Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Abstract

•LacNAc and keratan sulfate glycans were obtained by automated glycan assembly•Linear, branched, and sulfated oligosaccharides up to hexamers were generated•Glycan arrays revealed an interaction between keratan sulfate and AAV virus particles Mammalian glycans are crucial for many disease-relevant processes, including viral infections, but are inaccessible from natural sources in sufficient purity for fine structure-activity studies. Availability of well-defined mammalian-derived synthetic glycans is imperative for unveiling their impact on human physiology. Here, we disclose an automated glycan assembly (AGA) approach to synthesizing oligo-N-acetyllactosamines (LacNAc) and keratan sulfates (KS), two major classes of mammalian glycans. AGA furnished diverse, conjugation-ready LacNAc and KS glycans that helped uncover virus-glycan interactions by microarray and surface plasmon resonance studies. A disulfated KS tetrasaccharide was specifically recognized by the adeno-associated virus AAVrh10 gene-therapy vector and could thus confer viral entry into host cells. This study underlines AGA as a key technology for generating glycan probes with biological activity, in this case with possible implications for cell-type-specific gene delivery. Oligo-N-acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches. Oligo-N-acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches. 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The hydroxyl group that constitutes the next glycosylation site was protected with 9-fluorenylmethoxycarbonyl (Fmoc). The glucosamine C-6 hydroxyl was masked with a levulinoyl (Lev) ester that was efficiently cleaved under mild conditions followed by sulfation reactions. The C-6 hydroxyl group of Gal was temporarily blocked by a 2-naphthylmethyl (Nap) ether that was recently shown to be selectively removable in the presence of Fmoc,48Schmidt D. Schuhmacher F. Geissner A. Seeberger P.H. Pfrengle F. Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.Chemistry. 2015; 21: 5709-5713Crossref Scopus (81) Google Scholar in anticipation of branching or sulfation. Other hydroxyl groups were protected with either benzoyl (Bz) esters or benzyl (Bn) ethers, depending on whether participation from C-2 to selectively install 1,2-trans glycosidic linkages was required or not. All of these permanent protecting groups were removed either by methanolysis or by hydrogenolysis using Pd/C and H2 gas. To ensure C-2 participation, the amine of glucosamine was protected as a N-trichloroacetyl (TCA) group, which was converted to an acetyl (Ac) group during the hydrogenolysis step. Following these considerations, we prepared two differently protected building blocks of glucosamine (9 and 10) and galactose (11 and 12) on a multi-gram scale (Figure 2, Supplemental Experimental Procedures Section 2.2, and Figures S34–S41). Linear LacNAc glycans 1 and 2 were prepared with polystyrene resin equipped with photolabile linker 849Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar and building blocks 944Kröck L. Esposito D. Castagner B. Wang C.C. Bindschädler P. Seeberger P.H. Streamlined access to conjugation-ready glycans by automated synthesis.Chem. Sci. 2012; 3: 1617-1622Crossref Scopus (116) Google Scholar and 1150Hormann J. Hahm H.S. Seeberger P.H. Pagel K. Identification of carbohydrate anomers using ion mobility-mass spectrometry.Nature. 2015; 526: 241-244Crossref Scopus (242) Google Scholar (Figure S1). The monomers were added alternatingly by AGA using three reaction modules: acidic wash, glycosylation, and Fmoc deprotection (Table 1 and Scheme 1). The acidic wash module added consecutive washing steps with N,N-dimethylformamide (DMF), tetrahydrofuran, and dichloromethane (DCM) to remove any water from the reaction vessel, followed by a trimethylsilyl trifluoromethanesulfonate (TMSOTf) solution in DCM at −20°C to neutralize basic residues present on the resin from previous synthesizer cycles. The glycosylation module used 5 equiv of the respective building block and a solution of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH). First, the reaction was kept at the activation temperature (Ta) to allow for thorough mixing; then, it was increased to the incubation temperature (Ti) to complete the glycosylation (Table 1) according