Solution and solid-support synthesis of a potential leishmaniasis carbohydrate vaccine

Article abstract of DOI:10.1021/jo015521z

The synthesis of a potential carbohydrate vaccine for the parasitic disease leishmaniasis is described. New solution- and solid-phase synthetic strategies were explored for the assembly of a unique tetrasaccharide antigen found on the Leishmania lipophosphoglycan. An initial solution-phase synthesis relied on thioglycosides as building blocks and the establishment of the central disaccharide from lactal via an oxidation-reduction sequence. A second approach was completed both in solution and on solid support. The solid-phase synthesis relied on assembly from monosaccharide units and was used to evaluate different glycosylating agents in the efficient installation of the galactose β-(1-4) mannoside. Glycosyl phosphates proved most successful in this endeavor. This first solid-phase synthesis of the Leishmania cap provided rapid access to the tetrasaccharide in 18% overall yield while requiring only a single purification step. The synthetic cap tetrasaccharide was conjugated to the immunostimulator Pam₃Cys to create fully synthetic carbohydrate vaccine 1 and to the carrier protein KLH to form semisynthetic vaccine 2. Currently, both constructs have entered initial immunological experiments in mice targeted at the development of a vaccine against the parasitic disease leishmaniasis.

Full text of DOI:10.1021/jo015521z

J . Org. Chem. 2001, 66, 4233-4243
4233
Solu tion a n d Solid -Su p p or t Syn th esis of a P oten tia l Leish m a n ia sis
Ca r boh yd r a te Va ccin e
Michael C. Hewitt and Peter H. Seeberger*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
seeberg@mit.edu
Received J anuary 12, 2001
The synthesis of a potential carbohydrate vaccine for the parasitic disease leishmaniasis is described.
New solution- and solid-phase synthetic strategies were explored for the assembly of a unique
tetrasaccharide antigen found on the Leishmania lipophosphoglycan. An initial solution-phase
synthesis relied on thioglycosides as building blocks and the establishment of the central
disaccharide from lactal via an oxidation-reduction sequence. A second approach was completed
both in solution and on solid support. The solid-phase synthesis relied on assembly from
monosaccharide units and was used to evaluate different glycosylating agents in the efficient
installation of the galactose â-(1f4) mannoside. Glycosyl phosphates proved most successful in
this endeavor. This first solid-phase synthesis of the Leishmania cap provided rapid access to the
tetrasaccharide in 18% overall yield while requiring only a single purification step. The synthetic
cap tetrasaccharide was conjugated to the immunostimulator Pam3Cys to create fully synthetic
carbohydrate vaccine 1 and to the carrier protein KLH to form semisynthetic vaccine 2. Currently,
both constructs have entered initial immunological experiments in mice targeted at the development
of a vaccine against the parasitic disease leishmaniasis.
In tr od u ction
A potent vaccine that would facilitate destruction of
the parasite upon transfer from the sandfly into the
human host would be ideal. To date, several different
vaccine designs have been explored, each focusing on a
different part of the parasite in the hope of inducing a
specific immune response. Killed or attenuated parasites
did not result in valid vaccines, and leishmanial proma-
stigote surface protease gp63 vaccines are in various
phases of testing. Thus, no generally applicable vaccine
for leishmaniasis has yet emerged.7
Here we describe the preparation of fully synthetic and
semisynthetic immunogens that target leishmaniasis
based on the unique tetrasaccharide cap of the parasite’s
cell-surface lipophosphoglycan. New synthetic strategies
for the solution- and solid-phase synthesis of this carbo-
hydrate unit containing the rare galactose â-(1f4) man-
noside core are reported and different glycosylating
agents were explored. Two immunogens were prepared
by joining the tetrasaccharide antigen with the immu-
nostimulator tripalmitoyl-S-glycerylcysteine and with
keyhole limpet hemocyanin (KLH). Both the fully syn-
thetic and the semisynthetic immunogens are currently
undergoing immunological evaluation in mice.
Leishmaniasis is a tropical disease that afflicts over
12 million people worldwide, with 1.5-2 million new
cases estimated to occur each year.1 Three different forms
of the disease, spread by the bite of infected sandflies,
arise in humans. Visceral leishmaniasis, also known as
kala azar, causes swelling of the spleen and liver, and is
often lethal when left untreated. The most common form
of the disease, cutaneous leishmaniasis, results in de-
bilitating skin lesions, while mucocutaneous leishmania-
sis causes lesions in the mucous membranes, leading to
facial disfigurement. Although leishmaniasis is most
prevalent in tropical settings, the disease has recently
been diagnosed in overseas travelers and U.S. Gulf War
veterans,2 and has emerged as an opportunistic infection
of HIV patients.3 It is feared that the disease is currently
becoming endemic in the U.S.4
Leishmaniasis has been extensively studied in murine
models,5 but low parasite levels in humans render the
diagnosis of cutaneous and mucocutaneous leishmaniasis
difficult. Even after diagnosis, the mechanism of infection
poses a serious challenge for therapeutic approaches. The
parasite resides within macrophages, the very part of the
immune response that is designed to destroy it. Treat-
ment with pentavalent antimony medications such as
Pentostam and Glucantime is hampered by lengthy
treatment protocols (leading to low compliance) and toxic
side effects.6
Va ccin e Design Con sid er a tion s
In principle, any unique feature the parasite exposes
on its cell surface may be used for the development of a
leishmaniasis vaccine. Lipophosphoglycans (LPGs, Fig-
ure 1),8 which are ubiquitous on the cell surface of
Leishmania parasites, are composed of a glycosylphos-
phatidylinositol (GPI) anchor, a repeating phosphorylated
(1) For a review, see Herwaldt, B. L. Lancet 1999, 354, 1191.
(2) Magill, A. J .; Grogl, M.; Gasser, R. A.; Sun, W.; Oster, C. N. N.
Engl. J . Med. 1993, 328, 1383.
(3) Alvar, J .; Canavate, C.; Gutierrez-Solar, B.; et al. Clin. Microbiol.
Rev. 1997, 10, 298
(4) Enserink, M. Science 2000, 290, 1881.
(5) Reed, S. G.; Scott, P. Curr. Opin. Immunol. 1993 5, 524.
(6) Berman, J . D. Clin. Infect. Dis. 1997, 24, 684
(7) Modabber, F. Ann. Trop. Med. Parasitol. 1995, 89 (suppl 1), 83.
(8) For
a review, see Turco, S. J .; Descoteaux, A. Annu. Rev.
Microbiol. 1992, 46, 65.
10.1021/jo015521z CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/19/2001

4234 J . Org. Chem., Vol. 66, No. 12, 2001
Hewitt and Seeberger
F igu r e 1. Leishmania lipophosphoglycan.
disaccharide, and different cap oligosaccharides. The
LPGs have been examined as vaccine candidates since
the phosphoglycan portion of LPG was shown to be a
disease-promoting antigen,9 but the size (MW ca. 12000)
and heterogeneity of the diverse LPGs preclude their use.
The presence of unique oligosaccharide structures on the
surface of the parasite attracted our attention in con-
templating the use of synthetic carbohydrate chemistry
to create vaccines against tropical diseases. In particular,
a unique tetrasaccharide containing the unusual galac-
tose â-(1f4) mannosidic linkage piqued our interest from
a synthetic standpoint. Thus, we began to investigate the
feasibility of developing a structurally well-defined leish-
maniasis carbohydrate vaccine, based on this cap tet-
rasaccharide.
Different oligosaccharide vaccine constructs have re-
cently been investigated in the context of anticancer
vaccines based upon tumor-associated carbohydrate an-
tigens.10 Semisynthetic constructs were readily prepared
by covalent attachment of the synthetic oligosaccharide
antigen to a carrier protein such as KLH or BSA (bovine
serum albumin).11 Several semisynthetic constructs have
been tested in in vivo experiments and have entered
various phases of clinical trials against a host of can-
cers.12 The initial clinical results are quite promising and
validate this approach.
The structural ambiguity of the carbohydrate-protein
conjugates due to the ill-defined nature of the carrier
proteins prompted several groups to explore fully syn-
thetic carbohydrate vaccines that do not require ad-
ditional adjuvants. The lipopeptide tripalmitoyl-S-glyc-
erylcysteine (Pam3Cys) can be readily prepared,13 and
serves both as a carrier and adjuvant.14 Upon conjugation
to an antigen the result is a homogeneous, low molecular
weight construct that is readily taken up by antigen-
presenting cells. The stability of these synthetic vaccines
to heat, light, and different solvents renders them
particularly attractive for use in tropical settings.15
A
selective immune response against glycoproteins express-
ing a carbohydrate TN antigen (GalNAcR1 f O-Ser/
Thr)16 has been reported using a fully synthetic Pam3Cys-
TN vaccine.17
Given the success of semisynthetic and synthetic
carbohydrate vaccines against different diseases, we
designed two potential Leishmania vaccine constructs
(Figure 2). In one case the synthetic cap tetrasaccharide
was to be connected to Pam3Cys to fashion fully synthetic
vaccine 1, while a KLH-conjugate would provide semi-
synthetic vaccine 2.
Solu tion -P h a se Syn th esis of th e Leish m a n ia Ca p
Tetr a sa cch a r id e. Different sections of the Leishmania
LPG have served as targets for synthetic studies, includ-
ing the GPI heptasaccharyl myo-inositol.18 Synthetic
phosphorylated oligosaccharides19 of the Leishmania
Donovani LPG were analyzed as substrates for Leish-
(9) Mitchell, G. F.; Handman, E. Parasite Immun. 1986, 8, 255.
(10) Danishefsky, S. J .; Allen, J . R. Angew. Chem., Int. Ed. 2000,
39, 836.
(11) Francis, M. J . In Vaccines: Recent Trends and Progress;
Gregoriadis, G., Allison, A. C., Poste, G., Eds.; Plenum Press: New
York, 1991.
(13) Wiesmuller, H.-K.; Bessler, W. G.; J ung, G. Hoppe-Seylers Z.
