Synthesis of β-1,4-di-d-mannuronic acid glycosides as potential ligands for toll-like receptors

Article abstract of DOI:10.1016/j.tetlet.2007.02.086

Toll-like receptors (TLRs) play important roles in host immune defense, and synthetic TLR ligands are useful therapeutic agents for a variety of diseases including infection, inflammation, and cancers. Alginates are strong immune stimulants mediated by TL

Full text of DOI:10.1016/j.tetlet.2007.02.086

Tetrahedron Letters 48 (2007) 2915–2918
Synthesis of b-1,4-di-D-mannuronic acid glycosides
as potential ligands for toll-like receptors
Zi-Hua Jiang,* Rongsong Xu, Charlene Wilson and Agnes Brenk
Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ont., Canada P7B 5E1
Received 1 September 2006; revised 10 February 2007; accepted 16 February 2007
Available online 22 February 2007
Abstract—Toll-like receptors (TLRs) play important roles in host immune defense, and synthetic TLR ligands are useful therapeutic
agents for a variety of diseases including infection, inflammation, and cancers. Alginates are strong immune stimulants mediated by
TLR2/4. Reported here are the design and chemical synthesis of two glycosides (1 and 2) containing b-1,4-di-D-mannuronic acid
moiety derived from alginate. The synthesis features the preparation of b-1,4-D-mannobiose derivatives through diastereoselective
b-glycosylation of 4,6-di-O-benzylidene protected thiomannoside donor 7, followed by an oxidation step using TEMPO/BAIB to
provide the b-1,4-di-D-mannuronic acid moiety.
Ó 2007 Elsevier Ltd. All rights reserved.
The recently discovered toll-like receptors (TLRs) are
innate pattern recognition receptors, which play impor-
tant roles in host immune defense.1,2 Activation of
TLRs and downstream signaling pathways lead to the
expression of pro- and anti-inflammatory mediators
which have a great impact on human physiology. Syn-
thetic ligands capable of regulating TLR signaling path-
ways are useful therapeutic agents for treating various
diseases including infection, chronic inflammation, and
cancers.3–7 Our interest in synthetic vaccine develop-
ment8 has led us to the design and synthesis of a few
TLR4 ligands which are potent immune stimulatory
molecules potentially useful as vaccine adjuvants.9,10
Alginates are potent immune stimulating agents. In
terms of cytokine production by monocytes, the potency
increases with the content of M residues.13 Poly-G
blocks do not induce cytokine production from mono-
cytes, and they also reduce the cytokine production level
induced by poly-M blocks, indicating that poly-G
blocks may act as antagonists of poly-M blocks. In
contrast, other reports show that enzymatically depoly-
merized alginate oligosaccharides, both oligo-M and
oligo-G with a double bond (D4) in the nonreducing
end, cause cytotoxic cytokine production in human
mononuclear cells14 and tumor necrosis factor-alpha
(TNF-a) in RAW264.7 cells.15,16 TLR2 and TLR4 have
been shown to mediate the cytokine production induced
by alginate polymers as well as alginate oligosaccha-
rides,15,17 indicating that short oligosaccharides of algi-
nate may well serve as agonists or antagonists for
these receptors. Therefore, we have designed glycosides
1 and 2 containing b-1,4-di-D-mannuronic acid moiety
as potential ligands for TLR2/4 (Fig. 2).
Alginates are un-branched linear polysaccharides con-
sisting of 1,4-linked b-D-mannuronic acid (M) and its
C5-epimer a-L-guluronic acid (G) residues (Fig. 1) pro-
duced by marine macroalgae and some bacteria belong-
ing to the genera Azotobacter and Pseudomonas.11 Their
compositions are highly variable, including homo-poly-
meric (poly-M or poly-G) or hetero-polymeric (poly-
GM) structures, depending on the sources from which
they are isolated. Alginates from bacteria also display
certain degree of acetylation at O-2- and O-3-position
of D-mannuronic acid residues.12
Despite the various industrial applications18 and medi-
cal significance19 of alginates, the chemical synthesis of
alginate oligosaccharides has not been reported, possi-
bly due to the notorious challenge of accessing b-glyco-
sidic linkage of D-mannuronic acid and L-guluronic acid
building blocks. In the present Letter, we report the
syntheses of compounds 1 and 2.
