Fluorous supported modular synthesis of heparan sulfate oligosaccharides

Article abstract of DOI:10.1021/ol303270v

The modular synthesis of heparan sulfate fragments is greatly facilitated by employing an anomeric aminopentyl linker protected by a benzyloxycarbonyl group modified by a perfluorodecyl tag, which made it possible to purify highly polar intermediates by fluorous solid phase extraction. This tagging methodology made it also possible to perform repeated glycosylations to drive reactions to completion.

Full text of DOI:10.1021/ol303270v

ORGANIC
LETTERS
2013
Vol. 15, No. 2
342–345
Fluorous Supported Modular Synthesis of
Heparan Sulfate Oligosaccharides
Chengli Zong,†,‡ Andre Venot,† Omkar Dhamale,†,‡ and Geert-Jan Boons*,†,‡
Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend
Road, Athens, Georgia 30602, United States, and Department of Chemistry,
The University of Georgia, Athens, Georgia, Georgia 30602, United States
gjboons@ccrc.uga.edu
Received November 28, 2012
ABSTRACT
The modular synthesis of heparan sulfate fragments is greatly facilitated by employing an anomeric aminopentyl linker protected by a
benzyloxycarbonyl group modified by a perfluorodecyl tag, which made it possible to purify highly polar intermediates by fluorous solid phase
extraction. This tagging methodology made it also possible to perform repeated glycosylations to drive reactions to completion.
Heparan sulfates (HS) are highly N- and O-sulfated
polysaccharides involved in a number of important biolo-
gical processes such as embryo development, inhibition
of blood coagulation, organization of the extracellular
matrix, angiogenesis, the presentation of enzymes and
cytokines on cell surfaces, and as coreceptors for viral
infections.1 In general, it is difficult to determine oligosac-
charide sequences and sulfation patterns required for
binding of HS binding proteins.2 To address this difficulty,
we have developed a modular approach for the chemical
synthesis of HS oligosaccharides whereby a set of di-
saccharide building blocks, which resemble the different
disaccharide motifs found in HS, can repeatedly be used for
the assembly of a wide range of sulfated oligosaccharides.3
In this approach, levulinoyl esters (Lev)4 are employed for
the protection of hydroxyls that need sulfation. In HS, the
C-3 and C-6 of glucosamine and C-1 hydroxyls of uronic
acids can be sulfated, and therefore depending on the
sulfation pattern of a targeted disaccharide module, one
or more of these positions are protected as Lev esters. In
case the C-20 position of a disaccharide module does not
† Complex Carbohydrate Research Center.
‡ Department of Chemistry.
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r
10.1021/ol303270v
Published on Web 01/08/2013
2013 American Chemical Society

