A two-directional approach for the solid-phase synthesis of trisaccharide libraries

Article abstract of DOI:10.1002/(SICI)1521-3773(19980803)37:13/14<1898::AID-ANIE1898>3.0.CO;2-T

Without protecting groups, polymer-bound thioglycosyl sugars can undergo two successive glycosylation reactions, once as a donor and once as an acceptor, to give trisaccharides such as 1. Trisaccharide libraries prepared by this strategy have a known suga

Full text of DOI:10.1002/(SICI)1521-3773(19980803)37:13/14<1898::AID-ANIE1898>3.0.CO;2-T

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of a glycine-derivatized polymer. This amide bond formation
should offer a very reliable approach for immobilization.
However, a product can simply be removed from the solid
support by base-mediated cleavage of the ester linkage
between the carbohydrate and the succinimide residue.
Glycine-derivatized TentaGel hydroxyl resin (2; TG ˆ Ten-
taGel)was selected as the solid support so that the high
flexibility and mobility of its polyethylene glycol moieties
could be utilized, thus ensuring high reactivities of the
immobilized compounds.[5] A resin-bound saccharide 3 can
first act as a glycosyl donor, and on reaction with a glycosyl
acceptor 4 in solution will give a disaccharide 5. It is hoped
that oligomerization will be prevented by site isolation of the
immobilized substrates. The resulting disaccharide can imme-
diately be used in the next step as a glycosyl acceptor. When
coupled with a thioglycoside 6 that is in solution, 5 will give a
resin-bound trisaccharide 7. A library of trisaccharides can be
prepared by a mix-and-split approach. Thus, 3 can be coupled
in individual glycosylation reactions to a range of acceptors,
and the products can be mixed and split before each pool of
resin-bound acceptors are glycosylated with a range of donors.
After cleavage of the resin and deprotection, a library with a
known residue at the nonreducing end is generated (meth-
od A). Alternatively, compound 3 can act as an acceptor and
on coupling with donor 8 (X ˆ F, SOPh, or OCNHCCl3) will
give a resin-bound glycosyl donor 9, which in a subsequent
step can be coupled with an acceptor 4 to give a trisaccharide.
A mix-and-split approach will thus give a trisaccharide library
with a known residue at the reducing end of the saccharides
(method B). A key principle will be that each glycosylation
will be performed under conditions that give a mixture of
anomers. Recently, we established[6] that glycosylations[7] of
thioglycosides mediated by N-iodosuccinimide/trimethylsilyl
trifluoromethanesulfonate (NIS/TMSOTf) in dichlorome-
thane at room temperature consistently give mixtures of
anomers.
A Two-Directional Approach for the Solid-
Phase Synthesis of Trisaccharide Libraries
Tong Zhu and Geert-Jan Boons*
In recent years the search for novel pharmacological agents
has focused on the preparation of ªchemical librariesº as
potential sources of new lead compounds for drug discovery.[1]
Combinatorial chemistry has been developed mainly for the
preparation of peptide and nucleic acid libraries and some
small molecules, but only very few reports deal with combi-
natorial carbohydrate chemistry.[2] We report here the syn-
thesis of a combinatorial saccharide library on a solid support
whereby all glycosidic linkages are intentionally synthesized
as mixtures of anomers. This approach addresses problems
associated with classical oligosaccharide solid-phase synthe-
sis.[3] In addition, a novel two-directional approach[4] for the
assembly of the oligosaccharides on the solid support is used
in which each immobilized saccharides can act as a glycosyl
donor as well as an acceptor.
The strategic considerations for the preparation of trisac-
charide libraries are summarized in Scheme 1. The thioglyco-
syl building blocks 1 are immobilized onto a solid support
through the formation of an amide linkage between the
carboxylic acid of a succinic acid moiety and the amine group
Treatment of the dibutyltin acetal of diol 11 with succinic
anhydride afforded 12 in an excellent yield of 85%
(Scheme 2).[8] Attachment of
a Fmoc-protected glycine
(Fmoc ˆ fluorenyloxycarbonyl) to the TentaGel hydroxyl
1
resin 13 (0.37 mmolg resin) under standard conditions[9a]
and subsequent removal of the Fmoc group by treatment with
piperidine gave polymer 2. Compound 12 was immobilized by
amide bond formation with 2 in the presence of benzotriazol-
1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate
(PyBOP) to give the polymer bound monosaccharide
15(Scheme 2).[9b] Compound 15 was coupled with 16 in the
presence of NIS/TMSOTf to give the desired disaccharide 17,
which after washing was used as an acceptor. Glycosylation
with the perbenzylated thioglycosyl donor 18 gave the
trisaccharide 19 (Scheme 3). This latter coupling was repeated
due to an incomplete reaction (the 4'-OH group of 17 has a
low reactivity). Completion of each reaction step was
ascertained by thin-layer chromatography and matrix assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) of the crude product after cleavage of a
small amount of beads (3 ± 5 mg) by treatment with base.
