Preactivation-based, one-pot combinatorial synthesis of heparin-like hexasaccharides for the analysis of heparin-protein interactions

Article abstract of DOI:10.1002/chem.201000987

Heparin (HP) and heparan sulfate (HS) play important roles in many biological events. Increasing evidence has shown that the biological functions of HP and HS can be critically dependent upon their precise structures, including the position of the iduronic acids and sulfation patterns. However, unraveling the HP code has been extremely challenging due to the enormous structural variations. To overcome this hurdle, we investigated the possibility of assembling a library of HP/HS oligosaccharides using a preactivation-based, one-pot glycosylation method. A major challenge in HP/HS oligosaccharide synthesis is stereoselectivity in the formation of the cis-1,4-linkages between glucosamine and the uronic acid. Through screening, suitable protective groups were identified on the matching glycosyl donor and acceptor, leading to stereospecifie formation of both the cis-1,4- and trans-1,4-linkages present in HP. The protective group chemistry designed was also very flexible. From two advanced thioglycosyl disaccharide intermediates, all of the required disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified building-block preparation. Furthermore, the reactivity-independent nature of the preactivation-based, one-pot approach enabled us to mix the building blocks. This allowed rapid assembly of twelve HP/HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. The speed and the high yields achieved in glycoassembly without the need to use a large excess of building blocks highlighted the advantages of our approach, which can be of general use to facilitate the study of HP/HS biology. As a proof of principle, this panel of hexasaccharides was used to probe the effect of backbone sequence on binding with the fibroblast growth factor-2 (FGF-2). A trisaccharide sequence of 2-O-sulfated iduronic acid flanked by N-sulfated glucosamines was identified to be the minimum binding motif and N-sulfation was found to be critical. This provides useful information for further development of more potent compounds towards FGF-2 binding, which can have potential applications in wound healing and anticancer therapy.

Full text of DOI:10.1002/chem.201000987

FULL PAPER
DOI: 10.1002/chem.201000987
Preactivation-Based, One-Pot Combinatorial Synthesis of Heparin-like
Hexasaccharides for the Analysis of Heparin–Protein Interactions
Zhen Wang,[a] Yongmei Xu,[b] Bo Yang,[a] Gopinath Tiruchinapally,[a] Bin Sun,[a]
Renpeng Liu,[b] Steven Dulaney,[a] Jian Liu,[b] and Xuefei Huang*[a]
Abstract: Heparin (HP) and heparan
sulfate (HS) play important roles in
many biological events. Increasing evi-
dence has shown that the biological
functions of HP and HS can be critical-
ly dependent upon their precise struc-
tures, including the position of the idur-
onic acids and sulfation patterns. How-
ever, unraveling the HP code has been
extremely challenging due to the enor-
mous structural variations. To over-
come this hurdle, we investigated the
possibility of assembling a library of
HP/HS oligosaccharides using a preac-
tivation-based, one-pot glycosylation
method. A major challenge in HP/HS
oligosaccharide synthesis is stereoselec-
tivity in the formation of the cis-1,4-
linkages between glucosamine and the
uronic acid. Through screening, suita-
ble protective groups were identified
on the matching glycosyl donor and ac-
ceptor, leading to stereospecific forma-
tion of both the cis-1,4- and trans-1,4-
linkages present in HP. The protective
group chemistry designed was also very
flexible. From two advanced thioglyco-
syl disaccharide intermediates, all of
the required disaccharide modules for
library preparation could be generated
in a divergent manner, which greatly
simplified building-block preparation.
Furthermore, the reactivity-independ-
ent nature of the preactivation-based,
one-pot approach enabled us to mix
the building blocks. This allowed rapid
assembly of twelve HP/HS hexasac-
charides with systematically varied and
precisely controlled backbone struc-
tures in a combinatorial fashion. The
speed and the high yields achieved in
glycoassembly without the need to use
a large excess of building blocks high-
lighted the advantages of our approach,
which can be of general use to facili-
tate the study of HP/HS biology. As a
proof of principle, this panel of hexa-
saccharides was used to probe the
effect of backbone sequence on bind-
ing with the fibroblast growth factor-2
(FGF-2). A trisaccharide sequence of
2-O-sulfated iduronic acid flanked by
N-sulfated glucosamines was identified
to be the minimum binding motif and
N-sulfation was found to be critical.
This provides useful information for
further development of more potent
compounds towards FGF-2 binding,
which can have potential applications
in wound healing and anticancer thera-
py.
Keywords: carbohydrates · combi-
natorial chemistry · growth factors ·
heparin · oligosaccharides
Introduction
linear polyanionic polysaccharides.[1] The backbones of HP
and HS are composed of disaccharide subunits with d-glu-
cosamine (GlcN) and uronic acid (either l-iduronic (IdoA)
or d-glucuronic acid (GlcA)) joined by alternating a-1,4 and
b-1,4 linkages. The variation in uronic acid structures, the
modification of the amino group in GlcN (N-acetylation and
sulfation) and differential sulfations of hydroxyl groups (2-O
of the uronic acid, 3-O and 6-O of the GlcN) lead to tre-
mendous structural diversity in naturally existing HP/HS.
Through interactions with a large number of polypeptides
and proteins,[1–3] HP/HS play crucial roles in numerous phys-
iological processes such as viral infection, blood coagulation,
inflammatory response, cell adhesion, cell growth regulation,
and tumor metastasis.[1,4–9] The binding between HP/HS and
their receptors can be critically dependent on the saccharide
structures, as exemplified by the high-affinity pentasacchar-
Heparin (HP) and heparan sulfate (HS), a member of the
glycosaminoglycan (GAG) family, are structurally related
[a] Z. Wang, B. Yang, G. Tiruchinapally, B. Sun, S. Dulaney, X. Huang
Department of Chemistry, Michigan State University
East Lansing, MI 48824 (USA)
Fax : (+1)517-353-1793
E-mail: xuefei@chemistry.msu.edu
[b] Y. Xu, R. Liu, J. Liu
Division of Medicinal Chemistry and Natural Products
UNC Eshelman School of Pharmacy, University of North Carolina
Chapel Hill, NC 27599 (USA)
Supporting information (detailed experimental procedures and select-
ed H, 13C and 2D NMR spectra) for this article is available on the
1
WWW under http://dx.doi.org/10.1002/chem.201000987.
