Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease

Article abstract of DOI:10.1093/glycob/cwv081

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, is expressed on glycosylp

Full text of DOI:10.1093/glycob/cwv081

Glycobiology Advance Access published September 18, 2015
1
Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα-, and GlcNAcα-containing
neoglycoproteins and their immunological evaluation in the context of Chagas disease
Nathaniel S. Schocker1, Susana Portillo2, Carlos R. N. Brito2,3, Alexandre F. Marques3, Igor C.
Almeida2, and Katja Michael1,4
1Department of Chemistry, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968,
USA
2Department of Biological Sciences, University of Texas at El Paso, 500 W. University Ave., El Paso, TX
79968, USA
3Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,
Belo Horizonte, MG 31270-901, Brazil
4To whom correspondence should be addressed: e-mail: kmichael@utep.edu; Tel.: +1-915-747-
5240
© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-
mail: journals.permissions@oup.com

2
Abstract
The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease, has a cell
surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the
trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-
anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of
protective anti-α-Gal antibodies in infected individuals. Here, we have efficiently synthesized the
mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine
serum albumin (BSA). Chemiluminescent-ELISA revealed that Galα(1,3)Galβ(1,4)GlcNAcα-
BSA is recognized by purified anti-α-Gal antibodies from chronic Chagas disease patients ~230-
fold more strongly than by anti-α-Gal antibodies from sera of healthy individuals (NHS anti-α-
Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized
Galα(1,3)Galβ(1,4)GlcNAcα ~20-fold more strongly than pooled NHS. In contrast, the
underlying disaccharide Galβ(1,4)GlcNAcα, and the monosaccharide GlcNAcα or GlcNAcβ
conjugated to BSA are poorly or not recognized by purified anti-α-Gal antibodies or sera from
Chagasic patients or healthy individuals. Our results highlight the importance of the terminal
Galα moiety for recognition by Ch anti-α-Gal antibodies and the lack of antibodies against
nonself Galβ(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of
Ch vs. NHS anti-α-Gal antibodies to Galα(1,3)Galβ(1,4)GlcNAcα-BSA suggests that this
neoglycoprotein might be suitable for experimental vaccination. To this end, the
Galα(1,3)Galβ(1,4)GlcNAcα-BSA neoglycoprotein was then used to immunize α1,3-
galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared
to pre-immunization titers. Taken together, our results indicate that the synthetic

3
Galα(1,3)Galβ(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential
diagnostic and vaccine candidate for Chagas disease.
Keywords: Biomarkers/Carbohydrates/Chagas disease/Immunization/Neoglycoprotein

4
Introduction
The surface of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease
(ChD), is heavily coated by glycoproteins containing highly immunogenic glycans (Acosta-
Serrano, A., Hutchinson, C., et al. 2007, Travassos, L.R. and Almeida, I.C. 1993). An
immunodominant glycotope, Galα(1,3)Galβ(1,4)GlcNAcα, is abundantly expressed in the
mammal-dwelling T. cruzi trypomastigote stage (Almeida, I.C., Ferguson, M.A., et al. 1994) and
is not expressed on human cells, thus it is highly immunogenic to humans (Macher, B.A. and
Galili, U. 2008, Travassos, L.R. and Almeida, I.C. 1993). The Galα(1,3)Galβ(1,4)GlcNAcα
epitope contains a terminal, non-reducing αGal residue, which is highly conserved on
trypomastigote-derived GPI-mucins (tGPI-mucins) of at least four major T. cruzi genotypes
causing ChD in humans: TcI, TcII, TcV, and TcVI (Almeida, I.C., Krautz, G.M., et al. 1993,
Izquierdo, L., Marques, A.F., et al. 2013, Soares, R.P., Torrecilhas, A.C., et al. 2012, Travassos,
L.R. and Almeida, I.C. 1993). The Galα(1,3)Galβ(1,4)GlcNAcα glycotope contains the
disaccharide Galα1,3Galβ, which is strongly recognized by Chagasic (Ch) anti-α-Gal Abs and to
a much lesser extent by the natural anti-α-Gal Abs from healthy individuals (NHS anti-α-Gal)
(Almeida, I.C., Ferguson, M.A., et al. 1994, Ashmus, R.A., Schocker, N.S., et al. 2013), which
are produced mainly against Gram-negative enterobacteria of the human flora (Galili, U., Wang,
L., et al. 1999). These enterobacteria (e.g., E. coli, Enterobacter spp., Serratia spp., Salmonella
spp., Shigella spp., Klebsiella spp., and Citrobacter spp.) have various types of non-reducing,
terminal α-Gal-linked glycans, mostly Galα1,2-R, Galα1,4-R, and Galα1,6-R (where R is the
remaining side chain or core glycan) on the lipopolysaccharide (LPS) core oligosaccharides or
O-antigens (Wilkinson, S.G. 1996). The glycotope Galα(1,3)Galβ(1,4)GlcNAcα, so far not

5
reported in enterobacteria, and most likely other yet unidentified T. cruzi-specific cell surface
saccharides with terminal αGal moieties, induce the major lytic, protective antibodies (Ch anti-α-
Gal Abs) produced during both the acute and chronic stages of ChD (Almeida, I.C., Ferguson,
M.A., et al. 1994, Almeida, I.C., Milani, S.R., et al. 1991, Avila, J.L., Rojas, M., et al. 1989,
Gazzinelli, R.T., Pereira, M.E., et al. 1991, Milani, S.R. and Travassos, L.R. 1988, Travassos,
L.R. and Almeida, I.C. 1993). These studies strongly indicate that lytic Ch anti-α-Gal Abs could
be one of the main immunological mechanisms for controlling the parasitemia in both stages of
the disease in humans. Thus, Galα(1,3)Galβ(1,4)GlcNAcα offers a potential route toward a
carbohydrate-based vaccine against Chagas disease. Glycoconjugates are still unexplored as
vaccine targets in T. cruzi, although these molecules are the most abundant and immunogenic
antigens on the plasma membrane of all T. cruzi developmental stages (Acosta-Serrano, A.,
Hutchinson, C., et al. 2007, Buscaglia, C.A., Campo, V.A., et al. 2004, Frasch, A.C. 2000).
Here we describe the synthesis of glycosides of Galα(1,3)Galβ(1,4)GlcNAcα, and its
truncated versions Galβ(1,4)GlcNAcα and GlcNAcα, as well as its diastereomer GlcNAcβ, all
equipped with a thiol functionality (glycosides 1 – 4, Figure 1) for their conjugation to the carrier
protein bovine serum albumin (BSA). All neoglycoproteins (NGPs) were immunologically
evaluated by chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) (Almeida,
I.C., Covas, D.T., et al. 1997), using purified Ch anti-α-Gal Abs vs. NHS anti-α-Gal Abs, and Ch
human serum pool (ChHSP) vs. normal human serum pool (NHSP). Lastly, the NGP
Galα(1,3)Galβ(1,4)GlcNAcα-BSA was used to immunize α1,3-galactosyltransferase-knockout
(α1,3GalT-KO) mice, which do not express terminal αGal epitopes in their cells (Tearle, R.G.,
Tange, M.J., et al. 1996, Thall, A.D., Murphy, H.S., et al. 1996). These animals are able to

6
produce lytic anti-α-Gal Abs, mimicking therefore the human humoral immune response against
T. cruzi (Marques, Michael, Almeida et al., unpublished data).
The production of the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα and related analogs has
been previously accomplished for a variety of uses, and mostly involves chemoenzymatic
syntheses (Brinkmann, N., Malissard, M., et al. 2001, Fang, J., Li, J., et al. 1998, Qian, X.,
Sujino, K., et al. 1999, Vic, G., Hao Tran, C., et al. 1997), which are often efficient. However,
some research groups prefer its chemical synthesis due to reagent availability, scalability, and
derivatization options. For example, α-Gal trisaccharides have been chemically synthesized and
coupled to Sepharose (Dahmén, J., Magnusson, G., et al. 2002), attached to a lipid for non-
covalent association to target molecules (Litjens, R.E.J.N., Hoogerhout, P., et al. 2005), or
attached to linkers such as p-nitrophenol esters (Plaza-Alexander, P. and Lowary, T.L. 2013) and
3-aminopropyl groups (Hanessian, S., Saavedra, O.M., et al. 2001, Wang, Y., Yan, Q., et al.
2005) to allow for further functionalization.
The four key features of our approach to an Galα(1,3)Galβ(1,4)GlcNAcα-containing
NGP are: i) predominant use of acyl protecting groups that can be easily installed and cleanly
removed; ii) utilization of Kiso’s 4,6-di-tertbutylsilyl protected galactosyl donor (Imamura, A.,
Kimura, A., et al. 2006) to ensure a stereoselective α-galactosylation; iii) utilization of easily
accessible monosaccharide building blocks; and iv) use of an allyl glycoside at the non-reducing
end of the trisaccharide allowing for the installation of a thiol functional group, via a thiol-ene
reaction, for covalent conjugation to a carrier protein. Implementing these features, our strategy
involves the synthesis of an acyl-protected disaccharide (Galα1,3Galβ), its conversion into a
trichloroacetimidate donor, glycosylation of an appropriate allyl-glycoside GlcNAc acceptor to

