A Rapid and Mild Sulfation Strategy Reveals Conformational Preferences in Therapeutically Relevant Sulfated Xylooligosaccharides

Article abstract of DOI:10.1002/chem.202100527

Although sulfated xylooligosaccharides are promising therapeutic leads for a multitude of afflictions, the structural complexity and heterogeneity of commercially deployed forms (e. g. Pentosan polysulfate 1) complicates their path to further clinical development. We describe herein the synthesis of the largest homogeneous persulfated xylooligomers prepared to date, comprising up to eight xylose residues, as standards for biological studies. Near quantitative sulfation was accomplished using a remarkably mild and operationally simple protocol which avoids the need for high temperatures and a large excess of the sulfating reagent. Moreover, the sulfated xylooligomer standards so obtained enabled definitive identification of a pyridinium contaminant in a sample of a commercially prepared Pentosan drug and provided significant insights into the conformational preferences of the constituent persulfated monosaccharide residues. As the spatial distribution of sulfates is a key determinant of the binding of sulfated oligosaccharides to endogenous targets, these findings have broad implications for the advancement of Pentosan-based treatments.

Full text of DOI:10.1002/chem.202100527

Accepted Article
Accepted Article
Title: A Rapid and Mild Sulfation Strategy Reveals Conformational
Preferences in Therapeutically Relevant Sulfated
Xylooligosaccharides
Authors: Yen Vo, Brett D. Schwartz, Hideki Onagi, Jas S. Ward,
Michael G. Gardiner, Martin G. Banwell, Keats Nelms, and
Lara Malins
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202100527
Link to VoR: https://doi.org/10.1002/chem.202100527
01/2020

10.1002/chem.202100527
Chemistry - A European Journal
FULL PAPER
A Rapid and Mild Sulfation Strategy Reveals Conformational
Preferences in Therapeutically Relevant Sulfated
Xylooligosaccharides
Yen Vo,[a] Brett D. Schwartz,[a]* Hideki Onagi,[a] Jas S. Ward,[a] Michael G. Gardiner,[a] Martin G.
Banwell,[a] Keats Nelms[b] and Lara R. Malins [a]*
[a]
[b]
Dr. Y. Vo, Dr. B. D. Schwartz, Dr. Hideki Onagi, Dr. J S. Ward, Dr. M. G. Gardiner, Prof. M. G. Banwell, Dr. L. R. Malins
Research School of Chemistry
Australian National University
Canberra, ACT 2601, Australia
E-mail: brett.schwartz@anu.edu.au; lara.malins@anu.edu.au
Twitter: @Malins_Lab
Dr. K. Nelms
Beta Therapeutics Pty. Ltd.,
Level 6, 121 Marcus Clarke Street
Canberra, ACT 2601, Australia.
Supporting information for this article is given via a link at the end of the document.
Abstract: Although sulfated xylooligosaccharides are promising
therapeutic leads for multitude of afflictions, the structural
A) Sulfation as a key regulator of bioactivity
a
e.g. glycosaminoglycans (GAGs)
“Sulfation Code”
complexity and heterogeneity of commercially deployed forms (e.g.
Pentosan polysulfate 1) complicates their path to further clinical
development. We describe herein the preparation of the largest
homogeneous persulfated xylooligomers prepared to date—
comprising up to eight xylose residues—as standards for biological
OR
–OOC
•
•
Diverse sulfation patterns
Influence molecular
interactions/bioactivity
Implications for synthetic
GAG mimetics as
possible
sites of
sulfation
O
O
O
O
RO
RO
•
RO
RHN
n
therapies
heparan sulfate
Pentosan polysulfate (PPS) – a clinically relevant heparan sulfate mimetic
studies. Near quantitative sulfation was accomplished using
a
O
O
O
O
O
remarkably mild and operationally simple protocol which avoids the
need for high temperatures and a large excess of the sulfating
reagent. Moreover, the sulfated xylooligomer standards so obtained
enabled definitive identification of a pyridinium contaminant in a
sample of a commercially prepared Pentosan drug and provided
significant insights into the conformational preferences of the
constituent persulfated monosaccharide residues. As the spatial
distribution of sulfates is a key determinant of the binding of sulfated
oligosaccharides to endogenous targets, these findings have broad
implications for the advancement of Pentosan-based treatments.
O
O
R1O
–O3SO
O
–O3SO
–O3SO
–O3SO
–O3SO
O
–O3SO
–
OSO3
n
–
1
OSO3
OR2
O
O
O
R1 = H, Ac
R2 = CH3, SO3
–O3SO
–
COO–
–O3SO
n
B) This work: persulfated xylooligosaccharides via mild sulfation
ClSO3Me
or
O
O
O
[Sulfation]
Quant. @ 0 o
SO3.DMF
HO
HO
O
O
R
HO
HO
•
•
C
HO
HO
HO
Large excess of
reagent not required
n
n = 0, 1, 3, 6
[Xylooligosaccharides]
•
2–8 carbohydrate units
Quant. persulfation
Conformational
preference: 1C4 for non-
reducing sugars
–
–
OSO3
O
OSO3
•
•
O
–O3SO
–O3SO
O
O
O
–O3SO
OH
Introduction
–O3SO
Xyl2 – Xyl8 units
–O3SO
n
Sulfation is a powerful regulator of bioactivity.[1] For example,
glycosaminoglycans (GAGs), ubiquitous components of the
extracellular matrix broadly involved in signaling and
development, have complex and diverse carbohydrate sulfation
patterns that have been linked to their specific biological
functions.[2] The precise array of negatively charged sulfate
groups serves to mediate a variety of binding events largely
through targeted electrostatic interactions.[3] Accordingly,
elucidation of the so-called GAG “sulfation code,”[3,4] including
important work regarding 3-O-sulfation of heparan,[5] is crucial to
unlocking the complexities of GAG biochemistry and has fueled
considerable interest in sulfated GAG mimetics as useful
therapeutic leads.[6]
Figure 1. A) The importance of sulfation in glycosaminoglycans (GAGs) and
therapeutic GAG mimetics; B) The persulfated xylooligosaccharides accessed
in this work.
