One-pot strategies for the synthesis of the tetrasaccharide linkage region of proteoglycans

Article abstract of DOI:10.1021/ol200192d

A linker-attached tetrasaccharide corresponding to the linkage region of proteoglycans was synthesized via one-pot procedures from the silylated monosaccharide derivatives. Regioselective one-pot protection protocols were applied in generating the requisite monosaccharide building blocks whereas stereoselective one-pot glycosylation approaches were utilized to assemble the tetrasaccharide skeleton.

Full text of DOI:10.1021/ol200192d

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1506–1509
One-Pot Strategies for the Synthesis of the
Tetrasaccharide Linkage Region of
Proteoglycans
Teng-Yi Huang,†,‡ Medel Manuel L. Zulueta,† and Shang-Cheng Hung*,†,§
Genomics Research Center, Academia Sinica, 128 Sec.2 Academia Road, Taipei 115,
Taiwan, Department of Chemistry, National Tsing Hua University, 101 Sec. 2 Kuang-Fu
Road, Hsinchu 300, Taiwan, and Department of Applied Chemistry, National Chiao
Tung University, 1001 Ta-Hsueh Road, Hsinchu 300, Taiwan
schung@gate.sinica.edu.tw
Received January 21, 2011
ABSTRACT
A linker-attached tetrasaccharide corresponding to the linkage region of proteoglycans was synthesized via one-pot procedures from the silylated
monosaccharide derivatives. Regioselective one-pot protection protocols were applied in generating the requisite monosaccharide building
blocks whereas stereoselective one-pot glycosylation approaches were utilized to assemble the tetrasaccharide skeleton.
Proteoglycans are biologically important macromole-
cules widespread on the cell surface and in the extracellular
matrix.1 Theyinteractwithotherbiomolecules throughthe
distinct profile of the glycosaminoglycan (GAG) chains
decorating the protein core.2 GAG biosynthesis initiates
from the β-attachment of D-xylose (Xyl) to a serine residue
followed by consecutive additions of two D-galactose (Gal)
and one D-glucuronic acid (GlcA) unit in the respec-
tive β1f4, β1f3, and β1f3 manner generating the
tetrasaccharide linkage region (Figure 1).3 Further chain
elongation sorts GAGs into two major categories. Heparin
and heparan sulfate form by the alternate additions of N-
acetyl D-glucosamine and GlcA whereas chondroitin sulfate
and dermatan sulfate incorporate N-acetyl D-galactosamine
† Genomics Research Center, Academia Sinica.
‡ Department of Chemistry, National Tsing Hua University.
§ Department of Applied Chemistry, National Chiao Tung University.
(1) (a) Iozzo, R. V.; Schaefer, L. FEBS J. 2010, 277, 3864–3875. (b)
Ariga, T.; Miyatake, T.; Yu, R. K. J. Neurosci. Res. 2010, 88, 2303–2315.
(c) Esko, J. D.; Kimata, K.; Lindahl, U. In Essentials of Glycobiology,
2nd ed.; Varki, A., Cummings, V.; Esko, J. D.; Freeze, H. H.; Stanley, P.;
Bertozzi, C. R.; Hart, G. W.; Etzler, M. E., Eds; Cold Spring Harbor
Laboratory Press: New York, 2009; pp 229-248.
(2) Zhang, L. In Progress in Molecular Biology and Translational
Science; Zhang, L., Ed.; Elsevier: Amsterdam, 2010; Vol. 93, pp 1-17.
(3) (a) Nadanaka, S.; Kitagawa, H. J. Biochem. 2008, 144, 7–14. (b)
Bishop, J. R.; Schuksz, M.; Esko, J. D. Nature 2007, 446, 1030–1037. (c)
Silbert, J. E.; Sugumaran, G. IUBMB Life 2002, 54, 177–186.
Figure 1. Structures of proteoglycans.
r
10.1021/ol200192d
Published on Web 02/18/2011
2011 American Chemical Society

and GlcA.4 Intermittent N-deacetylation, uronic acid 5-C-
epimerization, and multiple N- and O-sulfonations deliver
the mature structure responsible for biological activity.
