Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach

Article abstract of DOI:10.1093/glycob/cwr109

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3 nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.

Full text of DOI:10.1093/glycob/cwr109

Glycobiology vol. 22 no. 1 pp. 96–106, 2012
doi:10.1093/glycob/cwr109
Advance Access publication on August 11, 2011
Directing the biological activities of heparan sulfate
oligosaccharides using a chemoenzymatic approach
Yongmei Xu2, Zhen Wang3, Renpeng Liu2, Arlene
S Bridges3, Xuefei Huang1,4, and Jian Liu1,2
2Division of Medicinal Chemistry and Natural Products, Eshelman School of
Pharmacy and 3Department of Pathology, School of Medicine, University of
North Carolina, Chapel Hill, NC 27599, USA; and 4Department of
Chemistry, Michigan State University, East Lansing, MI 48824, USA
unit of HS consists of a disaccharide repeat of glucuronic acid
(GlcUA) or iduronic acid (IdoUA) and glucosamine (GlcN)
carrying sulfo groups (Peterson et al. 2009). Sulfation types
and the distribution of IdoUA dictate the biological activity of
HS (Gama et al. 2006; de Paz and Seeberger 2008). Heparin, a
widely used anticoagulant drug, is a specialized highly sulfated
form of HS. The diverse biological functions present consider-
able opportunities for exploiting HS or HS-protein conjugates
for developing new classes of anticancer (Shriver et al. 2004),
antiviral (Baleux et al. 2009) and improved anticoagulant
drugs (Chen et al. 2007). A method to construct HS is, there-
fore, critical for developing HS-based therapeutics. Total
chemical synthesis is fully capable of preparing short frag-
ments of HS. The most successful example is the synthesis of
an antithrombin (AT)-binding heparin pentasaccharide analog
known as fondaparinux (Petitou and van Boeckel 2004).
However, the chemical synthesis of oligosaccharides larger
than a hexasaccharide is extremely challenging (Petitou and
van Boeckel 2004). In addition to the size, the number of
oligosaccharides with diverse sulfation patterns required for
structure–activity relationship studies presents another obstacle
because the syntheses do not usually follow a single route.
Typically, different sets of precursor structures are needed
depending on the sulfation patterns of the target compounds.
The preparations of an array of precursors are tedious and time-
consuming. It is highly desirable for a divergent approach,
where a single precursor can lead to various sulfated oligosac-
Received on January 2, 2011; revised on August 4, 2011; accepted on
August 5, 2011
Heparan sulfate (HS) and heparin are highly sulfated
polysaccharides exhibiting essential physiological func-
tions. The sulfation patterns determine the functional
selectivity for HS and heparin. Chemical synthesis of
HS, especially those larger than a hexasaccharide, remains
challenging. Enzymatic synthesis of HS has recently
gained momentum. Here we describe the divergent assem-
bly of HS heptasaccharides and nonasaccharides from a
common hexasaccharide precursor. The hexasaccharide
precursor was synthesized via a chemical method. The
subsequent elongation, sulfation and epimerization were
completed by glycosyltransferases, HS sulfotransferases
and epimerase. Using the synthesized heptasaccharides,
we discovered that the iduronic acid is critical for binding
to fibroblast growth factor-2. We also designed a synthetic
path to prepare a nonasaccharide with an antithrombin-
binding affinity of 3 nM. Our method demonstrated the
feasibility of combining chemical and enzymatic synthesis
to prepare structurally defined HS oligosaccharides with
desired biological activities.
charides. Chemo-enzymatic methods offer
solution.
a promising
The biosynthesis of HS involves numerous specialized
enzymes, including HS polymerase, epimerase and sulfotrans-
ferases (Figure 1). HS polymerase is responsible for building
the polysaccharide backbone, containing the repeating unit of
-GlcUA-GlcNAc-. The backbone is then modified by
N-deacetylase/N-sulfotransferase (NST) (having two separate
domains exhibiting the activity of N-deacetylase and NST,
respectively), C5-epimerase (C5-epi, converting GlcUA to
IdoUA), 2-O-sulfotransferase (2-OST), 6-OST and 3-OST to
produce the fully elaborated HS. Using HS sulfotransferases
and C5-epi, we previously developed a method to synthesize
HS polysaccharides (Chen et al. 2005, 2007; Zhang et al.
2008) as well as oligosaccharides (Liu et al. 2010).
Keywords: antithrombin / FGF / heparan sulfate /
oligosaccharides / sulfotransferases
Introduction
Heparan sulfate (HS) is a unique class of macromolecular
natural product that is present in large quantities on the mam-
malian cell surface and in the extracellular matrix. The roles of
HS in regulating blood coagulation, embryonic development
and the inflammatory responses and assisting viral/bacterial
infections have been documented extensively. The structural
In this article, we exploit to interface chemical and
enzymatic synthesis to prepare different biologically active HS
oligosaccharides from a single hexasaccharide precursor. The
synthesis of hexasaccharide precursor (GlcNH2-GlcUA-Glc
1To whom correspondence should be addressed: Tel: +1-919-843-6511;
Fax: +1-919-843-5432; e-mail: jian_liu@unc.edu (J.L.); Tel: +1-517-355-
9715; Fax: +1-517-353-1793; e-mail: xuefei@chemistry.msu.edu (X.H.)