Finally, treatment of 19 with MeONa/MeOH in 1,4-dioxane
gave trisaccharide 20 ( ꢀ 65% overall yield based on the resin
Scheme 1. A two-directional strategy for the syntheses of trisaccharide
libraries.
[*] Prof. G.-J. Boons, T. Zhu
School of Chemistry, The University of Birmingham
Edgbaston, Birmingham B15 2TT (UK)
Fax: (‡44)121-414-4403
Email: gjboons@chemistry.bham.ac.uk
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similar result was obtained when the more rigid Merrifield
resin (1% cross-linked) was employed as the solid support.
The hydroxyl group of 15 was therefore protected as a
tetrahydropyranyl (THP) ether to avoid oligomerization.
Thus, treatment of 15 with 3,4-dihydro-2-pyran (DHP) in
the presence of pyridinium p-toluenesulfonate (PPTS)[10] gave
21 in virtually quantitative yield. This product was successfully
coupled with 22 to give the disaccharide 24. The THP group of
24 was easily removed by treatment with acetic acid/water to
yield 23.
Immobilized 12 also proved to be a good glycosyl acceptor
when coupled with a perbenzylated trichloroacetimidate
donor. Thus, the TMSOTf-mediated coupling of 15 with an
excess of 25 gave resin-bound 26. This product was further
coupled with acceptor 16 to give trisaccharide 19 (60%
overall yield based on the resin loading) (Scheme 5).
Scheme 2. Synthesis of the polymer-bound thioglycoside 15. a) Trifluoro-
acetic acid (TFA), CH2Cl2, 95%; b) Bu2Sn(OMe)2, benzene, Dean ± Stark
conditions then succinic anhydride, pyridine, 85%; c) N,N'-diisopropylcar-
bodiimide (DIC), 4-dimethylaminopyridine (DMAP), Fmoc-GlyOH,
DMF; d) piperidine (20% in DMF), >90% overall yield; e) PyBOP,
diisopropylethylamine (DIPEA), DMF.
Scheme 5. Synthesis of polymer-bound trisaccharide 19 (method B).
a) TMSOTf, CH2Cl2, 4-Š molecular sieves; b) NIS/TMSOTf, CH2Cl2, 4-
Š molecular sieves.
To verify the proposed strategy, a relatively small library of
12 trisaccharides was prepared (Scheme 6). Three different
glycosyl acceptors 22, 27, and 28 were coupled in individual
Scheme 3. Synthesis of trisaccharide 20 (method A). a) NIS/TMSOTf,
CH2Cl2, 4-Š molecular sieves; b) MeONa, MeOH, 1,4-dioxane then
‡
DOWEX50 (H ) ion-exchange resin.
loading; Scheme 3). Analysis by NMR spectroscopy and mass
spectrometry showed that no oligomerization had occurred
and that all glycosidic linkages were formed as mixtures of
anomers (Glc(1-4)Glc: a/b ꢀ 2/1 and Glc(1-6)Gal: a/b ꢀ 1/1).
Thus, the NIS/TMSOTf-mediated glycosylation of thioglyco-
sides in dichloromethane is highly compatible with the solid
support and linker system employed. It is noteworthy that the
glycosylation rates are significantly decreased (4 h) compared
to those for similar reactions in solution (5 ± 10 min). How-
ever, this feature does not affect the reliability of the
glycosylation approach.
Encouraged by these results, we turned to the glycosyl
acceptor 22 that has an unreactive 4-hydroxyl group. Un-
fortunately, coupling of 15 with 22 in the presence of NIS/
TMSOTf gave a small amount of oligomeric products (5 ±
10%) in addition to the required product 23 (Scheme 4). A
Scheme 6. Synthesis of the trisaccharide library 36. a) NIS/TMSOTf,
CH2Cl2, 4-Š molecular sieves; b) HOAc/THF/H2O, 4/2/1, 508C; c) MeO-
Na, MeOH, 1,4-dioxane then DOWEX50 (H ) ion-exchange resin;
‡
d) Pd(OAc)2, H2, EtOH.
reactions with 21, with NIS/TMSOTf as the promoter, to give
three different disaccharides (24, 29, and 30, respectively) as
mixtures of anomers. The beads were combined, the THP
group removed by treatment with HOAc/THF/H2O (4/2/1),[11]
Scheme 4. Synthesis of polymer-bound disaccharide 23. a) NIS/TMSOTf,
CH2Cl2, 4-Š molecular sieves; b) DHP, PPTS; c) AcOH, THF, H2O.
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[2] a) O. Kanie, F. Barresi, Y. Ding, J. Labbe, A. Otter, L. S. Forsberg, B.