7
produce a Galα(1,3)Galβ(1,4)GlcNAcα allyl glycoside, and further derivatization into a
mercaptopropyl glycoside needed for protein conjugation.
BSA was chosen for the generation of NGPs because of its large number of conjugation
sites per BSA molecule, its superior solubility properties, and its suitability as a carrier protein
(Makela, O. and Seppala, I.J.T. 1986) and provider of T cell epitopes for the immunization of
mice (Atassi, M.Z., Long, P.M., et al. 1982), as well as its capability to attach non-covalently to
wells of microtiter plates. Previously, we discovered that Ch anti-α-Gal Abs recognize the
disaccharide Galα(1,3)Galβ, which comprises the two terminal sugars of the glycotope
trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, much more strongly than Galα alone (Ashmus, R.A.,
Schocker, N.S., et al. 2013). In order to obtain information on the importance of
Galβ(1,4)GlcNAcα or GlcNAc for antibody recognition, three additional BSA NPGs containing
Galβ1,4GlcNAcα, GlcNAcα, or GlcNAcβ were synthesized and tested by CL-ELISA.
Results and Discussion
The α-Gal disaccharide 11 was synthesized from the known allyl β-galactoside 5 (Stevenson,
D.E. and Furneaux, R.H. 1996), which was made from its peracetylated precursor following an
optimized procedure (Khamsi, J., Ashmus, R.A., et al. 2012). Disaccharide 11 was synthesized in
a 55% overall yield, starting with p-methoxybenzylation of allyl glycoside 5 at position 3 via its
tin acetal to give 6, followed by benzoylation of the three remaining hydroxyls to afford 7.
Oxidative cleavage of the p-methoxybenzyl group with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone furnished the β-Gal acceptor 8. This acceptor was glycosylated with the known di-
tert-butylsilylidene equipped α-Gal trichloroacetimidate donor 9 (Imamura, A., Kimura, A., et al.

8
2006), using trimethylsilyl trifluoromethanesulfonate catalysis to give disaccharide 10. The di-
tert-butylsilylidene group was cleaved with a large excess of 70% hydrogen fluoride in pyridine
in THF, followed by acetylation of the two hydroxyls to give the peracylated allyl disaccharide
11 (Scheme 1).
The α-Gal disaccharide 11 was then treated with palladium (II) chloride in methanol to
give the hemiacetal, which was filtered immediately after consumption of the starting material to
avoid the formation of a polar by-product that we observed after two hours of reaction, and
converted into the trichloroacetimidate 12 with trichloroacetonitrile in the presence of 1,8-
diazabicycloundec-7-ene (Scheme 2). This donor was first used to glycosylate the allyl GlcNAc
acceptor 13 with TMS-OTf, but produced a low-yielding mixture of anomers (1:4 α/β) likely due
to the well-known poor nucleophilicity of the 4-OH of GlcNAc acceptors. The separation of the
two diastereomeric trisaccharides proved to be difficult but could be accomplished by reversed
phase FPLC. Replacing the acceptor 13 by the allyl 2-deoxy-2-azido-Glc acceptor 14 produced
trisaccharide 15 in 46% yield, which could be purified by flash chromatography, and the azide
was then reduced to an N-acetyl group with neat thioacetic acid to give the trisaccharide 16.
Radical addition of thioacetic acid with azobisisobutyronitrile in tetrahydrofuran under UV light
gave the thioester 17, followed by saponification under Zemplén conditions to afford the target
trisaccharide 1 (Scheme 2).
The Galβ(1,4)GlcNAcα disaccharide 2 was synthesized as shown in Scheme 3 in a 70%
overall yield from the allyl GlcNAc acceptor 13. Through the use of a large excess of the known
acetylated trichloroacetimidate β-Gal donor 18 (Schmidt, R.R. and Michel, J. 1980), and the use
of boron trifluoride etherate at an unusual elevated temperature (Hendel, J.L., Wang, J.-W., et al.
2009), the Galβ(1,4)GlcNAcα disaccharide 19 was obtained in high yield (83%), followed by

9
radical addition of thioacetic acid to give the thioester 20. Saponification under Zemplén
conditions cleanly gave the target disaccharide 2. The mercaptopropyl glycoside of GlcNAcα
(3) was synthesized as previously described (Houseman, B.T., Gawalt, E.S., et al. 2003), while
the mercaptopropyl glycoside of GlcNAcβ (4) was synthesized by radical addition of thioacetic
acid to the known allyl glycoside 21 (Kiso, M. and Anderson, L. 1979) to give thioester 22,
followed by saponification to provide the target glycoside 4 (Scheme 3).
The mercaptopropyl glycosides oxidized to disulfides within hours-days of isolation,
which could easily be reduced by tris(2-carboxyethyl)phosphine before their conjugation to
BSA. The thiol groups on compounds 1-4 served as nucleophiles in the conjugate addition to
commercially available maleimide-derivatized BSA in aqueous buffer at pH 7.2, as shown in
Figure 2. This produced neoglycoproteins via thioether linkages, and the average number of
saccharides conjugated per BSA molecule was estimated by matrix-assisted laser desorption
ionization-time of flight mass spectrometry. The conjugation of 22-23 units of
Galα(1,3)Galβ(1,4)GlcNAcα and 23-24 units of Galβ(1,4)GlcNAcα per molecule of BSA are
shown in Figure 2. An average of 29 units of GlcNAcα and 25 units of GlcNAcβ were
conjugated to BSA (see Supplemental Material, Page S3).
The four NPGs Galα(1,3)Galβ(1,4)GlcNAcα-BSA, Galβ(1,4)GlcNAcα-BSA, GlcNAcα-
BSA, GlcNAcβ-BSA, and a BSA control conjugate in which the maleimide groups had been
blocked with cysteine (Cys-BSA), were immobilized in 96-well polystyrene Nunc Maxisorp
ELISA plates and antibody-binding responses were measured using CL-ELISA (Almeida, I.C.,
Covas, D.T., et al. 1997), with pooled Chagasic human sera (ChHSP) and normal human sera
(NHSP), as well as Ch anti-α-Gal Abs and NHS anti-α-Gal Abs, purified as described (Almeida,
I.C., Milani, S.R., et al. 1991). As shown in Figure 3A, Galα(1,3)Galβ(1,4)GlcNAcα-BSA

10
clearly displays a 20-fold differential between ChHSP and NHSP, whereas the NGPs
Galβ(1,4)GlcNAcα-BSA, GlcNAcα-BSA, and GlcNAcβ-BSA all show minimal binding to
either pooled sera. There was no significant difference between the weak antibody reactivity
observed with GlcNAcα and GlcNAcβ. Cys-BSA proved to be an effective negative control. As
shown in Figure 3B, Galα(1,3)Galβ(1,4)GlcNAcα-BSA displays a 230-fold differential between
purified Ch and NHS anti-α-Gal antibodies, while neoglycoproteins Galβ(1,4)GlcNAcα-BSA,
GlcNAcα-BSA, GlcNAcβ-BSA are practically not recognized by either antibodies. These results
emphasize that the terminal Galα residue is crucial for Chagasic antibody binding, and
demonstrates a convenient method to differentiate between T. cruzi-infected and non-infected
sera. In addition, they show that, although Galβ(1,4)GlcNAcα and GlcNAcα are nonself
glycotopes for humans, there is little or no antibody response against them in the sera of
Chagasic patients (Fig. 3A).
Next, the in vivo response to Galα(1,3)Galβ(1,4)GlcNAcα-BSA was evaluated in
C57Bl/6 α1,3galactosyltransferase-knockout (α1,3GalT-KO) mice. Akin to humans and in
contrast to wild-type mice, these animals lack terminal Galα1,3-linked residues on glycoproteins,
thus being able to produce high levels of anti-α-Gal antibodies (Tearle, R.G., Tange, M.J., et al.
1996, Thall, A.D., Murphy, H.S., et al. 1996). Sera collected from immunized and control
animals were pooled separately and analyzed by CL-ELISA (Ashmus, R.A., Schocker, N.S., et
al. 2013). As shown in Figure 4, sera from the Galα(1,3)Galβ(1,4)GlcNAcα-BSA-immunized
mice displayed a 22-fold higher antibody response to Galα(1,3)Galβ(1,4)GlcNAcα-BSA after
immunization as compared to pre-immunization levels, whereas mice immunized with BSA
alone showed minimal antibody reactivity before and after immunization.

