A practical solid-phase synthesis of glycosylphosphatidylinositol precursors

Article abstract of DOI:10.1002/anie.200351950

The conserved pseudo-pentasaccharide structure common to all known GPI-anchors (GPI=glycosylphosphatidylinositol) was assembled on a solid support by using a novel attachment procedure and a optimized deprotection/ glycosylation protocol. Orthogonal prote

Full text of DOI:10.1002/anie.200351950

Communications
Solid-Phase Synthesis of Glycolipids
A Practical Solid-Phase Synthesis of
Glycosylphosphatidylinositol Precursors**
Niels-Christian Reichardt and Manuel Martín-Lomas*
Glycosylphosphatidylinositol (GPI) anchors serve to attach
proteins to cellular membranes through a covalent linkage.[1]
These glycolipids have been a subject ofsustained interest for
the last fifteen years.[2] Besides the anchoring function, GPIs
are involved in important biological events. They act as a
targeting signal to the secretion and insertion ofproteins to
distinct membrane domains[3] and seem to play a role in
transmembrane signaling.[4] GPIs function as a malarial toxin
as well[5] and anti-GPI vaccination was shown to prevent
malaria in an animal model.[6]
As a general rule, all reported GPI structures present a
linear myoinositol containing oligosaccharide backbone.
(Figure 1).
While this general structure is conserved, there is a
considerable diversity within the GPI anchor family, which is
reflected on the nature and the location of branching groups
in this pseudo-pentasaccharide skeleton. These branching
groups are species-specific carbohydrate side chains, addi-
tional phosphoethanolamine units, and variations in the lipid
moiety. As for the location, positions 2 and 3 of the proximal
mannose unit and positions 2 and 6 ofthe distal mannose unit
seem to invariably constitute the branching points.[1]
Because oftheir biochemical signiifcance, their biomed-
ical interest, and the challenges involved in their total
synthesis, GPIs have received a lot ofattention from synthetic
chemists, and have been chosen as a target to test methods
and strategies in complex oligosaccharide synthesis. .[1–7]
Consequently, a number oftotal syntheses ofspeciifc GPIs
were reported.[7] However, a general strategy that supports
the synthesis ofa diversity ofGPI structures from a common
precursor has not been reported so far.
The development of conditions for the efficient solid-
phase synthesis ofcomplex oligosaccharides on automated
synthesizers may constitute a key step in deciphering many
outstanding questions in Glycobiology.[8] An effective strat-
egy for the synthesis of GPI on solid support that allows the
access to a diversity ofGPI structures rfom a common
pseudo-pentasaccharide construct is, therefore, highly desir-
able. With only one exception,[9] all described syntheses of
[*] Prof. Dr. M. Martín-Lomas, N.-C. Reichardt
Grupo de Carbohidratos
Instituto de Investigaciones Químicas, CSIC
AmØrico Vespucio s/n, Isla de La Cartuja, 41092 Sevilla (Spain)
Fax: (+34)95-446-0565
E-mail: manuel.martin-lomas@iiq.csic.es
[**] This work was supported by the Ministerio de Ciencia y Tecnología
(Grant BQU 2002–03734). N.-C. R. thanks the European Union for a
fellowship (Grant HRN-CT-2000–00001/Glycotrain).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200351950
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1) A polyethylene glycol (PEG)-grafted polystyrene resin
functionalized with a Wang-Cl linker. These flexible PEG
grafts provide a solutionlike environment for bound
molecules, which results in fast reaction rates and permits
[11]
the obtainment ofhigh resolution NMR spectra.
2) A Wang-type linker readily cleavable under acidic media
but stable to low-temperature-glycosylation conditions
with catalytic Lewis acid promoters, and highly stable to
basic conditions.[12]
3) A mild experimental procedure for the attachment of the
substrate to the solid support through stannylidene
activation.
4) O-Glycosyl trichloroacetimidates as donors that allow
glycosylation under the above conditions.[13]
5) Lev/Fmoc[14] and TBDPS/Lev[15] as orthogonal protecting
groups for the construction of the pseudo-pentasaccharide
backbone and the establishment ofbranching points.
