Synthesis of uridine 5′-diphosphoiduronic acid: A potential substrate for the chemoenzymatic synthesis of heparin

Article abstract of DOI:10.1021/jo801409c

(Chemical Equation Presented) An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5′-diphosphoiduronic acid (UDP-IdoA) and uridine 5′- diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5′-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.

Full text of DOI:10.1021/jo801409c

Synthesis of Uridine 5′-diphosphoiduronic Acid: A Potential
Substrate for the Chemoenzymatic Synthesis of Heparin
Michel Weïwer,† Trevor Sherwood,† Dixy E. Green,‡ Miao Chen,§ Paul L. DeAngelis,‡
Jian Liu,§ and Robert J. Linhardt*,†,|
Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, and
Department of Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180,
Department of Biochemistry and Molecular Biology, UniVersity of Oklahoma Health Sciences Center,
940 Stanton L. Young BlVd., Oklahoma City, Oklahoma, and UniVersity of North Carolina School of
Pharmacy, DiVision of Medicinal Chemistry and Natural Products, CB no. 7360 Beard Hall,
Room 309, Chapel Hill, North Carolina 27599-7360
linhar@rpi.edu
ReceiVed July 1, 2008
An improved understanding of the biological activities of heparin requires structurally defined heparin
oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases
that use UDP-sugar nucleotides as donors. Uridine 5′-diphosphoiduronic acid (UDP-IdoA) and uridine
5′-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine
5′-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-
natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to
better understand enzyme specificity.
Introduction
charge density of any known biological molecule.2 Pharmaceuti-
cal grade heparin is commonly derived from mucosal tissues
of slaughtered meat animals, such as porcine intestine or bovine
lung, and is a polydisperse, polycomponent, and polypharma-
cologic agent.3 Low molecular weight heparins (LMWHs) are
rapidly replacing heparin as the clinical anticoagulant of choice.
However, these LMWHs are derived from heparin and are also
polydisperse mixtures, making their controlled preparation and
analysis difficult. Thus, structurally defined heparin oligosac-
charides are still of great interest as potential anticoagulant
agents. Fondaparinux (Arixtra, GSK) and Idraparinux (Sanofi-
Aventis), homogeneous pentasaccharide analogues of the ATIII
binding pentasaccharide, have been chemically synthesized and
Glycosaminoglycans (GAGs) are an important class of
therapeutic agents that engage in specific interactions with a
wide variety of proteins in both the intracellular and extracellular
environment.1 Heparin, the most studied GAG, is widely used
as an injectable anticoagulant and has the highest negative
* To whom correspondence should be addressed.
† Department of Chemistry and Chemical Biology, Rensselaer Polytechnic
Institute.
‡ Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Sciences Center.
§ University of North Carolina School of Pharmacy, Division of Medicinal
Chemistry and Natural Products.
| Department of Chemical and Biological Engineering and Department of
Biology, Rensselaer Polytechnic Institute.
(1) Casu, B. Chapter 1, Structure and active domains of heparin. In Chemistry
and Biology of Heparin and Heparan Sulfate; Garg, H. G., Linhardt, R. J., Hales,
C. A., Eds.; Elsevier: Oxford, UK, 2005.
(2) Linhardt, R. J.; Dordick, J. S.; Deangelis, P. L.; Liu, J. Semin. Thromb.
Hemostasis 2007, 33 (5), 453–465.
(3) Linhardt, R. J.; Gunay, N. S. Semin. Thromb. Hemostasis 1999, 3, 5–16.
10.1021/jo801409c CCC: $40.75
Published on Web 08/30/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7631–7637 7631

Weïwer et al.
SCHEME 1. Retrosynthesis of UDP-IdoA
introduced into the marketplace. These drugs can reduce the
side effects associated with the use of heparin, particularly the
risk for heparin-induced thrombocytopenia (HIT). Unfortunately,
the chemical synthesis of Fondaparinux is both complicated and
costly.4 Therefore, the development of new strategies to enable
the efficient, high-yield, and stereocontrolled synthesis of
homogeneous heparin-like oligosaccharides is of major impor-
tance.
Heparin is composed of alternating (1f4) linked R-L-IdoA
or ꢀ-D-GlcA and ꢀ-D-GlcN units, which are diversely sulfonated.
The uronic acid portion of heparin consists of approximately
80-90% of R-L-IdoA units and only 10-20% of ꢀ-D-GlcA
units. The biosynthesis of heparin involves glycosyltransferases
that successively add a N-acetylglucosamine unit (GlcNAc) from
UDP-GlcNAc and a glucuronic acid unit (GlcA) from UDP-
GlcA. The GlcA residues of the polysaccharide precursor are
subsequently C-5 epimerized to form iduronic acid (IdoA)
through the action of a C-5 epimerase. C-5 epimerases involved
in heparin biosynthesis act on GlcA residues flanked by
N-sulfoglucosamine residues.5 This enzyme specificity reduces
access to well-defined, unnatural structures. Using the com-
pletely controlled, glucuronosyltransferase-based, introduction
of IdoA residues might be possible using the unnatural analogue
UDP-IdoA. The glucuronosyltransferase-based transfer of UDP-
IdoA would allow access to many natural and unnatural
sequences.
The availability of well-defined heparin oligosaccharides
should provide an improved understanding of the important
interactions that occur between heparin and proteins. Synthetic
chemists have developed a variety of synthetic methodologies
for accessing GAG oligosaccharides.6 However, while these
multistep chemical strategies display elegant chemistry, they
result in products that are simply too expensive. The chemoen-
zymatic synthesis of heparin oligosaccharides represents an
interesting alternative to the complex chemical syntheses and
purification processes. Indeed, enzymatic and chemoenzymatic
strategies have been developed in the past few years and proven
to be efficient, relying on the unique capacities of enzymes
(glycosyltransferases, sulfotransferases, epimerases, etc.) to
catalyze regiospecific and stereospecific transformations.7
During the past decade, several unnatural UDP-hexosamines
have been synthesized and used as substrates or inhibitors of
hexosamine glycosyltransferases.8 UDP-N-trifluoroacetylglu-
cosamine (UDP-GlcNTFA), for example, is accepted as a
substrate in place of UDP-GlcNAc by the “core-2” GlcNAc
transferase (EC 2.4.1.102) in the biosynthesis of O-linked
glycoproteins, and by the GlcNAcT-V transferase (EC 2.4.1.155),
a key biosynthetic enzyme controlling the branching pattern of
cell surface complex Asn-linked oligosaccharides.9 However,
unnatural analogues of UDP-GlcA have not been investigated
as potential substrates of glucuronosyltransferases.
direct and controlled enzymatic introduction of IdoA units into
a growing oligosaccharide. This unnatural substrate might also
be useful for the study of the specificity of various glucurono-
syltransferases.