11
In conclusion, the mercaptopropyl glycoside of Galα(1,3)Galβ(1,4)GlcNAcα was
efficiently synthesized in 12 steps from known monosaccharide building blocks. In contrast to
the published chemical syntheses, an important feature of this synthesis is the ease of
accessibility of the glycosyl acceptors, which are synthesized in only 2-3 steps from
commercially available starting materials. In addition, our synthesis utilizes common and
inexpensive glycosylation catalysts. The two key steps in this synthesis are the stereoselective
installation of the terminal Galα unit into disaccharide 10 in 92% yield, and the challenging
glycosylation of the 2-deoxy-2-azido acceptor 14 to give the correct stereoisomer (trisaccharide
15) in 46% yield. With the exception of the p-methoxy group introduced into galactose
derivative 6, the di-tert-butylsilylidene protecting group of the galactosyl donor 9, and the allyl
group as a precursor of a hemiacetal in compound 11, easily installable and removable acetyl and
benzoyl protecting groups were used throughout the synthesis. Utilizing anomeric allyl groups
allowed for the convenient conversion into mercaptopropyl glycosides that were needed for the
conjugation to maleimide-derivatized BSA. The mercaptopropyl group of these glycosides is
highly versatile as it is suitable for the conjugation to a large variety of other biomolecules and
surfaces by conjugate addition to maleimides, nucleophilic substitution, and thiol-ene reaction.
Finally, we showed that trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, which is an
immunodominant glycotope in infective T. cruzi trypomastigotes, is highly immunogenic in the
context of T. cruzi infection in both mice and humans. We propose that the
Galα(1,3)Galβ(1,4)GlcNAcα-BSA and its analogs containing different carrier proteins or
peptides could be further explored as potential biomarkers or tools for the diagnosis and follow-
up of chemotherapy of ChD, and as vaccine candidates.

12
Materials and Methods
Thin-layer chromatography was performed with silica gel on aluminum support, 8.0-12.0 µm,
Sigma-Aldrich, and visualized by UV light or with 2% H2SO4 in ethanol, followed by heating.
Flash chromatography was performed with silica gel, grade A, 32–63 µm, Dynamic Adsorbents.
1H NMR spectra were recorded on a JEOL 600 MHz NMR spectrometer using tetramethylsilane
or chloroform as an internal standard. 13C NMR spectra were recorded on the same JEOL NMR
spectrometer at 150 MHz. Optical rotations were recorded on an Atago AP300 automatic
polarimeter. Mass spectra were recorded on a JEOL Accu TOF mass spectrometer using
electrospray ionization, or on a Shimadzu Axima MALDI-TOF MS. Dichloromethane and
pyridine were refluxed over calcium hydride and distilled, methanol was refluxed over
magnesium and distilled. Reagents were purchased from Sigma-Aldrich, Acros Organics, Fisher
Scientific, and Alfa Aesar. 96-well polystyrene Nunc MaxiSorp ELISA plates and CL-ELISA
reagents were purchased from Thermo Scientific or Jackson ImmunoResearch, luminescence
was recorded on a Luminoskan Ascent, Thermo Scientific.
3-thiopropyl α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-deoxy-2-
acetamido-α-D-glucopyranoside (1)
To a flask containing 17 (0.027 g, 0.018 mmol), 3 mL of 0.5M NaOMe was added under argon,
and stirred at room temperature for 30 minutes. HRMS showed full removal of acyl protecting
groups, and all material was present as a mixture of thiol and disulfide. Amberlyst-15 ion-
exchange resin was added and stirred until the solution was neutral, followed by filtration
through Celite and evaporation of the solvent. The remainder was dissolved in water and
lyophilized to give 1 as a white powder (0.011 g, quant.). ESI-TOF HRMS [C23H41NO16S +
Na]+ calc. m/z = 642.2044, found 642.1980.

13
3-thiopropyl β-D-galactopyranosyl-(1→4)-2-deoxy-2-acetamido-α-D-
glucopyranoside (2)
To a flask containing 20 (0.045 g, 0.051 mmol), 4 mL of 0.5M NaOMe was added under argon,
and stirred at room temperature for 30 minutes. HRMS showed full removal of acyl protecting
groups, and all the material was present as a disulfide. Amberlyst-15 ion-exchange resin was
added and stirred until the solution was neutral, followed by filtration through Celite and
evaporation of the solvent. The remainder was dissolved in water and lyophilized to give 2 as a
white powder (0.024 g, quant.). ESI-TOF HRMS [C34H60N2O22S2 + Na]+ calc. m/z = 935.2977,
found 935.2836.
3-thiopropyl 2-deoxy-2-acetamido-β-D-glucopyranoside (4)
To a flask containing 22 (0.059 g, 0.127 mmol), 4 mL of 0.5M NaOMe was added under argon,
and stirred at room temperature for 2 hours. HRMS showed full removal of acyl protecting
groups, and most of the material was present as a disulfide. Amberlyst-15 ion-exchange resin
was added and stirred until the solution was neutral, followed by filtration through Celite and
evaporation of the solvent. The remainder was dissolved in water and lyophilized to give 4 as a
white powder (0.037 g, quant.). ESI-TOF HRMS [C22H40N2O12S2 + Na]+ calc. m/z = 611.1920,
found 611.1707.

14
Allyl 3-O-(4-methoxybenzyl)-β-D-galactopyranoside (6)
A solution of 5 (Stevenson, D.E. and Furneaux, R.H. 1996) (0.409 g, 1.86 mmol) and Bu2SnO
(0.693 g, 2.79 mmol) in 18 mL anhydrous MeOH was stirred and refluxed under argon for 8 h.
The solution was then quickly concentrated, and resuspended in 18 mL benzene. Bu4NBr (0.30
g, 0.93 mmol) was added, followed by 4-methoxybenzyl chloride (0.378 mL, 2.79 mmol), and
stirred at 80°C for 12 h. The solution was concentrated, and purified by column chromatography
on silica gel (CHCl3/MeOH 9:1) to give 6 as a white powder (0.430 g, 75%). Its 1H and 13C
NMR spectra matched the ones previously described for this compound (Yoshida, T., Chiba, T.,
et al. 2001).
Allyl 3-O-(4-methoxybenzyl)-2,4,6-tri-O-benzoyl-β-D-galactopyranoside (7)
A solution of 6 (0.380g, 1.22 mmol) in 5 mL anhydrous pyridine was cooled to 0°C under argon.
BzCl (0.854 mL, 7.35 mmol) was added dropwise, and stirred for 3h. The solution was diluted
with EtOAc, washed once with 1M HCl, once with a sat. NaHCO3 solution and once with brine,
dried over MgSO4, filtered, concentrated, and purified by column chromatography on silica gel
(hexanes/EtOAc 2:1) to give 7 as a white powder (0.658 g, 90%). [α] 28D 72.4 (c=1 in CHCl3);
1
Rf=0.38 (MeOH/CHCl3 1:9); H NMR (600 MHz, CDCl3, 300K) δ 8.18; 8.05; 7.97; 7.56-7.61;
7.43-7.50 (5m, 15H, 3 x Bz); 7.05; 6.59 (2m, 4H, 4-OMe-benzyl); 5.90 (m, 1H, H-4); 5.77 (m,
1H, OCH2CHCH2); 5.55 (dd, 1H, 3JH1/H2=8.9 Hz, 3JH2/H3=8.9 Hz, H-2); 5.18 (m, 1H,
OCH2CHCH2); 5.07 (m, 1H, OCH2’CHCH2); 4.60-4.67 (m, 3H, H-1, H-6, CH2PhOMe); 4.41-
4.47 (m, 2H, H-6’, CH2’PhOMe); 4.35 (m, 1H, OCH2CHCH2); 4.13 (m, 1H, OCH2CHCH2’);
4.08 (m, 1H, H-5); 3.79 (dd, 1H, 3JH3/H4=3.4 Hz, H-3); 3.70 (s, 3H, OCH3) ppm. 13C NMR