for the treatment of osteoarthritis. PPS is
a complex,
heterogeneous and semi-synthetic drug composed of sulfated,
β-1→4-linked xylooligosaccharides of varying lengths. The
structure is further complicated by intermittent C-2 substitution
with sulfated 4-O-methyl-α-D-glucuronic acid units. Difficulties
isolating the individual components and thereby establishing
precise structure-activity relationships represent a major barrier
to further clinical development of PPS-based treatments.
Moreover, recent reports of retinal toxicity[7] associated with
prolonged exposure to PPS warrant urgent examination of its
precise composition. Prompted by the immense therapeutic
potential of PPS and an on-going medicinal chemistry campaign
A notable example is Pentosan polysulfate 1 (PPS, Figure 1A), a
heparan sulfate mimic that is currently the only prescribed
therapeutic for interstitial cystitis and is in clinical development
1
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10.1002/chem.202100527
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in our own laboratory, we sought to prepare homogeneous, low
molecular weight sulfated xylooligomers lacking the glucuronic
acid branch so as to facilitate detailed biological studies of
unbranched oligosaccharides. It was envisioned that such a
collection of standards (Figure 1B) would prove valuable in
unravelling the Pentosan sulfation code.
axial” 1C4 conformation of b-D-substituted xylopyranosides
occurs with increasing degrees of sulfation (e.g. sulfation at
either O-2 and O-3 (11), or at O-2, O-3 and O-4 (12) (Figure 2B)
as evidenced by small JH1-H2 coupling constants. Moreover,
Nishida postulated that the characteristic 2,3-di-O-sulfate pattern
in the b-1→4-xyloglycan structure of Pentosan PS 1 may reflect
3
a
1C4 conformational preference among the constituent xylose
1
Herein, we describe the realization of such objectives through
the convergent synthesis of persulfated xylooligosaccharides
incorporating up to eight xylose units (Xyl2–Xyl8-SO3, Figure 1B)
and so representing the largest single-species sulfated
homologues reported to date.[8] This outcome was accelerated
by optimization of a mild and quantitative sulfation protocol using
methyl chlorosulfate, a largely overlooked reagent that also
enabled herein the rapid sulfation of amino acids, steroids, and
acid-sensitive substrates. Furthermore, as a result of rigorous
NMR spectroscopic studies, insights have been gained into the
conformational preferences of Pentosan-derived sulfated
xylooligosaccharides. Such information on the spatial orientation
of sulfate groups has important implications for computational
drug design and the ability of sulfated xylooligosaccharides to
engage biological targets for therapeutic benefit.
units.[14] However, the complexity of the H NMR spectral data
displayed by Pentosan PS mixtures limits the “readable”
coupling constant data and so this proposal remains to be
verified. Accordingly, elucidating the conformational preferences
of individual Pentosan-type persulfated xylooligosaccharides
emerged as a second aim of our work, with the targeted Xyl2-
SO3 8, Xyl3-SO3 13, Xyl5-SO3 14 and Xyl8-SO3 15 systems
expected to serve as highly informative reference standards
through which the structure of Pentosan PS itself could be better
understood (Figure 5, vide infra).
A) Initial sulfation attempts
O
O
HO
HO
HO
HO
OH
–
OMe
OSO3
O
SO3.Pyr
DMF
HO
HO
N
2
3
60 °C
18 h
O
O
–O3SO –O3SO
O
HO
HO
HO
HO
HO
OH
5
4
–
–
SO3.DMF
DMF
6
7: R’ = SO3
OSO3
–O3SO
O
unstable
O
O
H2O
–O3SO
Results and Discussion
8: R’ = H
-15 to 0 °C
2 h
–O3SO
OR'
–O3SO
Since uniform persulfation of carbohydrates can be challenging,
often requiring elevated temperatures and a large excess of the
sulfating agent,[9] conditions leading to the targeted persulfated
polysaccharides required careful consideration. Prompted by
reports[10] on the successful exhaustive sulfation of xylan with
SO3•pyridine to prepare PPS, we first examined this method.
Specifically, xylose 2, methyl-b-xyloside 3 and xylobiose 4 were
each subjected to reaction with SO3•pyridine at elevated
temperature (Figure 2A). Unfortunately, under these conditions,
clean persulfation of compounds 2–4 was compromised by the
covalent addition of pyridine to the reducing ends of these
systems and concomitant cleavage of the b-1→4 linkage of
xylobiose. The addition of pyridine to the reducing terminus of
xylans has recently been observed by others,[11] and our NMR
analysis of a commercial sample of a marketed Pentosan drug
supports the presence of pyridinium contaminants stemming
from the sulfation process (see Figure 6 and pages 56 and 239
–244 of the Supporting Information for further discussion).
