New Set of Orthogonal Protecting Groups for the Modular Synthesis of Heparan Sulfate Fragments

Article abstract of DOI:10.1021/ol0359261

(Equation Presented) Six strategically chosen monosaccharide building blocks, which are protected by a novel set of four orthogonal protecting groups (Lev, Fmoc, TBDPS, and All), can be employed for the efficient synthesis of the 20 disaccharide moieties

Full text of DOI:10.1021/ol0359261

ORGANIC
LETTERS
2003
Vol. 5, No. 26
4975-4978
New Set of Orthogonal Protecting
Groups for the Modular Synthesis of
Heparan Sulfate Fragments
Arati Prabhu, Andre Venot, and Geert-Jan Boons*
Complex Carbohydrate Research Center, The UniVersity of Georgia,
220 RiVerbend Road, Athens, Georgia 30602
gjboons@ccrc.uga.edu
Received October 1, 2003
ABSTRACT
Six strategically chosen monosaccharide building blocks, which are protected by a novel set of four orthogonal protecting groups (Lev, Fmoc,
TBDPS, and All), can be employed for the efficient synthesis of the 20 disaccharide moieties found in heparan sulfate. The properly protected
disaccharide building blocks can be converted into glycosyl donors and acceptors, which can be used for the modular synthesis of a wide
range of well-defined oligosaccharides that differ in sulfation pattern.
Heparan sulfates (HS) are highly sulfated polysaccharides
that are linked to a core protein, which are found on the cell
surfaces and the extracellular matrix of all eukaryotes, where
they play pivotal roles in a large number of biological
processes. For example, many enzymes, growth factors,
enzyme inhibitors, chemokines, cell adhesion molecules, and
microbial proteins require HS for their functions.1-6 The
biosynthesis of HS involves the initial formation of a simple
polysaccharide composed of alternating â-D-glucuronic acid
(GlcA) and R-N-acetyl-D-glucosamine (GlcNAc) units joined
by 1-4 anomeric linkages. This structure is then modified
by a series of enzymatic transformations involving N-
deacetylation followed by N-sulfation, C-5 epimerization of
GlcA to L-iduronic acid (IdoA), and finally O-sulfation.
Ultimately, these modifications result in the formation of an
IdoA(2-OSO3)-GlcNSO3(6-OSO3) sequence. Structural stud-
ies have, however, shown that HS contains 19 other
disaccharide subunits arising from incomplete or additional
enzymatic modifications. Combining these different disac-
charides into larger structures results potentially in enormous
structural diversity.1
Detailed structure-activity relationship studies are begin-
ning to unravel the biological significance of HS structural
diversity. However, progress is hampered by the difficulties
of identifying HS-binding motifs for specific proteins. It is
to be expected that this problem will be addressed by
screening a relatively large panel of well-defined HS
fragments.7-14 Organic synthesis provides the most powerful
approach for obtaining well-defined HS fragments. However,
no strategy for the preparation of a wide range of HS
(7) Kovensky, J.; Duchaussoy, P.; Bono, F.; Salmivirta, M.; Sizun, P.;
Herbert, J. M.; Petitou, M.; Sinay, P. Bioorg. Med. Chem. 1999, 7, 1567-
1580.
(8) Kovensky, J.; Duchaussoy, P.; Petitou, M.; Sinay, P. Tetrahedron:
Asymmetry 1996, 7, 3119-3128.
(9) van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. Engl.
1993, 32, 1671-1690.
(10) Dreef-Tromp, C. M.; Basten, J. E. M.; Broekhoven, M. A.; van
Dinther, T. G.; Petitou, M.; van Boeckel, C. A. A. Bioorg. Med. Chem.
Lett. 1998, 8, 2081-2086.
(1) Esko, J. D.; Selleck, S. B. Annu. ReV. Biochem. 2002, 71, 435-471.
(2) Gallagher, J. T.; Turnbull, J. E. Glycobiology 1992, 2, 523-528.
(3) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 391-
412.
(4) Spillmann, D.; Lindahl, U. Curr. Opin. Struct. Biol. 1994, 4, 677-
682.
(5) Rostand, K. S.; Esko, J. D. Infect. Immun. 1997, 65, 1-8.
(6) Sasisekharan, R.; Shriver, Z.; Venkataraman, G.; Narayanasami, U.
Natl. ReV. Cancer 2002, 2, 521-528.
(11) Petitou, M.; Herault, L. P.; Bernat, A.; Driguez, P. A.; Duchaussoy,
P.; Lormeau, J. C.; Herbert, J. M. Nature 1999, 398, 417-422.
(12) Westerduin, P.; Basten, J. E. M.; Broekhoven, M. A.; de Kimpe,
V.; Kuijpers, W. H. A.; van Boeckel, C. A. A. Angew. Chem., Int. Ed.
Engl. 1996, 35, 331-333.
(13) Wong, C. H.; Ye, X. S.; Zhang, Z. Y. J. Am. Chem. Soc. 1998,
120, 7137-7138.
10.1021/ol0359261 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/27/2003