15
(150MHz, CDCl3, 300K): δ 166.3; 166.0; 165.3; 159.3; 133.7; 133.5; 133.4; 133.1; 130.3; 129.6-
130.1; 129.5; 129.4; 128.5-128.7; 128.4; 117.7; 113.7; 100.2 (C-1); 75.8; 71.5; 71.3; 70.7; 70.1;
66.8; 62.8; 55.2 ppm. ESI-TOF HRMS [C38H36O10 + Na]+ calc. m/z = 675.2206, found 675.2001;
[C38H36O10 + K]+ calc. m/z = 691.1946, found 691.2022.
Allyl 2,4,6-tri-O-benzoyl-β-D-galactopyranoside (8)
To a solution of 7 (0.633 g, 0.97 mmol) in 20 mL CH2Cl2 and 1.1 mL H2O, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) (0.440 g, 1.94 mmol) was added in two portions, 30 minutes
apart, and stirred vigorously for 12 h. The red and green solution was filtered through Celite,
diluted with dichloromethane, and extracted with water (25 mL) and brine solution (25 mL),
dried over MgSO4, filtered, concentrated, and purified by column chromatography on silica gel
(EtOAc/hexanes 2:1) to give 8 as a white powder (0.504 g, 98%). Its 1H and 13C NMR spectra
matched the ones previously described for this compound (Sherman, A.A., Yudina, O.N., et al.
2001).
Allyl 4,6-di-O-tert-butylsilylene-2,3-di-O-benzoyl-α-D-galactopyranosyl-(1→3)-
2,4,6-tri-O-benzoyl-β-D-galactopyranoside (10)
A solution of acceptor 8 (0.175 g, 0.329 mmol) and 4,6-di-O-tertbutylsilyl-2,3-di-O-benzoyl-α-
D-galactopyranosyl trichloroacetimidate donor 9 (Imamura, A., Kimura, A., et al. 2006) (0.266 g,
0.395 mmol) in anhydrous dichloromethane (6 mL) was added to a 10 mL round bottomed flask
with freshly activated, crushed 4Ǻ molecular sieves and stirred under argon for 15 min. at 0°C.

16
TMSOTf (0.010 mL, 0.059 mmol) was added dropwise, and the mixture was gradually brought
to room temperature and stirred for 2 h. To quench the reaction, Et3N (0.010 mL, 0.072 mmol)
was added and stirred. The solution was diluted with dichloromethane (50 mL) and extracted
with water (2 × 25 mL) and brine solution (25 mL), dried over MgSO4, filtered, concentrated,
and purified by column chromatography on silica gel (hexanes/EtOAc 3:1) to give 10 as a white
powder (0.315 g, 92%). [α]28D 160.7 (c=1 in CHCl3); Rf=0.55 (EtOAc/hexanes 1:2); 1H NMR
(600 MHz, CDCl3, 300K) δ 8.09, 7.99, 7.84, 7.74, 7.60, 7.51, 7.41, 7.26, 7.13, 7.01 (10m, 25H,
5xBz); 5.79-5.87 (m, 2H, OCH2CHCH2, βGalH-4); 5.70-5.76 (m, 2H, αGalH-2, βGalH-2); 5.62
(d, 1H, 3JH1/H2=3.4 Hz, αGalH-1); 5.26 (m, 1H, OCH2CHCH2); 5.16 (m, 2H, OCH2’CHCH2,
αGalH-3); 4.77 (d, 1H, 3JH1/H2=8.3Hz, βGalH-1); 4.55 (dd, 1H, 3JH5/H6=11.7Hz, 2JH6/H6’= 6.9Hz,
βGalH-6); 4.40 (m, 1H, OCH2CHCH2); 4.22-4.30 (m, 3H, βGalH-3, βGalH-5, αGalH-4); 4.19
(m, 1H, OCH2CHCH2’); 4.03-4.14 (m, 2H, αGalH-5, βGalH-6’); 3.64-3.71 (m, 2H, αGalH-6,
αGalH-6’); 1.02 (s, 9H, t-butyl); 0.79 (s, 9H, t-butyl) ppm. 13C NMR (150MHz, CDCl3, 300K): δ
166.3, 166.1, 165.6, 165.4, 165.0, 133.6, 133.4; 133.4; 133.0; 132.9; 132.8; 129.5-129.9; 129.2;
128.8; 128.6; 128.3; 128.1; 118.0; 100.3 (βC-1); 94.2 (αC-1); 73.8; 71.5; 70.9; 70.7; 70.5; 70.2;
67.6; 67.1; 66.5; 65.9; 62.3; 27.4; 27.2; 25.4; 23.2; 20.7 ppm. ESI -
TOF HRMS [C58H62O16Si + Na]+ calc. m/z = 1065.3705, found 1065.3587;
[C58H62O16Si + K]+ calc. m/z = 1081.3444, found 1081.2728.

17
Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl-α-D-galactopyranosyl-(1→3)-2,4,6-tri-O-
benzoyl-β-D-galactopyranoside (11)
A solution of 10 (0.464 g, 0.444 mmol) in anhydrous THF (7 mL) was added to a 50 mL plastic
conical tube and stirred under argon at rt. A solution of HF-pyridine (70% HF, 30% pyridine)
(0.223 mL, 8.88 mmol) was added to the reaction mixture and stirred for 3 h, then quenched with
0.5 mL saturated NaHCO3. The solution was diluted with EtOAc and extracted with water and
brine, dried over MgSO4, and concentrated. The compound was then added to a 25 mL round
bottom flask in 5 mL anhydrous pyridine, and Ac2O was added (0.252 mL; 2.66 mmol) and
stirred for 12 h. The solvent was then co-evaporated with toluene, and the remainder was
purified by column chromatography on silica gel (hexanes/EtOAc 2:1) to give 11 as a white
powder (0.389 g, 89% in 2 steps). [α]28D 163.0 (c=1 in CHCl3); Rf=0.20 (EtOAc/hexanes 1:2); 1H
NMR (600 MHz, CDCl3, 300K) δ 8.15; 7.99; 7.70; 7.61; 7.51; 7.44; 7.39; 7.28; 7.24; 7.10; 7.01
(11m, 25H, 5 x Bz); 5.80-5.86 (m, 2H, OCH2CHCH2, βGalH-4); 5.76 (dd, 1H, 3JH2/H3=9.6 Hz,
βGalH-2); 5.67 (d, 1H, 3JH1/H2=4.1 Hz, αGalH-1); 5.61 (dd, 1H, 3JH2/H3=11.0 Hz, αGalH-2); 5.41
(dd, 1H, 3JH3/H4=3.4 Hz, αGalH-3); 5.26 (m, 1H, OCH2CHCH2); 5.09-5.17 (m, 2H,
OCH2’CHCH2, αGalH-4); 4.78 (d, 1H, 3JH1/H2= 7.6 Hz, βGalH-1); 4.57 (dd, 1H, 3JH5/H6=11.0 Hz,
2JH6/H6’=6.2 Hz, βGalH-6); 4.41 (m, 1H, OCH2CHCH2); 4.27-4.32 (m, 2H, βGalH-3, βGalH-5);
4.19 (m, 2H, OCH2CHCH2’, αGalH-5); 4.11 (dd, 1H, 3JH5/H6=6.6 Hz, βGalH-6’); 3.96 (m, 2H,
αGalH6, αGalH6’); 1.97-2.03 (m, 6H, 2x Ac) ppm. 13C NMR (150 MHz, CDCl3, 300K): δ
170.1; 169.8; 166.2; 166.0; 165.4; 165.1; 164.9; 133.6; 133.5; 133.4; 133.1; 133.0; 129.8; 129.8;
129.7; 129.5; 129.5; 129.3; 128.8; 128.6; 128.4; 128.3; 128.3; 128.2; 118.1; 100.3 (βC-1); 93.4
(αC-1); 73.4; 71.5; 70.6; 70.2; 67.9; 67.6; 66.8; 65.5; 62.3; 61.5; 20.8; 20.6 ppm. ESI-
TOF HRMS [C54H50O18 + NH4]+ calc. m/z = 1004.3341, found 1004.3070.