to the optimized glycosylation conditions reported previously.51Hahm S.H. Liang C.F. Lai C.H. Fair R.H. Schumacher F. Seeberger P.H. Automated glycan assembly of complex oligosaccharides related to blood group determinants.J. Org. Chem. 2016; 81: 5866-5877Crossref PubMed Scopus (23) Google Scholar The glycosylation module was performed twice to minimize the amount of unreacted glycosyl acceptor. Fmoc deprotection was achieved with 20% triethylamine (TEA) in DMF to liberate the hydroxyl group for subsequent chain elongation. These conditions replaced the conventional conditions used in AGA (20% piperidine in DMF49Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar) to suppress migration of Bz groups in the terminal galactose residue. The cycles consisting of glycosylation and Fmoc deprotection were repeated four times to assemble tetrasaccharide 1 and six times for hexasaccharide 2. After AGA, the fully protected linear LacNAc oligosaccharides 13 (Figure S2) and 14 (Figure S4) were released from the resin by UV irradiation in a continuous-flow reactor.49Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar Formation of the desired molecules without any deletion sequences was confirmed by analytical normal-phase high-performance liquid chromatography (HPLC) (Supplemental Experimental Procedures, Figures S2, and S4). Without further purification, 13 and 14 were subjected to methanolysis with sodium methoxide to remove Bz esters as well as to hydrogenolysis with Pd/C and H2 to both remove Bn ethers and reduce trichloroacetamide to acetoamide. This afforded 1 (5.1 mg, 6.1 μmol, 25% over 11 steps; Figures S3, S20, and S21) and 2 (4.8 mg, 4.0 μmol, 16% over 15 steps; Figures S5, S22, and S23), which were both purified by semi-preparative reverse-phase HPLC with Hypercarb columns.Table 1AGA ModulesModuleNumbers of CyclesReagentsConditionsAcidic wash1TMSOTf (2.5 equiv), DCM−20°C for 1 minGlycosylation 129 and 10 (5 equiv), NIS (5.5 equiv), TfOH (0.55 equiv), DCM, dioxane (v/v, 9:1)Ta = −30°C for 5 minTi = −10°C for 25 minGlycosylation 2211 and 12 (5 equiv), NIS (5.5 equiv), TfOH (0.55 equiv), DCM, dioxane (v/v, 9:1)Ta = −40°C for 5 minTi = −20°C for 25 minGlycosylation 3212 (7.5 equiv), NIS (8.3 equiv), TfOH (0.83 equiv), DCM, dioxane (v/v, 9:1)Ta = −40°C for 5 minTi = −20°C for 25 minFmoc deprotection320% of TEA in DMFroom temperature for 5 minNap deprotection3DDQ (8.0 equiv), DCE, MeOH, phosphate buffer (v/v/v, 64:16:1)room temperature for 30 minLev deprotection3NH2NH2·H2O (11.2 equiv), AcOH, pyridine (v/v, 3:2)room temperature for 30 minCapping3Ac2O (1 mL)room temperature for 30 minSulfation3SO3·pyridine (40 equiv), DMF, pyridine (v/v, 1:1)50°C for 30 min Open table in a new tab Hexasaccharide 3 was synthesized with resin 8 and building blocks 9, 11, and 12 (Table 1, Scheme 1, and Figure S6). The Nap-ether-protected galactose 12 allowed for branching at the C-6 position. In addition to the three modules used for linear LacNAc synthesis, the Nap protecting group was removed by a 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) solution in 1,2-dichloroethane (DCE), methanol, and phosphate buffer to introduce disaccharide branching during the Nap deprotection module (Scheme 1). The Fmoc-protected trisaccharide52Kandasamy J. Hurevich M. Seeberger P.H. Automated solid phase synthesis of oligoarabinofuranosides.Chem. Commun. (Camb.). 2013; 49: 4453-4455Crossref Scopus (39) Google Scholar was assembled with 9 and 12, and then Nap was removed. Glycosylation with glucosamine 9 installed branching at the C-6 position of Gal. Then, two hydroxyl groups were liberated by Fmoc removal before bis-glycosylation using the modified glycosylation module (2 × 7.5 equiv of 11) was performed and helped to reduce both the reaction time and the amount of building block used.52Kandasamy J. Hurevich M. Seeberger P.H. Automated solid phase synthesis of oligoarabinofuranosides.Chem. Commun. (Camb.). 