Fluorous-assisted chemoenzymatic synthesis of heparan sulfate oligosaccharides

Article abstract of DOI:10.1021/ol500738g

The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate (^FBoc) tag did not interfere with enzymatic recognition

Full text of DOI:10.1021/ol500738g

Letter
pubs.acs.org/OrgLett
Fluorous-Assisted Chemoenzymatic Synthesis of Heparan Sulfate
Oligosaccharides
Chao Cai,† Demetria M. Dickinson,† Lingyun Li,† Sayaka Masuko,† Matt Suflita,‡ Victor Schultz,†
Shawn D. Nelson,† Ujjwal Bhaskar,† Jian Liu,∥ and Robert J. Linhardt*,†,‡,§
‡
§
†Department of Chemistry and Chemical Biology, Department of Biology, and Departments of Chemical and Biological
Engineering and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,
Troy, New York 12180, United States
∥Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
North Carolina 27599, United States
S
* Supporting Information
ABSTRACT: The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag
attached at the reducing end is reported. The fluorous tert-butyl dicarbonate (FBoc) tag did not interfere with enzymatic
recognition for both elongation and specific sulfation, and flash purification was performed by standard fluorous solid-phase
extraction (FSPE). Based on an FBoc attached disaccharide as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate
oligosaccharides were successfully synthesized employing fluorous techniques.
lycosaminoglycans (GAGs) are anionic polysaccharides
fluorous surface through fluorous solid-phase extraction
Gfound in all animal cells that are composed of repeating (FSPE) and can be easily released through fluorophilic elution.
disaccharide units of hexuronic acid and hexosamine.1 Of all the
Reversible binding, ease of purification, broad reaction scope,
and an ability to be automated all make fluorous tagging
especially suitable for high-throughput combinatorial syn-
thesis.12
classes of GAGs, the heparan sulfate (HS) and heparin family
of GAGs are the most attractive therapeutic targets2 as they are
known to regulate a wide range of physiological processes3
through their interactions with biologically important proteins,
such as growth factors.4 However, even with recent advances in
synthetic carbohydrate chemistry,5 the preparation of HS and
heparin oligosaccharides remains a major challenge due to their
structural complexity and heterogeneity. Therefore, chemo-
enzymatic approaches, relying on biosynthetic enzymes for the
synthesis of highly sulfated GAG oligosaccharides, represent
powerful and efficient alternatives to traditional methods.6
Fluorous chemistry emerged as a new tool for solution-phase
high-throughput organic synthesis in the late 1990s.7 Fluorous
separation techniques rely on the high affinity of perfluoroalkyl
chains toward fluorous surfaces and solvents. Fluorous tag-
facilitated chemical synthesis has been developed extensively by
Curran8 over the past decade and applied to proteomics,9
peptide synthesis,10 and carbohydrate microarrays.11 In contrast
to the streptavidin−biotin system, fluorous tags bind to a
In recent years, fluorous techniques have been applied to
oligosaccharide synthesis and have significantly facilitated
purification. In their automated solid-phase oligosaccharide
synthesis, Seeberger and co-workers used a TIPS-like fluorous
linker to “cap” unreacted sugar residues and to remove
unwanted deletion sequences from the glycosylation mixture.13
Huang and co-workers reported a similar strategy, where the
fluorous “cap” is applied to the product, and synthesized linear
and branched oligosaccharides in a one-pot manner.14 Pohl and
co-workers attached a fluorous linker at the reducing end of a
mannoside to prepare linear and branched mannose
oligosaccharides15 and immobilized these onto fluorocarbon-
coated glass slides to study their binding with concanavalin A.16
Received: March 11, 2014
Published: April 4, 2014
© 2014 American Chemical Society
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Boons and co-workers reported a modular synthesis of heparan
sulfate tetrasaccharide with a fluorous tagged aminopentyl
linker at the reducing end of glucosamine substrates.17
Scheme 2. Synthesis of Heparosan Hexasaccharide Analogue
14
Although recent advances in chemoenzymatic synthesis of
heparan sulfate oligosaccharides have included one-pot
synthesis6d and the synthesis at the hundreds of milligram
scale,6c development of more efficient and less time-consuming
methodology is still necessary to speed up the purification
process.18 The application of fluorous techniques in enzymatic
reactions is still not well developed. In this paper, we report a
fluorous-assisted chemoenzymatic synthesis of heparan sulfate
oligosaccharides from a chemically synthesized disaccharide
acceptor, with a fluorous Boc (FBoc)19 on the glucosamine
nitrogen at the reducing end. Liu and co-workers synthesized a
similar heparan sulfate oligosaccharide with a fluorous linker
but with an unnatural anhydromannitol residue at the reducing
end.20 The newly designed disaccharide acceptor in this study
has an α-configured O-methyl glycoside at the reducing end,
which can serve as a potential starting point for chemo-
enzymatic synthesis of the heparin pentasaccharide anticoagu-
lant drug Arixtra (fondaparinux).
Our retrosynthetic analysis of the heparan sulfate oligosac-
charides (1 and 2) with fluorous-assisted methodology is
shown in Figure 1. These would be prepared from the fluorous