Physiol. Chem. 1983, 364, 593.
(14) Reichel, F.; Ashton, P. R.; Boons, G.-J . Chem. Commun. 1997,
2087;
(12) (a) Ragupathi, G.; Slovin, S. F.; Adluri, S.; Sames, D.; Kim, I.
J .; Kim, H. M.; Spassova, M.; Bornmann, W. G.; Lloyd, K. O.; Scher,
H. I.; Livingston, P. O.; Danishefsky, S. J . Angew. Chem., Int. Ed. 1999,
38, 563. (b) Ragupathi, G.; Park, T. K.; Zhang, S.; Kim, I. J .; Graber,
L.; Adluri, S.; Lloyd, K. O.; Danishefsky, S. J .; Livingston, P. O. Angew.
Chem., Int. Ed. 1997, 36, 125. (c) Slovin, S. F.; Ragupathi, G.; Adluri,
S.; Ungers, G.; Terry, K.; Kim, S.; Spassova, M.; Bornmann, W. G.;
Fazzari, M.; Dantis, L.; Olkiewicz, K.; Lloyd, K. O.; Livingston, P. O.;
Danishefsky, S. J .; Scher, H. I. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
5710.
(15) Muller, C. P.; J ung, G. Lancet 1988, 1424
(16) (a) Springer, G. F. Science 1984, 224, 1198. (b) Itzkowitz, S.
H.; Yuan, M.; Montgomery, C. K.; Kjeldsen, T.; Takahashi, H. K.;
Bigbee, W. L.; Kim, Y. S. Cancer Res. 1989, 49, 197.
(17) Toyokuni, T.; Dean, B.; Cai, S.; Boivin, D.; Hakomori, S.-I.;
Singhal, A. K. J . Am. Chem. Soc. 1994, 116, 395.
(18) Ruda, K.; Lindberg, J .; Garegg, P. J .; Oscarson, S.; Konradsson,
P. J . Am. Chem. Soc. 2000, 122, 11067.

Synthesis of Potential Leishmaniasis Carbohydrate Vaccine
J . Org. Chem., Vol. 66, No. 12, 2001 4235
F igu r e 2. Synthetic and semisynthetic leishmaniasis vaccine constructs.
Sch em e 1. Solu tion -P h a se Syn th esis of th e Leish m a n ia Ca p Tetr a sa cch a r id e
mania transferase.20 The cap tetrasaccharide itself has
double bond under the agency of 2,2′-dimethyldioxirane
and reaction with ethanethiol in the presence of trace
amounts of acid resulted in thioethyl lactose 4.25 Further
conversion by a two step oxidation-reduction process26
to invert the stereochemistry of the C2 position furnished
the desired thioethyl galactose-â-(1f4)-mannoside 5 after
pivaloylation. Thioethyl glycoside 5 served as glycosyl
donor in the union with N-Cbz protected aminohexanol27
employing methyl triflate (MeOTf) activation to yield 54%
of the desired R-glycoside and permanently installed the
protected amino tether. Removal of the C2-pivaloyl-
protected alcohol with sodium hydroxide provided disac-
charide acceptor 6. Coupling of 2-pivaloyl mannosyl
thiodonor 728 with disaccharide 6 provided protected
trisaccharide 8. Further elongation by repetition of the
deprotection/coupling steps furnished the fully protected
also been the subject of a previous synthesis.21 Fraser-
Reid et al. explored the utility of n-pentenyl glycosides22
as building blocks to create a fully protected cap structure
in a both a linear and convergent fashion. The modest
yield (35%) for the final coupling between a trisaccharide
acceptor and monosaccharide donor as well as several
protecting group manipulations during assembly left
room for improvement. The tetrasaccharide was not
liberated from the protecting groups but rather served
as a formidable synthetic challenge to explore new
methodology. A radiolabeled version of the cap tetrasac-
charide was also recently prepared by Upreti et al.23
Hexabenzyl lactal 324 served as starting point for the
solution synthesis (Scheme 1). Epoxidation of the glycal
(19) (a) Nikolaev, V. A.; Rutherford, T. J .; Ferguson, M. A. J .;
Brimacombe, J . S. J . Chem. Soc., Perkin Trans. 1 1996, 1559. (b)
Nikolaev, A. V.; Rutherford, T. J .; Ferguson, M. A. J .; Brimacombe, J .
S. J . Chem. Soc., Perkin Trans. 1 1995, 1977. (c) Nikolaev, A. V.;
Chudek, J . A.; Ferguson, M. A. J . Carbohydr. Res. 1995, 272, 179.
(20) Brown, G. M.; Millar, A. R.; Masterson, C.; Brimacombe, J . S.;
Nikolaev, A. V.; Ferguson, M. A. J . Eur. J . Biochem. 1996, 242, 410.
(21) Arasappan, A.; Fraser-Reid, B. J . Org. Chem. 1996, 61, 2401.
(22) Madsen, R.; Fraser-Reid, B. In Modern Methods in Carbohy-
drate Synthesis; Khan, S. H., O’Neil, R. A. Eds.; Harwood Academic
Publishers: Switzerland, 1995; Chapter 4.
(23) Upreti, M.; Ruhela, D.; Vishwakarma, R. A. Tetrahedron 2000,
6577.
(24) Kinzy, W.; Schmidt, R. R. Carbohydr. Res. 1987, 164, 265.
(25) Seeberger, P. H.; Eckhardt, M.; Gutteridge, C. E.; Danishefsky,
S. J . J . Am. Chem. Soc. 1997, 119, 10064.
(26) Albright, J . D.; Goldman, L. J . Am. Chem. Soc. 1967, 89, 2416.
(27) Muller, D.; Vic, G.; Critchley, P.; Crout, D. H. G.; Lea, N.;
Roberts, L.; Lord, J . M. J . Chem. Soc. Perkin Trans. 1 1998, 2287.
(28) Seeberger, P. H.; Eckhardt, M.; Gutteridge, C. E.; Danishefsky,
S. J . J . Am. Chem. Soc. 1997, 119, 10064

4236 J . Org. Chem., Vol. 66, No. 12, 2001
Hewitt and Seeberger
Sch em e 2. Syn th esis of Syn th etic a n d Sem isyn th etic Ca r boh yd r a te Va ccin e Con str u cts
Sch em e 3
cap tetrasaccharide 9 equipped with an aminohexyl
spacer on the reducing terminus. No evidence for forma-
1
tion of the undesired â-isomers were detectable by H
NMR for formation of 8 and 9. Global deprotection of 9
was achieved via a two-step protocol. Saponification of
the pivaloyl ester was followed by removal of the benzyl
ethers and the carbamate via palladium-catalyzed hy-
drogenation to yield tetrasaccharide 10.
With the linker-equipped Leishmania cap tetrasaccha-
ride in hand, we embarked on the preparation of the
synthetic and semisynthetic immunogens. The primary
amine served as attachment site in the union with the
immunostimulator Pam3Cys using the HATU/HOAt ac-
tivation method29 to fashion the amide linkage. The
coupling between the lipophilic amino acid and hydro-
philic carbohydrate furnished the desired vaccine con-
struct 1 in 60% yield. Purification of 1 was best accom-
plished by silica column chromatography before character-
ization by high-resolution mass spectrometry and 1H
NMR unequivocally confirmed the structure of the vac-
cine. It is worth pointing out that concentrated solutions
of 1 rapidly form micelles and complicate analysis of 1
significantly.
In addition to the fully synthetic immunogen 1, and
in light of the success achieved with carbohydrate-
protein constructs as antitumor agents (vide supra), we
focused next on the preparation of a semisynthetic
vaccine 2. Tetrasaccharide amine 10 was condensed with
S-acetylmercaptoacetate pentafluorophenyl ester to af-
ford 12 in 68% yield (Scheme 2). Similarly, KLH was
condensed with N-succidimidylbromoacetate to provide
bromoacetate-modified protein 13. Union of 12 and 13
in the presence of NH2OH was followed by capping of any
remaining bromoacetates with 2-aminoethanethiol to
furnish semisynthetic vaccine 2. The conjugation ratio
of cap tetrasaccharide to each KLH molecule was 55:1
as determined by carbohydrate and protein analysis.
Both the synthetic vaccine 1 and semisynthetic sugar-
protein conjugate 2 are currently undergoing initial
immunological evaluation in Balb-C mice.
Solid -P h a se Syn t h esis of t h e Leish m a n ia Ca p
Tetr a sa cch a r id e. In addition to establishing a reliable
new route for the synthesis of the Leishmania cap
tetrasaccharide, we intended to develop novel synthetic
strategies that would allow us to take advantage of the
solid-phase paradigm. Advantages offered by the syn-
thesis of carbohydrates on solid support are improved
yields due to the use of excess reagents to drive reactions
to completion (3 equiv of donor were used for each
glycosylation) and a single purification step at the end
of the synthesis rather than chromatographic purification
after each transformation in a multistep synthesis. While
convenient with respect to the ease of procurement of
starting glycals, our initial synthetic approach included
several steps that are difficult to carry out on solid
support and proceeded in relatively low yields. The
oxidation-reduction procedure for the conversion of a
glucoside into a mannoside had previously been success-
fully accomplished even on solid support but resulted in
less than quantitative yields.30 It was this difficult
transformation that rendered a synthesis from monosac-
charides in a stepwise fashion attractive.
(29) (a) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397. (b)
Carpino, L. A.; Ionescu, D.; El-Faham, A. J . Org. Chem. 1996, 61, 2460.
(30) Seeberger, P. H.; Danishefsky, S. J . Unpublished results.