18
Trichloroacetimidate 2,3-di-O-benzoyl-4,6-di-O-acetyl-α-D-galactopyranosyl-
(1→3)-2,4,6-tri-O-benzoyl-β-D-galactopyranoside (12)
To a solution of 11 (0.369 g, 0.374 mmol) in MeOH (6 mL), PdCl2 (0.0398 g, 0.225 mmol) was
added and stirred for 2 h at room temperature until consumption of most of the starting material.
After 2 h, a degradation product can be observed. The solution was filtered through Celite,
concentrated, and purified by column chromatography on silica gel (EtOAc/hexanes 2:3) to give
the α and β anomers (0.308 g, 87%). A recovered compound assumed to be remaining starting
material was actually the vinyl glycoside. The anomeric product mixture was then placed into a
round-bottomed flask, 10 mL anhydrous CH2Cl2 was added under argon, and the solution was
cooled to 0°C. CCl3CN (0.325 mL, 3.24 mmol) was added, followed by dropwise addition of
DBU (0.015 mL, 0.097 mmol) and the mixture was brought to rt over 3 h. The solution was
concentrated and purified by column chromatography on silica gel (EtOAc/hexanes 1:2) to give
12 as a white powder (0.295 g, 84%). [α]27D 186.2 (c=1 in CHCl3); Rf=0.65 (acetone/hexanes
1:1); 1H NMR (600 MHz, CDCl3, 300K) δ 8.64 (s, 1H, NH); 8.10; 7.94; 7.67-7.71; 7.53-7.63;
7.47; 7.36-7.41; 7.23-7.30; 7.12; 7.02 (9m, 25H, 5 x Bz); 6.90 (d, 1H, 3JH1/H2=3.4 Hz, αGalH-1)
5.99 (d, 1H, 3JH4/H5=2.8 Hz,αGalH-4); 5.94 (dd, 1H, 3JH2/H3=10.3 Hz, αGalH-2); 5.76 (d, 1H,
3JH1/H2=3.4 Hz, αGal’H-1); 5.65 (dd, 1H, 3JH2/H3=10.3 Hz, αGal’H-2); 5.49 (dd, 1H, 3JH3/H4=3.4
Hz,αGal’H-3); 5.28 (m, 1H, αGal’H-4); 4.76 (dd, 1H, 3JH3/H4=3.4 Hz, αGalH-3); 4.65 (m, 1H,
αGalH-5); 4.44 (dd, 1H, 7.6 Hz, 11.7 Hz, αGalH-6); 4.41 (dd, 1H, 3JH5/H6=11.7 Hz, αGal’H-5);
4.31 (dd, 1H, 5.5 Hz, 11.7 Hz, αGalH-6’); 4.09-4.14 (m, 1H, αGal’H-6); 4.02 (dd, 1H,
2JH6/H6’=6.2 Hz, αGal’H-6’); 1.98-2.06 (m, 6H, 2x Ac) ppm. 13C NMR (150 MHz, CDCl3,
300K): δ 170.2; 169.8; 166.0; 165.6; 165.3; 165.0; 160.5; 133.9; 133.0-133.3; 129.6-129.9;

19
129.4; 128.8; 128.5; 128.3; 128.2; 93.8 (αGalC-1); 93.2 (αGal’C-1); 90.9 (CCl3); 70.1; 69.5;
68.7; 67.8; 66.7; 66.0; 62.5; 60.9; 20.9; 20.6 ppm. ESI-TOF HRMS did not show a molecular ion
peak for [C53H46Cl3NO18 ]+
.
Allyl 2-deoxy-2-acetamido-3,6-di-O-benzoyl-α-D-glucopyranoside (13)
To a solution of allyl 2-deoxy-2-acetamido-α-D-glucopyranoside (Gavard, O., Hersant, Y., et al.
2003) (3.98 g, 15.24 mmol) in 80 mL anhydrous AcCN, 1-benzoylimidazole (5.46 mL, 36.56
mmol) was added via a plastic syringe, and was heated at 80°C for 12 h. After evaporation of the
solvent the remainder was dissolved in EtOAc and extracted twice with water and once with
brine solution, dried over MgSO4, filtered, concentrated, and purified by column
chromatography on silica gel (toluene/EtOAc 2:1) to give 13 as a white powder (4.79 g, 67%).
Its 1H and 13C NMR spectra matched the ones previously described for this compound (Danac,
R., Ball, L., et al. 2007). A minor byproduct with a higher Rf value was identified as the tri-O-
benzoylated compound.
Allyl 2-deoxy-2-azido-3,6-di-O-benzoyl-α-D-glucopyranoside (14)
Compound 14 was prepared similarly to a published synthesis with slight variations in the
solvent and the time period over which BzCl was added (Danac, R., Ball, L., et al. 2007): A
solution of allyl 2-deoxy-2-azido-α-D-glucopyranoside (Gavard, O., Hersant, Y., et al. 2003)
(0.30 g, 1.223 mmol) in 10 mL anhydrous pyridine was cooled to -20°C, and BzCl (0.350 mL,
3.01 mmol) was added dropwise in 3 portions of 0.117 mL each over 1 h, and stirred for an

20
additional 1 h. The solution was diluted with EtOAc and extracted twice with water and once
with brine solution, dried over MgSO4, filtered, concentrated, and purified by column
chromatography on silica gel (hexanes/EtOAc 5:2) to give 14 as a white powder (0.364 g, 66%).
[α]25D 161.0 (c=1 in CHCl3) Rf=0.48 (EtOAc/hexanes 1:2); 1H NMR (600 MHz, CDCl3, 300K) δ
8.05-8.11; 7.58; 7.43-7.48 (m, 10H, 2 x Bz); 5.95 (m, 1H, OCH2CHCH2); 5.64 (dd, 1H,
3JH3/H4=9.6 Hz, H-3); 5.36 (m, 1H, OCH2CHCH2); 5.25 (m, 1H, OCH2’CHCH2); 5.09 (d, 1H,
3JH1/H2=4.1 Hz, H-1); 4.73 (dd, 1H, 3JH5/H6=4.8 Hz, 2JH6/H6’=12.4 Hz, H-6); 4.60 (dd, 1H,
3JH5/H6’=2.1 Hz, H-6’); 4.29 (m, 1H, CH2CHCH2); 4.12 (m, 2H, H-5, CH2CHCH2’); 3.77 (dd, 1H,
3JH4/H5=9.6 Hz, H-4); 3.47-3.54 (broad, 1H, 4-OH); 3.44 (dd, 1H, 3JH2/H3=11.0 Hz, H-2) ppm. 13C
NMR (150 MHz, CDCl3, 300K): δ 167.2; 167.0; 133.7; 133.4; 133.0; 130.1; 129.7-130.0; 129.2;
128.6; 128.6; 118.5; 97.0 (C-1); 74.0; 70.7; 70.0; 69.0; 63.5; 61.2 ppm. ESI-
TOF HRMS [C23H23N3O7 + H]+ calc. m/z= 454.1614, found 454.1912. A minor byproduct of this
reaction was identified as the tri-O-benzoylated compound.
Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl-α-D-galactopyranosyl-(1→3)-2,4,6-tri-O-
benzoyl-β-D-galactopyranosyl-(1→4)-2-deoxy-2-azido-3,6-di-O-benzoyl-α-D-
glucopyranoside (15)
A solution of acceptor 14 (0.126 g, 0.279 mmol) and donor 12 (0.304 g, 0.279 mmol) in
anhydrous CH2Cl2 (6 mL) was placed in a 10 mL round bottomed flask with freshly activated,
crushed 4Ǻ molecular sieves and stirred under argon for 15 min at 0°C. TMS-OTf (0.015 mL,
0.0835 mmol) was added dropwise to the reaction mixture, which was gradually brought to room
temperature and stirred for 2 h. The reaction was quenched with Et3N (0.02 mL, 0.143 mmol),