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8365

ide sequence for antithrombin III (ATIII) interaction,[10,11]
with the removal of a single 3-O-sulfate in the sequence re-
ducing its activity by about 20000-fold. This knowledge led
to the development of Arixtra, a fully synthetic HP/HS pen-
tasaccharide, which is approved for the treatment of deep
vein thrombosis.[10,12] However, despite this success, in many
cases, it has been difficult to unravel the biological activities
encoded in the HP/HS structures, since it is extremely chal-
lenging to obtain homogeneous HP/HS oligosaccharides
from natural sources. Furthermore, pharmaceutical HP is
isolated from pig and bovine organs, which has the potential
risks of pathogen contamination and adulteration.[13,14] Syn-
thesis, therefore, provides a powerful means to access these
complex molecules for the establishment of detailed struc-
ture–activity relationships and analysis of HP/HS–protein in-
teractions.[10,15–17]
Nature synthesizes HP and HS by modifying a homopoly-
mer of heparosan GlcNAc-a-1,4-GlcA through the actions
of many enzymes, including N-deacetylase/N-sulfotransfer-
ase, C5-epimerase, and O-sulfotransferases.[9,18] Most of
these reactions are incomplete, thus explaining the structural
heterogeneity of native HP/HS. Attempts to harness the
power of enzymes for HP/HS synthesis have been report-
ed.[19–24] However, the substrate specificities of most of these
enzymes are not yet well characterized, which hinders the
application of enzymatic approaches to the assembly of a di-
verse array of HP/HS oligosaccharides.
As an alternative to using enzymes, chemical syntheses of
HP/HS oligosaccharides have been actively pursued,[15–17]
which can provide compounds with any sequences and sulfa-
tion patterns as well as unnatural analogues. However, there
are several challenges associated with chemical synthesis:
1) with the rich functionalities in the HP/HS oligosacchar-
ides, selection of appropriate protective groups is crucial for
backbone extension and oligosaccharide functionalization.
Even groups distant from the reactive sites can have a pro-
found effect on the outcome of glycosylation reactions.[25]
2) Stereochemical control is crucial for HP/HS oligosacchar-
ide synthesis, especially in the formation of the cis-1,4-link-
age between GlcN and uronic acid. Although, in general,
the cis linkage is preferred due to the anomeric effect, the
stereoselectivity can vary drastically with the building-block
structures as well as reaction conditions[26–29] and the separa-
tion of anomers can be tedious and at times impossible.[28]
Thus, it is highly desirable that conditions can be established
for stereospecific formation of the cis-1,4-linkage.
The aforementioned challenges notwithstanding, the
groups of van Boeckel, Petitou, and Sinaꢁ accomplished the
first total synthesis of the ATIII binding HP pentasacchar-
ide.[30–32] This landmark achievement required about 60 syn-
thetic steps, which served to highlight the power of synthetic
carbohydrate chemistry for saccharide-based drug develop-
ment.[10] To minimize the number of steps required for pro-
tective group manipulation and aglycon leaving group ad-
justment on intermediate oligosaccharides, strategies such as
anomeric-reactivity-based, one-pot synthesis[33] and selective
activation[34] have been applied for the synthesis of the
ATIII-binding pentasaccharide. Several other targeted syn-
theses of HP/HS pentasaccharides or longer have also been
accomplished.[29,35–46] Despite these significant progresses in
the field, the target-oriented approaches required a new
total synthesis for each HP/HS structure. This is not efficient
for structure–activity relationship (SAR) studies, in which a
number of HP/HS structures would be required to decipher
the HP code.
For SAR studies, a diversity-oriented combinatorial ap-
proach needs to be developed to access a wide range of HP/
HS structures. This can be accomplished by using modular
building blocks, in which a set of properly protected building
blocks can be converted to multiple glycosyl donors and ac-
ceptors.[29,34,43,47,48] Recently, this strategy has been applied
to the preparation of HP/HS tetrasaccharides, which were
subsequently used to elucidate structural requirements for
inhibition of b-secretase involved in Alzheimerꢂs disease de-
velopment.[35] To apply the modular strategy to higher oligo-
saccharides, the availability of robust glycosylation chemis-
try is crucial, which must give high yields in reactions of a
variety of building blocks without the need for time-consum-
ing individual optimization.
Herein, we report the establishment of a modular assem-
bly strategy for a panel of HP/HS hexasaccharides using the
preactivation-based, one-pot method.[49–53] This method
granted much more freedom in protective group selection,
enabling us to achieve high-yielding stereospecific glycosyla-
tions. Furthermore, the integration of several glycosylation
reactions into a single synthetic operation (one-pot) signifi-
cantly expedited the glyco-assembly processes, allowing the
rapid construction of the hexasaccharide library in a combi-
natorial fashion. The availability of these HP/HS hexasac-
charides with well-defined sulfation patterns and systemati-
cally varied backbone structures facilitated the SAR studies
towards binding with the fibroblast growth factor-2 (FGF-2),
an important protein involved in angiogenesis, cell prolifera-
tion, and embryonic development.[54,55]
Results and Discussion
Building-block design: There are two general routes to syn-
thesize HP/HS oligosaccharides through uronic acids or the
corresponding hexose building blocks. Although uronic acid
donors have been used directly in GAG synthesis,[34,56,57] the
presence of electron-withdrawing carboxylate moieties may
lower glycosylation yields.[58] Furthermore, the possibility of
epimerization limits the synthetic operations that can be
performed on these compounds. Therefore, we decided to
explore glucoside and idoside as building blocks, which
would be converted to the corresponding uronic acids after
oligosaccharide backbone formation.