Synthesis of Potential Leishmaniasis Carbohydrate Vaccine
J . Org. Chem., Vol. 66, No. 12, 2001 4237
Sch em e 4. Solu tion - a n d Solid -P h a se Syn th esis of th e Leish m a n ia Tetr a sa cch a r id e Usin g Mon osa cch a r id e
Bu ild in g Block sa
Mindful of the fact that a successful solid-phase ap-
proach would have to produce the tetrasaccharide in a
form that would allow for the attachment of a handle on
its reducing end for the adornment of different immu-
nostimulants, the installation of a pentenyl glycoside was
very attractive.31 Recently, we introduced a new linker
concept for solid-phase oligosaccharide synthesis based
on an octenediol linker, which after olefin cross-meta-
thesis under an atmosphere of ethylene reveals a termi-
nal pentenyl glycoside.32 The assembly of the cap tet-
rasaccharide from monosaccharides was accomplished
using an identical reaction path both in solution and on
the solid support (Scheme 4).
Preparation of the differentially protected mannose
core building block 16 commenced with known mannose
diol 14 (Scheme 3).33 Regioselective benzoylation of the
axial C2 hydroxyl group was followed by masking of the
less reactive C4-hydroxyl as a TBS-ether to furnish fully
differentiated 15. The temporary anomeric p-methoxy-
phenyl protection of 15 was oxidatively removed, and the
resulting hemiacetal of mannose was converted into
trichloroacetimidate 16 by reaction with trichloroaceto-
nitrile and DBU.34
fashioned n-pentenyl glycoside 19 upon activation of the
trichloroacetimidate with catalytic amounts of TBSOTf
in 1 h at room temperature (Scheme 4). Selective removal
of the C4 silyl ether by treatment with TBAF was
followed by glycosylation of the exposed C4 hydroxyl
group with glycosyl phosphate donor 20. Galactosyl
phosphate 20 was readily prepared in gram quantities
from tribenzyl galactal employing a straightforward and
high-yielding one-pot procedure.35 As expected, the use
of glycosyl phosphate donors facilitated the glycosylation
of the sterically encumbered C4 hydroxyl moiety without
complications and completely stereoselectively by virtue
of the C2 participating pivaloyl group.38 Selective removal
of the C2 benzoate protection revealed the axial acceptor
site for the coupling with trichloroacetimidate mannosyl
donor 23 to provide trisaccharide 25. Removal of the axial
acetate and renewed elongation by coupling with 23
furnished cap tetrasaccharide pentenyl glycoside 26. This
stepwise tetrasaccharide assembly proceeded without
problem but required the purification of intermediates
at seven steps throughout the synthesis.
To further simplify the preparation of the cap tetrasac-
charide and to maximize the overall yields, the assembly
of the target molecule on solid support was explored.
Following the same sequence of reactions as for the
solution-phase synthesis (Scheme 4), octenediol linker
functionalized Merrifield resin 17 served as the starting
point in place of n-pentenyl alcohol. At each step of the
synthesis a small portion of the resin was set aside and
the reaction products were cleaved by olefin cross-
metathesis36 using Grubbs catalyst under an atmosphere
of ethylene. Thus, the saccharide products were obtained
in the form of the terminal n-pentenyl glycosides37 and
compared to the samples obtained by solution-phase
With differentially protected core mannose building
block 16 at our disposal, coupling with pentenyl alcohol
(31) Buskas, T.; So¨derberg, E.; Konradsson, P.; Fraser-Reid, B. J .
Org. Chem. 2000, 65, 958.
(32) Andrade, R. B.; Plante, O. J .; Melean, L. G.; Seeberger, P. H.
Org. Lett. 1999, 1, 1811.
(33) Mori, M.; Ito, Y.,; Ogawa, T. Carbohydr. Res. 1989, 192, 131.
(34) (a) Rademann, J .; Schmidt, R. R. Tetrahedron Lett. 1996, 37,
3989, (b) Rademann, J .; Schmidt, R. R. J . Org. Chem. 1997, 62, 3989.
(c) Heckel, A.; Mross, E.; J ung, K.-H.; Rademann, J .; Schmidt, R. R.
Synlett 1998, 171, (d) Rademann, J .; Geyer, A.; Schmidt, R. R. Angew.
Chem., Int. Ed. 1998, 37, 1241. (e) Hunt, J . A.; Roush, W. R. J . Am.
Chem. Soc. 1996, 118, 9998. (f) Adinolfi, M.; Barone, G.; De Napoli,
L.; Iadonisi, A.; Piccialli, G. Tetrahedron Lett. 1996, 37, 5007. (g)
Adinolfi, M.; Barone, G.; De Napoli, L.; Iadonisi, A.; Piccialli, G.
Tetrahedron Lett. 1998, 39, 1953.
(35) Plante, O. J .; Andrade, R. B.; Seeberger, P. H. Org. Lett. 1999,
1, 211.

4238 J . Org. Chem., Vol. 66, No. 12, 2001
Hewitt and Seeberger
Eth yl 2,3,4,6-Tetr a-O-ben zyl-â-D-galactopyr an osyl-(1f4)-
3,6-d i-O-ben zyl-th io-â-D-glu cop yr a n osid e 4. Hexabenzyl
lactal 324 (256 mg, 0.29 mmol) was dissolved in CH2Cl2 (600
µL) and cooled to 0 °C. Dimethyldioxirane (15.0 mL in acetone,
0.87 mmol) was added via cannula, and the solution was
stirred for 10 min at 0 °C. The solvent was removed in vacuo.
After drying in vacuo for 30 min, the epoxide was dissolved in
CH2Cl2 (714 µL), and EtSH (714 µL) was added. The mixture
was cooled to -78 °C, and triflouroacetic anhydride (5 µL) was
added dropwise. The reaction mixture was allowed to warm
to room temperature over 16 h. The solvent was removed in a
stream of N2. Purification by flash silica column chromatog-
raphy (10 f 30% EtOAc/hexanes) afforded 4 (143 mg, 51%)
as a white solid. [R]24D: -3.92° (c 1.0, CH2Cl2); IR (thin film)
chemistry. Simple washing of the resin was used to
remove excess reagents. Cleavage of support-bound tet-
rasaccharide 25 yielded pure tetrasaccharide n-pentenyl
glycoside 27 after a single silica column in 18% overall
yield from 17. The solid-phase assembly took 4 days and
was significantly faster than the solution-phase synthesis
that required at least two weeks.
This solid-phase synthesis demonstrated that branched
oligosaccharides of biological interest are accessible in
good overall yields using glycosyl trichloroacetimidate
and glycosyl phosphate donors in concert. The terminal
n-pentenyl glycoside could be further elaborated into an
aldehyde or carboxylic acid that would allow for attach-
ment to immunostimulators such as proteins or lipopep-
tides.
1
3447, 3062, 3029, 2867, 1498 cm-1; H NMR (CDCl3) δ 7.32-
7.11 (m, 30H), 5.04 (d, J ) 11.1 Hz, 1H), 4.92 (d, J ) 11.5 Hz,
1H), 4.76-4.65 (m, 5H), 4.49-4.20 (m, 8H), 3.95-3.87 (m, 2H),
3.78-3.66 (m, 3H), 3.54-3.32 (m, 7H), 2.65 (q, J ) 4.1 Hz,
2H), 2.48 (s, 1H), 1.24 (t, J ) 4.2 Hz, 3H); 13C NMR (CDCl3) δ
138.7, 138.6, 138.4, 138.1, 138.0, 137.6, 128.1, 128.1, 128.0,
127.9, 127.8, 127.6, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1,
102.6, 85.3, 83.4, 82.2, 79.7, 79.5, 75.8, 75.1, 74.7, 74.5, 73.4,
73.3, 72.9, 72.4, 72.1, 68.1, 68.0, 24.0, 15.3; FAB MS m/z (M +
Na+) calcd 949.3956, found 949.3952.
Con clu sion s
In summary, we have introduced two new synthetic
routes for the preparation of the parasite-specific Leish-
mania cap tetrasaccharide. The first solution-phase
synthesis relied on thioglycosides as building blocks and
the establishment of the central disaccharide from lactal
via an oxidation-reduction sequence. The synthetic cap
tetrasaccharide was conjugated to the immunostimulator
Pam3Cys to create a fully synthetic carbohydrate vaccine
and to the carrier protein KLH to form a semisynthetic
vaccine. Currently, both vaccine constructs have entered
initial immunological experiments in mice targeted at the
development of a vaccine against the parasitic disease
leishmaniasis. The results of the immunological evalu-
ations will be reported in due course. The second syn-
thetic scheme built on assembly of the target structure
from monosaccharides and was readily executed both in
solution and on the solid support. It was demonstrated
that glycosyl trichloroacetimidates and glycosyl phos-
phates can be used together to create branched oligosac-
charides of biological significance. This first solid-phase
synthesis of the Leishmania cap provides rapid access
to synthetic material while requiring only a single
purification step.
Eth yl 2,3,4,6-Tetr a-O-ben zyl-â-D-galactopyr an osyl-(1f4)-
3,6-d i-O-ben zyl-2-O-p iva loyl-th io-â-D-m a n n op yr a n osid e
5. Thioethyl â-D-glucoside 4 (1.42 g, 1.48 mmol) was treated
with DMSO/Ac2O (10 mL/5 mL) for 72 h at room temperature.
The solvents were removed in vacuo. The residue was diluted
with EtOAc (50 mL) and washed with sat. NaHCO3 (2 × 100
mL), H2O (1 × 100 mL), and brine (1 × 100 mL). The crude
residue was dried with Na2SO4 and concentrated to dryness.
