Synthesis of the Carbohydrate Moiety of Glycoproteins from the Parasite Echinococcus granulosus and Their Antigenicity against Human Sera

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the carbohydrate moiety of glycoprotein from Echinococcus granulosus have been accomplished. Trisaccharide Galβ1-3Galβ1-3GalNAcα1-R (A), tetrasaccharide Galα1-4Galβ1-3Galβ1-3GalNAcα1-R (B), and pentasaccharide Galα1-4Galβ1-3Galβ1-3Galβ1-3GalNAcα1-R (C), (R = biotinylated probe) were synthesized by stepwise condensation and/or block synthesis by the use of 5-(methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. The synthesis of the tetrasaccharide and the pentasaccharide was improved from the viewpoint of reducing the number of synthetic steps and increasing the total yield by changing from stepwise condensation to block synthesis. Moreover, hexasaccharide E, which contains the oligosaccharide sequence which occurs in E. granulosus, was synthesized from trisaccharide D. We examined the antigenicity of these five oligosaccharides by an enzyme-linked immunosorbent assay (ELISA). Although compounds of C–E did not exhibit antigenicity against cystic echinococcosis (CE) patient sera, compounds B, D, and E showed good serodiagnostic potential for alveolar echinococcosis (AE).


Introduction
In the course of our studies on natural oligosaccharides from invertebrate animal species, we are interested in the unique glycosphingolipids (GSLs) and glycoproteins (GPs) found in various Protostomia phyla and we have synthesized these oligosaccharides in order to elucidate biological functions [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. In particular, our interests have been focused on the unique oligosaccharide structures of GSLs and GPs found in several parasites including Echinococcus multilocularis [2,7,8,[11][12][13]15], Schistosoma mansoni [5,10], Ascaris suum [1,17], and Toxocara canis [6]. Among them, GSLs and GPs from E. multilocularis have attracted our special attention. E. multilocularis is a parasite, which belongs to the class Cestoda of the phylum Platyhelminthes and causes alveolar echinococcosis (AE), a severe parasitic zoonosis that can be fatal without appropriate treatment. In 1992, Persat et al. reported [19] that sera from AE patients recognized a neutral glycosphingolipid fraction from E. multilocularis and determined the structures of some of the glycosphingolipids isolated from this fraction. Hülsmeier et al. reported [20] in 2002 that Em2, an antigen extracted from E. multilocularis, is a mucin-type glycoprotein. Based on this information, we synthesized four glycosphingolipids including Galβ1→6Galβ1-Cer as a core structure [15] and five carbohydrate tion from E. multilocularis and determined the structures of some of the glycosphingolipids isolated from this fraction. Hülsmeier et al. reported [20] in 2002 that Em2, an antigen extracted from E. multilocularis, is a mucin-type glycoprotein. Based on this information, we synthesized four glycosphingolipids including Galβ1→6Galβ1-Cer as a core structure [15] and five carbohydrate structures of glycoproteins including a reducing terminal Galβ1→3GalNAcα1-which is a core 1 of mucin-type O-glycans of E. multilocularis [8,11], and examined antigenicity of the pure compounds by ELISA for their serodiagnostic potential [7,12,13].
More recently, Díaz et al. reported [21] that the extracellular matrix of a related cestode Echinococcus granulosus contains a novel mucin-type O-glycan capping motif consisting of Galpα1-4Gal linkages at the non-reducing end (Figure 1). E. granulosus is a parasitic cestode causing cystic echinococcosis (CE) in intermediate hosts like humans. The adult worm lives in the small intestine of a carnivore (definitive host), and the intermediate larval stage can infect a wide range of mammal species-including humans-that acquire the infection through accidental ingestion of eggs. Díaz et al. elucidated in full the major glycan of the E. granulosus laminated layer (LL) and these are conventional core 1 and 2 O-glycans modified with Galα1-4Gal that are linked to three kinds of carbohydrates (Gal, GalNAc, and GlcNAc). Based on this information, we report here on the synthesis of the biotinylated glycan portions A-E (Figures 1 and 2) of the glycoprotein antigen of E. granulosus in order to elucidate the interactions between the oligosaccharides and sera, and the structure-activity relationships involved in the antigen recognition. Compound D is a synthetic intermediate derived from the process of synthesizing compound E.

Syntheses of the Target Oligosaccharides A, B, and C
The synthetic strategy for oligosaccharides A-C is shown in Figure 3. (NMR spectra provided in the Supplemental Data).

