Automated solid phase synthesis enables rapid access to the linear and
branched arabinofuranoside oligosaccharides. A simple purification step is
sufficient to provide the conjugation ready oligosaccharides in good yield

Hurevich, Mattan

Kandasamy, Jeyakumar

Seeberger, Peter H.

Kandasamy, J.

Hurevich, M.

Seeberger, P.

MPG.PuRe

Automated solid phase synthesis of oligoarabinofuranosides

Institutional Repository of the Freie Universität Berlin

ISSN 1359-7345Chemical Communicationswww.rsc.org/chemcomm Volume 49 | Number 40 | 18 May 2013 | Pages 4427–45741359-7345(2013)49:40;1-4COMMUNICATIONPeter H. Seeberger et al.Automated solid phase synthesis of oligoarabinofuranosidesThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4453--4455 4453Cite this: Chem. Commun.,2013,49, 4453Automated solid phase synthesis ofoligoarabinofuranosides†Jeyakumar Kandasamy,za Mattan Hurevichza and Peter H. Seeberger*abAutomated solid phase synthesis enables rapid access to the linear andbranched arabinofuranoside oligosaccharides. A simple purificationstep is suﬃcient to provide the conjugation ready oligosaccharidesin good yield.Arabinogalactan (AG) and lipoarabinomannan (LAM) polysac-charides are the major components of mycobacterial cell envelopesand contain multiple arabinose residues.1 Arabinogalactan consistsof three identical oligoarabinosides attached to a galactofuranose.2–4Each oligoarabinoside contains arabinofuranose (Araf) residues,linked together via a-(1–5), a-(1–3) and b-(1–2) glycosidic linkages.Synthetic oligoarabinofuranosides are useful tools for studyingthe biosynthetic enzymes involved in the mycobacterial cell wallsynthesis.5–9 Access to various arabinofuranose fragments andanalogues that can be immobilized on surfaces of glycan arraysand particles is instrumental in identifying potential ligands forthese enzymes and developing biological assays to study theirsubstrate specificities.10 Several solution phase strategies forsynthesis of arabinofuranosidic oligomers were established.11–17The groups of Lowary and Ito have accomplished the synthesis ofthe arabinan dodecasaccharide via a convergent fragmentapproach.18,19 These syntheses are challenging because theyrequire many discrete operations and multiple purification steps.Automated solid phase synthesis significantly reduces thetime-requirements and enables rapid access to structurally-defined oligosaccharides,20–24 as well as conjugation readyoligosaccharide analogues.25,26 A combination of new technologiesand synthetic methods was developed and adjusted to keepthe automation technique up to date with the most recentdevelopments in carbohydrate chemistry.20–26 The synthesis ofoligosaccharides such as Lewis X and Globo H demonstrated thatcomplex carbohydrates as well as challenging glycosidic linkagescan be accessed using an oligosaccharide synthesizer.21,22 To date,no automated oligofuranoside syntheses have been reported. Here,we describe the first automated synthesis of linear and branchedoligoarabinofuranosides (Fig. 1).The simple linear (1, 2) and branched (3, 4) oligoarabinofur-anoside targets were pursued in order to test the ability of theautomated route to produce both linear and branched structures.Linear a-D-(1–5)-Araf 1 and 2 have been prepared via iterativeglycosylations using arabinose building block 7. Thioglycoside 7was designed according to the requirements of the automatedapproach, with 9-fluorenylmethoxycarbonyl (Fmoc) as a temporaryprotecting group at C-5 and benzoates at C-2 andC-3 as participatingprotecting groups that are readily removed during cleavage from thesolid support. The synthesis of thioglycoside 7 was accomplished inthree steps starting from thioglycoside 5.27,28 Regioselective tritylprotection of the primary hydroxyl in 5 followed by the benzoylprotection of remaining hydroxyls and the removal of the trityl groupfurnished thioglycoside 6 (Scheme 1). Fmoc protection of the C-5hydroxyl produced the desired thioglycoside building block 7 inhigh yield.Linear a-D-(1–5)-Araf oligosaccharides 1 and 2 were assembledusing an oligosaccharide synthesizer (Scheme 2).25 Glycosylationswere carried out at temperatures between 40 1C and 20 1Cusing thioglycoside 7 and a mixture of N-iodosuccinimide (NIS)and trifluoromethanesulfonic acid (TfOH). Each cycle involvedtwo additions of a glycosylating agent (double coupling), usingfive equivalents of the building block. Removal of the Fmoc groupwith piperidine uncovered the hydroxyl group ready for elonga-tion. The glycosylation–deprotection cycle was repeated twice forthe synthesis of disaccharide 1 and six times for the preparationof hexasaccharide 2. Following oligosaccharide assembly on thesolid support, cleavage from the resin using sodium methoxidesolution furnished disaccharide 8 and hexasaccharide 9 thatwere purified by reverse phase preparative HPLC. Hydrogenolysisof 8 and 9 followed by a simple Sep-Pak purification aﬀordeddisaccharide 1 and hexasaccharide 2 in overall yield of 85% and65%, respectively, based on resin loading.a Department of Biomolecular Systems, Max-Planck-Institute of Colloids andInterfaces, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germanyb Freie Universita¨t Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, Germany. E-mail: peter.seeberger@mpikg.mpg.de;Fax: +49 331 567 9302; Tel: +49 331 567 9301† Electronic supplementary information (ESI) available: Experimental proce-dures, spectral (1H, 13C NMR and HSQC) and analytical (HPLC and MS) data.See DOI: 10.1039/c3cc00042g‡ Both authors contributed equally to this work.Received 3rd January 2013,Accepted 21st January 2013DOI: 10.1039/c3cc00042gwww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 22 January 2013. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:54:24. View Article OnlineView Journal  | View Issue4454 Chem. Commun., 2013, 49, 4453--4455 This journal is c The Royal Society of Chemistry 2013In addition to the formation of a-(1–5) linkages, the synth-esis of branched oligoarabinofuranosides 3 and 4 requiresintroduction of a-(1–3) branching. For this purpose buildingblock 11 was used. Featuring two Fmoc groups, building block11 enables the removal of temporary protection and glycosyla-tion of both C-3 and C-5 hydroxyls in a single deprotection–glycosylation cycle. En route to thioglycoside 11, thioglycoside 5was converted to silyl acetal 10 in two steps (Scheme 3).Selective removal of the silyl acetal followed by the Fmocprotection of the resulting diol provided building block 11 in85% yield.Upon loading the resin with building block 11 using aglycosylation–deprotection cycle, the first elongation in thesynthesis of oligoarabinofuranoside 3 was carried out withthioglycoside 7 using conditions established for the synthesisof 1 and 2 (Scheme 4). The following glycosylation to create thea-(1–5) linkage and install a-(1–3) branching was performedusing thioglycoside 7. Removal of Fmoc groups and cleavagefrom the resin yielded crude trisaccharide 12. A simple purifica-tion protocol involving adsorption on a pad of silica gel followedby a hexane and CH2Cl2 wash and elution with a mixture ofCH2Cl2 : MeOH was suﬃcient to obtain pure trisaccharide 12.Hydrogenolysis of partially protected trisaccharide 12 gavedeprotected trisaccharide 3 in 78% overall yield based on resinloading, without the need for further purification.The automated synthesis of branched hexasaccharide 4 wasaccomplished via four glycosylation cycles using building blocksFig. 1 Structures and retrosynthetic analysis of a-D-(1–5), a-D-(1–3) oligoarabinofuranosides 1–4.Scheme 1 Synthesis of 7. Reagents and conditions: (a) (i) TrCl, Py; then BzCl;(ii) PTSA, MeOH, 77% (two steps); (b) FmocCl, Py, 96%.Scheme 2 Automated synthesis of oligosaccharides 1 and 2. Reagents andconditions: (a) 7, NIS/TfOH, 40 1C to 20 1C; (b) 20% piperidine in DMF;(c) NaOMe in MeOH; (d) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.Scheme 3 Synthesis of building block 11. Reagents and conditions: (a) t-Bu2Si-(OTf)2, 2,6-lutidine, 60%; (b) BzCl, Py, 89%; (c) (i) HFPy, THF; (ii) FmocCl, Py, 85%(two steps).Scheme 4 Automated synthesis of branched trisaccharide 3. Reagents andconditions: (a) 11, NIS/TfOH, 40 1C to 20 1C, repeated twice; (b) 20%piperidine in DMF; (c) 7, NIS/TfOH, 40 1C to 20 1C, repeated four times;(d) NaOMe in MeOH; (e) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.Communication ChemCommPublished on 22 January 2013. