S -Glycosides: Synthesis of S -linked arabinoxylan oligosaccharides

Article abstract of DOI:10.1039/d0ob00470g

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this con

Full text of DOI:10.1039/d0ob00470g

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DOI: 10.1039/D0OB00470G
Organic and Biomolecular Chemistry
ARTICLE
S-Glycosides: Synthesis of S-linked Arabinoxylan Oligosaccharides
Cecilia Romanò,a Hao Jiang, a Irene Boos a and Mads H. Clausen a
Received 00th January 20xx,
Accepted 00th January 20xx
S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid
structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this
context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a
small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-
glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected L-arabinopyranoside triflate. The
resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively
protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation
reactions to introduce branching L-arabinofuranosides.
DOI: 10.1039/x0xx00000x
constituents, xylan and arabinose, is of great interest for the
production of advanced biofuels.11 Moreover, the partial
degradation of AXs to arabinoxylooligosaccharides (AXOs)
opens the possibility for their application as prebiotics.12,13 The
main enzymes required for the degradation of AXs are endo-1,4-
β-D-xylanases (EC 3.2.1.8), 1,4-β-D-xylosidase (EC 3.2.1.37), and
α-L-arabinofuranosidases (AFs) (EC 3.2.1.55).3,7,14 In order to
exploit these enzymes efficiently in the bioconversion of
hemicellulose feedstocks, there is a need for systematic
investigations of their substrate specificities.
Introduction
In an effort to shift our economy towards more sustainable
resources, research into the bioconversion of lignocellulosic
biomass to produce second generation bioethanol and value
added chemicals has flourished during the last two decades.1–4
This feedstock, an intricate structure of cellulose, hemicellulose,
and lignin, is cheap, abundant, and sustainable. In fact, it
represents the non-edible component of plant biomass and it is
generally obtained from agricultural waste, pulp and paper
industry waste, and deciduous woods, meaning that its
utilization does not interfere with the cultivation of food
supplies.5–7 Hemicellulose constitutes around 20–35% of
lignocellulosic biomass and is mainly composed of four
structurally diverse polysaccharides: xylans, mannans,
xyloglucans, and glucans, including mixed linkage glucans.8
Hemicellulosic arabinoxylans (AX) are an important xylan
subclass, where the β-(14)-linked D-xylopyranoside backbone
is substituted by α-(12) and/or α-(13) L-arabinofuranosyl
residues. AX is mostly found as the main polysaccharide in a
variety of tissues of commercial cereals, such as wheat, rye,
barley, oat, rice, and corn – and AXs from cereal-processing by-
products represent an attractive and underutilized feedstock
resource.8,9
OH
O
OH
O
O
O
HO
O
HO
HO
O
HO
S
O
OH
OH
HO
OH
O
1
HO
OH
OH
O
OH
O
O
O
O
HO
O
HO
HO
O
HO
S
HO
OH
OH
OH
O
HO
S
OH
2
OH
O
OH
O
The process from raw biomass to value-added products
requires pre-treatment of the material to break the lignin seal
and allow for further degradation to its monosaccharide
components.4,5,7 To this end, enzymatic bioconversion has been
investigated as
hydrothermal and chemical processes.3,8,10 The biodegradation
of hemicellulose, and in particular of AXs into their sugar
O
O
O
HO
O
HO
HO
O
HO
O
OH
OH
OH
O
OH
O
HO
OH
a valuable alternative to conventional
HO
S
OH
3
OH
O
OH
O
O
O
HO
O
HO
HO
O
HO
a. Center for Nanomedicine & Theranostics, Department of Chemistry, Technical
University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark. E-mail:
mhc@kemi.dtu.dk
Electronic Supplementary Information (ESI) available: experimental procedures;
NMR spectra. See DOI: 10.1039/x0xx00000x
HO
OH
OH
HO
4
Chart 1: Targeted AXs derivatives 1‒3 and tetraxylan 4
