Concise assembly of linear α(1→6)-linked octamannan fluorescent probe

Article abstract of DOI:10.1016/j.tetlet.2008.09.164

Synthesis of a fluorescently labelled (dansylated) linear α(1→6)-linked octamannan, using glycosyl fluoride donors and thioglycosyl acceptors, is described. A selective and convergent two-stage activation progression was executed to construct di-, tetra,

Full text of DOI:10.1016/j.tetlet.2008.09.164

Tetrahedron Letters 49 (2008) 7157–7160
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Concise assembly of linear
a(1?6)-linked octamannan fluorescent probe
*
Mohammad S. Aqueel, Vibha Pathak, Ashish K. Pathak
Department of Chemistry, Western IllinoisUniversity, Macomb, IL 61455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a fluorescently labelled (dansylated) linear a(1?6)-linked octamannan, using glycosyl fluo-
Received 11 September 2008
Revised 26 September 2008
Available online 2 October 2008
ride donors and thioglycosyl acceptors, is described. A selective and convergent two-stage activation pro-
gression was executed to construct di-, tetra, and octa-mannosyl thioglycosides in three glycosylation
steps with excellent yield. Further, a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl)
group was coupled to 1-azidoethyl octamannosyl thioglycoside. Global deprotection of the coupled prod-
uct afforded the desired dansylated homo-linear
a
(1?6)-linked octamannan.
Ó 2008 Elsevier Ltd. All rights reserved.
Mannans are constituents of many bacterial and fungal cell
walls.1 They are functional components of proteins, and play
important roles in defining the structures of proteins, their
stabilization under physiological conditions, tuning of enzymatic
activities, cell–cell recognition, and in the adhesion of the microor-
N
O
HO
O
HO
HO
HO
O
O
O
S
6
HO
HO
O
HO
HO
NH
O
ganism to host cells.2 The
a(1?6)-linked oligomannans are found
HO
O
HO
in the cell wall polysaccharides of yeast.3 Polysaccharides, espe-
cially oligomannan, can be used to deliver drugs, genes and anti-
gens through polysaccharide receptors present in cells and
macrophages.4 Macrophages are known to express high levels of
specific polysaccharide receptors, for example, mannan, glucan,
and galactin receptors, on their membranes that generally bind
neutral or charged polysaccharides and internalize these ligands.5
Receptor-mediated delivery of drug–polysaccharide conjugates is
an approach that can deliver small and effective amounts of drugs
specifically to target organisms, minimizing patient exposure and
potential toxic side effects. We have recently demonstrated the
selective, receptor–mediated delivery of an antibacterial drug to
macrophages infected with Mycobacterium tuberculosis via conju-
gation of moxifloxacin with 1,3-b-glucan.6
1
BzO
O
AcO
BzO
BzO
N
O
O
6
BzO
BzO
O
BzO
BzO
O
N3
HO
BzO
STol
SO2Cl
BzO
2
3
4
Figure 1. Target a(1?6)-linked octamannan fluorescent probe 1 and its precursors.
bly of dansylated oligomannan fluorescent probe
approached from synthetic homo-linear (1?6) thiooctamanosyl
glycoside 2, 1-azidoethanol 3, and commercially available dansyl
1
was
a
chloride 4 fragments as represented in Figure 1.
Branched oligomannan syntheses are reported in the literature
using several different methodologies.8 Specifically, the homo-
Drug loading on commercially available high molecular weight
polysaccharides is very low, and it is attributed to their poor solu-
bility in solvents during the drug conjugation reactions. Therefore,
we describe here a simple and efficient method for the synthesis of
linear
a(1?6) oligomannans were synthesized by glycosylation
using a trichloroacetimidate donor glycoside.9 The strategy of com-
bining the chemistry of thioglycosides with that of glycosyl
fluorides10 for the synthesis of oligosaccharides known as the
two-stage activation procedure (Fig. 2) was developed by Nicolaou
et al.11 Briefly, in the activation stage one, the stable thioglycoside
is converted to the more reactive glycosyl fluoride donor by treat-
ment with N-bromosuccinimide (NBS) and diethylaminosulfur tri-
fluoride (DAST). In stage two, the glycosyl fluoride is then reacted
with a thioglycoside acceptor to produce a thiodisaccharide for fur-
ther promulgation of the oligosaccharide chain. Reiteration of this
process of converting thiooligosaccharide to a glycosyl fluoride and
a low molecular weight homo-linear
a(1?6)-linked octamannan
fluorescent probe 1 (Fig. 1) that may be utilized to study uptake
by macrophages through the mannan receptor present on the plas-
ma membrane. Further, in the future, such conjugation can be
exploited for selective receptor-mediated delivery of drugs. We
had previously synthesized and reported dansylated disaccharide
fluorescent probes to study glycosyltransferases in the biosynthe-
sis of M. tuberculosis cell wall polysaccharides.7 Herein, the assem-
* Corresponding author. Tel.: +1 309 298 2261; fax: +1 309 298 2180.
E-mail address: AK-Pathak@wiu.edu (A. K. Pathak).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.09.164

