A new linker for solid-phase synthesis of heparan sulfate precursors by sequential assembly of monosaccharide building blocks

Article abstract of DOI:10.1039/c0cc04686h

A novel ester type linker which upon cleavage releases the glycans as carbamate protected aminoglycosides was successfully employed in the sequential assembly of l-idose and azido glucose monosaccharide building blocks to heparan sulfate precursors.

Full text of DOI:10.1039/c0cc04686h

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COMMUNICATION
A new linker for solid-phase synthesis of heparan sulfate precursors
by sequential assembly of monosaccharide building blocksw
Pawel Czechura,a Nerea Guedes,a Sebastian Kopitzki,a Naiara Vazquez,b
Manuel Martin-Lomasac and Niels-Christian Reichardt*a
Received 29th October 2010, Accepted 2nd December 2010
DOI: 10.1039/c0cc04686h
A novel ester type linker which upon cleavage releases the glycans
as carbamate protected aminoglycosides was successfully employed
in the sequential assembly of L-idose and azido glucose mono-
saccharide building blocks to heparan sulfate precursors.
A new linker based on a 4-hydroxymethylbenzyl N-(5-hydroxy-
pentyl)-N-benzyl carbamate spacer (compound 10, Scheme 1) and
attached via an ester linkage to the carboxy-polystyrene resin6 has
been specifically designed. This linker provides a stable handle
under Lewis acid, mild basic and nucleophilic conditions, but is
easily cleaved under basic conditions releasing the amine
protected as carbamate, compatible with the azide and phthal-
imide functions commonly used as N-protecting groups for
glucosamine building blocks. Other approaches for the immobi-
lisation of amino-functionalized glycoconjugates release highly
polar amines19,20 which are difficult to purify and are not
compatible with the orthogonal functionalization of glycosamines
e.g. N-sulfation or acetylation in HS synthesis.
Heparan sulfate (HS) is a linear highly sulfated polysaccharide
attached in the form of proteoglycans to the cell surface of
practically all animal cells. Non-template driven variations in
chain length and sulfation pattern lead to an enormous degree
of structural diversity which is thought to reflect the role of
heparan sulfate as a ‘multifunctional cell regulator’.1 Glycan
arrays of short HS fragments with systematic variations of
sequence and sulfation pattern have been proposed to
accelerate the analysis of the many functions and the
molecular basis of protein–HS interactions.2 While isolation
of pure fragments from natural sources is currently difficult
due to the microheterogeneity of the material, the chemical
synthesis of HS libraries is still a challenging and laborious
task especially for larger glycans.3,4
As shown in Scheme 1, N-benzylated aminopentanol 1 was
silylated with thexyldimethylsilylchloride (TDS–Cl) affording
benzylamine 2 in 80% yield. On the other hand, diol 3 was
mono-protected in 50% yield as trichloroethylcarbonate 4 and
activated as carbonate 5. Compound 5 was then reacted with 2
Solid-phase synthesis of HS fragments could in principle be a
convenient strategy for the efficient preparation of compound
libraries with complete control of sulfation pattern, mono-
saccharide sequence and chain length for microarray based
interaction studies. Although (automated) a solid-phase metho-
dology has been applied with success to the synthesis of other
oligosaccharides,5–7 only very few reports dealing with the solid
supported synthesis of glycosaminoglycans8–11 have been pub-
lished. With a notable exception from the group of van der
Marel,12 the synthesis of HS fragments in solution is usually based
on the assembly of highly elaborated disaccharides, reflecting the
major repeating units of the polymer.4,13–18 Here, we describe a
strategy for the assembly of monosaccharide building blocks on a
solid support which avoids the multi-step processing of elaborated
disaccharidic structures and introduces a higher degree of
flexibility for the generation of structurally diverse HS libraries.
a Biofunctional Nanomaterials Department, CICbiomaGUNE,
Paseo Miramon 182, 20009 San Sebastian, Spain.
E-mail: nreichardt@cicbiomagune.es
Scheme 1 (a) TDS-Cl, imidazole, DMF, 80%; (b) TrocCl, pyridine,
CH3CN, 0 1C–RT, 52%; (c) ClCOOPhNO2, pyridine, CH2Cl2; (d) 2,
DIPEA, DMF, 0 1C–RT, 78% over 2 steps; (e) Zn, AcOH/THF (1 : 1)
RT, 95%; (f) (i) BzCl, pyridine, (ii) HFÁpyridine, THF; (g) carboxy-
polystyrene 2.19 mmol gÀ1, DIC, DMAP, DCM, quantitative, capping:
Me3SiCHN2, THF, MeOH, RT, overnight; (h) HFÁpyridine, THF,
0 1C–RT, quantitative; (i) MeONa, MeOH, MW, 55 1C, quantitative.
b Molecular Imaging Unit, CICbiomaGUNE, Paseo Miramon 182,
20009 San Sebastian, Spain
c CIBER-BBN, 20009 San Sebastian, Spain
w Electronic supplementary information (ESI) available: Experimental
details, NMR spectra, LC-MS data. See DOI: 10.1039/c0cc04686h
c
2390 Chem. Commun., 2011, 47, 2390–2392
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Scheme 2 (a) 10-SP, TMSOTf, DCM; (b) MeONa, MeOH, 72%; (c) Pd black, 10% HCOOH in MeOH, H2, quantitative; (d) 8, NIS, TfOH,
DCM; (e) (i) HFÁpyridine, THF, 84%, (ii) TEMPO/BAIB, DCM/H2O (3 : 1) 80%; (f) Bu2SnO, DCM, MeOH, MW, 120 1C; (g) 10-SP, NIS,
TfOH, DCM, >95% conversion; (h) Ac2O, pyridine, DCM, RT, 96%.
to give the desired carbamate 6 in an excellent yield of 80%
over the two steps. After quantitative deprotection of the
Troc-group with Zn in acetic acid the free linker 7 was
immobilized onto commercial carboxy-polystyrene with DIC