need sulfation, an acetyl ester is employed as a permanent
protecting group, which is stable under the conditions used
for the removal of Lev esters. An azido group is used as an
amino-masking functionality because it does not perform
neighboring group participation thereby allowing the in-
troduction of R-glucosides.5 The C-40 hydroxyl, which is
required for extension, is protected as a 9-fluorenylmethyl
carbonate (Fmoc), and this protecting group can be re-
moved with a hindered base such as Et3N without affecting
the Lev ester, whereas the Lev group can be cleaved with
hydrazine bufferedwith acetic acidand these conditions do
not affect the Fmoccarbonate.4 The anomeric center of the
modular disaccharides is protected as TDS glycosides, and
this functionality can easily be removed by treatment with
HF in pyridine without affecting the other protecting
groups. The resulting lactol can then be converted into a
leaving group for glycosylations with appropriate accep-
tors. Comparedtoconventional approaches,3d,6 a modular
synthetic strategy makes it possible to rapidly assemble
libraries of HS oligosaccharides for structureꢀactivity
relationship studies.
synthesis9 because compounds tagged by a linear fluorous
tag can easily be separated from nonfluorous material
by solid phase extraction using silica gel modified by
fluorocarbons.10 This generic procedure, which resembles
more filtration than chromatography, depends primarily
on the presence or absence of a fluorous tag, and not on
polarity or other molecular features.
We envisaged that fluorous supported synthesis would
speedup modular synthesis of HS oligosaccharides and
would in particular be attractive for the final modifications
of the fully assembled oligosaccharides, as these proce-
dures are high yielding but require large excesses of
reagents and provide polar compounds that are difficult
to purify by conventional approaches. Previously,3d we
employed an N-(benzyl)benzyloxycarbonyl aminopenta-
nol linker for the modification of the reducing end of HS
oligosaccharides, and thus linker 4 was selected, which
contains a benzyloxycarbonyl protecting group modified
by a perfluorodecyl tag. Linker 4 could easily be prepared
by treatment of aminopentanol (1) with 2 in aqueous
sodium bicarbonate to give, after purification by fluorous
solid phase extraction, benzyloxycarbonyl protected 3 in
an 86% yield (Scheme 1). Selective N-benzylation of 3 to
give 4 was accomplished by a three-step procedure involv-
ing acetylation of the hydroxyl with acetic anhydride in
pyridine followed by N-benzylation by treatment with
benzyl bromide in the presence of NaH in DMF and then
saponication of the acetyl ester using NaOMe in methanol.
Scheme 1. Preparation of Fluorous Tagged Aminopentyl Linker
Scheme 2. Preparation of Fluorous Tagged Tetrasaccharide
Although modular oligosaccharide assembly is very
attractive,3,7 the endgame involving selective protecting
group removal, O- and N-sulfation, and global deprotec-
tion requires a relatively large number of steps providing
polar compounds that are difficult to purify by conven-
tional approaches thereby slowing down the preparation
of libraries of HS oligosaccharides. Several platforms have
been developed to speedup the process of oligosaccharide
assembly.8 We were attracted by light fluorous supported
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374.
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Krock, L.; Esposito, D.; Castagner, B.; Wang, C.-C.; Bindschadler, P.;
Seeberger, P. H. Chem. Sci. 2012, 3, 1617.
Having at hand linker 4 modified with a perfluorodecyl
tag, attention was focused on its installation into modular
disaccharides by glycosylation. Thus, glycosyl donor 6 was
prepared by removal of the anomeric TDS moiety of
modular disaccharide 54d with HF in pyridine to give a
lactol (Scheme 2), which was converted into trifluoro-N-
phenylacetimidate 6 by reaction with N-phenyltrifluoroa-
cetimidoyl chloride in the presence of NaH in DCM.11
Previously, we observed that glycosylations of modular
disaccharides such as 6 with a regular N-(benzyl)-
benzyloxycarbonyl aminopentanol led to mixtures of
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Takahashi, T. Tetrahedron 2011, 67, 10011. (d) Hogendorf, W. F.;
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Org. Lett., Vol. 15, No. 2, 2013
343

anomers that were difficult to separate by silica gel column
chromatography. Therefore, an additional set of modular
disaccharides, having a preinstalled linker, needed to be
prepared.3d A number of conditions were examined to
improve the anomeric selectivity of the glycosylation of
linker 4 with glycosyl donor 6. A TfOH-promoted glyco-
sylation of 4 with 6 in DCM at ꢀ20 °C gave 7 as a mixture
of anomers (R/β = 1/2, 70%), which surprisingly could
readily be separated by traditional silica gel column chro-
matography. Increasing the temperature or the addition of
thiophene12 or DMF13 did not have a notable effect on the
anomeric ratio (R/β ≈ 1/1). The use of diethyl ether to
improve the R anomeric selectivity14 led to a low yield of
product due to the poor solubility of linker 4. The use of a
mixture of dioxane and toluene at ambient temperature14
and TfOH as the promoter gave compound 6 in a good
yield of 75% as mainly the R anomer (R/β = 3/1)
(Scheme 2). Linker 3 was also examined for tagging
modular disaccharides, but the results were disappointing
due to poor solubility of this compound in commonly
employed solvents for glycosylation.
Scheme 3. Preparation of Target Tetrasaccharides
Next, the Fmoc protecting group of 7 was removed by
treatment with Et3N in DCM to give glycosyl acceptor 8 in
a near-quantitative yield after purification by fluorous
solid phase extraction. A TfOH promoted coupling of
glycosyl donor 6 with acceptor 8 led to the formation of
tetrasaccharide 9 as exclusively the R-anomer (Scheme 2).
As expected, fluorous solid phase extraction resulted in the
removal of hydrolyzed acceptor and other nonfluorous
byproducts. The resulting compound 9 was, however,
contaminated with glycosyl acceptor 8 due to an incom-
plete glycosylation. Therefore, the latter mixture was
resubjected to treatment with glycosyl donor 6 (0.5 equiv)
and a catalytic amount of TfOH, which led to complete
consumption of the remaining acceptor to provide, after
fluorous solid phase extraction, pure tetrasaccharide 9 in a
yield of 72%. Solid supported synthesis often exploits
repeated reaction cycles to drive reactions to completion,8f
and the results described here highlight that such an
approach is possible for fluorous supported synthesis.
The sulfate esters were installed following removal of the
Lev esters from 9 with hydrazine acetate in a mixture of
DCM and methanol, followed by sulfation of the hydrox-
yls of compound 10 using a large excess of the pyridinium
sulfur trioxideꢀpyridine complex to provide compound 11
in high yield after purification by fluorous solid phase
extraction (one reaction cycle, Scheme 3). Next, the Fmoc
and methyl esters of 11 were saponified by treatment with
LiOH in a mixture of hydrogen peroxide and THF to give
partially deprotected 12. Purification by fluorous solid
phase extraction was troublesome probably due to the
formation of micelles. However, the addition of a small
amount of 2,2,2-trifluoroethanol solved this problem
and pure fluorous-tagged 12 could readily be obtained.
The azido moiety of 12 was reduced with trimethylphos-
phine in THF in the presence of NaOH15 to give amine 13,
which was immediately sulfated with the sulfur trioxideꢀ
pyridine complex in the presence of triethylamine in
methanol to afford, after fluorous solid phase extraction
with 2,2,2-trifluoroethanol (to avoid micelle formation),
N-sulfate 14 in a yield of 86%. As expected, the modified
N-(benzyl)benzyloxycarbonyl tag was stable under the
applied basic conditions. Alternatively, acetylation of the
free amine of 13 with acetic anhydride in methanol pro-
vided acetamido derivative 15. Finally, the benzyl ethers
and benzyloxycarbamate of 14 and 15 were removed by a
two-step procedure16 involving hydrogenation over Pd/C
in a mixture of MeOH/H2O, which led to the removal of
the spacer protecting groups, followed by hydrogenation
over Pd(OH)2 in H2O which resulted in the removal of the
benzyl ethers to give HS oligosaccharides 16 and 17,
respectively. Addition of a small amount of acetic acid
was found to be necessary to accelerate the hydrogenation.
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344
Org. Lett., Vol. 15, No. 2, 2013