7158
M. S. Aqueel et al. / Tetrahedron Letters 49 (2008) 7157–7160
O
O
AcO
BzO
BzO
OBz
O
O
HO
PO
PO
SR
SR
O
F
a
O
OBz
O
5 + 6
BzO
BzO
O
O
O
9
HO
O
PO
O
PO
O
STol
O
O
O
O
SR
SR
F
b
c
HO
BzO
BzO
OBz
OBz
O
AcO
BzO
BzO
O
PO
O
O
OBz
O
O
O
OBz
O
O
O
BzO
BzO
BzO
O
R = alkyl or aryl
P = protecting group
BzO
SR
STol
F
10
11
Figure 2. Two-stage activation of thioglycoside for glycoside bond formation. Other
protected OH’s on sugars are omitted for simplification.
a
AcO
BzO
BzO
OBz
O
their coupling with acceptor thioglycosides straightforwardly pro-
duces chain elongation (Fig. 2).
O
OBz
O
BzO
BzO
We executed the synthesis of the desired p-thiotolyl
a(1?6)
2
octamannoside 2 using a convergent two-stage activation and
iterative orthogonal glycosylation technique starting from the cou-
pling of thioglycoside acceptor 5 and glycosyl fluoride donor 6
(Fig. 3). The synthesis of mannose building blocks 5 and 6 was
achieved from thioglycoside 7 as reported earlier by us.12 However,
the synthesis of thioglycoside 7 was accomplished from the
O
OBz
O
BzO
12
BzO
STol
b
c
AcO
BzO
BzO
HO
BzO
BzO
OBz
O
OBz
O
known synthon 1,6-di-O-acetyl-2,3,4-tri-O-benzoyl-
a-
D-mannose
(8) starting from commercially available
D-mannose in an 88%
overall yield.13 Selective de-acetylation of 7 to the acceptor glyco-
side 5 was achieved by the overnight reaction with an AcCl/MeOH/
CH2Cl2 (0.1:20:20 v/v, 12.0 mL/mmol) mixture.12
O
O
OBz
OBz
O
O
BzO
BzO
BzO
BzO
2
2
O
O
OBz
O
OBz
O
Following the synthesis of starting mannose synthons 5 and 6,
the synthesis of 2 was carried out as illustrated in Scheme 1. In
the first step of the glycosylation sequence, the p-thiotolyl disac-
charide 9 was synthesized in high yield by selective anomeric acti-
vation of glycosyl fluoride 6 by AgClO4 and SnCl2 in CH2Cl2 and its
BzO
BzO
BzO
BzO
13
14
F
STol
d
coupling with the acceptor 5.12 The exclusive formation of
a(1?6)-
BzO
O
AcO
BzO
BzO
linked disaccharide 6 was supported by its analytical and spectral
O
O
analysis.12
6
BzO
BzO
O
BzO
BzO
O
Next, for the assembly of a(1?6)-linked tetramannosyl thiogly-
BzO
BzO
coside 12, the disaccharide 9 was converted to acceptor thiodisac-
charide 10 and donor 1-fluorodisaccharide 11. Deacetylation of 9
by the overnight reaction with an AcCl/MeOH/CH2Cl2 mixture pro-
STol
2
0
Scheme 1. Reactions and Reagents: (a) AgClO4, SnCl2, dry CH2Cl2, 4 AÅ Mol sieves, rt,
overnight, 9: 97%, 12: 80%. (c) AcCl/MeOH/CH2Cl2 (1:20:20 v/v), rt, overnight, 10:
quantitative yield, 13: 86%. (b) DAST, NBS, dry CH2Cl2, ꢀ20 °C, 4 h, 11: 91%, 14: 84%.
0
BzO
O
(d) Cp2HfCl2, AgOTf, dry CH2Cl2, 4 ÅA Mol sieves, rt, overnight, 61%.
AcO
BzO
O
BzO
BzO
O
6
BzO
O
BzO
BzO
O
duced the desired acceptor disaccharide 10 in quantitative yield. In
the 1H and 13C NMR spectra of compound 10, the absence of signals
from the acetate group as seen in compound 9 confirmed the selec-
tive deacetylation. The disaccharide 9 was also converted to the
donor fluoride 11 in 91% yield using DAST and NBS at ꢀ20 °C.
The structure of 11 was supported by the 1H NMR spectra in which
two characteristic peaks in thiotolyl glycosides [doublet at d
7.20 ppm from two protons of tolyl ring and a singlet at d
BzO
BzO
STol
2
AcO
BzO
BzO
HO
BzO
BzO
OBz
O
OBz
O
5
6
STol
F
2.25 ppm from methyl] were not observed. The a-stereochemistry
at the anomeric carbon was supported by a C-1 signal at d
105.21 ppm (d, JC-1,F = 222.1 Hz) in the 13C NMR spectrum of 11,
whereas the signal at d 98.11 ppm was attributable to the other
anomeric carbon. In the 1H NMR spectrum of 11, the H-1 signal
was found obscured with other protons in the multiplet at d
5.92 ppm, however, the anomeric proton H-10 was observed as a
singlet at d 5.15 ppm (J = 1.4 Hz).
AcO
BzO
BzO
OBz
O
AcO
BzO
BzO
OBz
O
7
STol
8
OAc
Figure 3. Synthetic plan for the synthesis of
thioglycoside 2.
a(1?6)-linked octamannosyl