After azeotroping (3 × 20 mL toluene) and drying in vacuo
for 1 h, the orange oil was dissolved in CH2Cl2/MeOH (7 mL/7
mL) and cooled to 0 °C. NaBH4 (265 mg, 7.4 mmol) was added,
and the reaction mixture was allowed to warm to room
temperature over 2 h. The reaction was quenched with
NaHCO3 (30 mL) and diluted with EtOAc (30 mL), and NH4-
Cl (30 mL) was added to dissolve solids before the phases were
separated. Following extraction of the aqueous phase with
EtOAc (3 × 30 mL), the combined extracts were washed with
brine (50 mL), dried (Na2SO4), filtered, and concentrated. The
crude material was purified by flash silica column chroma-
tography (20 f 30% EtOAc/hexanes), affording ethyl 2,3,4,6-
tetra-O-benzyl-â-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-
thio-â-D-mannopyranoside (660 mg, 47%) as a clear oil. [R]24
:
D
- 11.5° (c 1.0, CH2Cl2); IR (thin film) 3447, 3029, 2922, 2866,
1496 cm-1; 1H NMR (CDCl3) δ 7.41-7.27 (m, 30H), 5.00 (d, J
) 11.6 Hz, 1H), 4.86 (d, J ) 7.0 Hz, 1H), 4.84 (d, J ) 6.7 Hz,
1H), 4.77-4.69 (m, 4H), 4.62-4.60 (m, 2H), 4.52 (d, J ) 7.9
Hz, 1H), 4.51 (d, J ) 11.9 Hz, 1H), 4.43 (d, J ) 11.9 Hz, 1H),
4.41-4.07 (m, 2H), 3.95 (d, J ) 2.7 Hz, 1H), 3.89-3.86 (m,
1H), 3.80-3.76 (m, 2H), 3.65-3.62 (m, 1H), 3.55 (dd, J ) 3.4,
8.8 Hz, 1H), 3.52-3.62 (m, 4H), 2.79-2.74 (m, 2H), 2.62 (d, J
) 1.5 Hz, 1H), 1.34 (t, J ) 7.3 Hz, 3H); 13C NMR (CDCl3) δ
138.8, 138.5, 138.3, 138.2, 138.1, 137.8, 128.3, 128.2, 128.1,
128.1, 128.0, 127.7, 127.7, 127.6, 127.6, 127.5, 127.4, 127.4,
127.3, 127.2, 127.2, 103.0, 83.5, 82.5, 80.6, 79.9, 79.8, 75.1, 74.5,
74.4, 73.4, 73.4, 73.1, 73.0, 72.5, 72.4, 70.3, 69.0, 25.6, 15.3;
FAB MS m/z (M + Na+) calcd 949.3956, found 949.3996.
2,3,4,6-Tetr a -O-ben zyl-â-D-ga la ctop yr a n osyl-(1f4)-3,6-
di-O-ben zyl-th io-â-D-m an n opyr an oside (883 mg, 0.92 mmol)
was dissolved in CH2Cl2 (10 mL), and DMAP (447 mg, 3.66
mmol) was added. After stirring for 30 min, pivaloyl chloride
(225 µL, 1.83 mmol) was added. The reaction mixture was
stirred at room temperature for 1 h. The reaction was
quenched with sat. NaHCO3 (30 mL), and the aqueous phase
was extracted with CH2Cl2 (3 × 30 mL). The combined extracts
were washed with brine (50 mL), dried (Na2SO4), filtered, and
concentrated. Purification by flash silica column chromatog-
raphy (20% EtOAc/hexanes) afforded 5 (900 mg, 95%) as a
clear oil. [R]24D: - 21.5° (c 1.0, CH2Cl2); IR (thin film) 2966,
Exp er im en ta l Section
All commercial materials were used without further puri-
fication unless otherwise noted. 10x phosphate-buffered saline
(PBS) was purchased from Boehringer Mannheim and diluted
to the desired concentration; Pd-10 columns (Sephadex G-25)
were purchased from Pharmacia. Dichloromethane (CH2Cl2)
was distilled from calcium hydride under N2. THF was distilled
from sodium/benzophenone under N2. Analytical thin-layer
chromatography was performed on E. Merck silica gel 60 F254
plates (0.25 mm). Compounds were visualized by dipping the
plates in a cerium sulfate-ammonium molybdate solution
followed by heating. Liquid column chromatography was
performed using forced flow of the indicated solvent on Silicycle
silica (230-400 mesh). 1H NMR spectra were obtained on
either a Varian VXR-300 (300 MHz) or VXR-500 (500 MHz)
and are reported in parts per million (δ) relative to chloroform
(7.27 ppm). Coupling constants (J ) are reported in hertz. 13C
NMR spectra were recorded on a VXR-500 (125 MHz) and are
reported in δ relative to CDCl3 (77.0 ppm) as an internal
reference.
(36) (a) Schwab, P.; Grubbs, R. H.; Ziller, J . W. J . Am. Chem. Soc.
1996, 118, 100. (b) Grubbs, R. H.; Miller, S. J .; Fu, G. C. Acc. Chem.
Res. 1995, 28, 446. (c) Schmalz, H. G. Angew. Chem., Int. Ed. Engl.
1996, 34, 1833.
(37) Buskas, T.; Soderberg, E.; Konradsson, P.; Fraser-Reid, B. J .
Org. Chem. 2000, 65, 958.
2925, 2867, 1733, 1496 cm-1 1H NMR (CDCl3) δ 7.40-7.24
;
(m, 25H), 5.68 (d, J ) 3.36 Hz, 1H), 5.01 (d, J ) 11.6 Hz, 1H),
4.87 (d, J ) 11.0 Hz, 1H), 4.79-4.62 (m, 8H), 4.54 (d, J ) 11.9

Synthesis of Potential Leishmaniasis Carbohydrate Vaccine
J . Org. Chem., Vol. 66, No. 12, 2001 4239
Hz, 1H), 4.34 (s, 2H), 4.16-4.12 (m, 1H), 3.96-3.79 (m, 4H),
3.68 (dd, J ) 3.4, 9.2 Hz, 1H), 3.61-3.58 (m, 2H), 3.51 (dd, J
) 2.8, 9.8 Hz, 1H), 3.38-3.34 (m, 2H), 2.78 (q, J ) 7.2 Hz,
2H), 1.36 (t, J ) 7.6 Hz, 3H), 1.25 (s, 9H); 13C NMR (CDCl3) δ
177.3, 138.7, 138.5, 138.4, 138.4, 138.2, 137.6, 128.2, 128.0,
127.9, 127.7, 127.5, 127.3, 127.3, 127.2, 127.2, 127.1, 126.8,
102.5, 82.6, 82.3, 79.9, 79.7, 79.3, 75.0, 74.5, 73.8, 73.3, 73.2,
72.8, 72.7, 72.4, 71.4, 69.7, 68.9, 68.0, 39.0, 27.2, 25.7, 15.1;
FAB MS m/z (M+ + H) calcd 1010.4639, found 1010.4671.
6-(Ben zyloxyca r bon yla m in o)h exyl-(2-O-p iva loyl-3,4,6-
tr i-O-ben zyl-r-D-m an n opyr an osyl)-(1f2)-O-[(2,3,4,6-tetr a-
O-ben zyl-â-D-ga la ctop yr a n osyl)-(1f4)]-3,6-d i-O-ben zyl-r-
D-m a n n op yr a n osid e 8. A mixture of 7 (110 mg, 0.19 mmol)
and disaccharide acceptor 6 (143 mg, 0.13 mmol) was azeo-
troped with toluene (3 × 5 mL) and dried under vacuum for 1
h. Freshly dried 4 Å molecular sieves (110 mg) and CH2Cl2 (3
mL) were added. The suspension was cooled to 0 °C, and
methyl triflate (60 µL, 0.53 mmol) was added dropwise. After
stirring at 0 °C for 10 h, the reaction mixture was warmed to
room temperature, and triethylamine (1 mL) was added. The
reaction was diluted with EtOAc (20 mL), filtered, and washed
with sat. NaHCO3 (2 × 30 mL) and brine (1 × 30 mL).
Following drying (Na2SO4) and concentration, the crude
product was purified by flash silica column chromatography
(20 f 30% EtOAc/hexanes) to afford 8 (136 mg, 66%) as a clear
oil. [R]24D: +19.7° (c 1.0, CH2Cl2); IR (thin film) 2926, 1731,
1496, 1453, 1364 cm-1; 1H NMR (CDCl3) δ 7.37-7.20 (m, 50H),
5.52-5.51 (m, 1H), 5.01 (s, 2H), 5.04 (d, J ) 1.5 Hz, 1H), 4.91
(d, J ) 11.9 Hz, 1H), 4.86 (d, J ) 11.6 Hz, 1H), 4.82-4.78 (m,
3H), 4.70 (d, J ) 11.3 Hz, 1H), 4.66 (d, J ) 11.6 Hz, 1H), 4.64-
4.59 (m, 3H), 4.53-4.48 (m, 4H), 4.44 (d, J ) 11.3 Hz, 1H),
4.39 (d, J ) 12.2 Hz, 1H), 4.31 (d, J ) 3.7 Hz, 1H), 4.29 (d, J
) 2.7 Hz, 1H), 4.20 (d, J ) 11.6 Hz, 1H), 4.12-4.08 (m, 1H),
4.00-3.92 (m, 4H), 3.88-3.85 (m, 2H), 3.80-3.69 (m, 8H),
3.64-3.55 (m, 1H), 3.53 (t, J ) 8.5 Hz, 1H), 3.46-3.36 (m, 4H),
3.31-3.27 (m, 2H), 3.14-3.11 (m, 2H), 1.52-1.26 (m, 12H),
1.15 (s, 9H); 13C NMR (CDCl3) δ 177.6, 157.1, 139.9, 139.6,
139.5, 139.4, 139.1, 139.1, 138.7, 137.4, 129.2, 129.0, 128.9,
128.9, 128.9, 128.8, 128.8, 128.7, 128.5, 128.4, 128.4, 128.2,
128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 128.0, 127.6, 127.5,
104.1, 100.0, 99.3, 83.4, 80.6, 79.2, 78.7, 77.9, 76.1, 75.8, 75.7,
75.6, 75.2, 74.9, 74.0, 73.9, 73.8, 73.7, 73.4, 73.1, 73.0, 72.4,
72.3, 72.0, 69.9, 68.7, 68.5, 68.2, 67.5, 41.7, 39.5, 39.4, 34.7,
30.6, 30.4, 30.0, 29.6, 27.9, 27.7, 26.6, 25.2; FAB MS m/z (M+
+ Na+) calcd 1668.8325, found 1668.8298.
