Synthesis of 48 disaccharide building blocks for the assembly of a heparin and heparan sulfate oligosaccharide library

Article abstract of DOI:10.1021/ol062464t

(Chemical Equation Presented) An efficient synthesis of the entire set of suitably protected 48 disaccharide building blocks for the assembly of a heparin and heparan sulfate oligosaccharide library is described here.

Full text of DOI:10.1021/ol062464t

ORGANIC
LETTERS
2006
Vol. 8, No. 26
5995-5998
Synthesis of 48 Disaccharide Building
Blocks for the Assembly of a Heparin
and Heparan Sulfate Oligosaccharide
Library
Lung-Dai Lu, Chi-Rung Shie, Suvarn S. Kulkarni, Guan-Rong Pan, Xin-An Lu, and
Shang-Cheng Hung*
Department of Chemistry, National Tsing Hua UniVersity, No. 101, Section 2,
Kuang-Fu Road, Hsinchu 300, Taiwan, and Genomics Research Center,
Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan
hung@mx.nthu.edu.tw
Received October 6, 2006
ABSTRACT
An efficient synthesis of the entire set of suitably protected 48 disaccharide building blocks for the assembly of a heparin and heparan sulfate
oligosaccharide library is described here.
Heparin (HP) and heparan sulfate (HS) are structurally related
linear polyanionic polysaccharides, belonging to the family
of glycosaminoglycans, covalently bound to the core proteins
of proteoglycans. These naturally occurring, highly sul-
fonated and acidic biopolymers play crucial roles in numer-
ous biological systems through their interaction with diverse
proteins.1 HP, which occurs exclusively in mast cells, is
widely used as an anticoagulant drug in the clinic owing to
its high affinity binding with antithrombin III.2 HS, being
ubiquitously distributed on the cell surface and in the
extracellular matrix, mediates various physiologically im-
portant processes such as viral and bacterial infection, growth
factor regulation, inflammatory response, angiogenesis, tumor
metastasis, cell adhesion, and lipid metabolism.3
HP and HS are both biosynthesized through a unique
pathway, which involves the formation of a polysaccharide
chain consisting of alternating N-acetyl-R-D-glucosamine
(GlcNAc) and â-D-glucuronic acid (GlcA) residues jointed
by 1f4 linkages. This backbone is modified through a series
of enzymatic processes, including C5 epimerization of GlcA
to L-iduronic acid (IdoA), N-deacetylation of GlcNAc to
D-glucosamine (GlcNH2), and sulfonation at the O2 position
of GlcA/IdoA and at the N, O3, and/or O6 positions of
GlcNH2.4 Variable substitution patterns of the polysaccharide
chain give rise to a large number of complex sequences
resulting in microheterogeneity, for example, 48 di-, 482
tetra-, 483 hexa-, 484 octasaccharides, and so on. Of these
theoretically possible 48 disaccharide units (Scheme 1), only
23 have been characterized so far.5 Homogeneous HP and
HS materials with well-defined configurations are essential
(1) (a) Conrad, H. E. Heparin-Binding Proteins; Academic Press: San
Diego, 1998. (b) Lindahl, U. Glycoconjugate J. 2000, 17, 597-605. (c)
Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 390-412.
(2) (a) van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. 1993,
32, 1671-1690. (b) Petitou, M.; He´rault, J.-P.; Bernat, A.; Driguez, P.-A.;
Duchaussoy, P.; Lormeau, J.-C.; Herbert, J.-M. Nature 1999, 398, 417-
422. (c) Petitou, M.; van Boeckel, C. A. A. Angew. Chem., Int. Ed. 2004,
43, 3118-3133. (d) de Kort, M.; Buijsman, R. C.; van Boeckel, C. A. A.
Drug DiscoVery Today 2005, 10, 769-779.
