Chemoenzymatic design of heparan sulfate oligosaccharides

Article abstract of DOI:10.1074/jbc.M110.159152

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan s

Full text of DOI:10.1074/jbc.M110.159152

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 44, pp. 34240–34249, October 29, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Chemoenzymatic Design of Heparan Sulfate
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Oligosaccharides*
Received for publication, June 28, 2010, and in revised form, August 9, 2010 Published, JBC Papers in Press, August 21, 2010, DOI 10.1074/jbc.M110.159152
Renpeng Liu‡1, Yongmei Xu‡1, Miao Chen‡, Michel Weïwer§, Xianxuan Zhou‡, Arlene S. Bridges¶, Paul L. DeAngelisʈ,
Qisheng Zhang‡, Robert J. Linhardt§, and Jian Liu‡2
From the ‡Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, and ¶Department of Pathology
and Laboratory of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599 , the §Department of Chemistry and
Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, and the ʈDepartment of Biochemistry and Molecular
Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
Heparan sulfate is a sulfated glycan that exhibits essential
physiological functions. Interrogation of the specificity of hepa-
ran sulfate-mediated activities demands a library of structurally
defined oligosaccharides. Chemical synthesis of large heparan
sulfate oligosaccharides remains challenging. We report the
synthesis of oligosaccharides with different sulfation patterns
and sizes from a disaccharide building block using glycosyl-
transferases, heparan sulfate C5-epimerase, and sulfotrans-
ferases. This method offers a generic approach to prepare hepa-
ran sulfate oligosaccharides possessing predictable structures.
trade name Arixtra for the treatment of venous thromboem-
bolic disorders. However, the chemical synthesis of oligosac-
charides larger than an octasaccharide is extremely difficult,
especially when multiple target structures are required for bio-
logical evaluation (8). An enzyme-based method offers a prom-
ising alternative approach to synthesize HS.
The HS biosynthetic pathway involves multiple enzymes,
including HS polymerase, epimerase, and sulfotransferases (Fig.
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-sulfotrans-
ferase (having two separate domains exhibiting the activity of
N-deacetylase and N-sulfotransferase, respectively), C5-epime-
rase (C5-epi, converting GlcUA to IdoUA), 2-O-sulfotrans-
ferase (2-OST), 6-O-sulfotransferase (6-OST) and 3-O-sulfo-
transferase (3-OST) to produce the fully elaborated HS. With
the exception of HS polymerase, all of these biosynthetic
enzymes have been expressed at high levels in Escherichia coli
(1), permitting easy access to an abundance of enzymes. Using
HS sulfotransferases and C5-epi, we previously developed a
method to synthesize HS from a bacteria capsular polysaccha-
ride with biological activities (6, 9, 10).
The synthetic HS products are a mixture of polysaccharides
with different sizes and sulfated monosaccharide sequences. A
mixture of polysaccharides can sometimes confound structure
and activity relationship studies. Methods for the synthesis of
structurally defined oligosaccharides have been reported (11,
12). These methods describe synthetic strategies that targeted a
single product from a specialized oligosaccharide starting
material. These approaches clearly lack the ability for rapid syn-
thesis of HS oligosaccharides differing in sizes and structures
due to the difficulty in obtaining the starting materials. There-
fore, a technique combining the oligosaccharide backbone syn-
thesis with saccharide modifications, using HS biosynthetic
enzymes, should expand the capability of HS oligosaccharide
synthesis. In this paper, we utilize bacterial glycosyltransferases
and an unnatural UDP-monosaccharide donor to build an oli-
gosaccharide backbone from a disaccharide. These backbone
oligosaccharides can be then selectively modified with different
HS sulfotransferases and C5-epi to prepare structurally defined
products with desired sulfation patterns. Further, this method
was employed to identify a novel HS structure that binds to
antithrombin.
Heparan sulfate (HS)3 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. HS partici-
pates in regulating blood coagulation, embryonic development,
and the inflammatory response and assists viral/bacterial infec-
tions. It consists of a repeating disaccharide unit of glucuronic
acid (GlcUA) or iduronic acid (IdoUA) and glucosamine, both
capable of carrying sulfo groups (1). The sulfation pattern of HS
dictates its biological activity (2, 3). Heparin, a widely used anti-
coagulant drug, is a specialized form of highly sulfated HS. The
diverse biological functions present considerable opportunities
for exploiting HS or HS-protein conjugates for developing new
classes of anticancer (4), antiviral (5), and improved anticoagu-
lant drugs (6). Furthermore, a recent worldwide outbreak of
contaminated heparin underscores the needs for synthetic
heparins to replace those isolated from animal tissues (7).
Chemical synthesis is a powerful tool to obtain structurally
defined heparin/HS oligosaccharides. The most successful
example is the total synthesis of an antithrombin-binding pen-
tasaccharide (8). This pentasaccharide is marketed under the
* This work is supported, in whole or in part, by National Institutes of Health
Grants AI50050, HL094463, HL62244, HL096972, and GM38060. This work
was also supported by Grant-in-aid 0855424E from American Heart Asso-
ciation, MidAtlantic Affiliate.
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The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–6 and Tables 1 and 2.
1 .Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Rm 303, Beard Hall, Univer-
sity of North Carolina, Chapel Hill, NC 27599. Tel.: 919-843-6511; Fax: 919-
843-5432; E-mail: jian_liu@unc.edu.
3 The abbreviations used are: HS, heparan sulfate; AT, antithrombin; C5-epi,
C5-epimerase; ESI, electrospray ionization; GlcNTFA, trifluoroacetylglu-
cosamine; MS/MS, tandem MS; NST, N-sulfotransferase; OST, O-sulfotrans-
ferase; PAPS, 3Ј-phosphoadenosine 5Ј-phosphosulfate.
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Synthesis of Heparan Sulfate
FIGURE 1. Biosynthetic pathway of HS. The biosynthetic pathway includes the biosynthesis of polysaccharide backbone as well as the modification steps. The
synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS
polymerase. The backbone polysaccharide is then modified via five enzymatic modification steps. The modification site at each step is highlighted in a blue box.