Keywords: Alginate; D-Mannuronic acid; b-1,4-Di-D-mannuronic acid;
Oligosaccharide; Glycolipid; TLR ligand.
*
Compound 1 is designed as a glycolipid for the reason
Corresponding author. Tel.: +1 807 766 7171; fax: +1 807 346
7775; e-mail: zjiang@lakeheadu.ca
that naked small carbohydrates are rapidly cleared away
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.02.086

2916
Z.-H. Jiang et al. / Tetrahedron Letters 48 (2007) 2915–2918
HOOC
HOOC
O
HOOC
O
OH
OR
O
OR
O
OR
O
HOOC
OH
O
OH
O
OH
O
HOOC
O
RO
HOOC
O
RO
O
R = H, Ac
O
H
RO
k
n
m
OH
OH
OH
Poly-D-mannuronic acid (poly-M)
Poly-GM
Poly-L-guluronic acid (poly-G)
Figure 1. Structures of alginates.
O
O
O
1
2
R =
OH
OH
O
HO
HO
HO
HO
O
O
O
OR
OH
HO
R = CH3
Figure 2. b-1,4-Di-D-mannuronic acid glycosides 1 and 2 designed as potential ligands for TLR2/4.
from the biological system,20 but the conjugation with
lipids may enhance their stability, rendering them better
drug candidates than naked carbohydrates. Glycolipids
are also suitable for liposome formulation, a drug deliv-
ery system proven to be efficient in reducing toxicity and
side effects.21 Thus, lipidated pentaerythritol derivative 6
(Pet–OH) is prepared as the lipid anchor (Scheme 1).
Two C16–lipid chains are introduced to the readily
available pentaerythritol derivative 322 by treating with
1-bromohexadecane and sodium hydride in DMF to
provide 4 in 59% yield. Trans-ketal reaction of 4 with
neopentyl glycol in the presence of camphorsulfonic acid
(CSA) liberates diol 5 in 95% yield. Monobenzylation of
5 through treatment with dibutyltin oxide (Bu2SnO)
followed by benzyl bromide and tetrabutylammonium
bromide gives compound 6 in 70% yield.
O
O
OH
OH
3
a
b
The strategy we employ to make the b-1,4-di-D-mannu-
ronic acid moiety is to first synthesize the b-1,4-D-mann-
obiose unit, followed by an oxidation step to convert
both mannose residues to the mannuronic acid residues.
Diastereoselective synthesis of b-D-mannopyranoside
has been a long standing challenge in carbohydrate
chemistry.23 The 4,6-di-O-benzylidene protected
methodology recently developed by Crich and co-work-
ers24,25 offers an efficient method for the direct synthesis
of b-D-mannosides, as demonstrated by the synthesis of
b-1,2-D- and b-1,4-D-mannan.26,27 Here we use a similar
strategy to construct the b-1,4-D-mannobiose moiety
(Schemes 2 and 3). Thus, activation of the readily acces-
sible thiomannoside 727 by 1-benzenesulfinyl piperidine
and trifluoromethanesulfonic anhydride (BSP/Tf2O)25
in the presence of the hindered base 2,6-di-tert-butyl-4-
methylpyridine (DTBMP) in dichloromethane at
ꢀ60 °C followed by the addition of acceptor 6 at
ꢀ78 °C provides the desired b-mannoside 8 in 85% yield.
The a-isomer is also formed in about 8% yield, but an
O
O
O
O
4 (59%)
5 (95%)
O
O
HO
HO
c
BnO
HO
O
O
Pet OH
6 (70%)
Scheme 1. Reagents and conditions: (a) n-C16H33Br, DMF, NaH,
50 °C; (b) neopentyl glycol, CSA, CH2Cl2, 35 °C; (c) (i) Bu2SnO,
benzene, 90 °C; (ii) BnBr, Bu4NBr, 90 °C.
Ph
+
OBn
O
OBn
O
OBn
O
O
Ph
OBn
O
BzO
HO
BnO
HO
HO
BnO
a
O
b or c
d
O
O
Pet OH
OR
OR
OR
BnO
BnO
6
SPh
12 R = Pet (70%)
13 R = CH3 (83%)
10 R = Pet (90%)
11 R = CH3 (98%)
7
8 R = Pet (85%)
9 R = CH3
CH3
N
O
S
O
N
Pet =
O
OBn
DTBMP
BSP
Scheme 2. Reagents and conditions: (a) BSP, DTBMP, Tf2O, CH2Cl2, ꢀ78 °C to 0 °C; (b) neopentyl glycol, CSA, CH2Cl2, 35 °C, for 10; (c) HOAc–
H2O (4:1), 75 °C, for 11; (d) BzCl, pyridine, DMAP, CH2Cl2, ꢀ25 °C to 0 °C.

