Dermatan sulfates are glycosaminoglycan polysaccharides that serve a multitude
of biological roles as part of the extracellular matrix. Orthogonally
protected D-galactosamine and L-iduronic acid building blocks and a photo-
cleavable linker are instrumental for the automated synthesis of dermatan
sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in
good yield

Hahm, Heung Sik

Kandasamy, Jeyakumar

Klein, James C.

Schuhmacher, Frank

Seeberger, Peter H.

Institutional Repository of the Freie Universität Berlin

This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 1875--1877 | 1875Cite this:Chem. Commun., 2014,50, 1875Modular automated solid phase synthesis ofdermatan sulfate oligosaccharides†Jeyakumar Kandasamy,a Frank Schuhmacher,ab Heung Sik Hahm,ab James C. Kleinaand Peter H. Seeberger*abDermatan sulfates are glycosaminoglycan polysaccharides that servea multitude of biological roles as part of the extracellular matrix.Orthogonally protected D-galactosamine and L-iduronic acid buildingblocks and a photo-cleavable linker are instrumental for the auto-mated synthesis of dermatan sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in good yield.Dermatan sulfate is a glycosaminoglycan (GAG) predominantlyfound in skin and present in many mammalian tissues.1 It iscomposed of disaccharide repeating units consisting of N-acetyl-D-galactosamine (GalNAc) and L-iduronic acid (IdoA). These alternatingdisaccharide units can be variably O-sulfated at the C-4 and C-6positions in GalNAc, and the C-2 position in IdoA.2 Depending onthe source, 63–97% of the dermatan sulfate polymer is made upof IdoA - GalNAc4SO3 disaccharide repeating units that areconsidered to be characteristic of dermatan sulfate (Fig. 1).3Dermatan sulfate binds to a variety of proteoglycans andmodulates various biological processes such as coagulation, angio-genesis, tumor migration and growth factor expression.4,5 Specificbiological roles of dermatan sulfate are poorly understood dueto the variations and heterogeneity in its polymer structure.6,7In order to establish structure–activity relationships for dermatansulfate sequences, to correlate specific sequences and sulfationpatterns with protein binding and specific biological activitiesaccess to structurally defined dermatan sulfate oligosaccharidesis required. Homogeneous samples of such fragments are acces-sible only via chemical synthesis.To date, syntheses of dermatan sulfate oligosaccharides have beenexecuted in solution phase relying on many manual operations.8–12With appropriate building blocks in hand, automated solid phasesynthesis enables rapid access to structurally-defined oligo-saccharides.13,14 Automated protocols for the synthesis of otherglycosaminoglycans such as hyaluronan15 and chondroitin sulfatehave been reported previously.16 Here, we describe the automatedsolid phase synthesis of conjugation ready dermatan sulfate oligo-saccharides aimed at developing additional tools for biologicalstudies of GAGs.The dermatan sulfate backbone may arise via assembly oforthogonally protected galactosamine (GalN) and iduronic acidbuilding blocks (1 and 2, Fig. 1) designed for automated solidphase synthesis on solid supported photo-cleavable linker 3.16The C-3 position of GalN and the C-4 position of IdoA are maskedFig. 1 Structure of dermatan sulfate oligosaccharides and retrosyntheticanalysis.a Department of Biomolecular Systems, Max-Planck-Institute of Colloids andInterfaces, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany.E-mail: peter.seeberger@mpikg.mpg.de; Fax: +49 30 838-59302;Tel: +49 30 838-59301b Freie Universita¨t Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, Germany† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc48860hReceived 20th November 2013,Accepted 24th December 2013DOI: 10.1039/c3cc48860hwww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 03 January 2014. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:01:31. View Article OnlineView Journal  | View Issue1876 | Chem. Commun., 2014, 50, 1875--1877 This journal is©The Royal Society of Chemistry 2014with the temporary 9-fluorenylmethoxycarbonyl (Fmoc) protectinggroup in anticipation of the regiospecific elongation of the oligo-saccharide chain. The C-4 position of GalN carries a levulinoyl ester(Lev) that can be selectively cleaved under mild conditions to allowfor sulfation of the free hydroxyl group. The GalN amino group wasprotected as trichloroacetamide to ensure good b-selectivities inglycosylation reactions and allow for the installation of the desiredacetamide by the direct reduction at the late stage of the synthesis.The C-2 hydroxyl group of the IdoA building block was protected as