EXPERIMENTAL PROCEDURES
(16). The resultant disaccharide 14 was dialyzed against water
using 1000 MWCO membrane (Spectrum).
Expression of HS Biosynthetic Enzymes—A total of nine
enzymes were used for the synthesis of HS oligosaccharides,
including HS sulfotransferases and glycosyl transferases. N-sul-
fotransferase (NST), C5-epi, 2-OST, 6-OST-1, 6-OST-3,
3-OST-1, 3-OST-5, and N-acetyl-D-glucosaminyl transferase of
E. coli K5 strain (KfiA) were expressed and purified as previ-
ously described (6, 13–16). PmHS2 was expressed as an N-ter-
minal fusion to His6 using a PET-15b vector (Novagen) (17).
The expression of pmHS2 was carried out in BL21 star (DE3)
cells (Invitrogen) coexpressing bacteria chaperone proteins,
GroEL and GroES.
To synthesize oligosaccharide backbone, disaccharide
(GlcUA-AnMan, 14) (4.5 ␮mol) was incubated with UDP-Glc-
NTFA (3.9 ␮mol) and KfiA (0.1 mg) in a 1 ml of buffer contain-
ing 25 mM Tris-HCl (pH 7.2) and 10 mM MgCl2. The reaction
was incubated at room temperature overnight. An aliquot of
reaction mixture was analyzed by a polyamine-based HPLC
column (from Waters) to ensure that Ͼ95% of UDP-GlcNTFA
was converted to UDP. Upon the complete consumption of
UDP-GlcNTFA, pmHS2 (0.5 mg) and UDP-GlcUA (4 ␮mol)
were added into the reaction mixture for additional 4–5 h at
room temperature. Another aliquot of pmHS2 (0.5 mg) and
UDP-GlcUA (4 ␮mol) was added to drive the transfer of GlcUA
Preparation of Disaccharide (GlcUA-AnMan, 14) and Oligo-
saccharide Backbones—The disaccharide 14 was prepared
from nitrous acid-degraded heparosan as previously described unit to completion. It is important to note that pmHS2 has both
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Synthesis of Heparan Sulfate
activities in transferring GlcNAc (or GlcNTFA) and GlcUA. 5 mM MgCl2, 200 ␮M dithiothreitol, 2.5 mM UTP, and 0.012
Without removal of UDP-GlcNTFA, pmHS2 led to polymeri- units/␮l of inorganic pyrophosphatase (Sigma-Aldrich).
zation or uncontrollable oligomerization of the saccharide, Recombinant GlmU was expressed in E. coli and purified by a
resulting in low yield of the tetrasaccharide product. The reac- Ni-agarose column (16). The UDP-GlcNTFA was purified by
tion mixture was resolved on a Bio-Gel P-10 column (0.75 ϫ removing proteins using centrifugal filters (10,000 MWCO;
200 cm), which was equilibrated with 20 mM Tris (pH 7.5) and Millipore) followed by the dialysis against water using 1000
1 M NaCl at a flow rate of 4 ml/h. The elution position of the MWCO membrane for 4 h. The product was confirmed by MS
3
tetrasaccharide was determined by a H-labeled tetrasaccha- analysis. The concentration was determined by a quantitative
ride. The product was dialyzed against water using 1000 analysis with PMAN-HPLC using UDP-GlcNAc as a standard.
MWCO membrane. The reaction cycle was repeated four and
Selective De-N-trifluoroacetylation of Oligosaccharides Car-
five times to prepare the decasaccharide 15, undecasaccharide rying GlcNTFA Units—Various amounts of oligosaccharides
(100–200 ␮g) were dried and resuspended in a solution (200 ␮l)
16, and dodecasaccharide 17.
Synthesis of Fluorous-tagged Disaccharide (GlcUA-AnMan- containing CH3OH, H2O, and (C2H5)3N (v/v/v ϭ 2:2:1). The
Rf, 13) and Tagged Oligosaccharide Backbones—A disaccharide reaction was incubated at room temperature (or at 37 °C for
(GlcUA-AnMannose) was also prepared from heparosan fol- fluorous-tagged octasaccharides) overnight. The samples were
lowed a procedure very similar to that described above, omit- dried and reconstituted in H2O to recover de-N-trifluoroacety-
ting the NaBH4 reduction step. To synthesize fluorous-tagged lated oligosaccharides.
disaccharide 13, the disaccharide (GlcUA-AnMannose) was
Preparation of Sulfated Oligosaccharide—N-Sulfation of oli-
incubated with 2 equivalent 4-(1H, 1H, 2H, 2H-perfluoropen- gosaccharide was carried out by incubating the de-N-trifluoro-
tyl) benzylamine hydrochloride (Fluorous Technologies) and acetylated oligosaccharide substrates with NST and 3Ј-phos-
NaBH3CN (10 eq) in MeOH overnight at room temperature. phoadenosine 5Ј-phosphosulfate. The reaction mixture
The resulting tagged disaccharide 13 was purified by a Flu- typically contained 6 ␮g de-N-trifluoroacetylated decasaccha-
oroFlash column, further purified using paper chromatography ride, undecasaccharide, and dodecasaccharide, 80 ␮M PAPS, 50
by Whatman 3MM chromatography paper (Fisher) developed mM MES, pH 7.0, 1% Triton X-100 (v/v), and 4 ␮g of NST in a
with 100% acetonitrile. Disaccharide 13 was finally purified by a total volume of 300 ␮l. The reaction mixture was incubated at
C18 column (0.46 ϫ 25 cm; Thermo Fisher Scientific) under 37 °C overnight.
reverse phase HPLC conditions. The column was eluted with a
The oligosaccharides were purified by a DEAE column. The
linear gradient from 90% solution A (0.1% trifluoroacetic acid in reaction mixture (300 ␮l) was mixed with 1 ml of 0.01% Triton
water) to 50% solution A for 40 min at a flow rate of 0.5 ml/min, X-100 buffer at pH 5.0 containing 150 mM NaCl, 50 mM
then followed by an additional wash for 20 min with 100% solu- NaOAc, 3 M urea, 1 mM EDTA, then followed by four washes
tion B (0.1% trifluoroacetic acid in acetonitrile) at a flow rate of with the same buffer, each time 1 ml, and was eluted with 1 M
0.5 ml/min. The product was confirmed by electrospray ioniza- NaCl in 0.001% Triton X-100 buffer. The purified oligosaccha-
tion (ESI) mass spectrometry.