Syntheses of the Target Oligosaccharides A, B, and C
The synthetic strategy for oligosaccharides A-C is shown in Figure 3 (NMR spectra provided in the Supplemental Data).

Syntheses of the Target Oligosaccharides A, B, and C
The synthetic strategy for oligosaccharides A-C is shown in Figure 3. (NMR spectra provided in the Supplemental Data). Suitably protected monosaccharide derivatives (4, 5, 9, and 12) were chosen as building blocks. The 5-(Methoxycarbonyl)pentyl group was chosen as the temporary protecting group for all the target compounds to ensure future conjugation with biotin for the ELISA assay as previously shown by us [8]. The synthetic routes for target compounds A-C are outlined in Schemes 1-6. Initially, disaccharide acceptor 8 was prepared in a way that Suitably protected monosaccharide derivatives (4, 5, 9, and 12) were chosen as building blocks. The 5-(Methoxycarbonyl)pentyl group was chosen as the temporary protecting group for all the target compounds to ensure future conjugation with biotin for the ELISA assay as previously shown by us [8]. The synthetic routes for target compounds A-C are outlined in Schemes 1-6. Initially, disaccharide acceptor 8 was prepared in a way that serves as a common acceptor for the syntheses of compounds A, B, and C. Disaccharide 8 was prepared from thioglycoside donor 3, which was obtained from phenyl-1-thio-β-D-galactopyranoside (1) by regioselective 2-naphthylbenzylation of the in situ prepared stannylidene derivative with 2-naphthylbenzyl bromide (NapBr) and tetrabutylammonium bromide followed by benzoylation (Scheme 1). The glycosylation of 3 with 5 [8] in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH) [22] and AW-300 molecular sieves (MS AW-300) in CH 2 Cl 2 afforded desired disaccharide 6 in 71% yield. The nature of the new β-glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1 of Gal, δ = 5.11 ppm, J = 7.9 Hz). Condensation of 5 with the 3-O-chloroacetyl (ClAc) donor 4, which was prepared by selective removal of the 3 -O-NAP group in 3 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) followed by chloroacetylation afforded desired disaccharide (7) in higher yield (89%). Oxidative removal of the 3 -O-NAP group in 6 with DDQ gave disaccharide acceptor 8 in 63% yield and removal of the 3 -O-ClAc group in 7 with thiourea produced the same acceptor 8 in 84% yield. Comparing the protecting groups at the 3'-position, the ClAc group consistently gave higher yields than the NAP group in both, glycosylation and deprotection steps in the synthesis of 8. The glycosylation of disaccharide acceptor 8 with thioglycosyl donor 9 in the presence of NIS/TfOH and MS AW-300 in CH 2 Cl 2 afforded desired trisaccharide (10) in 78% yield. The nature of the new glycosidic linkage was determined as β by the vicinal coupling constant of the anomeric proton (H-1 of Gal, δ = 4.69 ppm, J = 8.1 Hz). Selective removal of the 4 -O-ClAc protecting group from 10 was achieved by thiourea to produce trisaccharide acceptor 11 in 85% yield, which was the direct precursor of compound A and used directly for the next glycosylation step of compound B. The Glycosylation of 11 with 12 [23] using NIS/TfOH and MS AW-300 in CH 2 Cl 2 produced desired tetrasaccharide (13) in 85% yield. The newly formed α-glycosidic linkage (H-1 of Galc, δ = 4.86 ppm, J = 2.6 Hz) was confirmed by 1 H NMR spectroscopy. 84% yield. Comparing the protecting groups at the 3'-position, the ClAc group consistently gave higher yields than the NAP group in both, glycosylation and deprotection steps in the synthesis of 8. The glycosylation of disaccharide acceptor 8 with thioglycosyl donor 9 in the presence of NIS/TfOH and MS AW-300 in CH2Cl2 afforded desired trisaccharide (10) in 78% yield. The nature of the new glycosidic linkage was determined as β by the vicinal coupling constant of the anomeric proton (H-1 of Gal, δ = 4.69 ppm, J = 8.1 Hz). Selective removal of the 4″-O-ClAc protecting group from 10 was achieved by thiourea to produce trisaccharide acceptor 11 in 85% yield, which was the direct precursor of compound A and used directly for the next glycosylation step of compound B. The Glycosylation of 11 with 12 [23] using NIS/TfOH and MS AW-300 in CH2Cl2 produced desired tetrasaccharide (13) in 85% yield. The newly formed α-glycosidic linkage (H-1 of Galc, δ = 4.86 ppm, J = 2.6 Hz) was confirmed by 1 H NMR spectroscopy.