21
filtered through Celite, concentrated and purified by column chromatography on silica gel
(hexanes/EtOAc 2:1) to give 15 as a slightly yellow powder (0.175 g, 46%). [α]26D 106.1 (c=1 in
CHCl3) Rf=0.53 (EtOAc/hexanes 1:1); 1H NMR (600 MHz, CDCl3, 300K) δ 8.20; 8.02; 7.97;
7.68; 7.55-7.64; 7.36-7.52; 7.31; 7.23; 7.11; 6.97 (10m, 35H, 7 x Bz); 5.93 (m, 1H,
OCH2CHCH2); 5.87 (dd, 1H, 3JH2/H3=9.5 Hz, 3JH3/H4=9.5 Hz, αGlcH-3); 5.66 (dd, 1H, 3JH1/H2=9.5
Hz,βGalH-2); 5.56 (m, 1H, βGalH-4); 5.53 (d, 1H, 3JH1/H2=3.4 Hz, αGalH-1); 5.34 (m, 1H,
OCH2CHCH2); 5.30 (dd, 1H, 3JH2/H3= 10.3 Hz, αGalH-2); 5.24 (m, 1H, OCH2’CHCH2); 5.06 (d,
1H, 3JH1/H2=3.4 Hz, αGlcH-1); 4.95 (m, 1H, αGalH-3); 4.80 (d, 1H, 3JH1/H2=7.9 Hz, βGalH-1);
4.53-4.60 (m, 2H, βGalH-6, βGalH-6’); 4.26 (m, 1H, OCH2CHCH2); 4.05-4.18 (m, 5H,
OCH2CHCH2, βGalH-3, βGalH-5, αGlcH-4, αGalH-4); 4.01 (m, 1H, αGalH-5); 3.87 (dd, 1H,
3JH5/H6=11.0 Hz, 2JH6/H6’=6.9 Hz, αGalH-6); 3.82 (dd, 1H, 3JH5/H6’=11.7 Hz, αGalH-6’); 3.73-3.78
(m, 2H, αGlcH-5, αGlcH-6); 3.40-3.49 (m, 2H,αGlcH-2, αGlcH-6’); 1.95-1.99 (m, 6H, 2 x Ac)
ppm. 13C NMR (150 MHz, CDCl3, 300K): δ 170.3; 169.7; 166.0; 165.9; 165.7; 165.2; 164.8;
164.5; 133.8; 133.5; 133.2; 133.0-133.1; 132.9; 129.2-130.0; 128.5-128.9; 128.0-128.4; 118.7;
101.3 (βGalC-1); 96.9 (αGlcC-1); 92.9 (αGalC-1); 76.4; 73.2; 71.3; 70.8; 70.6; 69.1; 69.0; 67.9;
67.8; 67.3; 66.8; 64.6; 62.5; 61.8; 61.4; 61.1; 20.7; 20.5 ppm. ESI-TOF HRMS
[C74H67N3O24 + Na]+ calc. m/z = 1399.4458, found 1399.4391; [C74H67N3O24 + K]+ calc. m/z
= 1420.3752, found 1420.3016.

22
Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl-α-D-galactopyranosyl-(1→3)-2,4,6-tri-O-
benzoyl-β-D-galactopyranosyl-(1→4)-2-deoxy-2-acetamido-3,6-di-O-benzoyl-α-D-
glucopyranoside (16)
To a flask containing 15 (0.125 g, 0.0904 mmol), was added 8 mL of thioacetic acid, and was
stirred for 24 h at 40°C. The solution was concentrated by two co-evaporations with toluene,
and purified by column chromatography on silica gel (EtOAc/hexanes 1:1 → 3:1) to give 16 as a
white powder (0.097g, 77%). [α]26D 94.3 (c=1 in CHCl3); Rf=0.15 (hexanes/EtOAc 1:1); 1H
NMR (600 MHz, CDCl3, 300K) δ 8.20; 8.02; 7.96; 7.68; 7.55-7.63; 7.47-7.53; 7.36-7.45; 7.28-
7.35; 7.22; 7.15; 7.03; 6.95 (10m, 35H, 7 x Bz); 5.84-5.92 (m, 2H, OCH2CHCH2, NH); 5.52-
5.67 (m, 5H, βGalH-2, βGalH-4, αGalH-1, αGlcNAcH-3); 5.26-5.31 (m, 2H, αGalH-2,
OCH2CHCH2); 5.23 (m, 1H,OCH2CHCH2); 4.90-4.94 (m, 2H, αGlcNAcH-1, αGalH-3); 4.80 (d,
1H, 3JH1/H2= 7.6 Hz, βGalH-1); 4.50-4.59 (m, 3H, αGlcNAcH-2); 4.06-4.22 (m, 4H, βGalH-3,
OCH2CHCH2); 4.00 (m, 2H, OCH2CHCH2); 3.88 (dd, 1H); 3.78-3.84 (m, 2H); 3.66-3.72 (m,
2H); 1.96-2.00 (m, 6H, 2 x Ac); 1.86 (s, 3H, NHAc) ppm. 13C NMR (150 MHz, CDCl3, 300K): δ
170.3; 170.2; 169.7; 166.6; 166.1; 165.9; 165.7; 165.2; 164.8; 164.6; 133.9; 133.5; 133.3; 133.1;
133.1; 132.9; 130.0; 129.5-129.8; 129.4; 129.2; 128.9; 128.6-128.8; 128.0-128.4; 118.6; 101.3
(βGalC-1); 96.4 (αGlcNAcC-1); 92.9 (αGalC-1); 75.9; 73.2; 71.7; 71.4; 70.8; 69.0; 68.8; 67.9;
67.8; 67.3; 66.8; 64.6; 62.5; 61.8; 61.1; 52.1; 29.8; 23.3; 20.8; 20.5 ppm. ESI-
TOF HRMS [C76H71NO25 + H]+ calc. m/z =1398.4393,
found 1398.4308; [C76H71NO25 + Na]+ calc. m/z = 1420.4213,
found 1420.4487; [C76H71NO25 + K]+ calc. m/z = 1436.3935, found 1436.3893.

23
3-(acetylthio)propyl 2,3-di-O-benzoyl-4,6-di-O-acetyl-α-D-galactopyranosyl-(1→3)-
2,4,6-tri-O-benzoyl-β-D-galactopyranosyl-(1→4)-2-deoxy-2-acetamido-3,6-di-O-
benzoyl-α-D-glucopyranoside (17)
To a solution of 16 (0.030 g, 0.022 mmol) and AIBN (0.004 g, 0.022 mmol) in anhydrous THF
(3 mL), thioacetic acid (0.016 mL, 0.222 mmol) was added and stirred under argon for 5 min.
The solution was then placed in a Rayonet UV reactor (350 nm) and stirred for 12 h under water
cooling (~ rt). The solution was concentrated by two co-evaporations with toluene, and purified
by column chromatography on silica gel (EtOAc/Hex 2:1) to give 17 as a white powder (0.028g,
89%). [α]26D 94.2 (c=0.5 in CHCl3); Rf=0.48 (EtOAc/hexanes 2:1); 1H NMR (600 MHz,
CDCl3, 300K) δ 8.20; 8.01; 7.96; 7.68; 7.55-7.63; 7.47-7.53; 7.20-7.45; 7.13; 7.06; 6.95 (10m,
35H, 7 x Bz); 6.19 (d, 1H, 3JNH/H2=9.3 Hz, NH); 5.52-5.67 (m, 4H, βGalH-2, αGalH-2); 5.52 (d,
1H, 3JH1/H2=3.4 Hz, αGalH-1); 5.28 (dd, 1H, 3JH2/H3=10.3 Hz, 3JH3/H4=10.3 Hz, αGalH-3); 4.92
(m, 1H, αGalH-4); 4.83 (d, 1H, 3JH1/H2=3.4 Hz, αGlcNAcH-1); 4.79 (d, 1H, 3JH1/H2=8.3 Hz,
βGalH-1); 4.52-4.59 (m, 3H, αGlcNAcH-2); 4.09-4.16 (m, 2H); 4.04 (m, 1H); 4.00 (m, 1H,
αGalH-5); 3.87 (dd, 1H, 3JH5/H6=11.7 Hz, 2JH6/H6’=6.9 Hz, αGalH-6); 3.81 (dd, 1H, 3JH5/H6’=11.7
Hz, αGalH-6’); 3.68-3.78 (m, 3H, OCH2CH2CH2); 3.62 (dd, 1H); 3.44 (m, 1H, OCH2CH2CH2);
3.09 (m, 1H, OCH2CH2CH2); 2.95 (m, 1H, OCH2CH2CH2’); 2.32 (s, 3H, SAc); 1.97-1.99 (m,
6H, 2 x Ac); 1.85-1.92 (m, 5H, NHAc, OCH2CH2, OCH2CH2’) ppm. 13C NMR (150 MHz,
CDCl3, 300K): δ 195.8; 170.5; 170.3; 169.7; 166.5; 165.9; 165.7; 165.1; 164.6; 133.9; 133.5;
133.2; 133.1; 133.1; 133.0; 132.8; 129.3-130.0; 128.6-129.2; 128.0-128.4; 101.3 (βGalC-1); 97.4
(αGlcNAcC-1); 92.9 (αGalC-1); 76.1; 73.2; 71.8; 71.4; 70.8; 69.1; 67.9; 67.8; 67.3; 66.8; 66.0;
64.6; 62.5; 61.8; 61.1; 51.9; 30.7; 29.8; 29.3; 25.5; 23.2; 20.8; 20.5 ppm. ESI-
TOF HRMS [C78H75NO26S + H]+ calc. m/z = 1474.4376, found 1474.4222.