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Organic & Biomolecular Chemistry
Page 2 of 6
ARTICLE
Organic and Biomolecular Chemistry
OBz
View Article Online
DOI: 10.1039/D0OB00470G
O
BzO
HO
O
OBz
O
BzO
O
O
AcO
OBz
S
NH2
NH2
AcO
8
OAc
OBz
OAc
OAc
5
6
O
O
O
BzO
O
AcO
AcO
AcO
AcO
O
BzO
S
S
OBz
SPh
TfO
AllocO
O
AllocO
AllocO
O
OBz
OLev
OLev
O
SPh
7
9
OLev
Figure 1: Synthetic approach for targets 1‒3
While collections of synthetically defined xylo- and presence of different branching patterns of L-arabinofuranosyl
arabinoxylooligosaccharides obtained through chemical residues gives the opportunity to pinpoint both α-L-
synthesis15–20 or chemoenzymatic methodologies21 can greatly arabinofuranosidase activity and the selectivity of those
contribute to define specificities and preferences of some AX enzymes that are believed to have
degrading enzymes, unique structure-activity information can xylanases/arabinofuranosidase activity.14,39
be obtained by utilizing resistant substrates such as thio-
a
mixed
oligosaccharides. These unnatural compounds, where at least
one glycosidic oxygen atom is replaced by a sulphur atom, are
Results and discussion
mimics acting as competitive inhibitors of many glycosyl
hydrolases, thus representing excellent tools for biochemical
and structural investigations of carbohydrate processing
enzymes.22,23
Synthesis of branched S-linked arabinoxylans 1‒3
To access the desired branched target structures 1‒3, the key S-
linked tetrasaccharide 9 was designed (Figure 1). The structure
is 2’’-O-levulinoyl (Lev) and 3’’-O-allyloxycarbonyl (Alloc)
substituted, thus allowing the regioselective unmasking of
either 2’’-OH or 3’’-OH for further glycosylation with an L-
arabinofuranoside donor. Moreover, acetyl and benzoyl ester
groups are employed as global protecting groups to ease the
final deprotection of the assembled structures in the presence
of the S-linkage. For the synthesis of key intermediate 9, a [2+2]
building block strategy with the orthogonally protected S-linked
disaccharide donor 7 and 4’-OH acceptor 8 was employed. For
the synthesis of thio-linked disaccharide 7, an anomeric thiol
group S-alkylation methodology was selected, with isothiourea
Synthetically, a thio-linkage can be introduced in one of two
ways: 1) by a glycosylation reaction between a suitable glycosyl
donor and a thiol acceptor (via a classical acid-catalysed S-
glycosylation24–26 or via
a base-promoted glycosylation
consisting of a SN2 displacement of a glycosyl halide by a
thiolate27–30); or 2) through anomeric thiol S-alkylation,
consisting in the reaction of a saccharide electrophile with a
nucleophilic anomeric thiol moiety.31–35 Both strategies, which
have been extensively reviewed,36 have been exploited for the
synthesis of several S-linked oligosaccharides. Recently, we
have presented our synthetic approaches towards the synthesis
of an S-linked tetraxylan.37 Several strategies for the formation
of the thio-linkage were explored and methods involving 1-
thioglycosides were found to be preferable due to a reduction
of by-products. As a natural progression of this previous
research, we have been interested in the preparation of S-
linked arabinoxylan derivatives 1‒3, together with the
optimization of the synthesis of linear S-linked tetraxylan 4
(Chart 1). These targets are ideal substrates for the study of AXs
degrading enzymes: their backbone, with four D-xylopyranose
residues, has a suitable length for recognition by both endo-1,4-
β-D-xylanases and 1,4-β-D-xylosidases.14,38 Moreover, the
xyloside
5
and the 4-O-trifluoromethanesulphonyl L-
arabinopyranoside 6 as the coupling pair (Figure 1). This choice
of strategy is justified by both the ease of access of building
blocks 5 and 6 as well as the greater robustness of isothiourea-
based couplings compared to those involving the use of free
thiol substrates.