The formation of different GAGs withhigh fidelity from
a common protein-linked tetrasaccharide precursor has
intrigued many investigators. Occasional modifications,
such as 2-O-phosphorylation of Xyl and 4- and 6-O-
sulfonations of Gal residues, were suggested to influence
the divergent biosynthetic routes.5 Understanding the
nature of these modifications and their effects on the
specificity of biosynthetic enzymes require compounds
thatmimic thenatural substrates. Synthetic methodologies
that target the proteoglycan linkage region backbone have
been reported.6 Notable concerns therein include the
tedious generation of appropriately protected monosac-
charide building blocks, particularly that of Xyl,7 and the
stereocontrol in the Galβ1f3Gal glycosidic bond forma-
tion that often give low selectivity and yield.8 We recently
demonstrated one-pot strategies for regioselective protec-
tion of monosaccharides and stereoselective glycosylation
in order to simplify synthetic procedures and reduce time-
and resource-consuming workup and purification steps.9
Drawing from these methodologies, we present herein an
approach to the chemical synthesis of the linkage region
tetrasaccharide 1 fitted with an amine terminated linker as
an aid to deciphering the processes associated with the
modification of the linkage region together with its roles in
chain elongation.
Scheme 1. Retrosynthesis of Compound 1
The β-selectivity in forming all glycosidic bonds would rely
on neighboring group assistance by the acyl groups at 2-O
of4-6; solvent effectscouldbe exploited inpertinentcases.
Compounds 4-6 would be prepared through regioselec-
tive one-pot protection strategies starting from the sily-
lated thioglycosides 7-9, respectively.
Our retrosynthetic plan is depicted in Scheme 1. By
typical transformations, compound 1 is accessible follow-
ing the assembly of the fully protected tetrasaccharide 2
using stereoselective one-pot glycosylations of the mono-
saccharide building blocks 4-6 and the linker derivative 3.
The orthogonal TBS protection of the thiogalactoside 5
would facilitate chain elongation and is also expected to
enhance the donor reactivity during the coupling processes.
The nearly similar reactivities of the 2-C, 3-C, and 4-C
hydroxyls of D-xylopyranosides render their full differen-
tiation a challenging task. Delightfully, the TMSOTf-
catalyzed Et3SiH-reductive benzylation of the 2,3,4-tri-
O-TMS ether 9 primarily gave, after TBAF treatment,
the 3-O-benzylated 10. At -40 °C with 1.1 equiv of
benzaldehyde, 10 was obtained in 65% yield (Scheme 2).
Its 2- and 4-OBn isomers were alsoisolated at 10% and 6%
yields, respectively. We next focused on regioselective
benzoyl group installation at the 2-O position. Treatment
of 10 with BzCl in pyridine or Bz2O in the presence of
TMSOTf formed the 4-O-benzoylated isomer as the major
product in 40% and 30% yields respectively. Recently, we
found that Yb(OTf)3-catalyzed acylation was effective in
(4) Sugahara, K.; Kitagawa, H. Curr. Opin. Struct. Biol. 2000, 10,
518–527.
(5) (a) Tone, Y.; Pedersen, L. C.; Yamamoto, T.; Izumikawa, T.;
Kitagawa, H.; Nishihara, J.; Tamura, J.; Negishi, M.; Sugahara, K. J.
Biol. Chem. 2008, 283, 16801–16807. (b) Kitagawa, H.; Tsutsumi, K.;
Ikegami-Kuzuhara, A.; Nadanaka, S.; Goto, F.; Ogawa, T.; Sugahara,
K. J. Biol. Chem. 2008, 283, 27438–27443.
(6) (a) Tamura, J.; Nakamura-Yamamoto, T.; Nishimura, Y.;
Mizumoto, S.; Takahashi, J.; Sugahara, K. Carbohydr. Res. 2010, 345,
2115–2123. (b) Thollas, B.; Jacquinet, J. C. Org. Biomol. Chem. 2004, 2,
434–442. (c) Tamura, J.; Nishihara, J. J. Org. Chem. 2001, 66, 3074–
3083. (d) Allen, J. G.; Fraser-Reid, B. J. Am. Chem. Soc. 1999, 121, 468–
469. (e) Neumann, K. W.; Tamura, J.; Ogawa, T. Bioorg. Med. Chem.
1995, 3, 1637–1650. (f) Rio, S.; Beau, J. M.; Jacquinet, J. C. Carbohydr.
Res. 1993, 244, 295–313. (g) Goto, F.; Ogawa, T. Tetrahedron Lett. 1992,
33, 5099–5102.
Scheme 2. Synthesis of the Thioxyloside 6
(7) Shimawaki, K.; Fujisawa, Y.; Sato, F.; Fujitani, N.; Kurogochi,
M.; Hoshi, H.; Hinou, H.; Nishimura, S. I. Angew. Chem., Int. Ed. 2007,
46, 3074–3079.
(8) (a) McGill, N. W.; Williams, S. J. J. Org. Chem. 2009, 74, 9388–
9398. (b) Jacquinet, J.-C. Carbohydr. Res. 2004, 339, 349–359. (c) Belot,
F.; Jacquinet, J.-C. Carbohydr. Res. 2000, 325, 93–106.