rides were dialyzed using 2500 MWCO 3500 membrane and
To prepare the fluorous-tagged octasaccharides (1-4), the dried. The final product was further purified by a DEAE-NPR
synthesis was started with a fluorous-tagged disaccharide HPLC column (0.46 ϫ 7.5 cm; Tosohaas). For tagged octasac-
GlcUA-AnMan-Rf 13. In each monosaccharide incorporation charide (1–4), heptasaccharide 5, N-sulfo 6-O-sulfo decasac-
step, we supplied the reaction mixture with either KfiA or charide 9, and dodecasaccharide 10, and N-sulfo 6-O-sulfo
pmHS2 and appropriate UDP-monosaccharide donor. As a 3-O-sulfo decasaccharide 11 and dodecasaccharide 12, the pro-
result, only one sugar residue was transferred into the back- cedures were very similar to those for N-sulfo oligosaccharides
bone. The reaction mixture was assembled as for the unlabeled (6-8) as described above using appropriate enzymes.
oligosaccharide backbone synthesis as described above. A
HPLC Analysis—HPLC analysis of oligosaccharides followed
FluoroFlash column was used to separate the tagged oligosac- the procedures as previously described (6, 9).
charides from unreacted UDP-monosaccharides and enzymes.
Determination of the Binding Affinity of Oligosaccharides to
Briefly, Fluorous silica gel (40 ␮m; Fluorous Technologies) was Antithrombin (AT)—The dissociation constant (Kd) of each
washed with water and eluted with methanol. The reaction sample and AT was determined using affinity coelectrophoresis
cycle was repeated three times to prepare octasaccharide (18).
backbones.
Microdialysis of Oligosaccharides—The synthesized oligo-
Preparation of UDP-GlcNTFA—UDP-GlcNTFA was synthe- saccharides were subjected to microdialysis prior to the MS
sized using a chemoenzymatic approach, involved in preparing analysis. The dialysis was carried out using hollow fiber dialysis
a GlcNTFA 1-phosphate and coupling it with UDP. Briefly, 11 tubing (13,000 MWCO; Spectrum) against 20 mM ammonium
mg of GlcNH2 1-phosphate (Sigma-Aldrich) was dissolved in acetate.
200 ␮l of anhydrous methanol and mixed with 60 ␮l of
Liquid Chromatography-linked Mass Spectrometry (LC-MS)
(C2H5)3N and 130 ␮l of S-ethyl trifluorothioacetate (Sigma- Analysis and Mass Spectrometry (MS) Analysis—LC-MS anal-
Aldrich). The reaction was incubated at room temperature for yses were performed on an Agilent 1100 HPLC-MSD-Trap.
24 h. The resultant GlcNTFA 1-phosphate was then converted Nonsulfated backbone oligosaccharides were injected onto an
to UDP-GlcNTFA using glucosamine-1-phosphate acetyl- Aquasil C18 column (3 ␮m 2.1 ϫ 50 mm; Thermo Fisher). A
transferase/N-acetylglucosamine-1-phosphate
uridyltrans- gradient of acetonitrile with a flow rate of 0.4 ml/min was
ferase (GlmU) in a buffer containing 46 mM Tris-HCl (pH 7.0), directed into the ion trap mass spectrometer. The gradient con-
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Synthesis of Heparan Sulfate
FIGURE 2. Scheme for the synthesis of N-sulfo octasaccharides and a heptasaccharide carrying an IdoUA2S. A, steps involved in the synthesis of N-sulfo
octasaccharide library (1-4). The individual structure of 2, 1, 3, and 4 is also shown in Fig. 3C, supplemental Fig. 1, and supplemental Fig. 2, B and D,
respectively. B, steps involved in the synthesis of hepatasaccharide 5. The modification sites are either colored in blue or highlighted in filled boxes. The residue
thatisepimerizediscoloredinblue. Reagentsandrecoveryyieldofeachstepareasfollows:a, KfiA, UDP-GlcNAc(orUPD-GlcNTFA), pmHS2, andUDP-GlcUA. The
purification yield by fluorous column was 80%, whereas the purification yield without fluorous column (B) was about 40%. b, methanol/triethylamine/water
(2:1:2), NST, PAPS. Recovery yield was 40–50%. c, KfiA and UDP-GlcNTFA. Recovery yield was 50%. d, C5-epi/2-OST, PAPS. Recovery yield was 40%.
RESULTS
sisted of an initial 5-min hold at 90% aqueous (0.1% formic acid
in water), a change to 90% organic (0.1% formic acid in aceto-
nitrile) over 2 min, a 2-min hold at 90% organic, a change to 90%
aqueous over 2 min, and a 4-min reequilibration at 90% aque-
ous. Experiments were performed in positive ionization mode
for untagged backbone oligosaccharide. Alternatively, the anal-
yses were performed in negative ionization mode for tagged
backbone oligosaccharides. Under both conditions, the electro-
spray source set to 3000 V and 350 °C, and the compound sta-
bility was set to 30%. Nitrogen was used for both nebulizer (8
liters/min) and drying gas (45 p.s.i.). Helium was used for colli-
sion-induced dissociation. The MS and tandem MS (MS/MS)
data were acquired and processed using Bruker Trap software
4.1. All product ions in MS/MS data were labeled according to
the Domon-Costello nomenclature (19).
Enzymatic Synthesis of N-sulfo Oligosaccharides—A scheme
for the synthesis of octasaccharides (1–4) from a disaccharide
is shown in Fig. 2A, involving the use of glycosyltransferases,
UDP-monosaccharide donors, and NST. Elongation from the
disaccharide to the octasaccharide was achieved by two bacte-
rial glycosyltransferases: N-Acetylglucosaminyl transferase
(KfiA) from E. coli K5 (16) and heparosan synthase-2 (pmHS2)
from Pasteurella multocida (17). A fluorous affinity tag, 4-(1H,
1H, 2H, 2H-perfluoropentyl) benzylamine (Rf), at the reducing
end was introduced for the product purification. The fluorous
tag allowed easy isolation of the product with FluoroFlash affin-
ity chromatography and has absorbance at 260 nm that facili-
tated HPLC analysis during the preparation.