While isothiouronium salt 5 could be easily accessed in one step
from 2,3,4-tri-O-acetyl-β-D-xylopyranosyl bromide 1040 in 98%
yield, the preparation of building block 6 required more
protecting group manipulations (Scheme 1). 3,4-O-
Isopropylidene L-arabinopyranoside 1141 was treated with
levulinic acid, DCC, and DMAP in anhydrous CH2Cl2 to afford
O
O
a
AcO
AcO
AcO
AcO
S
NH2
NH2
98%
AcO
OAc
Br
10
5
TfO
RO
O
HO
HO
HO
RO
OAc
O
O
O
O
O
O
AcO
AcO
S
RO
d
e
c
f
SPh
OLev
SPh
SPh
SPh
SPh
O
89%
79%
94%
57%
OLev
OR
OLev
OLev
14: R = Alloc
(15: R = Allyl)
6: R = Alloc
(16: R = Allyl)
7: R = Alloc
(17: R = Allyl)
11: R = H
12: R = Lev, 84%
13
b
Scheme 1: Reagents and conditions: (a) thiourea, CH3CN, reflux, 10 min; (b) levulinic acid, DCC, DMAP, CH2Cl2, RT, 6 h; (c) 80% aq. AcOH, 60 °C, 3 h; (d) allyl chloroformate,
CH2Cl2/pyridine, ‒30 °C, 2 h; (e) Tf2O, CH2Cl2/pyridine, 0 °C, 15 min; (f) Et3N, DMF/CH3CN, RT, 2 h.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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OAc
O
AcO
AcO
S
Page 3 of 6
Organic & Biomolecular Chemistry
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SPh
OLev
AllocO
O
7
OBz
O
Organic and Biomolecular Che
B
m
zOistry
ARTICLE
a
O
OBz
75%
HO
BzO
O
OBz
8
test reaction with NIS/TfOH at low temperature (‒50 °C)
OBz
O
View Article Online
OAc
O
O
BzO
afforded by-product 18, evidence DoOfI: 1p0.o10st3-9g/lDy0cOoBsy0l0a4t7io0Gn
hydrolysis of the thio-linkage, followed by dimerization to form
the disulphide (entry 1). DMTST promoted glycosylation proved
to be incompatible with the Alloc functionality (entry 2), while
MeOTf did not afford any conversion over 24 hours (entry 3).
On the other hand, and to our delight, glycosylation with
NIS/AgOTf at 0 °C afforded the desired product 9 in a 77% yield
(entry 4). Glycosylation at lower temperatures (‒40 °C to ‒10
°C) did not afford activation of the phenyl thioglycoside.
Interestingly, the reaction proceeded smoothly and without
affecting the S-interglycosidic linkage, despite the use of
thiophilic NIS. With tetrasaccharide 9 in hand, orthogonal
deprotections to afford acceptors 19 and 20 were performed
(Scheme 3). Alloc removal proceeded in a 65% yield in the
presence of Pd(PPh3)4 to give 19. Similarly, Lev removal with
hydrazine acetate afforded 20 in 79% yield. Gratifyingly, both
compounds reacted quickly and in high yields in TMSOTf-
promoted glycosylation reactions with the 2,3,5-tri-O-benzoyl-
α-L-arabinofuranosyl trichloroacetimidate donor 2142 (22,
quant.; 23, 93%). The newly formed pentasaccharides 22 and
23 were then deprotected under standard conditions to afford
desired targets 1 and 2 in 80% and 45% yield, respectively. In a
similar manner, intermediate 9 was subjected to Alloc
deprotection, followed by Lev removal to obtain 2’’,3’’-diol
acceptor 24 in 72% yield over two steps. Compound 24 was
subsequently reacted with donor 21 (2.5 equiv.) in the presence
of TMSOTf to give hexasaccharide 25 in 97% yield. A Zemplén
reaction finally afforded desired target 3 in 82% yield.
AcO
AcO
O
BzO
S
OBz
O
AllocO
O
OBz
OLev
9
derivative 12 in 84% yield. After removal of the 3,4-O-
isopropylidene group under acidic conditions (13, 89%),
position 3 of the diol was regioselectively protected with an
allyloxycarbonyl group. The reaction, performed with allyl
chloroformate at ‒30 °C afforded 14 in 79% yield. Careful
Scheme 2: (a) NIS/AgOTf, CH2Cl2, 4 Å MS; ‒10 °C  0 °C, 1 h.
control of the temperature proved essential for the preparation
of the desired compound and only trace amounts of double
protected 3,4-O-allyloxycarbonyl were formed during the
reaction. Finally, compound 14 was reacted with
trifluoromethanesulphonic anhydride in pyridine/CH2Cl2 to
afford building block 6 in 94% yield. The coupling reaction of
building blocks 5 and 6 proceeded in a 33% yield in anhydrous
CH3CN. A more satisfactory yield of 57% was obtained when the
solvent was substituted with a 1:1 mixture of anhydrous CH3CN
and DMF and only 1.2 equivalents of 6 were employed. While
no significant by-products were observed and unreacted triflate
6 could be recovered from the reaction mixture, it was not
possible to isolate unreacted isothiouronium salt (the limiting
reagent). Albeit not particularly high yielding, this first report on
the formation of the key S-glycosidic linkage with an
orthogonally protected building block, as opposed to the use of
per-acetylated/per-benzoylated
substrates,31
allows
explorations into the synthesis of more complex S-glycosides. In
order to compare reactivity to the use of ether-type protecting
groups in position 3, allyl protected derivative 15 was also
synthesized on a test scale and converted to its triflate
derivative 16. Coupling of 16 with isothiouronium salt 5
afforded the desired product 17 in 41% yield when reacted
under the same conditions as 7. While proving that the
methodology can be extended to a larger set of protecting
groups, it is important to note that the later deprotection of
ether-type protecting groups, such as benzyl and allyl, could
prove difficult due to the presence of the thio-linkage.
