Tailored design and synthesis of heparan sulfate oligosaccharide analogues using sequential one-pot multienzyme systems

Article abstract of DOI:10.1002/anie.201305667

Heparan sulfate analogues: Highly efficient one-pot multienzyme (OPME) chemoenzymatic systems for the activation and transfer of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) have been developed. They were applied to the sequential tailored synt

Full text of DOI:10.1002/anie.201305667

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Angewandte
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DOI: 10.1002/anie.201305667
Enzymatic Synthesis
Tailored Design and Synthesis of Heparan Sulfate Oligosaccharide
Analogues Using Sequential One-Pot Multienzyme Systems**
Yi Chen, Yanhong Li, Hai Yu, Go Sugiarto, Vireak Thon, Joel Hwang, Li Ding, Liana Hie, and
Xi Chen*
Heparan sulfate (HS) and heparin are linear sulfated hetero-
polysaccharides that consist of alternating a1–4-linked
d-glucosamines (GlcN) and 1–4-linked uronic acids, with an
a-linkage for l-iduronic acid (IdoA) and a b-linkage for
d-glucuronic acid (GlcA). Possible modifications include 2-O-
sulfation on the uronic acid residues, and one or more
modifications on the glucosamine residues, including
N-sulfation, N-acetylation, 6-O-sulfation, and 3-O-sulfation.
Heparin and low-molecular-weight heparin (LMWH) are the
most commonly used anticoagulants or antithrombotic drugs.
Compared to HS, heparin has a higher level of sulfation and
a higher IdoA content.[1] Heparin is mostly produced by mast
cells, and heparan sulfates are produced by different cell types
in animals.[2] They are attractive synthetic targets because of
the therapeutic application of heparin, and the important
roles of HS and heparin in regulating cancer growth, blood
coagulation, inflammation, assisting against viral and bacte-
rial infections, signal transduction, lipid metabolism, and cell
differentiation.[3]
plex HS/heparin are still not fully understood.[2] A tailor-made
synthetic process is still lacking.
Early chemical syntheses of heparin fragments and
analogues[4] required many protection and deprotection
steps, making the synthesis of even relatively small oligosac-
charides time-consuming and rather inefficient. More
recently, various chemical synthetic approaches[5] including
target-oriented,[6] modular,[7] combinatorial,[8] one-pot,[9] and
solid-phase[10] syntheses, have been developed and used to
produce HS/heparin oligosaccharides that range from di- to
octasaccharides of different sequences and sulfation patterns.
Glycan microarrays have been developed to study heparin/
HS–protein interactions.[11] Nevertheless, the synthesis of HS
oligosaccharides of non-repetitive sequences is much more
challenging than that of oligosaccharides with repetitive
sequences. Synthetic efficiency decreases dramatically as the
length of the target molecule increases. Furthermore, varia-
tions in the structure and length of the target molecules can
completely change the whole synthetic design.
Synthetic heparins can eliminate the side effects caused by
inherently heterogeneous heparins purified from natural
sources. Their syntheses, however, present great synthetic
challenges owing to their structural complexity. Although
much progress has been made over the last decade in terms of
synthesis, analysis, and understanding of complex HS and
heparin, the mechanisms for the formation and regulation of
HS/heparin and the structure–function relationship of com-
Traditional methods of purifying oligosaccharides after
enzymatic digestion of GAG chains have provided useful
quantities of HS/heparin oligosaccharides for early structure–
activity relationship studies.[12] Cloning and characterization
of HS biosynthetic enzymes have allowed chemoenzymatic
syntheses of various HS/heparin derivatives.[13] Bioactive HS
structures that bind to antithrombin, fibroblast growth factor
FGF2, or herpes simplex virus glycoprotein D (HSV gD)
were enzymatically synthesized on milligram scales from
completely desulfated and N-sulfated heparin by multiple
O-sulfation reactions using immobilized O-sulfotransferases
with the regeneration of the activated sulfate donor
3’-phosphoadenosine 5’-phosphosulfate (PAPS).[13c] Heparin-
like polysaccharides with anticoagulant activity were
obtained from purified Escherichia coli K5 capsular polysac-
charide, followed by epimerization of the C5 position of the
uronic acid residues, chemical persulfation, and selective
desulfonation.[13b] A heparin polysaccharide library with
different sulfation patterns was obtained using different HS
biosynthetic enzymes.[13d] Recently, two homogeneous hep-
arin oligosaccharides were chemoenzymatically synthesized
on a milligram scale, and showed comparable activity as
Arixtra (fondaparinux sodium), a synthetic anticoagulant
heparin pentasaccharide.[14] Owing to the complex nature of
the HS biosynthesis and the involvement of multiple iso-
enzymes with overlapping substrate preferences, the existing
methods do not provide precise control for the syntheses of
many desired structures.
