Synthesis of a beta1,2-mannopyranosyl tetrasaccharide found in the phosphomannan antigen of Candida albicans.

Article abstract of DOI:10.1021/ol0061743

The synthesis of a portion of the challenging beta1,2-mannosyl polymer found in the cell walls of Candida albicans was undertaken to develop a conjugate vaccine against C. albicans and to facilitate NMR conformational studies of this unique polysaccharide

Full text of DOI:10.1021/ol0061743

ORGANIC
LETTERS
2000
Vol. 2, No. 19
2939-2942
Synthesis of a â1,2-Mannopyranosyl
Tetrasaccharide Found in the
Phosphomannan Antigen of Candida
albicans
Mark Nitz, Byron W. Purse, and David R. Bundle*
Department of Chemistry, UniVersity of Alberta, Edmonton, AB, Canada T6G 2G2
daVe.bundle@ualberta.ca
Received June 7, 2000
ABSTRACT
The synthesis of a portion of the challenging â1,2-mannosyl polymer found in the cell walls of Candida albicans was undertaken to develop
a conjugate vaccine against C. albicans and to facilitate NMR conformational studies of this unique polysaccharide. The novel approach to
the synthesis of tetrasaccharide 1 employed the modified ulosyl bromide 11 as the glycosyl donor which provided high diastereoselectivity.
A participating solvent as well as p-chlorobenzyl protection facilitated the new approach.
Candida albicans is the most common etiologic agent in
candidiasis,1 an infection commonly affecting immunocom-
promised patients and those undergoing long-term antibiotic
treatment.2 Recently it has been shown that monoclonal
antibodies raised against a portion of the C. albicans
phosphomannan in rats were protective against subsequent
infection.3 Further studies indicated the active epitope to be
a portion of the â1,2-mannan polymer found in the phos-
phomannan antigen.4 In addition to their immunochemical
interest, it was predicted almost 30 years ago that polymers
composed of â1,2-linked mannose or glucose residues would
present interesting conformational properties. Conformations
of such oligomers that adopt typical glycosidic torsional
angles expected for disaccharide elements would be expected
to show compact conformations that fold back on themselves
and show strong steric interactions between noncontiguous
monomer units.5 The synthesis of 1, a portion this polymer,
was undertaken to provide material for the development of
a carbohydrate-based conjugate vaccine against C. albicans
and to facilitate NMR conformational studies of a portion
of this unique polysaccharide.
The rational synthesis of â-mannosides is a longstanding
problem in glycoside synthesis, that lacks a general solution,
despite several novel approaches.6 In the construction of large
homooligomers such as 1, the separation of anomeric
mixtures would pose a major obstacle to efficient assembly
by either block or sequential chain extension reactions.
Consequently, only those methods known to give exclusively
the â-mannopyranosyl linkage were investigated. In elabora-
(5) Rees, D. A.; Scott, W. E. J. Chem. Soc. (B) 1971, 469-479.
(6) (a) Gridley, J. J.; Osborn H. M. I. J. Chem. Soc., Perkin Trans. 1
2000, 1471-1491. (b) Barresi, F.; Hindsgual, O. Synthesis of â-D-mannose
Containing Oligosaccharides. In Modern Methods in Carbohydrate Syn-
thesis; Khan, S. H., O’Neil, R. A., Eds.; Harwood Academic Publishers:
Amsterdam, 1996; pp 251-276. (c) Lergenmu¨ller, M.; Nukada, T.;
Kuramochi, K.; Akhito, D.; Ogawa, T.; Ito, Y. Eur. J. Org. Chem. 1999,
1367-1376. (d) Crich, D.; Sun, S. J. Am. Chem. Soc. 1998, 120, 435.
(1) Fleury, F. J. Clin. Microbiol. ReV. 1981, 9, 335-348.
(2) Sobel, J. D. Clin. Infect. Dis. 1992, 14 (Suppl. 1), S148-S153.
(3) (a) Han, Y.; Cutler J. E. Immun. 1997, 63, 2714-2719. (b) Han, Y.;
Riessleman, M. H.; Cutler J. E. Infect. Immun. 2000, 68, 1649-1654. (c)
Han, Y.; Ulrich, M. A.; Cutler J. E. J. Infect. Dis. 1999, 179, 1477-1484.
(4) Han, Y.; Morrison, R. P.; Cutler, J. E. Infect. Immun. 1995, 66, 5771-
5776.
10.1021/ol0061743 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/30/2000