2013; 49: 4453-4455Crossref Scopus (39) Google Scholar After UV cleavage, protected branched hexasaccharide 15 was obtained as the major product, as judged by analytical normal-phase HPLC (Figure S7). Methanolysis and hydrogenolysis of 15 followed by reverse-phase HPLC purification provided the deprotected branched oligosaccharide 3 in 18% yield (5.3 mg, 4.4 μmol) over 13 steps (Figures S8, S24, and S25). Unlike prior glycosaminoglycan synthesis that used each backbone to obtain each defined sulfated oligosaccharide,45Geissner A. Anish C. Seeberger P.H. Glycan arrays as tools for infectious disease research.Curr. Opin. Chem. Biol. 2014; 18: 38-45Crossref PubMed Scopus (56) Google Scholar, 53Kandasamy J. Schuhmacher F. Hahm H.S. Klein J.C. Seeberger P.H. Modular automated solid phase synthesis of dermatan sulfate oligosaccharides.Chem. Commun. (Camb.). 2014; 50: 1875-1877Crossref Scopus (33) Google Scholar KS oligosaccharides with defined sulfation patterns were obtained from the common tetrasaccharide backbone 16, which harbors three orthogonal protecting groups (Scheme 2). Compound 16 was synthesized by AGA with alternating building blocks 10 and 12. After a cycle of acidic washing, glycosylation, and Fmoc deprotection, 16 was converted to four differentially sulfated tetrasaccharides (17–20) with a combination of four additional modules (Scheme 2 and Figures S9, S10, and S44–S47). The capping module49Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar was designed to block the free hydroxyl group when C-3 sulfation of Gal was not desired. Nap ethers or Lev esters were selectively removed with a homogeneous solution of DDQ48Schmidt D. Schuhmacher F. Geissner A. Seeberger P.H. Pfrengle F. Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.Chemistry. 2015; 21: 5709-5713Crossref Scopus (81) Google Scholar or hydrazine,49Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar respectively. The sulfation module49Eller S. Collot M. Yin J. Hahm H.S. Seeberger P.H. Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.Angew. Chem. Int. Ed. Engl. 2013; 52: 5858-5861Crossref Scopus (130) Google Scholar introduced sulfate groups at the defined positions. Compounds 17 (Figure S11), 18 (Figures S13, S48, and S49), 19 (Figures S15, S50, and S51), and 20 (Figures S17, S52, and S53) were obtained after UV-mediated cleavage from the resin. Without further purification, 17 and 19 were subjected to methanolysis and hydrogenolysis to afford disulfated tetrasaccharides 4 (3.5 mg, 3.5 μmol, 14% over 14 steps; Figures S12, S26, and S27) and 6 (3.0 mg, 3.0 μmol, 12% over 14 steps; Figures S16, S30, and S31), which were both purified by reverse-phase HPLC using Hypercarb columns. Protected trisulfated tetrasaccharide 18 (21.9 mg, 10.1 μmol, 39% over 11 steps) and tetrasulfated tetrasaccharide 20 (19.9 mg, 9.5 μmol, 38% over 12 steps) were both purified by preparative reverse-phase HPLC. Deprotection of 18 and 20 afforded 5 (3.2 mg, 3.0 μmol, 30% over two steps; Figures S14, S28, and S29) and 7 (2.8 mg, 2.4 μmol, 25% over two steps; Figures S18, S32, and S33), respectively. The AGA approach described here allowed for the procurement of conjugation-ready sulfated KS tetrasaccharides within 3 days, including at most 16 hr required for automated synthesis. AGA uses excess of glycosyl building block, which resulted in 2.5–3 hr for glycosylation and Fmoc deprotection in total. With the optimized protocol, protected oligosaccharides 13, 14, 15, 17, and 19 were obtained without any notable side products, as judged by analytical HPLC, indicating high conversion rates at each step during AGA. Minor impurities observed in crude protected oligosaccharides 18 and 20 were removed by reverse-phase HPLC (RP-HPLC) before global deprotection. Fully deprotected oligosaccharides were obtained with two HPLC purification steps at most. The syntheses of sulfated tetrasaccharides provided proof of concept that AGA is able to rapidly generate KS oligosaccharides with the most common sulfation patterns from two orthogonally protected monosaccharide building blocks. For future syntheses, it would be an option to scale up synthesis of the common intermediate 16, followed by structural diversification on a smaller scale, which would contribute further to the rapid generation of sulfated oligosaccharide libraries. Additionally, different combinations of the four building blocks 9–12 would allow for the synthesis of monosulfated tetrasaccharides (five possible sulfation patterns), disulfated tetrasaccharides (ten possible sulfation patterns), trisulfated tetrasaccharides (six possible sulfation patterns), and one pentasulfated tetrasaccharide via the described AGA strategy. Custom glycan microarrays were fabricated by immobilization of RP-HPLC-purified oligosaccharides 1–7 via their primary amines on N-hydroxysuccinimide-ester-functionalized glass slides. The minor non-carbohydrate impurities from ion exchange and the resin observed in these preparations of 1–7 were not expected to influence glycan-binding signals. The microarrays were probed with fluorescence-labeled recombinant AAV particles representing serotypes AAV2, -7, -8, -rh10, and -12 to determine their binding specificities as previously described.21Mietzsch M. Broecker F. Reinhardt A. Seeberger P.H. Heilbronn R. Differential adeno-associated virus serotype-specific interactions patterns with synthetic heparins and other glycans.J. Virol. 