6-(Ben zyloxycar bon ylam in o) Hexyl-(2-O-pivaloyl-3,4,6-
t r i-O-b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-O-(3,4,6-t r i-O-
b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-O-[(2,3,4,6-t et r a -O-
ben zyl-â-D-ga la ctop yr a n osyl)-(1f4)]-3,6-d i-O-ben zyl-r-D-
m a n n op yr a n osid e 9. To a solution of 8 (130 mg, 0.078 mmol)
in THF (500 µL) was added 1.0 M NaOH/MeOH solution (500
µL). The mixture was allowed to stir at room temperature for
17 h and quenched with sat. NH4Cl (5 mL). Following
extraction with EtOAc (3 × 5 mL), the combined extracts were
washed with brine (10 mL), dried (Na2SO4), filtered, and
concentrated. Purification by flash silica column chromatog-
raphy (20 f 40% EtOAc/hexanes) afforded 6-(benzyloxycar-
bonylamino)hexyl-(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-
(1f2)-O-[(2,3,4,6-tetra-O-benzyl-â-D-galactopyranosyl)-(1f4)]-
3,6-di-O-benzyl-R-D-mannopyranoside (93.3 mg, 76%) as a clear
oil. [R]24D: +24.8° (c 1.0, CH2Cl2); IR (thin film) 2924, 1718,
1496, 1453, 1363 cm-1; 1H NMR (CDCl3) δ 7.36-7.18 (m, 50H),
5.14-5.13 (m, 1H), 5.09 (s, 2H), 4.94 (d, J ) 11.3 Hz, 1H),
4.88 (d, J ) 11.9 Hz, 1 H), 4.85 (d, J ) 2.1 Hz, 1H), 4.79 (d, J
) 11.0 Hz, 1H), 4.76 (t, J ) 11.3 Hz, 2H), 4.69 (d, J ) 11.9
Hz, 1H), 4.65 (d, J ) 11.9 Hz, 1H), 4.64-4.60 (m, 3H), 4.56 (d,
J ) 10.1 Hz, 1H), 4.50-4.48 (m, 3H), 4.46-4.38 (m, 4H), 4.33
(d, J ) 11.9 Hz, 1H), 4.22 (d, J ) 11.9 Hz, 1H), 4.12 (t, J ) 9.2
Hz, 1H), 4.05-4.00 (m, 2H), 3.92-3.66 (m, 11H), 3.60-3.58
(m, 1H), 3.54-3.50 (m, 1H), 3.44 (dd, J ) 3.1, 9.8 Hz, 1H),
3.41-3.37 (m, 2H), 3.29-3.26 (m, 1H), 3.12 (q, J ) 6.7 Hz,
2H), 1.91-1.62 (br s, 1H), 1.51-1.26 (m, 11H); 13C NMR
(CDCl3) δ 156.5, 139.4, 139.1, 139.0, 138.9, 138.6, 138.6, 138.5,
138.2, 138.2, 136.9, 128.7, 128.7, 128.6, 128.6, 128.6, 128.5,
128.4, 128.4, 128.3, 128.3, 128.1, 128.1, 128.0, 128.0, 128.0,
127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6,
127.5, 127.4, 127.3, 127.3, 103.3, 101.0, 98.9, 82.8, 80.2, 80.1,
78.1, 77.4, 76.8, 75.5, 75.4, 75.2, 75.1, 74.8, 74.6, 73.8, 73.6,
73.6, 73.6, 73.4, 73.2, 72.9, 72.8, 72.1, 71.9, 71.7, 69.4, 69.2,
68.8, 68.5, 67.7, 66.7, 41.2, 30.1, 29.9, 29.5, 26.7, 26.1; FAB
MS m/z (M+ + Na+) calcd 1570.7224, found 1570.7176. A
mixture of 7 (108 mg, 0.19 mmol) and trisaccharide acceptor
6-(benzyloxycarbonylamino)hexyl-(3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl)-(1f2)-O-[(2,3,4,6-tetra-O-benzyl-â-D-galactopy-
6-(Ben zyloxycar bon ylam in o)h exyl 2,3,4,6-Tetr a-O-ben -
zyl-â-D-ga la ctop yr a n osyl-(1f4)-3,6-d i-O-ben zyl-r-D-m a n -
n op yr a n osid e 6. A mixture of 5 (542 mg, 0.52 mmol) and
Cbz-protected aminohexanol (286 mg, 0.78 mmol) were azeo-
troped with toluene (3 × 5 mL) and dried under vacuum for 1
h. CH2Cl2 (11 mL), freshly dried 4 Å molecular sieves (500 mg),
and di-tert-butylpyridine (174 µL, 2.08 mmol) were added, and
the mixture was allowed to stir for 30 min at room tempera-
ture. After cooling to 0 °C, methyl triflate (234 µL, 2.08 mmol)
was added dropwise. After stirring at 0 °C for 20 h, the mixture
was warmed to room temperature, and triethylamine (1 mL)
was added. The reaction mixture was diluted with EtOAc (20
mL), filtered, and washed with sat. NaHCO3 (2 × 30 mL) and
brine (1 × 30 mL). Following drying (Na2SO4) and concentra-
tion the crude product was purified by flash silica column
chromatography (20 f 30% EtOAc/hexanes) to afford 6-(Ben-
zyloxycarbonylamino)hexyl 2,3,4,6-tetra-O-benzyl-â-D-galac-
topyranosyl-(1f4)-3,6-di-O-benzyl-2-O-pivaloyl-R-D-mannopy-
ranoside (245 mg, 54%) as a clear oil. [R]24D: -7.6° (c 2.40,
CH2Cl2); IR (thin film) 2824, 1727, 1454, 1243, 1097 cm-1; 1H
NMR (CDCl3) δ 7.37-7.17 (m, 35H), 5.32 (dd, J ) 1.8, 3.1 Hz,
1H), 5.01 (s, 2H), 4.92 (d, J ) 11.6 Hz, 1H), 4.82-4.78 (m,
2H), 7.72 (s, 2H), 4.70-4.66 (m, 3H), 4.62 (d, J ) 11.6 Hz,
2H), 4.56 (d, J ) 11.3 Hz, 1H), 4.53 (d, J ) 6.7 Hz, 1H), 4.42
(d, J ) 12.2 Hz, 1H), 4.26 (d, J ) 0.9 Hz, 2H), 4.15-4.13 (m,
1H), 3.91 (dd, J ) 3.4, 9.2 Hz, 1H), 3.87 (d, J ) 2.7 Hz, 1H),
3.81 (d, J ) 8.9 Hz, 2H), 3.75-3.67 (m, 3H), 3.53-3.49 (m,
1H), 3.42-3.39 (m, 2H), 3.26 (q, J ) 5.2 Hz, 2H), 3.20-3.16
(m, 2H), 1.60-1.34 (m, 11H), 1.13 (s, 9H); 13C NMR (CDCl3) δ
177.5, 156.2, 138.9, 138.8, 138.7, 138.5, 138.3, 137.4, 136.5,
128.4, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9,
127.9, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.5,
127.4, 127.3, 127.3, 127.2, 127.1, 127.1, 127.0, 126.9, 126.7,
102.7, 97.4, 82.6, 79.9, 76.2, 74.9, 74.4, 74.3, 73.2, 73.1, 72.8,
72.7, 72.6, 72.6, 72.4, 72.3, 71.3, 71.0, 68.8, 68.7, 68.0, 67.6,
66.4, 58.4, 40.9, 38.8, 29.8, 29.4, 29.1, 27.0, 27.0, 26.9, 26.4,
26.4, 25.7, 25.7; FAB MS m/z (M+ + H) calcd 1214.6569, found
1214.6587.
To a solution of 6-(benzyloxycarbonylamino)hexyl 2,3,4,6-
tetra-O-benzyl-â-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-
O-pivaloyl-R-D-mannopyranoside (408 mg, 0.34 mmol) in THF
(1.5 mL) was added 1.0 M NaOH/MeOH solution (2 mL). The
mixture was allowed to stir at room temperature for 20 h and
then quenched with sat. NH4Cl (5 mL). The reaction mixture
was extracted with EtOAc (3 × 5 mL). The combined extracts
were washed with brine (10 mL), dried (Na2SO4), filtered, and
concentrated. Purification by flash silica column chromatog-
raphy (20 f 40% EtOAc/hexanes) afforded 6 (289 mg, 76%)
as a clear oil. [R]24D: +25.6° (c 1.0, CH2Cl2); IR (thin film) 3349,
2924, 1718, 1496, 1454 cm-1 1H NMR (CDCl3) δ 7.37-7.20
;
(m, 35H), 5.10 (s, 2H), 4.97 (d, J ) 11.6 Hz, 1H), 4.92 (d, J )
11.3 Hz, 1H), 4.86 (d, J ) 1.2 Hz, 1H), 4.79 (d, J ) 11.3 Hz,
1H), 4.74 (d, J ) 11.0 Hz, 1H), 4.72 (d, J ) 11.9 Hz, 1H), 4.68
(d, J ) 11.9 Hz, 1H), 4.63 (d, J ) 11.6 Hz, 1H), 4.59-4.54 (m,
2H), 4.45 (d, J ) 7.9 Hz, 1H), 4.40 (d, J ) 12.2 Hz, 1H), 4.38
(d, J ) 11.6 Hz, 1H) 4.27 (d, J ) 11.6 Hz, 2H), 4.14-4.10 (m,
1H), 4.00 (s, 1H), 3.91 (d, J ) 2.7 Hz, 1H), 3.83-3.66 (m, 8H),
3.61-3.57 (m, 1H), 3.46-3.37 (m, 5H), 3.16 (q, J ) 6.7 Hz,
2H), 2.51 (d, J ) 1.2 Hz, 1H) 1.59-1.34 (m, 11H); 13C NMR
(CDCl3) δ 156.3, 138.9, 138.8, 138.7, 138.5, 138.0, 136.6, 128.5,
128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 127.8, 127.7, 127.6,
127.5, 127.5, 127.4, 127.3, 127.3, 103.1, 98.9, 82.5, 79.9, 78.1,
77.4, 75.1, 74.6, 74.3, 73.5, 73.4, 73.1, 73.0, 72.7, 72.6, 71.0,
69.4, 68.6, 68.4, 67.5, 66.6, 41.0, 29.9, 29.7, 29.2, 26.5, 25.8,
14.1; FAB MS m/z (M+ + H) calcd 1130.5993, found 1130.5968.