M. S. Aqueel et al. / Tetrahedron Letters 49 (2008) 7157–7160
7159
With the acceptor disaccharide 10 and fluoride donor disaccha-
ride 11 in hand, we next performed the [2+2] glycosylation reac-
tion to access (1?6)-linked tetramannosyl thioglycoside 12.
BzO
O
AcO
BzO
BzO
O
a
O
6
a
X
BzO
BzO
2
O
The coupling reaction between glycosides 10 and 11 was carried
BzO
BzO
O
out at room temperature with promotors AgClO4 and SnCl2 in
0
BzO
BzO
O
CH2Cl2 over 4 ÅA molecular sieves, and resulted in the desired glyco-
side 12 in 80% yield after purification by SiO2 column chromatog-
raphy. The 1H NMR spectrum of 12 in CDCl3 showed four anomeric
15. X = N3
b
16. X = NH2
c
protons at d 5.77, 5.25, 5.02, and 4.85 ppm as singlets suggesting a-
N
glycosylation. In the 13C NMR spectrum of 12, the four anomeric
carbons were observed at d 98.37, 97.99, 97.79, and 86.91 ppm.
The final structural confirmation was obtained by FABMS analysis
of 12, which showed a peak at 2085.0 [M+Na]+, corresponding to
the molecular formula C117H98O33SNa.
BzO
O
AcO
BzO
BzO
O
O
O
S
6
BzO
BzO
O
O
BzO
BzO
NH
O
BzO
O
BzO
The a(1?6)-linked tetramannosyl thioglycoside 12 was further
17
converted to thioglycosyl acceptor 13 and glycosyl fluoride donor
14 in 96% and 84% yields by similar reactions performed for the
syntheses of glycosides 10 and 11, respectively. The characteristic
acetate and thiotolyl groups were found absent in the 1H NMR and
13C NMR spectra of compounds 13 and 14, respectively. In the 13C
NMR spectra of glycosyl fluoride 14, the four anomeric carbon sig-
nals were observed at d 105.21 (d, JC-1,F = 223.0 Hz), 98.38, 97.92,
and 97.56 ppm. Finally, the FABMS analyses of glycosides 13 and
14 confirmed the formation of these products.
d
N
O
HO
O
HO
HO
HO
O
O
O
S
6
HO
HO
O
HO
HO
NH
O
HO
O
HO
1
Next, we pursued the [4+4] glycosylation reaction to access
0
a
(1?6)-linked octamannosyl p-tolylthioglycoside 2. The glycosyl
Scheme 2. Reactions and Reagents: (a) HOCH2CH2N3, NIS, AgOTf, dry CH2Cl2, 4 ÅA
Mol sieves, ꢀ4 °C, 45 min, 89%. (b) Pd/C, HCO2NH4, dry MeOH/CH2Cl2 (9:1), rt, 4 h,
82%. (c) Dansyl chloride, N-methylimidazole, dry CH2Cl2, 0 °C, overnight, 69%. (d)
satd NH3 in MeOH, dry CH2Cl2, rt, overnight, 71%.
donor 14 was treated with glycosyl acceptor 13 in the presence
of promoters AgClO4 and SnCl2 in CH2Cl2. Even after 48 h at room
temperature, only 20% yield of the desired octasaccharide 10 was
achieved along with the recovery of unreacted acceptor glycoside
13. The glycosylation reaction between 13 and 14 was next per-
formed with coupling reagents Cp2HfCl2 and AgOTf in CH2Cl2 over-
night at room temperature. Octamannoside 2 was produced in 61%
yield after purification by SiO2 column chromatography. The 1H
NMR spectrum of 2 in CDCl3 showed the anomeric protons at d
5.78, 5.24, 5.06, 5.01, 5.00, 4.99, and 4.82 ppm as singlets, and at
d 4.96 ppm (J1,2 = 1.4 Hz) as a doublet supporting the 1,2-trans gly-
cosylation. In the 13C NMR spectrum of 2, the anomeric carbons
were not well resolved and appeared as three signals at d 98.41,
97.99, and 86.89 ppm. Lastly, the final structural confirmation
was obtained by MALDI-TOF mass analysis of 2, which showed a
peak at 3983.9 [M+Na]+ corresponding to the molecular formula
C225H186O65SNa.
linked octamannosyl glycoside 17 in 69% yield. The 1H NMR spec-
