Extending the Scope of GTFR Glucosylation Reactions with Tosylated Substrates for Rare Sugars Synthesis

Article abstract of DOI:10.1002/cbic.201700320

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded

Full text of DOI:10.1002/cbic.201700320

Accepted Article
Title: Extending the scope of GTFR Glucosylation reactions with
tosylated substrates for rare sugars synthesis
Authors: Juergen Seibel, Christoph Sotriffer, Julian Görl, and Christian
Possiel
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To be cited as: ChemBioChem 10.1002/cbic.201700320
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10.1002/cbic.201700320
ChemBioChem
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Extending the scope of GTFR Glucosylation reactions with
tosylated substrates for rare sugars synthesis
Julian Görl[a], Christian Possiel[a], Christoph Sotriffer[b], Jürgen Seibel*[a]
Functionalized rare sugars were synthesized with 2-, 3- and 6-
tosylated glucose derivatives as acceptor substrates by
transglucosylation with sucrose and the glucansucrase GTFR
from Streptococcus oralis. The 2- and 3-tosylated glucose
derivatives yielded the corresponding 1,6-linked disaccharides
(isomaltose analogues) whereas the 6-tosylated glucose
derivatives resulted in 1,3-linked disaccharides (nigerose
analogue) with high regioselectivity in up to 95% yield. Docking
studies provide insights into the binding mode of the acceptors
suggesting two different orientations which are responsible for
the change in regioselectivity.
limitation protein- and substrate engineering is frequently used to
fine-tune enzymatic conversion.[3c, 10] In earlier works our research
group succeeded in altering the product diversification by
mutagenesis of the glucansucrase from Streptococcus oralis
ATCC10557[11] (GTFR, EC 2.4.1.5) shifting the product to more
α-(1,3) linked, insoluble polymer or enhancing isomaltose
production.[12] The transfer reaction of an enzyme can be strongly
influenced by substituents blocking certain hydroxyl groups of the
acceptor (substrate engineering). Hellmuth et al. used lactose as
an acceptor for the glucansucrase GTFR which mainly produces
a trisaccharide presenting an α-(1,2) linkage due to the steric
blockage of the 3- and 6-positions by the galactose-moiety.[1, 12]
The use of 6-tosylated glucose derivatives (e.g. 12, Scheme 2c)
as acceptors lead to the formation of α-(1,3)-linked disaccharides
in 62-95% yield. The tosyl group can be readily substituted by
various thio-sugars to form trisaccharides.[1] In this work we
extended this concept by the usage of 2- and 3-tosylated glucose
derivatives as acceptors yielding the corresponding α-(1,6)-
glucosylated products. Performed docking studies provide
insights into the basic principles of the glucansucrase’s acceptor
reactions suggesting that two different orientations are
responsible for its regioselectivity.
Glycoconjugates play a crucial role in biological recognition
processes in particular viral and bacterial infection.[2] Since the
synthesis of oligosaccharides is still a challenge for modern
science, researchers try to allocate modular assembly systems to
obtain tailor-made oligosaccharides.[3] The presence of several
chemically similar hydroxyl-groups results in many reaction steps
involving different strategies of orthogonal protection group
chemistry. Thus, the control of regio- and stereochemistry is the
key challenge in chemical synthesis of oligosaccharides.[4]
Glucansucrases have been shown to be promising tools for the
synthesis of glycoconjugates.[5] They belong to the glucosyl
hydrolase family GH70 (CAZy database)[6] and are expressed
extracellularly by bacteria such as Streptococcus mutans,
S. oralis or Lactobacillus reuteri.[7] Glucansucrases take part in
biofilm formation by synthesizing exopolysaccharides (EPS) from
sucrose 1 as donor (Scheme 1).[7] In contrast to glycosyl
transferases, most GH70 enzymes are readily available and their
substrate (sucrose 1) is cheap. They produce a variety of α-linked
glucosyl-polymers with different lengths and linkages among
which are dextran (2, mainly α-1,6), reuteran (α-1,4), alternan