B) Conformational effects
cf. Ref. 14 (Nishida)
OPNB
OPNB
–
–
OSO3
OSO3
O
O
HO
O
HO
HO
O
OPNB
OPNB
–O3SO
11
–
12
–
HO
4C1
HO
10
9
–
OSO3
OH
OSO3
OSO3
4C1
1C4
1C4
increasing degree of sulfation
Persulfated xylobioses:
J = 3.4 Hz, d
–
OSO3
OSO3
–
Hd
O
OSO3
–
–
O
–O3SO
–O3SO
OSO3
O
O
–O3SO
–O3SO
O
Hc
–
OSO3
O
–O3SO
–O3SO
Ha
–O3SO
Hb
J = <1 Hz, s
Ha'
Hb'
Hc'
Hd'
J = <1 Hz, s
α-7
J = <1 Hz, s
β-7
Figure 2. A) Initial sulfation attempts using SO3•pyridine and SO3•DMF; B)
Conformational effects of increasing sulfation and observed coupling
constants for persulfated xylobiose 7. Note: sulfate counterions are omitted
throughout for clarity.
On the basis of the foregoing, our attention turned to the use of
SO3•DMF 6, noted for its high reactivity as a sulfating agent,
including in the production of xylan sulfates from plant
isolates.[10a] Accordingly, xylobiose 4 was treated with this
reagent at –15 °C and after two hours the a- and b-anomeric
forms of xylobiose hexasulfate 7 were obtained together with
pentasulfate 8, a reducing sugar, resulting from hydrolysis of the
initially formed anomeric sulfate. Interestingly, in the 1H NMR
spectrum of the crude persulfated material, the C-1 (anomeric)
hydrogen associated with anomer b-7 (5.80 ppm) appeared as a
singlet (3JH1-H2 = 0-1 Hz) (Figure 2B), as did the C-1` hydrogens
of both compounds C-1a 7 and C-1b 7. The conformational
equilibria associated with sulfated monosaccharides have been
assessed by Wessel,[12] Nifantiev[13] and Nishida[14] through
careful analysis of 1H NMR coupling constants. These studies
In situ sulfation approach using methyl chlorosulfate
Although SO3•DMF 6 readily afforded persulfated 7, before
embarking on the synthesis of more complex polysulfated
oligosaccharides, we sought to revisit viable approaches to
exhaustive sulfation. A variety of SO3•amine complexes[15] are
commonly[16] exploited for this purpose. However, the majority of
these require the application of elevated temperatures (e.g.
>70 °C) and large excesses of the reagent (5-10 equiv. per OH
group) to ensure high degrees of sulfation,[9] prompting the
exploration of microwave-assisted[17]
and acid-catalyzed
approaches,[18] as well as the judicious use of co-solvents.[8b,19]
The reactivity of the SO3 complex can be roughly correlated to
the Lewis basicity of the amine, with stronger bases forming
more stable and thus less reactive complexes.[15] Given the
weak basicity of DMF, the efficacy of SO3•DMF at low
temperatures in our initial study is unsurprising. However, as
4
revealed that a shift from the typical C1 to the “inverted” or “all-
2
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SO3•DMF is typically prepared by treating DMF with sulfur
With optimal sulfation and purification procedures established, a
trioxide,[20] we sought to identify an operationally simpler protocol. small suite of substrates (Figure 4), including amino acids (Ser,
A seemingly neglected entry in a patent published in 1951[21] on
the sulfation of leuco dyes led us to explore this process for the
in situ production of SO3•DMF. Accordingly, dropwise addition of
methyl chlorosulfate 16[22,23] to ice-cold DMF resulted in an
exothermic reaction with notable gas evolution due to the
production of chloromethane.[24] Crystals were obtained from the
reaction mixture after 48 h at 4 °C and a single-crystal X-ray
analysis provided the first confirmation of the structure of
SO3•DMF (Figure 3).
21 and Tyr, 22), a disaccharide (23), and steroids (24 and 25)
was subjected to these conditions. No erosion of optical purity
was observed for Fmoc-L-21 (see Supporting Information, pages
16 and 57), and even acid-sensitive substrates (e.g. the
precursor to 26) could be sulfated in high yield using a slightly
modified protocol involving pre-treatment of the starting material
with DIEA. The successful sulfation of the bioisosteric cubane
motif[25] to afford product 27 was confirmed by single-crystal X-
ray analysis.
Methyl chlorosulfate: extended sulfation scope
•
•
•
alcohols, phenols
amino acids, steroids
acid-sensitive substrates
O
S
[Sulfation]
O
–
+
Cl
OMe
R
OH
R OSO3
O
S
O
DMF
O
S
O
DMF
0 ο
O
O
MeO
Cl
S
Me
Cl
NMe2
≡
O
Me2N
O
– MeCl(g)
C
O
O
–
OSO3
[x-ray]
methyl
chlorosulfate 16
6
–
OSO3
O
[x-ray]
–
OSO3
PGHN
*
O
27•H2O
BocHN
OSO3
O
Figure 3. Plausible mechanism of SO3•DMF formation from methyl
O
Boc-
Fmoc-
Fmoc-DL-21: 77%
L
L
-21: 90%
27: 93%
22: 87%
chlorosulfate in DMF.
