolated protein using
ammonium ferrothiocyanate. Anal. Biochem. 237, 224–231 (1996)
38. Navath, R.S., Wang, B., Kannan, S., Romero, R., Kannan, R.M.:
Stimuli-responsive star poly(ethylene glycol) drug conjugates for
improved intracellular delivery of the drug in neuroinflammation.
J. Control. Release. 142, 447–456 (2010)
39. Rabinovich, G.A., Cumashi, A., Bianco, G.A., Ciavardelli, D.,
Iurisci, I., D’Egidio, M., Piccolo, E., Tinari, N., Nifantiev, N.,
Iacobelli, S.: Synthetic lactulose amines: Novel class of anticancer
agents that induce tumor cell apoptosis and inhibit galectin-
mediated homotypic cell aggregation and endothelial cell mor-
phogenesis. Glycobiology 16, 210–220 (2006)