Instead of using the popular trichloroacetimidate
donors,[35,38,42] we chose thioglycosyl building blocks because
they give superior stability in storage and yet are readily ac-
tivated with thiophilic promoters.[59] We designed glucoside
1 and idoside 2, containing benzyl (Bn), p-methoxybenzyl
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Analysis of Heparin–Protein Interactions
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Table 1. Evaluation of glycosyl donors.[a]
(PMB), and benzoyl (Bz) pro-
tecting groups, as suitable build-
ing blocks. The 2-O-Bz group
can direct the formation of 1,2-
trans-glycosyl linkage through
neighboring group participation
and be selectively removed to
expose the hydroxyl group for
future sulfation. The PMB
group on the 6-O position can
be transformed for late-stage
oxidation-state adjustments. For
the GlcN donors, we examined
the azido moiety as a nonparti-
cipatory group (donors 3–6).
Previously, Kerns and co-work-
ers have discovered that the use
of an oxazolidinone moiety to
simultaneously protect the C2-
nitrogen and C3-OH of a GlcN
donor led to the exclusive for-
mation of a-glycoside.[60] How-
ever, later studies showed that
the oxazolidinone could cause
side reactions due to its nucleo-
philicity, thus limiting its wide
usage in HP/HS oligosaccharide
preparation.[26,61] Instead, the
azido group can not only facili-
tate the formation of a-linkag-
es,[62] but also be selectively re-
duced to amines in the presence
of other protective groups for
subsequent N-sulfation or N-
Entry
Donor
Acceptor
Disaccharide
Yield [%]
1
2
3
4
5
6
7
8
3
5
4
3
5
4
13
16
2
2
2
1
1
1
14
17
7
8
9
10
11
12
15
18
84 (a only)
84 (a only)
79 (a only)
73 (a/b=2.5:1)
89 (a only)
75 (a only)
53
64
[a] TTBP=2,4,6-tri-tertbutyl pyrimidine, pTolSCl=p-toluenesulfenyl chloride.
acetylation. However, the stereochemical outcome of using
donors with the azido group is dependent upon donor and
acceptor structures as well as reaction conditions.[26–28] Thus
suitable protective group chemistry needs to be established
for the construction of the cis-1,4-linkages prior to oligosac-
charide synthesis.
disaccharide isolated (Table 1, entry 1). The newly formed
a-1,4-linkage was confirmed by NMR spectroscopy analysis
ACTHNUTRGNEUNG
(1J(C1’,H1’)=172.1 Hz).[65] Examination of GlcN donors
with different protective groups (4 and 5) demonstrated that
this chemistry was robust because they all gave complete cis
selectivities (Table 1, entries 2 and 3).
It has been reported that acceptors with the gluco-config-
uration tend to produce stereoisomeric mixtures in reactions
with 2-azido GlcN donors.[29] Indeed, the reaction of the tri-
benzylated GlcN 3 with glucoside acceptor 1 led to the for-
mation of both a and b anomers of disaccharide 10 (a/b
ꢀ2.5:1) (Table 1, entry 4). The differential stereochemical
outcome of using donor 3 to glycosylate gluco-configured
acceptor 1 versus the idoside acceptor 2 could be attributed
to double stereodifferentiation.[66] Seeberger and co-workers
developed a clever approach that by locking the glucoside
Evaluation of building blocks for disaccharide formation:
The formation of a cis-1,4-linkage using GlcN donors was
evaluated first (Table 1). The donor 3 was preactivated[53,63]
in the absence of any acceptors with the powerful promoter
pTolSOTf (1 equiv), which was formed in situ by reaction of
pTolSCl with AgOTf at À788C. Upon the completion of
donor activation, as determined by TLC analysis, the glyco-
syl acceptor 2 was added together with a sterically hindered
base, TTBP,[64] leading to the formation of disaccharide
product 7 in an excellent 84% yield with no b-linked trans-
1
acceptor into the C4 conformation, thus converting the 4-
OH into the idose-like axial position, high a selectivity was
obtained.[29] However, several additional synthetic steps
were necessary to modify the disaccharide products with
suitable protective groups and aglycon leaving groups for
further chain elongation. Instead of changing the glycosyl
acceptor, we explored the effect of donor protective groups
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X. Huang et al.
on stereoselectivities. Interestingly, GlcN donor 5 led to the
exclusive formation of the cis-linked disaccharide (1J-
ACHTUNGTRENNUNG(C1’,H1’)=173.9 Hz) with no corresponding trans-glycoside
isolated (Table 1, entry 5). The levulinoyl acid (Lev)-protect-
ed donor 6 also gave exclusively a anomer, but the glycosy-
lation yield was lower (data not shown). With the 6-OAc
moiety on donor 5, it is possible that the acetate might facil-
itate the formation of the cis-glycosidic linkage through the
remote neighboring group participation by the ester carbon-
yl moiety.[67,68] To test this possibility, the 6-OAc was substi-
tuted with 6-OBn (donor 4). The reaction of donor 4 with
acceptor 1 maintained the exclusive cis selectivity with good
yield (Table 1, entry 6), thus demonstrating 6-OAc was not
responsible for the observed high stereoselectivity. Based on
these observations, the tert-butyldimethyl silyl (TBS) group
is not only useful as a temporary protective group, masking
the 4-OH of GlcN for backbone elongation, but also plays
an important role for facilitating the stereospecific forma-
tion of the cis-1,4 glycosidic bond. Furthermore, the possibil-
ity of having either Bn or Ac on the 6-O of GlcN without
affecting stereoselectivities bestows great flexibility for in-
stalling 6-O-sulfation in the future.
