Chemoenzymatic design of heparan sulfate oligosaccharides.

Abstract

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 sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Full text

Page 1

Chemoenzymatic Design of Heparan Sulfate
Oligosaccharides*□
Receivedforpublication,June28,2010,andinrevisedform,August9,2010 Published,JBCPapersinPress,August21,2010,DOI10.1074/jbc.M110.159152
Renpeng Liu‡1, Yongmei Xu‡1, Miao Chen‡, Michel Weïwer§, Xianxuan Zhou‡, Arlene S. Bridges¶, Paul L. DeAngelis?,
Qisheng Zhang‡, Robert J. Linhardt§, and Jian Liu‡2
Fromthe‡DivisionofMedicinalChemistryandNaturalProducts,EshelmanSchoolofPharmacy,and¶DepartmentofPathology
andLaboratoryofMedicine,UniversityofNorthCarolina,ChapelHill,NorthCarolina27599,the§DepartmentofChemistryand
ChemicalBiology,RensselaerPolytechnicInstitute,Troy,NewYork12180,andthe?DepartmentofBiochemistryandMolecular
Biology,UniversityofOklahomaHealthSciencesCenter,Oklahoma City, Oklahoma 73104
S
Heparan sulfate is a sulfated glycan that exhibits essential
physiologicalfunctions.Interrogationofthespecificityofhepa-
ransulfate-mediatedactivitiesdemandsalibraryofstructurally
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.Thismethodoffersagenericapproachtopreparehepa-
ran sulfate oligosaccharides possessing predictable structures.
Heparan sulfate (HS)3is 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-
patesinregulatingbloodcoagulation,embryonicdevelopment,
andtheinflammatoryresponseandassistsviral/bacterialinfec-
tions. It consists of a repeating disaccharide unit of glucuronic
acid (GlcUA) or iduronic acid (IdoUA) and glucosamine, both
capableofcarryingsulfogroups(1).ThesulfationpatternofHS
dictatesitsbiologicalactivity(2,3).Heparin,awidelyusedanti-
coagulant drug, is a specialized form of highly sulfated HS. The
diversebiologicalfunctionspresentconsiderableopportunities
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
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-
logicalevaluation(8).Anenzyme-basedmethodoffersaprom-
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-.
ThebackboneisthenmodifiedbyN-deacetylase/N-sulfotrans-
ferase (having two separate domains exhibiting the activity of
N-deacetylaseandN-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).Thesemethodsdescribesyntheticstrategiesthattargeteda
single product from a specialized oligosaccharide starting
material.Theseapproachesclearlylacktheabilityforrapidsyn-
thesis of HS oligosaccharides differing in sizes and structures
due to the difficulty in obtaining the starting materials. There-
fore,atechniquecombiningtheoligosaccharidebackbonesyn-
thesis with saccharide modifications, using HS biosynthetic
enzymes, should expand the capability of HS oligosaccharide
synthesis.Inthispaper,weutilizebacterialglycosyltransferases
and an unnatural UDP-monosaccharide donor to build an oli-
gosaccharide backbone from a disaccharide. These backbone
oligosaccharidescanbethenselectivelymodifiedwithdifferent
HSsulfotransferasesandC5-epitopreparestructurallydefined
products with desired sulfation patterns. Further, this method
was employed to identify a novel HS structure that binds to
antithrombin.
* 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.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Figs. 1–6 and Tables 1 and 2.
1 .Both authors contributed equally to this work.
2Towhomcorrespondenceshouldbeaddressed:Rm303,BeardHall,Univer-
sity of North Carolina, Chapel Hill, NC 27599. Tel.: 919-843-6511; Fax: 919-
843-5432; E-mail: jian_liu@unc.edu.
3The 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, 3?-phosphoadenosine 5?-phosphosulfate.
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.
34240 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 44•OCTOBER 29, 2010
at RENSSELAER POLYTECHNIC IN, on October 29, 2010
www.jbc.org
Downloaded from
http://www.jbc.org/content/suppl/2010/09/10/M110.159152.DC1.html
Supplemental Material can be found at:

Page 2

EXPERIMENTAL PROCEDURES
Expression of HS Biosynthetic Enzymes—A total of nine
enzymes were used for the synthesis of HS oligosaccharides,
includingHSsulfotransferasesandglycosyltransferases.N-sul-
fotransferase (NST), C5-epi, 2-OST, 6-OST-1, 6-OST-3,
3-OST-1,3-OST-5,andN-acetyl-D-glucosaminyltransferaseof
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 His6using 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.
Preparation of Disaccharide (GlcUA-AnMan, 14) and Oligo-
saccharide Backbones—The disaccharide 14 was prepared
from nitrous acid-degraded heparosan as previously described
(16). The resultant disaccharide 14 was dialyzed against water
using 1000 MWCO membrane (Spectrum).
To synthesize oligosaccharide backbone, disaccharide
(GlcUA-AnMan, 14) (4.5 ?mol) was incubated with UDP-Glc-
NTFA(3.9?mol)andKfiA(0.1mg)ina1mlofbuffercontain-
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 ?95% 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)wasaddedtodrivethetransferofGlcUA
unittocompletion.ItisimportanttonotethatpmHS2hasboth
FIGURE1.BiosyntheticpathwayofHS.Thebiosyntheticpathwayincludesthebiosynthesisofpolysaccharidebackboneaswellasthemodificationsteps.The
synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS
polymerase.Thebackbonepolysaccharideisthenmodifiedviafiveenzymaticmodificationsteps.Themodificationsiteateachstepishighlightedinabluebox.
SynthesisofHeparanSulfate
OCTOBER 29, 2010•VOLUME 285•NUMBER 44JOURNAL OF BIOLOGICAL CHEMISTRY 34241
at RENSSELAER POLYTECHNIC IN, on October 29, 2010
www.jbc.org
Downloaded from

