Tailormade Polysaccharides with Defined Branching Patterns: Enzymatic Polymerization of Arabinoxylan Oligosaccharides

Article abstract of DOI:10.1002/anie.201806871

The heterogeneous nature of non-cellulosic polysaccharides, such as arabinoxylan, makes it difficult to correlate molecular structure with macroscopic properties. To study the impact of specific structural features of the polysaccharides on crystallinity

Full text of DOI:10.1002/anie.201806871

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Accepted Article
Title: Tailor-Made Polysaccharides with Defined Branching Patterns by
Enzymatic Polymerization of Arabinoxylan Oligosaccharides
Authors: Deborah Senf, Colin Ruprecht, Saina Kishani, Aleksandar
Matic, Guillermo Toriz, Paul Gatenholm, Lars Wågberg, and
Fabian Pfrengle
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Angewandte Chemie International Edition
10.1002/anie.201806871
COMMUNICATION
Tailor-Made Polysaccharides with Defined Branching Patterns by
Enzymatic Polymerization of Arabinoxylan Oligosaccharides
[
a,b]
[a]
[c]
[a,d]
[e]
Deborah Senf , Colin Ruprecht , Saina Kishani , Aleksandar Matic , Guillermo Toriz , Paul
[
e]
[c]
[a,b]
Gatenholm , Lars Wågberg , and Fabian Pfrengle*
[
4]
Abstract: Polysaccharides from plant biomass are explored
extensively as renewable resources for the production of materials
and fuels. However, the heterogeneous nature of non-cellulosic
polysaccharides such as arabinoxylan makes it difficult to correlate
molecular structure with macroscopic properties. To study the impact
of specific structural features of the polysaccharides on e.g.
crystallinity or affinity to other cell wall components, collections of
polysaccharides with defined repeating units are required. Herein, a
chemo-enzymatic approach towards artificial arabinoxylan
polysaccharides with systematically altered branching patterns is
described. The polysaccharides were obtained by glycosynthase-
catalyzed polymerization of glycosyl fluorides derived from
arabinoxylan oligosaccharides that were procured either chemically,
chemo-enzymatically, or from a commercial source. X-ray diffraction
and adsorption experiments on cellulosic surfaces revealed that the
physico-chemical properties of the synthetic polysaccharides
strongly depend on the specific nature of their substitution patterns.
The presented strategy of combining sophisticated carbohydrate
synthesis with glycosynthase technology offers access to artificial
polysaccharides for structure-property relationship studies that are
not accessible by other means.
and as ingredients in functional foods. Non-food applications
[
5]
range from their use as oxygen barrier films for packaging to
[
6]
strengtheners in paper production.
The molecular composition of xylans has a strong impact
on their macroscopic properties. A decrease in arabinose-
substitution is accompanied by a decrease in solubility and
crystallinity as the substituents disrupt the interactions between
individual xylan chains. Adsorption of xylan to cellulose also
typically increases with lower degrees of substitution.
Interestingly, the substitution pattern in glucuronoxylans is not
random and recently it was shown by solid-state NMR
spectroscopy of intact plant cell walls and molecular dynamics
[7]
[
8]
[
7, 9]
[
10]
[
11]
simulations
that only xylan domains with evenly spaced
substituents interact with the hydrophilic surface of cellulose
microfibrils. This implies that the discrete substitution pattern in
xylan strongly affects the ability of xylan to bind to cellulose,
which determines the strength of the xylan-cellulose network in
the cell wall and thus of the respective plant material.
Detailed investigations into structure-property relationships
of complex polysaccharides are hampered by their
inhomogeneity as they are obtained by extraction from natural
sources. Polysaccharides with defined branching patterns can
only be obtained synthetically using a bottom-up approach.
Cellulose and xylan are the major polysaccharides in
lignocellulosic biomass and as such are promising renewable
[12]
Enzymatic polymerizations using glycosynthases
offer a fast
and convenient synthetic route towards artificial polysaccharides.
Glycosynthases are mutated glycosyl hydrolases in which the
catalytic nucleophile in the active site is exchanged for a non-
nucleophilic residue, thus inactivating the hydrolytic reaction.