tion of a 1,2 linked polymer, a ulosyl bromide7 provides first
the corresponding ulopyranoside, which can be reduced to
afford directly the selectively protected glycosyl acceptor of
the next glycosylation step (for example 7). The glycosyl
donor 6 was attractive because of its high diastereoselectivity
over both the glycosylation and reduction steps, and the
minimization of the number of protecting groups required.
Allyl 3,4,6-tri-O-benzyl-â-D-mannopyranoside (5) was
targeted as a desirable building block for the reducing
terminus of the tetrasaccharide. The allyl group served as a
persistent anomeric protecting group which could be trans-
formed into a variety of functionalities late in the synthesis
to provide a tether via which attachment to protein could be
achieved for synthesis of neoglycoconjugates.8
Lichtenthaler et al.11 The ulosyl bromide 6 was synthesized
in a similar fashion, although the yields for the thermal
rearrangement that converts ortho ester 2 to an acetoxyglycal
(cf. 10) were approximately 65%.
Using silver-exchanged zeolite12 to promote the glycosyl-
ation of 5 by 6, followed by reduction of the product with
L-Selectride gave the desired disaccharide (7) in excellent
yield after purification. No R-manno anomer or gluco
epimers were isolated from the reaction mixture. It was
necessary to use the sterically hindered L-Selectride for this
reduction since in contrast to the monosaccharide, the
disaccharide gave epimeric mixtures when sodium borohy-
dride was employed.
Scheme 2
Scheme 1
Introducing subsequent â-mannopyranosyl units proved
more difficult. The conditions employed for disaccharide
synthesis failed to yield significant amounts of trisaccharide.
Exploration of different activation protocols led to the use
of the soluble promoter, silver triflate, with 2,6-di-tert-butyl-
4-methylpyridine (DtBMP)as an acid scavenger,6d and aceto-
nitrile as a participating solvent. These conditions followed
by a similar L-Selectride mediated reduction gave a 40-
45% yield of the trisaccharide, and 10% yield of the R-gluco
epimer together with a significant portion of the 3,4-di-O-
benzyl-1,6-anhydro-â-D-mannopyranose.11 It was hypothe-
sized that stabilization of the protecting groups would
disfavor the formation of this anhydro sugar side product
and in turn increase the yield of the reaction. The p-
chlorobenzyl protecting group was explored for this purpose
since it has been shown to be more acid stable than the parent
benzyl group but to have similar properties.13 The ulosyl
bromide 11 was synthesized in an analogous fashion to the
related 3,4,6-tri-O-benzyl-R-D-arabino-hexopyranos-2-ulosyl
bromide 6 used earlier in the disaccharide synthesis.
Allyl-â-D-mannopyranoside (5) could be conveniently
synthesized on a large scale starting from the readily
accessible ortho ester 2.9 Lewis acid promoted glycosylation
in allyl alcohol gave high yield of a near 1:1 mixture of the
expected product 3 and its deacetylated counterpart 4. The
allyl-â-D-glucopyranoside 3 was treated under Zemple´n
deacylation conditions to give the desired alcohol 4. Oxida-
tion of the glucopyranoside (4) using acetic anhydride and
DMSO and reduction with sodium borohydride gave the
mannopyranoside 5 in good yield with high selectivity
(Scheme 1).10
The synthesis of the first 1,2-linked â-mannosyl unit was
accomplished using conditions similar to those employed by
(7) Lichtenthaler, F. W.; Schneider-Adams, T. J. Org. Chem. 1994. 59,
6728-6734.
(8) (a) van Seeventer, P. B.; van Dorst, J. A. L. M.; Siemerink, J. F.;
Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1997, 300, 396-
373. (b) Buskas, T.; So¨derberg, E.; Konradsson, P.; Fraser-Reid, B. J. Org.
Chem. 2000, 65, 958-963.
(9) Lemieux, R. U.; Morgan, A. R. Can. J. Chem. 1965, 43, 2199-
2201.
(10) (a) Bore´n, H. B.; Ekborg, G.; Eklind, K.; Garegg, P. J.; Pilotti, A.;
Swahn, CG. Acta Chem. Scand. 1973, 27, 2639-2644. (b) Auge, C.;
Warren, C. D.; Jealoz, R.-W.; Kiso, W.; Andersen, L. Carbohydr. Res. 1980,
82, 85-95.
Standard deacylation of the readily available ortho ester
8,9 followed by Williamson ether synthesis with p-chloro-
benzyl chloride gave the p-chlorobenzyl protected ortho ester
(11) Lichtenthaler, F. W.; Schneider-Adams, T.; Immel, S. J. Org Chem.
1994, 59, 6735-6738.
(12) Garegg, P. J.; Ossowki, P. Acta Chem. Scand. 1983, B37, 249-
250.
(13) (a) Yamashiro, D. J. Org. Chem. 1977, 42, 523-524. (b) Pohl, N.
L.; Kiessling, L. L. Tetrahedron Lett. 1997, 38, 6985-6988.
2940
Org. Lett., Vol. 2, No. 19, 2000