[*] Dr. Y. Chen, Dr. Y. Li, Dr. H. Yu, Dr. G. Sugiarto, Dr. V. Thon,
J. Hwang, Dr. L. Ding, L. Hie, Prof. Dr. X. Chen
Department of Chemistry, University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
E-mail: xiichen@ucdavis.edu
Homepage: http://chemgroups.ucdavis.edu/~chen/
Dr. V. Thon
Current address: Laboratory of Bacterial Polysaccharides
Food and Drug Administration, Bethesda, MD 20892 (USA)
Dr. L. Ding
Current address: College of Life Science, Northwest University
Xi’an, Shaanxi 710069 (China)
L. Hie
Current address: Department of Chemistry and Biochemistry,
University of California-Los Angeles, Los Angeles, CA 90095 (USA)
[**] This work was partially supported by the NSF (CHE-0548235 and
CHE1012511) and the NIH (R01 HD065122). A Bruker Avance-800
NMR spectrometer was funded by an NSF grant (DBIO-722538).
X.C. is a Camille Dreyfus Teacher-Scholar and a UC-Davis Chan-
cellor’s Fellow.
An efficient approach to synthesize carbohydrates that
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305667.
contain post-glycosylational modifications (PGMs),[15] includ-
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ing HS oligosaccharides and analogues, with precise control
starts from the corresponding structurally modified mono-
saccharides, and uses sequential one-pot multienzyme
(OPME) glycosylation reactions (Scheme 1). The simplest
Scheme 2. One-pot four-enzyme GlcNAc activation and transfer
system for the synthesis of disaccharide GlcNAca1-4GlcAb2AA and
derivatives. Tris-HCl buffer (100 mm, pH 7.5) was used for the syn-
thesis of disaccharides 5 and 7, and MES buffer (100 mm, pH 6.5) was
used for the synthesis of disaccharide 6. Ac=acetyl, NahK_ATCC-
55813=Bifidobacterium longum ATCC55813 N-acetylhexosamine 1-
kinase, PmGlmU=Pasteurella multocida strain P-1059 (ATCC15742)
N-acetylglucosamine-1-phosphate uridylyltransferase, PmHS2=Pas-
teurella multocida strain P-1059 heparosan synthase 2, PmPpA=Pas-
teurella multocida strain P-1059 inorganic pyrophosphatase,
TFA=trifluoroacetyl.
Scheme 1. Sequential one-pot multienzyme (OPME) chemoenzymatic
synthesis of heparan sulfate (HS) oligosaccharide analogues. GlyK=
glycokinase, GlyT=glycosyltransferase, NucT=monosaccharide-1-
phosphate nucleotidyltransferase, PpA=inorganic pyrophosphatase.
in significant amounts at pH 7.5, but this could be reduced by
lowering the pH to 6.5 and by shortening the reaction time.
In addition, using three previously synthesized UDP-
GlcNAc derivatives, including UDP-N-glycolylglucosamine
(UDP-GlcNGc, 8), UDP-N-azidoacetylglucosamine (UDP-
GlcNAcN3, 9), and UDP-6-deoxy-6-glycolylamido-N-acetyl-
glucosamine (UDP-GlcNAc6NGc, 10),[19] a PmHS2-catalyzed
GlcNAc transfer reaction produced disaccharides GlcNGca1-
4GlcAb2AA (11), GlcNAcN3a1-4GlcAb2AA (12), and
GlcNAc6NGca1-4GlcAb2AA (13) in good to excellent
yields (74–92%) (Scheme 3).
