We report the automated glycan assembly of oligosaccharide fragments related
to the hemicellulose xyloglucan (XG). Iterative addition of monosaccharide and
disaccharide building blocks to a solid support provided seven cellulose and
xyloglucan fragments including XXGG- and XXXG-type oligosaccharides

Dallabernardina, Pietro

Pfrengle, Fabian

Schuhmacher, Frank

Seeberger, Peter H.

Institutional Repository of the Freie Universität Berlin

Organic &Biomolecular ChemistryPAPERCite this: Org. Biomol. Chem., 2016,14, 309Received 28th October 2015,Accepted 3rd November 2015DOI: 10.1039/c5ob02226fwww.rsc.org/obcAutomated glycan assembly of xyloglucanoligosaccharides†Pietro Dallabernardina,a,b Frank Schuhmacher,a,b Peter H. Seebergera,b andFabian Pfrengle*a,bWe report the automated glycan assembly of oligosaccharide fragments related to the hemicellulosexyloglucan (XG). Iterative addition of monosaccharide and disaccharide building blocks to a solid supportprovided seven cellulose and xyloglucan fragments including XXGG- and XXXG-type oligosaccharides.IntroductionXyloglucans (XGs) are the most prevalent class of hemicellu-loses in the cell wall of higher plants.1 They are constructedfrom a cellulosic backbone consisting of β-(1→4)-linked D-glu-copyranoses highly substituted with α-D-xylopyranose residuesin the C6 position of the glucose units.2 Further monosacchar-ides such as galactose and fucose may be attached to thexylose substituents. Generally, xyloglucans occur in two majorforms. While the XXGG-type consists of two xylose-substitutedglucose moieties separated by two unsubstituted glucose resi-dues, in the XXXG-type three xylose-substituted residues areseparated by only one unsubstituted glucose (Fig. 1b).1,3Despite the structural diversity found in diﬀerent species,the functions of xyloglucan in plant growth and developmentare conserved among all species of flowering plants.4 Xyloglu-cans are strongly bonded through non-covalent interactions tothe cellulose microfibrils in the cell wall (Fig. 1a).2 These inter-actions are not only essential for the structural capacity of thecell wall but also its flexibility during synthesis.2,4–7 Xyloglu-cans store energy6 and are signaling molecules.4Homogeneous, well-defined oligosaccharide samplesobtained by chemical synthesis are essential molecular toolsfor investigating the structure and function of xyloglucans.Few chemical syntheses of XG oligosaccharides have beenreported.8–11 The multi-step total synthesis of an XG-derivednonasaccharide9 relied on a late-stage introduction of a Fuc-Gal-Xyl side chain. Large defined xyloglucan fragments have alsobeen prepared by enzymatic coupling of XG oligosaccharidesobtained from enzymatic degradation of xyloglucan polymers.12This approach is limited only in the diversity of the productsdue to restricted accessibility of the oligosaccharide buildingblocks. Recently, automated glycan assembly13–15 has emergedas a powerful tool for the facile and expedient generation ofplant carbohydrate libraries.16,17 Here, we report the automatedsynthesis of seven cellulose and XG oligosaccharides based onthe linear assembly of the glycans on solid support using mono-saccharide and disaccharide building blocks (BB).Results and discussionInitially we investigated the possibility that XG oligosacchar-ides could be assembled exclusively from monosaccharideFig. 1 (a) The hemicellulose xyloglucan interconnects cellulosemicroﬁbrils through hydrogen bonding; (b) XXXG-type of XGoligosaccharides.†Electronic supplementary information (ESI) available: Experimental proceduresfor solution- and automated solid-phase reactions and characterization data forall compounds. See DOI: 10.1039/c5ob02226faDepartment of Biomolecular Systems, Max-Planck-Institute of Colloids andInterfaces, Am Mühlenberg 1, 14476 Potsdam, GermanybFreie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, Germany. E-mail: Fabian.Pfrengle@mpikg.mpg.deThis journal is © The Royal Society of Chemistry 2016 Org. Biomol. Chem., 2016, 14, 309–313 | 309Open Access Article. Published on 03 November 2015. Downloaded on 28/09/2016 12:33:15.