Results and Discussion
Several strategies have been developed for the synthesis of
UDP-sugar nucleotides.10 Among these, the Khorana-Moffatt
procedure,11 involving the coupling of a sugar monophosphate
with uridine 5′-monophosphomorpholidate 4-morpholine-N,N′-
dicyclohexylcarboxamidine salt (UMP-morpholidate), is still the
preferred route. The retrosynthesis of UDP-IdoA is shown in
Scheme 1.
L-IdoA is a rare sugar and is not commercially available.
The efficient synthesis of L-IdoA has been a major concern
for researchers synthesizing heparin oligosaccharides, and
thus a wide variety of strategies have been developed for its
preparation. Inversion by nucleophilic substitution at C-5
starting from 3-O-benzyl-1,2-isopropylidene-R-D-glucofura-
nose12 or D-glucurono-6,3-lactone,13 radical reduction of
(4) Sinay, P.; Jacquinet, J.-C. Carbohydr. Res. 1984, 132, C5–C9.
(5) Kusche, M.; Hannesson, H. H.; Lindahl, U. Biochem. J. 1991, 275, 151–
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2335. (c) Van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. Engl. 1993,
32, 1671–1690. (d) Seeberger, P. H.; Haase, W.-C. Chem. ReV. 2000, 100, 4349–
4393. (e) Ojeda, R.; Terentí, O.; de Paz, J.-L.; Martín-Lomas, M. Glycoconj. J.
2004, 21, 179–195. (f) Prabhu, A.; Venot, A.; Boons, G.-J. Org. Lett. 2003, 5
(26), 4975–4978. (g) Yeung, B. K. S.; Chong, P. Y. C.; Petillo, P. A.
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chemistry to systems biology; Elsevier Science: Boston, MA, 2007; Vol. 1, pp
713-746. (b) Kuberan, B.; Lech, M. Z.; Beeler, D. L.; Wu, Z. L.; Rosenberg,
R. D. Nat. Biotechnol. 2003, 21 (11), 1343–1346. (c) Mun˜oz, E. M.; Xu, D.;
Avci, F.; Kemp, M. M.; Liu, J.; Linhardt, R. J. Biochem. Biophys. Res. Commun.
2006, 339, 597–602. (d) Chen, J.; Avci, F. Y.; Mun˜oz, E. M.; McDowell, L. M.;
Chen, M.; Pedersen, L. C.; Zhang, L.; Linhardt, R. J.; Liu, J. J. Biol. Chem.
2005, 280 (52), 42817–42825. (e) Lindahl, U.; Li, J.-P.; Kusche-Gullberg, M.;
Salmivirta, M.; Alaranta, S.; Veromaa, T.; Emeis, J.; Roberts, I.; Taylor, C.;
Oreste, P.; Zoppetti, G.; Naggi, A.; Torri, G.; Casu, B. J. Med. Chem. 2005, 48,
349–352. (f) Sismey-Ragatz, A. E.; Green, D. E.; Otto, N. J.; Rejzek, M.; Field,
R. A.; DeAngelis, P. L. J. Biol. Chem. 2007, 282 (39), 28321–28327.
(8) (a) Busca, P.; Martin, O. R. Tetrahedron Lett. 2004, 45, 4433–4436. (b)
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2002, 595–606. (b) Arlt, M.; Hindsgaul, O. J. Org. Chem. 1995, 60, 14–15. (c)
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Soc. 1961, 83, 659–663.
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S. F.; Van den Bosch, R. H.; Mertens, J. M. R.; Van der Vlugt, F. A.
J. Carbohydr. Chem. 1985, 4, 293–321.
In this work, we synthesize UDP-IdoA 1, a potential unnatural
substrate of glucuronosyltransferases, which would allow the
7632 J. Org. Chem. Vol. 73, No. 19, 2008

Synthesis of Uridine 5′-diphosphoiduronic Acid
SCHEME 2. Use of a Sulfide Iduronic Acid Type Donor
SCHEME 3. MacDonald’s Phosphorylation of Iduronic Acid Type Donor 5
5-bromouronate,14 functionalization of ∆4-uronic acid spe-
cies,15 stereoselective addition on D-xylo-dialose,16 epimer-
ization of D-GlcA derivatives,17 and diastereoselective hy-
droboration of exoglucals18 have shown good results.
Compound 4 (Scheme 2) was first synthesized as described
by Ke et al. in 31% overall yield over 5 steps from the
inexpensive D-glucurono-6,3-lactone.13
Monophosphate 3 was next approached, using the procedure
developed by MacDonald in 1962,21 which involves the use of
an anomeric acetate and crystalline phosphoric acid at 60 °C
under high vacuum (Scheme 3).
Iduronate donor 5 was treated with an excess of crystalline
phosphoric acid. This strategy gave a mixture of the desired
monophosphate 3 and the elimination product 3′ in 1:2 ratio.
From a mechanistic point of view, we rationalized the formation
of 3′ by a protonation of the C-4 acetyl group by H3PO4
followed by a ꢀ-elimination, which is favored in a 1,2-trans
axial position, leading to the formation of 3′ and acetic acid.
This mechanism is supported by the fact that phosphorylation
of methyl 1,2,3,4-tetra-O-acetyl-ꢀ-D-glucuronate using the same
conditions affords no traces of 3′. Compounds 3 and 3′ were
separated on silica gel affording monophosphate 3 as the
ꢀ-anomer in 21% yield.