Next, we examined the formation of the trans-1,4-linkage
using the hexose donors. The glycosylation of glucoside
donor 13 with acceptor 14 led to the formation of disacchar-
ide product 15 in 53% yield with some acceptor (40%) re-
covered (Table 1, entry 7). In a similar manner, idose donor
16 was preactivated and reacted with disaccharide 17, pro-
ducing trisaccharide 18 in 64% yield (Table 1, entry 8).
Donor 16 was obtained as a side product during preparation
of idoside 2, thus making full use of all available building
blocks.
Scheme 1. Synthesis of disaccharide acceptor 19 and evaluation of tetra-
saccharide formation.
PMB moiety.[52] The differential outcome in glycosylations
with monosaccharide versus disaccharide building blocks de-
spite similar protective groups used is probably due to the
lower reactivities of the disaccharides resulting from the
presence of additional glycosyl units.[70] The reduction in gly-
cosylation rate could favor the competing side reaction from
remote participation of the OPMB group.
To overcome this difficulty, the 6-OPMB group in disac-
charide 11 was replaced with OLev (donor 21), from which
two disaccharide acceptors 22 and 23 were generated in high
yields (Scheme 2a, b). Gratifyingly, glycosylation of 22 by 21
proceeded smoothly to afford the desired tetrasaccharide 24
Stepwise and one-pot synthesis of HP/HS hexasaccharides:
With the successful establishment of stereospecificity in dis-
accharide formation, we moved on to oligosaccharide as-
sembly. In the absence of neighboring group participation,
the size of the glycosylating agent and the nature of the nu-
cleophile can have unpredictable effects on the stereo-selec-
tivity.[28,50,69] For example, although a 2-azido GlcN donor re-
acted with an IdoA monosaccharide acceptor to form a cis-
1,4-linked HP trisaccharide exclusively, reaction between a
tetrasaccharide donor and disaccharide acceptor with similar
protective groups led to an inseparable anomeric mixture of
hexasaccharides.[28] Therefore, to avoid this potential compli-
cation in stereochemistry, for oligosaccharide chain exten-
sions, we chose disaccharide donors (e.g., 9 and 11) with the
idose or glucose at the reducing end and participating acyl
groups at the 2-O-positions. The TBS group in 11 was re-
moved to generate acceptor disaccharide 19 (Scheme 1a).
Interestingly, although donor 13 successfully reacted with ac-
ceptor 14, the glycosylation of disaccharide 11 by 19 failed
to yield the desired tetrasaccharide. Instead, the major side
product isolated was the anhydro compound 20 (40–50%
yield) (Scheme 1b). This side product is presumably formed
through the participation of electron-rich 6-OPMB to stabi-
lize the oxa-carbenium ion followed by cleavage of the
Scheme 2. Synthesis of disaccharide building blocks 21–23 and hexasac-
charide 25. Reagents and conditions: a) 2,3-dichloro-5,6-dicyano p-benzo-
quinone (DDQ), CH2Cl2/H2O, RT, 3 h; LevOH, N’-(3-diemthylamino-
propyl)-N-ethylcarbodiimide (EDC), 4-dimethylaminopyridine (DMAP),
CH2Cl2, RT, 2 h; b) HF in pyridine, pyridine, RT, overnight; c) pTolSCl/
AgOTf, CH2Cl2/Et2O, EtOH, À78 to À108C; d) pTolSCl/AgOTf, CH2Cl2/
Et2O, then 22, À78 to À108C; e) pTolSCl/AgOTf, 23, CH2Cl2/Et2O, À78
to À108C.
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Analysis of Heparin–Protein Interactions
FULL PAPER
in 64% isolated yield with exclusive
b
selectivity
timization to the one-pot assembly of hexasaccharide 26 by
sequential glycosylations of three idose-containing disac-
charides 27, 17, and 28 in 50% yield (Scheme 3b). The pres-
(Scheme 2c). Subsequent reaction of 24 with disaccharide
23 produced hexasaccharide 25 in 81% yield. The stereo-
chemistry of 25 was confirmed by NMR spectroscopy[65] and
ence of six a-linkages (1J
ACTHNUTRGNE(UNG C1,H1)=172.1 (ꢃ3), 171.6,
MS analysis with three a-glycosyl linkages (1J
G
171.1 Hz (ꢃ2)) unequivocally confirmed the stereochemistry
of compound 26. The TBS moieties at the nonreducing end
of 25 and 26 can be selectively removed to generate hexa-
saccharide acceptors for future synthesis of longer oligosac-
charides.
174.9, 173.9, 173.9 Hz) and three b-linkages (1J
ACHTUNGTRENNUNG
161.9, 161.9, 159.6 Hz).
Encouraged by the results of stepwise glycosylations, we
performed the one-pot assembly of 25 (Scheme 3a).[59] Pre-
activation of the disaccharide donor 21 (1 equiv) at À788C
with pTolSCl/AgOTf was followed by the addition of the
The speed and high overall yield using only close to stoi-
chiometric amounts of building blocks in the assembly of
hexasaccharides 25 and 26 highlighted the advantages of our
approach. In addition, the building blocks used have the
same protective group patterns, thus possessing similar
anomeric reactivities.[70,71] For the popular reactivity-based
armed–disarmed strategy, time-consuming protective group
manipulations of these building blocks would be required to
render the glycosyl donor much more reactive than the ac-
ceptor for selective donor activation.[70–72] As the preactiva-
tion-based protocol can be performed independent of the
anomeric reactivities, it allowed us to use the building
blocks directly without the need to fine-tune their anomeric
reactivities, thus simplifying building-block preparation. This
will be particularly advantageous in oligosaccharide library
preparation, in which a large number of structures need to
be prepared.