We designed a unique chemoenzymatic approach to build
the N-sulfo oligosaccharides using KfiA and pmHS2. An initial
attempt to transfer an N-unsubstituted glucosamine (GlcNH2)
residue from UPD-GlcNH2 failed because UDP-GlcNH2 was
not a substrate for KfiA. An unnatural monosaccharide donor,
UDP-GlcNTFA, was next used in the synthesis. We found that
UDP-GlcNTFA served as an excellent donor substrate for KfiA,
being efficiently incorporated. The product could be further
extended by pmHS2 following the backbone synthesis as shown
Oligosaccharides with sulfo groups were dissolved in 70%
acetonitrile and 10 ␮M imidazole. A syringe pump (Harvard
Apparatus) was used to introduce the sample via direct infusion
(10 ␮l/min) into an Agilent 1100 MSD Trap. Experiments were
performed 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 liters/min) and
drying gas (15 p.s.i.). The procedures for MS/MS analysis are
described above.
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FIGURE 3. Structural characterization of octasaccharide 2. A, HPLC of octasaccharide 2 using a C18 column under reverse-phase condition. B, MS of
octasaccharide 2. C, MS/MS of octasaccharide 2 (precursor ion selection at m/z 459.1). The fragmentation pattern is depicted in the top. The product ions in
MS/MS data were labeled according to the Domon-Costello nomenclature (19).
in Fig. 2A. The GlcNTFA residue was selectively deprotected C5-epi transforms a GlcUA residue to an IdoUA residue, and
under mild basic conditions, yielding GlcNH2 unit in the pres- 2-OST sulfates the 2-OH position of the IdoUA residue (step d,
ence of GlcNAc residues. The resulting GlcNH2 residue was Fig. 2B). It should be noted that it was essential to introduce a
converted to GlcNS using NST in the presence of PAPS, step b GlcNTFA residue at the nonreducing terminus of the heptasac-
Fig. 2A).
charide. This GlcNTFA residue blocks the action of C5-epi on
The efforts resulted in four different octasaccharide products the immediately adjacent GlcUA. C5-epi is known to act only
that differed by the location and the number of GlcNS residue: on GlcUA residues flanked by two GlcNS residues as in the
octasaccharide 1 (no GlcNS, supplemental Fig. 1), octasaccha- sequence, -GlcNS-GlcUA-GlcNS-, but does not act on the
ride 2 (two GlcNS residues, Fig. 3) and 3 and 4 (single GlcNS at GlcUA present in the sequence, -GlcNAc-GlcUA-GlcNS- (20).
different positions, supplemental Fig. 2). The structures of the
The purity analysis and MS spectrum of heptasaccharide 5
octasaccharides were determined by MS. For example, octasac- are shown in Fig. 4. A 35S-labeled heptasaccharide was initially
charide 2 was resolved as a symmetric peak by HPLC using a synthesized by incubating with 35S-labeled PAPS, and the prod-
C18 column (Fig. 3A). ESI-MS analysis revealed its molecular uct was resolved as a symmetric peak at the expected retention
mass to be 1839.6 Da in close agreement to the calculated mass time on anion exchange HPLC, suggesting that it was Ͼ90%
of 1839.5 Da (Fig. 3B). MS/MS analysis confirmed the position pure (Fig. 4A). Unlabeled heptasaccharide was then synthesized
of the GlcNS residues in 2 (Fig. 3C) from the two characteristic under identical conditions and purified by the HPLC. ESI-MS
daughter ions, Y5 (m/z, 1244.5) and B3 (m/z, 592.3), products of demonstrated that heptasaccharide 5 had a molecular mass of
the cleavage of an internal glycosidic linkage (see supplemental 1511.0 Da (calculated 1512.2 Da). These data are consistent
Figs. 1 and 2for structural analysis of 1, 3 and 4).
with a heptasaccharide carrying three sulfo groups and one Glc-
Synthesis of an Oligosaccharide Carrying an IdoUA2S Unit— NTFA residue.
The IdoUA2S residue is a critical structural motif involved in
The position of the IdoUA2S residue in heptasaccharide 5
binding to fibroblast growth factor to confer the cell prolifera- was confirmed by disaccharide analyses using 35S site-specifi-
tion activity of HS (6). Heptasaccharide 5 carrying one cally labeling techniques (supplemental Fig. 3, A and B). Only
IdoUA2S residue in its center was synthesized from a disaccha- IdoUA2S-AnMan disaccharide was observed from the nitrous
ride (Fig. 2B). The enzymatic synthesis of IdoUA2S residue acid-degraded 2-O-[35S]sulfated heptasaccharide, suggesting
involves the concerted action of C5-epi and 2-OST, where that the 2-O-sulfo group was present only at the IdoUA2S, and
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FIGURE4. Structuralcharacterizationofheptasaccharide5. A, elutionprofileofheptasaccharide5onpolyamine-basedHPLC. B, ESI-MSspectrumofpurified
heptasaccharide 5. Chemical structure of heptasaccharide 5 and the calculated molecular mass of the heptasaccharide are presented above A and B.
it was flanked by two GlcNS residues (supplemental Fig. 3A).