Page 3

activities in transferring GlcNAc (or GlcNTFA) and GlcUA.
Without removal of UDP-GlcNTFA, pmHS2 led to polymeri-
zation or uncontrollable oligomerization of the saccharide,
resulting in low yield of the tetrasaccharide product. The reac-
tion mixture was resolved on a Bio-Gel P-10 column (0.75 ?
200 cm), which was equilibrated with 20 mM Tris (pH 7.5) and
1 M NaCl at a flow rate of 4 ml/h. The elution position of the
tetrasaccharide was determined by a3H-labeled tetrasaccha-
ride. The product was dialyzed against water using 1000
MWCO membrane. The reaction cycle was repeated four and
five times to prepare the decasaccharide 15, undecasaccharide
16, and dodecasaccharide 17.
Synthesis of Fluorous-tagged Disaccharide (GlcUA-AnMan-
Rf,13)andTaggedOligosaccharideBackbones—Adisaccharide
(GlcUA-AnMannose) was also prepared from heparosan fol-
lowed a procedure very similar to that described above, omit-
ting the NaBH4reduction step. To synthesize fluorous-tagged
disaccharide 13, the disaccharide (GlcUA-AnMannose) was
incubated with 2 equivalent 4-(1H, 1H, 2H, 2H-perfluoropen-
tyl) benzylamine hydrochloride (Fluorous Technologies) and
NaBH3CN (10 eq) in MeOH overnight at room temperature.
The resulting tagged disaccharide 13 was purified by a Flu-
oroFlashcolumn,furtherpurifiedusingpaperchromatography
by Whatman 3MM chromatography paper (Fisher) developed
with100%acetonitrile.Disaccharide13wasfinallypurifiedbya
C18column (0.46 ? 25 cm; Thermo Fisher Scientific) under
reverse phase HPLC conditions. The column was eluted with a
lineargradientfrom90%solutionA(0.1%trifluoroaceticacidin
water)to50%solutionAfor40minataflowrateof0.5ml/min,
thenfollowedbyanadditionalwashfor20minwith100%solu-
tion B (0.1% trifluoroacetic acid in acetonitrile) at a flow rate of
0.5ml/min.Theproductwasconfirmedbyelectrosprayioniza-
tion (ESI) mass spectrometry.
To prepare the fluorous-tagged octasaccharides (1-4), the
synthesis was started with a fluorous-tagged disaccharide
GlcUA-AnMan-Rf 13. In each monosaccharide incorporation
step, we supplied the reaction mixture with either KfiA or
pmHS2 and appropriate UDP-monosaccharide donor. As a
result, only one sugar residue was transferred into the back-
bone. The reaction mixture was assembled as for the unlabeled
oligosaccharide backbone synthesis as described above. A
FluoroFlash column was used to separate the tagged oligosac-
charides from unreacted UDP-monosaccharides and enzymes.
Briefly, Fluorous silica gel (40 ?m; Fluorous Technologies) was
washed with water and eluted with methanol. The reaction
cycle was repeated three times to prepare octasaccharide
backbones.
PreparationofUDP-GlcNTFA—UDP-GlcNTFAwassynthe-
sized using a chemoenzymatic approach, involved in preparing
a GlcNTFA 1-phosphate and coupling it with UDP. Briefly, 11
mg of GlcNH21-phosphate (Sigma-Aldrich) was dissolved in
200 ?l of anhydrous methanol and mixed with 60 ?l of
(C2H5)3N and 130 ?l of S-ethyl trifluorothioacetate (Sigma-
Aldrich). The reaction was incubated at room temperature for
24 h. The resultant GlcNTFA 1-phosphate was then converted
to UDP-GlcNTFA using glucosamine-1-phosphate acetyl-
transferase/N-acetylglucosamine-1-phosphate
ferase (GlmU) in a buffer containing 46 mM Tris-HCl (pH 7.0),
uridyltrans-
5 mM MgCl2, 200 ?M dithiothreitol, 2.5 mM UTP, and 0.012
units/?lofinorganic pyrophosphatase
Recombinant GlmU was expressed in E. coli and purified by a
Ni-agarose column (16). The UDP-GlcNTFA was purified by
removing proteins using centrifugal filters (10,000 MWCO;
Millipore) followed by the dialysis against water using 1000
MWCO membrane for 4 h. The product was confirmed by MS
analysis. The concentration was determined by a quantitative
analysis with PMAN-HPLC using UDP-GlcNAc as a standard.
Selective De-N-trifluoroacetylation of Oligosaccharides Car-
rying GlcNTFA Units—Various amounts of oligosaccharides
(100–200?g)weredriedandresuspendedinasolution(200?l)
containing CH3OH, H2O, and (C2H5)3N (v/v/v ? 2:2:1). The
reaction was incubated at room temperature (or at 37 °C for
fluorous-tagged octasaccharides) overnight. The samples were
driedandreconstitutedinH2Otorecoverde-N-trifluoroacety-
lated oligosaccharides.
Preparation of Sulfated Oligosaccharide—N-Sulfation of oli-
gosaccharide was carried out by incubating the de-N-trifluoro-
acetylated oligosaccharide substrates with NST and 3?-phos-
phoadenosine 5?-phosphosulfate. The reaction mixture
typically contained 6 ?g de-N-trifluoroacetylated decasaccha-
ride,undecasaccharide,anddodecasaccharide,80?MPAPS,50
mM MES, pH 7.0, 1% Triton X-100 (v/v), and 4 ?g of NST in a
total volume of 300 ?l. The reaction mixture was incubated at
37 °C overnight.
The oligosaccharides were purified by a DEAE column. The
reaction mixture (300 ?l) was mixed with 1 ml of 0.01% Triton
X-100 buffer at pH 5.0 containing 150 mM NaCl, 50 mM
NaOAc, 3 M urea, 1 mM EDTA, then followed by four washes
with the same buffer, each time 1 ml, and was eluted with 1 M
NaCl in 0.001% Triton X-100 buffer. The purified oligosaccha-
rides were dialyzed using 2500 MWCO 3500 membrane and
dried. The final product was further purified by a DEAE-NPR
HPLC column (0.46 ? 7.5 cm; Tosohaas). For tagged octasac-
charide (1–4), heptasaccharide 5, N-sulfo 6-O-sulfo decasac-
charide 9, and dodecasaccharide 10, and N-sulfo 6-O-sulfo
3-O-sulfodecasaccharide11anddodecasaccharide12,thepro-
cedures were very similar to those for N-sulfo oligosaccharides
(6-8) as described above using appropriate enzymes.
HPLCAnalysis—HPLCanalysisofoligosaccharidesfollowed
the procedures as previously described (6, 9).
Determination of the Binding Affinity of Oligosaccharides to
Antithrombin (AT)—The dissociation constant (Kd) of each
sampleandATwasdeterminedusingaffinitycoelectrophoresis
(18).
Microdialysis of Oligosaccharides—The synthesized oligo-
saccharides were subjected to microdialysis prior to the MS
analysis.Thedialysiswascarriedoutusinghollowfiberdialysis
tubing (13,000 MWCO; Spectrum) against 20 mM ammonium
acetate.
Liquid Chromatography-linked Mass Spectrometry (LC-MS)
Analysis and Mass Spectrometry (MS) Analysis—LC-MS anal-
yses were performed on an Agilent 1100 HPLC-MSD-Trap.
Nonsulfated backbone oligosaccharides were injected onto an
Aquasil C18column (3 ?m 2.1 ? 50 mm; Thermo Fisher). A
gradient of acetonitrile with a flow rate of 0.4 ml/min was
directedintotheiontrapmassspectrometer.Thegradientcon-
(Sigma-Aldrich).
SynthesisofHeparanSulfate
34242 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 44•OCTOBER 29, 2010
at RENSSELAER POLYTECHNIC IN, on October 29, 2010
www.jbc.org
Downloaded from

