Automated Glycan Assembly of Oligosaccharides Related to Arabinogalactan Proteins

Article abstract of DOI:10.1021/acs.orglett.5b02185

Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is

Full text of DOI:10.1021/acs.orglett.5b02185

Letter
pubs.acs.org/OrgLett
Automated Glycan Assembly of Oligosaccharides Related to
Arabinogalactan Proteins
Max P. Bartetzko, Frank Schuhmacher, Heung Sik Hahm, Peter H. Seeberger, and Fabian Pfrengle*
Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Muhlenberg 1, 14476 Potsdam, Germany
̈
Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Arnimallee 22, 14195 Berlin, Germany
̈
S
* Supporting Information
ABSTRACT: Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II
arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic
chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14
oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features
of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.
α-(1,3)-linked L-arabinofuranoses, short arabinan oligosacchar-
ides, or less frequently, rhamnose, fucose, and glucuronic acid
residues.4
large number of the proteins embedded in the plant cell
1
Awall are glycoproteins and proteoglycans. Arabinogalac-
tan proteins (AGPs) are one major proteoglycan family2 that
consists of arabinogalactan (AG) polysaccharides attached to a
hydroxyproline-rich peptide backbone (Figure 1).3 The AG-
moieties are classified as type II, characterized by a β-(1,3)-D-
galactan backbone heavily branched with β-(1,6)-D-galactan
side chains. The galactan core may be further substituted with
Carbohydrate decoration is the basis for AGP heterogeneity
in different tissues and plants,5 impeding the assignment of
AGP function in essential biological processes such as cell
differentiation6 and organ development.7 Monoclonal antibod-
ies (mAbs) are powerful tools to locate AG polysaccharides in
immunolabeling studies of plant cell walls.8 However, since the
precise molecular structures bound by these antibodies are
mostly unknown,9 it remains uncertain how the molecular
structure of the AG influences the localization and function of
particular AGPs.10
A better understanding of AGP glycan structure and function
requires well-defined, homogeneous oligosaccharides that are
accessible exclusively by chemical synthesis. While several
chemical syntheses of type-II arabinogalactan oligosaccharides
have been reported,11 a general approach providing convenient
access to AGP-derived oligosaccharides is missing. Key to such
an approach is the ability to produce many complex
oligosaccharides from a limited set of building blocks.
Automated glycan assembly12 offers the possibility to construct
carbohydrate libraries in a timely manner provided that fully
orthogonal protecting groups and capping steps are used.13
Here, we report the automated glycan assembly of 14 type-II
Figure 1. “Wattle-blossom” model of an AGP.3 Highly branched type-
II arabinogalactan (yellow ovals with black circles) is attached to a
hydroxyproline rich protein backbone (blue).
Received: July 28, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02185
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Organic Letters
Letter
arabinogalactan glycans of different size and complexity based
on four monosaccharide building blocks (Figure 2).
variety of oligogalactans on solid support (Scheme 1). Hydroxyl
groups that did not need to be manipulated during the solid-
phase synthesis were permanently protected. In the C-2
position, benzoyl esters (Bz) provided neighboring group
participation in the glycosylation reactions. All remaining
positions were protected with benzyl ethers (Bn, Scheme 1).
A temporary fluorenylmethoxycarbonyl (Fmoc) protecting
group was installed in either the C-3- (BB1) or C-6-position
(BB3) depending on which position needed to be elongated.
To enable branching, a third building block (BB2) was
furnished with both Fmoc and levulinoyl (Lev) protecting
groups. Lev-esters are fully orthogonal to Fmoc groups and
have been successfully used in automated glycan assembly.12 By
using dibutyl phosphate as the anomeric leaving group in all the
galactose building blocks, we hoped to overcome the poor
stereoselectivities and low yields frequently observed in the
formation of Gal-β1,3-Gal linkages with other anomeric leaving
groups, even in the presence of participating 2-O-protecting
groups.11,14
Figure 2. Oligosaccharide fragments of type-II arabinogalactan as
potential targets for automated glycan assembly.
With the required building blocks in hand, we first tested
whether the challenging Gal-β1,3-Gal linkage could be
constructed on the solid phase. Three glycosylations on
linker-functionalized resin L with BB1 provided the protected
(β1,3)-trigalactoside with complete stereoselectivity and full
conversion after cleavage of the photolabile linker in a
We initially focused on the (3,6)-galactan backbone without
arabinose substitution and designed a set of three galactose
building blocks (BB1−BB3) that allowed for the assembly of a
a
Scheme 1. Automated Glycan Assembly of Type-II Galactan Fragments 1−7
a
The letter code below the structures represents the synthesizer modules and deprotection steps applied in the respective synthesis. Reagents and
conditions: (a) 2 × 3.75 equiv of BB1, BB2, or BB3, TMSOTf, DCM, −35 °C (5 min) → −20 °C (30 min); (b) three cycles of 20% NEt3 in DMF,
25 °C (5 min); (c) three cycles of 0.15 M hydrazine in Py/AcOH/H2O (4:1:0.25), 25 °C (30 min); (f) hν (305 nm); (g) NaOMe,THF, 16 h; (h)
H2, Pd/C, EtOAc/MeOH/H2O/AcOH, 16 h. Yields are based on resin loading.
B
DOI: 10.1021/acs.orglett.5b02185
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a
Scheme 2. Automated Glycan Assembly of Type-II Arabinogalactan Fragments 8−14
a