Instead, the enzyme is able to irreversibly catalyze the ligation of
activated donors, such as -glycosyl fluorides, to suitable
acceptor substrates, forming new glycosidic bonds. The yields
provided by glycosynthases are generally higher than with the
[1]
resources for the production of materials and fuels. While
cellulose is regularly utilized for these purposes, the exploration
[
2]
of xylan lags behind due to its degree of structural complexity.
In all higher plants xylans possess a backbone consisting of β-
,4-linked D-xylopyranoses that are decorated with arabinose,
1
glucuronic acid, and acetyl groups (Fig. 1). The degree and type
of substitution differs substantially among plant species. In
grasses and cereals for instance, L-arabinofuranose residues
are the major substituents, while in soft- and hardwoods D-
[7, 9a, 13]
respective wild-type glycoside hydrolases.
[
3]
glucuronic acids or its 4-O-methyl derivatives are mostly found.
Xylans are used in the food industry as stabilizers, additives,
[
a]
Deborah Senf, Dr. Colin Ruprecht, Aleksandar Matic, Dr. Fabian
Pfrengle, Department of Biomolecular Systems, Max Planck Institute
of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam,
Germany, E-mail: Fabian.Pfrengle@mpikg.mpg.de
Deborah Senf, Dr. Fabian Pfrengle, Institute of Chemistry and
Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin,
Germany
Saina Kishani, Prof. Dr. Lars Wågberg, Fibre and Polymer
Technology, Royal Institute of Technology, Stockholm 100 44,
Sweden and Wallenberg Wood Science Center, KTH Royal Institute
of Technology, Stockholm 100 44, Sweden
[
b]
c]
[
[
d]
e]
Aleksandar Matic, University of Potsdam, Department of Chemistry
Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany (current
address)
Prof. Dr. Paul Gatenholm, Prof. Dr. Guillermo Toriz, Wallenberg
Wood Science Center and Biopolymer Technology, Department of
Chemistry and Chemical Engineering, Chalmers University of
Technology, Gothenburg 412 96, Sweden
[
Figure 1. Schematic representation of (a)
a natural xylan with random
branching pattern and (b) artificial arabinoxylans with defined branching
patterns.
Supporting information for this article is given via a link at the end of the
document.
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Angewandte Chemie International Edition
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COMMUNICATION
When glycosynthases based on endo-glycosylases are
used, the activated glycosyl donor acts as both the donor and
acceptor, resulting in a polymerization reaction. In this way
amounts
commercially.
Larger
-1,3-arabinofuranosyl-
substituted xylan oligosaccharides 6 and 7 were obtained by a
chemo-enzymatic approach (Scheme 1), in which arabinoxylan
trisaccharide donor 13 was ligated to di- and trixyloside
[13a, 14]
polysaccharides of different classes,
with molar masses up
[
14a, 14b, 15]
[14d]
to 60 kDa, have been produced.
glycosynthases have also been reported and used to synthesize
Xylanase-derived
acceptors 14 and 15 using the glycosynthase XynAE265G.
To prevent enzyme-catalyzed self-condensation, 13 was
equipped with a tetrahydropyranyl (THP) ether in the C4-position
of the non-reducing xylose residue. The THP group had
successfully been used in the step-wise enzymatic synthesis of
[
14d-f]
homopolymers of xylan.
Substituted xylan polysaccharides
have not been synthetically prepared. Herein, we present the
synthesis of artificial arabinoxylan polysaccharides with defined
branching patterns by glycosynthase-catalyzed polymerization of
a number of different oligosaccharide fluorides.
[
19]
[20]
cellodextrins and xyloglucan oligosaccharides previously.
Glycosyl donor 13 was prepared from the monosaccharide
building blocks 8-10 that already served as starting materials for
the synthesis of oligosaccharides 1-3. After assembly of
trisaccharide 11, the Fmoc-protecting group was exchanged by
a THP-ether, and subsequent methanolysis of the benzoyl
esters and hydrogenolysis of the benzyl ethers provided 4’-O-
THP-protected trisaccharide 12. Peracetylation of 12 was then
followed by fluorination with HF/pyridine at low temperature to
To catalyze the polymerization of the arabinoxylan
oligosaccharide fluorides, we chose the glycosynthase
XynAE265G that is able to produce xylan homopolymers
[14d]
containing more than 100 monosaccharide units.
