Modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies

Article abstract of DOI:10.1021/ja907358k

Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activiti

Full text of DOI:10.1021/ja907358k

Published on Web 11/11/2009
Modular Synthesis of Heparan Sulfate Oligosaccharides for
Structure-Activity Relationship Studies
Sailaja Arungundram,†,‡ Kanar Al-Mafraji,† Jinkeng Asong,†,‡ Franklin E. Leach III,‡
I. Jonathan Amster,‡ Andre Venot,† Jeremy E. Turnbull,§ and Geert-Jan Boons*,†,‡
Complex Carbohydrate Research Center, UniVersity of Georgia, 315 RiVerbend Road, Athens,
Georgia 30602, Department of Chemistry, The UniVersity of Georgia, Athens,
Georgia 30602-2556, and Center for Glycobiology, School of Biological Sciences, UniVersity of
LiVerpool, Crown Street, LiVerpool L69 7ZB, United Kingdom
Received September 10, 2009; E-mail: gjboons@ccrc.uga.edu
Abstract: Although hundreds of heparan sulfate binding proteins have been identified and implicated in a
myriad of physiological and pathological processes, very little information is known about the ligand
requirements for binding and mediating biological activities by these proteins. This difficulty results from a
lack of technology for establishing structure-activity relationships, which in turn is due to the structural
complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides.
To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of
HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building
blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building
blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been
employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations
in activity with structural changes support the view that important functional information is embedded in
HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the
preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer’s disease.
Introduction
in proteoglycans surrounding the stroma of tumors, and it has
been suggested that these alterations may support tumor growth
Glycoaminoglycans (GAGs) such as heparin and heparan
sulfate (HS) are naturally occurring polydisperse linear polysac-
charides that are heavily O- and N-sulfated.1,2 The interaction
between GAGs and proteins can have profound physiological
effects on hemostasis, lipid transport and adsorption, cell growth,
and migration and development.3,4 Binding of GAGs can result
in the immobilization of proteins at their sites of production,
regulation of enzyme activity, binding of ligands to their
receptors, and protection of proteins against degradation.
Alteration in GAG expression has been associated with disease,
for example,5 significant structural changes have been reported
and invasion.
Currently, more than 100 heparan sulfate binding proteins
have been identified,4 and it is to be expected that in the near
future many more will be discovered. For a small number of
HS binding proteins, it has been established that a specific
sulfation pattern is required for mediating biological activity,
and the best-studied case represents the interaction of anti-
thrombin with heparin.6 Each of the sulfates of the pentasac-
charide GlcNAc6S-GlcA-GlcN3S-IdoA-GlcNS is essential for
high-affinity binding to antithrombin and anticoagulant activity.
Interestingly, the pentasaccharide contains a rare glucosamine
moiety that has a sulfate ester at C-3. The latter moiety is also
required for binding of Herpes simplex gD protein to HS, which
in turn is important for viral infection.7 On the other hand, it
has been proposed that for some HS binding proteins, the spatial
organization of clusters of negative charge in HS is an important
determinant of binding and biological activity. It appears that
in these cases the HS binding proteins have a relaxed selectivity
for short HS oligosaccharides, an example being thrombin,
† Complex Carbohydrate Research Center, University of Georgia.
‡ Department of Chemistry, The University of Georgia.
§ University of Liverpool.
(1) Esko, J. D.; Selleck, S. B. Annu. ReV. Biochem. 2002, 71, 435–471.
(2) Gandhi, N. S.; Mancera, R. L. Chem. Biol. Drug Des. 2008, 72, 455–
482.
(3) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. Engl. 2002, 41, 391–
412. Hacker, U.; Nybakken, K.; Perrimon, N. Nat. ReV. Mol. Cell
Biol. 2005, 6, 530–541. Whitelock, J. M.; Iozzo, R. V. Chem. ReV.
2005, 105, 2745–2764. Van Vactor, D.; Wall, D. P.; Johnson, K. G.
Curr. Opin. Neurobiol. 2006, 16, 40–51. Bishop, J. R.; Schuksz, M.;
Esko, J. D. Nature 2007, 446, 1030–1037.
(4) Ori, A.; Wilkinson, M. C.; Fernig, D. G. Front. Biosci. 2008, 13, 4309–
4338.
(6) Lindahl, U.; Backstrom, G.; Thunberg, L.; Leder, I. G. Proc. Natl.
Acad. Sci. U.S.A. 1980, 77, 6551–6555. Petitou, M.; van Boeckel, C. A.
Angew. Chem., Int. Ed. Engl. 2004, 43, 3118–3133. de Kort, M.;
Buijsman, R. C.; van Boeckel, C. A. A. Drug DiscoV. Today 2005,
10, 769–779.
(5) Johnson, Z.; Proudfoot, A. E.; Handel, T. M. Cytokine Growth Factor
ReV. 2005, 16, 625–636. Parish, C. R. Nat. ReV. Immunol. 2006, 6,
633–643. Taylor, K. R.; Gallo, R. L. FASEB J. 2006, 20, 9–22. Chen,
Y.; Gotte, M.; Liu, J.; Park, P. W. Mol. Cells 2008, 26, 415–426.
Zacharski, L. R.; Lee, A. Y. Expert Opin. InVestig. Drugs 2008, 17,
1029–1037.
(7) Shukla, D.; Liu, J.; Blaiklock, P.; Shworak, N. W.; Bai, X.; Esko,
J. D.; Cohen, G. H.; Eisenberg, R. J.; Rosenberg, R. D.; Spear, P. G.
Cell 1999, 99, 13–22.
9
17394 J. AM. CHEM. SOC. 2009, 131, 17394–17405
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Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
which requires a highly sulfated structure for binding.8 This
diversity of interactions emphasizes the need for more detailed
structure-activity studies on a wider range of HS binding
proteins.9
set of properly protected disaccharide building blocks that
resemble the different disaccharide motifs found in HS are
assembled by a parallel combinatorial manner into larger
structures.19,20 Our previous efforts suffered, however, from
difficulties in preparing key mono- and disaccharide intermedi-
ates, difficulties in removing temporary protecting groups,
unreliability in glycosylations, and difficulties in the final
deprotection. Others have attempted to develop modular ap-
proaches for HS synthesis;17,21 however, these methods provided
unnatural sulfation patterns, were unable to make structures
larger than disaccharides, or did not demonstrate the convenient
preparation of a wide range of structural motifs.
We report here a robust modular synthetic approach for the
preparation of a wide range of well-defined HS oligosaccharides
for SAR studies. The synthetic methodology is based on the
use of a relatively small number of properly protected disac-
charide donors and acceptors that in a parallel combinatorial
manner, using a standard set of reaction conditions, can be
assembled into a large number of HS oligosaccharides. The
compounds are equipped with an anomeric aminopentyl spacer,
which provides an opportunity for conjugation to a solid surface,
which, for example, is required for microarray technology
development.18 To illustrate the convenience of the modular
building blocks, a library of 12 oligosaccharides has been
prepared to probe the requirement for inhibition of the protease,
ꢀ-secretase (or BACE-1). The cleavage of amyloid precursor
protein (APP) by the protease BACE-1 is a key step in
generating amyloid plaques, which are a characteristic of
Alzheimer disease; synthetic compounds that inhibit this enzyme
have potential as novel agents to treat this disease.22,23
For most HS binding there is very little or no information
about the ligand requirements for binding and mediating
biological activity.10 This difficulty is due to a lack of technology
for establishing structure-activity relations (SAR), which in turn
is due to the structural complexity of natural HS and difficulties
of preparing well-defined compounds.11,12 Initial approaches to
establish structure-activity relations (SAR) employ modified
derivatives of heparin in which acetamido, sulfonamido, or
sulfate esters are chemically modified to produce polysaccha-
rides that have simpler compositions than the parent compound
have proved useful.13 In addition, HS has been sulfated at
specific positions using biosynthetic enzymes.14 Although these
approaches make it possible to draw some conclusions about
the requirement of particular functionalities for binding or
biological activity, they do not allow determination of the
structure of binding epitopes. Natural libraries of HS oligosac-
charides have been generated and screened,15 but sequencing
of identified hits is still a technical challenge.
In principle, synthetic and chemoenzymatic approaches have the
potential to provide a sufficiently large range of well-defined HS
oligosaccharides for SAR or array development. Elegant synthetic
approaches for heparin synthesis have been described;11,16-18
however, no efficient strategy for the synthesis of a wide range
of HS structures has been reported. In order to address this
problem, we proposed a modular approach for the chemical
synthesis of a wide range of HS oligosaccharides whereby a
Results and Discussion
(8) Grootenhuis, P. D.; Westerduin, P.; Meuleman, D.; Petitou, M.; van
Boeckel, C. A. Nat. Struct. Biol. 1995, 2, 736–739.
Synthetic Strategy for Modular HS Oligosaccharide
Synthesis. Heparan sulfate consists of 1,4-linked disaccharide
units of R-L-iduronic or ꢀ-D-glucuronic acid and either N-acetyl-
or N-sulfo-R-D-glucosamine. The principal positions of O-
sulfation are C-2 of iduronate and C-6 of glucosamine as well
as, more rarely, C-3 of glucosamine. Variable substitution during
biosynthesis results in the formation of at least 20 different
disaccharide motifs. Combining these different disaccharides
into larger structures potentially results in enormous structural
diversity.1
A modular approach that employs a set of properly protected
disaccharide building blocks that resemble the different disac-
charide motives found in HS and can easily and repeatedly be
used for the preparation of multiple targets has the potential to
provide a library of HS oligosaccharides for structure-activity
relationship studies. A key issue of such a modular approach is
the selection of a set of protecting groups that meet the following
requirements: (i) the C-2 hydroxyl-protecting groups of the
(9) Gama, C. I.; Tully, S. E.; Sotogaku, N.; Clark, P. M.; Rawat, M.;
Vaidehi, N.; Goddard, W. A., III; Nishi, A.; Hsieh-Wilson, L. C. Nat.
Chem. Biol. 2006, 2, 467–73.
(10) Kreuger, J.; Spillmann, D.; Li, J. P.; Lindahl, U. J. Cell Biol. 2006,
174, 323–327.
(11) Karst, N. A.; Linhardt, R. J. Curr. Med. Chem. 2003, 10, 1993–2031.
Poletti, L.; Lay, L. Eur. J. Org. Chem. 2003, 2999–3024. Noti, C.;
Seeberger, P. H. Chem. Biol. 2005, 12, 731–756. Linhardt, R. J.;
Dordick, J. S.; Deangelis, P. L.; Liu, J. Semin. Thromb. Hemost. 2007,
33, 453–465.
(12) van den Bos, L. J.; Codee, J. D. C.; Litjens, R. E. J. N.; Dinkelaar, J.;
Overkleeft, H. S.; van der Marel, G. A. Eur. J. Org. Chem. 2007,
3963–3976. Sun, Z. C.; Wei, Z.; Wei, K. M. Prog. Chem. 2008, 20,
1136–1142.
(13) Yates, E. A.; Guimond, S. E.; Turnbull, J. E. J. Med. Chem. 2004,
47, 277–280.
(14) Lindahl, U.; Li, J. P.; Kusche-Gullberg, M.; Salmivirta, M.; Alaranta,
S.; Veromaa, T.; Emeis, J.; Roberts, I.; Taylor, C.; Oreste, P.; Zoppetti,
G.; Naggi, A.; Torri, G.; Casu, B. J. Med. Chem. 2005, 48, 349–352.
Chen, J.; Jones, C. L.; Liu, J. Chem. Biol. 2007, 14, 986–993.
(15) Guimond, S.; Turnbull, J. E. Curr. Biol. 1999, 9, 1343–1346.