6) A fast and traceless cleavage of the target molecule and
intermediates.[12]
Under these general conditions pseudo-oligosaccharides
1, 2 and 3 have been prepared by sequential glycosylation
starting from pseudodisaccharide 4. Compound 4 can be
obtained in multigram quantities.[10b,c] The formation of the
glucosamine a1!6 myoinositol linkage with complete selec-
tivity is difficult to achieve.[10b,d,e] Therefore, building block 4
was synthesized in solution.
After attempting different strategies, we decided to attach
the pseudo-disaccharide glycosyl acceptor to the solid support
through the myoinositol moiety. The scarce differences of
reactivity among the myoinositol hydroxyl groups may
complicate the anchoring process, but results ofprevious
experiments prompted us to use this strategy instead ofan
alternative attachment through C6-OH ofthe d-glucosamine
unit. Thus, compound 4 was transformed into diol 7 by the
regioselective opening ofthe benzylidene acetal ( !5),
addition ofthe levulinate group ( !6) and removal ofthe l-
camphor ketal (!7). The attachment of 7 to the solid support
was performed by stannylidene acetal-mediated activation of
the diol system followed by the reaction with the linker
functionalized resin (!8) (Scheme 1); the loading was
0.31 mmolgꢀ1. Unreacted linker sites were capped by quench-
ing with methanol or by transforming the hydrolized benzyl
chloride functionality into the corresponding trichloroaceti-
midate and subsequent methylation with methanol in the
Figure 1. GPI-anchor structures, R1: Man or not-determined glycan, R2:
Galn, GalNAc, GalNAc-Gal-NANA, P-Man, PEtNH2, GlcNAc-Gal,
(GalNAc-Gal)9, GalNAc-Glc, R3 extra phosphoethanaolamine, R4:
GalNAc, NANA-Gal-GalNAc, R5: Acyl (not substrate to PI3-Kinase);
synthetic GPI-glycan-strucures 1, 2 and 3. Man=mannose,
Gal=galactose.
GPI structures have been carried out in solution. Because of
our long-standing interest in the role ofGPI-like molecules in
insulin signaling,[10] we have been aiming at an efficient solid-
phase step-by-step construction ofthe GPI pseudo-pentasac-
charide backbone. Such a synthesis should permit the
introduction ofa variety ofside chains at the main branching
positions by using commercially available linkers and
supports, and standard experimental conditions. We have
now developed such synthetic procedure, which is reported
herein.
After extensive investigations on the compatibility of all
parameters involved such as the type ofsolid support, linker,
attachment strategy, capping procedure, glycosylation
method, and protecting group strategy, we chose the following
system:
presence ofBF OEt2.[16] Acetylation (!9) and subsequent
3
1
removal of the levuline group afforded 10. H NMR analysis
ofa cleaved sample showed a 2:1 C1-OH/C2-OH regioselec-
tivity for the attachment process. This is of no consequence
for the solid phase synthesis of the oligosaccharide skeleton
herein described but has to be taken into account for the
further solution phase installation of the lipid moiety.
Mannopyranosyl trichloroacetimidates 11, 12[17] and
13[18a,b,c] have been sequentially used in the step-by-step
construction ofthe orthogonally protected pseudo-oligosac-
charide backbone 1. The highly functionalized glycosyl donor
11 was designed as to permit selective deprotection at C3, C4,
and C6 for further attachment of branching groups. It
contains a combination ofacetyl, silyl, levulinoyl and Fmoc
orthogonal protecting groups[14,15] and could be obtained in a
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The synthesis ofthe pseudopentasaccharide skel-
eton started with the glycosylation ofacceptor 10 with
donor 11 in the presence of TMSOTf to afford the
corresponding resin-bound trisaccharide. A prepara-
tive sample was cleaved from the resin, submitted to
treatment with triethylamine to release the Fmoc
group, and acetylated to give pseudotrisaccharide 3
that was fully characterized. Glycosyl acceptor 21 was
obtained after hydrolysis of the Fmoc group and then
glycosylated with glycosyl donor 12[17] in the previ-
ously established conditions. Again a preparative
Scheme 1. Preparation and attachment of pseudodisaccharide 5 to
resin. a) NaCNBH3, HCl (1m sol. in Et2O), THF, 10 min, 81%;
b) LevOH, DCC, DMAP, CH2Cl2, 1 h, 95%; c) TFA 20% in CHCl3,
80%; d) Bu2SnO, MeOH, reflux, 3 h; e) resin Argo-Gel-Wang-Cl, [21]
(0.43 mmolgꢀ1), TBAI, CsF, MeCN, 70–80%, 1-OH/2-OH 1:2;
f) Ac2O, py, DMAP, CH2Cl2, 1.5 h, quant.; g) N2H4:AcOH, CH2Cl2,
20 h, quant. Lev=RCOCH2CH2COCH3 DCC=dicyclohexylcarbodi-
imide, DMAP=4-dimethylaminopyridine, TFA=trifluoroacetic acid,
TBAI=tetrabutylammonium iodide, py=pyridine.