Synthesis of S-linked tetraxylan 4
Our previous approach towards the synthesis of 4 involved the
preparation of a trifluoroacetimidate donor and its reaction
with a thio-acceptor. In this work, a simpler route, inspired by
the one presented in the previous section, was devised. The S-
linkage was synthesised from xylothiouronium salt 5 and L-
arabinopyranoside triflate derivative 28, which was synthesized
in 4 steps from 10: acetylation under standard conditions and
cleavage of the 3,4-O-isopropylidene group (26, 91% over 2
steps) was followed by selective benzoylation to afford 3-OBz
derivative 27 in 63% yield.
Key disaccharide 7 was then reacted with acceptor 8, in the
presence of several promoting systems (Scheme 2, Table 1). A
Promoting system
Temperature
Product
Yield
57%
1
NIS (1.5 equiv.)/TfOH (0.15 equiv.)
‒50 °C
Decomposition – Alloc incompatible with DMTST
No reaction
‒
‒
75%
2
3
4
DMTST (3 equiv.)
MeOTf (5 equiv.)
NIS (1.5 equiv.)/AgOTf (0.25 equiv.)
0 °C  RT
RT
‒10 °C  0 °C
Table 1: Test glycosylations with donor 7 and acceptor 8
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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ARTICLE
Promoting system
Temperature
Product
Yield
57%
1
NIS (1.5 equiv.)/TfOH (0.15 equiv.)
‒50 °C
OBz
OLev
OBz
O
O
O
O
BzO
AllocO
BzO
BzO
O
BzO
O
BzO
S
S
OBz
O
O
AllocO
O
O
OBz
OBz
OLev
18
Decomposition – Alloc incompatible with DMTST
No reaction
‒
‒
75%
2
3
4
DMTST (3 equiv.)
MeOTf (5 equiv.)
NIS (1.5 equiv.)/AgOTf (0.25 equiv.)
0 °C  RT
RT
‒10 °C  0 °C
OBz
OAc
O
O
BzO
AcO
AcO
O
BzO
S
OBz
O
AllocO
O
O
OBz
OLev
9
OBz
O
OBz
O
BzO
O
OAc
O
OAc
O
O
O
O
BzO
BzO
AcO
AcO
O
BzO
AcO
AcO
a or b
S
S
OBz
OBz
O
O
R1O
AllocO
O
OR2
OBz
OBz
OLev
19: R1 = H, R2 = Lev, 65%
9
HN
20: R1 = Alloc, R2 = H, 79%
BzO
CCl3
O
O
c
BzO
OBz
21
OH
O
OBz
OH
O
OAc
O
O
O
O
O
HO
BzO
d or e
HO
HO
AcO
AcO
O
BzO
S
S
OH
OBz
O
O
R1O
R1O
HO
O
O
OR2
OR2
OH
OBz
22: R1 = 2,3,4-OBz--Araf, R2 = Lev, quant.
23: R1 = Alloc, R2 = 2,3,4-OBz--Araf, 93%
1: R1 = -Araf, R2 = H, 80%
2: R1 = H, R2 = -Araf, 45%
HN
BzO
O
CCl3
O
BzO
OBz
21
OR1
OBz
OAc
OR
R1O
O
O
O
OR1
O
O
BzO
AcO
AcO
O
BzO
RO
RO
O
R1O
OR1
S
g
S
OBz
O
R1O
O
O
O
O
O
OR2
97%
OBz
O
R1O
O
9: R1 = Alloc, R2 = Lev
24: R1 = H, R2 =H, 72%
25: R = Ac, R1 = Bz
3: R = H, R1 = H, 82%
f
R1O
R1O
h
R1O
R1O
O
OR1
Scheme 2: Reagents and conditions: for 19: (a) Pd(PPh3)4, AcOH, RT, 5h; for 20: (b) H2NNH2·HOAc, AcOH/pyridine, RT, 10 min; (c) TMSOTf, CH2Cl2, 4 Å, ‒40 °C, 15 min;
for 1: (d) (i) H2NNH2·HOAc, MeOH/CH2Cl2, RT, 3 h, (ii) MeONa, CH2Cl2/MeOH, RT, 16 h; for 2: (e) (i) Pd(PPh3)4, THF/H2O, RT, 3h, (ii) MeONa, CH2Cl2/MeOH, RT, 16 h; (f)
(i) Pd(PPh3)4, THF/H2O, RT, 3h, (ii) H2NNH2·HOAc, AcOH/pyridine, RT, 10 min; (g) TMSOTf, CH2Cl2, 4 Å, ‒60 °C, 20 min; (h) MeONa, CH2Cl2/MeOH, RT, 16 h.