(3) (a) Iozzo, R. V. Nat. ReV. Mol. Cell Biol. 2005, 6, 646-656. (b)
Ha¨cker, U.; Nybakken, K.; Perrimon, N. Nat. ReV. Mol. Cell Biol. 2005, 6,
530-541. (c) Fuster, M. M.; Esko, J. D. Nat. ReV. Cancer 2005, 5, 526-
542.
(4) Lindahl, U. Pure Appl. Chem. 1997, 69, 1897-1902.
(5) Esko, J. D.; Selleck, S. B. Annu. ReV. Biochem. 2002, 71, 435-471.
10.1021/ol062464t CCC: $33.50
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trans-glycosidic linkages during chain elongation, the same
ester functionality at C2 would be expected to offer anchi-
meric assistance, whereas benzyl ethers at such locations
might have the stereochemistry of glycosidation controlled
via solvent effects (e.g., CH3CN).7 In addition, a temporary
protecting group (TBDPS) is needed to mask the primary
hydroxyl on the D-glucopyranosyl subunit of 1 to allow
oxidation to the corresponding carboxylic acid. The 2-naph-
thylmethyl group (2-NAP),8 which was used to block the
C4 hydroxy group of the GlcNH2 subunit, would allow
mildly chemoselective deprotection for further elongation of
the sugar chain, and it could also be simultaneously removed
along with other permanent benzyl groups under hydro-
genolytic conditions at the final termination process. The C2
amino group of GlcNH2 would typically be protected as an
azide owing to its nonparticipating nature in coupling
reactions. It would be expected to predominantly lead to the
R-anomeric disaccharide building blocks and could be readily
transformed into the NHAc and NHCbz groups. At a later
stage, the -N3 could be selectively converted to the
-NHSO3- unit via a combination of Staudinger reaction and
N-sulfonation, whereas the -NHCbz could be expected to
reveal a free -NH2 upon hydrogenolysis. The 1,6-anhydro-
â-L-idopyranosyl sugars 5, which can be prepared from
D-glucose through C5 epimerization, serve as highly active
glycosyl acceptors because of the rigid conformation and
three equatorially substituted groups at C2, C3, and C4. The
1,6-anhydro ring of 2 can be opened, and further functional
group modification and glycosylation of the corresponding
L-idopyranosyl sugar at C6 and C1 can be carried out,
respectively. Thus, four D-glucosamine-derived glycosyl
donors 3 may be individually coupled with two D-glucopy-
ranosyl 4-alcohols 4 and two L-idopyranosyl 4-alcohols 5
via Schmidt’s trichloroacetimidate method9 to get two
sets of eight disaccharides. These 16 compounds can each
be converted into the N-acetylated and N-Cbz-protected
derivatives, generating a total of 48 disaccharide synthons 1
and 2.
Scheme 1
for determining structure-activity relationships. Such mol-
ecules are extremely difficult to acquire from natural sources,
and chemical synthesis may offer one of the best options
for securing them. Over the past few years, several ap-
proaches have been documented in the literature to prepare
specific HP and HS saccharide units.6 Herein, we report a
straightforward synthesis of the entire set of 48 disaccharide
building blocks needed for the assembly of HP and HS
oligosaccharide libraries.