One-pot modular synthesis of an HP-like hexasaccharide li-
brary for studying HP/HS FGF-2 interactions: The fibro-
blast growth factors (FGFs) are involved in many important
developmental and physiological processes.[54,55] FGFs bind
with endogenous HP/HS, which is essential for high-affinity
interactions with the membrane-bound FGF receptors sub-
Scheme 3. One-pot synthesis of HP-like hexasaccharides 25 and 26.
first disaccharide acceptor 22
(0.9 equiv). The reaction tem-
perature was raised to À108C
to expedite glycosylation and
the acceptor was 22 completely
consumed in 2 h based on TLC
analysis. The reaction tempera-
ture was cooled back to À788C,
followed by addition of the
second disaccharide acceptor 23
(0.8 equiv) and pTolSCl/AgOTf.
The fully protected hexasac-
charide 25 was obtained in
71% yield in one pot in 5 h and
its structure was identical with
that from the stepwise reac-
tions. The higher yield from the
one-pot reaction compared with
the stepwise synthesis was pre-
sumably due to less product
loss during workup and purifi-
cation. The one-pot reaction
Scheme 4. Divergent functionalization of disaccharide building blocks from common intermediates. Reagents
and conditions: a) HF in pyridine, pyridine, RT, overnight; b) BnBr, Ag2O, molecular sieves, RT, 5 h;
condition were general, which
were extended without any op- c) pTolSCl/AgOTf, CH2Cl2/Et2O, N-(benzyl)benzyloxycarbonyl 3-amino propanol, À78 to À108C.
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X. Huang et al.
sequently resulting in signal transduction for cell prolifera-
tion and growth.[73–76] Therefore, the identification of strong-
binding HP/HS oligosaccharides can lead to the develop-
ment of agents mediating the biological functions of FGFs,
which can have important therapeutic applications in areas
including wound healing, angiogenesis, and cancer thera-
py.[77,78]
dine to give the disaccharide acceptor 17 in 92% yield as
the disaccharide module for backbone elongation. The
newly liberated hydroxyl group in 17 was benzylated as pro-
moted by silver oxide leading to the capping disaccharide 29
in 94% yield. It should be emphasized that the mild basic
condition employed for benzylation did not affect the base-
sensitive Lev and Bz groups in the molecule. Glycosylation
of disaccharide 27 with N-(benzyl)benzyloxycarbonyl 3-
amino propanol followed by TBS removal gave the reducing
end module 30 in 89% yield with an exclusive trans selectiv-
ity due to neighboring group participation by the 2-OBz
moiety (Scheme 4a). In a similar manner, three glucose-con-
taining disaccharides 32, 33, and 34 were prepared diver-
gently from disaccharide 31 (Scheme 4b).
The basic fibroblast growth factor (FGF-2) is a member
of the FGF family. SAR studies on FGF-2 binding with HP/
HS have shown that the 3-O- and 6-O-sulfates on the GlcN
of HP/HS are not essential, whereas 2-O-sulfation of the
uronic acid is important.[24,38,79,80] The conversion of N-sulfa-
tion to acetamide significantly reduced the activity.[38] How-
ever, the activity lost by N-acylation could be partially re-
gained
by
O-sulfation.[80]
Whereas much study has been
devoted to examine the effects
of sulfation on FGF-2 binding,
the information regarding back-
bone sequence preference is
not very well established.[38,46]
The majority of studies per-
formed to date have used HP/
HS oligosaccharides mainly
containing of IdoA.[38,46,81,82]
Nevertheless, GlcA-containing
oligosaccharides have been
shown to bind and activate
FGF-2.[83] Therefore, a system-
atic investigation on the effects
of backbone structures can fur-
ther advance our understanding
of the FGF-2 activities.
Table 2. One-pot synthesis of HP/HS hexasaccharides.
Since HP/HS hexasaccharides
have been shown to be biologi-
cally active,[78] we decided to
focus on the preparation of a li-
brary of hexasaccharides. As 3-
O- and 6-O-sulfation of GlcN
are not essential for FGF-2
binding,[6,38,84] this library of 12
hexasaccharides contains sys-
tematically varied backbone se-
quences
and
2-O-sulfated
uronic acids (i.e., IdoA2S and
GlcA2S) to probe the effects of
the number and location of
IdoA as well as N-sulfation.
To acquire this library effi-
ciently, we developed a diver-
gent approach to access all of
the necessary modules from
two common thioglycosyl disac-
charides 27 and 31. Specifically,
the 4-OTBS group in disacchar-
ide 27 was selectively removed
by treatment with HF in pyri-
Entry
Donor
A1
A2
Product
Yield [%]
1
2
3
4
5
6
29
29
29
33
33
33
17
32
32
17
17
32
34
34
30
34
30
30
35
36
37
38
39
40
54
59
58
62
57
50
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Chem. Eur. J. 2010, 16, 8365 – 8375

Analysis of Heparin–Protein Interactions
FULL PAPER
With the disaccharide building blocks in hand, we ex-
plored the construction of the hexasaccharide library. Previ-
ously, Bonnaffꢄ and co-workers developed an interesting ap-
proach, in which three HP/HS tetrasaccharides have been
obtained by subjecting three glycosyl acceptors to simultane-
ous glycosylation in a mixture synthesis.[85] However, the
subsequent demixing required time-consuming HPLC sepa-
ration of the three differentially sulfated glycoside products.