-21: 93%
–
OSO3
–
OSO3
26: 71%a
–
THPO
–O3SO
H
To test the feasibility and efficiency of a one-pot sulfation
protocol with SO3•DMF generated in situ from methyl
chlorosulfate and DMF, methyl-a-glucoside 17 was chosen as a
model substrate and subjected to various conditions (Table 1).
In particular, addition of near stoichiometric amounts of methyl
chlorosulfate (1.1 equiv. per OH group) at 0 °C for 1 h afforded
47% of the tetrasulfate 18, with the remaining material
comprised of trisulfates 19 and 20. Increasing the number of
equivalents of ClSO3Me per OH residue to 1.5 afforded 93% of
product 18 after 1 h at 0 °C. Performing the reaction at room
temperature led to exhaustive persulfation, as did increasing the
equivalents of ClSO3Me to 2.0 per OH group at 0 °C. Drawbacks
of this process include the production of sodium methyl sulfate
and dimethylamine generated through exposure of the reagent
to adventitious moisture and quenching of the reaction mixture
with sodium bicarbonate. Nevertheless, dimethylamine bound as
the conjugate base can be readily removed by ion-exchange
and sodium methyl sulfate can be removed by either flash
chromatography or size-exclusion chromatography, especially
during desalting (see Supporting Information for details).
–
OSO3
O
H
H
–
OSO3
–O3SO
–O3SO
24: 62%
–O3SO
–O3SO
–O3SO
–
–
O
OSO3
–
H
H
OSO3
O
25: >95%b
OSO3
H
23: >95%b
–O3SO
OSO3
–
–O3SO
H
aAddition of DIEA (see SI for details); bPercent conversion determined by 1H NMR.
Figure 4. Scope of sulfation with methyl chlorosulfate. Note: sulfate
counterions are omitted throughout for clarity.
Preparation of pentasulfo-xylobiose (Xyl2-SO3) and
heptasulfo-xylotriose (Xyl3-SO3) standards
Having established an efficient sulfation protocol, we next turned
our attention to the synthesis of the target oligosaccharide
reference standards. Syntheses of Xyl2-SO3 8 and Xyl3-SO3 13
were greatly simplified by starting with an inexpensive,
commercially available xylooligomeric mixture (Xyl2 to Xyl5)
derived from corncobs.[26] Exhaustive acetylation of this
mixture[8a] followed by flash chromatography provided
substantial amounts of the xylobiose and xylotriose peresters 28
and 29, respectively (see Figure 5 and Supporting Information
for details).
Table 1. Optimization of sulfation conditions.
[Sulfation]
O
OSO3Na
O
OH
OSO3Na
O
Cl
S
O
OMe
With these suitable building blocks in hand, a protecting group
strategy was devised to overcome product instability resulting
from sulfation at C-1, as observed in our initial studies (see
Figure 2A). It was envisioned that global sulfation of
oligosaccharides bearing anomeric O-benzyl (path a) or S-tolyl
(path b) groups could be followed by selective deprotection to
deliver the target reducing sugars (Figure 5A). Following path a
in the first instance, anomeric deacetylation followed by Schmidt
glycosylation with benzyl alcohol provided compound 31 after
global deacetylation (Figure 5A). Treatment of 31 with ClSO3Me
(–30 to > 0 °C) in DMF in an operationally simple sulfation
process afforded the b-benzylated pentasulfo-species 32 that
was cleanly hydrogenolyzed to provide target 8.
19: R1 = H
R2 = SO3Na
O
R2O
R1O
NaO3SO
NaO3SO
HO
HO
DMF
20: R1 = SO3Na
R2 = H
NaO3SO
OH
NaO3SO
17
18
OMe
OMe
OMe
Conversion (%)a
Equiv. ClSO3Me
per OH group
Reaction
temp.
Entry
t (min)