© The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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Chemoenzymatic synthesis of heparan sulfate
Fig. 1. Biosynthetic pathway of HS. The HS biosynthetic pathway includes the biosynthesis of backbone and modifications. The synthesis of the backbone, a
repeating unit of -GlcUA-GlcNAc without sulfo groups nor IdoUA units, is not shown. The modification site at each step is highlighted in a blue box.
NH2-GlcUA-GlcNH2-GlcUA-OEt, hexasaccharide 1) was
achieved by chemical synthesis in high yields. Sulfation and
epimerization of GlcUA to IdoUA were achieved by enzymes.
We demonstrated the synthesis of heptasaccharides carrying
different sulfation patterns in less than three or four steps. The
heptasaccharides were used to determine the critical structural
elements for binding to fibroblast growth factor-2 (FGF-2)
having no affinity to AT. Enzymatically extending the hexasac-
charide precursor to a nonasaccharide, we synthesized a nona-
saccharide that binds to AT with high affinity. Our results
demonstrate that the combination of chemical and enzymatic
synthesis provides the flexibility for the synthesis of a wide
range of HS oligosaccharides. This technique could become a
powerful tool for the on-demand synthesis of HS oligosacchar-
ides to elucidate the structure and activity relationship of HS.
removal, saponification and hydrogenation provided the fully
deprotected hexasaccharide 1 in 64% overall yield. All the
synthetic hexasaccharides were fully characterized by NMR
and mass spectrometry (MS) (Supplementary data).
Enzymatic synthesis of heptasaccharides with different
sulfation patterns
Using hexasaccharide 1 as a starting material, we synthesized
five heptasaccharide derivatives differing in sulfations and the
presence of IdoUA residues (Figure 3). Hexasaccharide 1 was
first N-sulfated using NST to form N-sulfated hexasaccharide.
The resultant hexasaccharide was then elongated to heptasac-
charide 2 by Pasteurella multocida heparosan synthase-2
(pmHS2) using a UDP-GlcUA as a sugar donor. The
N-sulfation at the nonreducing end is necessary for extension
to a heptasaccharide as hexasaccharide 1 was unreactive to
pmHS2.
Results
Chemical synthesis of hexasaccharide 1
Heptasaccharide 2 was then subjected to additional modifi-
cations to yield different products. In one modification, C5-epi
was included at the 2-O-sulfation step to synthesize heptasac-
charide 3, containing two IdoUA2S residues. Heptasaccharide 3
was subsequently modified by 6-OST-1 and 6-OST-3 to form
heptasaccharide 4. In another modification, C5-epi was excluded
in order to prepare heptasaccharide 3′, containing two GlcUA2S
residues. Heptasaccharide 3′ was then modified by 6-OST-1 and
6-OST-3 to lead to heptasaccharide 4′.
Structural analyses of heptasaccharides 2, 3 and 4 were
completed using electrospray ionization (ESI) MS and disac-
charide analysis. After N-[35S]sulfation and extension by
pmHS2, heptasaccharide 2 displayed a major symmetric peak
Chemical synthesis of the fully protected hexasaccharide 10
was performed using the pre-activation-based one-pot oligo-
saccharide synthesis method (Figure 2; Huang et al. 2004;
Wang et al. 2010). Starting from three disaccharide building
blocks 11–13 (Wang et al. 2010), hexasaccharide 10 was
obtained in 71% yield in one pot. Deprotection of the hexa-
saccharide 10 was performed by first removing the three levu-
linoyl protecting groups with hydrazine in pyridine and acetic
acid, followed by 2,2,6,6-tetramethyl-1-piperidinyloxy and
sodium hypochloride oxidization (Lee et al. 2004; Huang and
Huang 2007) and benzyl ester formation producing compound
14 in 50% yield. Subsequent silyl ether protective group
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Y Xu et al.
Fig. 2. Synthetic scheme of hexasaccharide 1.
(30.5 min) by polyamine-based anion exchange (PAMN)-
high-performance liquid chromatography (HPLC) with minor
impurities (Figure 4A). We then resynthesized nonradioac-
tively labeled heptasaccharide 2 under the identical condition.