This
glycosynthase is derived from a xylanase belonging to the
glycoside hydrolase (GH) family 10 (Carbohydrate Active
[
16]
Enzyme database).
GH10 xylanases typically release small
[
21]
oligosaccharides that carry arabinose substituents in the 2-
and/or 3-position of the non-reducing xylose residue as
provide the corresponding glycosyl fluoride.
Reaction
temperature and time were carefully adjusted to ensure
formation of the thermodynamically favored -fluoride without
cleavage of the rather acid-labile arabinofuranose substituent.
The THP-group did not stay intact under these reaction
conditions and was subsequently reintroduced at the C4’-
position using 3,4-dihydro-2H-pyran (DHP) and catalytic
hydrolysis
products
from
natural
arabinoxylan
[
17]
polysaccharides. Based on this specificity, we designed seven
arabinoxylan oligosaccharides (Fig. 2) to serve as monomers for
the polymerization reactions.
[
22]
amounts of pTsOH.
Removal of the acetate groups by
treatment with 1 equiv sodium methoxide at 0 °C finally afforded
THP-protected glycosyl fluoride 13. Overnight-incubation of
glycosyl fluoride 13 with either acceptor 14 or 15 and the
Figure 2. Oligosaccharide monomers for the synthesis of arabinoxylan
polysaccharides. 1-3 and 6 and 7 were produced by chemical and chemo-
enzymatic synthesis, respectively. 4 and 5 were commercially available.
Xylobiose 1 and -1,3-arabinofuranosyl substituted xylan
tri- and tetrasaccharides 2 and 3 were chemically synthesized
following the same protecting group strategy as we have used
previously for the automated glycan assembly of arabinoxylan
Scheme 1. Chemo-enzymatic synthesis of penta- and hexaarabinoxylosides 6
and 7. Reagents and conditions: a) NEt
DCM, 4 h, rt; c) NaOMe, MeOH/DCM, 16 h, rt, 66% over 2 steps; d) H
MeOH/EtOAc/Na PO buffer, rt, 24 h; e) Ac O, py, 4 h, rt; f) HF/py, DCM, -
30 °C → -20 °C, 2 h, 60% over 3 steps; g) DHP, p-TsOH, DCM, 4 h, rt, 91%;
h) NaOMe, MeOH, 0 °C, 96%; i) 14 or 15, XynAE265G, rt, Na PO buffer,
6 h; j) 1 M HCl, rt, 30 min, 6: 78%, 7: 76% over 2 steps. For the synthesis of
14 and 15, see Supporting Information.
3
, DCM, 2 h, rt, 90%; b) DHP, p-TsOH,
2
, Pd/C,
x
H
x
4
2
[
17d, 18]
oligosaccharides
Arabinofuranosyl-
disubstituted oligosaccharides 4 and 5 were procured in limited
(see Supporting Information). -1,2-
x
H
x
4
and -1,2--1,3-arabinofuranosyl-
1
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COMMUNICATION
Scheme 2. Synthesis glycosyl fluoride donors 23–29. Reagents and conditions: a) Ac
2
O, py, rt,; b) only for 18 and 20: i) N
2
H
4
∙AcOH, DMF, 0 °C, 5 h, ii) DAST,
DCM, 0 °C, 1 h, c) NIS, cat. HF/py, -50 °C (for 21 and 22 only), HF/py, DCM, -30 °C → -10 °C, 2-5 h, 16: 79%, 17: 89%; 18: 77% over 3 steps, 19: 43%, 20: 44%
over 3 steps, 21: 53% over 2 steps, 22: 52% over 2 steps; h) NaOMe, MeOH, 0 °C, 23: quant., 24: quant.; 25: quant., 26: quant., 27: 96%, 28: 99%, 29: quant.
glycosynthase XynAE265G, followed by removal of the THP-
protecting group, gave the desired penta-and hexasaccharides 6
and 7 in good yields. The thioether moiety in 6 and 7 provided a
good hydrophobic handle for purification and permitted their later
conversion into the corresponding glycosyl fluorides.
thioglycosides 6 and 7 required the admixture of NIS to activate
[
23]
the thioether leaving group. A milder procedure was applied to
oligosaccharides 3 and 5, as the usage of a larger excess of
HF/pyridine provoked cleavage of the arabinofuranose. The
hydroxyl group at the anomeric center was selectively
deprotected using hydrazine acetate first, and the resulting
hydroxyl group converted with N,N’-diethylaminosulfur trifluoride
(DAST) into the corresponding -fluoride. The -fluorides were
finally equilibrated into the desired -fluorides 25 and 27 using
The conversion of arabinoxylan oligosaccharides 1-7 into
glycosyl fluorides 23-29 was performed by
a three step
sequence consisting of peracetylation, fluorination, and
[
21]
deacetylation (Scheme 2).