18
19
20
1
2
3
4
1.1
1.5
1.5
2.0
60
60
10
60
0 oC
0 oC
47
93
99
99
21
32
4
3
21 oC
0 oC
<0.5
<0.5
<0.5
<0.5
aDetermined by 1H NMR analysis; bStarting temperature before addition of
ClSO3Me
3
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FULL PAPER
A) Biose units
– R'
OSO3
1) BnOH, BF3OEt2
DCM; 66%
2) NaOMe, MeOH; 98%
[Sulfation]
1) DMAPA, THF; 73%
α:β 1:20
O
2) Cl3CCN, DBU
DCM; 59%
O
O
path a
O
O
AcO
AcO
O
ClSO3Me
HO
HO
O
OBn
STol
AcO
HO
–
DMF
49%
AcO
AcO
OH
OH
OSO3
O
–
O
CCl3
NH
30
31
OSO3
O
O
O
AcO
AcO
O
32: R’ = OBn
33: R’ = STol
AcO
AcO
AcO
1) AcBr, MeOH
AcOH
OAc
NaOMe
MeOH/THF
STol
O
O
O
O
–
ClSO3Me
–
28
OSO3
OSO3
AcO
AcO
O
AcO
HO
HO
O
HO
2) TolSH, DCM
DMF
87%
97%
AcO
AcO
OH
OH
path b
R’ = OBn: Pd/C, H2, D2O
Na2CO3, TBAHS
34
35
78%
56% (2 steps)
R’ = STol: AuCl3, D2O
81%
1) PNBCl, Me2SnCl2
DIEA,THF/DMF; 89%
2) PivCl, DMAP, NEt3, DCM; 89%
Xyl2 building block
–
OSO3
Mg(OMe)2
MeOH/THF
O
O
O
O
O
–O3SO
PNBO
PivO
O
O
HO
PivO
O
PivO
O
STol
–O3SO
STol
PivO
≡
–O3SO
PivO
PivO
78%
PivO
PivO
OH
–O3SO
[x-ray]
Xyl2-SO3
8
36
37
1) BnOH, BF3OEt2
DCM; 52%
2) NaOMe
B) Triose units
Xyl3 building block
1) DMAPA, THF; 77%
2) Cl3CCN, DBU
DCM; 90%
α:β6.5:1.0
α:β 1:15
O
O
O
O
O
O
O
O
O
O
MeOH; 56%
AcO
AcO
AcO
AcO
O
O
AcO
O
O
HO
O
OBn
AcO
AcO
AcO
HO
HO
HO
AcO
AcO
AcO
AcO
AcO
AcO
OLG
HO
HO
HO
OAc
CCl3
LG =
29
39
NH
38
C) NMR: sulfation with ClSO3Me
ClSO3Me, DMF
52%
[Sulfation]
–
–
–
OSO3
OSO3
OSO3
–O3SO
–O3SO
O
O
OBn
40
O
O
O
–
–
–
–
–
OSO3
OSO3
OSO3
OSO3
OSO3
O
Pd/C, H2, D2O
–O3SO
–O3SO
–O3SO
–O3SO
–O3SO
O
O
OBn
O
O
–O3SO
O
O
O
O
O
–O3SO
OH
65%
–O3SO
–O3SO
–O3SO
–O3SO
Xyl3-SO3
13
40
!-H1
D) Pentaose unit
Xyl3 building block
Xyl2 building block
O
O
O
O
O
TMSOTf, PhCH3
AcO
AcO
O
O
HO
PivO
O
PivO
STol
AcO
AcO
+
AcO
AcO
AcO
87%
PivO
PivO
OLG
CCl3
NH
LG =
37
38
–
OSO3
–
–
OSO3
OSO3
1) NaOH(aq)
MeOH/THF
56%
STol
O
O
–O3SO
–O3SO
O
O
O
–
–
OSO3
–
O
OSO3
O
O
42
OSO3
AcO
AcO
O
AcO
O
–O3SO
STol
STol
–O3SO
O
O
–O3SO
–O3SO
O
PivO
O
O
3
2) ClSO3Me
DMF; 63%
PivO
AcO
AcO
–O3SO
2
2
–O3SO
3
41
42
[Sulfation]
AuCl3
D2O
–
–
–
–
O
OSO3
OSO3
OSO3
OSO3
O –
–O3SO
–O3SO
O
66%
O
O
O3SO
O
O
O
O
–O3SO
OH
–O3SO
–O3SO
–O3SO
14
Xyl5-SO3
E) Octaose unit
TMSOTf
PhCH3; 64%
Xyl3 building block
[from 29 - see Supporting Information]
O
O
O
O
O
O
O
O
O
O
AcO
O
AcO
O
PivO
AcO
AcO
O
O
O
PivO
HO
O
PivO
O
PivO
STol
+
AcO
STol
AcO
AcO
PivO
AcO
AcO
PivO
AcO
AcO
AcO
OPiv
PivO
PivO
PivO
OLG
2
2
44
43
38
CCl3
NH
LG =
NIS, TMSOTf
PhCH3
46 Xyl2 building block
70%
1) NaOH, THF, MeOH
–
O
–
OSO3
OSO3
O
87%
O
O
O
O
–O3SO
O
AcO
O
O
PivO
O
–O3SO
–O3SO
O
O
O
–O3SO
O
AcO
AcO
2) SO3.DMF, DMF
AcO
AcO
AcO
AcO
PivO
45
AcO
OBn
OAc
76%
–O3SO
OBn
Pd/C, H2
2
3
6
D2O
47
[Sulfation]
O
93%
HO
HO
D
-Xylose
HO
OH
[see SI]
Xyl2 building block
–
–
–
–
–
–
–
OSO3
OSO3
OSO3
O
OSO3
OSO3
OSO3
OSO3
–O3SO
O
O
O
O
–O3SO
O
O
O
O
O
O
O
O
O
O
O
AcO
O
–O3SO
HO
O
OH
AcO
AcO
AcO
OBn
–O3SO
–O3SO
–O3SO
–O3SO
–O3SO
–O3SO
–O3SO
Xyl8-SO3 15
46
Figure 5. A) Synthesis of sulfated xylooligosaccharide standards; A) xylobiose units; B) xylotriose units; C) 1H and 13C NMR spectra of sulfated derivatives; D)
xylopentaose unit; E) xylooctaose unit. Note: sulfate counterions are omitted throughout for clarity.
4
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Access to the b-thiotolyl-xylobioside 35 was accomplished
through the intermediate glycosyl bromide (path b).