The product was purified by HPLC for MS analysis. ESI-MS
analysis revealed the molecular weight of the product to be
1474.6 0.2 Da, very close to the calculated molecular
(1474.2 Da; Figure 4B). The structure of heptasaccharide 2
was further proved by the disaccharide analysis of heparin
lyase digestion, a common approach used in analyzing the
structure of HS oligosaccharides (Venkataraman et al. 1999;
Zaia and Costello 2001; Lawrence et al. 2008; McCullough
et al. 2010; Wei et al. 2011). To this end, 35S-labeled hepta-
saccharide 2 was digested to disaccharides as illustrated in
Figure 4D. The resultant disaccharides were resolved by
reverse-phase ion pairing (RPIP)-HPLC, showing two
35S-labeled disaccharides (Figure 4C). Coeluting with the dis-
accharide standards, we confirmed the 35S-peak eluted at 35
min is GlcUA-GlcNS and the 35S-peak eluted at 40 min is
ΔUA-GlcNS. The measured ratio of GlcUA-GlcNS and
ΔUA-GlcNS was 1:1.9, which is close to the theoretical value
(1:2) of heptasaccharide 2 digested by heparin lyases. Taken
together, our results proved the structure of heptasaccharide 2
as shown in Figure 3. Using similar techniques, we proved
the successful synthesis of heptasaccharides 3 and 4. The data
for the structural characterization of heptasaccharides 3 and 4
are shown in Supplementary data, Figures 1S and 2S. A
35S-labeled disaccharide from nitrous acid-degraded 2-O-[35S]
sulfo heptasaccharide 3 coeluted with the disaccharide stan-
dard, IdoUA2S-AnMan (but not GlcUA2S-AnMan), confirm-
ing that heptasaccharide 3 contains IdoUA2S residues
(Supplementary data, Figure 1SE). Furthermore, we confirmed
the nonreducing terminus of heptasaccharide 4 to be a GlcUA
because the heptasaccharide was susceptible to the digestion
of β-glucuronidase (Supplementary data, Figure 3S).
The structures of heptasaccharides 3′ and 4′ were proved by
the disaccharide analysis. Here, we did not prepare sufficient
amount of samples for MS analysis. For heptasaccharide 3′, we
demonstrated the presence of GlcUA2S (Supplementary data,
Figure 4S). As expected, the disaccharide analysis of heptasac-
charide 4′ revealed the disaccharides GlcUA-GlcNS 6S and
ΔUA2S-GlcNS6S (Supplementary data, Figure 5S).
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Chemoenzymatic synthesis of heparan sulfate
Fig. 3. Synthetic scheme of heptasaccharides. Hexasaccharide 1 (hexasaccharide precursor) was first N-sulfated using NST followed by the extension using
pmHS2 and UDP-GlcUA to yield heptasaccharide 2. Heptasaccharide 2 was converted to heptasaccharide 3 by C5-epimerase and 2-OST and subsequently
converted to heptasaccharide 4 by 6-OST-1 and 6-OST-3. Heptasaccharide 2 was also converted to heptasaccharide 3′ by 2-OST (without C5-epimerase) and
subsequently converted to heptasaccharide 4′ by 6-OST-1 and 6-OST-3. The sites of modification at each step are highlighted in black (for the synthesis of
heptasaccharides 2–4) or blue (for the synthesis of heptasaccharides 3′ and 4′) background.
Determination of the contribution of sulfation types and
IdoUA residue to the binding of FGF-2
To compare the FGF-2 binding affinity of the synthetic
heptasaccharide to the full-length HS, we determined the
binding affinity (Kd) of heptasaccharide 4 that carries the
IdoUA residue as well as 6-O- and 2-O-sulfo groups
(Supplementary data, Figure 6S). The binding affinity was
determined to be 202 14 nM (average of two determinations
range) using affinity coelectrophoresis. The Kd of FGF-2 for
a full-length HS was determined to be 35 nM using a surface
plasmon resonance method (Chen et al. 2005). The data
suggest that heptasaccharide has somewhat lower affinity than
a full-length polysaccharide.
HS is known to play an important role in binding to growth
factors and growth factor receptors to regulate the cell growth
process (Bai and Esko 1996). It has been widely accepted that
sulfation patterns dominate the binding affinity between HS
and growth factors (Kreuger et al. 2006). Here, we examined
the binding of FGF-2 to the heptasaccharides with different
sulfation patterns using a filter-trapping method described by
Maccarana et al. (1993). In this experiment, 35S-labeled hepta-
saccharides were incubated with purified FGF-2 (Figure 5).
The mixture was then spotted onto a nitrocellulose membrane,
which binds to protein nonspecifically, permitting to capture
the complex of FGF-2 and heptasaccharides. The binding was
quantified via scintillation counting. As expected, both hepta-
saccharides 3 and 4 bound to FGF-2 very well, while neither
heptasaccharide 3′ nor heptasaccharide 4′ displayed signifi-
cant binding to FGF-2. It is important to note that heptasac-
charides 3 and 3′, as well as heptasaccharides 4 and 4′, carry
identical numbers of sulfo groups. The structural difference is
whether it contains the IdoUA residue. Thus, our data demon-
strate that the IdoUA residue is essential for binding to
FGF-2. We also observed ꢀ2-fold difference between hepta-
saccharides 3 and 4 in binding to FGF-2, suggesting that
additional 6-O-sulfo groups enhance the binding to FGF-2.
Synthesis of AT-binding nonasaccharide
Our next goal was to prepare an oligosaccharide that binds to
AT from heptasaccharide 2. The synthesis involved in exten-
sion to a nonasaccharide followed by the modifications by
C5-epimerase and sulfotransferases as illustrated in Figure 6. It
is worthwhile to note that the extension to nonasaccharide is
necessary to confer the AT-binding affinity as the modification
of heptasaccharide 4 with 3-OST-1 did not render AT-binding
affinity (Table I). This experiment exemplified the use of
enzymes to design the oligosaccharide structures with a desired
biological activity. The target oligosaccharide, nonasaccharide
9, should consist of a GlcUA residue at the U7 position and a
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Y Xu et al.