straightforward manner (Scheme 2). Stannylidene
acetal mediated silylation ofthioglycoside 14[19] gave
the 3-O-silyl derivative 15. Conventional acetylation
afforded 16. Cleavage ofthe benzylidene acetal in 16
yielded diol 17 that was selectively protected as a 6-
Fmoc ester to give 18. The levulinic ester group was
then installed (!19). Mild hydrolysis gave 20 that
was
activated
as
trichloroacetimidate
11
(Scheme 3).[20]
Scheme 2. Synthesis of key mannose donor 7. a) Bu2SnO, MeOH,
reflux, 1 h; b) TBDPSCl, DMF, TBAI, 16 h, 71%; c) Ac2O, py, DMAP,
2 h, 95%; d) BF3Oet2, EtSH, CH2Cl2, 92%; e) FmocCl, MeCN, py,
DMAP, 1 h, 85%; f) LevOH, DCC, DMAP, 1.5 h, 85%; g) NBS, ace-
tone, H2O, ꢀ208C, 2 h, 90%; h) Cl3CCN, NaH, 40 min, 79%.
TBDPS=tert-butyldiphenylsilyl, DMF=dimethylformamide, Fmoc=
9-fuorenylmethoxycarbonyl, NBS=N-bromosuccinimide.
Scheme 3. Assembly of glycan structure on solid support a) TMSOTf (0.1 equiv),
CH2Cl2, ꢀ208C, 2 h; b) Et3N, CH2Cl2, room temperature, 4 h; c) 1. TFA 10% in CHCl3,
room temperature, 2 h, 2. Ac2O, py, DMAP, 08C, 2 h. TMS=Trimetylsilyl, Tf=trifluoro-
methanesulfonate.
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[8] a) P. H. Seeberger, W.-C. Haase, Chem. Rev. 2000, 100, 4349 –
sample was cleaved and acetylated to give pseudotetrasac-
charide 2 the 1H NMR spectrum ofwhich was fully assigned.
The final assembly was performed by generating the glycosyl
acceptor as above and performing a further glycosylation step
with donor 13[18a–c] to afford 23. Compound 1 was obtained
after releasing from the resin and acetylation. The overall
yield for the target structure 1 was 20% after a ten steps
synthesis with an average 85% per step.
The above results describe a practical solid phase strategy
for the assembly of GPI precursor structures that combines a
solution like environment and a fully orthogonal protecting-
group pattern with the efficiency of the trichloroacetimidate
method for stereoselective glycosylation. The use of a PG-
grafted resin permits high resin loadings without affecting the
performance of the coupling reactions. The attachment of the
pseudodisaccharide unit to the resin using a mild stannylene
activation constitutes a convenient and original procedure for
highly functionalized derivatives. The biosynthetically
inspired linear construction ofthe GPI backbone permits to
access to GPI-like intermediates from the same precursor.
The described system allows for the construction of a diversity
ofstructures in a combinatorial manner and ofr the gen-
eration ofa small library ofGPI precursors.
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[15] TBDPS-cleavage was achieved on resin by repeated treatment
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Received: May 22, 2003
Revised: July 1, 2003 [Z51950]
Keywords: carbohydrates · glycolipids · glycosylation ·
.
protecting groups · solid-phase synthesis
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