(16) de Paz, J. L.; Angulo, J.; Lassaletta, J. M.; Nieto, P. M.; Redondo-
Horcajo, M.; Lozano, R. M.; Gimenez-Gallego, G.; Martin-Lomas,
M. Chembiochem 2001, 2, 673–685. Petitou, M.; Imberty, A.;
Duchaussoy, P.; Driguez, P. A.; Ceccato, M. L.; Gourvenec, F.; Sizun,
P.; Herault, J. P.; Perez, S.; Herbert, J. M. Chem.sEur. J. 2001, 7,
858–873. Codee, J. D. C.; Overkeelft, H. S.; van der Marel, G. A.;
van Boeckel, C. A. A. Drug DiscoVery Today: Technol. 2004, 1, 317–
326. Lubineau, A.; Lortat-Jacob, H.; Gavard, O.; Sarrazin, S.;
Bonnaffe, D. Chem.sEur. J. 2004, 10, 4265–4282. de Paz, J. L.;
Martin-Lomas, M. Eur. J. Org. Chem. 2005, 1849–1858. Zhou, Y.;
Lin, F.; Chen, J. F.; Yu, B. Carbohydr. Res. 2006, 341, 1619–1629.
Chen, J. F.; Zhou, Y.; Chen, C.; Xu, W. C.; Yu, B. Carbohydr. Res.
2008, 343, 2853–2862. Chen, C.; Yu, B. Bioorg. Med. Chem. Lett.
2009, 19, 3875–3879.
(19) Haller, M.; Boons, G. J. J. Chem. Soc., Perkin Trans. 1 2001, 814–
822. Haller, M. F.; Boons, G. J. Eur. J. Org. Chem. 2002, 2002, 2033–
2038.
(20) Prabhu, A.; Venot, A.; Boons, G. J. Org. Lett. 2003, 5, 4975–4978.
(21) Gavard, O.; Hersant, Y.; Alais, J.; Duverger, V.; Dilhas, A.; Bascou,
A.; Bonnaffe, D. Eur. J. Org. Chem. 2003, 3603–3620. Orgueira,
H. A.; Bartolozzi, A.; Schell, P.; Litjens, R.; Palmacci, E. R.;
Seeberger, P. H. Chem.sEur. J. 2003, 9, 140–169. Lu, L. D.; Shie,
C. R.; Kulkarni, S. S.; Pan, G. R.; Lu, X. A.; Hung, S. C. Org. Lett.
2006, 8, 5995–5998. Polat, T.; Wong, C. H. J. Am. Chem. Soc. 2007,
129, 12795–12800.
(17) Lee, J. C.; Lu, X. A.; Kulkarni, S. S.; Wen, Y. S.; Hung, S. C. J. Am.
Chem. Soc. 2004, 126, 476–477.
(22) Patey, S. J.; Edwards, E. A.; Yates, E. A.; Turnbull, J. E. J. Med.
Chem. 2006, 49, 6129–6132.
(18) Noti, C.; de Paz, J. L.; Polito, L.; Seeberger, P. H. Chem.sEur. J.
2006, 12, 8664–8686.
(23) Patey, S. J.; Edwards, E. A.; Yates, E. A.; Turnbull, J. E. Neurodegener.
Dis. 2008, 5, 197–199.
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A R T I C L E S
Arungundram et al.
Preparation of Modular Building Blocks. On the basis of
these considerations, the six strategically chosen monosaccharide
building blocks 1-6 were prepared, which were employed for
the preparation of the eight disaccharide modules 9-16 (Figure
2). It was observed that installment of an aminopentyl linker at
the disaccharide stage led to mixtures of anomers, which were
difficult to separate by traditional silica column chromatography,
and therefore, the spacer containing glycosyl acceptors 7 and 8
was employed for the preparation of disaccharide modules
17-24.
Figure 1. Orthogonal protecting groups for disaccharide building blocks.
Glycosyl acceptors 5-8 could be prepared form readily
available lactol 2527 (Scheme 1). Thus, for the preparation of 5
and 6, the anomeric center of 25 was protected as a TDS ether
by reaction with TDSCl in the presence of imidazole in DCM
to give 26 in an excellent yield of 90%. The chemical synthesis
of compounds 7 and 8 required the installation of an R-anomeric
N-(benzyl)benzyloxycarbonyl-protected aminopentenyl spacer,
which was achieved by conversion of lactol 25 into a trichlo-
roacetimidate 27, which was coupled with N-(benzyl)benzy-
loxycarbonyl aminopentanol in the presence of TMSOTf in a
mixture of dichloromethane and diethyl ether to give 28 as a
mixture of anomers (R/ꢀ ) 3/1), which could be separated by
silica gel column chromatography to afford the individual
anomers. A number of other anomeric spacers was examined,
and, for example, lower yields in subsequent glycosylations were
obtained when benzyloxycarbonyl amino propanol was em-
ployed. Furthermore, the use of N-(benzyl)benzyloxycarbonyl
propanol gave compounds having complex 1H NMR due to the
E/Z isomerism of the amino protecting groups.18 Fortunately,
the latter problem could be partially alleviated by employing
N-(benzyl)benzyloxycarbonyl-protected aminopentanol.
The acetyl esters of 26 and 28 were cleaved by treatment
with sodium methoxide in methanol, and the 4,6-diol of the
resulting compound was protected as a benzylidene acetal by
treatment with PhCH(OMe)2 in the presence of a catalytic
amount of camphorsulfonic acid in DMF to give 29 and 30,
respectively. The C-3 hydroxyl of 29 was benzylated with
benzyl bromide in the presence of Ag2O, and the benzylidene
acetal of the resulting compound was hydrolyzed by treatment
with TFA in DCM and water to afford diol 31. The more robust
compound 30 was benzylated with benzyl bromide and NaH in
DMF, and the benzylidene acetal of the resulting compound
was removed by TFA in DCM and water to give 32. The C-6
hydroxyl of 31 and 32 were regioselectively protected as an
acetyl ester by reaction with acetic acid and the activator
2-chloro-1-methyl-1-pyridinium iodide in the presence of
DABCO in DCM to give the target compounds 5 and 7,
respectively. Compounds 6 and 8, which have a Lev ester at
C-6, were prepared by a similar procedure using levulinic acid
instead of acetic acid.
glucuronic or iduronic acid moieties should allow the stereo-
selective introduction of 1,2-trans-glycosidic linkages, whereas
the C-2 amino group of the glucosamine derivatives needs to
be derivatized in such a way that 1,2-cis-glycosidic linkages
can be formed; (ii) temporary protecting groups for the anomeric
center and C-4 need to be selected for the preparation of glycosyl
donors and acceptors; (iii) a protecting group is required that
can selectively be removed to reveal those hydroxyls that need
sulfation; (iv) the removal of the permanent protecting groups
should be compatible with the presence of base- and acid-labile
sulfate esters; (v) the protecting group scheme should be
applicable to synthesize each of the disaccharide modules found
in natural HS; finally, (vi) a unified set of chemical conditions
is required for the preparation of the various disaccharide
modules, oligosaccharide assembly, sulfation, and deprotection.
The protecting group strategy that we developed for the
disaccharide modules is summarized in Figure 1. Thus, levuli-
noyl esters (Lev)24 will be employed for those hydroxyls that
need sulfation. In HS, the C-3 and C-6 of glucosamine and C-2′
hydroxyls of the hexuronic acid moiety can be sulfated, and
therefore, depending on the sulfation pattern of a targeted
disaccharide module, one or more of these positions will be
protected as Lev esters. An important feature of the Lev ester
is that when present at the C-2′ position it will be able to direct
the formation of 1,2-trans-glycosides by neighboring group
participation.25 In the case that the C-2′ position of a disaccha-
ride module does not need sulfation, an acetyl group is employed
as a permanent protecting group. This ester can also perform
neighboring group participation but is stable under the conditions
used for the removal of Lev esters. An azido group will be used
as an amino-masking functionality.26 This derivative does not
perform neighboring group participation and therefore allows
the introduction of R-glucosides. An azido group can easily be
reduced to an amine, which can be either acetylated or sulfated.
The C-4′ hydroxyl, which is required for extension, will be
protected as 9-fluorenylmethyl carbonate (Fmoc). The Fmoc
group can be removed with Et3N in dichloromethane or DMF
without affecting the Lev ester, whereas the Lev group can be
cleaved with hydrazine buffered with acetic acid, and these
conditions do not affect the Fmoc carbonate.24 The anomeric
center of the disaccharides will be protected as thexyldimethyl
silyl (TDS) glycosides, and this functionality can easily be
removed by treatment with HF in pyridine without affecting
the other protecting groups. The resulting lactol can then be
converted into a trichloroacetimidate by employing K2CO3 and
trichloroacetonitrile in DCM.20 Finally, benzyl ethers are used
as permanent protecting groups for the other hydroxyls.
Idosyl donors 1 and 2 were easily prepared by starting from
readily available 1,6-anhydro-idose 33 (Scheme 2).17,28 Thus,
the treatment of 3317 with ethanethiol and BF3 ·Et2O in DCM29
(27) Vasella, A.; Witzig, C.; Chiara, J. L.; Martinlomas, M. HelV. Chim.
Acta 1991, 74, 2073–2077. Alper, P. B.; Hung, S. C.; Wong, C. H.
Tetrahedron Lett. 1996, 37, 6029–6032.
(28) van Boeckel, C. A. A.; Beetz, T.; Vos, J. N.; de Jong, A. J. M.; van
Aelst, S. F.; van den Bosch, R. H.; Mertens, J. M. R.; van der Vlugt,
F. A. J. Carbohydr. Chem. 1985, 4, 293–321. Tabeur, C.; Machetto,
F.; Mallet, J. M.; Duchaussoy, P.; Petitou, M.; Sinay, P. Carbohydr.
Res. 1996, 281, 253–276. Tatai, J.; Osztrovszky, G.; Kajtar-Peredy,
M.; Fugedi, P. Carbohydr. Res. 2008, 343, 596–606.
(24) Zhu, T.; Boons, G. J. Tetrahedron: Asymmetry 2000, 11, 199–205.
(25) Boons, G. J. Contemp. Org. Synth. 1996, 3, 173–200. Demchenko,
A. V. Synlett 2003, 1225–1240.
(26) Bongat, A. F. G.; Demchenko, A. V. Carbohydr. Res. 2007, 342, 374–
406.
(29) Basten, J. E. M.; Dreef-Tromp, C. M.; de Wijs, B.; van Boeckel,
C. A. A. Bioorg. Med. Chem. Lett. 1998, 8, 1201–1206.
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Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
Figure 2. Modular mono- and disaccharide building blocks.
Scheme 1. Synthesis of 2-Azido-2-deoxy-R-D-glucopyranoside
Scheme 2. Synthesis of Thioethyl Gluco and Idosyl Donora
Acceptorsa
a Reagents and conditions: (a) (i) AcOH, Ac2O, TFA, r.t. (86%); (ii)
EtSH, BF3 ·Et2O, DCM, 0 °C to r.t. (90%); (b) (i) NaOMe, MeOH; (ii)
PhCH(OMe)2, CSA, DMF (70%, 2 steps); (c) Ac2O, Py (1: 93%, 3: 85%);
(d) LevOH, DCC, DMAP, DCM, r.t. (2: 89%, 4: 70%); (e) (i) NaOMe,
MeOH; (ii) PhCH(OMe)2, p-TsOH, DMF (61%, 2 steps); (iii) (Bu2Sn)O,
MeOH, 75-80 °C, BnBr, CsF, DMF, 16 hr, r.t. (61%).
a Reagents and conditions: (a) TDSCl, imidazole, DCM (90%); (b)
CCl3CN, DBU, DCM (90%); (c) HO(CH2)5N(Bn)Cbz, DCM:Et2O,
TMSOTf, molecular sieves, -20 °C (R-anomer, 62%); (d) (i) NaOMe,
MeOH; (ii) PhCH(OMe)2, CSA, DMF (29, 92%; 30, 76%, 2 steps); (e) (i)
BnBr, Ag2O, molecular sieves, DCM; (ii) DCM:TFA:H2O (31, 95%, 2
steps); (f) (i) NaH, BnBr, DMF; (ii) DCM:TFA:H2O (32, 93%, 2 steps);
(g) AcOH, 2-chloro-1-methyl-1-pyridinium iodide, DABCO, DCM (5, 65%,
7, 68%), (h) LevOH, 2-chloro-1-methyl-1-pyridinium iodide, DABCO,
DCM (6, 86%; 8, 82%).
of glucosyl donors 3 and 4 by protecting of the C-2 hydroxyl
as acetyl or Lev esters, respectively, using standard conditions.