Fig. 4. Structural characterization of heptasaccharide 2. (A) The HPLC chromatogram of 35S-labeled heptasaccharide 2 using a PAMN column. Solid bar
indicates the impurities. (B) The MS spectrum of HPLC-purified heptasaccharide 2. (C) The HPLC of the disaccharide analysis of heparin lyase-digested
35S-labeled heptasaccharide 2. The disaccharide analysis was carried out on a C18-column eluted under RPIP-HPLC conditions. The elution positions of the
disaccharides were confirmed with appropriate disaccharide standards. (D) The reactions involved in the digestions of 35S-labeled heptasaccharide 2 using
heparin lyases. The 35S-labeling sites are colored in red. A nonradioactive monosaccharide was also formed during the digestion; however, it was not detectable.
3-O-sulfo group at the G6 GlcN because both GlcUA and
3-O-sulfated GlcN residues are part of the AT-binding site
(Zhang et al. 1999). To avoid the conversion of the GlcUA
residue at the U7 position by C5-epi, a GlcNAc residue was
introduced at the G8 position (Liu et al. 2010). The GlcNAc
residue of G8 should also be 6-O-sulfated as it is known that
the 6-O-sulfation is required to display high affinity to
AT-binding in the pentasaccharide context (Atha et al. 1985).
We then introduced another GlcUA residue at the U9 position
to ensure the 6-O-sulfation at the G8 position by 6-OST. The
3-O-sulfation at the G6 position was carried out by 3-OST-1 as
it sulfates the GlcNS residue with the disaccharide sequence of
-GlcUA-GlcNS6S- (U7-G6), but not in the sequences of
-IdoUA2S-GlcNS6S- (I5-G4-I3-G2) or -GlcUA-GlcNAc6S-
(U9-G8; Xu et al. 2008).
A series of HPLC analysis and the disaccharide analysis
were conducted to prove the structures of the final product
and the intermediate products. The successful extension of
heptasaccharide 2 to octasaccharide 5 and nonasaccharide 6
was detected by observing the shift in retention time on
diethylaminoethyl (DEAE)-HPLC analysis (Supplementary
data, Figure 7S). The desired 2-O-sulfation/epimerization of
heparin lyase digested nonasaccharide 7 as shown in
Supplementary data, Figure 8S. Furthermore, for the
2-O-[35S]-labeled nonasaccharide 7, we proved that the
IdoUA residues carry 2-O-sulfo groups (Supplementary data,
Figure 9S). Taken together, our data confirmed the structure
of nonasaccharide 7.
The structure of nonasaccharide 8 was also proved by the
disaccharide analysis of the compound after the digestion
with heparin lyases. The 6-O-[35S]-labeled nonasaccharide
displayed a predominantly single 35S-labeled peak, suggesting
that the preparation was pure (Figure 7A). Digestion of the
nonasaccharide should yield three 35S-labeled disaccharides,
GlcUA-GlcNAc6S, ΔUA-GlcNS6S and ΔUA2S-GlcNS6S, as
illustrated in Figure 7D. As expected, the disaccharide
analysis of heparin lyases-digested nonasaccharide showed
three anticipated disaccharides with the ratio of 1.1:1:1.7, very
close to the theoretically calculated value of 1:1:2. Therefore,
the data confirmed the structure of nonasaccharide 8.
The synthesis of nonasaccharide 9 was accomplished using
3-OST-1 enzyme, which sulfates the saccharide residue G6.
DEAE-HPLC analysis of nonasaccharide 9 suggested that the
product was pure (Supplementary data, Figure 10SA). Because
3-O-sulfation is known to prevent the digestion of oligosac-
charides to disaccharides by heparin lyases (Yamada et al.
nonasaccharide
7
was confirmed by the site-specific
35S-labeling technique followed by a disaccharide analysis of
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Chemoenzymatic synthesis of heparan sulfate
1993; Sundaram et al. 2003), we chose to use two-step nitrous
acid degradation to convert nonasaccharide 9 to the disacchar-
ide as illustrated in Supplementary data, Figure 10SD. As
expected, we observed the presence of disaccharide of
GlcUA-AnMan3S6S.
The binding of nonasaccharide to AT was carried out using
an AT-affinity column (Table I). Here, two polysaccharide
controls were included, 3-O-[35S]-sulfated HS and N-[35S]-sulf
ated HS. As expected, ꢀ37% of the 3-O-[35S]-sulfated HS
binds to AT, while the N-[35S]-sulfated HS did not display sig-
nificant binding to AT due to the lack of the critical 3-O-sulfo
group as demonstrated in our previous publication (Xu et al.