The synthesis of four glycosyl donors 15-18 is shown in
Scheme 2. First, D-glucosamine-derived 1,3-diol 610 was
converted to the â-1,3-dibenzoate 7 (BzCl, Et3N, 92%). The
corresponding 3-OBn derivative 8 was obtained in 77%
overall yield via anomeric benzoylation (Bz2O, Et3N) fol-
lowed by O3 benzylation (Ag2O, BnBr). A highly regiose-
lective borane-reductive O6 ring opening of 4,6-O-naph-
thylidene acetals 7 and 8 in the presence of 5 mol % of
Our retrosynthesis of 48 disaccharide synthons 1 and 2
from two common sugars, D-glucose and D-glucosamine, is
outlined in Scheme 1. The O-sulfation pattern in the target
molecules called for strategic placement of acyl groups (Bz)
to protect those hydroxyls that would be ultimately sulfonated
and of permanent benzyl protecting groups (Bn) for those
that would remain free. For the generation of exclusive 1,2-
11
Cu(OTf)2 cleanly afforded the individual 6-alcohols 9
(6) (a) Tabeur, C.; Mallet, J.-M.; Bono, F.; Herbert, J.-M.; Petitou, M.;
Sinay¨, P. Bioorg. Med. Chem. 1999, 7, 2003-2012. (b) de Paz, J.-L.;
Angulo, J.; Lassaletta, J.-M.; Nieto, P. M.; Ridondo-Horcajo, M.; Lozano,
R. M.; Gimenez-Gallego, G.; Martin-Lomas, M. ChemBioChem 2001, 2,
673-685. (c) Prabhu, A.; Venot, A.; Boons, G.-J. Org. Lett. 2003, 5, 4975-
4978. (d) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Litjens, R. E. J. N.;
Palmacci, E. R.; Seeberger, P. H. Chem.-Eur. J. 2003, 9, 140-169. (e)
Poletti, L.; Lay, L. Eur. J. Org. Chem. 2003, 2999-3024. (f) Karst, N. A.;
Linhardt, R. J. Curr. Med. Chem. 2003, 10, 1993-2031. (g) Lee, J.-C.;
Lu, X.-A.; Kulkarni, S. S.; Wen, Y. S.; Hung, S.-C. J. Am. Chem. Soc.
2004, 126, 476-477. (h) Yu, H. N.; Furukawa, J.-I.; Ikeda, T.; Wong, C.-
H. Org. Lett. 2004, 6, 723-726. (i) Lubineau, A.; Lortat-Jacob, H.; Gavard,
O.; Sarrazin, S.; Bonnaffe´, D. Chem.-Eur. J. 2004, 10, 4265-4282. (j)
Code, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft, H. S.; van Boeckel,
C. A. A.; van Boom, J. H.; van der Marel, G. A. J. Am. Chem. Soc. 2005,
127, 3767-3773. (k) Noti, C.; Seeberger, P. H. Chem. Biol. 2005, 12, 731-
756.
(89%) and 10 (86%), which were subsequently benzoylated
to give the 6-OBz derivatives 11 and 12 in 95% and 92%
yields, respectively. Benzylation of 9 or 10 employing BnBr/
Ag2O or BnBr/NaH did not succeed. Alternatively, TMSOTf-
(7) Schmidt, R. R.; Behrendt, M.; Toepfer, A. Synlett 1990, 694-696.
(8) Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.; Alderfer, J. L.;
Matta, K. L. Tetrahedron Lett. 2000, 41, 169-172.
(9) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Chem. Biochem. 1994,
50, 21-123.
(10) Lu, X.-A.; Chou, C.-H.; Wang, C.-C.; Hung, S.-C. Synlett 2003,
1364-1366.
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Hung, S.-C. Angew. Chem., Int. Ed. 2005, 44, 1665-1668.
5996
Org. Lett., Vol. 8, No. 26, 2006

of the 4,6-O-benzylidene acetal in a single flask gave the
4,6-diol 23 (63%), which was similarly protected at the O6
position to afford the corresponding silyl ether 24 in 91%
yield.
Scheme 2
A practical route for the synthesis of rare 1,6-anhydro-â-
L-idopyranosyl 4-alcohols 27 and 30 is depicted in Scheme
4. Compound 25, generated from diacetone R-D-glucose in
Scheme 4
activated Et3SiH-reductive etherification21 of the correspond-
ing 6-O-trimethylsilyl derivatives with benzaldehyde led to
the expected 6-OBn compounds 13 (92%) and 14 (91%),
respectively. These four anomeric benzoates 11-14, upon
facile nucleophilic displacement with ammonia, were con-
comitantly transformed into the glycosyl trichloroacetimi-
dates 15-18 in high overall yields, respectively.