OPME system consists of a monosaccharide kinase (GlyK),
a nucleotidyltransferase (NucT), and a glycosyltransferase
(GlyT) to allow the formation of the desired glycosidic bonds
in a regio- and stereospecific manner. An inorganic pyro-
phosphatase (PpA) can also be used to remove the pyro-
phosphate by-product formed to drive the reaction towards
the formation of the sugar nucleotide. Further chemical
modifications can be performed to introduce additional
functionality after the formation of the oligosaccharides.
Herein, we describe such a method for the efficient synthesis
of HS oligosaccharide analogues with desired sulfation
patterns.
PmHS2 from the Gram-negative bacterium Pasteurella
multocida is a well-studied heparosan synthase.[16] It is
a bifunctional enzyme that acts as both an a1-4-N-acetylglu-
cosaminyltransferase (a1-4GlcNAcT) and a b1-4-glucuronyl-
transferase (b1-4GlcAT). PmHS2 cloned from the Pasteurella
multocida strain P-1059 (ATCC#15742, type A strain) has
a good expression level in Escherichia coli (ca. 17 mgLÀ1 cell
culture). The a1-4GlcNAcT activity of PmHS2 is more
promiscuous than that of Escherichia coli K5 a1-4GlcNAcT
(KfiA) when using UDP-GlcNAc derivatives as donor
substrates.[17]
Scheme 3. PmHS2-catalyzed synthesis of GlcNAca1-4GlcAb2AA disac-
charide derivatives 11–13 in Tris-HCl buffer (100 mm, pH 7.5).
Gc=glycolyl.
As shown in Scheme 2, using glucuronide GlcAb2AA (1)
with a fluorescent label as the acceptor substrate and taking
advantage of the substrate promiscuities of several enzymes
involved in GlcNAc activation and transfer, disaccharide
GlcNAca1-4GlcAb2AA (5) and its derivatives GlcNTFAa1-
4GlcAb2AA (6) and GlcNAc6N3a1-4GlcAb2AA (7) were
synthesized in high yields (84–95%) from N-acetylglucos-
The obtained disaccharides 5–7 and 11–13 were used in
a one-pot three-enzyme GlcA activation and transfer system
(Scheme 4) to test the acceptor–substrate specificity of the b1-
4GlcAT activity of PmHS2. In this system, a glucose-1-
phosphate uridylyltransferase cloned from Escherichia coli
K-12 (EcGalU)[21] was used to catalyze the formation of
UDP-Glc from Glc-1-P and UTP. A UDP-glucose dehydro-
genase, which was cloned from the Pasteurella multocida
strain P-1059 (PmUgd; see the Supporting Information for
cloning, purification, and DNA and protein sequences), was
used in the presence of the coenzyme nicotinamide adenine
dinucleotide (NAD+) to oxidize the C6 position of glucose
amine
(GlcNAc,
2),
N-trifluoroacetylglucosamine
(GlcNTFA, 3), and 6-azido-6-deoxy-N-acetylglucosamine
(GlcNAc6N3, 4), respectively, using a one-pot four-enzyme
reaction containing NahK_ATCC55813,[18] PmGlmU,[19]
PmPpA,[20] and PmHS2.[17] It was found that the cleavage of
the N-TFA (TFA = trifluoroacetyl) group in 3 and 6 occurred
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were further used for the formation of diverse trisaccharide
derivatives. In principle, the same strategy can be repeated for
the production of longer oligosaccharides and derivatives.
