Modular automated solid phase synthesis of dermatan sulfate oligosaccharides

Article abstract of DOI:10.1039/c3cc48860h

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.

Full text of DOI:10.1039/c3cc48860h

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Modular automated solid phase synthesis of
dermatan sulfate oligosaccharides†
Cite this: Chem. Commun., 2014,
50, 1875
Jeyakumar Kandasamy,a Frank Schuhmacher,ab Heung Sik Hahm,ab James C. Kleina
and Peter H. Seeberger*ab
Received 20th November 2013,
Accepted 24th December 2013
DOI: 10.1039/c3cc48860h
www.rsc.org/chemcomm
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 auto-
mated synthesis of dermatan sulfate oligosaccharides. Conjugation-
ready oligosaccharides were obtained in good yield.
Dermatan sulfate is a glycosaminoglycan (GAG) predominantly
found in skin and present in many mammalian tissues.1 It is
composed of disaccharide repeating units consisting of N-acetyl-D-
galactosamine (GalNAc) and L-iduronic acid (IdoA). These alternating
disaccharide units can be variably O-sulfated at the C-4 and C-6
positions in GalNAc, and the C-2 position in IdoA.2 Depending on
the source, 63–97% of the dermatan sulfate polymer is made up
of IdoA - GalNAc4SO3 disaccharide repeating units that are
considered to be characteristic of dermatan sulfate (Fig. 1).3
Dermatan sulfate binds to a variety of proteoglycans and
modulates various biological processes such as coagulation, angio-
genesis, tumor migration and growth factor expression.4,5 Specific
biological roles of dermatan sulfate are poorly understood due
to the variations and heterogeneity in its polymer structure.6,7
In order to establish structure–activity relationships for dermatan
sulfate sequences, to correlate specific sequences and sulfation
patterns with protein binding and specific biological activities
access to structurally defined dermatan sulfate oligosaccharides
is required. Homogeneous samples of such fragments are acces-
sible only via chemical synthesis.
Fig. 1 Structure of dermatan sulfate oligosaccharides and retrosynthetic
analysis.
synthesis enables rapid access to structurally-defined oligo-
To date, syntheses of dermatan sulfate oligosaccharides have been saccharides.13,14 Automated protocols for the synthesis of other
executed in solution phase relying on many manual operations.8–12 glycosaminoglycans such as hyaluronan15 and chondroitin sulfate
With appropriate building blocks in hand, automated solid phase have been reported previously.16 Here, we describe the automated
solid phase synthesis of conjugation ready dermatan sulfate oligo-
a Department of Biomolecular Systems, Max-Planck-Institute of Colloids and
saccharides aimed at developing additional tools for biological
studies of GAGs.
¨
Interfaces, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany.
E-mail: peter.seeberger@mpikg.mpg.de; Fax: +49 30 838-59302;
The dermatan sulfate backbone may arise via assembly of
orthogonally protected galactosamine (GalN) and iduronic acid
building blocks (1 and 2, Fig. 1) designed for automated solid
phase synthesis on solid supported photo-cleavable linker 3.16
The C-3 position of GalN and the C-4 position of IdoA are masked
Tel: +49 30 838-59301
b
¨
Freie Universitat Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,
14195 Berlin, Germany
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc48860h
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Chem. Commun., 2014, 50, 1875--1877 | 1875