2008). Comparing with the polysaccharide controls, nonasac-
charide 9 shows excellent binding (70%) to AT. In theory,
100% of nonasaccharide 9 should bind to AT. Lower than
100% binding is due to the fact the product recovery from an
AT-affinity column was not complete. A similar case has been
reported in our previous publication (Xia et al. 2002). In con-
trast, the 3-O-[35S]-sulfated heptasaccharide 4 does not bind to
AT (Table I). 3-O-sulfated heptasaccharide 4 is expected to
have the following structure: GlcA-GlcNS6S3S-Ido
A2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-Et. It is known that
the actual AT-binding site should include the following
structure: -GlcNS(or NAc)6S-GlcA-GlcNS6S3S-IdoA2S-
GlcNS6S-. Comparing these two structures, one can find that a
GlcNAc6S (or GlcNS6S) residue is missing from the nonredu-
cing end in 3-O-sulfated 4, which is known to be essential for
binding to AT (Atha et al. 1985). Furthermore, the binding affi-
nity (Kd) of nonasaccharide 9 to AT was determined to be 3.0
1.1 nM (average of three determination SD). The represen-
tative gel picture and data analysis are shown in Figure 8. A Kd
value of fondaparinux, an approved pentasaccharide anticoagu-
lant drug, was determined to be 5.9 1.5 nM using this
method, suggesting that nonasaccharide 9 has slightly higher
affinity than fondaparinux (10). The biological significance for
Fig. 5. The binding of 35S-labeled heptasaccharides to FGF-2. FGF-2 (1 μg)
was incubated with 5000 cpm of 35S-labeled oligosaccharides in the PBS
buffer. The mixture was spotted onto a nitrocellulose membrane. The
membrane was then washed with a PBS buffer containing 300 mM NaCl. The
spotted membrane was then mixed with scintillation fluid to measure the
amount 35S-counts in order to determine the percentage of 35S-labeled
oligosaccharides bound to FGF-2. The data presented represent the average of
two experiments. Error bars indicate the range.
Fig. 6. Scheme for the synthesis of AT-binding nonasaccharide 9. The reaction sites are colored in blue. The potential AT-binding domain in nonasaccharide 9 is
colored in red.
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Y Xu et al.
nonasaccharide 9, such as its in vitro and in vivo anticoagulant
activities, remains to be investigated largely because the study
requires substantial amount of sample.
enzymes, including glycosyltransferases, HS C5-epimerase
and sulfotransferases, to produce the oligosaccharides with
different sulfation patterns and sizes. Our method can elongate
an oligosaccharide to the desirable size via the action of gly-
cosyltransferase. The types of sulfation can be controlled by
selecting the number of modification steps. The position of
N-sulfo GlcN controls the position and number of the
IdoUA2S residues as demonstrated in the synthesis of hepta-
saccharide 4 and nonasaccharide 9. Consequently, the modifi-
cation directed the biological activities of the oligosaccharide
products. Clearly, the chemoenzymatic method offers the
flexibility and throughput to investigate the structure–function
relationship of HS in a given biological process. It should be
noted that the project was aimed to demonstrate the feasibility
of using HS biosynthetic enzymes to generate the oligosac-
charides with distinct biological functions from a single start-
ing hexasaccharide (1). Using this hexasaccharide (6 mg), we
synthesized up to 11 oligosaccharides, including the final pro-
ducts and intermediates. It is not completely surprising that
the scale of the synthesis demonstrated in this study was
small. In this study, we did not measure the yields at each
reaction step. As described in our previous publication (Liu
et al. 2010), the conversion to the desired product at each step
is nearly quantitative, and the product loss predominantly
occurs at the purification step with a yield of 30–50%. In a
more targeted synthesis, the scale of the synthesis can be
Discussion
In this manuscript, we describe a unique approach to prepare
HS oligosaccharides by combining chemical and enzymatic
synthesis. A hexasaccharide precursor was chemically syn-
thesized which was used as a key intermediate for divergent
synthesis. The hexasaccharide was further modified with
Table I. The binding of HS polysaccharide and oligosaccharides to AT
Samples
AT binding (%)a
3-O-[35S]-sulfated HSb
N-[35S]-sulfated HSc
37 13
1.5 1.5
Nonasaccharide 9
70
2.8 2.5
5
3-O-[35S]-sulfated heptasaccharide 4d
aThe binding percentage of the samples to AT was determined using an
affinity column as described in Experimental procedures.
bThe 3-O-[35S]-sulfated HS was prepared by incubating HS from bovine
kidney with purified 3-OST-1 and [35S]PAPS.
cThe N-[35S]-sulfated HS was prepared by incubating HS from bovine with
purified NST and [35S]PAPS.
dThe 3-O-[35S]-sulfated heptasaccharide 4 was prepared by incubating
heptasaccharide 4 with purified 3-OST-1 and [35S]PAPS.
Fig. 7. Structural characterization of nonasaccharide 8. (A) DEAE-HPLC chromatogram of 35S-labeled nonasaccharide. (B) The structure of nonasaccharide 8.
(C) The RPIP-HPLC chromatogram of the disaccharides of heparin lyase-digested N-[35S]sulfo nonasaccharide 8. (D) The reaction involved in the digestion of
nonasaccharide 8 by heparin lyases. A nonradioactive monosaccharide was formed during the digestion; however, it was not detectable.