We initially attempted to prepare the monophosphate 3
through the activation of the anomeric position of compound
4 as a phenyl sulfide.19 According to a procedure developed
by Bonnaffe´,20 peracetylation of compound 4 was performed
in the presence of acetyl chloride, pyridine, and a catalytic
amount of dimethylaminopyridine (DMAP) in DCM at -40
°C to limit the potential isomerization of the pyranose ring
to the corresponding furanose ring that is known to occur in
the presence of pyridine/acetic anhydride. Iduronate donor
5 was then treated with thiophenol in the presence of
BF3 · Et2O. The corresponding sulfide 6 was obtained in a
modest yield of 37% as a 1/3 R/ꢀ mixture after purification
on silica gel, but 52% of the starting material could also be
recovered and reused. Glycosylation of 6 with dibenzylphos-
phoric acid in the presence of N-iodosuccinimide (NIS) gave
the desired 7 in 91% yield (R/ꢀ ) 1/4). The ꢀ-anomer was
isolated by flash chromatography in 70% yield and subjected
to hydrogenolysis. During the hydrogenolysis reaction, a
ꢀ-elimination of the C-4 acetate group was observed and
compound 3′ was formed as the major product. Hydrogenoly-
sis in the presence of 2 equiv of NaHCO3 also afforded
compound 3′ as the major product. ꢀ-Elimination can be acid-
catalyzed or base-catalyzed, and thus neither of these reaction
conditions were suitable. When we tried to first remove the
ester protecting groups using Zemplen conditions, the cor-
responding methyl glycoside 8 was obtained.
In an attempt to increase the yield of the phosphorylation
step, another strategy was considered, in which monophosphate
3 would be obtained in two steps from 5 through the activation
of the anomeric position as a bromide (Scheme 4). The bromide
(13) Ke, W.; Whitfield, D. M.; Gill, M.; Larocque, S.; Yu, S.-H. Tetrahedron
Lett. 2003, 44, 7767–7770.
(14) (a) Chiba, T.; Sinay, P. Carbohydr. Res. 1986, 151, 379–389. (b) Yu,
H. N.; Furukawa, J.-I.; Ikeda, T.; Wong, C.-H. Org. Lett. 2004, 6 (5), 723–726.
(15) Bazin, H. G.; Kerns, R. J.; Linhardt, R. J. Tetrahedron Lett. 1997, 38,
923–926.
(16) Lubineau, A.; Gavard, O.; Alais, J.; Bonnaffe, D. Tetrahedron Lett. 2000,
41, 307–311.
(17) (a) Vlahov, I. R.; Linhardt, R. J. Tetrahedron Lett. 1995, 36, 8379–
8382. (b) Schell, P.; Orgueira, H. A.; Roehrig, S.; Seeberger, P. H. Tetrahedron
Lett. 2001, 42, 3811–3814.
(18) (a) Chiba, T.; Jacquinet, J.-C.; Sinay, P.; Petitou, M.; Choay, J.
Carbohydr. Res. 1988, 174, 253–264. (b) Rochepeau-Jobron, L.; Jacquinet, J.-
C. Carbohydr. Res. 1997, 303, 395–406.
(19) Deng, S.; Gangadharmath, U.; Chang, C.-W. T. J. Org. Chem. 2006,
71, 5179–5185.
(20) Gavard, O.; Hersant, Y.; Alais, J.; Duverger, V.; Dilhas, A.; Bascou,
A.; Bonnaffe´, D. Eur. J. Org. Chem. 2003, 3603–3620.
(21) MacDonald, D. L. J. Org. Chem. 1962, 27, 1107–1109.
J. Org. Chem. Vol. 73, No. 19, 2008 7633

Weïwer et al.
SCHEME 4. Synthesis of UDP-IdoA Using a Bromide Donor
SCHEME 5. Synthesis of UDP-Hexenuronic Acid 1′
donor 9 was obtained as the R-anomer 9-R in 95% purity by
1H NMR (see the Supporting Information) and in 89% yield.
The desired monophosphate 3 was obtained from the bromide
donor 9 in 41% yield by treatment with the tetrabutylammonium
salt of phosphoric acid. The reaction was stereospecific and only
the ꢀ-anomer of 3 was identified (see the Supporting Informa-
Enzymatic Assays. These unnatural analogues of UDP-GlcA
have been tested as substrates for glycosyltransferases isolated
from Pasteurella multocida (PmHS1 and PmHS2).7f UDP-IdoA
could ultimately serve as a substrate for the direct and specific
incorporation of IdoA units in a growing oligosaccharide to
enzymatically build the heparin backbone. UDP-HexUA could
serve as a potential chain terminator for the preparation of
polymers having a defined molecular weight range.
1
tion, H NMR J1,P ) 7.5 Hz and J1,2 ) 2.0 Hz). Traces of the
eliminated monophosphate 3′ could also be observed. Treatment
of compound 3 with a catalytic amount of sodium methoxide
in methanol afforded the unprotected monophosphate 2, which
was converted into its monopyridinium salt by treatment with
Amberlite resin (pyridinium form). The methyl ester was kept
intact to avoid problems during the coupling reaction. The
monophosphate 2 was then reacted with UMP-morpholidate
according to the procedure of Khorana and Moffatt. The
resulting mixture was then treated with a 2:2:1 mixture of
MeOH/H2O/Et3N to remove the methyl ester. Purification was
accomplished with a BioGel P2 with 0.25 M NH4HCO3 as
eluent, followed by desalting on BioGel P2 with water as eluent.
The fractions containing UDP-IdoA were lyophilized and further
purified by using a semipreparative SAX-HPLC column (linear
gradient from 0 to 1 M NaCl over 50 min.) required for the
removal of the uridine diphosphate dimer formed by self-
condensation of UMP-morpholidate, which is a classical
byproduct of the coupling reaction. After desalting on BioGel
P2, the UDP-IdoA 1 target was obtained in 26% yield over 2
steps.