(alternating α-1,3 and α-1,6) and mutan (α-1,3).[8] Beside polymer
formation and hydrolysis glucansucrases are able to glucosylate
numerous acceptors such as alditols, aliphatic alcohols and
natural aromatic compounds.[9] This reaction type makes them a
valuable tool in the chemo-enzymatic synthesis of
glycoconjugates since highly selective transfer reactions can be
accomplished without the need of protection groups. However,
their product promiscuity can be contra productive for the
synthesis of tailor-made carbohydrates. To overcome this
Scheme 1 GTFR from S. oralis produces a water soluble dextran with α-(1,6)
and α-(1,3) linkages (about 62 and 14%, respectively) with minor branching
(14%).[12]
For the synthesis of the 2-tosylated-glucoside 1-O-allyl-2-O-
p-toluenesulfonyl-α-D-glucopyranoside (allyl 2-Ts-α-D-Glcp, 7),
O-allyl-D-glucose was synthesized from glucose 3 according to
literature[13] (Scheme 2a). The crude mixture of α- and β-allyl D-
glucose was subsequently converted into 1-O-allyl-4,6-O-
benzylidene-D-glucopyranoside.[14] At this stage α- and β-anomer
were separated by column chromatography to yield 4 in 44%
overall yield (starting from glucose 3). The tosylation of 4 affords
a mixture of 2- and 3-tosylated products (5 and 6) which can be
isolated by column chromatography in yields of 50% and 12%
respectively. Subsequent deprotection of 5 with 70% AcOH
yielded 7 in quantitative yields.
[a]
[b]
J. Görl, C. Possiel, Prof. Dr. J. Seibel
Department of Organic Chemistry, Universität Würzburg,
Am Hubland, 97074 Würzburg Germany
E-mail: seibel@chemie.uni-wuerzburg.de
Prof. Dr. C. Sotriffer
Department of Pharmacy and Food Chemistry, Universität
Würzburg
Am Hubland, 97074 Würzburg, Germany
E-mail:.sotriffer@uni-wuerzburg.de
Supporting information for this article is given via a link at the end of
the document
Due to the low yields of the 3-tosylated derivate an alternative
way starting from di-O-isopropyliden α-D-glucofuranose 8 was
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1

10.1002/cbic.201700320
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sufficient yields (Scheme 3). However, the
acceptor without the allyl-group (3-Ts-D-Glcp, 10)
yielded the corresponding isomaltose-derivate α-
D-glucopyranosyl-(1,6)-3-O-p-toluoenesulfonyl-
D-glucopyranose (α-D-Glcp-(1,6) 3-Ts-D-Glcp 14)
in 88% yield (determined via HPLC, 40% isolated
yield, Scheme 3). Purification of 14 was not trivial
due to the easy loss of the tosyl group. The
obtained decomposition products could not be
fully characterized but ESI mass spectrometry
(m/z = 347.095 [M+Na]+, calc. 347.0949) suggest
an intramolecular substitution reaction forming an
epoxide.
The blockage of the favoured hydroxyl-group
of the acceptor with an orthogonal group (such as
other sugar moieties, azides, tosylates) probably
forces the acceptor to occupy different
orientations towards the donor causing a change
in the regioselectivity. For further investigation,
docking studies were performed to gain insights
into potential binding modes of the acceptors.
Since no crystal structure of GTFR is known to
date an N-truncated and mutated (D1025N)
variant of GTF-180a from Lactobacillus reuteri
180[15] was used as template (PDB identifier:
3HZ3, numbering in brackets) in the docking
process. GTF-180a has 47% sequence identity
(61% positives, 5% gaps, protein-BLAST tool)
and forms similar α-1,6 and 1,3-linked glucan with
branches[16]. In addition, Leemhuis et al. recently
compared the active site clefts of several
glucansucrases suggesting that α-(1,6)-linking
with minor branching GTFs exhibit similarly
formed clefts.[7] As a member of the α-amylase
Scheme 2 a) Synthesis of the acceptor 1-O-allyl-2-O-para-toloenesulphonyl-α-D-
glucopyranoside 7, b) Synthesis of 3-tosyl-D-glucopyranose 10 and subsequent allylation to 1-
O-allyl-3-tosyl-D-glucopyranoside 11, c) structure of the acceptor 1-O-allyl-6-O-para-
superfamily[17]
, the GTFR enzyme converts
sucrose via a double-displacement mechanism[18]
Glu554 (Glu1063) serves as proton source and
.
toloenesulphonyl-α-D-glucopyranoside 12 (previous work)[1]
.