group participation.18,19 In cases where the C-2′ position of
a building block does not need sulfation, an acetyl group
can be employed as a permanent protecting group. This ester
can also perform neighboring group participation but is stable
under the conditions needed for removing the Lev esters.
An azido group could be used as an amino-masking
functionality. This derivative does not perform neighboring
group participation and therefore allows the introduction of
R-glycosides.20 An azido-group can easily be reduced to an
amine, which can either be acetylated or sulfonated.
The C-4′ hydroxyl, which is required for extension, will
be protected as 9-fluorenylmethyl carbonate. The Fmoc group
can be removed with Et3N in dichloromethane without
affecting the levulinoyl ester, whereas the levulinoyl group
can be cleaved with hydrazine buffered with acetic acid,
conditions that do not affect the Fmoc carbonate.21
The anomeric center of the disaccharides will be protected
as allyl glycosides as this functionality can easily be removed
by isomerization to the vinyl glycoside and hydrolysis to
the hemiacetal. The resulting hemiacetal can be converted
into a trichloroacetimidate by employing NaH and trichlo-
roacetonitrile in dichloromethane.22
Figure 1. Orthogonal protecting groups for disaccharide building
blocks.
structures has been reported. To address this important issue,
we are developing a modular approach for the chemical syn-
thesis of a wide range of HS oligosaccharides whereby a set
of properly protected disaccharide building blocks, resem-
bling the different disaccharide motifs found in HS, can easily
and repeatedly be used for the assembly of a library of
sulfated oligosaccharides. As part of this program,15,16 we
described a strategy for HS synthesis whereby uronic acids
are formed at the end of a synthetic sequence by selective
oxidation of C-6 hydroxyls of idosides and glucosides using
a catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO) and sodium hypochloride as co-oxidant. This
approach avoids synthetic problems associated with use of
iduronic and glucuronic acids such as epimerization of C-5,
poor glycosyl-donating properties, and complications associ-
ated with protecting group manipulations.
Benzyl ethers could be used as protecting groups for the
primary hydroxyls that will be oxidized to the carboxylic
acids and for the secondary hydroxyls that will remain
unsulfated in the final product.
Finally, a TBDPS ether could be employed for the
protection of the C-6 position of the glucosamine residues
to avoid oxidation by TEMPO.23-25
Here we report, for the first time, the synthesis of a range
of properly protected disaccharides that resemble the different
disaccharides found in HS and can be used for the modular
synthesis of HS-oligosaccharides. A key strategic issue was
the use of a levulinoyl ester (Lev), 9-fluorenylmethyl
carbonate (Fmoc), a tert-butyldiphenylsilyl ether (TBDPS),
and an allyl ether (All) as a novel set of orthogonal protecting
groups. It is now shown that six strategically chosen
monosaccharides can be used in a parallel combinatorial
manner to prepare all structural elements found in HS.
The proposed generic protecting group scheme is shown
in Figure 1. Levulinoyl esters17 will be employed for those
hydroxyls that need sulfation in the final product. In HS,
the C-3 and C-6 of the glucosamine and C-2 hydroxyls of a
hexuronic acid moiety can be sulfated, and therefore,
depending on the sulfation pattern of a targeted disaccharide
building block, one or more of these positions will need to
be protected as levulinoyl groups. An important feature of
the Lev ester is that when present at the C-2′ position, it
directs the formation of 1,2-trans-glycosides by neighboring
On the basis of the protecting group strategy outlined
above, the monosaccharide building blocks 1-6 should allow
all of the disaccharide units found in HS to be prepared and
also give the capability for these disaccharides to be
assembled into larger structures (Figure 2).
(14) For other attempts to develop modular synthesis, see: (a) Orgueira,
H. A.; Bartolozzi, A.; Schell, P.; Litjens, R. E. J. N.; Palmacci, E. R.;
Seeberger, P. H. Chem.sEur. J. 2003, 9, 140-169. (b) de Paz, J. L.; Ojeda,
R.; Reichardt, N.; Martin-Lomas, M. Eur. J. Org. Chem. 2003, 3308-
3324. These approaches however have not established a set of protecting
groups that allow differential sulfation of the C-2 iduronic or glucuronic
moieties. In addition, the approach by Seeberger and co-workers led to
unnatural sulfation patterns.
Figure 2. Building blocks for modular HS synthesis.
The preparation of the key building blocks 3 and 4 are
summarized in Scheme 1. The C-2 hydroxyl of idoside 77
(15) Haller, M.; Boons, G. J. J. Chem. Soc., Perkin Trans. 1 2001, 814-
822.
(16) Haller, M. F.; Boons, G. J. Eur. J. Org. Chem. 2002, 2033-2038.
(17) Koeners, H. J.; Verhoeven, J.; van Boom, J. H. Tetrahedron Lett.
1980, 21, 381-382.
(18) Boons, G. J. Contemp. Org. Synth. 1996, 3, 173-200.
(19) Boons, G. J. Tetrahedron 1996, 52, 1095-1121.
(20) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 823-839.
4976
Org. Lett., Vol. 5, No. 26, 2003