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueBBs. Two glucose BBs (1 and 2) and one xylose BB (3) weredesigned for automated synthesis. Glucose BBs 1 and 2 wereequipped in the C2 position with benzoyl esters (Bz) as partici-pating protecting groups to favour the formation of β-linkages.Fluorenylmethoxycarbonyl (Fmoc) and levulinoyl (Lev) wereused as temporary protecting groups to enable either elonga-tion of the β-1,4-glucan backbone or substitution of the 6-posi-tion with xylose. All remaining positions were permanentlyprotected as benzyl ethers. Both building blocks contain phos-phate leaving groups to enable smooth chain elongation.18,19Xylose BB 3 was protected in all positions with benzyl groupsand used directly as the thioglycoside.With BBs 1–3 in hand,20–23 we explored their potential forthe automated glycan assembly of trisaccharide 5 (Scheme 1a).TMSOTf-promoted glycosylation of linker-functionalized resin4 with two times 3.7 equivalents BB 1 followed by Fmoc-de-protection and another glycosylation with BB 2 provided thedisaccharide for installation of the α-xyloside. After removal ofthe Lev-group we investigated the NIS/TfOH-promoted glycosy-lation of the second glucose unit with BB 3. At temperatures aslow as −35 °C, full conversion was achieved and the desired tri-saccharide obtained in 29% yield after cleavage of the photo-labile linker in a commercial flow photoreactor.17 Unfortu-nately the reaction resulted in a mixture of α/β-isomers with arelative ratio of 1 : 1 (Scheme 1b). The two stereoisomers wereunambiguously assigned by MALDI mass spectrometry andNMR-spectroscopy (Scheme 1c/d). With less of the desiredα-isomer being obtained than in similar solution–phase reac-tions,12 and since multiple α-xylosides have to be installed inthe synthesis of larger XG oligosaccharides, BB 3 was deemednot applicable for our purposes.Based on this result we decided to employ a disaccharideBB containing a preinstalled α-xylosidic linkage. We envi-sioned that the synthesis of disaccharide BB 10 would notrequire more steps than the synthesis of BB 2 (Scheme 2).Glucose derivative 7 was selectively glycosylated with xylopyra-nose imidate 6 to provide disaccharide 8 as the major product.The transformation proceeded in rather low yield due to theformation of the β isomer and the bis-glycosylated compoundsas side products.12 Subsequently, the C4-hydroxyl of glucosewas protected as Lev-ester and resulting thioglycoside 9 con-verted into corresponding phosphate 10. Installation of anFmoc-group at C4 failed due to steric hinderance.With disaccharide BB 10 in hand, xyloglucan fragmentswere produced using an automated oligosaccharide synthesi-zer (Scheme 3). Disaccharides had been successfully usedduring automated glycan assembly previously and iterativeScheme 2 Synthesis of disaccharide BB 10. Reagents and conditions:(a) TMSOTf, CH2Cl2, −78 °C → −10 °C, 39%; (b) LevOH, DIC, DMAP,CH2Cl2, 84%; (c) HOP(O)(OBu)2, NIS, TfOH, CH2Cl2, 0 °C, 57%.Scheme 1 (a) Automated glycan assembly of trisaccharide 5. Reagents and conditions: 2 × 3.7 equiv. BB 1 or BB 2, TMSOTf, CH2Cl2, −30 °C (5 min)or −35 °C (5 min) → −15 °C (30 min) (Module A); 3 cycles of 20% Et3N in DMF, 25 °C (5 min) (Module B); N2H4·OAc (155 mM) in pyridine/AcOH/H2O4 : 1 : 0.25, 25 °C (30 min) (Module C); 2 × 3.7 equiv. BB 3 NIS/TfOH, CH2Cl2/dioxane 2 : 1, −55 °C (5 min) → −35 °C (40 min) (Module D); (b) HPLC-chromatogram of the crude fully protected anomeric mixture; (c) MALDI-TOF analysis after separation of the diastereomers by preparative HPLC; (d)HSQC of the anomeric region of the two isolated stereoisomers.Paper Organic & Biomolecular Chemistry310 | Org. Biomol. Chem., 2016, 14, 309–313 This journal is © The Royal Society of Chemistry 2016Open Access Article. Published on 03 November 2015. Downloaded on 28/09/2016 12:33:15.