Conversion of the remaining GlcNTFA residues in decasac-
Two disaccharides of ⌬UA-GlcNS and ⌬UA2S-GlcNS were charide, undecasaccharide, and dodecasaccharide to GlcNS
observed from the N-[35S]sulfated heptasaccharide that was proceeded through detrifluoroacetylation and treatment with
degraded with heparin lyases (supplemental Fig. 3B). Further, NST using [35S]PAPS to afford N-[35S]sulfated products. The
the ratio of the resultant disaccharide (⌬UA-GlcNS/⌬UA2S- product afforded a single prominent peak at the expected reten-
GlcNS) was determined to be 1.0:0.8, very close to the theoret- tion time on high resolution DEAE-HPLC, suggesting that the
ical value of 1:1 expected for heparin lyase-degraded heptasac- products were of high purity (supplemental Fig. 4, A, C, and E).
charide 5. These data clearly demonstrate that the IdoUA2S The nonradioactive oligosaccharides (6-8) were next resynthe-
residue is located at the center of the heptasaccharide 5.
sized under identical conditions using NST and unlabeled
Synthesis of Oligosaccharides Carrying 6-O-Sulfo and 3-O- PAPS and purified by DEAE-HPLC. The ESI-MS analyses of the
Sulfo Groups—We next examined the introduction of 6-O- oligosaccharides (supplemental Fig. 4, B, D, and F) confirm that
sulfo and 3-O-sulfo groups into the HS oligosaccharides using all three compounds are fully N-sulfated with the structures
O-sulfotransferases (Fig. 5). To that end, oligosaccharides with shown in Fig. 5.
N-sulfo groups (6–8, Fig. 5) needed to be first synthesized.
Next, we introduced 6-O-sulfo groups using a mixture of
Disaccharide acceptor 14, a nontagged disaccharide primer, 6-OST isoform 1 (6-OST-1) and 6-OST isoform 3 (6-OST-3)
was extended to decasaccharide, undecasaccharide, and dode- affording the N-sulfo-6-O-sulfo decasaccharide 9 and N-sulfo-
casaccharide, respectively, using UDP-GlcNTFA and UDP- 6-O-sulfo dodecasaccharide 10 (Fig. 5). DEAE-HPLC analysis
GlcUA donors. The structures of the backbone oligosaccha- of the 35S-labeled oligosaccharide displayed a prominent sym-
rides were confirmed by LC-MS (supplemental Table 1). In metric 35S peak at the expected retention time, suggesting that
eight enzymatic steps at the milligram scale, a disaccharide was the preparation was pure (supplemental Fig. 5, A and C). Non-
converted to decasaccharide 15. Further extension to unde- radioactive decasaccharide 9 and dodecasaccharide 10 were
casaccharide 16 and dodecasaccharide 17 was also highly effec- next resynthesized under identical conditions using PAPS,
tive. Although some unexpected partial detrifluoroacetylation purified by DEAE-HPLC, and subjected to MS analysis. ESI-MS
occurred during product purification, this did not impact the analysis of each product revealed molecular masses of 2329.9
synthesis of N-sulfated oligosaccharides because the next step Da and 2828.1 Da, consistent with the structure of decasaccha-
required complete detrifluoroacetylation.
ride 9 having 8 sulfates and dodecasaccharide 10 having 10
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FIGURE 5. Scheme for the synthesis of deca-, undeca-, and dodecasaccharides. The reaction sites are either colored in blue or highlighted in filled boxes. Different
sizes of oligosaccharides are represented R, where R ϭ H (decasaccharide), R ϭ -GlcNTFA/NS (undecasaccharide), and R ϭ GlcUA-GlcNTFA/NS (dodecasaccharide).
Reagents and recovery yield of each step are as follows: a, KfiA, UDP-GlcNAc (or UPD-GlcNTFA), pmHS2, and UDP-GlcUA. The purification yield by was at 35% on
average in the first four cycles. When the oligosaccharide was extended beyond to decasaccharide, the recovery from the Bio-Gel P-10 reached 75–80%. b, methanol/
triethylamine/water (2:1:2), NST, PAPS. Recovery yield was 25–30%. d, 6-OST-1/6-OST-3, PAPS. Recovery yield was 30%. e, 3-OST-1/3-OST-5, PAPS. Recovery yield was
10%. Oligosaccharide 15 represents a N-detrifluoroacetylated decasaccharide, 16 represents an N-detrifluoroacetylated undecasaccharide, and 17 represents a
N-detrifluoroacetylated dodecasaccharide. Oligosaccharides 6, 7, and 8 represent N-sulfo decasaccharide, N-sulfo undecasaccharide, and N-sulfo dodecasaccharide,
respectively. Only decasaccharide and dodecasaccharide proceeded to make N-sulfo and 6-O-sulfo oligosaccharides (9 and 10).
sulfates (calculated 2329.9 Da and 2827.3 Da). Disaccharide tion of the synthesis of dodecasaccharide 12 (Fig. 5 and sup-
analyses of the labeled products, carrying 6-O-[35S]sulfo plemental Fig. 6).
groups, confirmed the structures of decasaccharide 9 and dode-
It should be noted that a domain consisting of multiple
casaccharide 10 (supplemental Table 2). Disaccharide analysis repeats of GlcUA-GlcNS3S6S in 11 and 12 has not been iden-
is a commonly used approach to analyze the structures of HS tified in the HS isolated from natural sources. A recent finding
oligosaccharides (21, 22).
of high abundance of 3-O-sulfated glucosamine residue of the
Incubation of decasaccharide 9 and dodecasaccharide 10 HS isolated from human follicular fluid raised the possibility
with both 3-OST isoform 1 (3-OST-1) and 3-OST isoform 5 that this particular HS contains domain structures similar to
(3-OST-5) afforded decasaccharide 11 and dodecasaccharide those found in 11 and 12 (23). Unlike HS, 11 and 12 do not
12, respectively (Fig. 5). In each case, a mixture of two major contain the IdoUA2S-GlcNS motif. It is known that 3-OST-1
products was obtained as analyzed by DEAE-HPLC (Fig. 6A and acts on GlcUA (or IdoUA)-GlcNS6S sequences, but not on
supplemental Fig. 6A), leading to a reduced yield for the target IdoUA2S-GlcNS sequences (15, 24, 25). It is possible that the
products. Consequently, we could not obtain sufficient amount presence of IdoUA2S plays a role in down-regulating the level
of these products for MS analysis. Instead, to assign structure of 3-O-sulfation.