The letter code below the structures represents the synthesizer modules and deprotection steps applied in the respective synthesis. Reagents and
conditions: (a) 2 × 3.75 equiv of BB1, BB2, or BB3, TMSOTf, DCM, −35 °C (5 min) → −20 °C (30 min); (b) three cycles of 20% NEt3 in DMF,
25 °C (5 min); (c) three cycles of 0.15 M hydrazine in Py/AcOH/H2O (4:1:0.25), 25 °C (30 min); (d) 2 × 3.75 equiv of BB4, NIS, TfOH, DCM/
dioxane, −40 °C (5 min) → −20 °C (40 min); (e) three cycles of 0.5 M Bz2O and 0.25 M DMAP in DCE, Py, 40 °C (30 min); (f) hν (305 nm);
(g) NaOMe, THF, 16 h; (h) H2, Pd/C, EtOAc/MeOH/H2O/AcOH, 16 h. Yields are based on resin loading.
commercial flow photoreactor.15 As expected, synthesis of a β-
(1,6)-linked trigalactoside proceeded equally well when using
BB3. Hence, the stage was set to assemble linear and branched
galactan oligosaccharides 1−7 by iterative glycosylation and
deprotection reactions. Glycosylations were performed twice
using 3.75 equiv of galactose building blocks BB1−3 and
equimolar amounts of TMSOTf. Notably, we did not observe
reduced conversion resulting from the use of slightly less excess
building block than the two times 5 equiv that are standard for
automated glycan assembly.12 Every glycosylation was followed
by either Fmoc or Lev removal to free the position on galactose
for subsequent elongation. All branched structures were
synthesized by assembling the linear β-(1,3)-linked backbone
first and subsequently attaching the β-(1,6)-linked side chain.
After completion of the automated synthesis, the protected
oligosaccharides were cleaved from the solid support and
treated with sodium methoxide in methanol. The resulting
semiprotected products were subjected to hydrogenolysis, and
the fully deprotected oligogalactans 1−7 were obtained in 5−
39% overall yield, based on the calculated loading of linker-
functionalized resin L (Scheme 2). The products were purified
via preparative reversed-phase HPLC at both the fully and
semiprotected stage. The purity of all products was ensured by
characterization with analytical HPLC, NMR spectroscopy, and
high-resolution mass spectrometry.
The synthesis of galactan fragments containing exclusively
galactose proceeded smoothly, and the attachment of L-
arabinofuranose substituents to this core structure was expected
to be straightforward. Addition of L-arabinose building block
BB413 to the previously used set of galactose building blocks
BB1−3 enabled us to produce seven additional type-II
arabinogalactan substructures (Scheme 2). In the synthesized
AG oligosaccharides, the arabinose unit is either attached to the
β-(1,3)-linked galactan backbone (8, 9, 11) or to the β-(1,6)-
linked galactan side chain (10, 12−14). The synthesis of the
more elaborate oligosaccharides 12−14 required capping steps
during the automated synthesis to ensure the selective
attachment of the arabinose to the desired galactose unit. For
that purpose, the terminal Fmoc group was removed after
assembly of the (1,3)-galactan backbone, and the resulting free
hydroxyl group was masked as an ester. We found that the
standard capping protocol previously applied in automated
glycan assembly,12 employing acetic anhydride in pyridine, was
not suitable for our purposes; the resulting acetyl groups do not
withstand the conditions required for the removal of Lev
groups. Thus, we decided to modify the established protocol in
order to obtain benzoyl esters as capping moieties due to their
C
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stability toward the hydrazine used for Lev deprotection.16
Quantitative capping was achieved by incubating the resin-
bound oligogalactan for 30 min at 40 °C with a solution of
benzoic anhydride and DMAP in DCE. After capping the
backbone, the side chain was introduced by iterative Lev
deprotection and glycosylation reactions. The arabinose
building block BB4 was selectively attached to the side chain
after Fmoc deprotection.
The glycosylation conditions established for the synthesis of
oligogalactans 1−7 were also applied for oligosaccharides 8−
14, except that thioglycoside BB4 was activated with N-
iodosuccinimide and catalytic amounts of TfOH in DCM. After
cleavage of the products from the solid support, global
deprotection, and purification, AG oligosaccharides 8−14
were obtained in yields between 9 and 17% based on the
calculated resin loading (Scheme 2).
In conclusion, we have established a general approach to
synthesize glycans found on AGPs. Automated glycan assembly
provided 14 type-II AG glycans that are valuable probes for
plant research. All products are equipped with an aminopentyl
linker at the reducing end and can be directly printed as
microarrays to determine the binding specificities of antibodies
that recognize plant cell wall glycans.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b02185.
(15) Using modified conditions from: Eller, S.; Collot, M.; Yin, J.;
Hahm, H. S.; Seeberger, P. H. Angew. Chem., Int. Ed. 2013, 52, 5858−
5861. See the Supporting Information for further details.
(16) Li, J.; Wang, Y. Synth. Commun. 2004, 34, 211−217.
Experimental procedures for solution- and automated
solid-phase reactions and characterization data for all
compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: Fabian.Pfrengle@mpikg.mpg.de
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the Max
Planck Society, the German Research Foundation (DFG,
Emmy Noether program PF850/1-1 to F.P.), the Fonds der
chemischen Industrie (Liebig-fellowship to F.P.), and an ERC
Advanced Grant (AUTOHEPARIN to P.H.S.). We thank Dr.
K. Gilmore (Max-Planck-Institute of Colloids and Interfaces)
for critically editing this manuscript.
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DOI: 10.1021/acs.orglett.5b02185
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