The oligosaccharide derivatives can be used as important
probes for investigating the acceptor-binding pocket of
PmHS2, which may bind to oligosaccharide acceptors that
are longer than monosaccharides or disaccharides, as dis-
cussed previously.[22]
A strategy for the synthesis of HS oligosaccharide
analogues on a preparative scale with controllable sulfation
patterns was also explored. Trisaccharide GlcAb1-
4GlcNTFAa1-4GlcAb2AA (15; > 50 mg) was synthesized
using the one-pot three-enzyme GlcA activation and transfer
system shown in Scheme 4. The loss of the N-TFA group was
minimized by carrying out the reaction at pH 7.0 instead of
pH 7.5, and the yield of the reaction was thus increased to
87%. As shown in Scheme 5, the obtained trisaccharide 15
Scheme 4. One-pot three-enzyme GlcA activation and transfer system
for the synthesis of trisaccharide GlcAb1-4GlcNAca1-4GlcAb2AA and
derivatives. EcGalU=Escherichia coli K-12 glucose-1-phosphate uridylyl-
transferase, PmUgd=Pasteurella multocida strain P-1059 UDP-glucose
dehydrogenase.
(Glc) in UDP-Glc to form UDP-glucuronic acid (UDP-
GlcA); in combination with the b1-4GlcAT activity of
PmHS2, this was used to form trisaccharides. The reactions
were analyzed by a high-performance liquid chromatography
(HPLC) system equipped with a fluorescence detector. As
shown in Scheme 4, all disaccharides (5–7 and 11–13) tested
were suitable acceptor substrates for the b1-4GlcAT activity
of PmHS2. Trisaccharides GlcAb1-4GlcNAca1-4GlcAb2AA
(14), GlcAb1-4GlcNAc6N3a1-4GlcAb2AA (16), and
GlcAb1-4GlcNAcN3a1-4GlcAb2AA (18) were formed in
quantitative or near quantitative yields (95–100%).
GlcAb1-4GlcNTFAa1-4GlcAb2AA (15) was formed in
a lower yield (72%), mainly because of the loss of the
N-TFA-group to form another trisaccharide, GlcAb1-
4GlcNH2a1-4GlcAb2AA. Disaccharide GlcNGca1-4GlcA-
b2AA (11), which posses an N-glycolyl group at the
C2 position of GlcN, was a good acceptor for the b1-
4GlcAT activity of PmHS2 to form GlcAb1-4GlcNGca1-
4GlcAb2AA (17) in 75% yield. In comparison, disaccharide
GlcNAc6NGca1-4GlcAb2AA (13) with the same N-glycolyl
group at the C6 position of the GlcNAc moiety was a poorer
acceptor, and trisaccharide GlcAb1-4GlcNAc6NGca1-
4GlcAb2AA (19) was formed in only 14% yield.
Scheme 5. One-pot four-enzyme synthesis of tetrasaccharide 20 and
subsequent chemical derivatization reactions for the formation of
tetrasaccharides 21–24. Reagents and conditions: a) K2CO3, H2O, RT,
overnight, 81%; b) pyridine–SO3 complex, NaOH (2m), H2O, 3 days,
70%; c) H2, Pd/C, MeOH, H2O, 1 h, quantitative.
was used as a substrate in the one-pot four-enzyme GlcNAc
activation and transfer system (the same one as shown in
Scheme 2) for the synthesis of tetrasaccharide GlcNAc6N3a1-
4GlcAb1-4GlcNTFAa1-4GlcAb2AA (20) in high yield
(93%). The N-TFA group and the N3 group were individually
converted into free amine groups, as shown for tetrasacchar-
ides 21 and 23, allowing sequential N-sulfation to generate HS
tetrasaccharide analogues 22–24. More specifically, the
N-TFA group at the C2 position of the internal GlcNTFA
residue in tetrasaccharide 20 was removed under mildly basic
conditions to produce tetrasaccharide GlcNAc6N3a1-
4GlcAb1-4GlcNH2a1-4GlcAb2AA’ (21) in 81% yield. As
the removal of the N-TFA group was accompanied by the
hydrolysis of the methyl carboxylate ester, tetrasaccharide 21
contains a free carboxylate in the 2AA’ motif instead of the
carboxylic ester in the 2AA moiety of tetrasaccharide 20.