Recently, Sismey-Ragatz et al. reported the enzyme-catalyzed
incorporation of unnatural UDP-glucosamine derivatives to
different heparosan type acceptors using the Pasteurella mul-
tocida heparosan synthases PmHS1 and PmHS2.7f These
enzymes both possess two active sites, a glucosaminyltransferase
site that transfers UDP-GlcNAc and a glucuronosyltransferase
site that transfers UDP-GlcA to an oligosaccharide acceptor.
Neither of these glycosyltransferases were able to transfer UDP-
HexUA. To build polymers having the backbone of heparin
(GlcN-IdoA), UDP-IdoA was used in combination with UDP-
GlcNAc, in the presence of PmHS1 or PmHS2, and A-F-AN
as acceptor.7f Radiochemical assays suggested that both PmHS1
and PmHS2 coincorporated UDP-IdoA and UDP-GlcNAc, albeit
much lower efficiency than with UDP-GlcA (∼4% rate of
authentic UDP-GlcA). Gel analysis of the PmHS2 polymeri-
zation reactions without acceptor demonstrated small amounts
of short polymers were generated by using the UDP-IdoA
preparation (Figure 1). UDP-GlcA yielded a much greater
amount of larger polymers. In another set of experiments, we
tested A-F-AN as acceptor and either UDP-GlcA or UDP-IdoA
as donor in combination with PmHS2 and monitored the
formation of the corresponding tetrasaccharide A-F-AN-UA.
Using the same strategy, the unsaturated analogue of UDP-
GlcA and UDP-IdoA, the UDP-HexUA 1′, was obtained in 31%
yield over 3 steps from 3′ (Scheme 5).
7634 J. Org. Chem. Vol. 73, No. 19, 2008

Synthesis of Uridine 5′-diphosphoiduronic Acid
FIGURE 1. Analysis of GAG polymers by polyacrylamide gel
electrophoresis and analysis of the A-F-AN-UA tetrasaccharide by
MALDI-ToF. PmHS2 was used in three polymerization reactions
containing UDP-GlcNAc and either UDP-GlcA (G), no second sugar
(0), or the UDP-IdoA preparation (I). Large molecular mass polymers
were formed in abundance with the authentic precursor, but the UDP-
IdoA analogue resulted in smaller amounts of short chains (bracketed).
The positions of hyaluronan standards are shown in sugar units (18-
mer ) ∼1.6 kDa; 40-mer ) ∼8 kDa) (note: the light staining band
marked with an asterisk (/) is the enzyme). MALDI-ToF spectra: top
spectrum ) A-F-AN + UDP-GlcA, bottom spectrum ) A-F-AN +
UDP-IdoA.
MALDI-ToF mass spectrometry allowed the detection of trace
amounts of tetrasaccharide with the expected mass when UDP-
IdoA was tested (Figure 1, A-F-AN-GlcA observed 1106.55
Da when using UDP-GlcA versus 1106.12 Da when using UDP-
IdoA; the mock reaction did not possess this same peak).
While a small portion of the UDP-IdoA incorporated, it
remains unclear whether the low levels of incorporation are due
to (a) either UDP-IdoA or its elongation products inhibiting the
synthase, or (b) the presence of trace UDP-GlcA contaminant,
or (c) the formation of UDP-GlcA from UDP-IdoA through
epimerization during synthase catalysis.
FIGURE 2. PAMN-HPLC profiles of PmHS2 modified 3H-labeled
trisaccharide. A 3H-labeled trisaccharide with a structure of (GlcN[3H]Ac-
GlcUA-AnMan) was incubated with PmHS2 and UDP-GlcA or UDP-
IdoA. The product was analyzed by PAMN-HPLC which was eluted
with a linear gradient of KH2PO4. Panel A shows the profile of
3H-labeled trisaccharide (GlcN[3H]Ac-GlcA-AnMan) was
used as acceptor in the presence of PmHS1 or PmHS2 to help
identify the uronic acid residue that is transferred when using
the UDP-IdoA preparation (Figure 2), and the reactions were
monitored by using PAMN-HPLC.22 3H-labeled trisaccharide
was synthesized from a disaccharide (GlcUA-AnMan) by using
E. coli derived KfiA and UDP-GlcNAc[3H], where AnMan
represents 2,5-anhydromannitol.22
3
3
unmodified H-trisaccharide. Panel B shows the profile of H-trisac-
charide modified with PmHS2 in the presence of UDP-GlcA for 2 h.
3
Panel C shows the profile of H-labeled trisaccharide modified with
PmHS2 in the presence of UDP-IdoA for 2 h. Panel D shows the profile
3
of H-labeled trisaccharide modified with PmHS1 in the presence of
UDP-IdoA overnight.
When UDP-GlcA was used (positive control), the desired
tetrasaccharide was obtained with a very good selectivity. When
UDP-IdoA was used as substrate, the conversion was very low,
but a small peak corresponding to the tetrasaccharide was
obtained. These products were treated with glucuronidase and
iduronidase to identify the nature of the uronic acid unit
incorporated.23 Both tetrasaccharide products were glucu-
ronidase-sensitive but were iduronidase-insensitive. Thus, the
UDP-IdoA could not be incorporated as IdoA into oligosac-
charides with use of PmHS1 or PmHS2. While trace amounts
of UDP-GlcA contaminant could be present in the synthetic
preparation, it could not be detected with NMR or SAX-HPLC.
Alternatively, UDP-GlcA might be formed from UDP-IdoA
through unexpected and hitherto never previously observed C-5
epimerization during synthase catalysis. A wider library of UDP-
GlcA analogues is needed to understand the specificity of these
glycosyltransferases. The current study constitutes a preliminary
study. Furthermore, it may be possible to incorporate IdoA units
in a growing oligosaccharide from UDP-IdoA by modifying the
glycosyltransferase to accept such an unnatural substrate. Work
is underway to generate a library of glycosyltransferase mutants
to test their ability to utilize UDP-IdoA to build IdoA containing
oligosaccharides.