Page 4

sistedofaninitial5-minholdat90%aqueous(0.1%formicacid
in water), a change to 90% organic (0.1% formic acid in aceto-
nitrile)over2min,a2-minholdat90%organic,achangeto90%
aqueous over 2 min, and a 4-min reequilibration at 90% aque-
ous. Experiments were performed in positive ionization mode
foruntaggedbackboneoligosaccharide.Alternatively,theanal-
yses were performed in negative ionization mode for tagged
backboneoligosaccharides.Underbothconditions,theelectro-
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).
Oligosaccharides with sulfo groups were dissolved in 70%
acetonitrile and 10 ?M imidazole. A syringe pump (Harvard
Apparatus)wasusedtointroducethesampleviadirectinfusion
(10?l/min)intoanAgilent1100MSDTrap.Experimentswere
performed in negative ionization mode with the electrospray
source set to 3000 V and 200 °C and the compound stability set
to30%.Nitrogenwasusedforbothnebulizer(5liters/min)and
drying gas (15 p.s.i.). The procedures for MS/MS analysis are
described above.
RESULTS
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
tagallowedeasyisolationoftheproductwithFluoroFlashaffin-
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-GlcNH2failed because UDP-GlcNH2was
not a substrate for KfiA. An unnatural monosaccharide donor,
UDP-GlcNTFA, was next used in the synthesis. We found that
UDP-GlcNTFAservedasanexcellentdonorsubstrateforKfiA,
being efficiently incorporated. The product could be further
extendedbypmHS2followingthebackbonesynthesisasshown
FIGURE2.SchemeforthesynthesisofN-sulfooctasaccharidesandaheptasaccharidecarryinganIdoUA2S.A,stepsinvolvedinthesynthesisofN-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,stepsinvolvedinthesynthesisofhepatasaccharide5.Themodificationsitesareeithercoloredinblueorhighlightedinfilledboxes.Theresidue
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%.
SynthesisofHeparanSulfate
OCTOBER 29, 2010•VOLUME 285•NUMBER 44JOURNAL OF BIOLOGICAL CHEMISTRY 34243
at RENSSELAER POLYTECHNIC IN, on October 29, 2010
www.jbc.org
Downloaded from