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:54:24. View Article OnlineThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4453--4455 44557 and 11 (Scheme 5). The automated synthesis of hexasaccharide13 required about 14 hours. Partially protected hexasaccharide13 was purified by passing the crude mixture over a silica pad.Hydrogenolysis provided the amine functionalized hexasaccharide4 in 63% overall yield based on resin loading.Automated solid phase oligosaccharide synthesis drasticallyreduces the overall synthesis time from the building block tothe final compound. Table 1 provides an overview of the timerequired for the synthesis of oligosaccharides 1–4. The overalltime to reach branched hexamer 4 was slightly shorter thanthat for hexamer 2 owing to the simplified purification protocol(Table 1, step III).In conclusion, linear and branched conjugation-ready oli-goarabinofuranosides 1–4 were assembled via automated solidphase synthesis. This robust and reproducible route constitutesthe first automated solid phase synthesis of oligofuranosides.This method will provide straightforward access to a largenumber of diﬀerent arabinogalactan fragments for biologicalstudies.We acknowledge the Max-Planck Society and the Swiss NationalScience Foundation (200020-117889) as well as a Korber Prize(to P. H. Seeberger) for generous financial support. M.H. thanksthe Max-Planck Society for a Minerva postdoctoral fellowship. Wethank Dr I. Vilotijevic, Dr C. Rademacher, Dr S. G. Parameswarappa,Dr J. Hudon, Dr C. Anish, Dr C. L. Pereira and Mr H. S. Hahm fortheir help in editing this paper. Cover image courtesy of scientificillustrator, Melanie Burger (melanie.burger@mail.utoronto.ca).Notes and references1 P. J. Brennan and H. Nikaido, Annu. Rev. Biochem., 1995, 64, 29–63.2 G. S. Besra, K. H. Khoo, M. R. Mcneil, A. Dell, H. R. Morris andP. J. Brennan, Biochemistry, 1995, 34, 4257–4266.3 L. B. Shi, S. Berg, A. Lee, J. S. Spencer, J. 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Soc.,2007, 129, 2770–2771.23 M. T. C. Walvoort, H. van den Elst, O. J. Plante, L. Kro¨ck,P. H. Seeberger, H. S. Overkleeft, G. A. van der Marel and J. D. C.Code´e, Angew. Chem., Int. Ed., 2012, 51, 4393–4396.24 M. T. C. Walvoort, A. G. Volbeda, N. R. M. Reintjens, H. van den Elst,O. J. Plante, H. S. Overkleeft, G. A. van der Marel and J. D. C. Code´e,Org. Lett., 2012, 14, 3776–3779.25 L. Kro¨ck, D. Esposito, B. Castagner, C. C. Wang, P. Bindscha¨dler andP. H. Seeberger, Chem. Sci., 2012, 3, 1617–1622.26 D. Esposito, M. Hurevich, B. Castagner, C. C. Wang andP. H. Seeberger, Beilstein J. Org. Chem., 2012, 8, 1601–1609.27 C. S. Callam and T. L. Lowary, J. Chem. Educ., 2001, 78, 73–74.28 D. J. Hou, H. A. Taha and T. L. Lowary, J. Am. Chem. Soc., 2009, 131,12937–12948.Scheme 5 Automated synthesis of branched hexasaccharide 4. Reagents and con-ditions: (a) 7, NIS/TfOH,40 1C to20 1C, repeated twice; (b) 20% piperidine in DMF;(c) 11, NIS/TfOH,40 1C to20 1C, repeated twice; (d) 7, NIS/TfOH,40 1C to20 1C,repeated four times; (e) NaOMe in MeOH; (f) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.Table 1 Time required for the synthesis of oligosaccharides 1–4aCompoundStepsTotal time (h) Overall yield (%)I II III IV V1 6 6 6 16 3 37 852 16 6 6 16 3 47 653 8 6 3 16 3 36 784 14 6 3 16 3 42 63a Time given in hours for all steps, (I) automated synthesis (glycosylation–deprotection); (II) cleavage from resin (NaOMe); (III) first purificationstep (HPLC or silica pad); (IV) global deprotection (Pd/C, H2); (V) finalpurification (Sep-Pak, optional).ChemComm CommunicationPublished on 22 January 2013. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:54:24. View Article Online

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Automated solid phase synthesis of oligoarabinofuranosides

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