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Chemoenzymatic synthesis of heparan sulfate
Fig. 8. Determination of the binding affinity of nonasaccharide 9 to AT. (A) The representative autoradiography of the agarose gel in which purified
3-O-[35S]-sulfated nonasaccharide 9 was subjected to electrophoresis through separation zones containing AT at concentrations indicated. Approximately 8000
cpm/lane of nonasaccharide was loaded into each zone. (B) The plot of R/[AT] vs R, where R = (M0 − M)/M0. M0 is the migration of free 35S-labeled
nonasaccharide and M is the observed migration of the nonasaccharide in the presence of AT at various concentrations. The plot yields a straight line with a slope
of −1/Kd. The linear coefficient (r2) was determined to be 0.97.
readily enlarged to milligrams or even higher as demonstrated
in our recent publications (Sheng et al. 2011; Xu et al. 2011).
Using enzymatically synthesized oligosaccharides, we
revealed the structural selectivity of HS oligosaccharides for
binding to FGF-2. The interaction of HS and FGF-2 has been
implicated in regulating cell growth and angiogenesis pro-
cesses (Kreuger et al. 2006). Although the contribution of
sulfo groups to the binding affinity to FGF-2 has been
reported (Maccarana et al. 1993; Ashikari-Hada et al. 2004,
2009; Wang et al. 2010), the binding affinity between HS and
FGF-2 is perceived to be mainly governed by the density of
negative charges, rather by specific monosaccharide sequences
(Kreuger et al. 2006). In this study, five heptasaccharides were
successfully prepared. These oligosaccharides permitted us to
dissect the contribution of 2-O-sulfation, 2,6-O-sulfation as
well as the presence of an IdoUA residue to the binding to
FGF-2. We observed that the presence of IdoUA significantly
enhances the percentage of the binding to FGF-2, despite the
fact that the oligosaccharides carry identical number of sulfo
groups. Indeed, structural analysis of the binding of FGF-2
and oligosaccharides using NMR revealed that an IdoUA
residue provides the structural flexibility to an oligosaccharide
(Guglieri et al. 2008). Such structural flexibility allows the
oligosaccharides to position the sulfo groups toward FGF-2 to
enhance the binding affinity (Raman et al. 2003). Using
chemically synthesized HS hexasaccharides, we previously
demonstrated that hexasaccharides containing a trisaccharide
domain containing -GlcNS-IdoUA2S-GlcNS- inhibited the
binding of FGF-2 to HS. Replacing the IdoUA2S residue with
GlcUA2S abolished the inhibition effect on the binding of HS
to FGF-2 (Wang et al. 2010). Therefore, our data are consist-
ent with the previous findings, namely, an IdoUA residue
plays a critical role for binding to FGF-2.
proliferation, consistent with its contribution to the
FGF-binding (Chen et al. 2007). However, a reported pub-
lished by Borgenstrom et al. (2003) concluded that heavily sul-
fated heparosan binds to FGF-1 and FGF-2, despite the fact
that this polysaccharide contains no IdoUA residues. It should
be noted that a significant level of the 3-O-sulfated GlcUA
residue, an unnatural structural unit, has been found in chemi-
cally sulfated heparosan (Lindahl et al. 2005). Therefore,
whether the presence of GlcUA3S in those chemically sulfated
heparosan could contribute to the binding to FGF-1 or FGF-2
remains to be answered. We also noted that chemically sulfated
heparosan is a polysaccharide (>50 saccharide residues),
whereas the effect of IdoUA on FGF-2 binding was observed
on a heptasaccharide. The size of the polysaccharide may con-
tribute to the binding affinity to FGF-2.
Our method also demonstrated the synthesis of an
AT-binding oligosaccharide from the common intermediate
hexasaccharide 1. The synthesis was achieved by introducing
a GlcNAc residue, which is used to control the extent of
epimerization and serve as an acceptor site for 6-O-sulfation.
This effort is an excellent example of designing oligosacchar-
ide structures for specific biological activities. Because the
AT-binding site consists of a pentasaccharide sequence
of -GlcNAc (or NS)6S-GlcUA-GlcNS3S6S-IdoUA2S-GlcNS6
S-, both GlcNAc(or NS)6S and GlcUA are essential for the
affinity to AT (Figure 6). The GlcNAc residue (of G8 in nona-
saccharides) preserves the GlcUA at the U7 position in nona-
saccharide 8 after the modification by C5-epi and 2-OST. The
GlcNAc residue of G8 is also important to serve as the accep-
tor for the 6-O-sulfation because the GlcNAc6S is part of the
AT-binding site (Atha et al. 1985). Without the GlcNAc
residue of G8, the resultant oligosaccharide does not bind to
AT even it carries a 3-O-sulfo GlcN unit.