In conclusion, two new unnatural analogues of UDP-GlcA,
UDP-IdoA 1 and UDP-HexUA 1′, were synthesized and used
to study the specificity of different glucuronosyltransferases.
UDP-HexUA did not serve as a substrate for the glucurono-
syltransferases tested. When UDP-IdoA was used as the
substrate, sugar residues were transferred but only GlcA was
incorporated into the products formed. This result could be
explained by the contamination of synthetic UDP-IdoA with a
small amount of UDP-GlcA. Alternatively, UDP-IdoA might
be isomerized to UDP-GlcA by the synthase upon transfer by
a not known yet understood enzymatic process.
Experimental Section
Methyl 1,2,4-Tri-O-acetyl-3-O-pivaloyl-ꢀ-L-idopyranuronate
(5). Methyl 3-O-pivaloyl-L-iduronate (4) (3.1 g, 10.6 mmol) was
suspended in anhydrous CH2Cl2 (56 mL) under argon atmosphere
at -40 °C (acetone/dry ice) and 2,4-dimethylaminopyridine (130
(22) Chen, M.; Bridge, A.; Liu, J. Biochemistry 2006, 45, 12358–12365.
(23) Chen, J.; Duncan, M. B.; Carrick, K.; Pope, R. M.; Liu, J. Glycobiology
2003, 13, 785–794.
J. Org. Chem. Vol. 73, No. 19, 2008 7635

Weïwer et al.
mg, 1.06 mmol), pyridine (8.66 mL, 106 mmol), and acetyl chloride
(4.54 mL, 63.7 mmol) were added. After being stirred at this
temperature for 10 h, the mixture was diluted with CH2Cl2 (150
mL) and the resulting organic phase was washed with saturated
NaHCO3, water, 1 M HCl, and water. The organic layer was dried
over Na2SO4, filtered, and concentrated under diminished pressure.
After purification on silica gel with use of a gradient of petroleum
ether/ethyl acetate (85/15 to 50/50), methyl 1,2,4-tri-O-acetyl-3-
O-pivaloyl-ꢀ-L-idopyranuronate (5) (2.88 g, 65%) was obtained.
An additional mixture of 5r/5ꢀ (302 mg, 7%) was also recovered
zylphospho-2,4-di-O-acetyl-3-O-pivaloyl-R-L-idopyranuronate (7r)
1
(12 mg, 18%) were obtained. 7ꢀ: H NMR (500 MHz, CDCl3) δ
(ppm) 7.37-7.28 (10H, HAr), 5.84 (1H, dd, J1,P ) 6.4 Hz, J1,2
)
0.6 Hz, H-1), 5.15-5.00 (6H, m, H-3, H-4, 2CH2 of OBn), 4.89
(1H, d, J ) 2.4 Hz, H-5), 4.84 (1H, m, H-2), 3.70 (3H, s, CO2Me),
2.08, 2.07 (6H, 2 s, 2 OAc), 1.17 (9H, s, Piv). 13C NMR (125 MHz,
CDCl3) δ (ppm) 177.3 (d, JC,P ) 6.6 Hz, Piv), 169.2 (d, JC,P ) 2.6
Hz, OAc), 168.9 (d, JC,P ) 2.4 Hz, OAc), 167.3 (CO2Me), 135.3,
135.2 (2d, JC,P ) 7.3 Hz and JC,P ) 7.3 Hz, 2 quaternary Ar), 128.6,
128.5, 128.4, 128.0, 127.8 (10 CHAr), 94.9 (d, JC,P ) 5.6 Hz, C-1),
69.8 (d, JC,P ) 5.2 Hz, C-5), 69.5 (d, JC,P ) 5.2 Hz, C-3), 67.5 (d,
JC,P ) 3.9 Hz, C-2), 66.4 (d, JC,P ) 2.5 Hz, C-4), 65.9, 65.4 (2d,
JC,P ) 11.0 Hz and JC,P ) 3.7 Hz, 2CH2 OBn), 52.6 (d, JC,P ) 2.9
Hz, CO2Me), 38.7 (quaternary C of Piv), 26.8 (d, JC,P ) 5.7 Hz, 3
Me of Piv), 20.6, 20.5 (2d, JC,P ) 2.8 Hz and JC,P ) 2.7 Hz, 2
OAc). HRMS m/z calcd for C30H37O13PNa [M + Na]+ 659.1864,
found 659.1860.
1
(combined yield of 5r/5ꢀ: 72%). H NMR (500 MHz, CDCl3) δ
(ppm) 6.03 (1H, d, J ) 2.0 Hz, H-1), 5.32 (1H, t, J ) 4.1 Hz,
H-3), 5.10 (1H, m, H-4), 5.01 (1H, ddd, J ) 4.1 Hz, J ) 2.0 Hz,
J ) 0.7 Hz, H-2), 4.68 (1H, d, J ) 2.7 Hz, H-5), 3.79 (3H, s,
CO2Me), 2.14, 2.12, 2.09 (9H, 3 s, 3 OAc), 1.26 (9H, s, Piv). 13C
NMR (125 MHz, CDCl3) δ (ppm) 175.7, 169.4, 169.2, 168.5, 166.8
(Piv, 3 OAc and CO2Me), 89.9 (C-1), 73.2 (C-5), 66.8 (C-3), 66.5
(C-2), 65.6 (C-4), 52.7 (CO2Me), 38.9 (quaternary C of Piv), 27.0
(3 Me of Piv), 20.7, 20.6, 20.5 (3 Me of 3 OAc). HRMS m/z calcd
for C18H26O11Na [M + Na]+ 441.1367, found 441.1369.