facilitates the attack of Asp516 (D1025N) as
developed (Scheme 2b). The 3-position was tosylated in 68%
yields. The subsequent acidic deprotection of 9 was performed
under reduced pressure to shift the equilibrium to the product side
by removing acetone from the reaction mixture while maintaining
the reaction temperature at 40 °C (the product quickly
decomposes at high temperatures or basic conditions). 3-O-p-
toluenesulphonyl glucopyranoside 10 (3-Ts-D-Glcp) was
obtained in 87% yield. An optional allylation gave 1-O-allyl-3-O-
p-toluenesulphonyl glucopyranoside 11 (allyl 3-Ts-D-Glcp) as a
mixture of α- and β-anomers in 50% yields. 3-Ts-D-Glcp is readily
soluble in water whereas for the allylated acceptors, enzymatic
reactions were performed in 10% DMSO.
nucleophile on the anomeric centre of the glcp-moiety of sucrose.
A covalent enzyme-substrate complex (ES-complex) is formed
and the product is obtained by nucleophilic attack of an acceptor
on C1 releasing the free enzyme.[18] In order to imitate the attack
of the acceptor on the bound glucose, an ES-complex was
simulated from the crystal structure 3HZ3 which contains sucrose
The glucansucrase GTFR was incubated with allyl 2-Ts-α-D-
Glcp 7 and sucrose 1 (10 eq.) as a donor substrate. The enzyme
glucosylated allyl 2-Ts-α-D-Glcp 7 to give the isomaltose derivate
allyl α-D-Glcp-(1,6) allyl 2-Ts-α-D-Glcp 13 (Scheme 3) in 91%
yield (determined via HPLC, 62% isolated yield). The GTFR
prefers the transfer of glucose to the tosylated acceptors to the
production of the dextran polymer. After the consumption of the
acceptor the enzyme continues with the dextran formation while
13 is not degraded in the process. In contrast to the previous
reaction, using allyl 3-Ts-D-Glcp 11 as an acceptor gave only very
poor yields of glucosylated product which could not be isolated in
Scheme 3 Overview of the performed reactions with the GTFR enzyme and
the tosylated acceptor substrates. Reaction and isolated (in brackets) yields
are given.
2
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10.1002/cbic.201700320
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COMMUNICATION
bound in the active site due to a single mutation of Asp1025 to
asparagine. The fruf-moiety was deleted (PyMOL) and the
resulting structure was subsequently used as a template in
AutoDock 3.0. The dockings were then compared with the
binding (-6.61 kcal·mol-1) corresponds to a position in which O3 is
pointing to C1 of the protein-bound glucose, at a distance of 3.0 Å.
The next-closest group of the tosylated ligand is 4-OH, with a 3.1
Å distance to C1. Both OH-groups of the ligand appear to be
interacting favorably with Glu554 (Glu1063, general acid/base).
The 2-OH is forming a hydrogen bond with Asn520 (Asn1029) and
the tosyl group is placed in a shallow pocket (Figure 1A, S1B)
formed by the residues Aps627 (Asp1136) to Val633 (Gln1142)
which was identified as the +2 subsite before (Figure S1A).[15a]
Docking of allyl 2-Ts-α-D-Glcp 7 provided essentially three
different orientations of the ligand among the top-ranked results:
two in which the tosyl-group is placed in the +1 subsite and a third
in which the 6-OH of the allyl 2-Ts-α-D-Glcp is pointing towards
the C1 of the protein-bound glucose (Figure 1B, S1C). In terms of
the docked-energy score, the latter is only marginally (0.1 to 0.5
kcal·mol-1) less favorable than the former two and may thus be
considered as an energetically degenerate top-ranked result. The
orientation suggests that productive binding for reaction is readily
possible. In this binding mode (Figure 1B), the distance between
the O6 of the incoming ligand and the C1 of the bound glucose is
2.9 Å, which is close to the lower limit that can be expected in non-
covalent docking. In comparison to the 6-tosylated sugar, the
tosyl-group of allyl 2-Ts-Glcp occupies a different pocket (+2'
subsite, Figure 1B, S1C). The ligand appears well accommodated
in a cleft between Asn520 (Asn1029) and Trp556 (Trp1065), with
the sulfone group forming potential hydrogen bonds with the
Tyr469 (Ala978) backbone and the Asn520 (Asn1029) side chain
which was suggested by Vujicic-Zagar et al. to be the +2’
subsite.[15a]
Docking of allyl 3-Ts-Glcp 11 (α- and ß-anomer) with the
same settings as the other ligands did not provide any binding
orientation with a free OH-group in proximity to C1 of the protein-
bound glucose. The closest distance of a hydroxyl-group was 3.3-
3.5 Å and the calculated binding energies are considerably less
favorable (about 2 kcal·mol-1). The observations are in agreement
with the experiment yielding only low amounts of glucosylated
product. In contrast to the docking with the corresponding
allylated derivate, docking of 3-Ts-Glcp 10 (α-anomer) provided
two different docking modes: one in which the tosyl group is
pointing towards the anomeric carbon of bound glucose (not
shown) and one with the 6-OH pointing towards C1 at a distance
of 2.8-2.9 Å (-5.68 kcal·mol-1). The latter docking mode occupies
the same cleft as the best docking modes for allyl 6-Ts-Glcp (+2
subsite, Figure 1C, S1D). Thus it is reasonable to assume that the
allyl-group causes an additional steric hindrance that disables
effective glucosylation of the allylated derivate.