Having glycosyl donors 1-4 and acceptors 5-8 in hand,
attention was focused on the parallel combinatorial synthesis
of a range of disaccharide modules (Scheme 3). Thus, a NIS/
TMSOTf-mediated coupling30 of each of the thioglycosyl donors
1-4 with each the glycosyl acceptors 5 and 6 gave eight
different disaccharides (37-40 and 49-52) having either a
glucoside or an idoside at the nonreducing end and a Lev ester
at C-6 or C-2′ or at both hydroxyls. In each glycosylation, only
a 1,2-trans-glycoside was formed due to neighboring group
participation by the C-2 acetyl or Lev ester of the glycosyl
donors, giving the disaccharides in excellent yields ranging from
70% to 95%. Next, the disaccharides 37-40 and 49-52 were
converted into glycosyl acceptors 41-44 and 53-56 and
glycosyl donors 45-48 and 57-60 by a unified set of reaction
conditions. Thus, the benzylidene acetals of 37-40 and 49-52
were removed by treatment with p-toluenesulfonic acid in the
presence of ethanethiol or by a mixture of TFA, DCM, and
gave thio-idoside 34, which was deacetylated to give a triol,
which was protected as 4,6-O-benzyldine acetal to give 35. The
C-2 hydroxyl of 35 was protected as an acetyl or Lev ester by
reaction with acetic anhydride in pryidine or levulinic acid in
the presence of the activator DCC to provide the required idosyl
donors 1 and 2. Glucosyl donors 3 and 4 were readily obtained
by saponification of the acetyl ester of ethyl 2,3,4-tri-O-acetyl
thioglucoside using standard conditions followed by selective
protection of the 4,6-diol of the resulting compound as a
benzylidene acetal by treatment with PhCH(OMe)2 and cam-
phorsulfonic acid in DMF and then regioselective benzylation
of the C-3 hydroxyl by first preparing a stannylidene acetal by
treatment with dibutyl tin oxide followed by reaction with benzyl
bromide in the presence of CsF in DMF to give 36. The latter
compound was a convenient starting material for the preparation
(30) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron
Lett. 1990, 31, 1331–1334.
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Arungundram et al.
Scheme 3. Synthesis of Glucuronyl and Idouronyl Disaccharidesa
Scheme 4. Synthesis of Hexasaccharide 69a
a Reagents and conditions: (a) NIS, TMSOTf, 0 °C, DCM (37, 81%;
38, 75%; 39, 92%; 40, 75%; 49, 90%; 50, 66%; 51, 95%; 52, 80%); (b) (i)
EtSH, p-TsOH, DCM, or DCM:TFA:H2O; (ii) TEMPO, BAIB, DCM, H2O,
1 h; (iii) CH2N2, THF (3 steps 65-85%); (c) (i) FmocCl, Py,DMAP, 0 °C
to room temperature; (ii) HF · Py, 18 h; (iii) K2CO3, CCl3CN, DCM
(70-90%).
a Reagents and conditions: (a) TMSOTf, -20 to +5°C, 4 Å sieves, 61,
64%; 63, 65%; (b) Et3N, DCM, 82%; (c) NH2NH2 ·HOAc, toluene/EtOH,
90%; (d) Py·SO3, DMF; (e) (i) Et3N, DMF; (ii) LiOH, H2O2, THF; (iii) 4
M NaOH, MeOH (58%, 3 steps); (f) PMe3, THF, NaOH, 65%; (g)
N-sulfation: Py ·SO3, MeOH, Et3N, 0.1 M NaOH, 50%; (h) (i) Pd/C, H2,
MeOH:H2O; (ii) Pd(OH)2/C, H2, H2O, 67%.
water to give the corresponding diols, which were oxidized with
2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) in the presence
of iodobenzene diacetate (BAIB) as the cooxidant.31,32 The
resulting carboxylic acids were protected as methyl esters by
treatment with diazomethane to give compounds 41-44 and
53-56 in an overall yield ranging from 65% to 90%. Interest-
ingly, the use of sodium hypochloride as the co-oxidant in the
TEMPO oxidation31 led to a lower yield of product due to partial
oxidation of the secondary hydroxyl. The disaccharides were
also the starting material for the preparation of glycosyl donors
45-48 and 57-60 by protection of the C-4′ alcohols as an Fmoc
carbonate by treatment with FmocCl in pyridine in the presence
of DMAP followed by removal of the anomeric TDS with HF
in pyridine and installation of the anomeric trichloroacetimidate
using trichloroacetonitrile and K2CO3 in DCM. The latter
reaction conditions did not affect the base-labile Fmoc protecting
group. Each reaction was high yielding regardless of the
chemical composition of the disaccharide. The spacer-containing
disaccharide acceptors 17-24 (Figure 2) were prepared by a
similar strategy by coupling glycosyl donors 1-4 with glycosyl
acceptors 7 and 8 followed by benzylidene acetal removal,
selective oxidation of the C-6′ hydroxyl of the resulting
compound, and protection of the resulting carboxylic acid as a
methyl ester (for details, see Supporting Information).
acceptors 41-44 and 53-56 (or the corresponding spacer
containing acceptors 17-24) will give 64 different tetrasaccha-
rides. These compounds can easily be converted into glycosyl
acceptors by removal of the Fmoc carbonate, and each of the
resulting compounds can then again be coupled with the 8
glycosyl donors to provide 512 different hexasaccharides. After
the assembly of the oligosaccharides, the azido moieties can
be converted into acetamido or N-sulfate derivatives, further
increasing the structural diversity of synthetic compounds. It
may also be possible to obtain additional compounds by
converting the azido moiety of the disaccharides into NHAlloc
and then employ it in a glycosylation with an azido-containing
disaccharide. The Alloc and azido offer a convenient set of
orthogonal amino protecting groups that allow selective modi-
fication of each function.33
Preparation of a Hexasaccharide Using Modular Building
Blocks. To demonstrate that the disaccharide acceptors and
donors can be employed for modular HS oligosaccharide
synthesis, hexasaccharide 69 was prepared from disaccharide
modules 19, 47, and 59 (Scheme 4). Thus, a TMSOTf-mediated
glycosylation of trichloroacetimidate 59 with spacer-containing
glycosyl acceptor 19 gave tetrasaccharide 61 in a yield of
approximately 60% as only the R-anomer. The Fmoc protecting
group of 61 was removed by standard conditions using Et3N in
DCM, and the resulting glycosyl acceptor 62 was coupled with
glycosyl donor 47 to give the fully protected hexasaccharide
63 in a good yield, and in this case, also only the R-anomer
In principle, parallel combinatorial coupling of the eight
glycosyl donors 45-48 and 57-60 with the eight glycosyl
(31) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181.
(32) DeMico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G.
J. Org. Chem. 1997, 62, 6974–6977. van den Bos, L. J.; Codee, J. D.;
van der Toorn, J. C.; Boltje, T. J.; van Boom, J. H.; Overkleeft, H. S.;
van der Marel, G. A. Org. Lett. 2004, 6, 2165–2168.
(33) Zhang, Y.; Gaekwad, J.; Wolfert, M. A.; Boons, G. J. Chem.sEur. J.
2008, 14, 558–569.
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Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
was observed. Next, sulfate esters were installed by removal of
the Lev esters of 63 by treatment with hydrazine acetate in a
mixture of toluene and ethanol followed by sulfation of the
resulting hydroxyls of 64 with pyridinium sulfur trioxide to give
compound 65. Next, the acetyl and methyl esters of 65 were
saponified by a two-step procedure employing first LiOH in a
mixture of hydrogen peroxide and THF34 and then sodium
hydroxide in methanol to give partially deprotected 66. The
azido moiety of 66 was reduced with trimethyl phosphine in
THF in the presence of NaOH18,35 to give amine 67, which
was immediately sulfated with pyridinium sulfur trioxide in the
presence of triethylamine in methanol to give N-sulfate 68.
Finally, the benzyl ethers and benzyloxycarbamate of 68 were
removed by a two-step procedure36 involving hydrogenation
over Pd/C in a mixture of MeOH/H2O, which led to the removal
of the spacer protecting groups followed by hydrogenation over
Pd(OH)2, which led to the removal of the benzyl ethers to give
HS oligosaccharides 69. Interestingly, a one-step hydrogenation
procedure proceeded very sluggishly and did not provide the
target compound.
Figure 3. Putative synthetic HS ligands for BACE-1.
active site without interfering with APP processing by R- or
γ-secretases.39 Systematic modification of porcine intestinal
mucosal heparin was used to demonstrate a critical importance
of 6-O-sulfates for inhibition of BACE-1.22 Furthermore,
replacement of N-sulfate groups by acetamido moieties slightly
impairs activity,22,23 and derivatives containing N-acetyl and
2-O- and 6-O-sulfates had the highest anti-BACE-1 to anti-Xa
activity ratio,22 demonstrating opportunities for optimizing
therapeutic activities.
To probe the requirement of HS oligosaccharides for inhibi-
tion of BACE-1, a library of 12 tetrasaccharides (76-86, Figure
3) was prepared employing the disaccharide modules. Thus,
tetrasaccharides 76-83, which contain C-6 sulfate esters but
differ in the modification of the C-2 amino groups and the
presence of glucuronic acid or idouronic acid moieties, were
prepared. These compounds should reveal the importance of
the nature of the uronic acid moiety and the presence of an
acetamido or N-sulfate group for inhibitory activity. Further-
more, compounds 84 and 85 are derived from 80 and 78,
respectively, but have an additional sulfate ester at C-2 of an
iduronic acid moiety.
The target HS fragments could easily be prepared by
employing the disaccharide modules 19, 23, 24, 47, and 59
combined with the sulfation and deprotection protocols described
for the preparation of hexasaccharide 69. Thus, standard
TMSOTf-mediated glycosylation of glycosyl donors 19, 23, and
24 with glycosyl acceptor 47 and 59 in DCM gave the
tetrasaccharides 70-75 in yields of approximately 60%, and
fortunately, in each case only the R-anomer was obtained
(Scheme 5). Our preliminary studies had indicated that 2-deoxy-
2-azido-glucopyranosyl trichloroacetimidates give excellent
R-anomeric selectivities when modified with an acyl protecting
group at C-6 and employed in glycosylations with glycosyl
acceptors of relatively low reactivity. This observation may be
due to remote neighboring group participation of the C-6 ester.40
O-Sulfation, deprotection, and N-acetylation or N-sulfation was
performed by standard procedures to give the target tetrasac-
charides 76-86.
1
The H NMR spectra of the oligosaccharides were fully
assigned by 1D and 2D NMR spectroscopy. The R-anomeric
configuration of 2-azido-glucosides was confirmed by J1,2
coupling constants and 13C chemical shifts of C-1 (∼97 ppm).