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ARTICLE
Interestingly, attempted selective acetylation of the 3,4-diol as a thioglycoside donor in a [2+2] glycosylation for the
under similar conditions (1.1 equiv. AcCl, pyridine, ‒30 °C) gave synthesis of the linear tetraxylan when AgOTf/NIS was
an inseparable 1:1 mixture of 3-OAc and 4-OAc products, employed as promoting system. While some examples of
possibly because of a higher rate of 3-O-acetyl ester migration hydrolysis of phenyl thioglycosides with inner S-glycosidic
from position 3 to 4, compared to the 3-O-benzoyl ester. linkages can be found in the literature,37,43 to the best of our
Reaction of 27 with trifluoromethanesulphonic anhydride and knowledge this is the first report on the activation of a phenyl
pyridine gave compound 28 in 93%. The subsequent coupling thioglycoside in the presence of an interglycosidic S-linkage for
reaction with isothiouronium salt 5 afforded S-disaccharide 29 a subsequent glycosylation. The reactivity of the two sulphur
atoms in donors 7 and 29 is likely to be influenced similarly by
the protecting groups at C-2/C-2’ as well as elsewhere, since
O
HO
RO
TfO
BzO
they are all disarming.44,45 Instead, the chemoselectivity
observed may stem from variation in nucleofugicity46 or steric
deactivation.46,47 Moreover, glycosylation reactions with the L-
arabinofuranosyl imidate donor in the presence of TMSOTf
proved to not affect the S-interglycosidic linkage.
O
O
O
c
a
SPh
SPh
SPh
O
91%
93%
OH
OAc
OAc
11
26: R = H
27: R = Bz, 61%
28
b
OAc
O
AcO
AcO
S
d
SPh
BzO
O
55%
OAc
29
Conflicts of interest
75%
e
There are no conflicts to declare.
OR1
OR
O
R1O
O
O
O
RO
RO
O
OR1
S
R1O
R1O
O
OR1
OR
Acknowledgements
30: R = Ac, R1 = Bz
4: R = H, R1 = H, 87%
f
We are grateful to the Villum Foundation (grant no. 9283), the
Novo Nordisk Foundation (grant no. NNF18OC0053048), and
the Danish Council for Independent Research (grant no. 9041-
00080B) for supporting our research. C. R. acknowledges
financial support from the European Commission’s ARGONAUT
project (grant agreement no. 841278). The 800 MHz NMR data
Scheme 3: Reagents and conditions: (a) (i) Ac2O, pyridine, RT, 16 h, (ii) aq. 80%
AcOH, 60 °C, 4 h; (b) BzCl, pyridine, ‒35 °C, 1 h; (c) Tf2O, CH2Cl2/pyridine, 0 °C, 45
min; (d) 5, Et3N, CH3CN, RT, 16 h; (e) 8, NIS/AgOTf, CH2Cl2, 4 Å MS; ‒15 °C  0 °C,
30 min; (f) MeONa, CH2Cl2/MeOH, RT, 16 h.
in 55% yield. Although similar in yield to the reaction described were recorded at the NMR Center at DTU supported by the
in our previous work with a trifluoroacetimidate donor and a Villum Foundation.
thio-acceptor,37 the conversion, although incomplete, is cleaner
and affords the possibility of isolating unreacted 28 for recycling
as little elimination of the triflate is observed. Glycosylation of
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newly synthesized 29 with acceptor 8 afforded tetrasaccharide
30 in 75% yield. Again, the promoting system NIS/AgOTf
uniquely activated the thioglycoside at the reducing end of 29,
without affecting the S-interglycosidic linkage. Final
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afforded
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A synthetic route towards the preparation of four substrates for
the study of AX degrading enzymes has been developed. The
key unnatural S-linkage was introduced by coupling with an
orthogonally protected L-arabinopyranoside, and proved to be
compatible with deprotection reactions under mild conditions
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