Saponification of the resulting penta-acetate 34 gave polyol 35
that was itself smoothly persulfated with methyl chlorosulfate in
DMF to give 33. The anomeric hydroxyl was installed through
hydrolysis of the glycosidic C–S bond using Au(III) chloride,[27]
forming the reducing sugar 8. The p-toluene thiol-derived by-
products produced in this final step, though initially troublesome,
were removed using extraction, trituration and size-exclusion
chromatography techniques. The spectral data derived from
product 8 obtained by either route were identical, in all respects,
and confirmed the near quantitative conversion in the key
sulfation step, thus validating the utility of our in situ sulfation
approach. The presence of the persulfated product was
supported by mass spectrometry (ESI–, see Supporting
Information for details) and the homogeneity of the product as
determined by NMR spectroscopic analysis, is indicative of a
high degree of sulfation. Having established this proof of
concept, access to the heptasulfo-xylotriose Xyl3-SO3
homologue 13 was readily achieved from peracetate 29 using a
O-benzyl protecting group strategy and in situ sulfation with
methyl chlorosulfate in DMF (Figure 5B; see Figure 5C for
representative NMR spectral data for the key sulfation step).
benzyl-a-xylobioside acceptor 46[33] afforded the protected Xyl8
45 in 70% yield. Saponification of the associated acetate and
pivalate groups provided the corresponding polyol that was
sulfated directly with SO3•DMF, owing to the availability of the
reagent in the laboratory, to give product 47. To complete the
synthesis, hydrogenolysis of an aqueous solution of compound
47 under standard conditions afforded the heptadecasulfo-Xyl8
1
reducing sugar 15, H and 13C NMR spectral analysis of which
provided data consistent with those reported for sulfated, non-
glucuronic acid branched pentosans.[11a,b,34]
Sulfation of pyridinium xylotriose and a pentosan dosing
study
With the ultimate goal of deconvoluting the complex structure-
activity relationships of individual components of Pentosan PS-
based therapies, and given the troubling identification of
pyridinium artefacts in commercial Pentosan treatments, we next
turned our attention to the synthesis of a persulfated pyridinium
standard (Figure 6). Prior NMR studies have established a
covalent linkage of the pyridinium motif to the reducing end of
sulfated xylans,[11a,b,d] resulting from global sulfation of xylan
using SO3•pyridine complex. Nevertheless, a discrete sulfated
xylooligosaccharide standard bearing an anomeric pyridinium
moiety has not been reported. Given that minor impurities such
as these may be implicated in the emerging retinal toxicity
associated with long-term PPS exposure,[7] we envisaged that
such a synthetic standard could facilitate detailed biological
assays and safety studies.
Preparation of undecasulfo-xylopentaose (Xyl5-SO3)
Syntheses of homologues of xylose higher than xylotriose have
rarely been pursued.[28,29,30] We envisaged that the five-unit β-
1→4-xylooligomer could be accessed through union of the Xyl2
acceptor 37 and the Xyl3 donor 38 (Figure 5D). Conversion of
tolyl-1-thio-b-xylobioside (35) to the acceptor 37 was achieved
via a selective protection of the C-4’-OH as the p-nitrobenzoyl
ester followed by pivaloyl protection of the remaining hydroxyl
groups to give polyester 36 (Figure 5A). Selective deprotection
of the C-4’-p-PNB group using Mg(OMe)2 afforded target 37, the
structure of which was confirmed by single-crystal X-ray analysis.
Glycosylation of compound 37 with the Xyl3 donor 38 under
Schmidt conditions then provided the fully protected
Synthesis of pyridinium triose and Pentosan dosing studies
O
O
O
AcBr
MeOH, AcOH
AcO
AcO
O
O
AcO
AcO
29
AcO
AcO
AcO
34%
48
Br
1) Pyridine, acetone; 72%
2) NaOMe, MeOH; 84%
OH
ClSO3Me, DMF
39%
O
O
HO
HO
O
O
O
HO
HO
HO
[Sulfation]
49
HO
N
–
–
–
OSO3
OSO3
OSO3
1
–O3SO
–O3SO
O
O
O
O
O
xylopentaose 41 (Figure 5D). Analysis of the H NMR spectrum
Ha
• pyridyl-α-Xyl-SO3
detected in Pentosan 1
via NMR dosing studies
of compound 41 revealed that the five H-1/H-2 hydrogens at the
anomeric centers displayed coupling constants in the range of
5.9 to 9.3 Hz, consistent with their di-axial dispositions and so
confirming b-1→4 linkages throughout the length of the
xylopentaose scaffold. Compound 41 was deacylated under
standard conditions and the ensuing polyol sulfated in situ using
ClSO3Me/DMF. Under such conditions the undecasulfo-
derivative 42 was obtained, and after treatment with Au(III)
chloride, the desired reducing sugar 14 was isolated in high
purity and with a high degree of sulfation (see Figure 5C for
select NMR data).
–O3SO
–O3SO
N
pyridyl-α-Xyl3-SO3
50
pyridyl signals
1H NMR dosing studies
Ha
Compound 50
Pentosan 1
Ha
Ha
50 + Pentosan 1
Figure 6 Synthesis of persulfated pyridinium xylotriose 50 and confirmation of
pyridinium artefacts in a commercial sample of a Pentosan drug. Note: sulfate
counterions are omitted throughout for clarity.