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineaddition of BBs 1 and 10 to linker-functionalized resin 4 pro-vided the protected form of 13 with full conversion after cleav-age of the oligosaccharide from the solid support.24 However,oligosaccharides containing two α-xyloside substituents wereformed with reduced eﬃciency and the desired products wereaccompanied by deletion sequences missing one of the disac-charide units. Still, fully deprotected compounds 14, 16 and 17were isolated in overall yields of 9–16% after cleavage from thesolid support, treatment with sodium methoxide and a finalhydrogenation reaction. Octasaccharide 15 containing threedisaccharide units was assembled in 2% overall yield whenusing three instead of two times 3.7 equivalents BB 10 for thetwo key glycosylations. Due to steric hindrance the assembly ofchains containing more than three disaccharide units is notpractical. To complete the set of molecular tools for xyloglucanbiology, two cellulose fragments (11, 12) were prepared. Coup-lings using 3.7 equivalents of BB 1 just once were suﬃcient toensure complete glycosylations.ConclusionsIn conclusion, seven oligosaccharides related to cellulose andxyloglucan were prepared by automated glycan assembly. Theuse of a disaccharide BB enabled the synthesis of oligosacchar-ides containing up to three α-xylosidic linkages. These XGglycans will be used as molecular probes to investigate thespecificities of xyloglucan-modifying enzymes and cell wallglycan-directed antibodies.Scheme 3 Automated glycan assembly of XG oligosaccharides. Reagents and conditions: (a) 1 or 2 × 3.7 equiv. BB 1, TMSOTf, CH2Cl2, −30 °C(5 min) → −15 °C (30 min) (Module A); (b) 2 or 3 × 3.7 equiv. BB 10, TMSOTf, CH2Cl2, −35 °C (5 min) → −15 °C (35 min) (Module A); (c) 3 cycles of20% NEt3 in DMF, 25 °C (5 min) (Module B); (d) N2H4·OAc (155 mM) in pyridine/AcOH/H2O 4 : 1 : 0.25, 25 °C (30 min) (Module C); (e) CH2Cl2, hν(305 nm); (f ) NaOMe, THF/MeOH, 12 h; (g) H2, Pd/C, EtOAc/MeOH/H2O/HOAc, 12 h. 11: 28%; 12: 23%; 13: 15%; 14: 16%; 15: 2%; 16: 10%; 17: 9%.The letter code below the structures refers to a common nomenclature of XG oligosaccharides.25Organic & Biomolecular Chemistry PaperThis journal is © The Royal Society of Chemistry 2016 Org. Biomol. Chem., 2016, 14, 309–313 | 311Open Access Article. Published on 03 November 2015. Downloaded on 28/09/2016 12:33:15.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineExperimental sectionSynthesizer modules and conditionsLinker-functionalized resin 4 (16.9 μmol of hydroxyl groups)was placed in the reaction vessel of the automated oligosac-charide synthesizer and swollen for at least 30 min in DCM.Before every synthesis the resin was washed with DMF, THFand DCM. Subsequently the glycosylation (Module A and D)and deprotection (Module B and C) steps were performed.Mixing of the components was accomplished by bubblingArgon through the reaction mixture.Module A: glycosylation with glycosyl phosphatesThe resin (16.9 μmol of hydroxyl groups) was swollen in DCM(2 mL) and the temperature of the reaction vessel was adjustedto −30 °C. Prior to the glycosylation reaction the resin waswashed with TMSOTf in DCM and then DCM only. For the gly-cosylation reaction the DCM was drained and a solution ofphosphate BB (3.7 equiv. in 1 mL DCM) was delivered to thereaction vessel. After the set temperature was reached, the reac-tion was started by the addition of TMSOTf in DCM (3.7 equiv.in 1 mL DCM). The glycosylation was performed for 5 min at−30 °C or −35 °C and then at −15 °C for 30 or 35 minutes.Subsequently the solution was drained and the resin waswashed three times with DCM. The whole procedure was per-formed once, twice or three times depending on the conver-sion of the acceptor sites. Afterwards the resin was washedthree times with DCM at 25 °C.Module B: Fmoc deprotectionThe resin was washed with DMF, swollen in 2 mL DMF andthe temperature of the reaction vessel was adjusted to 25 °C.Prior to the deprotection step the DMF was drained and theresin was washed with DMF three times. For Fmoc de-protection 2 mL of a solution of 20% Et3N in DMF was deli-vered to the reaction vessel. After 5 min the solution wasdrained and the whole procedure was repeated another twotimes. After Fmoc deprotection was complete the resin waswashed with DMF, THF and DCM.Module C: Lev deprotectionPrior to the deprotection step the resin was washed with DCMthree times, swollen in 1.3 mL DCM and the temperature of thereaction vessel was adjusted to 25 °C. For Lev deprotection0.8 mL of a solution of 150 mM N2H4·AcOH in Pyridine/AcOH/H2O 4 : 1 : 0.25 was delivered to the reaction vessel. After 30 minthe solution was drained and the deprotection step was repeatedtwo times. After Lev deprotection was complete the resin waswashed with DCM, DMF, THF and again DCM