24
Allyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2-deoxy-2-acetamido-3,6-
di-O-benzoyl-α-D-glucopyranoside (19)
To a solution of acceptor 13 (Danac, R., Ball, L., et al. 2007) (3.60 g, 7.63 mmol) and donor 18
(Schmidt, R.R. and Michel, J. 1980) (14.0g, 28.42 mmol) in 60 mL anhydrous CH2Cl2, BF3-OEt2
(1.93 mL, 15.25 mmol) was added and immediately brought to 35-40°C. After 3 h, Et3N (2.35
mL, 16.87 mmol) was added. The solution was washed one time with a saturated NaHCO3
solution, and the aqueous layer was extracted with CH2Cl2. The organic phases were combined,
dried over MgSO4, filtered, concentrated, and purified by column chromatography on silica gel
(EtOAc/Hex 2.3:1) to give 19 as a white powder (5.10 g, 83%). [α]22D 56.6 (c=1 in CHCl3);
Rf=0.30 (EtOAc/hexanes 2:1); 1H NMR (600 MHz, CDCl3, 300K) δ 8.07; 7.61; 7.52; 7.47 (4m,
10H, 2 x Bz); 5.91 (m, 1H, OCH2CHCH2); 5.85 (d, 1H, 3JNH/H2=9.6 Hz, NH); 5.62 (dd, 1H,
3JH2/H3=11.0 Hz, 3JH3/H4=8.3 Hz, αGlcNAcH-3); 5.30 (m, 1H, OCH2CHCH2); 5.25 (m, 1H,
OCH2’CHCH2); 5.13 (m, 1H, βGalH-4); 5.10 (dd, 1H, 3JH2/H3=10.3 Hz, βGalH-2); 4.91 (d, 1H,
3JH1/H2=3.4 Hz, αGlcNAcH-1); 4.82 (dd, 1H, 3JH3/H4=3.4 Hz, βGalH-3); 4.69 (m, 1H, αGlcNAcH-
6); 4.61 (d, 1H, 3JH1/H2=8.3 Hz, βGalH-1); 4.47 (m, 1H, αGlcNAcH-2); 4.41 (dd, 1H, 2JH6/H6’=4.1
Hz, 3JH5/H6’=11.7 Hz, αGlcNAcH-6’); 4.22 (m, 1H, OCH2CHCH2); 4.07-4.15 (m, 2H,
αGlcNAcH-4, αGlcNAcH-5); 4.03 (m, 1H, OCH2CHCH2’); 3.64 (dd, 1H, 3JH5/H6=8.3 Hz,
2JH6/H6’=11.0 Hz, βGalH-6); 3.48 (dd, 1H, 3JH5/H6’=5.5 Hz, βGalH-6’); 3.36 (dd, 1H, 6.2 Hz, 8.3
Hz, βGalH-5); 1.80-2.10 (m, 15H, 4 x Ac, NHAc) ppm. 13C NMR (150 MHz, CDCl3, 300K): δ
170.2; 169.9; 169.4; 166.4; 166.1; 133.6; 133.5; 133.2; 129.8; 129.7; 128.7; 128.6; 118.6; 101.1
(βGalC-1); 96.5 (αGlcNAcC-1); 76.4; 72.2; 71.0; 70.6; 69.4; 68.9; 68.8; 66.3; 62.6; 60.0; 52.2;
23.2; 20.5-20.8 ppm. ESI-TOF HRMS [C39H45NO17 + H]+ calc. m/z = 800.2766,

25
found 800.2864; [C39H45NO17 + Na]+ calc. m/z
= 822.2585, found 822.2022; [C39H45NO17 + K]+ calc. m/z = 838.2325, found 838.1110.
3-(acetylthio)propyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2-deoxy-2-
acetamido-3,6-di-O-benzoyl-α-D-glucopyranoside (20)
To a solution of 19 (0.050 g, 0.063 mmol) and AIBN (0.010 g, 0.063 mmol) in anhydrous THF
(3 mL), thioacetic acid (0.045 mL, 0.63 mmol) was added and stirred under argon for 5 min. The
solution was then placed in a Rayonet UV reactor (350 nm) stirred for 12 h under water cooling
(~ rt). The solution was concentrated by two co-evaporations with toluene, and purified by
column chromatography on silica gel (EtOAc/Hex 2:1) to give 20 as a white powder (0.046g,
84%). [α]22D 45.6 (c=0.9 in CHCl3); Rf=0.25 (EtOAc/hexanes 2:1); 1H NMR (600 MHz, CDCl3,
300K) δ 8.08; 7.61; 7.52; 7.47 (4m, 10H, 2 x Bz); 6.15 (d, 1H, 3JNH/H2=9.6 Hz, NH); 5.58 (dd,
1H, 3JH2/H3=10.3 Hz, 3JH3/H4=8.3 Hz, αGlcNAcH-3); 5.13 (m, 1H, βGalH-4); 5.10 (dd, 1H,
3JH2/H3=10.3 Hz, βGalH-2); 4.80-4.84 (m, 2H, αGlcNAcH-1, βGalH-3); 4.70 (m, 1H,
αGlcNAcH-6); 4.60 (d, 1H, 3JH1/H2=8.3 Hz, βGalH-1); 4.48 (m, 1H, αGlcNAcH-2); 4.40 (dd, 1H,
2JH6/H6’=4.1 Hz, 3JH5/H6’=11.7 Hz, αGlcNAcH-6’); 4.09 (m, 2H, αGlcNAcH-4, αGlcNAcH-5);
3.80 (m, 1H, OCH2CH2CH2); 3.61 (dd, 1H, 3JH5/H6= 8.3 Hz, 2JH6/H6’=11.0 Hz, βGalH-6); 3.47 (m,
2H, OCH2’CH2CH2, βGalH-6’); 3.37 (m, 1H, βGalH-5); 3.10 (m, 1H, OCH2CH2CH2); 2.96 (m,
1H, OCH2CH2CH2’); 2.35 (s, 3H, SAc); 1.85-2.05 (m, 17H, NHAc, 4 x Ac, OCH2CH2CH2) ppm.
13C NMR (150 MHz, CDCl3, 300K): δ 195.7; 170.5; 170.1; 169.9; 169.4; 166.3; 166.1; 133.5;
129.8; 129.7; 128.7; 128.6; 101.1 (βGalC-1); 97.4 (αGlcNAcC-1); 76.4; 72.2; 71.0; 70.6; 69.4;
68.9; 66.3; 66.1; 62.6; 60.0; 52.1; 30.7; 29.8; 29.3; 25.6; 23.1; 20.5-20.8 ppm. ESI-