Furthermore, a downfield shifts of 0.5 ppm of H-6 was observed
for O-sulfation of C-6 hydroxyls and 0.4 ppm of H-2 for
N-sulfation.
Synthesis of a Library of HS Oligosaccharides to Probe
Inhibition of BACE-1. Alzheimer’s disease is a progressive
neurodegenerative disorder of the central nervous system that
is characterized by the formation of ꢀ-amyloid peptides, which
accumulate as perivascular or parenchymal deposits in brains.
Cleavage of amyloid precursor protein (APP) by the aspartyl
protease ꢀ-site APP-cleaving enzyme 1 (BACE-1) generates a
membrane-bound protein, which is further processed by the
γ-secretase enzyme complex to generate the neurotoxic amyloid
ꢀ-peptide. Selective inhibition of ꢀ-amyloid peptide formation
could potentially slow or even reverse the devastating conse-
quences of the disease.37 Indeed, experimental data from
transgenic mouse models of Alzheimer’s disease, BACE-1
knockout mice, and pharmacological studies corroborate the
potential usefulness of drugs that interfere with BACE-1
expression and/or enzymatic activity for the treatment of
Alzheimer’s disease.38
Heparan sulfate, which is a constituent of amyloid plaques,
can interact with amyloid proteins, peptides, and fibrils, promote
aggregation, and enhance the stability of fibrils. Soluble heparin
and heparin analogues have been shown to inhibit these
processes both in vitro and in vivo. Recently, however, it was
shown that HS can inhibit the proteolytic activity of BACE-1;
the putative mechanism is by blocking access to the enzyme
The ability of the HS oligosaccharides to inhibit BACE-1
cleavage of APP was assessed using a fluorescent resonance
energy transfer (FRET) peptide cleavage assay employing the
Swedish amino acid variant FRET peptide 5-FAM-Glu-Val-
Asn-Leu-Asp-Ala-Phe-Lys(QXL520)-OH. When intact, the
amino terminal fluorophore is quenched, but upon enzymatic
cleavage, it is released from quencher and fluoresces at 520 nm.
(34) Lucas, H.; Basten, J. E. M.; van Dinther, T. G.; Meuleman, D. G.;
van Aelst, S. F.; van Boeckel, C. A. A. Tetrahedron 1990, 46, 8207–
8228. Rochepeau-Jobron, L.; Jacquinet, J. C. Carbohydr. Res. 1998,
305, 181–191.
(35) Brewer, M.; Rich, D. H. Org. Lett. 2001, 3, 945–948.
(36) Venot, A.; Swayze, E. E.; Griffey, R. H.; Boons, G. J. Chembiochem
2004, 5, 1228–1236.
(37) Golde, T. E. J. Clin. InVest. 2003, 111, 11–18.
(38) Chang, W. P.; Koelsch, G.; Wong, S.; Downs, D.; Da, H.; Weerasena,
V.; Gordon, B.; Devasamudram, T.; Bilcer, G.; Ghosh, A. K.; Tang,
J. J. Neurochem. 2004, 89, 1409–1416. Asai, M.; Hattori, C.; Iwata,
N.; Saido, T. C.; Sasagawa, N.; Szabo, B.; Hashimoto, Y.; Maruyama,
K.; Tanuma, S.; Kiso, Y.; Ishiura, S. J. Neurochem. 2006, 96, 533–
540.
(39) Scholefield, Z.; Yates, E. A.; Wayne, G.; Amour, A.; McDowell, W.;
Turnbull, J. E. J. Cell Biol. 2003, 163, 97–107.
(40) Demchenko, A. V.; Rousson, E.; Boons, G. J. Tetrahedron Lett. 1999,
40, 6523–6526. Crich, D.; Hu, T. S.; Cai, F. J. Org. Chem. 2008, 73,
8942–8953.
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A R T I C L E S
Arungundram et al.
Scheme 5. Synthesis of a Library of Tetrasaccharidesa
Figure 4. Dose response inhibition curves for selected compounds
compared to porcine mucosal heparin in the FRET peptide cleavage assay
(9, heparin; 2, compound 83; b, compound 77; 0, compound 81).
contain two glucuronic acid moieties, displayed reasonable
activity, and in this case, HS oligosaccharide 76 having
acetamido moieties was somewhat more active than compound
77 modified by N-sulfates. Previous studies have indicated that
the binding cleft of BACE-1 can accommodate relatively large
HS oligosaccharides,22 and thus, it is conceivable that derivatives
76/77 and 82/83 bind in different regions of the cleft, explaining
the difference in the structure-activity relationship. Finally, a
sulfate ester at C-2 of an uronic acid moiety, as in compounds
84-86, had low or no inhibitory activity.
The synthetic HS oligosaccharides are less active than full-
length HS polysaccharide, which was expected because previous
observations with natural oligosaccharides suggest that the
binding site of BACE-1 can accommodate relatively large HS
oligosaccharides.22 However, the fact that a tetrasaccharide
displayed considerable inhibitory activity indicates that a library
of such compounds is appropriate for identifying lead com-
pounds, which provide an attractive starting point for the
synthesis of a focused library of larger oligosaccharides. The
attraction of such an approach is that the preparation of a
representative library of tetrasaccharides is an achievable task,
whereas preparation of a library of larger HS oligosaccharides
remains a considerable challenge.
a Reagents and conditions: (a) TMSOTf, DCM, -20 to 5 °C, molecular
sieves (70, 61%; 71, 62%; 72, 64%; 73, 62%; 74, 51%; 75, 59%).
Table 1. Inhibitory Activities of Synthetic Compounds and Heparin
for Cleavage of FRET Peptide by BACE-1
HS
IC50 (µg/mL)
IC50 (µmol)
heparin
76
77
0.02
39
97
35
78
78
79
80
81
>300
240
104
290
35
195
94
236
32
82
83
4.6
3.7
84
85
86
69
>300
167
>300
64
Conclusions
138
36
The modular synthetic approach reported here utilizes a
relatively small number of selectively protected disaccharide
building blocks that can easily be converted into glycosyl donors
and acceptors, which in turn can be employed for the convenient
preparation of libraries of well-defined HS oligosaccharides.
Such a collection of compounds can be employed for
structure-activity relationship studies for HS binding proteins.
Key features of the approach include the use of Lev esters for
those hydroxyls that need sulfation, an Fmoc carbonate as a
temporary protecting group for the C-4′ hydroxyl for the
preparation of glycosyl acceptors, an anomeric TDS group for
glycosyl donor synthesis, and acetyl esters and benzyl ethers
as permanent protecting groups. The trichloroacetimidate meth-
odology41 was employed for reliable oligosaccharide assembly,
As can be seen in Table 1 and Figure 4, HS oligosaccharide
83 was able to inhibit the cleavage of the peptide by BACE-1
with relatively high potency. Several derivatives such as 76,
77, and 82 displayed modest activity, whereas compounds 78,
79, 81, 84, and 86 had low or no inhibitory activity. Compound
83 is a tetrasaccharide composed of two iduronic acid moieties
that are sulfated at the C-6 hydroxyls and two glucosamine
moieties modified by N-sulfates. Interestingly, compound 82,
which has acetamido moieties instead of N-sulfates, has 10-
fold reduced potency highlighting the importance of the
N-sulfates of 83 for optimal inhibitory activity. Replacement
of one of the iduronic acid moieties by a glucuronic acid
derivative, as in compounds 79 and 81, led to a large reduction
in inhibitory activity. Surprisingly, compounds 76 and 77, which
(41) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Chem. Biochem. 1994,
50, 21–123.
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Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
until the reaction mixture stayed yellow. The excess diazomethane
was quenched by the addition of AcOH until the reaction mixture
became colorless. The mixture was concentrated in vacuo and
coevaporated with toluene, and the residue was purified by silica
gel column chromatography to yield a methyl ester.
General Procedure for Synthesis of Fmoc-Protected Disaccha-
rides. To a 0.03 M solution of disaccharide in DCM at 0 °C was
added FmocCl (10 equiv) and DMAP (0.01 equiv). The reaction
mixture was brought to room temperature, and stirring was
continued until TLC indicated complete consumption of the starting
material (∼2 h). After quenching the reaction with MeOH (50 µL),
the mixture was diluted with DCM (50 mL) and washed with
saturated aqueous sodium bicarbonate (2 × 50 mL) and brine (50
mL). The organic phase was dried (MgSO4) and filtered, and the
filtrate was concentrated in vacuo. The residue was chromato-
graphed over silica gel using a gradient of hexanes and EtOAc to
give Fmoc carbonate-protected disaccharide.
General Procedure for Silyl Ether Cleavage. A disaccharide was
dissolved in THF (0.05 M) followed by the addition of HF ·pyridine
(100 equiv). After stirring for 18 h, the reaction mixture was diluted
with DCM (50 mL) and washed with water, (50 mL), saturated
aqueous sodium bicarbonate (50 mL), and brine (50 mL). The
organic phase was dried (MgSO4) and filtered, and the filtrate was
concentrated in vacuo. The residue was chromatographed over silica
gel using a gradient of hexanes and EtOAc to give pure lactol.
General Procedure for Preparation of Trichloroacetimidates
(45-48, 57-60). To a solution of the lactol in DCM (2 mL for
0.08 mmol) was added finely powdered anhydrous K2CO3 (2 equiv).
After stirring at room temperature for 1.5 h, the reaction mixture
was filtered and the filtrate concentrated in vacuo. The residue was
chromatographed over silica gel using a mixture of hexanes and
EtOAc containing 0.01% pyridine to yield a trichloroacetimidate
donor.
General Procedure for Preparation of Tetrasaccharides (70-75).
Disaccharide trichloroacetimidate donor (1.2 equiv based on
acceptor) and disaccharide acceptor (1.0 equiv) were combined in
a flask, coevaporated with toluene (3 × 3 mL), and dissolved in
DCM to maintain a concentration of 0.04-0.05 M. Powdered
freshly activated 4 Å molecular sieves (weight of sieves equal to
the combined weight of donor and acceptor) were added, and the
mixture was stirred for 30 min at ambient temperature and then
cooled to -20 °C. TMSOTf (0.1 equiv) was added, and stirring
was continued until TLC indicated the disappearance of donor (∼15
min). The reaction was allowed to warm to +5 °C and then
quenched by the addition of pyridine (5 µL) after 1 h. The mixture
was filtered, the filtrate was concentrated in vacuo, and the residue
was purified by silica gel column chromatography using a gradient
of toluene and EtOAc to give pure tetrasaccharide.
and in each glycosylation, only the required R-anomer was
obtained. Furthermore, it was found that installation of the uronic
acid moieties could best be performed at the disaccharide stage
by selective TEMPO/BIAB-mediated oxidation of the C-6
hydroxyl of a glucoside or idoside to the corresponding
carboxylic acid. The utility of the modular building blocks has
been illustrated by the preparation of a library of 12 oligosac-
charides, and importantly, a standard set of reaction conditions
could be employed for the preparation of all target compounds.
The HS oligosaccharides were employed to probe structural
features of HS for inhibition of the protease, BACE-1. The
significant and complex variations in activity with structural
changes observed in this study support the view that important
functional information is embedded in HS sequences.10,15
Furthermore, the most active derivative identified in this study
provides an attractive lead compound for the preparation more
potent compounds for BACE-1, which may find use as a
therapeutic agent for Alzheimer’s disease. The synthetic com-
pounds are also equipped with an artificial aminopentyl spacer,
which offers an opportunity for HS oligosaccharide array
development. Such an array is expected to provide a unique
tool for rapid ligand identification for HS binding proteins.