we decided to use the site-specific [35S]sulfo-labeled technique
Determination of AT-binding Affinity of O-Sulfooligo-
followed by disaccharide analysis as illustrated in Fig. 6. The saccharides—We next measured the binding affinity of the syn-
6-O-[35S]sulfated decasaccharide 9 was first prepared. The 3- thesized oligosaccharides to AT. The AT binding correlates to
O-sulfo groups were then introduced using 3-OST-1 and HS anticoagulant activity. We previously demonstrated that an
3-OST-5 with unlabeled PAPS to afford 6-O-[35S]sulfo-labeled AT-binding HS does not require the presence of iduronic acid
decasaccharide 11 (Fig. 6D). The two 35S-labeled products, or 2-O-sulfoiduronic acid residues (6), which greatly simplifies
labeled as Peak I and Peak II, were separated by the DEAE- the synthesis of anticoagulant HS. However, the minimum
HPLC (Fig. 6A). Nitrous acid resulted in disaccharides that length and the precise structure of this novel AT-binding
were analyzed by RPIP-HPLC (Fig. 6, B and C). Nitrous acid domain are not known. We hypothesized that decasaccharide
treatment of Peak I (Fig. 6E) afforded two 35S-labeled disaccha- 11 and dodecasaccharide 12 would provide insights on the
rides assigned to GlcUA-AnMan6S and GlcUA-AnMan3S6S in structural requirement for this AT-binding site.
the ratio of 1:3.4, suggesting that Peak I carried three 3-O-
The AT-binding affinities of oligosaccharides 10, 11, and 12
sulfo groups. Nitrous acid-treated Peak II (Fig. 6F) afforded a were determined (Table 1). Dodecasaccharide 10, having no
single type of 35S-labeled disaccharide, assignable to GlcUA- 3-O-sulfo groups, exhibited a Kd of Ͼ100 ␮M. Dodecasaccha-
AnMan3S6S, suggesting that Peak II was the anticipated ride 12, having 3-O-sulfo groups, showed the Kd to be 145 nM,
fully 3-O-sulfo decasaccharide shown in Fig. 5. Using the close to that of a full-length HS polysaccharide (57 nM) (6). The
same analytical approach, we also demonstrated the comple- size dependence of this interaction was clearly demonstrated by
34246 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 44•OCTOBER 29, 2010

Synthesis of Heparan Sulfate
FIGURE 6. Determination of the structures of 3-O-sulfo decasaccharide (11). A, HPLC of 6-O-[35S]sulfo-labeled decasaccharide 11 using a DEAE-HPLC
column. Thedecasaccharidewaspreparedbyincubatingdecasaccharide9with3-OST-1and3-OST-5asdepictedinD. Twomajorproducts, designatedasPeak
I and Peak II, were observed. Both components were purified and subjected to disaccharide analysis. B, HPLC of disaccharide analysis of Peak I using a C18
column under RPIP-HPLC conditions. The chemical reaction involved in the disaccharide analysis of Peak I is shown in E. C, HPLC of the disaccharide analysis of
Peak II using a C18 column under RPIP-HPLC conditions. F, reaction involved in the disaccharide analysis of Peak II. The radioactively labeled 6-O-sulfo group is
colored in red. The potential modification sites modified by 3-OSTs are highlighted in blue boxes. Only the disaccharide products carrying 6-O-[35S]sulfo groups
were detected from nitrous acid degradation and are shown in E and F.
TABLE 1
dependence of this interaction (26). Dodecasaccharide 12 binds
Binding affinity of oligosaccharides to AT
AT with lower affinity than the commercial pentasaccharide
The binding affinity of the oligosaccharides to AT was determined using affinity
drug, Arixtra. It is possible that a lower binding affinity to AT is
coelectrophoresis.
because an IdoUA2S residue is absent in the structure.
Substrates
Proposed structure
Kd
nM
DISCUSSION
11
(GlcUA-GlcNS3S6S)4-GlcUA-AnMan
(GlcUA-GlcNS3S6S)5-GlcUA-AnMan
(GlcUA-GlcNS6S)5 -GlcUA-AnMan
515 Ϯ 40
145 Ϯ 24
Ͼ100,000
33
12
In this paper, we demonstrated the feasibility of synthesizing
structurally defined oligosaccharides from a simple starting
material. The critical advance is the ability to be able to convert
a disaccharide to oligosaccharides carrying N-sulfo groups. The
placement of the GlcNS residue is believed to be critical in
modulating the susceptibilities to the subsequent O-sulfation
10
Arixtraa
GlcNS6S-GlcUA-GlcNS3S6S-IdoUA2S-GlcNS6S-OMe
Recomparinb Polysaccharide, no defined structure
57
The 35S-labeled Arixtra was prepared as described in a paper published previously (33).
a
b
The binding affinity of Recomparin to AT was taken from our previous publication
(6).
decasaccharide 11 having a Kd of 515 nM, representing consid- and epimerization (27). Although there are numerous ways to
erably weaker AT-binding affinity. These results both confirm prepare the oligosaccharide with the repeating unit of
the critical role of 3-O-sulfo groups for AT-binding and the size (-GlcUA-GlcNAc-) (16, 28), the product cannot be used
OCTOBER 29, 2010•VOLUME 285•NUMBER 44
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Synthesis of Heparan Sulfate
directly for the subsequent enzymatic modifications to prepare site (15, 33). Unlike 3-O-sulfotransferase, 6-O-sulfotransferase
HS due to the lack of N-sulfoglucosamine (GlcNS) residue. In
vivo, the GlcNS residue is synthesized by N-deacetylase/N-sul-
fotransferase (29). However, it is unclear how to selectively con-
vert a GlcNAc residue to a GlcNS residue when multiple
GlcNAc residues are present in an oligosaccharide using
N-deacetylase/N-sulfotransferase in vitro. Therefore, synthesis
of oligosaccharides having defined N-sulfation positions is a
critical step in developing a route for the controlled enzymatic
synthesis of HS oligosaccharides and polysaccharides. As an
alternative approach to the NST-PAPS method, N-sulfation
can be also carried out with a chemical approach using
SO3ꢀpyridine. We observe that NST-PAPS system affords com-
plete N-sulfation with absolute selectivity for the amino group.