Z.-H. Jiang et al. / Tetrahedron Letters 48 (2007) 2915–2918
2917
OBn
O
OBn
O
Ph
OBn
O
Ph
OBn
O
BzO
O
BnO
12
or
13
O
O
HO
O
BnO
16 R = Pet (100%)
17 R = CH3 (100%)
b
a
7
+
O
O
OR
OR
BnO
BnO
14 R = Pet (67%)
15 R = CH3 (71%)
c or d
O
O
OBn
O
OBn
O
HO
O
BnO
HO
HO
BnO
g or h
1 (90%)
2 (92%)
BnO
BnO
OBn
O
OBn
O
18 R = Pet (90%)
19 R = CH3 (86%)
e, f
O
BnO
HO
BnO
OR
OR
O
N
20 R = Pet (55%)
21 R = CH3 (63%)
O
OAc
OAc
I
Pet =
O
OBn
BAIB
TEMPO
Scheme 3. Reagents and conditions: (a) BSP, DTBMP, Tf2O, CH2Cl2, ꢀ78 °C to 0 °C; (b) NaOMe, MeOH–CH2Cl2 (1:1), rt; (c) neopentyl glycol,
CSA, CH2Cl2, 35 °C, for 18; (d) HOAc–H2O (4:1), 75 °C, for 19; (e) BAIB, TEMPO, CH2Cl2–H2O (2:1), rt; (f) BnBr, KF, DMF, rt; (g) H2, Pd/C,
THF–H2O (4:1), rt, for 1; (h) H2, Pd/C, MeOH–H2O (3:1), rt, for 2.
analytically pure material has not been obtained. The
removal of 4,6-di-O-benzylidene group in 8 is effected
by treating with neopentyl glycol in the presence of
CSA to give compound 10 in 90% yield. Compound
10 is converted to 6-O-benzoate 12 through regio-
selective benzoylation with benzoyl chloride and pyri-
dine in the presence of 4-N,N-dimethylpyridine (DMAP)
in 70% yield. The b-glycosidic linkage, originally formed
in compound 8, has been confirmed by the value of
1JC,H coupling constant of the anomeric carbon in
12 (1JC1,H1 = 154.6 Hz).28 Similarly, the previously
reported methyl b-D-mannoside 927 is treated with acetic
acid–water (4:1) at 75 °C to give diol 11,27 which is then
converted to its 6-O-benzoate 13 in 83%.
products 1 and 2 in good yield. The structures of 1 and
2 have been confirmed by spectroscopy data.33,34
The initial biological evaluation of compounds 1 and 2
with respect to the immune stimulating and modulating
properties is under way. The result will be reported in
due course.
In brief summary, we have described an efficient route
for the synthesis of b-1,4-di-D-mannuronic acid glyco-
sides 1 and 2 designed as potential ligands for TLR2/
4. The method shall be applicable for synthesizing glyco-
conjugates containing larger mannuronic acid oligo-
saccharides. Further research is being devoted to the
synthesis and biological investigation of neo-glycoconju-
gates containing oligo-D-mannuronic acid as well as
oligo-L-guluronic acid.
By using the same glycosylation protocol, both 12 and
13 are glycosylated with 7 to provide disaccharides 14
and 15 in 67% and 71% yield, respectively (Scheme 3).
Again the a-isomers for both reactions are formed in
negligible amount and therefore are not characterized.
The newly formed b-linkage in 14 and 15 is confirmed29
by the characteristic H-5 signal24,27 of the sugar residue
Acknowledgments
This work was supported by the Natural Sciences and
Engineering Research Council of Canada, NSERC
(312630), and Lakehead University.