4240 J . Org. Chem., Vol. 66, No. 12, 2001
Hewitt and Seeberger
ranosyl)-(1f4)]-3,6-di-O-benzyl-R-D-mannopyranoside (193 mg,
0.12 mmol) were azeotroped with toluene (3 × 5 mL) and dried
under vacuum for 1 h. Freshly dried 4 Å molecular sieves (100
mg) and CH2Cl2 (3 mL) were added. The suspension was cooled
to 0 °C, and methyl triflate (60 µL, 0.53 mmol) was added
dropwise. After stirring at 0 °C for 10 h, the reaction mixture
was warmed to room temperature, and triethylamine (1 mL)
was added. The reaction was diluted with EtOAc (20 mL),
filtered, and washed with sat. NaHCO3 (2 × 30 mL) and brine
(1 × 30 mL). Following drying (Na2SO4), filtration and
concentration the crude product was purified by silica gel
chromatography (15 f 20% EtOAc/hexanes) to afford 9 (180
mg, 70%) as a clear oil. [R]24D: +14.8° (c 1.0, CH2Cl2); IR (thin
film) 2922, 1730, 1454, 1362, 1108 cm-1;1H NMR (CDCl3) δ
7.36-7.12 (m, 75H), 5.49-5.48 (m, 1H), 5.09 (s, 2H), 4.93 (d,
J ) 11.3 Hz, 1H), 4.90 (dd, J ) 1.8, 9.7 Hz, 1H), 4.83-4.76
(m, 4H), 4.73 (s, 1H), 4.72-4.62 (m, 6H), 4.58-4.53 (m, 6H),
4.51 (s, 2H), 4.48-4.40 (m, 6H), 4.36 (d, J ) 11.0 Hz, 1H),
4.28 (d, J ) 12.2 Hz, 1H), 4.23 (d, J ) 11.6 Hz, 1H), 4.16 (d,
J ) 11.6 Hz, 1H), 4.12-4.08 (m, 1H), 4.01 (s, 1H), 3.98-3.95
(m, 3H), 3.90 (d, J ) 2.7, 1H), 3.88-3.85 (m, 2H), 3.82-3.78
(m, 4H), 3.76-3.61 (m, 6H), 3.58-3.55 (m, 3H), 3.46-3.43 (m,
3H), 3.36 (q, J ) 4.9 Hz, 2H), 3.26-3.22 (m, 2H), 3.14-3.10
(m, 2H), 1.54-1.26 (m, 12H), 1.18 (s, 9H); 13C NMR (CDCl3) δ
177.9, 157.0, 140.0, 139.7, 139.5, 139.5, 139.3, 139.2, 139.2,
139.2, 139.1, 139.1, 139.1, 138.6, 137.4, 129.2, 129.0, 129.0,
129.0, 128.9, 128.9, 128.8, 128.8, 128.7, 128.7, 128.6, 128.6,
128.5, 128.5, 128.4, 128.2, 128.2, 128.1, 128.1, 127.9, 127.9,
127.8, 127.7, 103.6, 101.7, 100.0, 99.2, 83.4, 80.8, 80.4, 78.9,
78.4, 75.9, 75.8, 75.7, 75.7, 75.5, 75.5, 75.4, 75.3, 74.8, 74.0,
73.9, 73.7, 73.7, 73.4, 73.2, 73.0, 72.9, 72.7, 72.6, 72.3, 72.1,
70.3, 69.7, 69.6, 69.0, 68.8, 68.2, 67.3, 54.2, 41.7, 39.6, 30.6,
30.4, 30.1, 27.9, 27.7, 27.2, 26.6; FAB MS m/z (M+ + Na+) calcd
2086.9736, found 2087.0706.
6-(a m in o)h exyl-(r-D-m a n n op yr a n osyl)-(1f2)-O-(r-D-
m an n opyr an osyl)-(1f2)-O-[(â-D-galactopyr an osyl)-(1f4)]-
r-D-m a n n op yr a n osid e 10. Tetrasaccharide 9 (181 mg, 0.087
mmol) was dissolved in THF (500 µL), and a 1.0 M solution of
NaOH in MeOH (500 µL) was added. The mixture was allowed
to stir at room temperature for 31 h and quenched with sat.
NH4Cl (5 mL). The reaction mixture was extracted with EtOAc
(3 × 5 mL). The combined extracts were washed with brine
(10 mL), dried (Na2SO4), and concentrated. Purification by
flash silica column chromatography (20 f 40% EtOAc/hex-
anes) afforded 6-benzyloxycarbonylamino) hexyl-(3,4,6-tri-O-
benzyl-R-D-mannopyranosyl)-(1f2)-O-(3,4,6-tri-O-benzyl-R-
D-mannopyranosyl)-(1f2)-O-[(2,3,4,6-tetra-O-benzyl-â-D-
galactopyranosyl)-(1f4)]-3,6-di-O-benzyl-R-D-mannopyrano-
side (85.5 mg, 50%) as a clear oil. [R]24D: +23.6° (c 1.0, CH2-
Cl2); IR (thin film) 2923, 2360, 1716, 1454, 1362 cm-1; 1H NMR
(CDCl3) δ 7.36-7.01 (m, 65H), 5.09 (s, 2H), 5.03 (d, J ) 1.8
Hz, 1H), 4.91 (d, J ) 11.0 Hz, 2H), 4.87 (d, J ) 10.1 Hz, 1H),
4.83 (d, J ) 9.8 Hz, 1H), 4.81 (d, J ) 11.3 Hz, 1H), 4.78-4.68
(m, 4H), 4.66-4.58 (m, 4H), 4.56-4.46 (m, 9H), 4.43-4.39 (m,
3H), 4.33 (d, J ) 11.0 Hz, 1H), 4.29-4.24 (m, 4H), 4.19 (d, J
) 11.6 Hz, 1H), 3.96-3.90 (m, 4H), 3.89-3.84 (m, 5H), 3.78-
3.64 (m, 10H), 3.61-3.55 (m, 4H), 3.41 (dd, J ) 2.7, 9.8 Hz,
1H), 3.33-3.25 (m, 4H), 3.14-3.11 (m, 4H), 1.50-1.11 (m,
14H); 13C NMR (CDCl3) δ 157.1, 139.9, 139.7, 139.5, 139.5,
139.3, 139.2, 139.2, 139.0, 138.8, 138.7, 137.4, 129.5, 129.3,
129.3, 129.2, 129.2, 129.2, 129.2, 129.2, 129.1, 129.1, 129.0,
129.0, 129.0, 129.0, 128.9, 128.9, 128.8, 128.7, 128.6, 128.6,
128.6, 128.5, 128.5, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2,
128.1, 128.1, 128.0, 127.9, 127.8, 103.6, 101.7, 99.3, 83.4, 80.8,
80.6, 80.3, 78.5, 78.3, 78.0, 78.0, 77.8, 77.5, 76.0, 75.9, 75.7,
75.7, 75.6, 75.5, 75.3, 75.0, 74.2, 74.0, 73.9, 73.9, 73.7, 73.5,
73.4, 73.2, 73.0, 72.8, 72.8, 72.2, 72.2, 70.3, 69.7, 69.6, 69.2,
69.0, 68.2, 68.0, 67.3, 41.7, 30.6, 30.1, 27.2, 26.6; FAB MS m/z
(M+ + H) 1994.9897, found 1994.9816. 10% Pd/C (50 mg) was
suspended in ethanol (10 mL). The flask was purged with H2,
and a hydrogen balloon was attached. After 20 min, tetrasac-
charide (benzyloxycarbonylamino) hexyl-(3,4,6-tri-O-benzyl-R-
D-mannopyranosyl)-(1f2)-O-(3,4,6-tri-O-benzyl-R-D-mannopy-
ranosyl)-(1f2)-O-[(2,3,4,6-tetra-O-benzyl-â-D-galactopyranosyl)-
(1f4)]-3,6-di-O-benzyl-R-D-mannopyranoside (40 mg, 0.02 mmol)
in EtOAc (1 mL) was added dropwise. After 48 h, the solution
was filtered and concentrated. Lyophilization (H2O) afforded
the leishmania cap tetrasaccharide 10 (9 mg, 53%) as a white
powder. [R]24D: +38.7° (c 0.71, MeOH); IR (thin film) 3383,
1
2928, 1653, 1127, 1063 cm-1; H NMR (D2O) δ 7.41 (s, 1H),
5.29 (d, J ) 1.2 Hz, 1H), 5.09 (d, J ) 1.2 Hz, 1H), 5.01 (d, J )
1.5 Hz, 1H), 4.41 (d, J ) 7.9 Hz, 1H), 4.11 (dd, J ) 1.8, 3.1 Hz,
1H), 4.05-4.04 (m, 1H), 4.00-3.50 (m, 24H), 2.99-2.95 (m,
2H), 1.65-1.60 (m, 5H), 1.38 (br s, 4H); 13C NMR (D2O) δ 103.7,
102.8, 101.2, 98.4, 79.3, 79.1, 77.3, 75.9, 73.8, 73.1, 72.0, 71.6,
70.9, 70.5, 70.5, 69.6, 69.2, 68.6, 67.7, 67.5, 61.8, 61.8, 61.7,
60.9, 40.0, 28.8, 27.2, 26.0, 25.5; FAB MS m/z (M+ + H) calcd
766.3345, found 766.3327.