Furthermore, the costs associated with enzymatic N- and
O-sulfation might be substantially reduced through the enzy-
matic synthesis of PAPS.
isoforms appear to have same substrate specificities (34), sug-
gesting than an additional strategy will be needed to introduce a
6-O-sulfo group in a specific position.
The synthetic scale of our method is ultimately determined
by the amount of enzymes and substrates. A large scale enzy-
matic synthesis of a targeted oligosaccharide is underway. We
have observed no limitation in the scale-up of the enzymes and
substrates, particularly in the laboratory scale synthesis. Fur-
thermore, the conversion at each enzymatic step is nearly quan-
titative, and the loss of the sample largely occurs in the purifi-
cation steps. Thus, the yield will undoubtedly be improved by
combining several sulfotransferases in one pot to reduce the
number of purification steps.
The structural analysis of HS oligosaccharides still remains a
major roadblock for the complex HS synthesis. We demon-
strated the use of MS/MS to conduct the sequence analysis of
N-sulfo octasaccharides. However, this method is not suffi-
ciently reliable in pinpointing the sulfo groups in an oligosac-
charide having a complex sulfation pattern. The analysis of HS
oligosaccharides by MS is further complicated as desulfation
occurs often during analysis (35, 36). It should be noted that the
lack of structurally defined oligosaccharide standards also hin-
ders the efforts for developing the techniques for analyzing HS.
In summary, the current study demonstrates the feasibility of
total synthesis of structurally defined HS oligosaccharides
using a chemoenzymatic approach. This method is capable of
synthesizing oligosaccharides with different sulfation patterns
and sizes by transferring UDP-sugars onto a readily available
disaccharide acceptor. A key advance involves the utilization of
an unnatural UDP-monosaccharide donor that allows the con-
trolled placement of GlcNS and GlcNAc residues throughout
the oligosaccharide backbone. A recent report describes 12 HS
tetrasaccharides prepared using a modular chemical synthesis
(37). It is somewhat difficult to compare the efficiency of chem-
ical synthesis and chemoenzymatic synthesis quantitatively
because each method targeted to different oligosaccharide
products. However, it is noteworthy that the chemical synthesis
of IdoUA2S requires at least eight synthetic steps (37). Only a
single step is required utilizing C5-epi and 2-OST with about
40% yield (step d, Fig. 2B). Moreover, the targets synthesized in
the current study are considerably larger than those synthe-
sized chemically. However, chemical synthesis offers the bene-
fits of the preparation of unnatural structural motif, which can-
not be accomplished by enzymes due to the restrictions in the
substrate specificity. Therefore, an effective combination of
chemical and chemoenzymatic methods will further optimize
We demonstrate a convincing example for utilizing an
unnatural UDP-monosaccharide to expand the capability of
using glycosyltransferase to synthesize the specialized oligosac-
charides. A review by Boons and colleagues suggests that the
scope of the use of glycosyltransferases for the synthesis of oli-
gosaccharide is limited due to the high donor and acceptor
substrate specificities (30). We demonstrate for the first time
that KfiA is capable of using UDP-GlcNTFA as a donor sub-
strate in high efficiency. The use of the unnatural monosaccha-
ride donor empowers the placement of a GlcNS residue at any
desired position in the oligosaccharide backbone. We also dem-
onstrate that pmHS-2 can catalyze the extension of an oligosac-
charide that has a GlcNTFA unit at its nonreducing end. We
have developed an effective approach to introduce N-sulfoglu-
cosamine units into specific locations within a given octasac-
charide. Furthermore, we demonstrate the synthesis of defined
heparan sulfate oligosaccharides from these N-sulfo oligosac-
charides. Although the use of UDP-GlcNTFA and KfiA for the
synthesis of HS is novel, it should be noted that the demonstra-
tion of the synthesis of UDP-GlcNTFA and use as a donor sub-
strate for the core-2 GlcNAc transferase was previously
reported by Sala et al. (31). Our method can also be used to
conduct parallel synthesis through the introduction of a
fluorous affinity tag. The fluorous affinity-tagged technique has
been previously employed for preparing tagged monosaccha-
ride conjugates for carbohydrate-based microarray to deter-
mine the structural specificities of the interactions of proteins
and carbohydrates (32).
Whether our method is able to control all sulfation types
remains to be investigated. The control of the N-sulfation can
be readily achieved. The method also appears to be capable of
placing an IdoUA2S residue based on the distribution of GlcNS the synthesis. Structurally defined HS will be employed to inter-
residue. Although we demonstrated that 6-O- and 3-O-sulfa-
tion can proceed to completion in a given oligosaccharide, it is
unclear how to place a single 6-O- or 3-O-sulfo group in the
context of repeating unit of -GlcUA-GlcNS-. We suspect that
the presence of IdoUA2S influences the placement of 3-O-sulfo
group. This may ultimately be controlled by relying on the sub-
strate specificities of different 3-O-sulfotransferase isoforms.
Each isoform is believed to exhibit unique substrate specificities
to recognize the saccharide structures around the modification
rogate structure and activity relationship studies in the new
field of HS glycomics. These results also open up the possibility
of discovering novel HS-based anticoagulant drugs as well as
other HS-based therapeutic agents.
Acknowledgments—We thank H. N. Bethea and E. Pempe for review-
ing the manuscript and Q. Shi for assistance in the initial preparation
of fluorous-tagged disaccharide.
34248 JOURNAL OF BIOLOGICAL CHEMISTRY
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Synthesis of Heparan Sulfate
REFERENCES
19. Domon, B., and Costello, C. E. (1988) Glycoconj. J. 5, 397–409
20. Conrad, H. E. (1998) Heparin-binding Proteins, pp. 34–47, Academic
Press, San Diego, CA
1. Peterson, S. P., Frick, A., and Liu, J. (2009) Nat. Prod. Rep. 26, 610–627
2. Gama, C. I., Tully, S. E., Sotogaku, N., Clark, P. M., Rawat, M., Vaidehi, N.,
Goddard, W. A., 3rd, Nishi, A., and Hsieh-Wilson, L. C. (2006) Nat. Chem.