abenzoate ester to ensure a-selectivity in glycosylation reactions. Theremaining hydroxyl groups that were not modified during theassembly were permanently protected as benzyl ethers.Owing to the high cost of galactosamine, these building blocksare accessed from galactose via formation of galactal followed byazido-selenation16,17 or from glucosamine via epimerization of theC-4 position.8,9 Taking the latter approach into account, we pre-pared building block 1 in nine steps from glucosamine via migra-tion of a-ester with epimerization as the key step (Scheme 1).18,19Thioglycoside 11 was synthesized in four steps from glucosaminehydrochloride (see ESI†).20 Levulination of the free hydroxyl groupin 11 followed by regioselective ring opening of benzylidine acetalusing triethylsilane/trifluoroacetic acid yielded thioglycoside 12 in70% yield over two steps. Migration of the C-3 Lev ester to the C-4hydroxyl with epimerization was eﬀected by transforming the freehydroxyl group in 12 to the corresponding triflate to producegalactose thioglycoside 13 in 78% yield. Fmoc protection of thefree hydroxyl group in 13 followed by the conversion of thioglyco-side to glycosyl phosphate using dibutyl hydrogen phosphate andN-iodosuccinimide–triflic acid yielded differentially protected build-ing block 1 in 86% yield.Synthesis of IdoA building block 2 was achieved in five stepsfrom thioglycoside 1421 (Scheme 2). Tetrakis(triphenylphosphine)-palladium(0) was employed for the deprotection of the allyl groupin thioglycoside 14 and the resulting hydroxyl group was protectedas a benzoyl ester to obtain thioglycoside 15 in 89% yield (over twosteps). Removal of the naphthyl group in 15 using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) followed by Fmoc protection ofthe resulting alcohol provided thioglycoside 16 in 82% overall yield.Thioglycoside 16 was then converted to the correspondingphosphate glycoside 2 in 92% yield.The simple non-sulfated dermatan oligosaccharides werepursued first in order to test the ability of the automated routeto deliver the glycan backbone without deletion sequences. Di- andtetra-saccharides 4 and 5 were prepared using the oligosaccharidesynthesizer via iterative glycosylations with building blocks 1 and 2and linker 3 (Scheme 3). Glycosylations were carried out at tem-peratures between 10 1C and 0 1C using trimethylsilyl trifluoro-methanesulfonate (TMSOTf). Each cycle involved addition of aglycosylating agent (double coupling) twice, using five equivalentsof building blocks. Removal of the Fmoc protecting group withtriethylamine uncovered the hydroxyl group ready for elongation.Synthesis of disaccharide 4 commencedwith the first glycosylation–deprotection cycle using building block 1 followed by the secondglycosylation–deprotection cycle using building block 2. The resultingfree hydroxyl group on the terminal sugar was then acetylated on thesynthesizer employing acetic anhydride in pyridine. Disaccharide 4wasreleased from the resin by applying UV light using a photo flowreactor.16 The crude product was purified by HPLC to obtain 4 in 66%overall yield for six steps based on resin loading. Tetrasaccharide 5was assembled similarly via four glycosylation–deprotection cyclesby alternating use of building blocks 1 and 2, followed by theacetylation of the free hydroxyl group on the terminal sugar. Uponcleavage from the resin, the crude product was purified by HPLC toobtain 6 in 28% overall yield (ten steps). No deletion sequences wereobserved in the automated synthesis of 4 and 5 as judged by HPLC.Automated synthesis of sulfated dermatan oligosaccharidesrequired two additional steps for installation of sulfates: selectiveremoval of the Lev protecting groups and sulfation of the resultinghydroxyl groups. A solution of hydrazine acetate in pyridine–aceticacid effected removal of the Lev groups at 25 1C. The pyridine sulfurtrioxide complex in dimethylformamide–pyridine was used for thesulfation of free hydroxyl groups at 50 1C on the synthesizer.16Synthesis of sulfated monosaccharide 6 and disaccharide 7 wasachieved on a solid support in five and seven automated steps,respectively, under glycosylation–deprotection conditions estab-lished for compounds 4 and 5, followed by automated acetylation,Lev deprotection and sulfation as described above. After the cleavageof the photo-sensitive linker, crude products were analysed byreverse phase (RP)-HPLC and purified by size exclusion chromato-graphy (Sephadex LH-20) to obtain monosaccharide 6 in 43% anddisaccharide 7 in 32% overall yield based on resin loading.Automated assembly of sulfated tetrasaccharide 8 under theconditions established for the synthesis of 6 and 7 yielded aScheme 1 Synthesis of galactosamine building block 1. Reagents and condi-tions: (a) Lev2O, Py, 91%. (b) Et3SiH, TFA, MS (4 Å), 77%. (c) Tf2O, Py, 10 1C,15 minutes; addition of H2O, reflux atB80 1C for 5 h, 78%. (d) FmocCl, Py, 93%.