Scheme 1. Preparation of tetrasaccharide derivative 13.
Global deprotection of the precursors for A and B was performed by a combination of protection/deprotection steps. Initially, we attempted the simultaneous reduction of the azido group and removal of the benzyl protecting groups in 13 by catalytic hydrogenoly-Scheme 1. Preparation of tetrasaccharide derivative 13.
Molecules 2021, 26, x FOR PEER REVIEW 5 of 32 sis with 10% Pd/C. However, since this conversion was not successful, we studied stepwise conversion. Selective hydrogenolysis using 10% Pd/C of the azido group in the presence of ammonia followed by hydrogenolytic cleavage of the benzyl groups over 10% Pd/C in acetic acid and exposure to Ac2O in pyridine resulted in the O-and N-acylation. However, the benzylidene acetal was not removed under these conditions. Thus, the benzylidene acetal was removed with TsOH followed by O-acetylation with Ac2O in pyridine to afford 14 in 51% yield after five steps. After deacetylation of 14 under Zemplén conditions, 5-(methoxycarbonyl)pentyl glycoside 15 was converted into the ethylenediamine monoamide by exposure to ethylenediamine and conjugated to biotin using our previously established methodology [8] to afford tetrasaccharide-biotin conjugate A in 67% yield after column chromatographic purification on Sephadex LH-20 (Scheme 2).