Scheme 1
Scheme 2
was protected as an acetyl ester using acetic anhydride and
pyridine to give fully protected 8. Treatment of 8 with
NaCNBH3 and HCl26 opened the benzylidene acetal regi-
oselectively to give alcohol 9. The C-4 hydroxyl of 9 was
protected as an Fmoc ester by reaction with FmocCl in
dichloromethane and pyridine to give target compound 10.
It was found that 9 was susceptible to acetyl migration and
therefore needed immediate protection as an Fmoc carbonate.
Idosides can adopt either a 1C4 or a skewed boat 1S0
sponding disaccharide building blocks should be easily
accessible by replacement of the TBDPS group of disac-
charides 15-18 by a Lev ester. To investigate the compat-
ibility of TBDPS removal with the other protecting groups,
disaccharide 16 was subjected to a range of reaction
conditions. It was found that treatment of 16 with HF‚
pyridine in acetonitrile resulted in clean formation of 19.
Other conditions such as HF‚pyridine in THF or TBAF in
THF with or without buffering with acetic acid resulted in
removal of the Fmoc group or gave mixtures of compounds.
The hydroxyl of 19 was protected as a Lev ester using
standard conditions to give the modular building block 20
in an almost quantitative yield. In a similar way, compounds
15, 17, and 18 can be converted into derivatives that have a
levulinoyl ester at C-6 to give an additional three disaccha-
rides for modular oligosaccharide synthesis.
Next, the possibility of converting the disaccharides into
glycosyl acceptors and donors was investigated. Thus,
treatment of 16 with Et3N in dichloromethane for 18 hours21
resulted in clean removal of the Fmoc group without affecting
the acetyl and Lev ester, and after purification by silica gel
column chromatography, glycosyl acceptor 21 was isolated
in a yield of 91%. Fortunately, compound 21 was stable for
a prolonged period of time and no acyl migration was
observed.
1
conformation. In the C4 conformation, the C-2 acetyl and
C-4 hydroxyl occupy axial orientations and are sufficiently
close in space to allow acyl migration. Fully protected 10
was the precursor of Lev-protected 12. It was prepared by
treating 10 with dilute HCl in methanol to give 11 and
protecting the C-2′ hydroxyl as a levulinoyl ester using
standard conditions. Compounds 10 and 12 were converted
into trichloroacetamidates 3 and 4 by a two-step procedure
involving allyl ether cleavage with Wilkinson’s catalyst to
13 and 14 and treatment with trichloroacetonitrile and NaH
in dichloromethane. The latter condition did not affetct the
base-labile Fmoc and Lev ester.
Monosaccharides 1-4 were employed for the preparation
of properly protected disaccharides 15-18. Thus, TMSOTf-
mediated coupling22 of 115,16 with 3 gave disaccharide 15 in
a yield of 85% as only a 1,2-trans-glycoside. In a similar
fashion, the use of glycosyl donor 4 and acceptor 1 gave
disaccharide 16, which has a Lev ester at C-2′. TMSOTf-
mediated coupling of 215,16 with either 3 or 4 conveniently
gave disaccharides 17 and 18, which have a Lev ester at
C-3. Surprisingly, glycosylation with acceptor 2 gave only
good yields of coupling product when performed in dichlo-
roethane. Disaccharides 15-18 have different combinations
of Lev protecting groups at C-3 and C-2′, reflecting different
sulfation patterns found in HS. Several naturally occurring
HS-disaccharide moieties possess an additional sulfate at C-6
of a glucosamine moiety. It was expected that the corre-
Compound 16 could also be converted into glycosyl donor
23 by first removing the allyl moiety using PdCl2 and sodium
acetate in aqueous acetic acid followed by treatment of the
resulting hemiacetal 22 with NaH and trichoroacetonitrile.27
It is to be expected that the disaccharides 15, 17 and 18 can
also be converted into corresponding glycosyl donors and
acceptors using similar procedures. Finally, the deprotection,
sulfonation, and oxidation steps were investigated. Treatment
of 16 with hydrazine hydrate buffered with acetic acid
(21) Zhu, T.; Boons, G. J. Tetrahedron: Asymmetry 2000, 11, 199-
205.
(22) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212-235.
(23) de Nooy, A. E. J.; Besemer, A. C.; van Bekkum, H. Carbohydr.
Res. 1995, 269, 89-98.
(24) de Nooy, A. E. J.; Besemer, A. C.; van Bekkum, H. Recl. TraV.
Chim. Pays-Bas 1994, 113, 165-166.
(25) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181-1184.
(26) Garegg, P. J. Pure Appl. Chem. 1984, 56, 845-858.
(27) Roussel, F.; Knerr, L.; Grathwohl, M.; Schmidt, R. R. Org. Lett.
2000, 2, 3043-3046.
Org. Lett., Vol. 5, No. 26, 2003
4977

