Automated solid-phase synthesis of a branched Leishmania cap tetrasaccharide.

Article abstract of DOI:10.1021/ol016631v

[reaction--see text] Described is the first automated solid-phase synthesis of a branched oligosaccharide by stepwise assembly from monosaccharides. Cap tetrasaccharide 1, found as part of the cell surface lipophosphoglycan (LPG) of the protozoan parasite

Full text of DOI:10.1021/ol016631v

ORGANIC
LETTERS
2001
Vol. 3, No. 23
3699-3702
Automated Solid-Phase Synthesis of a
Branched Leishmania Cap
Tetrasaccharide
Michael C. Hewitt and Peter H. Seeberger*
Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
seeberg@mit.edu
Received August 22, 2001
ABSTRACT
Described is the first automated solid-phase synthesis of a branched oligosaccharide by stepwise assembly from monosaccharides. Cap
tetrasaccharide 1, found as part of the cell surface lipophosphoglycan (LPG) of the protozoan parasite Leishmania, was readily prepared using
glycosyl phosphate and glycosyl trichloroacetimidate building blocks.
Oligopeptides1 and oligonucleotides2 are routinely prepared
by automated solid-phase synthesis. Oligosaccharides con-
stitute the third major class of repeating biopolymers found
in nature and have been much more difficult to prepare.
Automated solid-phase synthesis and one-pot solution-phase
synthesis approaches for the preparation of these molecules
have only very recently been reported.3,4 Unlike peptides and
oligonucleotides, oligosaccharides are commonly encoun-
tered in highly branched form. In the first automated
oligosaccharide synthesis the branching points were installed
by incorporation of disaccharide building blocks to main-
tain the linear mode of assembly.3 The common occurrence
of branched motifs in carbohydrates of biochemical and bio-
medical significance prompted us to address this challenge.
endemic in the U.S.6,7 Lipophosphoglycans (LPG),8 which
are ubiquitous on the cell surface of Leishmania parasites,
are composed of a glycosylphosphatidylinositol (GPI) anchor,
a repeating phosphorylated disaccharide, and different cap
oligosaccharides (Figure 1). The branched tetrasaccharide at
the terminus of the LPG constitutes an attractive vaccine
target and has been previously synthesized in solution9 and
on solid support.9c While the initial immunological results
using the synthetic carbohydrate vaccine are promising,10
more rapid access to the carbohydrate portion of potential
vaccines would greatly facilitate such ventures.
(4) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong,
C.-H. J. Am. Chem. Soc. 1999, 121, 734.
(5) For a review, see: Herwaldt, B. L. Lancet 1999, 354, 1191.
(6) Magill, A. J.; Grogl, M.; Gasser, R. A.; Sun, W.; Oster, C. N. N.
Engl. J. Med. 1993, 328, 1383.
Leishmaniasis is a tropical disease that afflicts over 12
million people worldwide5 and is on the verge of becoming
(7) Alvar, J.; Canavate, C.; Gutierrez-Solar, B. et al. Clin. Microbiol.
ReV. 1997, 10, 298
(8) For a review, see: Turco, S. J.; Descoteaux, A. Annu. ReV. Microbiol.
(1) Atherton, A.; Sheppard, R. C. Solid-Phase Peptide Synthesis:
Practical Approach; Oxford University Press: Oxford, U.K., 1989.
(2) Caruthers, M. H. Science 1985, 230, 281.
A
1992, 46, 65.
(9) (a) Arasappan, A.; Fraser-Reid, B. J. Org. Chem. 1996, 61, 2401.
(b) Upreti, M.; Ruhela, D.; Vishwakarma, R. A. Tetrahedron 2000, 6577.
(c) Hewitt, M. C.; Seeberger, P. H. J. Org. Chem. 2001, 66, 4233.
(10) Schofield, L. Personal communication.
(3) Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001, 291,
1523.
10.1021/ol016631v CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/25/2001