Scheme 2. Synthesis of target trisaccharide A.
Tetrasaccharide B was synthesized in a multi-step sequence as outlined in Scheme 3. At first, selective removal of the DTBS group in 13 was achieved with HF/pyridine and the benzylidene acetal was removed by acidic hydrolysis followed by acetylation with acetic anhydride in pyridine to afford 16 in 65% yield. Then, the azido-group was converted to an acetamido moiety by reduction with PPh3 followed by N-acylation with Ac2O in pyridine and debenzylation by catalytic hydrogenolysis using Pearlman's catalyst followed by O-acetylation to produce protected tetrasaccharide 17 in 65% yield. The deacetylation of 17 under Zemplén conditions yielded unprotected tetrasaccharide 18 in 60% yield. Compound 18 was then used for ligation to biotin to provide target tetrasaccharide B after column chromatographic purification on Sephadex LH-20 (Scheme 3).
Originally, it was planned to prepare pentasaccharide C by elongation from previously prepared disaccharide acceptor 8 and thioglycoside donor 4. However, preparation of the intermediate trisaccharide 19 by glycosylation of thioglycoside donor 4 with disaccharide acceptor 8 by NIS/TfOH-promoted activation was not successful. Similarly, the glycosylation of donor 20 [5] as well as donor 22 [24] with disaccharide acceptor 8 was Global deprotection of the precursors for A and B was performed by a combination of protection/deprotection steps. Initially, we attempted the simultaneous reduction of the azido group and removal of the benzyl protecting groups in 13 by catalytic hydrogenolysis with 10% Pd/C. However, since this conversion was not successful, we studied stepwise conversion. Selective hydrogenolysis using 10% Pd/C of the azido group in the presence of ammonia followed by hydrogenolytic cleavage of the benzyl groups over 10% Pd/C in acetic acid and exposure to Ac 2 O in pyridine resulted in the Oand N-acylation. However, the benzylidene acetal was not removed under these conditions. Thus, the benzylidene acetal was removed with TsOH followed by O-acetylation with Ac 2 O in pyridine to afford 14 in 51% yield after five steps. After deacetylation of 14 under Zemplén conditions, 5-(methoxycarbonyl)pentyl glycoside 15 was converted into the ethylenediamine monoamide The failure to generate trisaccharide intermediates 19, 21, and 23 forced us to modify our original synthetic strategy as outlined in Figure 4. Compound 20 was selected as a new glycosyl donor of the di-and trisaccharides and the elongation of the carbohydrate chain was repeated by glycosylation and dechloroacetylation as shown in Schemes 5 and 6. The glycosylation of the acceptor 5 with 20 in the presence of NIS/TfOH provided disaccharide 25 in 76% yield. The disaccharide acceptor 26 was obtained in 76% yield from 25 after treatment with thiourea, which was used directly for the next glycosylation step. Trisaccharide derivative 27 was obtained by glycosylation of glycosyl donor 20 with 26. The deprotection of the ClAc group in 27 was performed as described for compound 26 to provide trisaccharide acceptor 28 (Scheme 5). Tetrasaccharide B was synthesized in a multi-step sequence as outlined in Scheme 3. At first, selective removal of the DTBS group in 13 was achieved with HF/pyridine and the benzylidene acetal was removed by acidic hydrolysis followed by acetylation with acetic anhydride in pyridine to afford 16 in 65% yield. Then, the azido-group was converted to an acetamido moiety by reduction with PPh 3 followed by N-acylation with Ac 2 O in pyridine and debenzylation by catalytic hydrogenolysis using Pearlman's catalyst followed by O-acetylation to produce protected tetrasaccharide 17 in 65% yield. The deacetylation of 17 under Zemplén conditions yielded unprotected tetrasaccharide 18 in 60% yield. Compound 18 was then used for ligation to biotin to provide target tetrasaccharide B after column chromatographic purification on Sephadex LH-20 (Scheme 3).
Originally, it was planned to prepare pentasaccharide C by elongation from previously prepared disaccharide acceptor 8 and thioglycoside donor 4. However, preparation of the intermediate trisaccharide 19 by glycosylation of thioglycoside donor 4 with disaccharide acceptor 8 by NIS/TfOH-promoted activation was not successful. Similarly, the glycosylation of donor 20 [5] as well as donor 22 [24] with disaccharide acceptor 8 was also not successful in the presence of NIS and TfOH. The latter reaction afforded undesired α-glycosylated trisaccharide 24 in 67% yield (Scheme 4). These results suggest that 4 and 20 are unreactive (mismatched) donors to react with disaccharide acceptor 8. On the other hand, donor 22 displays superior reactivity but exclusively produces undesired α-anomer because of the absence of a C-2 acyl neighboring group participation. The coupling of acceptor 28 to thioglycoside donor 9 afforded protected tetrasaccharide 29 in 53% yield. Selective removal of the 4-O-ClAc group in 29 by thiourea afforded tetrasaccharide acceptor 30 in 97% yield. In order to prepare the Galα1-4Gal-sequence with high α-stereoselectivity, we selected 4,6-O-di-tert-butylsilylene (DTBS)-protected galactose donor 12. Previous studies have indicated that DTBS-protected galactose donors induce high α-selectivity in glycosylation reactions [23]. NIS/TfOH-promoted activation of thioglycoside donor 12 and coupling to tetrasaccharide acceptor 30 generated protected pentasaccharide 31 in 68% yield (Scheme 6).
Although we were able to achieve the synthesis of the desired protected target pentasaccharide using the stepwise elongation approach, the amount obtained was not sufficient to undergo global deprotection. As a result, we investigated a block synthesis approach in which a non-reducing terminal disaccharide derivative was synthesized in advance and condensed with the reducing end terminal di-and tri-saccharide derivatives to synthesize the tetra-and the penta-saccharides ( Figure 5). The failure to generate trisaccharide intermediates 19, 21, and 23 forced us to modify our original synthetic strategy as outlined in Figure 4. Compound 20 was selected as a new glycosyl donor of the di-and trisaccharides and the elongation of the carbohydrate chain was repeated by glycosylation and dechloroacetylation as shown in Schemes 5 and 6. The glycosylation of the acceptor 5 with 20 in the presence of NIS/TfOH provided disaccharide 25 in 76% yield. The disaccharide acceptor 26 was obtained in 76% yield from The coupling of acceptor 28 to thioglycoside donor 9 afforded protected tetrasaccharide 29 in 53% yield. Selective removal of the 4-O-ClAc group in 29 by thiourea afforded tetrasaccharide acceptor 30 in 97% yield. In order to prepare the Galα1-4Gal-sequence with high α-stereoselectivity, we selected 4,6-O-di-tert-butylsilylene (DTBS)-protected galactose donor 12. Previous studies have indicated that DTBS-protected galactose donors induce high α-selectivity in glycosylation reactions [23]. NIS/TfOH-promoted activation of thioglycoside donor 12 and coupling to tetrasaccharide acceptor 30 generated protected pentasaccharide 31 in 68% yield (Scheme 6).
Although we were able to achieve the synthesis of the desired protected target pentasaccharide using the stepwise elongation approach, the amount obtained was not sufficient to undergo global deprotection. As a result, we investigated a block synthesis approach in which a non-reducing terminal disaccharide derivative was synthesized in advance and condensed with the reducing end terminal di-and tri-saccharide derivatives to synthesize the tetra-and the penta-saccharides ( Figure 5).   The coupling of acceptor 28 to thioglycoside donor 9 afforded protected tetrasaccharide 29 in 53% yield. Selective removal of the 4-O-ClAc group in 29 by thiourea afforded tetrasaccharide acceptor 30 in 97% yield. In order to prepare the Galα1-4Gal-sequence with high α-stereoselectivity, we selected 4,6-O-di-tert-butylsilylene (DTBS)-protected galactose donor 12. Previous studies have indicated that DTBS-protected galactose donors induce high α-selectivity in glycosylation reactions [23]. NIS/TfOH-promoted activation of thioglycoside donor 12 and coupling to tetrasaccharide acceptor 30 generated protected pentasaccharide 31 in 68% yield (Scheme 6).
Although we were able to achieve the synthesis of the desired protected target pentasaccharide using the stepwise elongation approach, the amount obtained was not sufficient to undergo global deprotection. As a result, we investigated a block synthesis approach in which a non-reducing terminal disaccharide derivative was synthesized in advance and condensed with the reducing end terminal di-and tri-saccharide derivatives to synthesize the tetra-and the penta-saccharides ( Figure 5).
Disaccharide donor 36 was obtained by using the synthetic strategy outlined in Scheme 7. At first, the glycosylation of the known monosaccharide acceptor 32 [25] with monosaccharide donor 33 (commercially available) afforded benzylgroup-protected disaccharide 34 in 79% yield. Removal of the benzyl protecting groups of 34 by catalytic hydrogenation over 10% Pd-C in THF-MeOH followed by O-acetylation produced protected   Disaccharide donor 36 was obtained by using the synthetic strategy outlined in Scheme 7. At first, the glycosylation of the known monosaccharide acceptor 32 [25] with monosaccharide donor 33 (commercially available) afforded benzylgroup-protected disaccharide 34 in 79% yield. Removal of the benzyl protecting groups of 34 by catalytic hydrogenation over 10% Pd-C in THF-MeOH followed by O-acetylation produced protected disaccharide 35.   Pentasaccharide C was obtained in moderate yields from trisaccharide acceptor 28 and disaccharide donor 36 in a similar manner (Scheme 9). Deprotection and biotinylation of pentasaccharide 40 were performed as described for compound B to provide target trisaccharide C in an excellent 85% yield (Scheme 9). The glycosylation of acceptor 26 with donor 36 in the presence of TMSOTf and MS AW-300 in CH2Cl2 afforded desired tetrasaccharide 37 in 34% yield. The nature of the new β-glycosidic linkage was determined by the vicinal coupling constant of the anomeric proton (H-1 of Galb, δ = 5.01 ppm, J = 8.1 Hz). The deprotection and biotinylation of 37 proceeded in excellent yield to give target tetrasaccharide B (Scheme 8).