Figure 1 Docking studies of tosylated acceptor substrates with the GTFR
enzyme. A) The best docking result of allyl 2-Ts-α-D-Glcp (-6.11 kcal·mol-1)
occupies the +2 subsite, with a distance of 2.9 Å between C1 of the simulated
ES complex and O6; B) the best docking mode of 3-Ts-α-D-Glcp (-5.68
kcal·mol-1) occupies the +2' subsite instead; C) the best docking mode of
ally 6-Ts-α-D-Glcp (-6.61 kcal·mol-1) occupies the same subsite as the 3-
tosylated sugar; figure generated with PyMOL (PyMOL Molecular Graphics
System, Version 1.8; Schrꢀdinger, LLC; 2015)
Our goal was to utilize the glucansucrase GTFR as a tool in
chemo-enzymatic synthesis for rare sugars and sugar building
blocks. The decoration of the acceptor glucose with p-
toluenesulfonyl groups in 2-, 3- and 6-positions allows the control
of the regioselectivity of the transglucosylation reaction. The
synthesized nigerose and isomaltose derivatives equipped with p-
toluenesulfonyl groups are ideal functional building blocks for
follow-up reactions via SN2 substitutions with different
nucleophiles i.e. thioacetate[15b], azides, hydroxylates and for
synthesis of bacterial rare sugar building blocks.[19] In addition the
docking studies provided a plausible model of putative binding
modes of the acceptors that may be responsible for the strict
regioselectivity of the enzymatic reactions.
subsites identified through analysis of the GTF-180a crystal
structure in complex with maltose by Vujivic-Zahar et al.[15a] (PDB
identifier: 3KLL, Figure S1A).
In a first docking study the goal was to confirm the results
achieved experimentally by Hellmuth et al.[1] using 6-tosylated
glucose derivatives as substrates yielding α-1,3 linked
disaccharides. Docking of allyl 6-Ts-α-D-Glcp 12 provided a
variety of different overlapping orientations among the top ranks
in which the glucosyl-scaffold occupies the +1 subsite (Figure 1A,
S1B). The best result in terms of the estimated free energy of
3
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Experimental Section
Experimental details can be found in the Supporting Information
Acknowledgements
The authors acknowledge the financial support by the Federal
Ministry of Education and Research of Germany in the framework
of MEX15WTZ (project number 01DN16034).
Keywords: biocatalysis • substrate engineering • glucansucrase
• substrate promiscuity • rare sugar
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4
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COMMUNICATION
COMMUNICATION
Julian Görl, Christian Possiel, Christoph
Sotriffer, Jürgen Seibel*
Page No. – Page No.
Extending the scope of GTFR
Glucosylation reactions with
tosylated substrates for rare sugars
synthesis
Substrate directed synthesis of rare sugars. Tosylated glucose derivates were
used as acceptors of the glucansucrase R from S. oralis. The tosyl groups allow
only distinct orientations of the acceptor in the active cleft (docking experiments)
resulting in highly regioselective glucosylated products.
For internal use, please do not delete. Submitted_Manuscript
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