Instead of relying on mixture synthesis, we adopted a paral-
lel reaction approach, in which a single desired product was
to be obtained from each reaction. Since the preactivation
method was independent of the anomeric reactivities, it al-
lowed us to mix the available building blocks rapidly to
create sequence diversity. For example, sequential glycosyla-
tions using three disaccharide modules 29, 17, and 34 follow-
ing the one-pot procedure gave hexasaccharide 35 with the
idose, idose, and glucose backbone structure in 54% yield
within just a few hours (Table 2, entry 1). Simple substitu-
tion of 17 by 32 in the one-pot synthesis generated hexasac-
charide 36 with the idose, glucose, and glucose backbone
structure (Table 2, entry 2). In a similar manner, hexasac-
charides 37–40 were produced in good yields with potential
N- and 2-O-sulfation sites and precisely positioned idose
moieties (Table 2, entries 3 to 6). Combined with hexasac-
charides 25 and 26, these compounds covered all of the pos-
sible hexasaccharide backbone
Scheme 5. Deprotection and selective N-derivatization led to two HP/HS
hexasaccharides (42, 43) divergently from 35. Reagents and conditions:
a) NH2NH2, acetic acid, pyridine, 08C, TEMPO, NaOCl, KBr, Bu4NBr,
NaHCO3, CH2Cl2/H2O, 0 8C to RT, NaClO2, then PhCHN2; b) LiOH,
H2O2, THF, RT, then NaOH, Me3P, NaOH, THF, H2O; c) SO3·Et3N,
DMF, 558C, SO3·Py, Et3N/pyridine, H2, Pd(OH)2; d) Ac2O, MeOH, Et3N,
RT; SO3·Et3N, DMF, 558C, H2, Pd(OH)2.
the four steps. Sulfation of the free hydroxyls and amines in
41 with subsequent catalytic hydrogenation over the Pearl-
man catalyst produced HP-like hexasaccharide 42. The ami-
nopropyl group at the reducing end should not interfere
with FGF-2 binding;[38] it can be useful for future develop-
ment of multivalent constructs[87] as well as microarrays.[38]
Alternatively, the structurally related hexasaccharide 43 was
obtained by selective amine acetylation of 41 followed by
O-sulfation and catalytic hydrogenation.[33,42] Hexasacchar-
sequences with GlcN at the
nonreducing end. Although the
glycosylation
outcome
can
often be highly dependent on
building-block structures, it is
remarkable that all of the one-
pot reactions tested here pro-
ceeded well without individual
reaction optimization. This
highlighted the robustness of
our protective group chemistry
and glycosylation technology,
which should be applicable for
future preparation of larger
HP/HS oligosaccharide library.
Deprotection of the hexasac-
charide 35 was performed by
first selectively removing the
three Lev protective groups
with hydrazine, followed by oxi-
dation employing 2,2,6,6-tetra-
methyl-1-piperidinyloxy
(TEMPO)/NaOCl and acid pro-
tection to facilitate product
characterization
(Scheme 5).[42,52,86] Saponifica-
tion of the hexasaccharide by
LiOH, H2O2, and KOH, fol-
lowed by Staudinger reduction
of the azido group provided
compound 41 in 75% yield for
Chem. Eur. J. 2010, 16, 8365 – 8375
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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8371

X. Huang et al.
ides 44 to 53 were prepared analogously, which contain sys-
tematically varied backbones, the 2-O-sulfates on the uronic
acid units and the amino groups are either sulfated or acety-
lated.
placement of GlcNS with N-acetylated GlcN (hexasacchar-
ides 43, 45, 48, 50, and 52) led to the complete loss of FGF-
2 binding, which was consistent with previous reports albeit
on different backbone structures.[38] 2-O-Sulfation of the
uronic acids could not compensate for the loss of N-sulfates
in these hexasaccharides. With the large groovelike binding
site on FGF-2 to accommodate the polysaccharide,[74] the
potency of the oligosaccharides can be further enhanced by
elongating their length.
The binding between the synthetic HP/HS hexasacchar-
ides and FGF-2 was examined through a competition
assay[24] with radiolabeled HS polysaccharides.[24] 35S-labeled
HS was harvested from Chinese hamster ovary cells that
were grown in the medium containing sodium [35S]sulfate.
The complex formed between [35S]HS and FGF-2 could be
captured on a nitrocellulose membrane. For the binding
assay, the synthetic hexasaccharides or unlabeled HS were
incubated with [35S]HS and FGF-2. The FGF-2 binding com-
pounds would compete with [35S]HS, resulting in its dissocia-
tion from FGF-2 and consequently lower radioactivity on
the membrane.
Conclusion
We have successfully developed a methodology to assemble
a library of HP/HS oligosaccharides. Matched donor and ac-
ceptor pairs were identified to allow stereospecific forma-
tion of the disaccharide building blocks, including those con-
taining the challenging cis-1,4-linkages. Preactivation-based,
one-pot sequential glycosylations using the disaccharides led
to the rapid construction of
The panel of hexasaccharides was assayed and found to
exhibit differential potencies in FGF-2 binding (Figure 1).
Although compound 51 contained the same number and lo-
hexasaccharides in high yields.
To test the generality of the
methodology, the synthesis of
an HP/HS hexasaccharide li-
brary was then explored. As
our reaction protocol does not
require the glycosyl donor to
have higher anomeric reactivi-
ties than the acceptor, building
blocks with similar protective
groups, hence similar anomeric
reactivities, can be employed
without the need for time-con-
suming protective group adjust-
ment to achieve exact anomeric
reactivities. Thus, mixing six
Figure 1. FGF2 (1 mg) was incubated with 10 (empty bar) or 40 mg (filled bar) unlabeled hexasaccharides 42–53
or HS (1.6 mg) in PBS buffer (200 mL) at room temperature for 30 min. Then, [35S]HS (4500 cpm) was added
to the mixture followed by incubation at 378C for 90 min. The data represent the average of four or more ex-
periments with the error bars showing standard deviations. The control was the sample without any hexasac-
charides or unlabeled HS. The percentage of residual [35S]HS binding was calculated by dividing the residual
35S counts on the membrane with sample incubation by control counts. For more details, see the Experimental
Section.