and catalytic DMAP affording the resin 9-SP with a capacity
adjusted to 0.2 mmol gÀ1. Remaining carboxylic acid was
capped as methylester with TMS–diazomethane.21 Cleavage of
the TDS–ether in 9-SP with hydrogen fluoride–pyridine
complex in THF afforded the resin bound alcohol 10-SP
within 3 h. A clean analytical sample for MALDI-Tof analysis
was produced by treating a resin aliquot with MeONa in
DCM/MeOH for five minutes under microwave irradiation.
Preparative cleavage of the deprotected linker 10 from the
resin was performed as reported by Roussel et al.22 We then
tested the utility of our resin for the preparation of
C5-aminolinked glycoconjugates (Scheme 2). Reaction of 5
equivalents of mannose N-phenyltrifluoracetimidate 1123 with
resin 10-SP under TMSOTf catalysis afforded the resin bound
glycoconjugate 12-SP after two glycosylation cycles. Cleavage
from the resin with sodium methoxide produced glycoconjugate
12 in 79% yield, which was completely deprotected to the
aminopentyl glycoside 13 by catalytic transfer hydrogenation
with Pd black.24,25 Next we turned our attention to the solid-
phase synthesis of HS precursors. A major challenge in the
synthesis of heparan sulfate and heparin fragments is the low
reactivity of uronic acid derivatives in glycosylation reactions.26
We sought to overcome this problem by employing more
reactive L-idose glycosyl donors. Oxidation of L-idose residues
to iduronic acid would be performed after cleavage from the
resin on the assembled oligosaccharide.27–30 This strategy was
evaluated in solution on model compound 15 obtained from
condensation of linker 8 with donor 14 (Schemes 1 and 2). The
6-O TBDPS group in 15 was easily cleaved with HF/pyridine
and the resulting alcohol oxidized with TEMPO/AIBN27,31 to
the uronic acid 17 in good yield without affecting other
protecting groups. NIS/TfOH promoted glycosylation of
10-SP with thioglycoside 1429,32 afforded a-neoglycoconjugate
18-SP with complete conversion in a single cycle. Removal of
the levulinate ester in 18-SP was difficult to monitor as cleavage
from the resin resulted in concomitant delevulination (19).
However, dibutyltinoxide (DBTO) mediated transesterification
of carboxylic acid esters33 on model compound 15 allowed to
cleave the primary benzoate ester with high selectivity affording
alcohol 16. Likewise, microwave irradiation of the resin bound
idose 18-SP with 5 eq. of DBTO at 120 1C afforded the idose
derivative 20 with all protecting groups in place after 10 min
only. Therefore, the hydrazine-acetate promoted hydrolysis of
the levulinate group on 18-SP could be monitored after DBTO
mediated cleavage of an analytical sample from the resin (22) as
indicated in Scheme 3. Acceptor 22-SP was then glycosylated
with 5 equivalents of the azido glucose donor 21 under
TMSOTf catalysis affording the resin bound disaccharide
23-SP in 53% conversion after 2 cycles as determined by
LC-MS analysis (23). This yield could be raised to 82% after
three additional glycosylation cycles (see ESIw). In comparison,
the solution phase reaction with equimolar amounts of reac-
tants produced the disaccharide in only 42% yield. Treatment
of 23-SP with hydrazine then afforded the free acceptor 24-SP.
The glycosylation of 24-SP with 14 under NIS/TfOH activation
never produced the trisaccharide 26-SP in more than 50% yield.
Attempts to increase the conversion in subsequent glycosylation
cycles led to substantial cleavage of both a-idose–glycosidic
linkages. When glycoconjugate 15 was subjected to the same
NIS/TfOH excess in solution, anomerisation and partial
cleavage of the anomeric bond next to iodination was observed
by LC-MS. DMTST as activator, on the other hand, did not
c
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grant, and the European Union, ITN-Euroglycoarrays grant is
gratefully acknowledged. We would like to acknowledge help
with LC-MS by Javier Calvo, CICbiomaGUNE.
Notes and references
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Scheme 3 (a) Hydrazine acetate, MeOH, DCM; (b) Bu2SnO, DCM,
MeOH, MW, 120 1C; (c) TMSOTf, DCM, –40 1C; (d) MeONa,
MeOH, MW, 50 1C; (e) Ac2O, pyridine.
alter the idose–glycosidic linkage, but was less effective as only
40% of glycoconjugate 18-SP was obtained after one cycle.
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following known procedures, however, produced cleanly the
trisaccharide 26-SP in 53% conversion after 4 cycles with no
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A novel ester type linker incorporating a
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carbamate protected C5-spacer for later ligand immobilisation
proved stable throughout the synthesis and was easily cleaved
from the resin by a choice of two complementary methods. We
have found that the lability of the idose residues under NIS/TfOH
activation makes thioglycoside donors less attractive for the solid-
phase synthesis of heparan sulfate oligosaccharide precursors.
Idose- and azido glucose trichloroacetimidates however, were
activated without any cleavage of glycosidic bonds. Work in
progress in our laboratory addresses the excess of glycosyl donors
currently needed for the glycan assembly and the application of
this strategy to the synthesis of a library of diverse HS oligo-
saccharide structures as ligands for glycan arrays.
Funding from Ministerio de Ciencia e Innovacion, CTQ2008-
´
04444/BQA grant, Government of the Basque Country, Etortek
c
2392 Chem. Commun., 2011, 47, 2390–2392
This journal is The Royal Society of Chemistry 2011