Chemo-enzymatic synthesis of oligosaccharides using a dendritic soluble support

Article abstract of DOI:10.1016/S0040-4039(00)01599-9

A new soluble support with high loading capacity is described. This support was used for chemical sulfatation and enzymatic synthesis of the trisaccharide Lewis(x). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01599-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8887–8891
Chemo-enzymatic synthesis of oligosaccharides using a
dendritic soluble support
Andr e´ Lubineau, Annie Malleron and Christine Le Narvor*
Laboratoire de Chimie Organique multifonctionnelle, associ e´ au CNRS, Institut de Chimie Mol e´ culaire d’Orsay,
Universit e´ Paris Sud, 91405 Orsay Cedex, France
Received 1 August 2000; accepted 15 September 2000
Abstract
A new soluble support with high loading capacity is described. This support was used for chemical
X
sulfatation and enzymatic synthesis of the trisaccharide Lewis . © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: oligosaccharides; chemo-enzymatic synthesis; soluble support; dendritic support.
Besides the development of polymer-supported synthesis of oligosaccharides using purely
1
chemical glycosylation methods, a chemo-enzymatic approach based on the use of both
2
chemical methods and glycosyltransferases has started to develop. In fact, glycosyltransferases
have become valuable reagents for glycosylation due to their high regio- and stereoselectivity
3
and increasing availability via recombinant DNA technology. They appear to be quite
appropriate reagents for use in solid phase synthesis too. However, enzymatic solid phase
synthesis raises extra problems, such as accessibility to the interior of the solid matrix of the
4
enzyme, or for chemo-enzymatic synthesis, compatibility of the support with both aqueous and
organic solvents.
A potential solution to these problems would be the use of soluble polymers, which enjoy the
advantages of both liquid (for the accessibility) and solid phase (for the purification) syntheses.
Although polyethylene glycol (PEG) is among the most studied soluble polymer supports for
5
organic chemistry, it suffers from a severe drawback, that is its very low loading capacity;
indeed there are only one or two attachment points per polymer molecule. This problem could
6
be overcome by combining principles of dendrimer chemistry with those of PEG chemistry to
7
produce new PEG supports with expanded functional group capacity.
*
Corresponding author. Fax: 33 (0) 1 69 15 47 15; e-mail: chlenarv@icmo.u-psud.fr
0
040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01599-9

8888
In this paper, we report the use of a new, easily available, PEG derivative 1 with high loading
capacity (1 mmol per g) as a soluble support for oligosaccharide enzymatic synthesis. This
capacity is similar to that of most of the widely used commercial resins such as the well-known
8
Merrifield resin.
Compound 1 was prepared by alkylation of PEG 6000 with the pentaerythritol derivative 3
Scheme 1) in 80% isolated yield after dialysis. Compound 3 was readily prepared according to
9
(
1
0,11
literature procedures
in two steps from pentaerythritol. NMR spectroscopy showed nearly
12
complete conversion of the PEG to compound 1 with less than 5% of free hydroxyl groups. In
fact, we choose allyl functional groups in view of using the readily available thioglycosides
derivatives for grafting sugars through the smooth, high yielding, radical coupling of the thiol
1
3
group onto double bonds. This linkage has the advantage of being resistant to most chemical
1
4
15
conditions. Furthermore, it can be rapidly cleaved by thiophilic reagents or in an electro-
1
6
chemical procedure.
Scheme 1.
17
First, lactose was installed on the new PEG support 1 using 1-thiolactose 4. The radical
coupling onto compound 1 was carried under 254 nm lamp irradiation (Scheme 2) using 0.05 M
Scheme 2.

8889
18
solutions of 1 in water and 1.3 equiv. of sugar per allyl group. Purification was easily carried
out by dialysis. Subsequent cleavage using mercury(II) trifluoroacetate in the presence of
barium carbonate afforded, after gel filtration, 0.77 mmol of lactose from 1 g of support.
Alternatively, sugar can be cleaved by electrohydrolysis in an one-compartment three-electrode
cell under potentiostatic control in acetonitrile containing water (5%) and 0.2 M lithium
16
tetrafluoroborate using woven carbon or platinum as working electrode.
Then we tried the same conditions as for lactose, the coupling of 2-acetamido-2-deoxy-1-
1
9
thio-b- -glucopyranoside 5. We obtained after dialysis and similar cleavage using mercury(II)
D
trifluoroacetate, 0.67 mmol of N-acetylglucosamine per gram of PEG. These results were
confirmed by mass spectrometry. Indeed, MALDI-TOF analysis showed that the compound 6
contains an average of 5.5 lactose residues/mol of PEG and 4 GlcNAc residues/mol of PEG
2
0
for compound 7.
Our support was then tested for chemical manipulations. In view of the synthesis of sulfated
2
1
oligosaccharides as ligands of selectins, we tried the sulfatation of compound 6 using the
2
2
stannylene methodology. In fact, the dried compound 6 was dissolved in MeOH and treated
with dibutyltin oxide (3 equiv./lactose) one night at reflux. After careful evaporation, sulfata-
tion was performed using SO ꢀNEt complex (1.2 equiv./lactose) in THF. Cleavage using
3
3
23
mercury(II) trifluoroacetate afforded a (2.7:1) mixture of 3%-sulfated lactose and lactose from
which the sulfated derivative could be easily separated using anionic exchange resin.
Then, we tested the ability of the new support toward enzymatic glycosylations. First,
enzymatic galactosylation of 7 (1.5 g) was carried out using bovine milk b(1–4) galactosyl-
24
transferase (2 U) and UDP–glucose/UDP–glucose 4-epimerase. The reaction could be easily
1
followed using H NMR analysis or alternatively by TLC after chemical cleavage of an
aliquot. After 3 days, the reaction was still incomplete as judged by the presence of both
N-acetyllactosamine and N-acetyl glucosamine. So, we took advantage of the use of supported
synthesis: after a simple dialysis, the support was recycled using fresh enzymes (0.4 U of
GalTase). Then, cleavage in the usual way gave pure N-acetyllactosamine without contamina-
tion with N-acetyl glucosamine. In this way, we obtained 230 mg of N-acetyllactosamine per
gram of support. Having in hand a small quantity of recombinant Fuc TIII (0.2 U), and
25
although N-acetyllactosamine is known to be a poor substrate for Fuc TIII we tested the
fucosylation of an aliquot of 7 (100 mg after galactosylation). After 3 days, cleavage by
X
mercury salts as described previously afforded a 1.5/1 (without recycling) mixture of Lewis
trisaccharide and N-acetyllactosamine (18 mg). NMR data were identical with those
2
6
reported.
In summary, it was clearly demonstrated that a high loading capacity of polyethyleneglycol
can be useful for the enzyme-assisted synthetic strategy. Studies are in progress to extend the
use of this PEG derivative to various chemo-enzymatic syntheses.
Acknowledgements
This work has been achieved in the frame of the French GTrec Network, supported by
MENRT (ACSV no. 954111) and CNRS (Programmes Interdisciplinaires Physique et Chimie
du Vivant et G e´ nie des Proc e´ d e´ s). We thank Dr. C. Aug e´ for helpful discussions and Professor
P. Le Marechal for the MALDI-TOF spectra analyses.

8890
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2
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