Sulfation of tetrasaccharide 21 for the formation of tetrasac-
charide 22 required a larger excess of sulfation reagent
(60 equiv) and a prolonged reaction time (3 days) to achieve
Taken together, these results indicate that aside from the
previously identified donor–substrate promiscuity of the a1-
4GlcNAcT activity of PmHS2,[16b,17] the b1-4GlcAT activity of
PmHS2 also shows a broader acceptor–substrate promiscuity
than previously described.[22] These substrate promiscuities
allowed the synthesis of GlcNAca1-4GlcAb2AA disacchar-
ide derivatives containing modified GlcNAc residues, which
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PmHS2-catalyzed synthesis of disaccharides 11–13: GlcAb2AA
a reasonable 70% yield. Catalytic hydrogenation of the azido
group at the C6 position of the non-reduced GlcNAc6N3 end
in tetrasaccharide 22 produced tetrasaccharide 23. Tetrasac-
charide 24 was subsequently synthesized by N-sulfation.
These N-sulfated oligosaccharide analogues mimic naturally
occurring HS oligosaccharides and may have improved
therapeutic potentials, as they will most likely be more
resistant to the activities of HS/heparin lyases and hydrolases.
1 (5–10 mg, 1 equiv) and UDP-GlcNAc derivatives (1.2 equiv) were
dissolved in water in a 15 mL centrifuge tube containing MgCl2
(10 mm) in Tris-HCl buffer (100 mm, pH 7.5). After adding PmHS2
(1–2 mg), water was added to bring the volume of the reaction
mixture to 10 mL. The reaction mixture was incubated for 12–36 h at
378C with gentle shaking.
Product formation was monitored by thin layer chromatography
(EtOAc/MeOH/H2O = 4:2:1, v/v), using
a p-anisaldehyde sugar
staining solution. The reaction was stopped by adding the same
volume of ice-cold ethanol and incubating at 48C for 30 min. The
mixture was concentrated and passed through a BioGel P-2 gel
filtration column to obtain the desired product. Purification by
column chromatography on silica gel (EtOAc/MeOH/H2O = 5:2:1)
was used when necessary to achieve further purification.
Small-scale one-pot three-enzyme synthesis of trisaccharides 14–
19: Typical reactions were performed in a total volume of 20 mL in
Tris-HCl buffer (100 mm, pH 7.5) containing MgCl2 (10 mm), UTP
(7.5 mm), disaccharides (5 mm), Glc-1-P (6 mm), NAD+ (12 mm),
GalU (2.5 mg), PmUgd (8 mg), and PmHS2 (11.5 mg). Reactions were
allowed to proceed for 12 h at 378C and quenched by adding ice-cold
ethanol (20 mL) and water (1.96 mL) to achieve 100-fold dilution.
Indeed, idraparinux,
a synthetic fully O-sulfated and
O-methylated analogue of heparin pentasaccharide, has
a higher anti-Xa activity and a longer duration of action
that fondaparinux (or Arixtra), an FDA-approved synthetic
compound, which contains an unmodified antithrombin-
binding heparin pentasaccharide for the prevention and
treatment of venous thromboembolism (Scheme 6).[23] The
N-sulfated analogues of HS oligosaccharides may also have
unexpected or improved functions.
Received: July 1, 2013
Published online: September 13, 2013
Keywords: carbohydrates · chemoenzymatic systems ·
.
enzymes · heparin · structure–activity relationships
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Experimental Section
One-pot four-enzyme synthesis of disaccharides 5–7: GlcAb2AA
1
(5–30 mg, 1 equiv), GlcNAc or derivatives (1.5 equiv), ATP
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pH 7.5) or MES (100 mm, pH 6.5) buffer. After adding Nan-
K_ATCC55813 (0.5–2.1 mg), PmGlmU (1–3 mg), PmPpA (0.5–
1.5 mg), and PmHS2 (1–6 mg), water was added to bring the
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