In the case of 16, 17 and 19,
[
24]
fluorination was performed using HF/pyridine at low temperature. only small amounts of HF/pyridine.
Activation of the peracetylated oligosaccharides derived from
Scheme 3. Synthesis of artificial arabinoxylan polysaccharides by enzymatic polymerization of arabinoxylan oligosaccharide fluorides. The molecular mass
distribution of the resulting polysaccharides was analyzed by HPSEC chromatography using a TSK gel 3000 column and an ELSD detector. Chromatograms with
two ELSD traces were obtained when a water-insoluble polysaccharide fraction (dashed line) and a water-soluble fraction (solid line) was obtained. These
fractions were analyzed separately.
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Angewandte Chemie International Edition
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COMMUNICATION
[
10a, 11, 28]
With the desired arabinoxylan glycosyl fluorides in hand,
enzymatic polymerizations were performed by overnight
incubation of the respective glycosyl fluoride with XynAE265G,
and arabinoxylan polysaccharides in amounts up to 55 mg (31)
in one batch were obtained (Scheme 3). In some cases,
precipitates of water-insoluble polysaccharides (30a, 32a, and
cellulose microfibrils.
An even pattern of xylan
substitution with acetyl and/or glucuronic acid residues appears
to be essential for this interaction to occur and consequently for
[
29]
the development of normal plant secondary cell walls. In order
to study the ability of synthetic xylan polysaccharides 30-32 to
adsorb and bind to cellulose, we performed QCM-D experiments
on spin-coated cellulose surfaces (Fig. 3b). As expected,
unsubstituted xylan 30 showed the highest adsorption of the
synthetic polysaccharides to the cellulose surface. More
interestingly, we observed that arabinoxylan 31 adsorbed
irreversibly, i.e. only a minor desorption upon rinsing, to the
cellulose surface while the water-soluble fraction of arabinoxylan
32, despite the fact that it is less substituted, only adsorbed
33a) were formed during the reactions while in the other cases
the reaction products remained fully soluble. When precipitated
polysaccharides were formed, they were collected by
centrifugation and the remaining solution was passed through a
short C18 cartridge to isolate the soluble polysaccharide fraction
(
30b, 32b, and 33b). Water-soluble polysaccharide products
were directly subjected to C18 reversed-phase chromatography.
MALDI mass spectrometry was used to confirm the formation of
oligomers of the starting oligosaccharides (see Supporting
Information). Further conformation was obtained by digestion of
weakly with
a
large desorption upon rinsing (both
polysaccharides having similar molecular weights). Since both
these polymers should have similar driving forces for their
adsorption, this result indicates that, once adsorbed, 31 has
experienced a conformational change from a threefold to a
twofold helical screw, permitting strong interactions between 31
and the cellulose surface. Unlike 31, 32 cannot adopt a twofold
helical screw conformation with the substituents all pointing in
the same direction. Natural rye arabinoxylan, which was
enzymatically hydrolyzed to a similar size as the synthetic xylan
polysaccharides, showed stronger adsorption properties than 32
(more comparable to synthetic xylan 30), indicating that it does
not contain large amounts of unevenly substituted xylan
domains. Our study provides direct support by physical in vitro
experiments outside the cell wall for the observation that specific
substitution patterns are required for strong xylan-cellulose
[
25]
the synthetic polysaccharides with xylanases, which resulted,
unlike in the case of natural rye arabinoxylan, in the formation of
very simple and defined hydrolysis products, a fact that may be
exploited for determining the substrate specificity of newly
discovered xylan-degrading enzymes
Information). The molecular mass distribution of the products
was derived from high-performance size-exclusion
chromatography (HPSEC) calibrated with pullulan standards
Fig. 2) since it cannot be deduced from mass spectrometry
experiments due to the size dependent molecule desorption
(see Supporting
(
[
26]
from the MALDI matrix. We found that the molecular weight at
the peak maximum (M ) of the different polysaccharide products
ranged from 4.7 kDa for the water-soluble fraction of linear xylan
P
30 to 29.6 kDa for the water-insoluble fraction of arabinoxylan 33. interactions and the first hint that, besides acetylation and
These values correspond to 36 (n = 18) and 224 (n = 56)
monosaccharides, respectively. The observed differences in the
degree of polymerization (DP) of the artificial polysaccharides
are most likely a result of the substrate specificity of the enzyme
that transfers some of the arabinoxylan oligosaccharides more
efficiently than others.