The role of the IdoUA residue in polysaccharides in stimu-
lating FGF-2/fibroblast growth factor receptors (FGFR)-
mediated cell proliferation was reported. It was shown that the
enzymatically synthesized polysaccharide lacking the IdoUA
has significantly reduced activity in promoting cell
In summary, we demonstrate the feasibility of chemoenzy-
matic synthesis of multiple oligosaccharides from a single
hexasaccharide precursor. The divergent design of our
approach allowed us to access diverse HS oligosaccharide
structures with the desired binding affinities to the target
103

Y Xu et al.
proteins. Although higher binding affinities are not always
equal to stronger biological activities, our results clearly
demonstrate the structural selectivity of HS toward the inter-
actions with AT and FGF-2. Our next goal is to prepare a
large number of oligosaccharides with different sulfation pat-
terns and to synthesize targeted oligosaccharides in large
quantity to construct HS-based microarray and conduct
further biological evaluation.
NaOAc, 3 M urea, 1 mM ethylenediaminetetraacetic acid
(EDTA), then followed by four times wash with the same
buffer, each time 1 mL. The column was eluted with 1 M
NaCl in 0.001% Triton X-100 buffer. The purified oligosac-
charides were dialyzed using molecular weight cutoff
(MWCO) 3500 membrane and dried. Alternatively, the pro-
ducts were purified by the DEAE-HPLC method as described
in HPLC analysis of oligosaccharides.
To prepare the oligosaccharides containing an IdoUA2S
residue, the substrate (30 μg) was incubated with C5-epi
(80 μg) in 300 μL buffer containing 50 mM MES (pH 7.0) and
1% Triton X-100, 2 mM calcium chloride. After the reaction
proceeded for 30 min, 2-OST (80 μg) and PAPS (100 μM)
were added and incubated overnight at 37°C. The product was
recovered by a DEAE column as described in Enzymatic prep-
aration of sulfated oligosaccharides.
Experimental procedures
Chemical synthesis of hexasaccharide 1
The synthetic scheme for hexasaccharide 1 is shown in
Figure 1. Briefly, a one-pot glycosylation reaction involved
three disaccharide precursors, 11, 12 and 13, was performed
to yield the fully protected hexasaccharide 10. The hexasac-
charide 10 was then deprotected to produce hexasaccharide 1.
The detailed procedures for the synthesis of hexasaccharide 1
and NMR and MS analysis data are described in
Supplementary data.
HPLC analysis of oligosaccharides
Both DEAE-HPLC and PAMN-HPLC were used to purify the
oligosaccharides. For DEAE-HPLC, method
1 used a
DEAE-nonporous (NPR) column (Tosohaas, Japan) that was
eluted with 20 mM, pH 7.0, Tris–HCl buffer for 10 min, then
with a linear gradient of NaCl in 20 mM, pH 7.0, Tris–HCl
from 0 to 1 M in 60 min at a flow rate of 0.4 mL/min.
Method 2 used the same column that was eluted with 0.35 M
NaCl and 20 mM, pH 7.0, Tris–HCl buffer for 10 min, then
with a linear gradient of NaCl in 20 mM, pH 7.0, Tris–HCl
buffer from 0.35 to 0.65 M in 90 min at a flow rate of 0.4
mL/min. Method 2 was used for the separation described in
Supplementary data, Figures 1SA and 2SA and B, while
method 1 was used for the other DEAE-HPLC. For
PAMN-HPLC, a YMC-pack Polyamine II column (250 × 4.6
mm, YMC, Kyoto, Japan) was eluted with 0.4 M KH2PO4 for
10 min, then with a linear gradient of KH2PO4 from 0.4 to 1
M in 45 min at a flow rate of 0.5 mL/min.
Expression and purification of HS biosynthetic enzymes
In total, eight enzymes were employed for the synthesis of
sulfated oligosaccharides, including NST, C5-epi, 2-OST,
6-OST-1, 6-OST-3, 3-OST-1, N-acetylglucosaminyltransferase
(KfiA) from Escherichia coli K5 and pmHS2. All of these
enzymes were expressed in E. coli using different fusion pro-
teins as described previously (Chen et al. 2005, 2007). For
C5-epi, 2-OST, 6-OST-1 and 6-OST-3, maltose-binding fusion
proteins were used, and the fusion proteins were purified by
an amylose column (New England Biolab, MA, USA). For
NST, a glutathione S-transferase fusion was prepared and puri-
fied by a glutathione Sepharose column (GE Healthcare,
Uppsala, Sweden; Kakuta et al. 1997). For 3-OST-1, KfiA
and pmHS2, an N-terminal 6× His-tagged protein was pre-
pared and purified by a Ni-agarose column (GE Healthcare;
Edavettal et al. 2004; Liu et al. 2010).
Disaccharide analysis
The oligosaccharides were digested with a mixture of heparin
lyases or by nitrous acid to yield disaccharides. For heparin
lyase digestion, the oligosaccharide (100,000 cpm) was incu-
bated with a mixture of heparin lyases I, II and III (each at
ꢀ20 μg) in 200 μL of 50 mM Na2HPO3, pH 7.0, at 37°C
overnight. The recombinant heparin lyases were expressed
and purified as described previously (Duncan et al. 2006). For
nitrous acid degradation, the oligosaccharide (100,000 cpm)
was incubated with 20 μL of H2O and 40 μL of 1 M HNO2,
which was prepared from a fresh mixture (v/v 1:1) of 0.5 M
H2SO4 and 0.5 M Ba(NO2)2. The reaction was incubated on
ice for 30 min with occasional vortex. The reaction was
quenched by a mixture of 20 μL of 1 M Na2CO3 and 1 M
NaHCO3 (v/v 1:1). Then, 20 μL of 0.5 M NaBH4 was added
and incubated at 50°C for 30 min, 20 μL of 10 M acetic acid
was added to stop reduction. The resultant disaccharide was
mixed with 0.5 μL of 5% phenol red and purified by BioGel
P-2 (BioRad) column, which was equilibrated with 0.1 M
ammonium bicarbonate at a flow rate of 4 mL/h. The resultant
disaccharides were analyzed using the RPIP-HPLC method as
described previously (Liu et al. 1999). The identities of the
disaccharides were determined by coeluting with standards.