6-(N-P alm itoyl-S-[2,3-bis(palm itoyloxy)-(2R,2S)-pr opyl]-
L-cysten yl)h exyl-(r-D-m an n opyr an osyl)-(1f2)-O-(r-D-m an -
n op yr a n osyl)-(1f2)-O-[(â-D-ga la ctop yr a n osyl)-(1f4)]-r-
D-m a n n op yr a n osid e 1. Amino-tetrasaccharide 10 (8.6 mg,
11.2 µmol), palmitoyl-cysteinyl ((RS)-2,3-di(palmitoyloxy)-pro-
pyl)-OH 11 (26 mg, 28.5 µmol), and O-(7-azabenzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (8.5
mg, 22.4 µmol) were dissolved in 2.25 mL of CH2Cl2:DMF (2:
1). Diisopropylethylamine (5 µL, 28.5 µmol) was added, and
the resulting yellow solution was allowed to stir at room
temperature for 20 h. The mixture was concentrated in vacuo,
and purified by silica gel column chromatography (2f50%
MeOH/CH2Cl2) to afford 1 (11.2 mg, 60%) as a white powder.
1H NMR (CDCl3/D2O) δ 8.61 (s, 1H), 8.31 (d, J ) 7.9 Hz, 1H),
7.39-7.19 (m, 2H), 5.23-4.73 (m, 4H), 4.18-2.74 (m, 43H),
2.38-2.12 (m, 12H), 1.58-1.42 (m, 13H), 1.26-1.00 (s, 98H),
0.89-0.86 (m, 14H); FAB MS m/z (M+ + Na+) calcd 1680.0513,
found 1680.0468; MALDI-TOF [M + Na]+ 1681.
6-(Am in o)h exyl-(r-D-m a n n op yr a n osyl)-(1f2)-O-(r-D-
m an n opyr an osyl)-(1f2)-O-[(â-D-galactopyr an osyl)-(1f4)]-
r-D-m a n n op yr a n osid e-KLH 2. A solution of N-succidimidyl
bromoacetate (3.5 mg, 15 µmol) in anhydrous DMF (250 µL)
was mixed with a solution of KLH (11.8 mg) in 2 mL of 1 x
PBS, pH 7.4. After 2 h, the reaction mixture was subjected to
gel filtration using a Pd-10 column that was equilibrated with
1 x PBS, pH 7.4. The protein was collected in 3.5 mL eluent.
The solution of bromoacetylated KLH (3.5 mL) thus obtained
was added to the S-acetylmercaptoacetyl-modified carbohy-
drate 12 (1.7 mg, 1.92 µmol) and incubated with 100 mL of
0.2 M hydroxylamine. After 24 h, the remaining bromoacetyl
groups were blocked by the addition of 2-aminoethanethiol (3
mg, 20 µmol). After an additional 24 h, the mixture was diluted
up to 5 mL with 1 x PBS and run on two Pd-10 columns. The
protein was collected in 3.5 mL of eluent (for each column).
Any nonconjugated carbohydrate was removed on a Centriprep
YM-30 filter (Millipore, MWCO 30,000). The concentrated
sugar-KLH conjugate 2 was stored at 4 °C.
p -Met h oxyp h en yl 2-O-Ben zoyl-3,6-d i-O-b en zyl-4-O-
ter t-bu tyld im eth ylsilyl-r-D-m a n n op yr a n osid e 15. Dihy-
droxy mannoside 1433 (1.15 g, 2.47 mmol) was dissolved in
pyridine (20 mL) and cooled to -20 °C. Benzoyl chloride (745
µL, 6.41 mmol) was added, and the mixture was stirred at -20
°C. After 15 min TLC analysis showed complete consumption
of the starting material. Methanol (3 mL) was added, and the
solution was allowed to warm to room temperature and then
diluted with EtOAc. The organic layer was separated and
washed with H2O (2 × 50 mL), sat. NaHCO3 (2 × 50 mL), and
brine (1 × 50 mL), dried (Na2SO4), filtered, and concentrated.
Flash silica column chromatography (20 f 30% EtOAc/
hexanes) gave p-methoxyphenyl 2-O-benzoyl-3,6-di-O-benzyl-
R-D-mannopyranoside as a clear oil (1.13 g, 80%). [R]24
:
D
+11.7° (c 0.29, CH2Cl2); IR (thin film) 1721, 1507, 1452, 1362,
1
1270 cm-1; H NMR (CDCl3) δ 8.08 (dd, J ) 1.2, 8.2 Hz, 2H),
7.58 (t, J ) 7.5 Hz, 1H), 7.42 (t, J ) 7.6 Hz, 2H), 7.42-7.26
(m, 9H), 7.06 (d, J ) 9.2 Hz, 2H), 6.81 (d, J ) 9.2 Hz, 2H),
5.78 (dd, J ) 2.1, 3.1 Hz, 1H), 5.59 (d, J ) 1.8 Hz, 1H), 4.87
(d, J ) 11.3 Hz, 1H), 4.67 (d, J ) 11.9 Hz, 1H), 4.59 (d, J )
11.0 Hz, 1H), 4.56 (d, J ) 11.9 Hz, 1H), 4.26 (td, J ) 1.5, 9.5
Hz, 1H), 4.12 (dd, J ) 3.4, 9.5 Hz, 1H), 4.06-4.02 (m, 1H),
3.87 (dd, J ) 4.6, 10.7 Hz, 1H), 3.81 (dd, J ) 2.7, 10.7 Hz,
1H), 3.78 (s, 3H), 2.60 (d, J ) 2.1 Hz, 1H); 13C NMR (CDCl3)
δ 165.5, 155.0, 149.8, 138.1, 137.5, 133.2, 129.8, 129.4, 128.4,

Synthesis of Potential Leishmaniasis Carbohydrate Vaccine
J . Org. Chem., Vol. 66, No. 12, 2001 4241
128.3, 128.2, 128.0, 127.8, 127.4, 127.3, 117.9, 114.5, 97.2, 77.5,
73.5, 71.9, 69.6, 68.2, 67.1, 55.6; FAB MS m/z (M+ + Na+) calcd
593.2151, found 593.2145.To a solution of p-methoxyphenyl
2-O-benzoyl-3,6-di-O-benzyl-R-D-mannopyranoside (1.56 g, 2.73
mmol) in CH2Cl2 (27 mL) was added 2,6-lutidine (794 µL, 6.82
mmol), followed by the addition of tert-butyldimethylsilyl
trifluoromethanesulfonate (941 µL, 4.10 mmol). After 20 min,
the reaction mixture was diluted with CH2Cl2 (50 mL) and
washed with sat. NaHCO3 (2 × 50 mL) and brine (1 × 50 mL),
dried (Na2SO4), filtered, and concentrated. Flash silica column
chromatography (20 f 30% EtOAc/hexanes) yielded 15 as an
oil (1.79 g, 96%). [R]24D: +30.0° (c 4.08, CH2Cl2); IR (thin film)
(CDCl3) δ 8.05-8.03 (m, 2H), 7.58-7.55 (m, 2H), 7.40-7.19
(m, 14H), 5.89-5.81 (m, 1H), 5.58 (t, J ) 2.1 Hz, 1H), 5.09-
5.01 (m, 2H), 4.96 (s, 1H), 4.78 (d, J ) 11.3 Hz, 1H), 4.68-
4.63 (m, 2H), 4.45 (d, J ) 11.0 Hz, 1H), 4.19-4.15 (m, 1H),
3.87-3.76 (m, 5H), 3.48 (dt, J ) 6.1, 9.5 Hz, 1H), 2.19-2.15
(m, 2H), 1.78-1.72 (m, 2H), 0.82 (s, 9H), 0.0 (s, 3H), -0.06 (s,
3H); 13C NMR (CDCl3) δ 166.5, 139.5, 138.7, 138.6, 133.8,
130.7, 130.5, 129.1, 129.0, 128.7, 128.5, 128.0, 127.9, 115.7,
98.4, 78.9, 77.9, 73.9, 73.7, 71.6, 70.2, 69.3, 68.6, 68.0, 31.0,
29.3, 26.7, 18.9, -3.2, -4.5; FAB MS m/z (M+ + Na+) calcd
669.3218, found 669.3226.
n -P en ten yl 3,4,6-Tr i-O-ben zyl-2-O-p iva loyl-â-D-ga la c-
top yr a n osyl-(1f4)-2-O-ben zoyl-3,6-d i-O-ben zyl-r-D-m a n -
n op yr a n osid e 22. To a solution of 19 (18 mg, 0.028 mmol) in
THF (1 mL) was added a 1.0 M solution of TBAF in THF (56
µL, 0.056 mmol). The resulting orange solution was stirred at
room temperature for 1.5 h and then purified by silica gel
column chromatography (30% EtOAc/hexanes) to afford n-
pentenyl 2-O-benzoyl-3,6-di-O-benzyl-R-D-mannopyranoside (14.4
mg, 97%) as a yellow oil. [R]24D: -21.8° (c 0.19, CH2Cl2); IR
(thin film) 3488, 2920, 1721, 1452, 1269 cm-1; 1H NMR (CDCl3)
δ 8.07-8.05 (m, 2H), 7.58-7.56 (m, 1H), 7.42-7.25 (m, 1H),
5.87-5.79 (m, 1H), 5.60-5.59 (m, 1H), 5.08-4.98 (m, 2H), 4.81
(d, J ) 11.3 Hz, 1H), 4.70 (d, J ) 11.9 Hz, 1H), 4.61 (d, J )
12.2 Hz, 1H), 4.51 (d, J ) 11.0 Hz, 1H), 4.15 (t, J ) 9.2 Hz,
1H), 3.91 (dd, J ) 3.4, 9.5 Hz, 1H), 3.89-3.81 (m, 3H), 3.79-
3.74 (m, 1H), 3.48 (dt, J ) 6.4, 9.8 Hz, 1H), 2.52 (s, 1H), 2.18-
2.13 (m, 2H), 1.76-1.70 (m, 2H); 13C NMR (CDCl3) δ 166.5,
139.0, 138.6, 138.4, 133.9, 130.6, 130.4, 129.2, 129.1, 129.0,
128.8, 128.6, 128.1, 115.8, 98.7, 78.4, 74.3, 72.1, 70.6, 69.1, 68.0,
68.0, 31.0, 29.3; FAB MS m/z (M+ + Na+) calcd 555.2353, found
555.2356. A mixture of galactosyl phosphate 20 (25 mg, 0.034
mmol) and acceptor n-pentenyl 2-O-benzoyl-3,6-di-O-benzyl-
R-D-mannopyranoside (15 mg, 0.029 mmol) were azeotroped
with toluene (3 × 5 mL) and dried under vacuum for 1 h. The
mixture was dissolved in CH2Cl2 (1 mL) and cooled to -78 °C.