Experimental Section
General Glycosylation Procedure for Synthesis of Disaccha-
rides (37-40, 49-52). Glycosyl thioethyl donor (1.2 equiv based
on acceptor) and 2-azido-2-deoxy-R-D-glucopyranoside acceptor
(1.0 equiv) were combined in a flask, coevaporated with toluene
(3 × 3 mL), and dissolved in DCM to maintain a concentration of
0.02 M (based on donor). Powdered freshly activated 4 Å molecular
sieves (weight of sieves equal to the combined weight of donor
and acceptor) were added, and the mixture was stirred for 30 min
at ambient temperature and then cooled to 0 °C. NIS (1.2 equiv)
and TMSOTf (0.1 equiv) were added to the mixture, and stirring
was continued until TLC indicated disappearance of glycosyl donor
(∼15 min). The reaction mixture was allowed to warm to +5 °C
and then quenched by the addition of DTBMP. The mixture was
filtered, the filtrate was concentrated in vacuo, and the residue was
purified by silica gel column chromatography using a stepwise
gradient of toluene and EtOAc (90/10 f 65/35, v/v).
General Procedure for Benzylidene Acetal Cleavage of Disac-
charides. Method A. To a solution of disaccharide (37, 49) in DCM
was added ethanethiol (6 equiv) and p-TsOH (0.2 equiv), and the
resulting solution was stirred at ambient temperature for 1 h. The
reaction mixture was quenched by the addition of Et3N and
concentrated in vacuo, and the residue was purified by silica gel
column chromatography to give pure product.
Method B. A solution of a disaccharide (38-40, 50-52) in a
mixture of DCM:TFA:H2O (0.06 M, 10/1/0.1, v/v/v) was stirred
at ambient temperature for 30 min. The reaction mixture was
concentrated in vacuo, and the residue was coevaporated with
toluene. The residue was purified by silica gel column chromatog-
raphy using a mixture of toluene and EtOAc to give pure product.
General Procedure for TEMPO/BAIB-Mediated Oxidation
and Esterification by Diazomethane (41-44, 53-56). To a vigor-
ously stirred solution of the diol (0.3 M solution) in a mixture of
DCM:H2O (2/1, v/v) was added TEMPO (0.2 equiv) and BAIB
(2.5 equiv). Stirring was continued until TLC indicated complete
conversion of the starting material to a spot of lower Rf (∼45 min).
The reaction mixture was quenched by the addition of aqueous
Na2S2O3 (10%, 10 mL). The mixture was extracted with EtOAc (2
× 10 mL), and the combined aqueous layers were back-extracted
with EtOAc (10 mL). The combined organic layers were dried
(MgSO4) and filtered, and the filtrate was concentrated in vacuo.
The oily residue was dissolved in THF (0.1 M) and treated with
an excess of freshly prepared ethereal solution of diazomethane
General Procedure for Fmoc Cleavage of Tetrasaccharide. A
tetrasaccharide was dissolved in a mixture of DCM (2.4 mL for
0.12 mmol) and triethylamine (0.6 mL). After stirring for 2 h, the
reaction mixture was concentrated in vacuo and the residue was
purified by silica gel column chromatography using a gradient of
hexanes and EtOAc to afford a tetrasaccaharide acceptor.
General Procedure for Cleavage of Lev Esters. Anhydrous
hydrazine acetate (5 equiv per Lev group) was added to a solution
of the starting material in a mixture of ethanol and toluene (2/1,
v/v, 5 mL for 150 mg). Stirring was continued until TLC analysis
(toluene/EtOAc 1/1, v/v, or hexanes/EtOAc 1/1, v/v) indicated
disappearance of starting material (∼2 h). The reaction mixture
was diluted with DCM (30 mL), washed with water (3 × 25 mL)
and brine (25 mL), dried (MgSO4), and filtered. The filtrate was
concentrated, and the residue was purified by silica gel column
chromatography using a gradient of hexanes or toluene and EtOAc
to afford product.
General Procedure for O-Sulfation. Sulfur trioxide pyridine
complex (10 equiv per OH) was added to a solution of the starting
material in DMF (1.0 mL for 0.02 mmol). The mixture was stirred
at ambient temperature for 2-4 h until TLC (CHCl3, CH3OH 90/
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A R T I C L E S
Arungundram et al.
10, v/v) indicated completion of the reaction. After the addition of
pyridine (0.2 mL) and CH3OH (0.5 mL) stirring was continued for
30 min. The mixture was concentrated in vacuo (bath temperature
20 °C), and the residue was applied to a column of Iatrobeads (1.5
g), which was eluted with a gradient of CH3OH in CHCl3 (96/4 f
88/12 v/v, containing 0.2% pyridine). The fractions containing
product were concentrated in vacuo (bath temperature 20 °C), and
the residue was immediately passed through a column of Biorad
50 × 8 Na+ resin (0.6 × 5 cm) using CH3OH as eluent, providing
the product as sodium salt.
General Procedure for Fmoc Cleavage. Et3N (0.1 mL) was added
to a solution of the starting material in DMF (1.0 mL for 0.02
mmol). The reaction mixture was stirred for ∼1.5 h until TLC
(CHCl3/CH3OH, 85/15, v/v) indicated disappearance of starting
material. The reaction mixture was concentrated in vacuo (bath
temperature 20 °C), and the residue was passed through a column
of Biorad 50 × 8 Na+ resin (0.6 × 5 cm) using CH3OH as eluent.
Fractions containing product were concentrated in vacuo, and the
residue was chromatographed over Iatrobeads (1.2 g) using a
gradient of CH3OH in CHCl3 (94/5 f 88/12, v/v) as eluent.
Appropriate fractions were concentrated in vacuo, providing
product, which was directly used in the next step.
with water and applied to a small RP-18 column (20 times the
weight of starting material), which was eluted with a stepwise
gradient of H2O and CH3OH (from 90/10 to 40/60, v/v). The
appropriate fractions were concentrated in vacuo to obtain N-
acetylated product.
General Procedure for Selective N-Sulfation. SO3 ·Py (5 equiv
per NH2) was added to the starting material in CH3OH (1 mL for
0.006 mmol) in a mixture of triethylamine (0.3 mL) and 0.1 M
NaOH (2 equiv per NH2) at 0 °C. The progress of the reaction was
monitored by TLC (silica gel TLC, EtOAc/pyridine/water/
CH3CO2H, 8/5/3/1, v/v/v/v; RP-18 TLC, H2O/CH3OH, 60/40, v/v).
Two additional portions of SO3 ·Py were added at 0 °C after 1 and
2 h. After stirring for an additional 8 h, the reaction mixture was
coevaporated with water (bath temperature 20 °C) and the residue
passed through a short column of Biorad 50 × 8 Na+ resin (0.6 ×
5 cm) using CH3OH and H2O (90/10, v/v) as eluent. Appropriate
fractions were concentrated in vacuo, and the residue was vortexed
with water and applied to small RP-18 silica gel column (20 times
the weight of starting material), which was then eluted with a
stepwise gradient of H2O and CH3OH (90/10 f 40/60, v/v). The
appropriate fractions were concentrated in vacuo to provide
N-sulfated product.
General Procedure for Global Debenzylation. Pd/C (10%, 1.5
times the weight of starting material) was added to a solution of
the starting material in CH3OH and H2O (1/1, v/v, 1 mL for 5 mg).
The mixture was placed under an atmosphere of hydrogen, and
the progress of the reaction was monitored by TLC (silica gel,
CHCl3/CH3OH/H2O 60/40/10, v/v/v; EtOAc/pyridine/water/
CH3CO2H, 3/5/3/1, v/v/v). The hydrogenation was stopped when
TLC indicated the disappearance of the starting material and the
presence of a ninhydrin-positive main spot (2 h). The mixture was
filtered through a PTFE syringe filter (0.2 mm, 13 mm) and washed
with a mixture of CH3OH and H2O (1/1, v/v, 2 mL), and the
solvents were concentrated in vacuo. The residue was dissolved in
distilled water (1.5 mL), and palladium hydroxide on carbon
(Degussa type, 20%, 1.5 times the weight of starting material) was
added. The resulting mixture was placed under an atmosphere of
hydrogen, and after 12 h, TLC (EtOAc/pyridine/water/CH3CO2H
4/5/3/1, v/v/v/v) indicated the completion of the reaction. The
mixture was filtered through a PTFE syringe filter, and the residue
was washed with H2O (2 mL). The filtrate was freeze dried, the
residue was passed through a short column of Biorad 50 × 8 Na+
resin (0.6 × 2.5 cm) using H2O as the eluent, and the appropriate
fractions were freeze dried to provide the final product.
5-Aminopentyl [(ꢀ-D-Glucopyranosyluronate)-(1f4)-(2-deoxy-
2-sulfoamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(r-L-
idopyranosyluronate)-(1f4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonate-
r-D-glucopyranoside)-(1f4)-(ꢀ-D-glucopyranosyluronate)]-(1f4)-
O-2-deoxy-N-sulfoamino-6-O-sulfonate-r-D-glucopyranoside Nonaso-
dium Salt (69). Hexasaccharide 63 (73.5 mg, 0.028 mmol) was
subjected to the sequence of deprotection steps including delevuli-
noylation, O-sulfation, Fmoc cleavage, saponification, deacetylation,
azide reduction, N-sulfation, and global debenzylation according
to the general procedures to provide hexasaccharide 69 (4.8 mg).
[R]D25: +126.4 (c ) 0.33, H2O). 1H NMR (800 MHz, D2O): δ 5.58
(d, 1H, J ) 3.7 Hz, H1c), 5.33 (d, 1H, J ) 3.6 Hz, H1E), 5.13 (d,
1H, J ) 3.6 Hz, H1A), 4.99 (bs, 1H, H1E), 4.80 (d, 1H, J ) 2.17
Hz, H5D), 4.59 (s, 1H, J ) 8.0 Hz, H1F), 4.58 (s, 1H, J ) 8.0 Hz,
H1B), 4.46 (bd, 1H, J ) 9.7 Hz, H6aE), 4.41 (bd, 1H, J ) 9.6 Hz,
H6aA), 4.31(bd, 1H, J ) 10.4 Hz, H6aC), 4.25 (dd, 1H, J ) 6.0
Hz, J ) 11.1 Hz, H6bA), 4.17 (m, 2H, H6bE, H6bC), 4.10 (t, 1H,
J ) 3.6 Hz, H3D), 4.04-4.01 (m, 3H, H4D, H5A, H5E), 3.97 (bd,
1H, J ) 10.04 Hz, H5C), 3.84 (t, 1H, J ) 9.1 Hz, H3B), 3.80-3.65
(m, 10H, H5B, H5F, H4E, H2D, H4B, H3A, OCHH Linker, H4C, H3E,
H4A), 3.62 (t, 1H, J ) 9.7 Hz, H3C), 3.56-3.47 (m, 3H, OCHH
Linker, H3F, H4F), 3.36 (t, 1H, J ) 8.6 Hz, H2B), 3.32 (t, 1H, J )
8.5 Hz, H2F), 3.27 (dd, 1H, J ) 3.6 Hz, J ) 10.2 Hz, H2A), 3.26
(dd, 1H, J ) 3.6 Hz, J ) 10.0 Hz, H2C), 3.24 (dd, 1H, J ) 3.6 Hz,
J ) 10.5 Hz, H2E) 3.0 (m, 2H, CH2N Linker), 1.72-1.62 (m, 4H,
General Procedure for Saponification of Methyl Esters and
De-O-acetylation. A premixed solution of 30% solution of H2O2 in
water (100 equiv per CO2Me) and 1 M LiOH (50 equiv per CO2Me)
were added to a solution of the starting material in THF (0.02 M).