Preparation of heptadecasulfo-xylooctaose (Xyl8-SO3)
A Xyl3 + Xyl3 + Xyl2 building-block approach was devised to
allow rapid access to the Xyl8 system (Figure 5E). The latent[31]
thioglycoside acceptor 43,[32] which acts as a linchpin for
accessing the Xyl8 system, was prepared in a fashion analogous
to congener 37 from peracetylated xylotriose 29 (see Supporting
Information for details). Reaction of compound 43 with the Xyl3
trichloroacetimidate 38 employing Schmidt glycosylation
conditions provided the hexasaccharide 44. Activation of this
Accordingly, we carried out the synthesis of sulfated pyridinium
triose 50 from peracetate 29. Conversion of the latter into the a-
bromide 48, followed by reaction of this with pyridine[11c] and
global deacetylation readily afforded the anomeric pyridinium
species 49. Sulfation of this last compound with ClSO3Me in
DMF afforded the persulfated a-pyridinium compound 50 that
was subject to full characterization. Dosing studies, undertaken
with
N-iodosuccinimide
and
trimethylsilyl
trifluoromethanesulfonate in toluene followed by addition of the
5
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10.1002/chem.202100527
Chemistry - A European Journal
FULL PAPER
using NMR spectroscopic methods and in which a commercial
sample of a Pentosan-based drug was treated with an authentic
sample of sulfated standard 50, confirmed the presence of an a-
pyridinium contaminant in Pentosan PS, as indicated by an
increase in intensity of the resonance attributed to the anomeric
H-1 proton (see Ha in Figure 6 and Supporting Information for
further details). As such, using compound 50 as a reference
standard provides a valuable means for identifying impurities
present in samples of Pentosan PS currently used in the clinic.
for a summary of all anomeric H1-H2 couplings of sulfated
xylooligomers prepared in this study) as well as small vicinal
couplings (< 2 Hz) for discernable resonances due to the
remaining ring protons. The all axial orientations of the sulfate
groups implied by such a series of couplings likely serves to
minimize electrostatic repulsion that would otherwise be
associated with negatively charged sulfates occupying
neighboring equatorial positions in a 4C1 conformation.[37] Such
results also align with observations made by others for
monosaccharide systems[12-14] and provide strong evidence in
support of Nishida’s hypothesis that the b-1→4-xyloglycan
Conformational analysis of sulfated xylooligosaccharides in
D2O
1
structure of Pentosan PS is dominated by a C4 conformational
The number and distribution of sulfate groups in
glycosaminoglycans (GAGs) is an important determinant of
biological activity that follows, inter alia, from the impact of such
residues on molecular conformation. Such matters have
attracted significant attention, particularly as they pertain to
heparin and heparan sulfate.[35] The conformational impacts of
sulfate groups in sugars, which have been largely elucidated
through 1H-1H coupling constant analyses of 1H NMR spectra,[12-
preference.
The situation is distinct for the third pyranose ring, namely the
reducing end of a-Xyl3-SO3 13. A coupling constant between H-1
(Ha, Figure 7) and H-2 (Hb) of JH1-H2 = 3.0 Hz is in accord with an
all equatorial arrangement of the sulfate residues in an a-
configured 4C1 system that is presumably dictated by the
anomeric effect (cf. a-xylobiose 4 in the Supporting Information;
JH1-H2 = 3.7 Hz). Coupling constants between H-2 and H-3 (7.7
Hz, Hb-Hc), and H-4 and H-5a (7.4 Hz) also support an
equilibrium favoring the 4C1 conformer. Such findings are
consistent with observations made for the analogous,
persulfated methyl-a-xyloside.[12] In the homologous a-Xyl5 14
and a-Xyl8 15 systems, the reducing end anomeric H-1-H-2
couplings were also found to be consistently ~3.0 Hz (see Table
2).
14,35b,36]
reveal intriguing conformational consequences arising
from sulfation. Using high-field, 2D NMR spectroscopic
techniques, it was possible to fully assign the 1H and 13C
resonances observed for the sulfated xylobiose and xylotriose
standards, these serving as robust models for their higher
homologues (see Supporting Information for full assignments).
1
For example, the 700 MHz H NMR spectrum of a-Xyl3-SO3 13
(Figure 7) revealed that the non-reducing xylose units exist
1
preferentially in the C4 conformation as indicated by a series of
Table 2. Selected coupling constants for xylooligosaccharide derivatives.
resonances with small to negligible couplings arising from the
ring hydrogens [with the exception of geminal couplings between
the xylose methylene hydrogens (e.g. Hg, Hh, Figure 7)]. For
example, a coupling of 1.2 Hz was observed between H-1’ (Hd)
and H-2’ (He) while H-1’’ (Hi) appeared as an apparent singlet.
The signal attributed to H-2’ (He) was observed as an apparent
triplet and exhibited a coupling with H-3’ (Hf) of 2.4 Hz. Sufficient
dispersion of signals allowed for detection of the anticipated W-
couplings between H-5a’ (Hh) and H-3’ (Hf) (1.7 Hz, app. dt) and
between H-5a’’ (Hn) and H-3’’(Hk) (1.6 Hz, app. dt) (Figure 7).
JH-1/H-2
(Hz)
JH-1/H-2
(Hz)
Compound
Residue
Compound
Residue
a-xylobiose 4
b-xylobiose 4
1
1
2
1
3.7
7.9
Bn-b-Xyl3-SO3
1
<1.0
<1.0
3.0
40
2–3
1
7.8
Xyl3-SO3 13
Bn-b-Xyl2-SO3 32
<1.0
2–3
<1.0
2
1
2
1
<1.0
<1.0
<1.0
2.9
STol-b-Xyl5-SO3
1
<1.0
<1.0
3.0
42
STol-b-Xyl2-SO3 33
a-Xyl2-SO3 8
2–5
1
a-Xyl5-SO3 14
2–5
<1.0
2
1
2
<1.0
3
Bn-a-Xyl8-SO3
1
3.5
1.0
47
b-Xyl2-SO3 8
2
<1.0
3–8
<1.0
1
<1.0
<1.7
a-Xyl8-SO3 15
1
2.9
pyridyl-a-Xyl3-SO3
50
2–3
3–8
<1.0
Interestingly, the minor, H-1 b-anomer of Xyl3 13 exhibited a
coupling between H-1 and H-2 of 3.0 Hz, while couplings of 3.0
Hz between H-4 and either H-5a or H-5b were also observed.