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[3] For the recent advances in solid-support oligosaccharide synthesis, see
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1997, 38, 7653 ± 7656.
and the mixture of disaccharide acceptors coupled with the
glycosyl donor 33, again with NIS/TMSOTf as the promoter.
The trisaccharide library was released from the polymer after
thin-layer chromatography and MALDI-TOF MS had shown
that all the disaccharides had been consumed. The library was
purified by size-exclusion column chromatography (LH-20;
CH2Cl2/MeOH, 1/1), and the benzyl groups removed by
hydrogenation over Pd(OAc)2 to give the deprotected library
36 (55% overall yield based on the resin loading).[12]
To further examine the quality of this library, a mono-
saccharide compositional analysis was performed. A portion
of the trisaccharide library was treated with aqueous trifluro-
acetic acid (2m) at 1008C for 4 h. Analysis of the resulting
mixture of monosaccharides by HPLC (Dionex with a PA1
column and pulsed amperometric detection) showed that
galactose, glucose, and mannose were present in approxi-
mately the required ratio.[13]
In conclusion, a highly efficient approach for the synthesis
of oligosaccharide and saccharide libraries on a solid-support
has been described. Saccharides can be reliably immobilized
by amide bond formation between a succinimide linker and a
glycine-derivatized TentaGel resin. The products can be
analyzed after each reaction step by MALDI-TOF MS and
thin-layer chromatography by cleavage of the base-labile
ester linker from a small amount of resin. The glycosylation
strategy is two-directional: The immobilized thioglycoside
acts first as a donor, and the product bearing a free hydroxyl
group is used in subsequent glycosylation as an acceptor and
glycosylated with a thioglycosyl donor. A mix-and-split
approach gave a library with a known monosaccharide residue
at the nonreducing end. Immobilized 12 can also act first as a
glycosyl acceptor and the resulting disaccharides can be used
as a glycosyl donor.
[4] a) G. J. Boons, T. Zhu, Synlett 1997, 7, 809 ± 811; b) G. J. Boons, S.
Bowers, D. M. Coe, Tetrahedron Lett. 1997, 38, 3773 ± 3776; c) T. Zhu,
G. J. Boons, ibid. 1998, 39, 2187 ± 2190.
[5] E. Bayer, Angew. Chem. 1991, 103, 117-133; Angew. Chem. Int. Ed.
Engl. 1991, 30, 113 ± 129.
[6] A. Demchenko, T. Stauch, G. J. Boons, Synlett 1997, 7, 818 ± 820.
[7] a) G. H. Veeneman, S. H. van Leeuwen, J. H. van Boom, Tetrahedron
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[8] G. J. Boons, G. H. Castle, J. A. Calse, P. Crice, S. V. Ley, C. Pinel,
Synlett 1993, 12, 913 ± 914.
[9] a) The degree of glycine attachment was estimated photometrically
from the amount of Fmoc chromophore released upon treatment of 15
with piperidine/DMF. b) The progress of the coupling reactions was
monitored by the Kaiser test; for details, see Novabiochem Catalog &
Peptide Synthesis Handbook 97/98.
[10] a) D. Tanner, P. Somfai, Tetrahedron 1987, 43, 4395 ± 4406; b) M.
Miyashita, A. Yoshikoshi, P. A. Grieco, J. Org. Chem. 1977, 42, 3772 ±
3774.
Experimental Section
General procedure for NIS/TMSOTf-mediated glycosylation on a solid
support: The polymer-bound thioglycosyl donor (0.1 mmol) was placed in a
round-bottom flask just covered with dichloromethane and allowed to
swell for 15 min. Glycosyl acceptor (0.5 mmol) in dichloromethane (4 mL)
and 4-Š molecular sieves (200 mg, beads) were added, and the suspension
was shaken at room temperature for 15 min. NIS (0.5 mmol) and TMSOTf
(0.05 mmol) were then added, and the mixture was shaken at room
temperature for 2 ± 4 h. After removal of the molecular sieves by decanting,
the resin was washed successively with DMF (3 Â 20 mL), dichloromethane
(3 Â 20 mL), and methanol (3 Â 20 mL), and dried in vacuo over P2O5 for
24 hours.
[11] K. F. Bernardy, M. B. Floyd, J. Poletto, M. J. Weiss, J. Org. Chem. 1979,
44, 1438 ± 1447.
[12] All saccharides in the library were prepared as individual compounds,
and each trisaccharide was formed as a mixture of anomers in a range
of a/b ˆ 1/1 ± 2/1.
[13] Expected product ratios: Gal/Glc/Man: 1/1/0.25, found: 1/0.9/0.25.
Received: February 27, 1998 [Z11529IE]
German version: Angew. Chem. 1998, 110, 2000 ± 2003
Keywords: combinatorial chemistry ´ glycosylations ´ oligo-
saccharides ´ solid-phase synthesis
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