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

Abstract

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 glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) 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-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galβ(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs 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 Abs 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 Abs and the lack of Abs against nonself Galβ(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galβ(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galβ(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galβ(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

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 (Travassos and Almeida 1993; Acosta-Serrano et al. 2007). An immunodominant glycotope, Galα(1,3)Galβ(1,4)GlcNAcα, is abundantly expressed in the mammal-dwelling T. cruzi trypomastigote stage (Almeida et al. 1994) and is not expressed on human cells, thus it is highly immunogenic to humans (Travassos and Almeida 1993; Macher and Galili 2008). 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 et al. 1993; Travassos and Almeida 1993; Soares et al. 2012; Izquierdo et al. 2013). 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 et al. 1994; Ashmus et al. 2013), which are produced mainly against gram-negative enterobacteria of the human flora (Galili 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 1996). The glycotope Galα(1,3)Galβ(1,4)GlcNAcα, so far not 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 (Milani and Travassos 1988; Avila et al. 1989; Almeida et al. 1991, 1994; Gazzinelli et al. 1991; Travassos and Almeida 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 ChD. 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 (Frasch 2000; Buscaglia et al. 2004; Acosta-Serrano et al. 2007).

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 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 (α1,3-GalT-KO) mice, which do not express terminal αGal epitopes in their cells (Tearle et al. 1996; Thall et al. 1996). These animals are able to produce lytic anti-α-Gal Abs, mimicking therefore the human humoral immune response against T. cruzi (Almeida et al. unpublished data).

Fig. 1.

Target mercaptopropyl saccharides of Galα(1,3)Galβ(1,4)GlcNAcα (1), Galβ(1,4)GlcNAcα (2), GlcNAcα (3) and GlcNAcβ (4).

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 (Vic et al. 1997; Fang et al. 1998; Qian et al. 1999; Brinkmann et al. 2001), 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 et al. 2002), attached to a lipid for non-covalent association to target molecules (Litjens et al. 2005) or attached to linkers such as p-nitrophenol esters (Plaza-Alexander and Lowary 2013) and 3-aminopropyl groups (Hanessian et al. 2001; Wang 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 as follows: (i) predominant use of acyl protecting groups that can be easily installed and cleanly removed; (ii) utilization of Kiso's 4,6-di-tert-butylsilyl protected galactosyl donor (Imamura 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 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 and Seppala 1986) and provider of T cell epitopes for the immunization of mice (Atassi 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 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 and Furneaux 1996), which was made from its peracetylated precursor following an optimized procedure (Khamsi 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 (PMB) group with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) furnished the β-Gal acceptor 8. This acceptor was glycosylated with the known di-tert-butylsilylidene equipped α-Gal trichloroacetimidate donor 9 (Imamura et al. 2006), using trimethylsilyl trifluoromethanesulfonate (TMS-OTf) catalysis to give disaccharide 10. The di-tert-butylsilylidene group was cleaved with a large excess of 70% hydrogen fluoride in pyridine (HF-pyr) in tetrahydrofuran (THF), followed by acetylation of the two hydroxyls to give the peracylated allyl disaccharide 11 (Scheme 1).

Scheme 1.

Synthesis of disaccharide 11. a: Bu₂SnO, MeOH, reflux; PMB-Cl, Bu₄NBr, benzene, reflux (75%); b: BzCl, pyr (91%); c: DDQ, CH₂Cl₂/H₂O (98%); d: TMS-OTf, DCM, 0°C, molecular sieves 4 Å (92%); e: HF-pyr, THF and f: Ac₂O, pyr (89%, 2 steps).

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 2 h of reaction, and converted into the trichloroacetimidate 12 with trichloroacetonitrile in the presence of 1,8-diazabicycloundec-7-ene (DBU) (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 (Crich and Dudkin 2001). The separation of the two diastereomeric trisaccharides proved to be difficult but could be accomplished by reversed-phase fast protein liquid chromatography (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 (AcSH) to give the trisaccharide 16. Radical addition of AcSH with azobisisobutyronitrile (AIBN) in THF under UV light gave the thioester 17, followed by saponification under Zemplén conditions to afford the target trisaccharide 1 (Scheme 2).

Scheme 2.

Synthesis of mercaptopropyl trisaccharide 1. a: PdCl₂, MeOH (87%); b: CCl₃CN, DBU, CH₂Cl₂ (84%); c: TMS-OTf, molecular sieves 4 Å, CH₂Cl₂ (30% of 1:4 α/β anomers, FPLC separable); d: TMS-OTf, molecular sieves 4 Å, CH₂Cl₂ (46%); e: AcSH (77%); f: AcSH, AIBN, THF, UV light (350 nm) (89%) and g: NaOMe, MeOH (quant.).

