Linear Synthesis of De novo Oligo-Iduronic Acid

Article abstract of DOI:10.1002/ejoc.201900540

L-Iduronic acid (IdoA) plays a pivotal role in glycosaminoglycan (GAG) protein interactions. However, the structural microheterogeneity of GAG appears to impede the systematic investigation of IdoA functions. Under such conditions, oligo-Iduronic acid (Oligo-IdoA) are ideal and straightforward heparin mimetics to unravel the relationship between IdoA structure and functions. Herein, we report for the first-time linear synthesis of rare oligo-IdoA precursor utilizing anhydrous β-L-idopyranosyl and IdoA thiophenol building block. After screening various synthetic strategies, we have Installed successive IdoA by 5 step reactions with 25–26 % overall yield. These oligo-IdoA are expected to be excellent probes to understand conformational plasticity of IdoA and fine tune carbohydrate–protein interactions.

Full text of DOI:10.1002/ejoc.201900540

A Journal of
Accepted Article
Title: Linear Synthesis of De novo Oligo-Iduronic Acid
Authors: Chethan Shanthamurthy and Raghavendra V. Kikkeri
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900540
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Supported by

European Journal of Organic Chemistry
10.1002/ejoc.201900540
COMMUNICATION
Linear Synthesis of De novo Oligo-Iduronic Acid
[a]
[a]
Chethan D. Shanthamurthy, and Raghavendra Kikkeri*
Abstract: L-Iduronic acid (IdoA) plays
a
pivotal role in
in-depth knowledge of IdoA. Because of these conditions,
developing the library of oligo-IdoA is envisioned as a potent
model. However, the synthesis of such oligosaccharides is
highly challenging, as IdoA is not commercially available. Herein,
we have succeeded developing the first synthetic protocol for
oligo-IdoA.
glycosaminoglycan (GAG) protein interactions. However, the
structural microheterogeneity of GAG appears to impede the
systematic investigation of IdoA functions. Under such conditions,
oligo-Iduronic acid (Oligo-IdoA) are ideal and straightforward heparin
mimetics to unravel the relationship between IdoA structure and
functions. Herein, we report for the first-time linear synthesis of rare
oligo-IdoA precursor utilizing anhydrous β-L-idopyranosyl and IdoA
thiophenol building block. After screening various synthetic
strategies, we have Installed successive IdoA by 6 step reactions
with 25-26% overall yield. These oligo-IdoA are expected to be
excellent probes to understand conformational plasticity of IdoA and
fine tune carbohydrate-protein interactions.
Results and Discussion
Our synthetic strategy is based on identifying a suitable protocol
for IdoA-disaccharides synthesis and extending the strategy for
oligosaccahrides. The pioneer work from the lab of Hung,
Seeberger, Boons, Liu, Gardiner, and others yielded gram scale
synthesis of IdoA building blocks from one-pot and de novo
8
Introduction
methods. In the present study, we employed 1,6-anhydro-β-L-
idopyranosyl 4-alcohol (5) and an iduronic acid-thiophenol donor
(9) as important precursors to synthesize oligosaccharides. The
initial synthetic strategy was based on use of IdoA derivatives 9
and 1 as “donor” and “acceptor,” circumvents the need for
deprotection and oxidation steps. We used an amine-linker to
block the reducing end IdoA residue for further conjugation.
Compound 5 was synthesized with a total yield of 27% from
L-Iduronic acid (IdoA) is key hexuronic acid component of
heparin (HP), heparan sulfate (HS), and dermatan sulfate (DS).
It is classified under the glycosaminoglycan (GAG) family. The
biosynthesis of HS and HP is initiated by the attachment of D-
xylose to the prescribed serine residue of proteoglycan core
proteins, followed by glycosylation of two galactose and
1
glucuronic acid residues, leading to the formation of
a
1,2:5,6-di-O-isopropylidene-α-D-glucofuranose by
reaction.8f
regioselective ring opening of
trimethyl(phenylthio)silane in the presence of ZnCI
temperature, led to thioglycoside 7 in a 75% yield. One-pot
oxidation of 7 with catalytic amounts of 2,2,6,6-tetramethyl-1-
a six-step
tetrasaccharide linker region. In the Golgi complex, the linker
region undergoes a series of enzymatic modifications, including
deacetylation, sulfation, and epimerization of D-glucuronic acid
A
5
with
2
at room
2
into L-iduronic acid. This results in highly complex and diverse
3
HS/HP structures. NMR and theoretical studies of HP and HS
piperidinyloxyl
bis(acetoxyiodo]benzene (BAIB) followed by esterification with
benzyl bromide yielded in 88% yield. It was further
(TEMPO)
in
the
presence
of