three times each.Module D: glycosylation with thioglycosidesThe resin (16.9 μmol of hydroxyl groups) was swollen in DCM(2 mL) and the temperature of the reaction vessel was adjustedto −30 °C. Prior to the glycosylation reaction the resin waswashed with TMSOTf in DCM and DCM. For the glycosylationreaction the DCM was drained and a solution of the thioglyco-side BB (3.7 equiv. in 1 mL DCM) was delivered to the reactionvessel. After the set temperature was reached, the reaction wasstarted by the addition of NIS (4.44 equiv.) and TfOH(0.44 equiv.) in 1 mL DCM/dioxane (2 : 1). The glycosylationwas performed for 5 min at −55 °C and then for 40 min at−30 °C. Subsequently the solution was drained and the resinwas washed with DCM. The whole procedure was repeatedonce to ensure full conversion of all acceptor sites. Afterwardsthe resin was washed three times with DCM at 25 °C.Cleavage from the solid supportAfter assembly of the oligosaccharides cleavage from the solidsupport was accomplished by modification of a previouslypublished protocol,18 using the Vapourtec E-Series UV-150photoreactor Flow Chemistry System. The medium pressuremetal halide lamp is filtered using the commercially availablered filter. The resin, suspended in DCM, was loaded into aplastic syringe. The suspension was then pumped using asyringe pump (PHD2000, Harvard Aparatus) at 1 mL min−1through a 10 mL reactor, constructed of 1/8 inch o.d. FEPtubing. The total volume within the photoreactor was 9 mL.The temperature of the photoreactor was maintained at 20 °Cand the lamp power was 80%. The exiting flow was depositedin a 10 mL syringe containing a filter, with a collection flaskbeneath the syringe.Global deprotection of the assembled oligosaccharidesThe protected oligosaccharide was dissolved in THF (3 mL)and NaOMe (0.5 M in MeOH, 0.5 mL) was added. The reactionmixture was stirred overnight and subsequently neutralized byaddition of prewashed Amberlite IR-120 resin. The resin wasfiltered oﬀ and the solvents were removed in vacuo. The crudeproduct was purified by preparative HPLC, dissolved in amixture of EtOAc/MeOH/AcOH/H2O (4 : 2 : 2 : 1, 3 mL) and theresulting solution was added to a round-bottom flask contain-ing Pd/C (10% Pd, 10–20 mg). The suspension was saturatedwith H2 for 30 min and stirred under an H2-atmosphere over-night. After filtration of the reaction mixture through a syringefilter the solvents were evaporated to provide the fully depro-tected oligosaccharide.AcknowledgementsWe gratefully acknowledge financial support from the MaxPlanck Society, the German Research Foundation (DFG, EmmyNoether program PF850/1-1 to FP), the Fonds der chemischenIndustrie (Liebig-fellowship to FP) and an ERC AdvancedGrant (AUTOHEPARIN to PHS). We thank Dr K. Gilmore forcritically proofreading this manuscript.Notes and references1 A. Ebringerova, Z. Hromadkova and T. Heinze, Adv. Polym.Sci., 2005, 186, 1–67.Paper Organic & Biomolecular Chemistry312 | Org. Biomol. 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Brasch, D. S. Larsen, L. D. Melton andJ. Simpson, Carbohydr. Res., 1996, 286, 1–15.12 R. Faurè, M. Saura-Valls, H. Brumer, A. Planas, S. Cottazand H. Driguez, J. Org. Chem., 2006, 71, 5151–5161.13 L. Kröck, D. Esposito, B. Castagner, C.-C. Wang,P. Bindschädler and P. H. Seeberger, Chem. Sci., 2012, 3,1617–1622.14 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science,2001, 291, 1523–1527.15 P. H. Seeberger, Acc. Chem. Res., 2015, 48, 1450–1463.16 D. Schmidt, F. Schumacher, A. Geissner, P. H. Seebergerand F. Pfrengle, Chem. – Eur. J., 2015, 21, 5709–5713.17 M. P. Bartetzko, F. Schumacher, H. S. Hahm,P. H. Seeberger and F. Pfrengle, Org. Lett., 2015, 17, 4344–4347.18 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger,Angew. Chem., Int. Ed., 2013, 52, 5858–5861.19 O. J. Plante, R. B. Andrade and P. H. Seeberger, Org. Lett.,1999, 1, 211–214.20 S. David, A. Malleron and C. Dini, Carbohydr. Res., 1989,188, 193–200.21 T. Polat and C.-H. Wong, J. Am. Chem. 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Automated glycan assembly of xyloglucan oligosaccharides

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Automated glycan assembly of xyloglucan oligosaccharides

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