There is an inherent limitation for the maximum achievable
lengths of the synthetic xylan polysaccharides that is determined
p
by their solubility. Thus, the M of the linear xylan polysaccharide
30 for example did not exceed 9.2 kDa. Besides for the linear
xylan, water-insoluble polysaccharide fractions were obtained for
arabinoxylans 32 and 33 which carry the arabinofuranosyl
substituents at the 2- and 3-position of every third xylose residue,
respectively. In line with this, for arabinoxylans 32 and 33
significant crystallinity was observed whereas 31, 35, 36, as well
as natural rye arabinoxylan having differently spaced
substituents were completely amorphous, as qualitatively
determined by X-ray diffraction (Fig. 3a). The specific
substitution pattern of arabinoxylans 32 and 33 might favor a
stable threefold helical screw conformation, with the substituents
[
27]
all pointing in the same direction. Such a conformation would
enable interactions between the xylan backbones of individual
molecules, explaining the low solubility and the crystallinity of 32
and 33. This is contradicting the commonly accepted notion that
solubility and crystallinity of xylans directly correspond with the
Figure 3. (a) Influence of the arabinose substitution patterns of the synthetic
polysaccharides on crystallinity, as determined by x-ray diffraction; rye AX: rye
arabinoxylan. (b) Adsorbed amounts (g/m ) (including immobilized water) of
[
7, 9a]
degree of substitution.
2
Recent studies show that in intact plant cell walls xylan
partially flattens from the typical threefold helical screw into a
twofold helical screw ribbon to enable intimate binding to
30b, 31, 32b, and partly digested rye arabinoxylan (MP = 5.5 kDa) on spin-
coated model cellulose surfaces, as determined by QCM-D.
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Angewandte Chemie International Edition
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COMMUNICATION
substitution by glucuronic acid, also the patterning of arabinose
substituents on the xylan backbone may be important. These
interactions influence both strength and digestibility of plant cell
walls.
In summary, we have explored glycosynthase technology
for the polymerization of seven mostly chemically synthesized
arabinoxylan oligosaccharides into artificial polysaccharides with
well-defined branching patterns. The obtained polysaccharides
contained polysaccharide chains with molecular masses up to
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2
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Acknowledgements
We gratefully acknowledge financial support from the Max
Planck Society and the German Research Foundation (DFG,
Emmy Noether program PF850/1-1 to FP and SFB 765). The
Knut and Alice Wallenberg Foundation is acknowledged for
funding the Wallenberg Wood Science Center. We thank Maiko
Schulze and Suvrat Chowdhary for experimental help. We thank
Ingrid Zenke for recording X-ray diffractograms and Janete
Rodriguez and Dr. Luca Bertinetti for helpful discussions. The
plasmid for expression of XynAE265G was kindly provided by
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Keywords: Carbohydrates • Chemo-enzymatic synthesis • Plant
cell wall • Xylan • Glycosynthases
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This article is protected by copyright. All rights reserved.

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10.1002/anie.201806871
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COMMUNICATION
Deborah Senf, Colin Ruprecht, Saina
Kishani, Aleksandar Matic, Guillermo
Toriz, Paul Gatenholm, Lars Wågberg,
and Fabian Pfrengle*
Page No. – Page No.
Tailor-Made Polysaccharides with
Defined Branching Patterns by
Enzymatic Polymerization of
Arabinoxylan Oligosaccharides
Synthetic designer polysaccharides: Enzymatic polymerization of synthetic
arabinoxylan oligosaccharides provided artificial polysaccharides with
systematically altered branching patterns. These well-defined arabinoxylans
constitute a unique set of tools for structure-property relationship studies.
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