disaccharide modules divergent-
ly derived from two common
intermediate disaccharides al-
lowed combinatorial synthesis
of 12 hexasaccharides with sys-
tematically varied backbone,
precisely positioned IdoA units,
cation of sulfates, it was a much weaker binder than other 2-
O- and N-sulfated hexasaccharides (compounds 42, 44, 46,
47, 49, and 53). This suggests that FGF-2 binding is not a
sole reflection of charge density; rather the specific HS se-
quence is important. From the SAR analysis, it is clear that
the trisaccharide domain N-sulfated GlcN (GlcNS)-IdoA2S-
GlcNS represents a minimum FGF-2 binding motif, which
can form a kink in polysaccharide chains as induced by FGF
binding.[88] The absence of the GlcNS unit at the reducing
end of IdoA2S as in hexasaccharide 51 significantly reduced
the FGF-2 affinity. The IdoA2S structure is also important
because the corresponding GlcNS-GlcA2S-GlcNS was not
effective in binding. This is presumably due to the confor-
mational plurality of the IdoA ring.[89] Furthermore, the re-
and well-defined amine functionalization. The availability of
such a library allowed the establishment of important struc-
tural features for HS interactions with the growth factor
FGF-2. It was found that N-sulfation and trisaccharide motif
GlcNS-IdoA2S-GlcNS is important for FGF-2 binding and
GlcA2S cannot substitute IdoA2S effectively. This knowl-
edge can provide valuable leads for further development of
novel HP/HS-based therapeutics targeting FGF-2.
The successful synthesis of the hexasaccharide library and
the high synthetic efficiency achieved herein showcased the
power of our synthetic methodology. With further develop-
ment, this can provide a promising option for the rapid as-
sembly of HP/HS oligosaccharides. With their well-defined
structures, the synthetic oligosaccharides can greatly aid in
8372
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Chem. Eur. J. 2010, 16, 8365 – 8375

Analysis of Heparin–Protein Interactions
FULL PAPER
5 mL), followed by addition of Lev (1.4 equiv), EDC·HCl (1.6 equiv) and
DMAP (0.1 equiv). The mixture was stirred at room temperature over-
night and then was diluted with CH2Cl2 (100 mL). The organic phase was
washed with saturated NaHCO3 and then dried over Na2SO4. The solvent
was concentrated in vacuo and the compound was purified by silica gel
column chromatography.
the analysis of many HP–protein interactions and decipher-
ing the structural information encoded in HP sequences.
Experimental Section
General procedure for deprotection of TBS: The TBS-protected com-
pound was dissolved in pyridine (for 100 mg of compound, 1.5 mL). The
mixture was cooled to 48C, followed by addition of HF in pyridine
(0.75 mL, 65–70% in pyridine). The mixture was stirred at room temper-
ature overnight and then the residue was diluted in EtOAc (100 mL),
washed with saturated NaHCO3, and then the organic layer was dried
over Na2SO4. The solvent was concentrated in vacuo and the compound
was purified by silica gel column chromatography.
General procedure for preactivation-based single-step glycosylation: A
solution of donor (60 mmol) and freshly activated 4 ꢅ molecular sieves
(200 mg) in CH2Cl2 (2 mL) was stirred at room temperature for 30 min,
and cooled to À788C, which was followed by the addition of AgOTf
(47 mg, 180 mmol) dissolved in Et2O (1 mL) without touching the wall of
the flask. After 5 min, orange-colored pTolSCl (9.5 mL, 60 mmol) was
added to the solution through a microsyringe. Since the reaction temper-
ature was lower than the freezing point of pTolSCl, pTolSCl was added
directly into the reaction mixture to prevent it from freezing on the flask
wall. The characteristic yellow color of pTolSCl in the reaction solution
dissipated within a few seconds, indicating depletion of pTolSCl. After
the donor was completely consumed, according to TLC analysis (about
5 min at À788C), a solution of acceptor (54 mmol) in CH2Cl2 (0.2 mL)
was slowly added dropwise by using a syringe (one equivalent of TTBP
was added with acceptor if the donor or acceptor contained the PMB
protecting group). The reaction mixture was warmed to À208C under
stirring over 2 h. Then, the mixture was diluted with CH2Cl2 (20 mL) and
filtered over Celite. The Celite was further washed with CH2Cl2 until no
organic compounds were observed in the filtrate by TLC analysis. All
solutions in CH2Cl2 were combined and washed twice with a saturated
aqueous solution of NaHCO3 (20 mL) and twice with water (10 mL). The
organic layer was collected and dried over Na2SO4. After removal of the
solvent, the desired oligosaccharide was purified from the reaction mix-
ture by silica gel flash chromatography.
General procedure for deprotection of Lev: The Lev-protected com-
pound (1 equiv) was dissolved in pyridine (for 150 mg of compound,
2.4 mL) and acetic acid (1.6 mL). The mixture was cooled to 08C, fol-
lowed by addition of hydrazine monohydrate (5 equiv for each Lev). The
mixture was stirred at 08C for 2 h and then was quenched with acetone
(0.28 mL). The mixture was stirred at room temperature for 1 h and the
acetone was evaporated under vacuum. The residue was diluted with
EtOAc (50 mL) and washed with saturated NaHCO3, 10% HCl, and
water, and the organic phase was dried over Na2SO4. The solvent was
concentrated in vacuo and the compound was purified by silica gel
column chromatography.
General procedure for 6-OH oxidation to carboxylic acid and benzyl
ester formation: H2O (2 mL), 1m KBr (1.5 equiv per OH), 2,2,6,6-tetra-
methyl-1-piperidinyloxy (TEMPO) (1 equiv for each OH), 0.5m NaHCO3
(10 equiv per OH) and Bu4NBr (10 equiv per OH) were consecutively
added to a solution of 6-OH-containing compound (for 100 mg of com-
pound, 1 equiv) in CH2Cl2 (2 mL) at room temperature. The mixture was
cooled to 08C, followed by slow addition of NaOCl (100 equiv per OH).