26
TOF HRMS [C41H49NO18S + H]+ calc. m/z = 876.2749, found 876.3192;
[C41H49NO18S + Na]+ calc. m/z = 898.2568, found 898.2413.
3-(acetylthio)propyl 2-deoxy-2-acetamido-3,4,6-tri-O-acetyl-β-D-glucopyranoside
(22)
To a solution of 21 (Kiso, M. and Anderson, L. 1979) (0.081 g, 0.209 mmol) and AIBN (0.034 g,
0.209 mmol) in anhydrous THF (5 mL), thioacetic acid (0.149 mL, 2.09 mmol) was added and
stirred under argon for 5 min. The solution was then placed in a Rayonet UV reactor (350 nm)
and stirred for 12 h under water cooling (~ rt). The solution was concentrated by two co-
evaporations with toluene, and purified by column chromatography on silica gel (CHCl3/MeOH
25:1) to give 22 as a white powder (0.082g, 85%). [α]26D 11.9 (c=1 in CHCl3); Rf=0.30
1
(MeOH/CHCl3 1:9) H NMR (600 MHz, CDCl3, 300K) δ 6.20 (d, 1H, 3JNH/H2=8.9 Hz, NH);
5.17 (dd, 1H, 3JH2/H3=8.9 Hz, 3JH3/H4=10.3 Hz, H-3); 5.02 (dd, 1H, 3JH4/H5=9.62 Hz, H-4); 4.50 (d,
1H, 3JH1/H2=8.3 Hz, H-1); 4.20 (dd, 1H, 3JH5/H6=12.4 Hz, 2JH6/H6’=4.8 Hz, H-6); 4.06 (dd, 1H,
3JH5/H6’=12.4 Hz, H-6’); 3.92 (m, 1H, H-2); 3.85 (m, 1H, OCH2CH2CH2); 3.64 (m, 1H, H-5);
3.42 (m, 1H, OCH2’CH2CH2); 3.00 (m, 1H, OCH2CH2CH2); 2.75 (m, 1H, OCH2CH2CH2’); 2.28
(s, 3H, SAc); 1.80-2.10 (m, 13H, NHAc, 3 x Ac, OCH2CH2CH2); 1.69 (m, 1H, OCH2CH2’CH2)
ppm. 13C NMR (150 MHz, CDCl3, 300K): δ 196.7; 171.0; 170.8; 170.6; 169.5; 100.8 (C-1);
72.9; 71.8; 68.7; 67.5; 62.2; 54.4; 30.7; 29.4; 25.4; 23.3; 20.6-20.9 ppm. ESI-
TOF HRMS [C19H29NO10S + H]+ calc. m/z = 464.1590, found 464.1340; [C19H29NO10S +
Na]+ calc. m/z = 486.1410, found 486.1100; [C19H29NO10S + K]+ calc. m/z = 502.1149, found
502.0768.

27
Immunization Protocol
Groups of five female C57Bl/6 α1,3GalT-KO mice (Tearle, R.G., Tange, M.J., et al. 1996, Thall,
A.D., Murphy, H.S., et al. 1996) were immunized subcutaneously with 20 µg
Galα(1,3)Galβ(1,4)GlcNAcα-BSA in 200 µl PBS/dose/immunization or 20 µg BSA alone in 200
µl PBS . All animals were immunized four times at 7-day intervals and sacrificed 14 days after
the last immunization. Blood was collected by cardiac puncture and serum was separated through
centrifugation for analysis by CL-ELISA. All animal procedures were performed according to
the vertebrate animal protocols A-201211-1 and A-201411-1, approved by the University of
Texas at El Paso’s Institutional Animal Care and Use Committee.
Supplementary data
Supplementary data for this article are available online at http://glycob.oxfordjournals.org/.
Funding
This work was supported by a University of Texas at El Paso College of Science
Multidisciplinary Pilot Projects and Collaborations grant, and by NIH grant 1R21AI115451-01
(to ICA and KM). ICA is a Special Visiting Researcher scholar from the Science Without
Borders Program, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Brazil. NSS is supported by the “Bridge to the Doctorate” scholarship (NSF grants HRD-
1139929). AFM is supported by the CNPq grant # 470737/2013-1. The BACF is supported by
the Research Centers in Minority Institutions (RCMI) program, grant 2G12MD007592, to the

28
Border Biomedical Research Center (BBRC) at UTEP, from the National Institutes on Minority
Health and Health Disparities (NIMHD), a component of the NIH.
Acknowledgements
We are very grateful to Dr. Joaquim Gascon and Dr. Montserrat Gállego (ISGlobal/Hospital
Clinic/Universitat de Barcelona) for kindly providing human sera without identifiers used in this
study. We are thankful to the Biomolecule Analysis Core Facility (BACF) at the University of
Texas at El Paso (UTEP) for the access to the microplate luminometer and other instruments
used in this study.
Conflict of interest statement
None declared.
Abbreviations
α1,3-GalT-KO, α1,3-galactosyltransferase-knockout; Abs, antibodies; AcSH, thioacetic acid;
AIBN, azobisisobutyronitrile; BF3-Et2O, boron trifluoride etherate; BSA, bovine serum albumin;
Ch anti-α-Gal, anti-α-Gal antibodies purified from sera of patients with chronic Chagas disease;
ChD, Chagas disease; CL-ELISA, chemiluminescent-ELISA; ChHSP, pooled sera of chronic
Chagas disease patients; DBU, 1,8-diazabicycloundec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone; DTBS, di-tertbutylsilyl; DTBS(OTf)2, di-tertbutylsilyl

29
bis(trifluoromethanesulfonate); FPLC, fast protein liquid chromatography; HF-pyr, hydrogen
fluoride in pyridine; MALDI-TOF, Matrix-assisted laser desorption ionization Time-of-Flight;
NGP, neoglycoprotein; NHSP, normal human serum pool; NHS anti-α-Gal, anti-α-Gal
antibodies from sera of healthy individuals; NMR, nuclear magnetic resonance; RLU, relative
luminescence units; TCEP, tris(2-carboxyethyl)phosphine; tGPI-mucins, trypomastigote-derived
GPI-mucins; TMSOTf, trimethylsilyl trifluoromethanesulfonate
References
Acosta-Serrano A, Hutchinson C, Nakayasu ES, Almeida IC, Carrington M. 2007. Comparison
and evolution of the surface architecture of trypanosomatid parasites In African Trypanosomes:
After the Genome. In: Barry JD MR, Mottram JC & Acosta-Serrano A editor. Norwich, UK:
Horizon Scientific Press. p. 319-337.
Almeida IC, Covas DT, Soussumi LM, Travassos LR. 1997. A highly sensitive and specific
chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma
cruzi infection. Transfusion, 37:850-857.
Almeida IC, Ferguson MA, Schenkman S, Travassos LR. 1994. Lytic anti-alpha-galactosyl
antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides
on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. The
Biochemical journal, 304 ( Pt 3):793-802.
Almeida IC, Krautz GM, Krettli AU, Travassos LR. 1993. Glycoconjugates of Trypanosoma
cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl

30
antibodies from patients with chronic Chagas disease. Journal of clinical laboratory analysis,
7:307-316.
Almeida IC, Milani SR, Gorin PA, Travassos LR. 1991. Complement-mediated lysis of
Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies. Journal of
immunology, 146:2394-2400.
Ashmus RA, Schocker NS, Cordero-Mendoza Y, Marques AF, Monroy EY, Pardo A, Izquierdo
L, Gallego M, Gascon J, Almeida IC, et al. 2013. Potential use of synthetic alpha-galactosyl-
containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas
disease. Organic & biomolecular chemistry, 11:5579-5583.
Atassi MZ, Long PM, Beisel K, Sakata S, Peters T, Jr., David CS. 1982. Immune recognition of
serum albumin--XIV. Cross-reactivity by T-lymphocyte proliferation of subdomains 3, 6 and 9
of bovine serum albumin. Mol Immunol, 19:313-321.
Avila JL, Rojas M, Galili U. 1989. Immunogenic Gal alpha 1----3Gal carbohydrate epitopes are
present on pathogenic American Trypanosoma and Leishmania. Journal of immunology,
142:2828-2834.
Brinkmann N, Malissard M, Ramuz M, Römer U, Schumacher T, Berger EG, Elling L, Wandrey
C, Liese A. 2001. Chemo-Enzymatic synthesis of the galili epitope
Galα(1→3)Galβ(1→4)GlcNAc on a homogeneously soluble PEG polymer by a multi-Enzyme
system. Bioorganic & Medicinal Chemistry Letters, 11:2503-2506.
Buscaglia CA, Campo VA, Di Noia JM, Torrecilhas AC, De Marchi CR, Ferguson MA, Frasch
AC, Almeida IC. 2004. The surface coat of the mammal-dwelling infective trypomastigote stage