The reaction mixture was stirred at room temperature for 8 h. A 4 N
solution of NaOH (1.0 mL) was added until pH 14. The reaction
mixture was left stirring for 18 h at room temperature. In the case that
the reaction had not gone to completion, stirring was continued at 35
°C for an additional 12 h. The mixture was then brought to pH 8-8.5
by addition of AcOH, and the mixture was concentrated in vacuo (bath
temperature 20 °C). The residue was vortexed with water and applied
to a RP-18 column (10 times the weight of starting material), which
was eluted with a stepwise gradient of H2O and CH3OH (from 90/10
f 70/30, v/v). The appropriate fractions were concentrated in vacuo
(bath temperature 20 °C), and the residue was passed through a column
of Biorad 50 × 8 Na+ resin (0.6 × 5 cm) using CH3OH as eluent,
providing product.
General Procedure for Reduction of Azide Group. A 1 M
solution of PMe3 in THF (8 equiv per azide group) was added to the
solution of the starting material in THF (1.0 mL for 0.013 mmol). A
0.1 M NaOH solution (10 equiv per azido group) was added, and the
mixture was stirred at room temperature for 5 h. The progress of the
reaction was monitored by TLC (CHCl3/CH3OH/H2O 70/30/5, v/v/v,
and RP-18 plates with H2O/CH3OH 40/60, v/v). The presence of amino
groups was confirmed using ninhydrin as visualizing agent (In some
cases, an additional amount of PMe3 solution was added to achieve
completion of the reaction). The pH was then adjusted to 8.5 by careful
addition of AcOH, and the mixture was concentrated in vacuo (bath
temperature 20 °C). The residue was vortexed with water and applied
to a small RP-18 silica gel column (10 times the weight of starting
material), which was eluted with a stepwise gradient of H2O and
CH3OH (from 90/10 to 40/60, v/v). The appropriate fractions were
concentrated in vacuo, and the residue was passed through a column
Biorad 50 × 8 Na+ resin (0.6 × 5 cm) using CH3OH as eluent,
providing product.
General Procedure for Selective N-Acetylation. Acetic anhydride
(10 equiv per NH2) was added to a solution of the starting material
in a mixture of anhydrous CH3OH (500 µL for 0.011 mmol) and
Et3N (20 equiv per NH2) at 0 °C. The progress of the reaction was
monitored by TLC (silica gel, CHCl3/CH3OH/H2O, 60/30/3, v/v/
v; RP18 silica gel, H2O/CH3OH, 40/60, v/v). After 5 h, another
portion of Et3N and Ac2O was added at 0 °C. After stirring for 1 h
at room temperature, the mixture was coevaporated with toluene
in vacuo (bath temperature 20 °C) and the residue passed through
a short column of Biorad 50 × 8 Na+ resin (0.6 × 5 cm) using a
mixture of CH3OH and H2O (90/10, v/v) as eluent, and appropriate
fractions were concentrated in vacuo. The residue was vortexed
9
17402 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
2 × CH2 Linker), 1.52-1.45 (m, 2H, CH2 Linker). ESI-MS: m/z
3.82 (dd, J ) 8.7 Hz, H3A, J ) 10.7 Hz), 3.78-3.73 (m, 3H, H3C,
H4C, H5D), 3.72-3.65 (m, 3H, H2B, H4A, OCHH Linker),
3.53-3.47 (m, 3H, H3D, H4D, OCHH Linker), 3.33 (dd, 1H, J )
7.9 Hz, J ) 9.4 Hz, H2D), 2.98 (t, 2H, J ) 7.6 Hz, CH2NH2 Linker),
2.01, 1.98 (2s, 3H each, 2 × CH3, NHAc), 1.70-1.58 (m, 4H, 2 ×
CH2 Linker), 1.48-1.40 (m, 2H, CH2 Linker). ESI-MS: m/z calcd.
for C33H54N3O29S2, 1020.2290; found, 1020.2273 [M - H]1-; calcd
calcd for C41H68N4O49S6, 796.0644; found, 796.0634 [M - 2H]2-
;
calcd for C41H67N4O49S6, 530.3738; found, 530.3721 [M - 3H]3-
.
5-Aminopentyl [(ꢀ-D-Glucopyranosyluronate)-(1f4)-(2-aceta-
mido-2-deoxy-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(ꢀ-D-
glucopyranosyluronate-(1f4)-O-2-acetamido-2-deoxy-6-O-sulfonate-
r-D-glucopyranoside Tetrasodium Salt (76). Tetrasaccharide 70
(101 mg, 0.052 mmol) was subjected to the sequence of deprotec-
tion steps including delevulinoylation, O-sulfation, Fmoc cleavage,
saponification, deacetylation, and azide reduction to give a diamino
tetrasaccharide. Part of this material was subjected to N-acetylation
and global debenzylation according to the general procedures to
give the tetrasaccharide 76 (3.0 mg). [R]D25: +70 (c ) 1, H2O). 1H
NMR (800 MHz, D2O): δ 5.40 (d, 1H, J ) 3.8 Hz, H1C), 4.85 (d,
1H, J ) 3.2 Hz, H1A), 4.55-4.54 (m, 2H, H1B, H1D), 4.42 (bd,
2H, H6aC, H6aA), 4.23 (dd, 1H, J ) 6.2 Hz, J ) 11.4 Hz, H6bA),
4.16 (dd, 1H, J ) 1.9 Hz, J ) 11.1 Hz, H6bC), 4.04-4.00 (m, 2H,
H5C, H5A), 3.96 (m, 3H, H2C, H2A, H3A), 3.82-3.78 (m, 2H, H3C,
H5B), 3.74-3.67 (m, 6H, H4B, H4C, H5D, OCHH Linker, H3B,
H4A), 3.51 (t, 1H, J ) 9.3 Hz, H3D), 3.50-3.46 (m, 2H, OCHH
Linker, H4D), 3.32 (dd, 2H, J ) 7.9 Hz, J ) 9.3 Hz, H2B, H2D),
2.98 (t, 2H, J ) 7.7 Hz, CH2N Linker), 2.02, 2.00 (2s, 3H each, 2
× CH3, NAc), 1.69-1.59 (m, 4H, 2 × CH2 Linker), 1.46-1.43
(m, 2H, CH2 Linker). ESI-MS: m/z calcd for C33H54N3O29S2,
1020.2290; found, 1020.2312 [M - H]1-; calcd for C33H53N3O29S2,
for C33H53N3O29S2, 509.6109; found, 509.6119 [M - 2H]2-
;
calcd for C33H52N3O29S2, 339.4048; found, 339.4051 [M - 3H]3-
.
5-Aminopentyl [ꢀ-D-Glucopyranosyluronate-(1f4)-(2-deoxy-2-
sulfoamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-r-L-idopy-
ranosyluronate)]-(1f4)-2-deoxy-2-sulfoamino-6-O-sulfonate-r-D-
glucopyranoside Hexasodium Salt (79). The second portion of the
diamino tetrasaccharide obtained above was subjected to N-sulfation
and global debenzylation according to the general procedure
25
described above to obtain the tetrasaccharide 79 (4.1 mg). [R]D
:
1
+40 (c ) 0.5, H2O). H NMR (800 MHz, D2O): δ 5.36 (d, 1H, J
) 3.6 Hz, H1C, 5.13 (d, 1H, J ) 3.7 Hz, H1A), 4.99 (d, 1H, J )
2.4 Hz, H1B), 4.72 (d, 1H, J ) 2.3 Hz, H5B), 4.57 (d, 1H, J ) 8.0
Hz, H1D), 4.46 (dd, 1H, J ) 2.0 Hz, J ) 11.0 Hz, H6aC), 4.32 (dd,
1H, J ) 1.7 Hz, J ) 11.0 Hz, H6aA), 4.23 (dd, 1H, J ) 5.5 Hz, J
) 11.0 Hz, H6bA), 4.18 (dd, 1H, J ) 1.7 Hz, J ) 11.0 Hz, H6bC),
4.10 (dd, 1H, J ) 3.5 Hz, J ) 4.4 Hz, H3B), 4.05 (bt, 1H, J ) 4.0
Hz, H4 ), 4.04-4.00 (m, 2H, H5A, H5C), 3.77-3.64 (m, 7H, H2B,
B
H3A, H3C, H4A, H4C, H5D, OCHH Linker), 3.57-3.51 (m, 2H, H3D,
OCHH Linker), 3.48 (t, 1H, J ) 9.3 Hz, H4D), 3.33 (dd, 1H, J )
8.0 Hz, J ) 9.1 Hz, H2D), 3.27 (dd, 1H, J ) 3.7 Hz, J ) 9.9 Hz,
H2A), 3.24 (dd, 1H, J ) 10.5 Hz, H2C), 3.02 (t, 2H, J ) 7.4 Hz,
CH2NH2), 1.70-1.60 (m, 4H, 2 × CH2 Linker), 1.52-1.42 (m,
2H, CH2 Linker). ESI-MS: m/z calcd for C29H50N3O33S4, 1096.1215;
found, 1096.1247 [M - H]1-; calcd for C29H49N3O33S4, 547.5571;
found, 547.5591 [M - 2H]2-; calcd for C29H48N3O33S4, 364.7023;
509.6109; found, 509.6118 [M - 2H]2-
.
5-Aminopentyl [(ꢀ-D-Glucopyranosyluronate)-(1f4)-(2-deoxy-
2-N-sulfoamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(ꢀ-
D-glucopyranosyluronate-(1f4)-O-2-deoxy-2-N-sulfoamino-2-deoxy-
6-O-sulfonate-r-D-glucopyranoside Hexasodium Salt (77). A diamino
tetrasaccharide obtained by partial deprotection of 70 was subjected
to N-sulfation and global debenzylation according to the general
procedures to give tetrasaccharide 77 (6.8 mg). [R]D25: +33 (c )
found, 364.7034 [M - 3H]3-
.