The pH value of the mixture was calibrated with 0.5m NaOH to maintain
at pH 10 and the resulting solution was warmed slowly to room tempera-
ture (maintain at pH 10). After stirring for 3 h, the CH2Cl2 was evaporat-
ed under vacuum and the residue was diluted with EtOAc (50 mL) and
washed with 10% HCl, saturated NaHCO3, and water. The organic
phase was dried over Na2SO4, filtered, and the solvents were removed in
vacco. Without separation, the resulting residue was dissolved in CH2Cl2
(5 mL), followed by addition of phenyldiazomethane in diethyl ether
until the color turned red. The mixture was stirred at room temperature
for 1 h and then was diluted with CH2Cl2 (50 mL), and the organic phase
was washed with saturated NaHCO3 and dried over Na2SO4. The solvent
was concentrated in vacuo and the compound was purified by silica gel
column chromatography.
General procedure for preactivation-based three-component one-pot gly-
cosylation: A solution of donor (60 mmol) and freshly activated 4 ꢅ mo-
lecular sieves (200 mg) in CH2Cl2 (2 mL) was stirred at room tempera-
ture for 30 min, and cooled to À788C, which was followed by addition of
AgOTf (47 mg, 180 mmol) dissolved in Et2O (1 mL) without touching the
wall of the flask. After 5 min, orange-colored pTolSCl (9.5 mL, 60 mmol)
was added to the solution through a microsyringe. Since the reaction
temperature was lower than the freezing point of pTolSCl, pTolSCl was
added directly into the reaction mixture to prevent it from freezing on
the flask wall. The characteristic yellow color of pTolSCl in the reaction
solution dissipated within a few seconds, indicating depletion of pTolSCl.
After the donor was completely consumed, according to TLC analysis
(about 5 min at À788C), a solution of acceptor (54 mmol) in CH2Cl2
(0.2 mL) was slowly added dropwise by using a syringe. The reaction mix-
ture was warmed to À208C under stirring in 2 h and then the mixture
was cooled to À788C, followed by sequential additions of AgOTf (16 mg,
60 mmol) in Et2O (1 mL), the second acceptor (48 mmol) in CH2Cl2
(1 mL). The mixture was stirred for 5 min at À788C and then pTolSCl
(7.6 mL, 48 mmol) was added to the solution. The reaction mixture was
warmed to À208C under stirring in 2 h. Then the mixture was diluted
with CH2Cl2 (20 mL) and filtered over Celite. The Celite was further
washed with CH2Cl2 until no organic compounds were observed in the
filtrate by TLC analysis. All solutions in CH2Cl2 were combined and
washed twice with a saturated aqueous solution of NaHCO3 (20 mL) and
twice with water (10 mL). The organic layer was collected and dried over
Na2SO4. After removal of the solvent, the desired oligosaccharide was
purified from the reaction mixture by silica gel flash chromatography.
General procedure for saponification: The mixture of compound (for
100 mg of compound, 1 equiv), THF (2.5 mL), and 1m LiOH (13 equiv
per COOBn) was cooled to À58C, followed by addition of H2O2
(150 equiv per COOBn, 30%). The mixture was stirred at room tempera-
ture for 16 h and then MeOH (6 mL) and 3m KOH (80 equiv per
COOBn) were added to the solution. The mixture was stirred for another
24 h and then was acidified with 10% HCl, concentrated to dryness. The
resulting residue was purified by quickly passing through a short silica
gel column (4:1, CH2Cl2-MeOH).
General procedure for azide reduction: 1m PMe3 solution in THF
(5 equiv per N3), 0.1m aqueous solution of NaOH (2.6 equiv per N3), and
H2O (2 mL) were added consecutively to a solution of azide-containing
compound (for 50 mg of compound, 1 equiv) in THF (7 mL). The mix-
ture was stirred at room temperature overnight and neutralized with
0.1m HCl until pH 7. The mixture was concentrated to dryness and the
resulting residue was purified by quickly passing through a short silica
gel column (4:1, CH2Cl2/MeOH).
General procedure for deprotection of PMB: The PMB-protected com-
pound (1.0 equiv) was dissolved in a mixture of CH2Cl2/H2O (for 0.5 g of
compound, 9 mL/1 mL) and the solution was cooled to 08C. DDQ
(1.1 equiv) was added to the solution and the mixture was stirred at
room temperature for 3 h. The mixture was filtered, diluted with CH2Cl2
(50 mL), and the organic phase was washed with H2O until the solution
became colorless. The solvent was concentrated in vacuo and the com-
pound was purified by silica gel column chromatography.
General procedure for O-sulfation: The mixture of OH-containing com-
pound (for 20 mg of compound, 1 equiv), DMF (1 mL), and SO3·NEt3
(5 equiv per OH) was stirred at 558C for 24 h. The mixture was quenched
by adding Et3N (0.2 mL) and then diluted with CH2Cl2/MeOH (1 mL/
1 mL). The resulting solution was layered on the top of a Sephadex LH-
20 chromatography column that was eluted with CH2Cl2/MeOH (1/1, v/
General procedure for protection of 6-OH with Lev: The compound con-
taining 6-OH (1 equiv) was dissolved in CH2Cl2 (for 0.5 g of compound,
Chem. Eur. J. 2010, 16, 8365 – 8375
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8373

X. Huang et al.
v). The solvent was evaporated to dryness under vacuo without further
purification.
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2 mL), and Pd(OH)2 (100 mg) was stirred under H2 at room temperature
overnight and then filtered. The filtrate was concentrated to dryness
under vacuum and then diluted with H2O (15 mL). The aqueous phase
was further washed with CH2Cl2 (3ꢃ5 mL) and EtOAc (3ꢃ5 mL) and
then the aqueous phase was dried under vacuum. The crude product was
further purified by size exclusion chromatography (G-15) and then
eluted from a column of Dowex 50WX4-Na+ to convert the compound
into the sodium salt form.
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Received: April 16, 2010
Published online: July 7, 2010
Chem. Eur. J. 2010, 16, 8365 – 8375
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www.chemeurj.org
8375