Prolonged hydrogenation in the absence of AcOH caused
a loss of sulfate groups. The 1H NMR spectra of the sulfated
oligosaccharides were fully assigned by 1D and 2D NMR
spectroscopy. The R-anomeric configuration of 2-azido-
glucosides was confirmed by the J1,2 coupling constants
and by the 13C chemical shift of C-1 (∼97 ppm). Further-
more, a downfield shift of 0.5 ppm of H-6 was observed for
O-sulfation of C-6 hydroxyls and 0.4 ppm of H-2 for
N-sulfation.
The studies reported here highlight the appealing fea-
tures of fluorous supported modular synthesis of HS
oligosaccharides. During oligosaccharide assembly, the
attraction of the technology is that two or more reaction
cycles can easily be performed to drive the reaction to
completion and thus early installation of the fluorous tag is
attractive.17 Unlike solid supported synthesis, light fluo-
rous technology does not require large excesses of reagents
to drive the reactions to completion. The fluorous-tagged
compounds could easily be analyzed by standard spectro-
scopic methods thereby providing control over the synthesis.
Furthermore, the final modifications involving selective
protecting group removal, O- and N-sulfation, and global
deprotection were much faster because these reactions
proceed with high efficiency. The resulting intermediates
are normally difficult to purify by traditional chromato-
graphic approaches. We observed, however, that polar
carbohydrates modified by a fluorous tag tend to aggre-
gate complicating chemical transformations and solid
phase extraction. This problem could easily be addressed
by employing 2,2,2-trifluoroethanol as a cosolvent, which
18
is more suitable than the use of EtOC4F9 or PhCF3 to
solve this problem.19 Pohl and co-workers are developing a
liquid handler to automate fluorous supported oligosac-
charide synthesis,20 and it is to be expected that such a
system will be very attractive for modular synthesis of HS
oligosaccharides.
Acknowledgment. This research was supported by the
National Institute of General Medicine (NIGMS) of
the National Institutes of Health (NIH) (Grant No
2R01GM065248). We acknowledge Mrs. Tiantian Sun
(CCRC, UGA) and Dr. LinLiu (CCRC, UGA) for helpful
discussions.
Supporting Information Available. 1H and HSQC
NMR spectra and experimental procedures for the pre-
paration of compounds 3ꢀ5, 7ꢀ13, and 15ꢀ17. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Tanaka, H.; Tateno, Y.; Takahashi, T. Org. Biomol. Chem. 2012,
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Satoh, A.; Goto, K.; Murakami, Y.; Imai, N.; Inazu, T. Tetrahedron:
Asymmetry 2005, 16, 3.
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R.; Pohl, N. L. B. Abstr. Pap. Am. Chem. Soc. 2011, 242.
The authors declare no competing financial interest.
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