Scheme 3
Scheme 4
resulted in removal of the Lev ester without affecting the
Fmoc carbonate or acetyl ester, and the resulting alcohol 24
was sulfated using SO3‚pyridine to give O-sulfate 25.
Catalytic hydrogenation of 25 using Pd(OH)2 in a mixture
of water, ethanol, and acetic acid resulted in removal of the
benzyl ethers and Fmoc carbonate, and conversion of the
azido moiety into an amine to give 26. Compound 26 was
immediately N-sulfated using SO3‚pyridine, and the primary
hydroxyl of the resulting 27 was selectively oxidized using
TEMPO and NaClO2 as co-oxidant23-25 to give 28. Finally,
treatment of 29 with HF‚pyridine in pyridine to cleave the
TBDPS ether gave, after purification by C-18 column
chromatography, target compound 29. Alternatively, N-
acetylation of compound 26 followed by selective oxidation
with TEMPO and NaClO2 and removal of the TBDPS group
should give an acetamido derivative. In conclusion, we have
described a new set of four orthogonal protecting groups that
is ideally suited for the preparation of a library of heparan
sulfate fragments. The synthetic strategy was inspired by the
observation that heparan sulfate is composed of 20 disac-
charide units that differ in sulfation pattern and the presence
of either iduronic or glucuronic acid. Properly protected
disaccharide building blocks that resemble these different
disaccharide motifs can now be prepared from our six
strategically chosen monosaccharides. These monosaccha-
rides are protected by four orthogonal protecting groups
(Fmoc, Lev, TBDPS, and All) and could repeatedly be used
for the preparation of different disaccharide building blocks.
In addition, the resulting disaccharides (e.g., 15-17) could
easily be converted into another set of modular building
blocks by replacement of the TBDPS ether at C-6 by a Lev
ester. It is shown that the disaccharides can easily be
converted into glycosyl donors and acceptors for the prepara-
tion of larger fragments. It is to be expected that the set of
orthogonal protecting groups reported here can be applied
for the synthesis of other classes of complex oligosaccharides.
Acknowledgment. This research was supported by the
NIH Research Resource Center for Biomedical Complex
Carbohydrates (P41-RR-5351).
Supporting Information Available: Experimental pro-
1
cedures and H and 13C NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0359261
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Org. Lett., Vol. 5, No. 26, 2003