Figure 1. Retrosynthetic analysis of heparan sulfate tetrasaccharide (1)
and hexasaccharide (2).
Scheme 1. Synthesis of Fluorous-Tagged Disaccharide 3
acceptor 3 through a repetition of enzymatic backbone
elongation followed by N-sulfation, 6-O-sulfation, and depro-
F
tection of the Boc tag. Fluorous disaccharide 3 would be
chemically synthesized through introduction of the FBoc tag to
the free amino group on the reducing end of disaccharide 4,
which would be obtained through a chemical glycosylation of
acceptor 7 with donor 6 and global deprotection and
hydrogenation of the fully protected disaccharide 5.
The synthesis of donor 6 and acceptor 7 followed a
conventional synthetic route described in the Supporting
Information (Schemes SI1 and SI2). As shown in Scheme 1,
the glycosylation of 6 with 7 afforded disaccharide 8 in
moderate yield. With the fully protected disaccharide in hand,
the TBDPS group was removed initially by treatment with HF·
Py21 to afford disaccharide 9. The unprotected C6-hydroxyl
group was then oxidized to a carboxylic acid using TEMPO-
BAIB22 to afford 5. The Bz groups were removed by strong
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Scheme 3. Chemoenzymatic Synthesis of Heparan Sulfate
Tetrasaccharide and Hexasaccharide
Figure 2. Illustration of extracted ion chromatograms (EIC)
corresponding to the HS backbone oligosaccharides indicated: (A)
disaccharide (3); (B) trisaccharide (11); (C) tetrasaccharide (12);
(D) pentasaccharide (13); and (E) hexasaccharide (14).
base treatment followed by hydrogenation for 3 d using Pd/C
as the catalyst. All deprotection steps proceeded smoothly to
give the disaccharide 4 in high yields (Scheme 1).
Fluorous-tagged disaccharide acceptor 3 was next elongated
with Escherichia coli glycosyltransferase KfiA and uracil
diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA)
to construct the trisaccharide 11 (Scheme 2). After flash
elution through FSPE with water and methanol, respectively,
the pure trisaccharide 11 was collected in methanol and
identified by LC−MS and NMR spectroscopy. One cycle of
fluorous-assisted purification generally takes <0.5 h and affords
a relatively pure product. The GlcNTFA residue is an unnatural
analogue of GlcNAc and should allow the selective introduction
of N-sulfate groups in the future synthesis of HS
oligosaccharides. Following the same protocol as above,
Pasteurella multocida heparan synthase (PmHS2) and uracil
diphosphate−glucuronic acid (UDP−GlcA) were employed to
construct the tetrasaccharide (12). These steps were repeated
one more time to afford the pentasaccharide (13) and
hexasaccharide (14) with the GlcA−GlcNTFA repeating unit.
LC−MS data analysis of unsulfated HS backbone from
disaccharide to hexasaccharide is shown in Figure 2.
Na2CO3 to form N-sulfated tetrasaccharide (15) (Scheme 3). A
high-field shift of 0.5 ppm for H-2 was observed in the H and
1
2D COSY NMR (2H2O, 600 MHz) in the glucosamine residue
that had been N-sulfated. The heparan sulfate 6-O-
sulfotransferase isoforms -1 and -3 (6-OST-1 and 6-OST-3)
were incubated together with 15 and PAPS to obtain the 6-O-
sulfate group containing tetrasaccharide 17, and excess PAPS
and buffer salts were easily removed by FSPE. 1H NMR (2H2O,
600 MHz) showed that the peaks at 3.67 and 3.78 ppm,
corresponding to the protons on the C6 of the internal
glucosamine residue in 15, shifted to 4.09 and 4.37 ppm in the
product 17. 2D COSY and HMQC NMR spectroscopy also
confirmed the formation of 17. The glucosamine residue at the
reducing end of 15 was not 6-O-sulfated, suggesting that
additional studies are required to more fully understand the
specificity of the 6-O-sulfotransferases.23 Subsequently, hex-
asaccharide 18 was obtained using the same protocol, including
N-sulfation, 6-O-sulfation, and FSPE steps. We found that some
product was retained on the FSPE column after four sulfate
groups had been added on the HS chain, but it could be easily
released by employing trifluoroethanol as a cosolvent.
Deprotection of FBoc was initially attempted, based on a
literature method,19 using either 50% aqueous TFA at room
With the HS backbone constructed and in hand, we
subsequently sulfated these substrates to check their perform-
ance with FSPE separation. Base-catalyzed (MeOH/NEt3/
H2O, 2/1/2) deprotection of the trifluoroacetamide group was
followed by the chemical N-sulfation with SO3·MeN3 and
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temperature or 3 N aqueous HCl at 60 °C for 2 h. We observed
that while 3 N HCl removed the fluorous tag from
tetrasaccharide (17), it also resulted in complete sulfate loss.
Treatment of the model substrate 3 with 50% aqueous TFA
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F
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F
In conclusion, we have successfully applied the Boc linker
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
1
1H, 13C, H−1H COSY, H−13C HMQC NMR, and ESI MS
spectra and experimental procedures for the preparation of
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: linhar@rpi.edu. Tel: 518-276-3404. Fax: 518-276-
3405.
Notes
(21) Lohman, G. J. S.; Hunt, D. K.; Ho, J. A.; Seeberger, P. H. J. Org.
Chem. 2003, 68, 7559.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We gratefully acknowledge support from the National Institutes
of Health in the form of Grant Nos. HL62244, HL094463,
HL096972, and GM102137.
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