(9) (a) Chang, K.-L.; Zulueta, M. M. L.; Lu, X.-A.; Zhong, Y.-Q.;
Hung, S.-C. J. Org. Chem. 2010, 75, 7424–7427. (b) Wang, C.-C.;
Kulkarni, S. S.; Lee, J.-C.; Luo, S.-Y.; Hung, S.-C. Nat. Protoc. 2008,
3, 97–113. (c) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.;
Huang, Y.-W.; Lee, C.-C.; Chang, K.-L.; Hung, S.-C. Nature 2007, 446,
896–899. (d) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Fan, H.-F.; Pai, C.-L.;
Yang, W.-C.; Lu, L.-D.; Hung, S.-C. Angew. Chem., Int. Ed. 2002, 41,
2360–2362.
Org. Lett., Vol. 13, No. 6, 2011
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the desymmetrization of myo-inositol 1,3,5-orthofor-
mate.10 Benzoylation with 5 mol % Yb(OTf)3 and 1.05
equiv of Bz2O provided good regioselectivity, and the
4-alcohol 6 was isolated in 81% yield. The metal ion
perhaps coordinates with both 2-O and 4-O atoms forcing
Scheme 4. Synthesis of the Thioglucoside 4
1
the C4 conformation in the intermediate 11, enhancing
regioselective Bz group introduction at the more nucleo-
philic 2-O (path I) rather than the 4-O position (path II).
When benzylation (PhCHO, Et3SiH, TMSOTf) and ben-
zoylation [Bz2O, Yb(OTf)3] were carried out in one pot
from 9, product 6 was obtained in 45% isolated yield. To
our knowledge, this is, by far, the most straightforward
preparation of xylosyl derivatives of identical protecting
group pattern.
6-tetra-O-TMS ether
7 in 77% yield. Through
TMSOTf catalysis, 7 underwent 4,6-O-benzylidenation
followed by reductive 3-O-benzylation to furnish the
intermediate 16. In the same flask, the 6-O-ring-open-
ing of the benzylidene acetal still catalyzed by
TMSOTf, employing BH3•THF as the reductant, gen-
erated the 2,6-diol intermediate 17. Then, the reaction
mixture was treated with Et3N to pave the way for the
2,6-di-O-acetylation in the presence of DMAP.
Scheme 3. Synthesis of the Thiogalactoside 5
Scheme 5. Synthesis of the Disaccharide Acceptor 20
Unlike the similar case for D-glucopyranosides where 2-O-
acylations predominate,11 one-pot benzylidenation and
monobenzoylation of p-methylphenyl 2,3,4,6-tetra-O-TMS-
1-thio-β-D-galactopyranoside gave us the unwanted 3-O-
benzoyl derivative as the major isomer (62%). Alternatively,
we performed the protecting group installation through the
TBDPS functionalized 8 (Scheme 3). With catalytic
TMSOTf, 8 was subjected to 3,4-O-benzylidenation, 2-O-
benzoylation, and 6-O-desilylation in one pot to afford the
6-alcohols 12 (68%, exo/endo = 1/1.1). Treatment of 12 with
10-camphorsulfonic acid (CSA) led to benzylidene migration
quantitatively providing the 3-alcohol 13 which, in turn, was
masked with the TBS group to form the fully protected 14 in
91% yield. Although 14 carries applicable protecting groups
for chain assembly, our preliminary trials for the β1f3 bond
formation between the Gal residues gave low yields, which
might be caused by the rigid and bulky 4,6-O-benzylidene
group. Accordingly, regioselective acetal ring-opening using
BH3•THF and TMSOTf was performed furnishing the
6-alcohol 15 (95%), which underwent Williamson etherifica-
tion to generate the galactosyl donor 5 in 91% yield. It was
noted, in this special example, that the 2-O-benzoyl group
tolerated the basic conditions.
With the key building blocks 4-6 in hand, the construc-
tion of the target sugar backbone was initiated (Scheme 5).
Following N-iodosuccinimide (NIS) and TfOH treatment,
selective activation of the thiogalactoside 5 in the presence
Scheme 4 illustrates the regioselective one-pot synthesis
of the D-gluco β-thiopyranoside 4 from the 2,3,4,
(12) (a) Noti, C.; de Paz, J. L.; Polito, L.; Seeberger, P. H. Chem.;
Eur. J. 2006, 12, 8664–8686. (b) Mong, T. K.-K.; Lee, H.-K.; Duron,
S. G.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 797–802.
(13) (a) King, S. A.; Pipik, B.; Thompson, A. S.; Decamp, A.;
Verhoeven, T. R. Tetrahedron Lett. 1995, 36, 4563–4566. (b) Nicolaou,
K. C.; Bockovich, N. J.; Carcanague, D. R.; Hummel, C. W.; Even, L. F.
J. Am. Chem. Soc. 1992, 114, 8701–8702.
(10) Padiyar, L. T.; Wen, Y.-S.; Hung, S.-C. Chem. Commun. 2010,
46, 5524–5526.