Scheme 3 illustrates our efficient preparation of thiogly-
cosides 21 and 24. Tetraol 19 underwent sequential 4,6-O-
benzylidenation (91%) and 2,3-di-O-benzylation (90%) to
yield the ether derivative 20, which was subjected to acid
hydrolysis followed by regioselective silylation at O6 to
furnish the 4-alcohol 21 (92%). A three-step protocol from
tetraol 19 was employed for the synthesis of compound 24.
First, one-pot 4,6-O-benzylidenation and 2,3-di-O-silylation
of 19 provided the bis-OTMS ether 22 (72%). Regioselective
O3 benzylation12 of 22 followed by O2 benzoylation under
the prevailing acidic environment and concomitant hydrolysis
three known steps,6g upon treatment with t-BuOK followed
by addition of a 1:2 mixture of 0.2 N H2SO4(aq) and diglyme
and subsequent heating at 160 °C in a one-pot manner
afforded the 2,4-diol 26 (52%), which was regioselectively
benzoylated to yield the 2-OBz 27 (85%). Direct benzylation
of 26 under various conditions gave a mixture of 2,3-di-
OBn 30 (low yield), 3,4-di-OBn, and 2,3,4-tri-OBn, which
was tedious for separation of the first two regioisomers.
Alternatively, methanolysis of the ketal 25 furnished the
methyl glucofuranoside 28 (81%), which underwent benzy-
lation at O2 to get the product 29 in 94% yield. Similar
conversion of compound 29 into the 1,6-anhydro sugar was
carried out, and the desired product 30 (45%) was obtained.
The assembly of eight monosaccharide units to prepare
the entire set of 48 disaccharide synthons is summarized in
Scheme 5. AgOTf- or TMSOTf-promoted coupling of the
donors 15-18 with the acceptors 21, 24, 27, and 30 led to
the disaccharides 31-46, respectively. The O6 benzoyl group
had a marked influence on the stereoselectivity of the
glycosylation reaction, yielding the D-gluco-disaccharides
(31, 33, 35, and 36) in only R-form and the L-ido-
disaccharides (39, 41, 43, and 44) as major R-isomers. In
contrast, the benzyl ethers at the C6 position of 17 and 18
reflected lower R/â selectivity. Finally, treatment of 31-46
with thioacetic acid13 gave the N-acetylated products
47-62 in excellent yields, respectively. A two-step trans-
formation of 31-46, individually, via consecutive Staudinger
Scheme 3
(12) 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. 8, No. 26, 2006
5997

Scheme 5
reaction and Cbz protection afforded the corresponding
N-Cbz derivatives 63-78 in good yields.
Transformations of two representative synthons into the
linker-attached HP/HS disaccharides are exemplified in
Schemes 6 and 7. Coupling of 47 with the alcohol 79
In conclusion, we have synthesized 48 HP- and HS-related
disaccharide building blocks. These synthons can be used
to prepare HP and HS oligosaccharides in chemically pure
form for screening against various proteins.
Scheme 7
Scheme 6
furnished the â-isomer 80, which underwent desilylation,
TEMPO oxidation, debenzoylation, O-sulfonation, and hy-
drogenolysis to give the desired molecule 81. Acetolysis of
39 followed by anomeric conversion and subsequent coupling
with 79 led to the R-isomer 82, which was subjected to
deacetylation, TEMPO oxidation, debenzoylation, azido
reduction, N- and O-sulfonation, and hydrogenolysis to yield
the expected disaccharide 83.
Acknowledgment. This work was supported by the
National Science Council (NSC 94-2113-M-007-021, NSC
94-2627-M-007-002, NSC 95-2752-B-007-002-PAE) and the
Academia Sinica (AS-92-TP-A04).
Supporting Information Available: Experimental pro-
cedures and 1H NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
(13) Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J. J.
Am. Chem. Soc. 2003, 125, 7754-7755.
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Org. Lett., Vol. 8, No. 26, 2006