1
5-Aminopentyl [(r-L-Idopyranosyluronate)-(1f4)-O-(2-aceta-
mido-2-deoxy-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(ꢀ-D-
glucopyranosyluronate)]-(1f4)-(2-acetamido-2-deoxy-6-O-sulfonate-
r-D-glucopyranoside Tetrasodium Salt (80). Tetrasaccharide 72 (89
mg, 0.045 mmol) was subjected to the sequence of deprotection
steps including delevulinoylation, O-sulfation, Fmoc cleavage,
saponification, deacetylation, and azide reduction to obtain the
diamino tetrasaccharide. One portion of the diamino tetrasaccharide
was subjected to N-acetylation and global debenzylation according
to the general procedure described above to give tetrasaccharide
1, H2O). H NMR (800 MHz, D2O): δ 5.62 (d, 1H, J ) 3.7 Hz,
H1C), 5.12 (d, 1H, J ) 3.7 Hz, H1A), 4.58 (d, 1H, J ) 7.9 Hz,
H1B), 4.56 (d, 1H, J ) 7.9 Hz, H1D), 4.43 (dd, 1H, J ) 1.9 Hz, J
) 11.0 Hz, H6aC), 4.41 (dd, 1H, J ) 2.0 Hz, J ) 11.2 Hz, H6aA),
4.23 (dd, 1H, J ) 6.0 Hz, J ) 11.2 Hz, H6bA), 4.15 (dd, 1H, J )
1.9 Hz, J ) 11.0 Hz, H6bC), 4.01 (ddd, 1H, J ) 2.0 Hz, J ) 6.0
Hz, J ) 10.0 Hz, H5A), 3.99 (bd, 1H, J ) 10.0 Hz, H5C), 3.84(t,
1H, J ) 8.8 Hz, H3B), 3.80-3.71(m, 6H, H5B, H4B, H4C, H5D,
OCHH Linker, H3A), 3.66-3.64 (m, 2H, H3C, H4A), 3.56-3.54
(m, 1H, OCHH Linker), 3.53-3.46 (m, 2H, H3D, H4D), 3.35 (dd,
1H, J ) 7.9 Hz, J ) 9.4 Hz, H2B), 3.32 (dd, 1H, J ) 8.0 Hz, J )
9.2 Hz, H2D), 3.26 (dd, 1H, J ) 3.7 Hz, J ) 8.2 Hz, H2A), 3.23(dd,
1H, J ) 3.7 Hz, J ) 10.8 Hz, H2C), 2.99 (t, 2H, J ) 7.4 Hz, CH2N
Linker), 1.70-1.61 (m, 4H, 2 × CH2 Linker), 1.50-1.44 (m, 2H,
CH2 Linker). ESI-MS: m/z calcd for C29H50N3O33S4, 1096.1215;
found, 1096.1237 [M - H]1-; calcd for C29H49N3O33S4, 547.5571;
1
80 (5.8 mg). [R]D25: +140 (c ) 0.5, H2O). H NMR (800 MHz,
D2O): δ 5.38 (d, 1H, J ) 3.8 Hz, H1C), 4.87-4.85 (m, 2H, H1A,
H1D), 4.57 (d, 1H, J ) 3.8 Hz, H5D), 4.55 (d, 1H, J ) 7.9 Hz,
H1B), 4.42 (dd, 1H, J ) 1.9 Hz, J ) 11.2 Hz, H6aA), 4.35 (dd, 1H,
J ) 2.0 Hz, J ) 11.0 Hz, H6aC), 4.25 (dd, 1H, J ) 6.0 Hz, J )
11.2 Hz, H6bA), 4.17 (dd, 1H, J ) 1.8 Hz, J ) 11.0 Hz, H6bC),
4.05-4.02 (m, 1H, H5A), 4.01-3.98 (m, 1H, H5C), 3.94-3.88 (m,
3H, H2A, H2C, H3A), 3.87 (dd, 1H, J ) 4.2 Hz, J ) 5.6 Hz (H4D),
3.79 (d, 1H, J ) 9.5 Hz, H5B), 3.77-3.66 (m, 6H, H3B, H3C, H3D,
H4B, H4C, OCHH Linker), 3.64 (dd, 1H, J ) 8.0 Hz, J ) 10.0 Hz,
H4A), 3.56 (dd, 1H, J ) 2.6 Hz, J ) 7.1 Hz, H2D), 3.50 (m, 1H,
OCHH Linker), 3.33 (dd, 1H, J ) 9.6 Hz, J ) 9.6 Hz, H2B), 2.98
(t, 2H, J ) 7.7 Hz, CH2NH2), 2.05 and 2.03 (2s, 3H each, 2 ×
CH3, NHAc), 1.74-1.60 (m, 4H, 2 × CH2 Linker), 1.50-1.42 (m,
2H, CH2 Linker). Calcd for C33H54N3O29S2, 1020.2290; found,
1020.2319 [M - H]1-; calcd for C33H53N3O29S2, 509.6109; found,
509.6129 [M - 2H]2-; calcd for C33H52N3O29S2, 339.4048;
found, 547.5580 [M - 2H]2-
.
5-Aminopentyl [ꢀ-D-Glucopyranosyluronate-(1f4)-(2-acetamido-
2-deoxy-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-r-L-idopyrano-
syluronate)]-(1f4)-2-acetamido-2-deoxy-6-O-sulfonate-r-D-glu-
copyranoside Tetrasodium Salt (78). Tetrasaccharide 71 (53 mg,
0.027 mmol) was subjected to the sequence of deprotection steps
including delevulinoylation, O-sulfation, Fmoc cleavage, saponi-
fication, deacetylation, and azide reduction to obtain a diamino
tetrasaccharide. Part of this material was subjected to N-acetylation
and global debenzylation according to the general procedure to give
tetrasaccharide 78 (2.5 mg). [R]D25: +22 (c ) 0.5, H2O). 1H NMR
(800 MHz, D2O): δ 5.16 (d, 1H, J ) 3.8 Hz, H1C), 4.96 (d, 1H, J
) 3.3 Hz, H1B), 4.86 (d, 1H,. J ) 3.6 Hz, H1A), 4.70 (d, 1H, J )
2.9 Hz, H5B), 4.56 (d, 1H, J ) 7.9 Hz, H1D), 4.43 (dd, 1H, J ) 2.6
Hz, J ) 11.2 Hz, H6aC), 4.33 (dd, 1H, J ) 2.0 Hz, J ) 11.2 Hz,
H6aA), 4.25 (dd, 1H, J ) 5.7 Hz, H6bA), 4.01 (dd, 1H, J ) 1.9 Hz,
H6bC), 4.08 (m, 1H, H5C), 4.06 (t, 1H, J ) 3.4 Hz, H4B), 4.03 (m,
1H, H5A), 3.96 (dd, 1H, J ) 10.4 Hz, H2C), 3.94 (dd, 1H, J ) 3.6
Hz, J ) 10.7 Hz, H2A), 3.88 (dd, J ) 3.4 Hz, J ) 5.9 Hz, H3B),
found, 339.4056 [M - 3H]3-
.
5-Aminopentyl [(L-r-Idopyranosyluronate)-(1f4)-O-(2-deoxy-
2-sulfoamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(ꢀ-D-
glucopyranosyluronate)]-(1f4)-2-deoxy-2-sulfoamino-6-O-sulfonate-
r-D-glucopyranoside Hexasodium Salt (81). The second portion of
the diamino tetrasaccharide obtained above was subjected to
N-sulfation and global debenzylation according to the general
procedure described above to give tetrasaccharide 81 (5.7 mg).
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17403

A R T I C L E S
Arungundram et al.
[R]D25: -5.4 (c ) 0.5, H2O). 1H NMR (800 MHz, D2O): δ 5.61 (d,
1H, J ) 3.8 Hz, H1C), 5.13 (d, 1H, J ) 3.6 Hz, H1A), 4.86 (d, 1H,
J ) 4.7 Hz, H1D), 4.58-4.54 (m, 2H, H1B, H5D), 4.43 (dd, 1H, J
) 1.8 Hz, J ) 11.1 Hz, H6aA), 4.35 (dd, 1H, J ) 2.0 Hz, J ) 11.1
Hz, H6aC), 4.24 (dd, 1H, J ) 6.1 Hz, J ) 11.1 Hz, H6bA), 4.17
(dd, 1H, J ) 2.0 Hz, J ) 11.1 Hz, H6bC), 4.04-4.01 (m, 1H, H5A),
3.97-3.92 (m, 1H, H5C), 3.88 (dd, 1H, J ) 4.0 Hz, J ) 6.0 Hz,
H4D), 3.84 (dd, 1H, J ) 8.7 Hz, J ) 9,3 Hz, H3B), 3.80 (d, 1H, J
) 9.6 Hz, H5B), 3.76 (dd, 1H, J ) 8.6 Hz, J ) 9.3 Hz, H4B),
3.75-3.70 (m, 4H, H3A, H3D, H4C, OCHH Linker), 3.65 (dd, 1H,
J ) 9.0 Hz, J ) 10.0 Hz, H4A), 3.62 (dd, 1H, J ) 9.2 Hz, J )
10.3 Hz, H3C), 3.57-3.53 (m, 2H, H2D, OCHH Linker), 3.36 (dd,
1H, J ) 8.0 Hz, H2B), 3.29-3.26 (m, 2H, H2A, H2C), 3.00 (t, J )
7.6 Hz, CH2NH2), 1.72-1.60 (m, 4H, 2 × CH2 Linker), 1.52-1.42
(m, 2H, CH2 Linker). ESI-MS: m/z calcd for C29H50N3O33S4,
1096.1215; found, 1096.1251 [M - H]1-; calcd for C29H49N3O33S4,
547.5571; found, 547.5590 [M - 2H]2-; calcd for C29H48N3O33S4,
for C29H49N3O33S4, 547.5571; found, 547.5563 [M - 2H]2-; calcd
for C29H48N3O33S4, 364.7023; found, 364.7012 [M -3H]3-
.
5-Aminopentyl [(r-L-Idopyranosyluronate)-(1f4)-O-(2-aceta-
mido-2-deoxy-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(2-O-
sulfonate-r-L-idopyranosyl-uronate)]-(1f4)-2-deoxy-2-acetamido-
6-O-sulfonate-r-D-glucopyranoside Pentasodium Salt (84). Tetras-
accharide 74 (17 mg, 0.0085 mmol) was subjected to the sequence
of deprotection steps including delevulinoylation, O-sulfation, Fmoc
cleavage, saponification, deacetylation, azide, N-acetylation, and
global debenzylation according to the general procedure described
above to give tetrasaccharide 84 (1.6 mg). [R]D25: 26 (c ) 0.5, H2O).
1H NMR (500 MHz, D2O): δ 5.22 (bs, 1H, H1B), 5.14 (d, 1H, J )
3.6 Hz, H1C), 4.87 (d, 1H, J ) 3.6 Hz, H1A), 4.85 (d, 1H, J ) 5.2
Hz, H1D), 4.80 (d, 1H, J ) 2.3 Hz, H5B), 4.57 (d, 1H, J ) 4.1 Hz,
H5D), 4.40 (dd, 1H, J ) 1.5 Hz, J ) 11.5 Hz, H6aAorC), 4.36 (dd,
1H, J ) 3.0 Hz, J ) 11.0 Hz, H6aAorC), 4.29 (bs, 1H, H2B),
4.28-4.20 (m, 3H, H6bA, H6bC, H3B), 4.09-4.01 (m, 4H, H2C,
H4B, H5A, H5C), 3.94 (dd, 1H, J ) 3.6 Hz, J ) 10.5 Hz, H2A),
3.89-3.83 (m, 2H, H3A, H4D), 4.78-4.67 (m, 5H, H3C, H3D, H4A,
H4C, OCHH Linker), 3.55 (dd, 1H, J ) 7.3 Hz, H2D), 3.53-3.48
(m, 1H, OCHH Linker), 3.10 (t, 2H, J ) 7.4 Hz, CH2NH2) 2.05
and 2.02 (2s, 3H each, 2 × CH3, NHAc) 1.74-1.5 (m, 2H, 2 ×
CH2 Linker), 1.59-1.48 (m, 2H, CH2 Linker). ESI-MS: m/z calcd
for C33H53N3O32S3, 549.5893; found, 549.5890 [M - H]2-; calcd
364.7023; found, 364.7030 [M - 3H]3-
.
5-Aminopentyl [(r-L-Idopyranosyluronate)-(1f4)-(2-acetamido-
2-deoxy-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(r-L-idopy-
ranosyluronate-(1f4)-O-2-acetamido-2-deoxy-6-O-sulfonate-r-D-
glucopyranoside Tetrasodium Salt (82). Tetrasaccharide 73 (148
mg, 0.076 mmol) was subjected to the sequence of deprotection
steps including delevulinoylation, O-sulfation, Fmoc cleavage,
saponification, deacetylation, and azide reduction to obtain the
diamino tetrasaccharide. One portion of the diamino tetrasaccharide
was subjected to N-acetylation and global debenzylation according
to the general procedure described above to give tetrasaccharide
for C33H52N3O32S3, 366.0571; found, 366.0582 [M - 3H]3-
.