These data imply an equilibrium favoring the “inverted” 1C4
conformer for the reducing end of the minor C-1-b anomer.
Collectively, these findings provide considerable insight into the
spatial
display
of
sulfate
groups
in
persulfated
Figure 7. Selected region of the 1H NMR spectrum of compound 13. Note:
xylooligosaccharides and are likely to have important
implications for biological and computational studies on the
binding interactions of these molecules.
negative charges and counterions on sulfates omitted for clarity.
Furthermore, the sulfated, b-configured precursors (32, 33, 40,
and 42) all exhibited apparent singlets for the anomeric
hydrogens throughout the oligomer (JH1-H2 = < 1 Hz) (see Table 2
6
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10.1002/chem.202100527
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[3]
(a) C. I. Gama, S. E. Tully, N. Sotogaku, P. M. Clark, M. Rawat, N.
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83, 129-157.
Conclusion
The present study describes an efficient synthetic route to the
largest single-species sulfated xylooligomers reported to date.
As key constituents of the therapeutically-relevant sulfated
xylooligosaccharide drug Pentosan PS, these synthetic
standards were targeted as valuable tools for deconvoluting the
composition of the complex, heterogeneous nature of this
therapeutically deployed material. Towards this objective, near-
quantitative sulfation of xylooligomers was accomplished
through the use of methyl chlorosulfate in DMF as a mild and
operationally simple approach to the in situ generation of
SO3•DMF. This sulfation protocol was extended to a variety of
aliphatic and aromatic alcohols, encompassing amino acids,
steroids and acid-sensitive substrates. Its use in the preparation
of a novel sulfated and pyridinium-ion containing xylotriose
facilitated the identification of anomeric a-pyridinium
contaminants in a commercial source of Pentosan PS 1—a
finding which may have important implications for enhancing the
safety profile of treatments employing this material. Finally,
detailed conformational analyses of certain sulfated
[4]
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a
preference for the 1C4
conformer in all associated non-reducing residues. Given the
importance of sulfate arrays in GAG binding interactions, this
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development of PPS-type drugs with improved therapeutic
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We are grateful to Beta Therapeutics Pty. Ltd. for providing
funding for this work. We thank Dr. Tristan Reekie (ANU) for
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acknowledge Ms. Anitha Jeyasingham and Mr. Joseph Boileau
(ANU) for assistance with mass spectrometry, Dr. Chris Blake
(ANU) for support with NMR spectroscopy, Mr. Daniel Bartkus
and Dr. Nicholas Kanizaj (ANU) for technical assistance, and Mr.
Christopher Fitzgerald and Assoc. Prof. Malcolm McLeod for
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Keywords: carbohydrates • conformation analysis • pentosan •
persulfation • xylooligosaccharides
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Supporting Information.
- See
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[36] (a) A. G. Gerbst, V. B. Krylov, D. A. Argunov, A. S. Solovev, A. S.
Dmitrenok, A. S. Shashkov, N. E. Nifantiev, Carbohydr. Res. 2016, 436,
20-24; (b) H. P. Wessel, J. Carbohydr. Chem. 1992, 11, 1039-1052.
[37] Wessel and Bartsch postulated that b-methyl tetrasulfoglucopyranoside
exists preferentially in a 1C4 conformation but that replacement of the
negatively charged sulfates with sulfamoyl groups (as neutral sulfate
mimics) leads to a favoring of the 4C1 conformation: (a) H. P. Wessel, S.
Bartsch, Carbohydr. Res. 1995, 274, 1-9; Later computational studies
suggest that b-methyl tetrasulfoglucopyranoside exists as an
equilibrium of 1C4 and skew-boat conformers: (b) A. G. Gerbst, V. B.
Krylov, D. A. Argunov, A. S. Dmitrenok, N. E. Nifantiev, ACS Omega
2019, 4, 1139–1143.
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10.1002/chem.202100527
Chemistry - A European Journal
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Entry for the Table of Contents
[Single-species persulfated xylooligomers]
[Therapeutic leads]
–
–
[Sulfation]
0 o
OSO3
O
OSO3
glycosaminoglycan
(GAG) mimetics
e.g. Pentosan
polysulfate
(OH)n
O
C
–O3SO
–O3SO
O
O
O
–O3SO
O
–O3SO
OH
structural
insights
–O3SO
Xyl2 – Xyl8 units
xylooligomers
n
1C4 conformational
preference
Preparation of the largest homogeneous persulfated xylooligomers reported to date is facilitated by a rapid and quantitative one-pot
sulfation protocol based on the in situ generation of SO3•DMF. The synthesized oligosaccharide standards afforded unique structural
insights into persulfated xylooligomers, including a 1C4 conformational preference for all associated non-reducing residues.
Application of the method to the synthesis of a sulfated pyridyl-a-xylotriose also enabled identification of a pyridinium contaminant in
a commercial sample of pentosan polysulfate, a widely-employed sulfated xylooligosaccharide-based therapy.
Institute and/or researcher Twitter usernames: @Malins_Lab
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