2014; 88: 2991-3003Crossref PubMed Scopus (89) Google Scholar AAVrh10 particles selectively and dose-dependently recognized KS oligosaccharide 6 (Figures 3A and 3B ). Binding to 6 was serotype specific, given that the other AAVs did not recognize this glycan (Figure 3C). As observed before,21Mietzsch M. Broecker F. Reinhardt A. Seeberger P.H. Heilbronn R. Differential adeno-associated virus serotype-specific interactions patterns with synthetic heparins and other glycans.J. Virol. 2014; 88: 2991-3003Crossref PubMed Scopus (89) Google Scholar AAV2 bound to different heparin and heparan sulfate oligosaccharides (Figure S19). Successful spotting of LacNAc and KS glycans on the microarray slides was qualitatively verified with the Bandeiraea (Griffonia) simplicifolia lectin-I (GS-I), which recognizes the terminal Gal residues that are present in 1–7.54Wu A.M. Wu J.H. Chen Y.Y. Song S.C. Kabat E.A. Further characterization of the combining sites of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4).Glycobiology. 1999; 9: 1161-1170Crossref PubMed Scopus (31) Google Scholar The lectin bound to 1–7 and appeared to favor the non-sulfated (1–3) over the sulfated (4–7) glycans. However, weaker binding signals observed for 4–7 could also be a result of less efficient immobilization of these glycans to the microarray surface. The binding specificity of AAVrh10 observed by microarray was further studied by SPR using sensor chips functionalized with 5 or 6 (Figure 3D). SPR has been used before to characterize interactions of virus particles with their receptor molecules.55Alba R. Bradshaw A.C. Parker A.L. Bhella D. Waddington S.N. Nicklin S.A. van Rooijen N. Custers J. Goudsmit J. Barouch D.H. et al.Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer.Blood. 2009; 114: 965-971Crossref PubMed Scopus (151) Google Scholar, 56Meng B. Marriott A.C. Dimmock N.J. The receptor preference of influenza viruses.Influenza Other Respir. Viruses. 2010; 4: 147-153Crossref PubMed Scopus (25) Google Scholar Confirming the microarray studies, non-labeled AAVrh10 particles dose-dependently recognized 6 but not 5, which differs from 6 only by its sulfation pattern. We disclose the automated synthesis of LacNAc and KS glycans from monosaccharide building blocks carrying three orthogonal protecting groups. AGA provided access to diverse oligosaccharides with different sulfation patterns and varying lengths by sequential combination of building blocks 9–12 and selective deprotection reactions, followed by further chemical modification. Common tetrasaccharide 16 was transformed into four differently sulfated KS tetrasaccharides 4–7 by orthogonal modifications. KS tetrasaccharide 6 was identified as a specific interaction partner of AAVrh10 and represents a novel candidate glycan receptor of this virus. Of note, AAVrh10 has a distinct tropism for cells of the brain,57Hordeaux J. Dubreil L. Deniaud J. Iacobelli F. Moreau S. Ledevin M. Le Guiner C. Blouin V. Le Duff J. Mendes-Madeira A. et al.Efficient central nervous system AAVrh10-mediated intrathecal gene transfer in adult and neonate rats.Gene Ther. 2015; 22: 316-324Crossref PubMed Scopus (37) Google Scholar a tissue that is known to express KS.58Krusius T. Finne J. Margolis R.K. Margolis R.U. Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain.J. Biol. Chem. 1986; 261: 8237-8242Abstract Full Text PDF PubMed Google Scholar LacNAc and KS glycans, now available through AGA, will serve as molecular tools for investigating biological processes involving these structures. In a broader sense, this study highlights the power of AGA to provide access to versatile, conjugation-ready glycan probes suitable for biological interaction studies.