(11) Lu, X.-A.; Chou, C.-H.; Wang, C.-C.; Hung, S.-C. Synlett 2003,
1364–1366.
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Org. Lett., Vol. 13, No. 6, 2011

of the thioxyloside 6 was achieved at -78 °C giving the β-
0
0
disaccharide 18 (65%, J1 ,2 = 8.1 Hz). Further condensa-
tion with the linker derivative 312 formed the adduct 19
(J1,2 = 7.1 Hz) in 77% yield. When this sequence was done
in one pot, 19 was acquired in an isolated yield of 48%. For
the necessary TBS group cleavage, acidic hydrolysis13 was
utilized to prevent the base-mediated Bz migration. Con-
sequently, the 3-alcohol 20 (91%) was efficiently acquired
after treatment of 19 with BF3•Et2O. Extension of the one-
pot procedure to the TBS deprotection via TfOH treat-
ment gave the required compound 20 in 32% yield.
Scheme 6. Synthesis of Compound 1a
We next turned to the alternative reducing end-to-non-
reducing end route. Taking advantage of the higher re-
activity of the primary alcohol in 3, its glycosylation at -40
°C by the thioxyloside 6 delivered the β-linked 21 (J1,2
=
7.0 Hz) in 71% yield. Coupling of 21 with the thiogalacto-
side 5 led to compound 19 (71%). Glycosylation of 3 with 6
followed by coupling with the donor 5 in one pot at the
same temperature provided 19 in 37% yield. Unfortu-
nately, inclusion of the TfOH-mediated cleavage of the
TBS group in the one-pot method resulted in products
which are difficult to purify.
The preparation of the target tetrasaccharide is depicted
in Scheme 6. The stereoselectivity of the NIS/TfOH-acti-
vated coupling of the thiogalactoside 5 and the disaccharide
acceptor 20 was improved from a β/R ratio of 3/1 with
CH2Cl2 as solvent to 14/1 when the CH2Cl2/CH3CN (1/3)
00 00
solvent mixture14 was utilized; the trisaccharide 22 (J1 ,2
=
7.7 Hz) was obtained in 79% yield. When this glycosylation,
followed by acidic desilylation, was carried out in one pot,
the 300-alcohol 23 was isolated in 63% yield. The thiogluco-
side 4activation with NIS and TfOH was further included in
the one-pot process which led to the linkage region tetra-
saccharide skeleton 2 in a one-pot yield of 37%. Removal of
a BAIB: [bis(acetoxy)iodo]benzene.
ꢀ
all ester groups in 2 using Zemplen’s deacylation provided
the pentaol 24 (84%), which underwent regioselective
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation
of the primary hydroxyl in the D-glucosyl unit to give the
carboxylate 25 in 73% yield.15 Global deprotection by
hydrogenolysis finally furnished the target compound 1 in
98% yield. The structure of 1 was confirmed through 1D
and 2D NMR and HRMS experiments.
the oligosaccharide assembly. The generated linker-at-
tached tetrasaccharide is designed for the examination of
the biosynthetic pathway involved in the modification of
the sugar units as well as its effects in chain elongation and
sorting of GAGs. These explorations will be carried out in
the future.
In conclusion, we have developed regioselective one-pot
preparations of the requisite monosaccharide building
blocks for the synthesis of the tetrasaccharide linkage
region of proteoglycans. Stereoselective one-pot glycosida-
tion protocols were also advanced which further simplified
Acknowledgment. This work was supported by the Na-
tional Science Council (NSC 97-2113-M-001-033-MY3,
NSC 98-2119-M-001-008-MY2, NSC 98-3112-B-007-005,
NSC 98-2627-B-007-008) and Academia Sinica.
(14) Chao, C.-S.; Li, C.-W.; Chen, M.-C.; Chang, S.-S.; Mong, K.-K.
T. Chem.;Eur. J. 2009, 15, 10972-10982.
(15) (a) Lu, L.-D.; Shie, C.-R.; Kulkarni, S. S.; Pan, G.-R.; Lu, X.-A.;
Hung, S.-C. Org. Lett. 2006, 8, 5995–5998. (b) Epp, J. B.; Widlanski,
T. S. J. Org. Chem. 1999, 64, 293–295.
Supporting Information Available. Experimental pro-
cedure and NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 6, 2011
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