(e) Dibutyl hydrogen phosphate, NIS-TfOH, MS (4 Å), 93%.Scheme 2 Synthesis of iduronic acid building block 2. Reagents andconditions: (a) Pd(Ph3)4, 1,3-dimethylbarbituric acid, MeOH, 91%. (b) BzCl,Py, 98%. (c) DDQ, MeOH, 88%. (d) FmocCl, Py, 94%. (e) Dibutyl hydrogenphosphate, NIS-TfOH, MS (4 Å), 92%.Communication ChemCommPublished on 03 January 2014. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:01:31. View Article OnlineThis journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 1875--1877 | 1877mixture of compounds resulting from incomplete removal ofLev esters with hydrazine acetate at 25 1C. Raising the tem-perature for Lev deprotection to 40 1C in the automated runimproved the purity and yield of the desired tetrasaccharide 8that was isolated in 12% yield over 12 steps.After assembly of the protected oligosaccharides on the solidsupport, deprotection of sulfated disaccharide 7 was tested insolution phase (Scheme 4). Hydrolysis of acetate, benzoate andmethyl esters was achieved using lithium hydroxide/hydrogenperoxide in THF–MeOH to obtain disaccharide 9 in 79% yield.Hydrogenolysis of 9 provided fully deprotected, conjugationready disaccharide 10 in 80% yield.In conclusion, an eﬃcient automated solid phase synthesisof dermatan oligosaccharides was achieved using orthogonallyprotected building blocks and a photo-cleavable linker as wellas an automated oligosaccharide synthesizer. Two-step depro-tection in solution aﬀords the conjugation ready dermatansulfate oligosaccharides that can be directly immobilized onmicroarrays or conjugated to proteins.We thank the Max-Planck Society and the European ResearchCouncil (ERC Advanced Grant AUTOHEPARIN to PHS) for generousfinancial support. We thank Dr I. Vilotijevic and Dr F. Pfrenglefor their help in editing this communication.Notes and references1 J. M. Trowbridge and R. L. Gallo, Glycobiology, 2002, 12, 117R–125R.2 S. A. Osborne, R. A. Daniel, K. Desilva and R. B. Seymour, Glycobiol-ogy, 2008, 18, 225–234.3 A. M. Halldorsdottir, L. J. Zhang and D. M. Tollefsen, Glycobiology,2006, 16, 693–701.4 S. Yamada and K. Sugahara, Curr. Drug Discovery Technol., 2008, 5,289–301.5 R. J. Linhardt and R. E. Hileman, Gen. Pharmacol., 1995, 26, 443–451.6 N. S. Gandhi and R. L. Mancera, Chem. Biol. Drug Des., 2008, 72,455–482.7 A. Raghuraman, P. D. Mosier and U. R. Desai, ACS Med. Chem. Lett.,2010, 1, 281–285.8 N. Barroca and J. C. Jacquinet, Carbohydr. Res., 2000, 329, 667–679.9 L. Rochepeau-Jobron and J. C. Jacquinet, Carbohydr. Res., 1997, 305,181–191.10 J. C. Jacquinet and P. Sinay, Carbohydr. Res., 1987, 159, 229–253.11 A. Marra, D. Xia, M. Petitou and P. Sinay, Carbohydr. Res., 1989, 195,39–50.12 N. A. Karst and R. J. Linhardt, Curr. Med. Chem., 2003, 10, 1993–2031.13 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science, 2001, 291,1523–1527.14 M. T. Walvoort, H. van den Elst, O. J. Plante, L. Krock, P. H. Seeberger,H. S. Overkleeft, G. A. van der Marel and J. D. Codee, Angew. Chem.,Int. Ed. Engl., 2012, 51, 4393–4396.15 M. T. Walvoort, A. G. Volbeda, N. R. Reintjens, H. van den Elst,O. J. Plante, H. S. Overkleeft, G. A. van der Marel and J. D. Codee,Org. Lett., 2012, 14, 3776–3779.16 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger, Angew.Chem., Int. Ed., 2013, 52, 5858–5861.17 Y. V. Mironov, A. A. Sherman and N. E. Nifantiev, Tetrahedron Lett.,2004, 45, 9107–9110.18 R. W. Binkley, J. Org. Chem., 1991, 56, 3892–3896.19 N. Laurent, D. Lafont, P. Boullanger and J. M. Mallet, Carbohydr.Res., 2005, 340, 1885–1892.20 L. Krock, D. Esposito, B. Castagner, C. C. Wang, P. Bindschadler andP. H. Seeberger, Chem. Sci., 2012, 3, 1617–1622.21 P. Bindschadler, A. Adibekian, D. Grunstein and P. H. Seeberger,Carbohydr. Res., 2010, 345, 948–955.Scheme 3 Automated synthesis of dermatan sulfate oligosaccharide backbones. Reagents and conditions: (a) TMSOTf, DCM, 10 to 0 1C. (b) 25% Et3Nin DMF, 25 1C. (c) Ac2O, Py, 25 1C. (d) NH2NH2HOAc, Py, AcOH 25 1C or 40 1C. (e) PySO3, DMF, Py, 50 1C. (f) UV irradiation using continuous flow reactor,DCM–MeOH, RT.Scheme 4 Deprotection of sulfated disaccharide 7. Reagents and conditions:(a) 1 M LiOH, H2O2, THF–MeOH, 79%. (b) Pd–C, H2, 80%.ChemComm CommunicationPublished on 03 January 2014. Downloaded by Freie Universitaet Berlin on 28/09/2016 12:01:31. View Article Online

Modular automated solid phase synthesis of dermatan sulfate oligosaccharides

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