oligosaccharides confirmed that IdoA is predominantly presents
1
2
with C
as a 4
4
-chair and S
-chair conformation.4 This conformation flexibility adds
0
skew-boat geometry and less abundance
8
C
1
glycosylated with an azide-linker to yield 78% of 1. The C4-OH
of 8 was protected with levulinic acid using DCC as a coupling
additional structural diversity in HP and HS active domains and
thereby fine-tunes its protein interactions. The interaction
between fondaparinux (a pentasaccharide heparin mimetic) and
antithrombin III is a well-studied example of the conformation
plasticity of IdoA and its critical role in inhibiting blood
agent, yielding the donor
9 (Scheme.1). An attempt to
glycosylate 9 and 1 in the presence of N-iodosuccinimide (NIS)
and a catalytic amount of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) in DCM at -20 °C ended with 1,2-unsaturated glycosyl
product 13 instead of the desired di-IdoA 2. The use of a strong
5
coagulation. Nieto and coworkers synthesized an HP
trisaccharides library and showed that 6-O-sulfated and N-
thiophilic
promoter
condition,
such
as
NIS/
sulfated glucosamine (GlcN) residues next to IdoA significantly
trifluoromethanesulfonic acid (TfOH), in a range of temperatures
and time periods failed to yield di-idoA 2 (Scheme. 2). These
results lead us to conclude that the neighboring 2-benzoyl
2
6
contribute to
S
0
conformation. Liu and his coworkers
synthesized HP hexasaccharide and proved that 2-O- and 3-O-
sulfation of GlcN and 2-O-sulfation of IdoA are also crucial for
substitution of donor
9 stabilizes the benzyl oxonium-ion
2
7
stabilizing S
0
geometry in oligosaccharides. Even though many
intermediate in the presence of a thiophilic agent and facilitates
1,2-trans glycosylation due to the weak nucleophilicity of 1,
steroselective glycosylation did not proceed and led to lactal.
To improve the glycosylation, we have synthesized IdoA-donors
with good leaving groups, such as trichloroacetimidate (10) and
phosphorimidate (11). Under mild acidic conditions (catalytic
amount of TMSOTf), IdoA-trichloroacetimidate (10) yielded 2 in
reports have confirmed the conformational flexibility of IdoA, the
considerable microheterogeneity of GAG chains has limited our
[
a]
Mr. C. D. Shanthamurthy, Dr. R. Kikkeri
Indian Institute of Science Education and Research
Dr. Homi Bhabha Road,
5
% yield. Encouraged by this minor step forward, we examined
other trichloroacetimidate promoters, such as silver(I) triflate
(AgOTf), triflic acid (TfOH), and BF .Et O. These, unfortunately,
did not improve glycosylation yield and resulted eliminated side-
Pune-411008, India
E-mail: rkikkeri@iiserpune.ac.in
http://ragavendrakv.wixsite.com/kikkeri
3
2
Supporting information for this article is given via a link at the end
of the document.
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European Journal of Organic Chemistry
10.1002/ejoc.201900540
COMMUNICATION
product 13. Similarly, IdoA-phosphorimidate (11) failed to yield
the desired 2 (Scheme. 2). Based on these unsuccessful efforts,
we concluded that the electron withdrawing C5-carboxylic ester
in 1 reduced the nucleophilicity of the axial C4-OH, and that 1 is
a poor nucleophile for 1,2-trans glycosylation.
To improve the glycosylation of IdoA precursors, we proposed
using nucleophiles 15 as donors to synthesize L-Idose
disaccharide 19 and explore TEMPO oxidation of primary
hydroxyl groups to yield the desired product. To achieve this,
1,2-trans L-Idose disaccharide 18 was synthesized from L-idose-
tricholorimidate donor 15 and 1,6-anhydro-β-L-idopyranosyl 4-
alcohol 5 using a catalytic amount of TMSOTf. This was,
followed by acetolysis, and linker glycosylation to obtain di-L-
Idose 19 in 23% (4 steps) overall yield. The removal of acyl
groups of 18 by using sodium methoxide yielded di-L-Idose 20
(
Scheme. 3). Finally, 20 was oxidized with TEMPO in the
presence of BAIB, yielding unexpected lactonized IdoA
monosaccharide 21 as a major product. Attempts with different
TEMPO co-oxidants such as sodium hypochlorite (NaOCl) and
2
sodium chlorite (NaClO ) met similar fate. This indicates that
TEMPO/BAIB/NaOCl mediated oxidation is selective for primary
alcohol, but the alkaline reaction condition and radical species
formed during the oxidation resulted in cleavage of the
glycosidic bond, presumably by an E1CB elimination
Scheme 1: Synthesis of acceptor molecules 1 and donor 9-11: Reagents and
conditions: (a) BzCl, DCM/Py, 85%; (b) TMSSPh, ZnCI
2
, CH
O (1/1); BnBr, TBAI, NaHCO , DMF,
2steps), 88% (8); (d) LevOH, DCC, DMAP, CH Cl 88% (9); (e)
M.S, -5 °C-RT, 78% (1); (f) NBS,