Figure 1. Leishmania LPG.
Here we report the first automated solid-phase synthesis
of Leishmania tetrasaccharide 1 from monosaccharide build-
ing blocks. This automated synthesis utilized both glycosyl
phosphates and glycosyl trichloroacetimidate building blocks
and different ester protecting groups to accomplish branching.
Examination of the tetrasaccharide revealed that it can be
accessed using two suitably protected mannose building
blocks and one galactose monomer (Figure 2). Mannosyl
After the three required monosaccharide building blocks
had been identified, preparation of core mannose unit 2
commenced from known ortho ester triol 5 (Scheme 1).13
Selective 3,6-di-benzylation of 514 was followed by protec-
tion of the remaining 4-hydroxyl group as a levulinate ester
to afford 6 in 89% yield. Treatment of the ortho ester with
aqeuous acetic acid furnished a mixture of anomeric lactols,
before reaction with DBU and trichloroacetonitrile provided
differentially protected mannosyl trichloroacetimidate 2.
A key aspect of the synthesis was the ability to introduce
branching from the central mannose unit by selective
protecting group manipulations. To examine this issue, a
solution-phase model study was conducted (Scheme 2).
Reaction of glycosyl donor 2 with pentenyl alcohol upon
activation with catalytic TMSOTf generated n-pentenyl
glycoside (NPG)15 7. NPGs are also the products of
automated oligosaccharide syntheses on polymer support
using our octenediol linker16 (vida supra). Removal of the
4-levulinate group by treatment with hydrazine in the
presence of the 2-acetate ester proceeded in 71% yield to
furnish the 4-hydroxyl-containing monosaccharide 8. Instal-
lation of the challenging â-1f4 linkage between galactose
and mannose was readily accomplished by reaction with
galactose phosphate 3. Treatment with sodium methoxide
to remove the axial acetate moiety in the presence of a 2′-
pivaloyl group provided disaccharide acceptor 9 in 79% yield
over two steps. These results confirmed the validity of our
protecting group strategy en route to the automated synthesis
of 1.
Figure 2. Monosaccharide building blocks required for automated
synthesis.
trichloroacetimidate 2 would provide the core mannose unit
with branching points in the 2- and 4-positions. Glycosyl
phosphate 311 would supply the galactose moiety, and
mannosyl trichloroacetimidate 412 would be used twice to
build the 1f2-linked mannose region.
With the three monosaccharide building blocks 2, 3, and
4 in hand, the automated synthesis was carried out on a
Scheme 1. Synthesis of Mannosyl Trichloroacetimidate 2
3700
Org. Lett., Vol. 3, No. 23, 2001

Scheme 2. Synthesis of Disaccharide Acceptor 9
modified ABI 433A peptide synthesizer3 using octenediol
functionalized Merrifields resin 10 (Scheme 3). Each cou-
pling cycle (Table 1) relied on double glycosylations to
alcohol with phosphate 3 using stoichometric amounts of
TMSOTf. The 2′-pivaloyl ester ensured complete stereo-
control during this coupling. Selective removal of the acetate
ester from the support-bound disaccharide with sodium
methoxide preceded coupling with mannosyl trichloro-
acetimidate 4 using catalytic TMSOTf. Repetition of acetate
cleavage and mannosylation with 4 completed the assembly
of resin-bound tetrasaccharide 1. The total time for assembly
of the carbohydrate skeleton of 1 on the automated synthe-
sizer was 9 h.
Cleavage of the octenediol linker using Grubbs catalyst17
in an atmosphere of ethylene provided crude n-pentenyl
glycoside tetrasaccharide 1. HPLC analysis of the crude
reaction products showed three major peaks (Figure 3):
Styrene, the trisaccharide (n-1) deletion sequence (19%
relative peak area), and the desired tetrasaccharide 1 (50%
relative peak area). Comparison with pure standards of these
compounds from solution-phase studies9c and ESI mass spec-
trometry was used to identify tetrasaccharide 1. The desired
product was purified by preparative HPLC and analyzed by
NMR.
Table 1. Conditions and Reagents for the Automated Synthesis
of 1
function
reagent
time (min)
glycosylation
wash
glycosylation
wash
5 equiv of donor and TMSOTfa
dichloromethane
20
9
20
9
5 equiv of donor and TMSOTfa
dichloromethane
deprotection
wash
wash
2 × 10 equiv of NaOMe or N2H4
0.2 M AcOH/0.2 M MeOH/THF
tetrahydrofuran
60
9
9
wash
dichloromethane
9
a Using 0.5 equiv for building blocks 2 and 4, 5.0 equiv for building
block 3.
ensure high coupling efficiencies and a single deprotection
event. Coupling of 2 to resin 10 using catalytic TMSOTf
was followed by removal of the levulinate ester upon
exposure to hydrazine. Formation of the central â-(1f4)
linkage was achieved by reaction of the support-bound
This first automated synthesis of a branched oligosaccha-
ride was demonstrated on the example of a Leishmania cap
tetrasaccharide. The desired tetrasaccharide was obtained in
less than 4 days starting from the monosaccharide building
Scheme 3. Automated Synthesis of Tetrasaccharide 1
Org. Lett., Vol. 3, No. 23, 2001
3701

available for organic synthesis on solid support. Work in
this area is in progress and will be reported in due course.
Acknowledgment. Financial support from the donors of
the Petroleum Research Fund, administered by the American
Chemical Society (ACS-PRF 34649-G1) for partial support
of this research; the NSF (CHE-9808061 and DBI-9729592)
for providing NMR facilities; the Mizutani Foundation for
Glycoscience; and the NIH (Biotechnology Training Grant
for M.C.H.) is gratefully acknowledged. We also thank Dr.
O. J. Plante for help with the automated synthesizer.
Supporting Information Available: Spectral data for all
new compounds and intermediates in automated synthesis
is included. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 3. HPLC trace of crude materials obtained after automated
synthesis. Conditions: 15-20% EtOAc/hexanes (30 min).
blocks, and showed that acetate and levulinate esters could
be used in concert to assemble branched structures. The
construction of complex carbohydrate motifs is now limited,
in theory, only by the orthogonality of protecting groups
OL016631V
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