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Scheme 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, À10 1C,
15 minutes; addition of H2O, reflux at B80 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 and
conditions: (a) Pd(Ph3)4, 1,3-dimethylbarbituric acid, MeOH, 91%. (b) BzCl,
Py, 98%. (c) DDQ, MeOH, 88%. (d) FmocCl, Py, 94%. (e) Dibutyl hydrogen
phosphate, NIS-TfOH, MS (4 Å), 92%.
with the temporary 9-fluorenylmethoxycarbonyl (Fmoc) protecting
group in anticipation of the regiospecific elongation of the oligo- to deliver the glycan backbone without deletion sequences. Di- and
saccharide chain. The C-4 position of GalN carries a levulinoyl ester tetra-saccharides 4 and 5 were prepared using the oligosaccharide
(Lev) that can be selectively cleaved under mild conditions to allow synthesizer via iterative glycosylations with building blocks 1 and 2
for sulfation of the free hydroxyl group. The GalN amino group was and linker 3 (Scheme 3). Glycosylations were carried out at tem-
protected as trichloroacetamide to ensure good b-selectivities in peratures between À10 1C and 0 1C using trimethylsilyl trifluoro-
glycosylation reactions and allow for the installation of the desired methanesulfonate (TMSOTf). Each cycle involved addition of a
acetamide by the direct reduction at the late stage of the synthesis. glycosylating agent (double coupling) twice, using five equivalents
The C-2 hydroxyl group of the IdoA building block was protected as a of building blocks. Removal of the Fmoc protecting group with
benzoate ester to ensure a-selectivity in glycosylation reactions. The triethylamine uncovered the hydroxyl group ready for elongation.
remaining hydroxyl groups that were not modified during the
assembly were permanently protected as benzyl ethers.
Synthesis of disaccharide 4 commenced with the first glycosylation–
deprotection cycle using building block 1 followed by the second
Owing to the high cost of galactosamine, these building blocks glycosylation–deprotection cycle using building block 2. The resulting
are accessed from galactose via formation of galactal followed by free hydroxyl group on the terminal sugar was then acetylated on the
azido-selenation16,17 or from glucosamine via epimerization of the synthesizer employing acetic anhydride in pyridine. Disaccharide 4 was
C-4 position.8,9 Taking the latter approach into account, we pre- released from the resin by applying UV light using a photo flow
pared building block 1 in nine steps from glucosamine via migra- reactor.16 The crude product was purified by HPLC to obtain 4 in 66%
tion of a-ester with epimerization as the key step (Scheme 1).18,19 overall yield for six steps based on resin loading. Tetrasaccharide 5
Thioglycoside 11 was synthesized in four steps from glucosamine was assembled similarly via four glycosylation–deprotection cycles
hydrochloride (see ESI†).20 Levulination of the free hydroxyl group by alternating use of building blocks 1 and 2, followed by the
in 11 followed by regioselective ring opening of benzylidine acetal acetylation of the free hydroxyl group on the terminal sugar. Upon
using triethylsilane/trifluoroacetic acid yielded thioglycoside 12 in cleavage from the resin, the crude product was purified by HPLC to
70% yield over two steps. Migration of the C-3 Lev ester to the C-4 obtain 6 in 28% overall yield (ten steps). No deletion sequences were
hydroxyl with epimerization was effected by transforming the free observed in the automated synthesis of 4 and 5 as judged by HPLC.
hydroxyl group in 12 to the corresponding triflate to produce
Automated synthesis of sulfated dermatan oligosaccharides
galactose thioglycoside 13 in 78% yield. Fmoc protection of the required two additional steps for installation of sulfates: selective
free hydroxyl group in 13 followed by the conversion of thioglyco- removal of the Lev protecting groups and sulfation of the resulting
side to glycosyl phosphate using dibutyl hydrogen phosphate and hydroxyl groups. A solution of hydrazine acetate in pyridine–acetic
N-iodosuccinimide–triflic acid yielded differentially protected build- acid effected removal of the Lev groups at 25 1C. The pyridine sulfur
ing block 1 in 86% yield.
trioxide complex in dimethylformamide–pyridine was used for the
Synthesis of IdoA building block 2 was achieved in five steps sulfation of free hydroxyl groups at 50 1C on the synthesizer.16
from thioglycoside 1421 (Scheme 2). Tetrakis(triphenylphosphine)- Synthesis of sulfated monosaccharide 6 and disaccharide 7 was
palladium(0) was employed for the deprotection of the allyl group achieved on a solid support in five and seven automated steps,
in thioglycoside 14 and the resulting hydroxyl group was protected respectively, under glycosylation–deprotection conditions estab-
as a benzoyl ester to obtain thioglycoside 15 in 89% yield (over two lished for compounds 4 and 5, followed by automated acetylation,
steps). Removal of the naphthyl group in 15 using 2,3-dichloro-5,6- Lev deprotection and sulfation as described above. After the cleavage
dicyano-1,4-benzoquinone (DDQ) followed by Fmoc protection of of the photo-sensitive linker, crude products were analysed by
the resulting alcohol provided thioglycoside 16 in 82% overall yield. reverse phase (RP)-HPLC and purified by size exclusion chromato-
Thioglycoside 16 was then converted to the corresponding graphy (Sephadex LH-20) to obtain monosaccharide 6 in 43% and
phosphate glycoside 2 in 92% yield.
disaccharide 7 in 32% overall yield based on resin loading.
Automated assembly of sulfated tetrasaccharide 8 under the
The simple non-sulfated dermatan oligosaccharides were
pursued first in order to test the ability of the automated route conditions established for the synthesis of 6 and 7 yielded a
1876 | Chem. Commun., 2014, 50, 1875--1877
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Scheme 3 Automated synthesis of dermatan sulfate oligosaccharide backbones. Reagents and conditions: (a) TMSOTf, DCM, À10 to 0 1C. (b) 25% Et3N
in DMF, 25 1C. (c) Ac2O, Py, 25 1C. (d) NH2NH2ÁHOAc, Py, AcOH 25 1C or 40 1C. (e) PyÁSO3, DMF, Py, 50 1C. (f) UV irradiation using continuous flow reactor,
DCM–MeOH, RT.
We thank the Max-Planck Society and the European Research
Council (ERC Advanced Grant AUTOHEPARIN to PHS) for generous
financial support. We thank Dr I. Vilotijevic and Dr F. Pfrengle
for their help in editing this communication.
Notes and references
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Lev esters with hydrazine acetate at 25 1C. Raising the tem-
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