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 and Michel 1980) and the use of boron trifluoride etherate (BF₃-Et₂O) at an unusual elevated temperature (Hendel et al. 2009), the Galβ(1,4)GlcNAcα disaccharide 19 was obtained in high yield (83%), followed by radical addition of AcSH 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 et al. 2003), while the mercaptopropyl glycoside of GlcNAcβ (4) was synthesized by radical addition of AcSH to the known allyl glycoside 21 (Kiso and Anderson 1979) to give thioester 22, followed by saponification to provide the target glycoside 4 (Scheme 3).

Scheme 3.

Synthesis of mercaptopropyl glycosides 2 and 4. a: BF₃-Et₂O, CH₂Cl₂, 35–40°C (83%); b: AcSH, AIBN, THF, UV light (350 nm) (84–85%) and c: NaOMe, MeOH (quant.).

The mercaptopropyl glycosides oxidized to disulfides within hours–days of isolation, which could easily be reduced by tris(2-carboxyethyl)phosphine (TCEP) 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 NGPs via thioether linkages, and the average number of saccharides conjugated per BSA molecule was estimated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) 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 Supplementary data, p. S3).

Fig. 2.

(Top) Schematic representation of conjugation of NGPs to BSA. a: Tris-(2-carboxyethyl)phosphine, phosphate buffer pH 7.2 and maleimide-activated BSA. (Bottom left and right) MALDI-TOF mass spectra of Galα(1,3)Galβ(1,4)GlcNAcα-BSA and Galβ(1,4)GlcNAcα-BSA, respectively.

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 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 et al. 1991). As shown in Figure 3A, Galα(1,3)Galβ(1,4)GlcNAcα-BSA 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 Abs, while NGPs Galβ(1,4)GlcNAcα-BSA, GlcNAcα-BSA and GlcNAcβ-BSA are practically not recognized by either Abs. These results emphasize that the terminal Galα residue is crucial for Ch 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 Ch patients (Figure. 3A).

Fig. 3.

(A) CL-ELISA reactivity of NHSP vs. ChHSP to NGP. (B) CL-ELISA reactivity of purified normal human sera anti-α-Gal Abs (NHS anti-α-Gal) vs. Chagasic anti-α-Gal Abs (Ch anti-α-Gal) to NGPs. RLU, relative luminescence units.

Next, the in vivo response to Galα(1,3)Galβ(1,4)GlcNAcα-BSA was evaluated in C57Bl/6 α1,3-galactosyltransferase-knockout (α1,3-GalT-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 Abs (Tearle et al. 1996; Thall et al. 1996). Sera collected from immunized and control animals were pooled separately and analyzed by CL-ELISA (Ashmus 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 with pre-immunization levels, whereas mice immunized with BSA alone showed minimal antibody reactivity before and after immunization.

Fig. 4.

CL-ELISA reactivity of α1,3GalT-KO mouse serum to Galα(1,3)Galβ(1,4)GlcNAcα-BSA and BSA before (unfilled bar) and after (filled bar) immunization with the NGP or control (BSA).

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 two or three 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 PMB 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 the 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.

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-nuclear magnetic resonance (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, and 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, and 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 0.5 M NaOMe was added under argon, and stirred at room temperature (RT) for 30 min. High resolution mass spectrometry (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.

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 0.5 M NaOMe was added under argon, and stirred at RT for 30 min. 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 the disulfide form of 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 0.5 M NaOMe was added under argon, and stirred at RT for 2 h. 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 mostly the disulfide form of 4 as a white powder (0.037 g, quant.). ESI-TOF HRMS [C₂₂H₄₀N₂O₁₂S₂ + Na]⁺ calc. m/z = 611.1920, found 611.1707.

Allyl 3-O-(4-methoxybenzyl)-β-d-galactopyranoside (6)

A solution of 5 (Stevenson and Furneaux 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 et al. 2001).