Methyl 1-Phospho-2,4-di-O-acetyl-3-O-pivaloyl-ꢀ-L-idopyra-
nuronate (3). Phosphoric acid (30 mg, 0.3 mmol) was treated with
a 40 wt % aqueous solution of tetrabutylammonium hydroxide (0.6
mL, 0.9 mmol) at 0 °C in an ice bath. After 10 min the mixture
was freeze-dried to afford the teatrabutylammonium phosphate (246
mg, 0.3 mmol). Methyl 1-bromo-2,4-di-O-acetyl-3-O-pivaloyl-ꢀ-
L-idopyranuronate (9) (101 mg, 0.23 mmol) dissolved in acetonitrile
(7 mL), triethylamine (32 µL, 0.23 mmol) dissolved in acetonitrile
(4 mL), and molecular sieves 3Å (250 mg) were added. This
mixture was heated in refluxing acetonitrile (80 °C) for 30 min.
After the mixture had cooled to rt, the molecular sieves was filtered
off and washed with acetonitrile. The filtrate was evaporated under
diminished pressure to afford a brown oil. After purification on
silica gel with a gradient of EtOAc/MeOH (1/0 to 1/1), methyl
1-phospho-2,4-di-O-acetyl-3-O-pivaloyl-ꢀ-L-idopyranuronate (3)
was obtained. To facilitate NMR assignment, it was then treated
with Amberlite (NH4+ form) in methanol to obtain the correspond-
ing ammonium salt of 3 (46 mg, 41%). 1H NMR (500 MHz, MeOD)
δ (ppm) 5.66 (1H, dd, J1,P ) 7.5 Hz, J1,2 ) 2.0 Hz, H-1), 5.14-5.05
(4H, m, H-2, H-3, H-4, H-5), 3.76 (3H, s, CO2Me), 2.09 and 2.04
(6H, 2 s, 2 OAc), 1.25 (9H, s, Piv). 13C NMR (125 MHz, MeOD)
δ (ppm) 178.2, 171.0, 170.8, 170.3 (Piv, 2 OAc and CO2Me), 95.1
(d, J1,P ) 4.9 Hz, C-1), 69.1 (d, J2,P ) 10.3 Hz, C-2), 69.0 (C-4),
68.9 (C-3), 68.2 (C-5), 52.9 (CO2Me), 39.9 (quaternary C of Piv),
27.4 (3 Me of Piv), 20.7, 20.5 (2 Me of 2 OAc). HRMS m/z calcd
for C16H24O13P [M - H]- 455.0960, found 455.0956.
Methyl 1-Bromo-2,4-di-O-acetyl-3-O-pivaloyl-r-L-idopyra-
nuronate (9). Methyl 1,2,4-tri-O-acetyl-3-O-pivaloyl-ꢀ-L-idopy-
ranuronate (5) (120 mg, 0.287 mmol) was suspended in anhydrous
CH2Cl2 (1 mL) at 0 °C and water (31 µL, 1.72 mmol) followed by
PBr3 (46 µL, 0.49 mmol) were added. After being stirred at this
temperature for 10 min, the mixture was warmed to rt and
vigorously stirred for 3 h. The resulting mixture was then diluted
with CH2Cl2 (15 mL) and washed with water and saturated NaHCO3
solution. The organic layer was dried over Na2SO4, filtered, and
concentrated under diminished pressure to give the methyl 1-bromo-
2,4-di-O-acetyl-3-O-pivaloyl-R-L-idopyranuronate (9r) (112 mg,
89%). This compound was used in the next step without further
purification after 1H NMR showed purity higher than 95%. 1H NMR
(500 MHz, CDCl3) δ (ppm) 6.46 (1H, br s, H-1), 5.19 (1H, br s,
H-4), 5.08-5.06 (2H, m, H-3 and H-2), 4.97 (1H, d, J ) 1.7 Hz,
H-5), 3.82 (3H, s, CO2Me), 2.12, 2.10 (6H, 2 s, 2 OAc), 1.30 (9H,
s, Piv).
Methyl 1-Phenylthio-2,4-di-O-acetyl-3-O-pivaloyl-L-idopyra-
nuronate (6). Methyl 1,2,4-tri-O-acetyl-3-O-pivaloyl-ꢀ-L-idopy-
ranuronate (5) (1.0 g, 2.39 mmol) was suspended in anhydrous
CH2Cl2 (5 mL) under argon atmosphere at room temperature and
freshly activated MS 4Å (200 mg), thiophenol (0.3 mL, 2.91 mmol),
·
and BF3 Et2O (0.45 mL, 3.59 mmol) were added. After being stirred
at this temperature for 1 day, the mixture was diluted with CH2Cl2
(150 mL) and the resulting organic phase was washed with cold
water then saturated NaHCO3. The organic layer was dried over
Na2SO4, filtered, and concentrated under diminished pressure. After
purification on silica gel with a gradient of petroleum ether/ethyl
acetate (85/15 to 50/50), methyl 1-phenylthio-2,4-di-O-acetyl-3-
O-pivaloyl-L-idopyranuronate (6) (413 mg, R/ꢀ ) 1/3, 37%) was
obtained. Further elution allowed recovery of the starting material
5 (520 mg, 52%) that could be reused.
1
6r: H NMR (500 MHz, CDCl3) δ (ppm) 7.60-7.58 (2H, m,
HAr), 7.33-7.31 (3H, m, HAr), 5.13 (1H, t, H-4), 5.08-5.04 (3H,
m, H-2, H-3, H-5), 4.46 (1H, d, J ) 1.8 Hz, H-1), 3.78 (3H, s,
CO2Me), 2.16, 2.09 (6H, 2 s, 2 OAc), 1.21 (9H, s, OPiv). 13C NMR
(125 MHz, CDCl3) δ (ppm) 175.1, 169.2, 169.1, 167.1 (Piv, 2 OAc
and CO2Me), 133.4 (C-1′), 132.1 (2C-2′), 129.1 (2C-3′), 128.1 (C-
4′), 85.1 (C-1), 74.6 (C-5), 67.6 (C-3), 66.1 (C-2), 65.6 (C-4), 52.6
(CO2Me), 38.7 (quaternary C of Piv), 27.0 (3 Me of Piv), 20.6,
20.5 (2 Me of 2 OAc). HRMS m/z calcd for C22H28O9SNa [M +
Na]+ 491.1346, found 491.1353.