1
with the 4,6-di-O-benzylidene group and the JC,H
coupling constant of the anomeric carbon. Compounds
14 and 15 are converted to triols 18 and 19, respectively,
in high yield through deacylation with sodium
methoxide in dichloromethane–methanol (!16, 17)
and removal of the 4,6-di-O-benzylidene group. Regio-
selective oxidation of the primary hydroxyl function in
18 and 19 to carboxylic acid is achieved by the combina-
tion of 2,2,6,6-tetramethylpiperidinyloxy free radical
(TEMPO) and [bis(acetoxyiodo]benzene (BAIB)30,31
(Scheme 3). Thus, triols 18 and 19 are converted into
their corresponding dicarboxylic acids, which are subse-
quently treated with benzyl bromide in the presence of
potassium fluoride in DMF to give benzyl esters 2032
and 21 in 55% and 63% yield, respectively. The TEM-
PO/BAIB oxidation system seems to be effective in
simultaneous oxidation of multiple primary hydroxyl
groups to carboxylic acid functions in the presence of
secondary hydroxyl groups. Hydrogenolytic debenzyl-
ation of 20 and 21 in the presence of palladium on
charcoal under hydrogen atmosphere produces the final
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52H, 26CH2), 1.48 (m, 4H, 2CH2), 2.77 (d, J = 2.5 Hz,
1H, OH), 3.10 (dd, J = 10.0, 3.0 Hz, 1H, H-3), 2.28–3.49
(m, 13H), 3.64 (d, J = 3.0 Hz, 1H, H-2), 3.65 (d,
J = 3.0 Hz, 1H, H-2), 3.81 (d, J = 9.5 Hz, 1H, H-5), 4.00
(d, J = 9.5 Hz, 1H, H-5), 4.18 (ddd, J = 10.0, 9.5, 2.5 Hz,
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4.46 (s, 1H, H-1), 4.49 (d, J = 12.0 Hz, 1H), 4.47 (d,
J = 12.0 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 4.68 (d,
J = 12.0 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.85
(d, J = 12.0 Hz, 1H), 5.00 (d, J = 12.5 Hz, 1H), 5.01
(d, J = 12.0 Hz, 1H), 5.10 (d, J = 12.0 Hz, 1H), 5.20 (d,
J = 12.5 Hz, 1H), 7.25 (m, 35 H). 13C NMR (125 MHz,
CDCl3): d 169.1, 168.1, 138.9, 138.7, 138.6, 138.0, 135.3,
135.1, 128.6, 128.5, 128.4, 128.3, 128.27, 128.2, 128.1,
128.0, 127.8, 127.7, 127.6, 127.4, 127.3, 127.2, 102.9, 102.1,
80.1, 79.4, 75.1, 75.0, 74.8, 74.6, 74.2, 73.7, 73.2, 72.3, 71.6,
70.5, 69.6, 68.0, 67.1, 66.9, 45.2, 31.9, 29.7, 29.6, 29.5, 29.3,
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33. Compound 1: Rf = 0.46 (CHCl3–CH3OH–H2O–HOAc,
4:2:0.4:0.4). MS (MALDI) calcd for C49H92O16 936.63
[M]+, found 959.63 [M+Na]+, 981.62 [MꢀH+2Na]+. A
well resolved NMR spectrum was not obtained.
34. Compound 2: Rf = 0.44 (isopropanol–H2O–HOAc, 4:2:1).
ESI-MS (negative mode) calcd for C13H20O13 384.08
1
[M]ꢀ, found 383.10 [MꢀH]ꢀ. H NMR (500 MHz, D2O):
d 3.54 (s, 3H, OCH3), 3.68 (dd, J = 9.5, 3.5 Hz, 1H, H-3),
3.79 (dd, J = 10.0, 9.5 Hz, 1H, H-4), 3.81 (dd, J = 8.5,
3.5 Hz, 1H, H-3), 3.92 (d, J = 10.0 Hz, 1H, H-5), 3.95 (d,
J = 9.0 Hz, 1H, H-5), 3.99 (dd, J = 9.0, 8.5 Hz, 1H, H-3),
4.00 (d, J = 3.0 Hz, 1H, H-2), 4.05 (d, J = 3.0 Hz, 1H, H-
2), 4.66 (s, 1H, H-1), 4.74 (s, 1H, H-1). 13C NMR
(125 MHz, D2O): d 173.2, 173.1, 101,3, 100.4, 78.3, 74.9,
74.3, 72.5, 71.5, 70.3, 69.6, 68.0, 57.3.