Allyl 3-O-(4-methoxybenzyl)-2,4,6-tri-O-benzoyl-β-d-galactopyranoside (7)

A solution of 6 (0.380 g, 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 3 h. The solution was diluted with EtOAc, washed once with 1 M 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%). ${[\alpha {]}^{28}}_{\mathrm{D}}72.4$ (c = 1 in CHCl₃); R_f = 0.38 (MeOH/CHCl₃ 1:9); ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 8.18; 8.05; 7.97; 7.56–7.61; 7.43–7.50 (5m, 15H, 3 × 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, ³J_H1/H2 = 8.9 Hz, ³J_H2/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, ³J_H3/H4 = 3.4 Hz, H-3); 3.70 (s, 3H, OCH₃) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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, DDQ (0.440 g, 1.94 mmol) was added in two portions, 30 min 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 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-tert-butylsilyl-2,3-di-O-benzoyl-α-d-galactopyranosyl trichloroacetimidate donor 9 (Imamura 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 4 Å molecular sieves and stirred under argon for 15 min at 0°C. TMS-OTf (0.010 mL, 0.059 mmol) was added dropwise, and the mixture was gradually brought to RT 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%). ${[\alpha {]}^{28}}_{\mathrm{D}}160.7$ (c = 1 in CHCl₃); R_f = 0.55 (EtOAc/hexanes 1:2); ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 8.09, 7.99, 7.84, 7.74, 7.60, 7.51, 7.41, 7.26, 7.13, 7.01 (10m, 25H, 5 × Bz); 5.79–5.87 (m, 2H, OCH₂CHCH₂, βGalH-4); 5.70–5.76 (m, 2H, αGalH-2, βGalH-2); 5.62 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGalH-1); 5.26 (m, 1H, OCH₂CHCH₂); 5.16 (m, 2H, OCH₂′CHCH₂, αGalH-3); 4.77 (d, 1H, ³J_H1/H2 = 8.3 Hz, βGalH-1); 4.55 (dd, 1H, ³J_H5/H6 = 11.7 Hz, ²J_H6/H6′ = 6.9 Hz, 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 (150 MHz, CDCl₃, 300 K): 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.

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). ${[\alpha {]}^{28}}_{\mathrm{D}}163.0$ (c = 1 in CHCl₃); R_f = 0.20 (EtOAc/hexanes 1:2); ¹H-NMR (600 MHz, CDCl₃, 300 K): 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 × Bz); 5.80–5.86 (m, 2H, OCH₂CHCH₂, βGalH-4); 5.76 (dd, 1H, ³J_H2/H3 = 9.6 Hz, βGalH-2); 5.67 (d, 1H, ³J_H1/H2 = 4.1 Hz, αGalH-1); 5.61 (dd, 1H, ³J_H2/H3 = 11.0 Hz, αGalH-2); 5.41 (dd, 1H, ³J_H3/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, ³J_H1/H2 = 7.6 Hz, βGalH-1); 4.57 (dd, 1H, ³J_H5/H6 = 11.0 Hz, ²J_H6/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, ³J_H5/H6 = 6.6 Hz, βGalH-6′); 3.96 (m, 2H, αGalH6, αGalH6′); 1.97–2.03 (m, 6H, 2 × Ac) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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.