Scheme 3
(9). Subsequent thermal rearrangement in bromobenzene
We hypothesized that this product must result from attack
of the acceptor on the nitrile carbon of the proposed
R-nitrilium intermediate (e.g., 15) (path b),14 instead of
reaction at the anomeric center (path a) of the donor. This
postulate was supported by the isolation of the chloroacetyl-
ated acceptor (e.g., 17) when chloroactonitrile was employed
as the solvent (Scheme 4). The resulting imidate intermediate
(16) hydrolyses to give the chloroacetylated acceptor (17).
Although reaction at the nitrile carbon of nitrilium intermedi-
ates has been seen with water and carboxylic acids,15 to the
authors knowledge no acylated glycosyl acceptors have
previously been isolated as a result of using nitriles as a
participating solvent. In this case the resulting side product
must be favored due to a sterically hindered acceptor as well
as the electron-deficient nitrilium uloside (e.g., 15). When
gave a high yield of the acetoxyglycal (10). Further treatment
with N-bromosuccinimide and ethanol in dichloromethane
gave the desired ulosyl bromide (11) (Scheme 2).
Glycosylation with this donor, in the presence of silver
triflate, and 2,6-di-tert-butyl-4-methyl pyridine in acetonitrile
followed by reduction with L-Selectride gave a 60-65%
yield of the desired trisaccharide (12) (Scheme 3) along with
15% of the corresponding R-gluco epimer, which was easily
separated by column chromatography.
Attempted introduction of the fourth â-mannopyranosyl
residue was met by the observation of an interesting and
unexpected product. Using the same conditions as those
employed to make the trisaccharide, up to 20% of the
unexpected 2-O-acetyl trisaccharide acceptor was isolated.
Scheme 4
Org. Lett., Vol. 2, No. 19, 2000
2941