Page 5

in Fig. 2A. The GlcNTFA residue was selectively deprotected
under mild basic conditions, yielding GlcNH2unit in the pres-
ence of GlcNAc residues. The resulting GlcNH2residue was
converted to GlcNS using NST in the presence of PAPS, step b
Fig. 2A).
Theeffortsresultedinfourdifferentoctasaccharideproducts
that differed by the location and the number of GlcNS residue:
octasaccharide 1 (no GlcNS, supplemental Fig. 1), octasaccha-
ride 2 (two GlcNS residues, Fig. 3) and 3 and 4 (single GlcNS at
different positions, supplemental Fig. 2). The structures of the
octasaccharidesweredeterminedbyMS.Forexample,octasac-
charide 2 was resolved as a symmetric peak by HPLC using a
C18column (Fig. 3A). ESI-MS analysis revealed its molecular
mass to be 1839.6 Da in close agreement to the calculated mass
of 1839.5 Da (Fig. 3B). MS/MS analysis confirmed the position
of the GlcNS residues in 2 (Fig. 3C) from the two characteristic
daughterions,Y5(m/z,1244.5)andB3(m/z,592.3),productsof
the cleavage of an internal glycosidic linkage (see supplemental
Figs. 1 and 2for structural analysis of 1, 3 and 4).
Synthesis of an Oligosaccharide Carrying an IdoUA2S Unit—
The IdoUA2S residue is a critical structural motif involved in
binding to fibroblast growth factor to confer the cell prolifera-
tion activity of HS (6). Heptasaccharide 5 carrying one
IdoUA2Sresidueinitscenterwassynthesizedfromadisaccha-
ride (Fig. 2B). The enzymatic synthesis of IdoUA2S residue
involves the concerted action of C5-epi and 2-OST, where
C5-epi transforms a GlcUA residue to an IdoUA residue, and
2-OSTsulfatesthe2-OHpositionoftheIdoUAresidue(stepd,
Fig. 2B). It should be noted that it was essential to introduce a
GlcNTFAresidueatthenonreducingterminusoftheheptasac-
charide. This GlcNTFA residue blocks the action of C5-epi on
the immediately adjacent GlcUA. C5-epi is known to act only
on GlcUA residues flanked by two GlcNS residues as in the
sequence, -GlcNS-GlcUA-GlcNS-, but does not act on the
GlcUA present in the sequence, -GlcNAc-GlcUA-GlcNS- (20).
The purity analysis and MS spectrum of heptasaccharide 5
are shown in Fig. 4. A35S-labeled heptasaccharide was initially
synthesizedbyincubatingwith35S-labeledPAPS,andtheprod-
uct was resolved as a symmetric peak at the expected retention
time on anion exchange HPLC, suggesting that it was ?90%
pure(Fig.4A).Unlabeledheptasaccharidewasthensynthesized
under identical conditions and purified by the HPLC. ESI-MS
demonstrated that heptasaccharide 5 had a molecular mass of
1511.0 Da (calculated 1512.2 Da). These data are consistent
withaheptasaccharidecarryingthreesulfogroupsandoneGlc-
NTFA residue.
The position of the IdoUA2S residue in heptasaccharide 5
was confirmed by disaccharide analyses using35S site-specifi-
cally labeling techniques (supplemental Fig. 3, A and B). Only
IdoUA2S-AnMan disaccharide was observed from the nitrous
acid-degraded 2-O-[35S]sulfated heptasaccharide, suggesting
that the 2-O-sulfo group was present only at the IdoUA2S, and
FIGURE 3. Structural characterization of octasaccharide 2. A, HPLC of octasaccharide 2 using a C18column 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).
SynthesisofHeparanSulfate
34244 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 44•OCTOBER 29, 2010
at RENSSELAER POLYTECHNIC IN, on October 29, 2010
www.jbc.org
Downloaded from