Enzymatic preparation of sulfated oligosaccharides
The oligosaccharides were subjected to various sulfation and
epimerization steps depending on the structures of the target
compound. For the N-sulfation step, the oligosaccharide sub-
strate (20–30 μg) was incubated with NST (80 μg) and 3′-
phosphoadenosine 5′-phophosulfate (PAPS) (2 equiv.) in
300 μL buffer containing 50 mM 2-(N-morpholino)ethanesul-
fonic acid (MES) (pH 7.0) and 1% Triton X-100 overnight at
37°C. To prepare N-[35S]-sulfated oligosaccharides, [35S]
PAPS (1–5 × 106 cpm) was included in the reaction mixture.
To prepare the 2-O-, 6-O- or 3-O-sulfated oligosaccharides,
nearly identical procedures were followed except for replacing
NST with 2-OST, 6-OST-1 (and 6-OST-3) and 3-OST-1,
respectively. The N-sulfated products were purified by a
BioGel P-10 column (0.75 × 200 cm, BioRad, CA, USA)
which was eluted with a buffer containing 20 mM Tris and 1
M NaCl (pH 7.5) at a flow rate of 8 mL/h. For the 2-O-, 6-O-
or 3-O-sulfated oligosaccharides, the products were purified
by a DEAE column after the reaction as follows. The reaction
mixture (300 μL) was mixed with 1 mL of 0.01% Triton
X-100 buffer at pH 5.0 containing 150 mM NaCl, 50 mM
104

Chemoenzymatic synthesis of heparan sulfate
Microdialysis of oligosaccharides
Supplementary data
In order to improve the sensitivity of the analysis by MS, the
oligosaccharides were subjected to microdialysis to remove
the salt. The dialysis was carried out using hollow fiber dialy-
sis tubing (MWCO 13,000 Da, Spectrum, CA, USA) against
20 mM ammonium acetate.
Supplementary data for this article is available online at
http://glycob.oxfordjournals.org/.
Funding
This work is supported in part by National Institutes of
Health (AI50050, HL094463, AI074775 and GM072667).
MS analysis
The analyses were performed at the ADME Mass
Spectrometry Center on an Agilent 1100 MSD-trap. The oligo-
saccharides were dissolved in 70% acetonitrile and 10 mM
imidazole. A syringe pump (Harvard Apparatus, MA, USA)
was used to introduce the sample via direct infusion (10 μL/
min). Experiments were carried out in negative ionization
mode with the electrospray source set to 3000 V and 200°C
and the compound stability set to 30%. Nitrogen was used for
both nebulizer (5 L/min) and drying gas (15 psi). The MS data
were acquired and processed using Bruker Trap Software 4.1.
Conflict of interest
None declared.
Abbreviations
AT, antithrombin; DEAE, diethylaminoethyl; EDTA, ethylene-
diaminetetraacetic acid; ESI, electrospray ionization; FGF-2,
fibroblast growth factor-2; FGFR, fibroblast growth factor
receptors; GlcN, glucosamine; GlcUA, glucuronic acid; HPLC,
high-performance liquid chromatography; HS, heparan sulfate;
IdoUA, iduronic acid; KfiA, N-acetylglucosaminyltransferase;
MES, 2-(N-morpholino)ethanesulfonic acid; MS, mass spec-
trometry; MWCO, molecular weight cutoff; NPR, nonporous;
NST, N-sulfotransferase; 2-OST, 2-O-sulfotransferase; PAMN,
polyamine-based anion exchange; PAPS, 3′-phosphoadenosine
5′-phophosulfate; PBS, phosphate buffered saline; pmHS2,
Pasteurella multocida heparosan synthase-2; RPIP, reverse-
phase ion pairing.
FGF-binding assay
The binding of the oligosaccharides and FGF-2 (a gift from
Dr. Linhardt, Rensselaer Polytechnic Institute) was carried out
using a “filter-trapping” assay (Maccarana et al. 1993).
Briefly, FGF-2 (1 μg) was incubated with 35S-labeled oligo-
saccharides (5000 cpm) in 200 μL of phosphate buffered
saline (PBS) buffer at room temperature for 30 min. The
mixture was then spotted onto nitrocellulose membrane (GE
Healthcare). The spots were washed three times with 500 μL
of PBS buffer containing 300 mM NaCl. The spot was cut
out and mixed with scintillation fluid to measure [35S]
radioactivity.
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