, DBU, 76% (10) (2 steps); (g) Dibutyl phosphate,
, NIS, TfOH, 4 Å M.S, -20 °C, 92% (11).
2
Cl
2
, RT, 75% (7);
9
mechanism. Overall, we concluded that oligo-L-Idose could be
(c) TEMPO, BAIB, CH
2
Cl
2
/H
2
3
60 °C,
easily generated from 5, but TEMPO oxidation of multiple
primary hydroxy groups may be detrimental to the process and
could result in cleavage of glycosidic bonds.
(
2
2
,
Azidoethoxyethanol, NIS, TfOH,
Acetone/H O (10/1); NCCCl
CH Cl
4 Å
2
3
2
2
Scheme 2. Synthesis of 2 from 9-11 and 1 as donors and acceptor. Promotor:
: NIS/TMSOTf or NIS/TfOH; 10: TMSOTf or AgOTf, TfOH, BF .Et O; 11:
TMSOTf.
9
3
2
Scheme 4: Synthesis of oligo-IdoA (I-1 to I-3): Reagents and conditions: a)
NIS, TMSOTf, CH
Cu(OTf) , Ac O, 0 °C, 94% (23), 85% (28),
CH Cl , RT, 89% (24), 80% (29), 80% (34); d) P-TsOH, CH
2% (25), 76% (30), 66% (35); e) TEMPO, BAIB, CH
BnBr, TBAI, NaHCO DMF, 60 °C, 65% (26), 63% (31), 58% (36); f)
Azidoethoxyethanol, NIS, TfOH, 4 Å M.S, -5 °C, 81% (2), 76% (3), 76% (4). g)
LiOH, THF/H O (4/1); Pd(OH) , MeOH, 57% ( I-1), 53% (I-2), 48% (I-3).
2
Cl
2
, 4 Å M.S, -10 °C, 64% (22), 62% (27), 62% (32); b)
85% (33); c) TMSSPh, ZnI
Cl /MeOH(1/1),
Cl /H O (1/1);
2
2
2
,
2
2
2
2
9
2
2
2
3
,
2
2
To tackle the TEMPO mediated oxidation-degradation of
oligosaccharides, we have decided to synthesize heterogeneous
L-IdoA-L-idose disaccharide 23 composition and oxidize a single
primary hydroxyl moiety at a time. We started the synthesis by
glycosylating 9 and 5 with NIS and TMSOTf and obtained
disaccharide 22 in 64% yield after 30 minutes (Scheme. 4). We
have corroborated the α-configuration of glycoside 22 by H and
13C-NMR, showing peaks at 5.29 and 95.17 ppm, respectively.
By acetolysis of the anhydro-ring of 22, in the presence of
Scheme 3: Synthesis of di-L-Idose precursor (19): Reagents and conditions:
a) Cu(OTf) , Ac O, 0 °C, 82% (14), 78% (17); (b) Bu SnO, MeOH, 55 °C;
NCCCl , DBU, CH Cl 53% (15), 43% (18) (2 steps); (c) TMSOTf, CH Cl , 4
Å M.S, -78 °C 59% (16), 68% (19); (d) NaOMe, MeOH, 88% (20); (e) TEMPO,
BAIB, CH Cl /H O (1/1), 26% (21).
(
2
2
2
3
2
2
2
2
1
2
2
2
2
copper (II) trifluoromethanesulfonate [Cu(OTf) ] and acetic
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European Journal of Organic Chemistry
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COMMUNICATION
anhydride, we have synthesized 23 in 94% yield. Successive
thioglycosylation, mild deacetylation, one-pot TEMPO oxidation,
and esterification of 23 yielded the target disaccharide molecule
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azide-linker yielded di-IdoA derivative in 81% yields.
2
Encouraged by the overall yield and stereoselectivity of the di-
IdoA, we extended the protocol to synthesize different chain
lengths of oligo-IdoA (Scheme. 4). As an example, 26 or 31
were reacted with 5 using NIS and TMSOTf. These were
followed by a series of reactions, including acetolysis of the
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esterification, and linker glycosylation. This sequence of
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yields of different lengths of oligo-IdoA precursors. Finally, the
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Conclusions
We report for the first time a new linear approach for the
synthesis of oligo-IdoA using an IdoA-thiophenol as the
donor and a β-L-idopyranosyl derivative as the nucleophile.
Sequential modifications of the L-Idose residue yielded
oligo-IdoA derivatives in moderate overall yields. After
screening various synthetic conditions, this is the best
possible method to synthesize oligo-IdoA. This synthetic
route enables us to synthesize different sulfation patterns of
oligo-IdoA to study conformation plasticity of IdoA,
secondary oligosaccharide structures, and thereby
modulate carbohydrate-protein interactions.
[
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Keywords: Carbohydrate • Oligosaccharide • Iduronic acid •
Heparin • Glycosylation
7
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(
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Entry for the Table of Contents (Please choose one layout)
Layout 1: Oligo-Iduronic acid
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Chethan D. Shanthamurthy,
Raghavendra Kikkeri*
Page No. – Page No.
Linear Synthesis of De novo Oligo-
Iduronic Acid
A rare iduronic acid oligosaccharides have been synthesized utilizing anhydrous β-
L-idopyranosyl and IdoA thiophenol building block. Installation of successive IdoA
residue required 6 step cycle allowed the synthesis of oligo-iduronic acid.
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