1
the sterically more hindered pivaloyl nitrile was chosen as
the solvent, the glycosidic linkage was synthesized in 48%
yield to give the desired tetrasaccharide 13 and 10% of the
R-gluco epimer along with small amounts of the trisaccharide
pivaloyl ester also being formed. Further studies into the
solvent effects are ongoing.
A terminal amine was chosen as a versatile handle from
which glycoconjugates could be readily generated.16 This was
installed via a photoaddition of 2-aminoethanthiol to the allyl
glycoside giving 14. Using longwave ultraviolet irradiation,
365 nm, and a quartz vessel, it was possible to carry out
this addition in the presence of the aromatic protecting
groups. Previous reports of this photoaddition used shortwave
UV irradiation, conditions under which the benzyl and
chlorobenzyl protecting groups were labile, resulting in a
complex mixture of products.17
ment of the H NMR was possible through GCOSY and
GTOCSY experiments aided by TROESY18 experiments to
confirm the order of the ring systems. The presence of four
â-mannopyranosyl residues was confirmed by heteronuclear
1JC,H coupling constants, that fell within the range 160.1-
163.4 Hz.19 The order of the chemical shifts of the ring
systems agree well with the NMR data published for manno-
oligosaccharides previously isolated from C. albicans phos-
phomannan.20 TROESY experiments18 indicate a potentially
interesting tertiary structure as long-range contacts between
the nonreducing terminus (fourth residue) and the second
residue are evident. More detailed investigations of the
conformation of this unique polysaccharide are ongoing.
The tetrasaccharide 1 has been coupled to a protein carrier,
and the glycoconjugate will be used to immunize mice. The
resulting polyclonal antibodies will be tested for anti-Candida
activity.
Deprotection was accomplished using dissolving metal
conditions at -78 °C. It was necessary to minimize the
reaction time for this reduction as the thioether proved labile
to reduction resulting in the propyl glycoside
Acknowledgment. M. Nitz was the recipient of a Natural
Sciences and Engineering Research Council postgraduate
research scholarship. Financial support for the research was
provided by the University of Alberta and a research
operating grant to D.R.B. from the Natural Science and
Engineering Research Council of Canada.
Despite the repetitive structure of the homooligomer 1 the
1
majority of the resonances in the H NMR spectrum of 1
are well resolved at 600 MHz, with only H-5s and H-6s
resonances exhibiting extensive overlap. Complete assign-
Supporting Information Available: Spectroscopic, ana-
lytical data and experimental procedures for compounds 1,
7, 12-14 are available. This material is available free of
charge via the Internet at http://pubs.acs.org.
(14) (a) Vankar, Y. D.; Vankar P. S.; Behrendt, M.; Schmidt, R. R.
Tetrahedron 1991, 47, 9985. (b) Rattcliffe, A. J.; Fraser-Reid, B. J. Chem.
Soc., Perkin Trans. 1 1990, 747. (c) Braccini, I.; Derouet, C.; Esnault, J.;
Herve´ du Penhoat, C.; Mallet, J.-M.; Michon, V.; Sinay¨, P. Carbohydr.
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Synlett 1990, 694. (e) Hashimoto, S.; Honda, T.; Ikegami, S. J. Chem. Soc.,
Chem. Commun. 1989, 685. (f) Ito, Y.; Ogawa, T. Tetrahedron Lett. 1987,
28, 4701.
(15) (a) Lemieux, R. U.; Ratcliff, R. M. Can. J. Chem. 1979, 57, 1244-
1251. (b) Pavia, A. A.; Ung-Chhun, S. N.; Durand, J.-L. J. Org. Chem.
1981, 46, 3158-3160. (c) Klemer, A.; Kohla,. J. Carbohydr. Chem. 1988,
7, 785-797. (d) Gordon, D. M.; Danishefsky, S. J. J. Org. Chem. 1991,
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OL0061743
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Shaka, A. J. J. Am. Chem. Soc. 1992, 114, 3157-3159. (c) Hwang, T.-L.;
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(19) Bock, K.; Pedersen, C. J. J. Chem Soc. Perkin Trans. 2 1974, 293-
297.
(20) (a) Shibata, N.; Hisamichi K.; Kobayashi, H.; Suzuki, S. Arch.
Biochem. Biophys. 1993, 302, 113-117. (b) Shibata, N.; Hisamichi, K.;
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315-319.
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