1
6ꢀ: H NMR (500 MHz, CDCl3) δ (ppm) 7.51-7.49 (2H, m,
HAr), 7.32-7.27 (3H, m, HAr), 5.68 (1H, d, J ) 2.6 Hz, H-1), 5.22
(1H, d, H-5), 5.18 (1H, t, H-3), 5.13, (1H, t, H-2), 4.99 (1H, t,
H-4), 3.80 (3H, s, CO2Me), 2.09, 2.08 (6H, 2 s, 2 OAc), 1.30 (9H,
s, OPiv). 13C NMR (125 MHz, CDCl3) δ (ppm) 176.1, 169.3, 169.2,
168.2 (Piv, 2 OAc and CO2Me), 134.0 (C-1′), 131.4 (2C-2′), 129.1
(2C-3′), 127.8 (C-4′), 85.5 (C-1), 68.5 (C-5), 67.7 (C-3), 66.9 (C-
2), 66.2 (C-4), 52.6 (CO2Me), 38.9 (quaternary C of Piv), 27.0 (3
Me of Piv), 21.0, 20.8 (2 Me of 2 OAc). HRMS m/z calcd for
C22H28O9SK [M + K]+ 507.1086, found 507.1087.
Methyl 1-Dibenzylphospho-2,4-di-O-acetyl-3-O-pivaloyl-L-
idopyranuronate (7). Methyl 1-phenylthio-2,4-di-O-acetyl-3-O-
pivaloyl-L-idopyranuronate (6) (47 mg, 100 µmol), dibenzyl
phosphoric acid (57 mg, 200 µmol), and freshly activated MS 4Å
(60 mg) were suspended in anhydrous dichloroethane (1 mL) at
room temperature. N-Iodosuccinimide (45 mg, 200 µmol) was added
and the mixture was stirred overnight. The molecular sieves was
then filtered off and washed with CH2Cl2. The resulting organic
phase was washed with saturated NaHCO3, water, saturated
Na2S2O3, then water. The organic layer was dried over Na2SO4,
filtered, and concentrated under diminished pressure. After purifica-
tion on silica gel with a gradient of petroleum ether/ethyl acetate
(7/3 to 1/1), methyl 1-dibenzylphospho-2,4-di-O-acetyl-3-O-piv-
aloyl-ꢀ-L-idopyranuronate (7ꢀ) (45 mg, 73%) and methyl 1-diben-
Uridine 5′-Diphosphoiduronic Acid (UDP-IdoA) (1). Methyl
1-phospho-2,4-di-O-acetyl-3-O-pivaloyl-R-L-idopyranuronate (3)
(18 mg, 36 µmol) was dissolved in anhydrous MeOH (1 mL),
cooled to 0 °C, and treated with a 0.5 M solution of MeONa in
MeOH (0.5 mL). After 3 h at rt, the solution was cooled to 0 °C
and Amberlite (H+ form, 250 mg) was added. After filtration,
MeOH was evaporated and the resulting mixture was redissolved
in water before treatment with Amberlite (PyH+ form). The
corresponding monopyridinium salt of 2 (14 mg) was obtained after
7636 J. Org. Chem. Vol. 73, No. 19, 2008

Synthesis of Uridine 5′-diphosphoiduronic Acid
freeze-drying. The monopyridinium salt of methyl 1-phospho-R-
L-idopyranuronate (2) (14 mg, 36 µmol) was dissolved in anhydrous
pyridine (1 mL) and evaporated under diminished pressure. This
operation was repeated 3 times. A solution of 4-morpholine N,N′-
dicyclohexylcarboxamidinium uridine 5′-phosphomorpholidate (34
mg, 49 µmol) in anhydrous pyridine (2 mL) was concentrated to
dryness in vacuo. This operation was also repeated 3 times. A
solution of the 4-morpholine N,N′-dicyclohexylcarboxamidinium
uridine 5′-phosphomorpholidate in anhydrous pyridine (1 mL) was
then added to the dry monopyridinium salt of methyl 1-phospho-
R-L-idopyranuronate (6) followed by concentration in vacuo. The
mixture was then redissolved in anhydrous pyridine (800 µL) and
stirred at rt for 5 days under argon atmosphere. Pyridine was then
evaporated under reduced pressure and the mixture was treated with
MeOH/H2O/Et3N 2:2:1 (1.5 mL) for 24 h at RT, in order to
deprotect the methyl ester. The mixture was then concentrated under
reduced pressure and redissolved in water. Purification was achieved
on a P2 BioGel column with a 0.25 M NH4HCO3 solution as eluent.
The fractions containing UDP-IdoA (UV at 262 nm) were pooled
and lyophilized. Further purification was achieved with use of a
semipreparative SAX column (20 × 250 mm), using a linear
gradient of NaCl (0 to 1 M over 50 min) at pH 3.5. After desalting
on P2 BioGel, UDP-IdoA 1 (6 mg, 9.3 µmol, 26%) was obtained
storage as aliquots at -80 °C. Maltose binding protein fusions of
PmHS1 or PmHS2, two isozymes of heparosan synthase, were
employed as the catalysts.7f Sugar addition reactions typically
contained 50 mM Tris, pH 7.2, 1 mM MnCl2, PmHS1 or PmHS2,
various amounts of UDP-GlcNAc, and either (a) UDP-GlcA (the
authentic uronic acid) or (b) UDP-IdoA as noted. To measure
relative incorporation rates of the two UDP-uronic acids (1 mM),
radioactive UDP-[3H]GlcNAc (0.27 µM; 0.2 µCi/reaction) addition
into polymer by enzyme (12-15 µg; 24 h at 30 °C) was monitored
by paper chromatography.7f For another proof of polymerization,
reactions with nonradioactive UDP-sugars (10-25 mM each) and
enzyme (46 µg; 22 h at 30 °C) were analyzed on polyacrylamide
gels with combined Alcian Blue/Silver staining.7f Single sugar
addition reactions with A-F-AN, a synthetic acceptor (0.5-1 mM),
and synthase (6-15 µg; 18-60 h at 22-30 °C) were used under
similar conditions as polymerization assays, but only a single UDP-
uronic acid (0.75-1.5 mM) was employed. Products were analyzed
by MALDI-ToF mass spectrometry with ATT matrix in negative
ion mode.7f
Enzymatic Synthesis of a Tetrasaccharide. PmHS2 (50 µg)
was incubated with trisaccharide (50,000 cpm) in a buffer containing
25 mM Tris (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, and 1 µM
UDP-GlcUA or UDP-IdoUA at 37 °C for 2 h or overnight. The
presence of anticipated 3H-labeled tetrasaccharide (GlcUA-
GlcN[3H]Ac-GlcUA-AnMan) was determined by determining the
retention time of the 3H-peak on polyamine-based anion exchange
HPLC.22 Briefly, the polyamine II column (YMC) was equilibrated
with ddH2O for 10 min, and then eluted with a linear gradient of
0 to 1 M KH2PO4 in 60 min at 0.5 mL/min. 3H-labeled trisaccharide
and tetrasaccharide were respectively eluted at around 33 and 83
mM of KH2PO4.