Biol. 2, 467–473
21. Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R. (1999)
Science 286, 537–542
22. Lawrence, R., Lu, H., Rosenberg, R. D., Esko, J. D., and Zhang, L. (2008)
Nat. Methods 5, 291–292
3. de Paz, J. L., and Seeberger, P. H. (2008) Mol. Biosyst. 4, 707–711
4. Shriver, Z., Raguram, S., and Sasisekharan, R. (2004) Nat. Rev. Drug Discov.
3, 863–873
23. de Agostini, A. I., Dong, J. C., de Vante´ry Arrighi, C., Ramus, M. A., Den-
tand-Quadri, I., Thalmann, S., Ventura, P., Ibecheole, V., Monge, F., Fis-
cher, A. M., HajMohammadi, S., Shworak, N. W., Zhang, L., Zhang, Z., and
Linhardt, R. J. (2008) J. Biol. Chem. 283, 28115–28124
24. Liu, J., Shworak, N. W., Sinay¨, P., Schwartz, J. J., Zhang, L., Fritze, L. M.,
and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185–5192
25. Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J. P., Malmstro¨m, A., Shukla, D., and
Liu, J. (2002) J. Biol. Chem. 277, 37912–37919
5. Baleux, F., Loureiro-Morais, L., Hersant, Y., Clayette, P., Arenzana-Seisd-
edos, F., Bonnaffe´, D., and Lortat-Jacob, H. (2009) Nat. Chem. Biol. 5,
743–748
6. Chen, J., Jones, C. L., and Liu, J. (2007) Chem. Biol. 14, 986–993
7. Liu, H., Zhang, Z., and Linhardt, R. J. (2009) Nat. Prod. Rep. 26, 313–321
8. Petitou, M., and van Boeckel, C. A. (2004) Angew. Chem. Int. Ed. Engl. 43,
3118–3133
9. Chen, J., Avci, F. Y., Mun˜oz, E. M., McDowell, L. M., Chen, M., Pedersen,
L. C., Zhang, L., Linhardt, R. J., and Liu, J. (2005) J. Biol. Chem. 280,
42817–42825
26. Atha, D. H., Lormeau, J. C., Petitou, M., Rosenberg, R. D., and Choay, J.
(1985) Biochemistry 24, 6723–6729
27. Esko, J. D., and Selleck, S. B. (2002) Annu. Rev. Biochem. 71, 435–471
28. Sigulinsky, C., Babu, P., Victor, X. V., and Kuberan, B. (2010) Carbohydr.
Res. 345, 250–256
10. Zhang, Z., McCallum, S. A., Xie, J., Nieto, L., Corzana, F., Jime´nez-Bar-
bero, J., Chen, M., Liu, J., and Linhardt, R. J. (2008) J. Am. Chem. Soc. 130,
12998–13007
29. Liu, J., and Pedersen, L. C. (2007) Appl. Microbiol. Biotechnol. 74, 263–272
30. Boltje, T. J., Buskas, T., and Boons, G. J. (2009) Nat. Chem. 1, 611–622
31. Sala, R. F., MacKinnon, S. L., Palcic, M. M., and Tanner, M. E. (1998)
Carbohydr. Res. 306, 127–136
11. Kuberan, B., Lech, M. Z., Beeler, D. L., Wu, Z. L., and Rosenberg, R. D.
(2003) Nat. Biotechnol. 21, 1343–1346
12. Copeland, R., Balasubramaniam, A., Tiwari, V., Zhang, F., Bridges, A.,
Linhardt, R. J., Shukla, D., and Liu, J. (2008) Biochemistry 47, 5774–5783
13. Kakuta, Y., Sueyoshi, T., Negishi, M., and Pedersen, L. C. (1999) J. Biol.
Chem. 274, 10673–10676
32. Jaipuri, F. A., Collet, B. Y., and Pohl, N. L. (2008) Angew. Chem. Int. Ed.
Engl. 47, 1707–1710
33. Pope, M., Raska, C. S., Thorp, S. C., and Liu, J. (2001) Glycobiology 11,
505–513
14. Edavettal, S. C., Lee, K. A., Negishi, M., Linhardt, R. J., Liu, J., and Pedersen,
L. C. (2004) J. Biol. Chem. 279, 25789–25797
34. Smeds, E., Habuchi, H., Do, A. T., Hjertson, E., Grundberg, H., Kimata, K.,
Lindahl, U., and Kusche-Gullberg, M. (2003) Biochem. J. 372, 371–380
35. Zaia, J., and Costello, C. E. (2003) Anal. Chem. 75, 2445–2455
36. Przybylski, C., Gonnet, F., Bonnaffe´, D., Hersant, Y., Lortat-Jacob, H., and
Daniel, R. (2010) Glycobiology 20, 224–234
15. Xu, D., Moon, A. F., Song, D., Pedersen, L. C., and Liu, J. (2008) Nat. Chem.
Biol. 4, 200–202
16. Chen, M., Bridges, A., and Liu, J. (2006) Biochemistry 45, 12358–12365
17. Sismey-Ragatz, A. E., Green, D. E., Otto, N. J., Rejzek, M., Field, R. A., and
DeAngelis, P. L. (2007) J. Biol. Chem. 282, 28321–28327
18. Lee, M. K., and Lander, A. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,
2768–2772
37. Arungundram, S., Al-Mafraji, K., Asong, J., Leach, F. E., 3rd, Amster, I. J.,
Venot, A., Turnbull, J. E., and Boons, G. J. (2009) J. Am. Chem. Soc. 131,
17394–17405
OCTOBER 29, 2010•VOLUME 285•NUMBER 44
JOURNAL OF BIOLOGICAL CHEMISTRY 34249

Glycobiology and Extracellular Matrices:
Chemoenzymatic Design of Heparan
Sulfate Oligosaccharides
Renpeng Liu, Yongmei Xu, Miao Chen,
Michel Weïwer, Xianxuan Zhou, Arlene S.
Bridges, Paul L. DeAngelis, Qisheng Zhang,
Robert J. Linhardt and Jian Liu
J. Biol. Chem. 2010, 285:34240-34249.
doi: 10.1074/jbc.M110.159152 originally published online August 21, 2010
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