Scheme 8. Synthesis of target tetrasaccharide B.
Pentasaccharide C was obtained in moderate yields from trisaccharide acceptor 28 and disaccharide donor 36 in a similar manner (Scheme 9). Deprotection and biotinylation of pentasaccharide 40 were performed as described for compound B to provide target trisaccharide C in an excellent 85% yield (Scheme 9).

Syntheses of the Target Oligosaccharides D and E
We next devised a synthetic strategy for trisaccharide D and hexasaccharide E as shown in Figure 6 (NMR spectra provided in the Supplemental Data). Trisaccharide D constitutes the partial structure of hexasaccharide E. Trisaccharide 44 served as starting material for the preparation of D. Trisaccharide 45 was prepared by condensation of 2,6-dimethyl-thiophenyl-trisaccharide donor 44 [11] with 5-(methoxycarbonyl)pentyl alcohol in the presence of NIS/TfOH and MS AW-300 in CH2Cl2 in 89% yield. The reduction and Nacetylation of the Troc groups of 45 with Zn-Cu THF/AcOH/Ac2O followed by debenzylation with catalytic hydrogenolysis over 10% Pd/C in MeOH and acetylation afforded 46. Deacylation of 46 followed by biotinylation of 47 was performed as described above to provide target trisaccharide D (Scheme 10). Scheme 9. Synthesis of target tetrasaccharide C.