1
as a white powder. H NMR (500 MHz, D2O) δ (ppm) 7.86 (1H,
d, J ) 8.1 Hz, H-6), 5.95-5.85 (2H, m, H-1′ and H-5), 5.46 (1H,
dd, J1′′,P ) 7.9 Hz, J1,2 ) 2.9 Hz, H-1′′), 4.55 (1H, d, J ) 2.8 Hz,
H-5′′), 4.30-4.10 (5H, m, 2H-5′, H-4′, H-3′, H-2′), 3.92 (1H, dd,
J ) 3.9 Hz, J ) 2.8 Hz, H-4′′), 3.79 (1H, t, J ) 4.8 Hz, H-3′′),
3.69 (1H, dd, J ) 5.1 Hz, J ) 2.9 Hz, H-2′′). 13C NMR (125 MHz,
D2O) δ (ppm) 175.9 (C-6′′), 166.3 (d, J ) 22.1 Hz, C-4), 151.9 (d,
J ) 21.9 Hz, C-2), 141.6 (C-6), 102.7 (d, J ) 10.1 Hz, C-5), 97.2
(d, J ) 6.5 Hz, C-1′′), 88.2 (d, J ) 6.3 Hz, C-1′), 83.4 (d, J ) 9.3
Hz, C-4′), 83.3 (d, J ) 9.0 Hz, C-5′′), 73.8 (d, J ) 21.1 Hz, C-3′),
70.6 (d, J ) 7.1 Hz, C-3′′), 70.3 (C-4′′), 69.7 (C-2′′), 66.0 (d, J )
6.1 Hz, C-2′), 64.9 (d, J ) 5.8 Hz, C-5′). 31P NMR (202.34 MHz,
D2O) δ (ppm) -10.2 (d, J ) 20 Hz), -12.2 (d, J ) 20 Hz). HRMS
m/z, calcd for C15H21N2O18P2 [M - H]- 579.0270, found 579.0255.
Uridine 5′-Diphosphohex-4,5-enuronic Acid (1′). Following the
same procedure as described above, uridine 5′-diphosphohex-4,5-
enuronic acid (1′) was obtained (10 mg, 31%). 1H NMR (500 MHz,
D2O) δ (ppm) 7.87 (1H, d, J ) 8.1 Hz, H-6), 5.93 (1H, d, J ) 4.5
Hz, H-1′), 5.90 (1H, d, J ) 8.1 Hz, H-5), 5.82 (1H, d, J ) 3.9 Hz,
H-4′′), 5.60 (1H, dd, J1′′,P ) 8.4 Hz, J1,2 ) 5.3 Hz, H-1′′), 4.30
(1H, m, H-2′), 4.21 (1H, m, H-3′), 4.16 (1H, m, H-4′), 4.13-4.10
(2H, m, H-3′′, H-5′), 3.84 (1H, t, J ) 5.0 Hz, H-2′′). 13C NMR
(125 MHz, D2O) δ (ppm) 168.9 (C-6′′), 166.63 and 166.56 (C-2
and C-4), 144.6 (C-5′′), 141.5 (C-6), 107.3 (C-4′′), 102.7 (C-5),
95.3 (C-1′′), 88.2 (C-1′), 83.4 (C-3′), 83.3 (C-4′), 70.0 (C-2′′), 69.7
(C-2′), 66.0 (C-5′), 65.1 (C-3′′). 31P NMR (202.34 MHz, D2O) δ
(ppm) -9.98 (d, J ) 20 Hz), -11.95 (d, J ) 20 Hz). HRMS m/z
calcd for C15H19N2O17P2 [M - H]- 561.0164, found 561.0158.
Enzymatic Assays. Polymerization Reactions with Pasteurella
multocida Heparosan Synthases (PmHS1 and PmHS2). UDP-
sugars were dissolved at 15 mM in 10 mM Tris, pH 7.2, buffer for
Glucuronidase/Iduronidase Treatment of the Tetrasaccha-
ride. The resulting 3H-labeled tetrasaccharide was purified by
PAMN-HPLC and desalted by dialyzing against water, using
MWCO 1000 membrane. The 3H-labeled tetrasaccharide was
digested with either ꢀ-glucuronidase or R-iduronidase as described
previously.23 The digested samples were reanalyzed by PAMN-
HPLC to determine if the tetrasaccharide was susceptible to the
digestion with either ꢀ-glucuronidase or R-iduronidase.
Acknowledgment. This work was supported by the National
Institute of Health (NIH HL062244 to R.J.L.). The authors
would like to thank Nigel J. Otto for the preparation of the
heparosan synthases, Dr. Zhenqing Zhang for his help in
acquiring NMR data, and Dr. Dmitri Zagorevski for HRMS
analyses.
Supporting Information Available: 1H and 13C NMR data
of compounds 1, 1′, 3-7, and 9 and 31P NMR, COSY, and
HMQC of compounds 1 and 1′. This material is available free
of charge via the Internet at http://pubs.acs.org.
JO801409C
J. Org. Chem. Vol. 73, No. 19, 2008 7637