trum of 17 indicated formation of the desired product as it showed
signals due to a dansyl group in the aromatic region as well as at d
2.79 ppm [N(CH3)2]. In the APT 13C NMR spectrum of 17 in CDCl3,
the carbons of CH2NH and N(CH3)2 were observed at d 42.67 and
45.39 ppm, respectively. The MALDI-TOF mass analysis of 17 gave
a peak at 4155.8 [M+Na]+ corresponding to the molecular formula
C232H196N2O68SNa.
Finally, global de-protection of 17 was accomplished by over-
night reaction with satd NH3 solution in MeOH at room tempera-
ture. TLC in CHCl3/MeOH/H2O (65:35:10, lower layer) showed
one fluorescent spot under a long wave UV lamp. The reaction mix-
ture was concentrated to dryness, and the syrup was washed sev-
eral times with CH2Cl2 and finally with EtOAc. The syrup was
purified by column chromatography on Sephadex LH-20 using
MeOH as the mobile phase to furnish the desired fluorescent probe
After the successful synthesis of 2, dansylated octamannan fluo-
rescent probe 1 was synthesized in four steps (Scheme 2). In step
one,
a(1?6)-linked octamannosyl thioglycoside 2 was reacted
with 1-azidoethanol 314 in the presence of activator NIS and Lewis
acid AgOTf at ꢀ4 °C for 45 min. Usual workup of the coupling reac-
tion followed by purification of the reaction product by column
a(1?6)-linked octamannosyl glycoside 1 in 71% yield. The signals
in the 1H NMR spectrum of 1 in D2O were not well resolved at
300 MHz, but showed protons in aromatic and sugar regions along
with a singlet at d 3.11 ppm (CH2NH) and d 2.29 ppm [N(CH3)2].
Similarly, in the 75 MHz 13C NMR spectrum of 1 in D2O, the pres-
ence of signals at d 46.82 ppm [N(CH3)2] and d 43.56 ppm (CH2NH)
supported the structure. The final structural confirmation was ob-
tained by FABMS analysis of 1, which showed a peak at 1613.1
[M+Na]+, corresponding to the molecular formula C62H98N2O43SNa.
In conclusion, we have reported a successful and efficient syn-
chromatography gave the desired 1-azidoethyl
a(1?6)-linked
octamannosyl glycoside 15 in 89% yield. In the 1H NMR and 13C
NMR spectra of 15, the signals from p-thiotolyl group were found
absent. The 13C NMR spectrum of 15 clearly indicated the signals
at d 50.60 and 62.46 ppm attributable to CH2N3 and OCH2 carbons,
respectively. The MALDI-TOF mass analysis of 15 showed a peak at
3947.1 [M+Na]+ corresponding to the molecular formula
C220H183N3O66Na.
thesis of a dansylated homolinear
a(1?6)-linked octamannan
In step two, the azido functionality in 15 was reduced using
ammonium formate (HCO2NH4) over Pd/C in CH2Cl2/MeOH (9:1),
and was monitored by TLC. Upon completion of the reaction, con-
fluorescent probe for its future biochemical potential use as a
delivery vehicle for drugs via mannan receptors present in the
infected macrophages.
centration and the usual workup produced 1-aminoethyl a(1?6)-
linked octamannosyl glycoside 16. Without further purification,
the crude 16 was reacted overnight at 0 °C in the dark with dansyl
chloride 3 and N-methylimidazole. TLC showed a major fluorescent
spot in long wave (365 nm) UV lamp, and purification by column
Acknowledgments
Authors acknowledge College of Arts and Science, Western Illi-
nois University for faculty start-up funds and NIH Grant
R21AI059270.
chromatography gave the desired blocked dansylated
a(1?6)-

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