Syntheses of the Target Oligosaccharides D and E
We next devised a synthetic strategy for trisaccharide D and hexasaccharide E as shown in Figure 6 (NMR spectra provided in the Supplemental Data). Trisaccharide D constitutes the partial structure of hexasaccharide E. Trisaccharide 44 served as starting material for the preparation of D. Trisaccharide 45 was prepared by condensation of 2,6dimethyl-thiophenyl-trisaccharide donor 44 [11] with 5-(methoxycarbonyl)pentyl alcohol in the presence of NIS/TfOH and MS AW-300 in CH 2 Cl 2 in 89% yield. The reduction and N-acetylation of the Troc groups of 45 with Zn-Cu THF/AcOH/Ac 2 O followed by debenzylation with catalytic hydrogenolysis over 10% Pd/C in MeOH and acetylation afforded 46. Deacylation of 46 followed by biotinylation of 47 was performed as described above to provide target trisaccharide D (Scheme 10).
Compound E is a hexasaccharide which combines two trisaccharide components: Galα1-4Galβ1-4GlcNAc and Galβ1-3Galβ1-3GalNAc. The former component can be conveniently installed using synthetic intermediate 44 while the latter component 48 was obtained from the regioselective reductive ring-opening of the benzylidene acetal in compound 10 as a glycosyl acceptor (Scheme 11). The glycosylation of 44 with 48 in the presence of NIS/TfOH and MS AW-300 in CH 2 Cl 2 afforded desired disaccharide (49) in 94% yield. The new β-glycosidic linkage was confirmed by 1 H NMR using the coupling constant of H-1 of GlcN (δ 4.62 J 1,2 7.0 Hz) as a diagnostic tool as well as from the 13 C-NMR value for C-1 of GlcN (δ 100.9). The removal of the Troc-protecting group of 49 was achieved with Zn in an Ac 2 O and AcOH mixture to produce protected N-acylated hexasaccharide 50. The removal of benzyl protecting groups in 50 was initially attempted by hydrogenolysis using Pd-C. However, this reaction failed and resulted in side reactions involving the ClAc protecting group leading to an intractable mixture of products. In contrast, significantly improved yields were obtained by deacylation under Zemplén conditions followed by hydrogenolytic cleavage of the benzyl protecting group to produce the desired hexasaccharide 51. Biotinylation was performed as the usual method to provide target hexasaccharide E in 79% yield. Scheme 9. Synthesis of target tetrasaccharide C.

Syntheses of the Target Oligosaccharides D and E
We next devised a synthetic strategy for trisaccharide D and hexasaccharide E as shown in Figure 6 (NMR spectra provided in the Supplemental Data). Trisaccharide D constitutes the partial structure of hexasaccharide E. Trisaccharide 44 served as starting material for the preparation of D. Trisaccharide 45 was prepared by condensation of 2,6-dimethyl-thiophenyl-trisaccharide donor 44 [11] with 5-(methoxycarbonyl)pentyl alcohol in the presence of NIS/TfOH and MS AW-300 in CH2Cl2 in 89% yield. The reduction and Nacetylation of the Troc groups of 45 with Zn-Cu THF/AcOH/Ac2O followed by debenzylation with catalytic hydrogenolysis over 10% Pd/C in MeOH and acetylation afforded 46. Deacylation of 46 followed by biotinylation of 47 was performed as described above to provide target trisaccharide D (Scheme 10).

Antigenicity of Oligosaccharides by ELISA
The reactivity of the five oligosaccharides A-E(NMR spectra provided in the Suppl mental Data). to alveolar echinococcosis (AE) patient sera and cystic echinococcosis (CE was examined using microplates coated with streptavidin. Contrary to expectations, th antibody response of the CE patient group was not significantly different from that of th normal healthy (NH) group (Figure 7).
Although A-E display oligosaccharide structures specific to E. granulosus, only com pounds A and B showed a modest effect of antigenicity to CE patient serum while n effect was observed with saccharides C-E. This suggests that the presence of the termin Galα1-4Gal sequence in oligosaccharides B-E of E. granulosus may suppress a host im mune response or the cell surface oligosaccharides on E. granulosus may be associate with lower host specificity than E. multirocularis [26]. Interestingly, we previously r ported that the trisaccharide sequence Galα1-4Galβ1-3GalNAc found on the surface of E multilocularis showed the strongest antigenic response to the AE group among a series o oligosaccharides [8]. Rather unexpected is our finding that oligosaccharides B, D, and that occur on E. granulosus display strong antigenicity to AE patient sera. Scheme 11. Synthesis of target tetrasaccharide E.