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 RT 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%). ${[\alpha {]}^{27}}_{\mathrm{D}}186.2$ (c = 1 in CHCl₃); R_f = 0.65 (acetone/hexanes 1:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): 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 × Bz); 6.90 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGalH-1); 5.99 (d, 1H, ³J_H4/H5 = 2.8 Hz, αGalH-4); 5.94 (dd, 1H, ³J_H2/H3 = 10.3 Hz, αGalH-2); 5.76 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGal′H-1); 5.65 (dd, 1H, ³J_H2/H3 = 10.3 Hz, αGal′H-2); 5.49 (dd, 1H, ³J_H3/H4 = 3.4 Hz, αGal′H-3); 5.28 (m, 1H, αGal′H-4); 4.76 (dd, 1H, ³J_H3/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, ³J_H5/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, ²J_H6/H6′ = 6.2 Hz, αGal′H-6′); 1.98–2.06 (m, 6H, 2 × Ac) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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; 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 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 et al. 2007). A minor by-product with a higher R_f 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 et al. 2007): a solution of allyl 2-deoxy-2-azido-α-d-glucopyranoside (Gavard 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 three portions of 0.117 mL each over 1 h, and stirred for an 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%). ${[\alpha {]}^{25}}_{\mathrm{D}}161.0$ (c = 1 in CHCl₃) R_f = 0.48 (EtOAc/hexanes 1:2); ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 8.05–8.11; 7.58; 7.43–7.48 (m, 10H, 2 × Bz); 5.95 (m, 1H, OCH₂CHCH₂); 5.64 (dd, 1H, ³J_H3/H4 = 9.6 Hz, H-3); 5.36 (m, 1H, OCH₂CHCH₂); 5.25 (m, 1H, OCH₂′CHCH₂); 5.09 (d, 1H, ³J_H1/H2 = 4.1 Hz, H-1); 4.73 (dd, 1H, ³J_H5/H6 = 4.8 Hz, ²J_H6/H6′ = 12.4 Hz, H-6); 4.60 (dd, 1H, ³J_H5/H6′ = 2.1 Hz, H-6′); 4.29 (m, 1H, CH₂CHCH₂); 4.12 (m, 2H, H-5, CH₂CHCH₂′); 3.77 (dd, 1H, ³J_H4/H5 = 9.6 Hz, H-4); 3.47–3.54 (broad, 1H, 4-OH); 3.44 (dd, 1H, ³J_H2/H3 = 11.0 Hz, H-2) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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 by-product 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 4 Å 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 RT and stirred for 2 h. The reaction was quenched with Et₃N (0.02 mL, 0.143 mmol), 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%). ${[\alpha {]}^{26}}_{\mathrm{D}}106.1$ (c = 1 in CHCl₃) R_f = 0.53 (EtOAc/hexanes 1:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): 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 (10 m, 35H, 7 × Bz); 5.93 (m, 1H, OCH₂CHCH₂); 5.87 (dd, 1H, ³J_H2/H3 = 9.5 Hz, ³J_H3/H4 = 9.5 Hz, αGlcH-3); 5.66 (dd, 1H, ³J_H1/H2 = 9.5 Hz, βGalH-2); 5.56 (m, 1H, βGalH-4); 5.53 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGalH-1); 5.34 (m, 1H, OCH₂CHCH₂); 5.30 (dd, 1H, ³J_H2/H3 = 10.3 Hz, αGalH-2); 5.24 (m, 1H, OCH₂′CHCH₂); 5.06 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGlcH-1); 4.95 (m, 1H, αGalH-3); 4.80 (d, 1H, ³J_H1/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, ³J_H5/H6 = 11.0 Hz, ²J_H6/H6′ = 6.9 Hz, αGalH-6); 3.82 (dd, 1H, ³J_H5/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 × Ac) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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.

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 AcSH, 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 (0.097 g, 77%). ${[\alpha {]}^{26}}_{\mathrm{D}}94.3$ (c = 1 in CHCl₃); R_f = 0.15 (hexanes/EtOAc 1:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): 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 × 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, ³J_H1/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 × Ac); 1.86 (s, 3H, NHAc) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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.

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), AcSH (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 (0.028 g, 89%). ${[\alpha {]}^{26}}_{\mathrm{D}}94.2$ (c = 0.5 in CHCl₃); R_f = 0.48 (EtOAc/hexanes 2:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): 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 × Bz); 6.19 (d, 1H, ³J_NH/H2 = 9.3 Hz, NH); 5.52–5.67 (m, 4H, βGalH-2, αGalH-2); 5.52 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGalH-1); 5.28 (dd, 1H, ³J_H2/H3 = 10.3 Hz, ³J_H3/H4 = 10.3 Hz, αGalH-3); 4.92 (m, 1H, αGalH-4); 4.83 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGlcNAcH-1); 4.79 (d, 1H, ³J_H1/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, ³J_H5/H6 = 11.7 Hz, ²J_H6/H6′ = 6.9 Hz, αGalH-6); 3.81 (dd, 1H, ³J_H5/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 × Ac); 1.85–1.92 (m, 5H, NHAc, OCH₂CH₂, OCH₂CH₂′) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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.