5-Aminopentyl [(ꢀ-D-Glucopyranosyluronate)-(1f4)-O-(2-ac-
etamido-2-deoxy-6-O-sulfonato-r-D-glucopyranoside)-(1f4)-O-(2-
O-sulfonate-r-L-idopyranosyluronate)]-(1f4)-2-deoxy-2-acetamido-
6-O-sulfonate-r-D-glucopyranoside Pentasodium Salt (85). Tetrasac-
charide 75 (159 mg, 0.079 mmol) was subjected to the sequence
of deprotection steps including delevulinoylation, O-sulfation, Fmoc
cleavage, saponification, deacetylation, and azide reduction to obtain
the diamino tetrasaccharide. One portion of the diamino tetrasac-
charide was subjected to N-acetylation and global debenzylation
according to the general procedure described above to give
tetrasaccharide 85 (9.3 mg). [R]D25: +26 (c ) 0.5, H2O). 1H NMR
(800 MHz, D2O): δ 5.14 (bs, 1H, H1B), 5.10 (d, 1H, J ) 3.7 Hz,
H1C), 4.83 (d, 1H, J ) 3.7 Hz, H1A), 4.78 (d, 1H, J ) 2.3 Hz,
H5B), 4.54 (d, 1H, J ) 7.9 Hz, H1D), 4.41 (dd, 1H, J ) 2.7 Hz, J
) 11.0 Hz, H6aC), 4.36 (d, 1H, J ) 1.8 Hz, J ) 11.2 Hz, H6aA),
4.26 (bd, J ) 3.2 Hz, H2B), 4.25-4.21 (m, 2H, H6bA, H6bC), 4.18
(bt, 1H, H3B), 4.09-4.05 (m, 1H, H5C), 4.05-4.01 (m, 2H, H4B,
H5A), 3.99 (dd, 1H, J ) 3.7 Hz, J ) 10.2 Hz, H2C), 3.92 (dd, 1H,
J ) 3.7 Hz, J ) 10.6 Hz, H2A), 3.82 (dd, 1H, J ) 8.8 Hz, J )
10.4 Hz, H3A), 3.76-3.72 (m, 3H, H3C, H4C, H5D), 3.71-3.68 (m,
2H, H4A, OCHH Linker), 3.53-3.46 (m, 3H, H3D, H4D, OCHH
Linker), 3.32 (dd, 1H, J ) 9.2 Hz, H2D), 2.98 (t, 2H, J ) 7.6 Hz,
CH2NH2), 2.03 and 2.00 (2s, 3H each, 2 × CH3, NHAc), 1.70-1.58
(m, 4H, 2 × CH2 Linker), 1.48-1.42 (m, 2H, CH2 Linker). ESI-
MS: m/z calcd for C33H53N3O32S3, 549.5893; found, 549.5879 [M
- H]2-; calcd for C33H52N3O32S3, 366.0571; found, 366.0559 [M
1
82 (4.32 mg). [R]D23: +70 (c ) 0.35, H2O). H NMR (800 MHz,
D2O): δ 5.14 (d, 1H, J ) 3.7 Hz, H1C), 4.94 (d, 1H, J ) 3.2 Hz,
H1B), 4.85 (d, 1H, J ) 3.7 Hz, H1A), 4.83 (d, 1H, J ) 4.6 Hz,
H1D), 4.68 (d, 1H, J ) 2.7 Hz, H5B), 4.55 (d, 1H, J ) 3.9 Hz,
H5D), 4.34-4.31 (m, 2H, H6aC, H6aA),4.23 (dd, 1H, J ) 6.6 Hz,
J ) 11.2 Hz, H6bA), 4.2 (dd, 1H, J ) 1.7 Hz, J ) 11 Hz, H6bC),
4.05 (t, 1H, J ) 3.2 Hz, H4B), 4.04-4.01 (m, 2H, H5C, H5A), 3.96
(dd, 1H, J ) 3.7 Hz, J ) 10.2 Hz, H2C), 3.91 (dd, 1H, J ) 3.7 Hz,
J ) 10.7 Hz, H2A), 3.87(dd, 1H, J ) 3.7 Hz, J ) 5.9 Hz, H3B),
3.85 (dd, 1H, J ) 4.1 Hz, J ) 6.1 Hz, H4D), 3.80 (t, 1H, J ) 9.8
Hz, H3A), 3.74-3.64 (m, 7H, H4C, H3C, H3D, H2B, H4A, H2B,
OCHH Linker), 3.53 (dd, 1H, J ) 4.6 Hz, J ) 7.3 Hz), 3.50-3.47
(m, 1H, OCHH Linker), 2.97 (t, 2H, J ) 7.6 Hz, CH2N Linker),
2.00, 1.98 (2s, 3H each, 2 × CH3, NHAc), 1.69-1.56 (m, 4H, 2 ×
CH2 Linker), 1.45-1.41 (m, 2H, CH2 Linker). Calcd for
C33H54N3O29S2, 1020.2290; found, 1020.2279 [M - H]1-; calcd
for C33H53N3O29S2, 509.6109; found, 509.6113 [M - 2H]2-; calcd
for C33H52N3O29S2, 339.4048; found, 339.4046 [M - 3H]3-
.
5-Aminopentyl [(r-L-Idopyranosyluronate)-(1f4)-(2-deoxy-2-
N-sulfoamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(r-L-
idopyranosyluronate-(1f4)-O-2-deoxy-2-N-sulfoamino-6-O-sulfonate-
r-D-glucopyranoside Hexasodium Salt (83). The second portion of
the diamino tetrasaccharide obtained above was subjected to
N-sulfation and global debenzylation according to the general
procedure described above to give tetrasaccharide 83 (6.5 mg).
- 3H]3-
.
5-Aminopentyl [(ꢀ-D-Glucopyranosyluronate)-(1f4)-O-(2-deoxy-
2-sulfamino-6-O-sulfonate-r-D-glucopyranoside)-(1f4)-O-(2-O-sul-
fonate-r-L-idopyranosyluronate)-(1f4)-2-deoxy-2-sulfoamino-6-O-
sulfonate-r-D-glucopyranoside Heptasodium Salt (86). A second
portion of the diamino tetrasaccharide obtained above was subjected
to N-sulfation and global debenzylation according to the general
procedure described above to give tetrasaccharide 86 (5.0 mg).
[R]D25: +79 (c ) 1.3, H2O). 1H NMR (800 MHz, D2O): δ 5.40 (d,
1H, J ) 3.3 Hz, H1C), 5.23 (d, 1H, J ) 2.7 Hz, H1B), 5.10 (d, 1H,
J ) 3.3 Hz, H1A), 4.70 (d, 1H, J ) 2.7 Hz, H1B), 4.57 (d, 1H, J
) 7.9 Hz, H1D), 4.45 (bd, 1H, J ) 11.0 Hz, H6aC), 4.35 (bd, 1H,
J ) 11.0 Hz, H6aA), 4.30-4.25 (m, 2H, H2B, H6bA), 4.20 (m, 1H,
H6bC), 4.16 (dd, 1H, J ) 4.0 Hz, J ) 5.7 Hz, H3B), 4.09-4.06
(m, 2H, H4B and H5C), 4.00 (m, 1H, H5A), 3.77-3.68 (m, 5H,
H3A, H4A, H4C, H5D, OCHH Linker), 3.66 (dd, 1H, J ) 9.1 Hz, J
) 9.5 Hz, H3C), 3.56-3.53 (m, 1H, OCHH Linker), 3.51 (t, 1H, J
1
[R]D23: +24.4 (c ) 0.3, H2O). H NMR (800 MHz, D2O): δ 5.33
(d, 1H, J ) 3.7 Hz, H1C), 5.11 (d, 1H, J ) 3.7 Hz, H1A), 4.97 (d,
1H, J ) 2.0 Hz, H1B), 4.85 (d, 1H, J ) 4.9 Hz, H1D), 4.71 (d, 1H,
J ) 2.2 Hz, H5B), 4.57 (d, 1H, J ) 3.9 Hz, H5D), 4.35 (dd, 1H, J
) 2.2 Hz, J ) 11.2 Hz, H6aC), 4.29 (dd, 1H, J ) 1.7 and 11 Hz,
H6aA), 4.23 (dd, 1H, J ) 5.4 Hz, J ) 11.2 Hz, H6bA), 4.17 (dd,
1H, J ) 1.7 Hz, J ) 11 Hz, H6bC), 4.08 (t, 1H, J ) 4.0 Hz, H3B),
4.04 (t, 1H, J ) 2.6 Hz, H4B), 4.00-3.98 (bm, 2H, H5A, H5C),
3.85 (dd, 1H, J ) 3.9 Hz, J ) 5.9 Hz, H4D), 3.74-3.61 (m, 7H,
OCHH Linker, H2B, H4C, H3D, H4A, H3A, H3C), 3.55-3.52 (m,
2H, OCHH Linker, H2D), 3.25 (dd,1H, J ) 3.9 Hz, J ) 10.3 Hz,
H2A), 3.23 (dd, 1H, J ) 3.7 Hz, J ) 10.8 Hz, H2C), 2.98 (t, 2H,
J ) 7.5 Hz, CH2N Linker), 1.69-1.62 (m, 4H, 2 × CH2 Linker),
1.49-1.44 (m, 2H, CH2 Linker). ESI-MS: m/z calcd for
C29H50N3O33S4, 1096.1215; found, 1096.1251 [M - H]1-; calcd
9
17404 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Synthesis of Heparan Sulfate Oligosaccharides
A R T I C L E S
) 9.2 Hz, H3D), 3.47 (t, 1H, J ) 9.2 Hz, H4D), 3.32 (dd, 1H, J )
8.4 Hz, J ) 9.2 Hz, H2D), 3.27-3.23 (m, 2H, H2A, H2C), 3.00 (t,
2H, J ) 7.4 Hz, CH2NH2), 1.72-1.55 (m, 4H, 2 × CH2 Linker),
1.50-1.40 (m, 2H, CH2 Linker). ESI-MS: m/z calcd for
C29H49N3O36S5, 587.5355; found, 587.5347 [M - H]2-; calcd for
nologies, U.K.), and data were analyzed by plotting log concen-
tration of inhibitor against percent inhibition and using a logistic
sigmoidal curve-fitting function using OriginPro 8 software (Orig-
inLab Corp., MA).
C29H48N3O36S5, 391.3546; found, 391.3535 [M - 3H]3-
.
Acknowledgment. This research was supported by the Center
for Research Resource of the National Institutes of Health (grant
no. 2 P41 RR005351 (G.-J.B.), Human Frontier Science Program
(grant RGP0062, G.-J.B. and J.E.T.), Medical Research Council
UK (Senior Research Fellowship to J.E.T.), and the Biotechnology
and Biological Sciences Research Council (grant to J.E.T). We
thank Elizabeth Edwards and Alexandra Holme for technical
assistance with the BACE inhibition assays.
BACE Inhibition Assays. The ability of the compounds to inhibit
BACE-1 cleavage of APP was assessed using a fluorescent
resonance energy transfer (FRET) peptide cleavage assay employing
the FRET peptide HiLyteFluor488-Glu-Val-Asn-Leu-Asp-Ala-Glu-
Phe-Lys(QXL520)-OH (Anaspec Inc., San Jose, CA) containing
the Swedish amino acid variant. Assays were performed in triplicate
in 96-well black plates (Greiner Bio-One Ltd.) in a total volume
of 100 µL of 20 mM sodium acetate, 0.1% Triton-X-100, pH 4.5
using 114 pmol of FRET peptide/well, and 0.1 µg of recombinant
human BACE-1 (R & D Systems; specific activity > 3.5 pmol/
min/µg). Inhibitors were added at 0.01-50 µg/mL and mixed with
enzyme prior to addition of substrate. Appropriate controls for
enzyme activity and background fluorescence were employed, and
plates were incubated (2 h, 25 °C) with the reaction stopped by
addition of 100 µL of 2.5 M sodium acetate. Fluorescence 480 ex/
520 em was measured on a Polarstar plate reader (BMG LabTech-
Supporting Information Available: Copies of NMR spectra
of synthetic intermediates and final products, and synthetic
procedures and NMR assignments for intermediates. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA907358K
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17405