Antigenicity of Oligosaccharides by ELISA
The reactivity of the five oligosaccharides A-E (NMR spectra provided in the Supplemental Data) to alveolar echinococcosis (AE) patient sera and cystic echinococcosis (CE) was examined using microplates coated with streptavidin. Contrary to expectations, the antibody response of the CE patient group was not significantly different from that of the normal healthy (NH) group (Figure 7).
Although A-E display oligosaccharide structures specific to E. granulosus, only compounds A and B showed a modest effect of antigenicity to CE patient serum while no effect was observed with saccharides C-E. This suggests that the presence of the terminal Galα1-4Gal sequence in oligosaccharides B-E of E. granulosus may suppress a host immune response or the cell surface oligosaccharides on E. granulosus may be associated with lower host specificity than E. multirocularis [26]. Interestingly, we previously reported that the trisaccharide sequence Galα1-4Galβ1-3GalNAc found on the surface of E. multilocularis showed the strongest antigenic response to the AE group among a series of oligosaccharides [8]. Rather unexpected is our finding that oligosaccharides B, D, and E that occur on E. granulosus display strong antigenicity to AE patient sera. ules 2021, 26, x FOR PEER REVIEW 13 of 32

Conclusions
We have developed an efficient synthetic strategy for the preparation of five oligosaccharide-biotin conjugates A-E which display carbohydrate structures that occur on the surface of E. granulosus. The oligosaccharide-biotin conjugates were prepared to study the antigenicity of the compounds to detect antibodies in patient sera infected with E. granulosus the cause of CE. Surprisingly, none of the oligosaccharide structures C-E was able to detect antibodies in sera from patients suffering from CE using our ELISA assay while only a modest response was seen with compounds A and B. However, glycoconjugates B, D, and E showed good serodiagnostic potential to recognize antibodies in AE patients. Although the oligosaccharide sequence of compound E has not been reported in E. multilocularis, it showed strong antigenicity to the serum of AE patients. Overall

Conclusions
We have developed an efficient synthetic strategy for the preparation of five oligosacch -aride-biotin conjugates A-E which display carbohydrate structures that occur on the surface of E. granulosus. The oligosaccharide-biotin conjugates were prepared to study the antigenicity of the compounds to detect antibodies in patient sera infected with E. granulosus the cause of CE. Surprisingly, none of the oligosaccharide structures C-E was able to detect antibodies in sera from patients suffering from CE using our ELISA assay while only a modest response was seen with compounds A and B. However, glycoconjugates B, D, and E showed good serodiagnostic potential to recognize antibodies in AE patients. Although the oligosaccharide sequence of compound E has not been reported in E. multilocularis, it showed strong antigenicity to the serum of AE patients. Overall, our results suggest that oligosaccharide-based structures on the cell surface of E. granulosus may serve as a diagnostic tool to detect AE. The reasons for this phenomenon are currently not understood. Possible explanations for this observation are: (i) E. granulosus induces a suppressed host immune response when compared to E. multilocularis; (ii) the presence of Galα1-4Gal terminal capping linkage in E. granulosus reduces a host immune response and (iii) oligosaccharide structures present in E. granulosus may also be present in E. multilocularis. Overall, our investigation encourages future studies in the development of carbohydratebased antigens as serodiagnostic tools to detect parasitic infections. In particular, our findings that oligosaccharide-biotin probes D and E can differentiate between sera from AE and CE patients warrant further studies toward the development of serodiagnostic tools to detect parasite-specific infections in humans.

Biotinylated tetrasaccharide (B)
Compound 18 (23.6 mg, 28.2 µmol) was dissolved in neat anhydrous ethylenediamine (4.8 mL) and heated at 70 • C for 64 h. The mixture was concentrated with toluene and the product was purified by Sephadex LH-20 column chromatography in H 2 O to give an amine intermediate. The amine derivative was dissolved in DMF (6 mL), and the pH was adjusted to 8-9 using DIPEA. Biotin-NHS (14.4 mg, 41.4 µmol) was added and the reaction was stirred at room temperature for 19 h. Toluene was added to and evaporated from the residue several times. The product was purified by Sephadex LH-20 column chromatography in H 2 O to give B (29.0 mg, 94%). Spectral data is described above B.