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 et al. 2007) (3.60 g, 7.63 mmol) and donor 18 (Schmidt and Michel 1980) (14.0 g, 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%). ${[\alpha {]}^{22}}_{\mathrm{D}}56.6$ (c = 1 in CHCl₃); R_f = 0.30 (EtOAc/hexanes 2:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 8.07; 7.61; 7.52; 7.47 (4 m, 10H, 2 × Bz); 5.91 (m, 1H, OCH₂CHCH₂); 5.85 (d, 1H, ³J_NH/H2 = 9.6 Hz, NH); 5.62 (dd, 1H, ³J_H2/H3 = 11.0 Hz, ³J_H3/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, ³J_H2/H3 = 10.3 Hz, βGalH-2); 4.91 (d, 1H, ³J_H1/H2 = 3.4 Hz, αGlcNAcH-1); 4.82 (dd, 1H, ³J_H3/H4 = 3.4 Hz, βGalH-3); 4.69 (m, 1H, αGlcNAcH-6); 4.61 (d, 1H, ³J_H1/H2 = 8.3 Hz, βGalH-1); 4.47 (m, 1H, αGlcNAcH-2); 4.41 (dd, 1H, ²J_H6/H6′ = 4.1 Hz, ³J_H5/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, ³J_H5/H6 = 8.3 Hz, ²J_H6/H6′ = 11.0 Hz, βGalH-6); 3.48 (dd, 1H, ³J_H5/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 × Ac, NHAc) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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, 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), AcSH (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 (0.046 g, 84%). ${[\alpha {]}^{22}}_{\mathrm{D}}45.6$ (c = 0.9 in CHCl₃); R_f = 0.25 (EtOAc/hexanes 2:1); ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 8.08; 7.61; 7.52; 7.47 (4 m, 10H, 2 × Bz); 6.15 (d, 1H, ³J_NH/H2 = 9.6 Hz, NH); 5.58 (dd, 1H, ³J_H2/H3 = 10.3 Hz, ³J_H3/H4 = 8.3 Hz, αGlcNAcH-3); 5.13 (m, 1H, βGalH-4); 5.10 (dd, 1H, ³J_H2/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, ³J_H1/H2 = 8.3 Hz, βGalH-1); 4.48 (m, 1H, αGlcNAcH-2); 4.40 (dd, 1H, ²J_H6/H6′ = 4.1 Hz, ³J_H5/H6′ = 11.7 Hz, αGlcNAcH-6′); 4.09 (m, 2H, αGlcNAcH-4, αGlcNAcH-5); 3.80 (m, 1H, OCH₂CH₂CH₂); 3.61 (dd, 1H, ³J_H5/H6 = 8.3 Hz, ²J_H6/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 × Ac, OCH₂CH₂CH₂) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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-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 and Anderson 1979) (0.081 g, 0.209 mmol) and AIBN (0.034 g, 0.209 mmol) in anhydrous THF (5 mL), AcSH (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 (0.082 g, 85%). ${[\alpha {]}^{26}}_{\mathrm{D}}11.9$ (c = 1 in CHCl₃); R_f = 0.30 (MeOH/CHCl₃ 1:9) ¹H-NMR (600 MHz, CDCl₃, 300 K): δ 6.20 (d, 1H, ³J_NH/H2 = 8.9 Hz, NH); 5.17 (dd, 1H, ³J_H2/H3 = 8.9 Hz, ³J_H3/H4 = 10.3 Hz, H-3); 5.02 (dd, 1H, ³J_H4/H5 = 9.62 Hz, H-4); 4.50 (d, 1H, ³J_H1/H2 = 8.3 Hz, H-1); 4.20 (dd, 1H, ³J_H5/H6 = 12.4 Hz, ²J_H6/H6′ = 4.8 Hz, H-6); 4.06 (dd, 1H, ³J_H5/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 × Ac, OCH₂CH₂CH₂); 1.69 (m, 1H, OCH₂CH₂′CH₂) ppm. ¹³C-NMR (150 MHz, CDCl₃, 300 K): 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.

Immunization protocol

Groups of five female C57Bl/6 α1,3-GalT-KO mice (Tearle et al. 1996; Thall et al. 1996) were immunized subcutaneously with 20 µg Galα(1,3)Galβ(1,4)GlcNAcα-BSA in 200 µL phosphate-buffered saline (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 is 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, a Robert J. Kleberg Jr. and Helen C. Kleberg Foundation grant (to John VandeBerg and ICA), and by NIH grant 1R21AI115451-01 (to ICA and KM). ICA and CRNB are, respectively, Special Visiting Researcher and Visitor PhD (Sandwich) Scholar of the Science Without Borders Program, 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 Border Biomedical Research Center (BBRC) at UTEP, from the National Institutes on Minority Health and Health Disparities (NIMHD), a component of the NIH.

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-enzyme-linked immunosorbent assay; 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(OTf)₂, di-tert-butylsilyl bis(trifluoromethanesulfonate); FPLC, fast protein liquid chromatography; HF-pyr, hydrogen fluoride in pyridine; HRMS, high resolution mass spectrometry; 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; PBS, phosphate-buffered saline; PMB, p-methoxybenzyl; RLU, relative luminescence units; RT, room temperature; TCEP, tris(2-carboxyethyl)phosphine; tGPI-mucins, trypomastigote-derived GPI-mucins; THF, tetrahydrofuran; TMS-OTf, trimethylsilyl trifluoromethanesulfonate.