Following the addition of TMSOTf (6.3 µL, 0.034 mmol), the
solution was stirred at -78 °C for 40 min and then triethyl-
amine (3 mL) was added. After warming to room temperature,
the reaction mixture was diluted with CH2Cl2 (50 mL), washed
with sat. NaHCO3 (2 × 50 mL) and brine (1 × 50 mL), dried
(Na2SO4), filtered, and concentrated. Flash silica column
chromatography (20 f 30% EtOAc/hexanes) yielded 22 (20 mg,
66%) as an oil. [R]24D: -4.9° (c 0.34, CH2Cl2); IR (thin film)
1
3063, 2855, 1724, 1507, 1360 cm-1; H NMR (CDCl3) δ 8.06-
8.05 (m, 2H), 7.61-7.56 (m, 2H), 7.42-7.21 (m, 14H), 7.10 (dd,
J ) 2.5, 9.1 Hz, 2H), 6.83 (dd, J ) 2.2, 9.1 Hz, 2H), 5.78 (dd,
J ) 2.2, 3.0 Hz, 1H), 5.58 (d, J ) 1.9 Hz, 1H), 4.86 (d, J )
11.0 Hz, 1H), 4.65 (d, J ) 11.8 Hz, 1H), 4.59 (d, J ) 12.1 Hz,
1H), 4.54 (d, J ) 10.3 Hz, 1H), 4.32 (t, J ) 9.3 Hz, 1H), 4.10-
4.02 (m, 2H), 3.91 (dd, J ) 4.7, 10.7 Hz, 1H), 3.85-3.78 (m,
4H), 0.86 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H); 13C NMR (CDCl3)
δ 165.6, 155.1, 150.2, 138.6, 137.7, 133.1, 129.8, 129.8, 128.4,
128.1, 128.0, 127.7, 127.3, 127.2, 127.1, 118.1, 114.5, 97.1, 78.0,
73.4, 73.0, 71.0, 69.3, 68.3, 67.6, 55.5, 25.9, 18.2, -3.9, -5.2;
FAB MS m/z (M+ + Na+) calcd 707.3016, found 707.3029.
2-O-Ben zoyl-3,6-d i-O-b en zyl-4-O-ter t-b u t yld im et h yl-
silyl-r-D-m a n n op yr a n ose Tr ich lor oa cetim id a te 16. To a
solution of mannoside 15 (22.1 mg, 0.032 mmol) in CH3CN/
H2O (1 mL, 4/1) was added cerium ammonium nitrate (53 mg,
0.096 mmol). The resulting clear orange solution was stirred
at room temperature for 10 min and then loaded onto a silica
column. Elution with 30% EtOAc/hexanes gave 2-O-benzoyl-
3,6-di-O-benzyl-4-O-tert-butydimethylsilyl-R-D-mannopyra-
nose as an orange oil (18.4 mg, 99%). [R]24D: -28.6° (c 1.51,
CH2Cl2); IR (thin film) 3421, 3927, 1723, 1452, 1271 cm-1; 1H
NMR (CDCl3) δ 8.03 (dd, J ) 1.2, 8.5 Hz, 1H), 7.59-7.56 (m,
1H), 7.42-7.19 (m, 12H), 5.58 (dd, J ) 2.1, 3.1 Hz, 1H), 5.37
(s, 1H), 4.75 (d, J ) 11.3 Hz, 1H), 4.66 (d, J ) 12.2 Hz, 1H),
4.60 (d, J ) 12.2 Hz, 1H), 4.44 (d, J ) 11.0 Hz, 1H), 4.12-
4.08 (m, 1H), 4.05-4.02 (m, 1H), 3.91 (dd, J ) 3.1, 8.9 Hz,
1H), 3.80 (dd, J ) 1.83, 10.1 Hz, 1H), 3.75-3.71 (m, 1H), 3.16
(s, 1H), 0.79 (s, 9H), -0.04 (s, 3H), -0.07 (s, 3H); 13C NMR
(CDCl3) δ 165.9, 138.3, 133.0, 130.1, 130.0, 128.6, 128.6, 128.2,
128.0, 127.9, 127.8, 127.5, 92.7, 77.9, 77.5, 73.6, 73.0, 71.2, 69.1,
68.5, 26.3, 18.5, -3.5, -4.8; FAB MS m/z (M+ + Na+) calcd
601.2597, found 601.2608.To a solution of 2-O-benzoyl-3,6-di-
O-benzyl-4-O-tert-butydimethylsilyl-R-D-mannopyranose (100
mg, 0.173 mmol) in CH2Cl2 (1 mL) at -20 °C was added Cl3-
CCN (173 µL, 1.73 mmol), followed by DBU (3 µL, 0.017 mmol).
The resulting dark green solution was stirred for 5 min and
then filtered through a pad of silica. The silica was washed
with 40% EtOAc/hexanes (100 mL), and the combined washes
were concentrated to yield 16 as an orange oil (87 mg, 70%).
[R]24D: +7.1° (c 2.51, CH2Cl2); IR (thin film) 2928, 1728, 1674,
1
2924, 2359, 1723, 1454, 1268 cm-1; H NMR (CDCl3) δ 7.98
(d, J ) 8.2 Hz, 2H), 7.48-7.47 (m, 2H), 7.34-7.22 (m, 37H),
7.15-7.11 (m, 5H), 5.84-5.76 (m, 1H), 5.54 (d, J ) 1.8 Hz,
1H), 5.43-5.39 (m, 1H), 5.04-4.96 (m, 2H), 4.93-4.85 (m, 2H),
4.81 (d, J ) 12.2 Hz, 1H), 4.72 (s, 2H), 4.68-4.62 (m, 2H),
4.51 (d, J ) 11.9 Hz, 1H), 4.47 (t, J ) 11.9 Hz, 2H), 4.31-4.24
(m, 3H), 4.05 (dd, J ) 2.7, 9.2 Hz, 1H), 3.91-3.82 (m, 4H),
3.76 (d, J ) 10.4 Hz, 1H), 3.72-3.68 (m, 1H), 3.57-3.54 (m,
1H), 3.47-3.37 (m, 4H), 2.13-2.08 (m, 2H), 1.71-1.65 (m, 2H),
1.15 (s, 9H); 13C NMR (CDCl3) δ 177.5, 166.5, 139.4, 139.3,
139.2, 138.6, 138.5, 133.6, 130.7, 130.5, 129.0, 128.9, 128.8,
128.7, 128.7, 128.5, 128.3, 128.3, 128.2, 128.1, 127.7, 115.7,
100.9, 98.3, 81.9, 77.9, 76.9, 75.1, 74.2, 74.1, 73.0, 72.8, 72.6,
71.9, 70.6, 69.8, 69.1, 68.0, 39.5, 30.9, 29.2, 28.0; FAB MS m/z
(M+ + Na+) calcd 1071.4871, found 1071.4854.
1
1263, 1105 cm-1; H NMR (CDCl3) δ 8.71 (s, 1H), 8.05 (dd, J
) 1.2, 8.2 Hz, 2H), 7.59-7.56 (m, 1H), 7.39-7.21 (m, 14H),
6.40 (d, J ) 2.1 Hz, 1H), 5.71 (dd, J ) 2.1, 3.1 Hz, 1H), 4.77
(d, J ) 11.3 Hz, 1H), 4.67 (d, J ) 11.9 Hz, 1H), 4.61 (d, J )
11.9 Hz, 1H), 4.51 (d, J ) 11.3 Hz, 1H), 4.37-4.33 (m, 1H),
4.00-3.97 (m, 1H), 3.93-3.90 (m, 1H), 3.79-3.77 (m, 1H), 0.84
(s, 9H), 0.04 (s, 3H), -0.01 (s, 3H); 13C NMR (CDCl3) δ 165.3,
159.9, 138.6, 137.3, 133.2, 129.9, 129.5, 128.4, 128.2, 128.1,
127.5, 127.2, 127.1, 95.3, 90.9, 77.5, 76.1, 73.2, 71.2, 68.7, 67.1,
67.0, 26.0, 25.9, 18.2, -3.9, -5.2; FAB MS m/z (M+ + Na+)
calcd 601.2597, found 601.2608.
n -P en ten yl (2-O-Acetyl-3,4,6-tr i-O-ben zyl-r-D-m an n opy-
r a n osyl)-(1f2)-[(3,4,6-t r i-O-b en zyl-2-O-p iva loyl-â-D-ga -
la ctop yr a n osyl)-(1f4)]-3,6-d i-O-ben zyl-r-D-m a n n op yr a -
n osid e 25. To a solution of 22 (23 mg, 0.022 mmol) in 4:1
CH2Cl2:MeOH (1 mL) was added a 0.5 M solution of NaOMe
in MeOH (260 µL, 0.13 mmol). After being stirred at room
temperature for 1 h, the crude mixture was purified by flash
silica column chromatography (20 f 40% EtOAc/hexanes) to
afford n-pentenyl 3,4,6-tri-O-benzyl-2-O-pivaloyl-â-D-galacto-
pyranosyl-(1f4)-3,6-di-O-benzyl-R-D-mannopyranoside (18.8
mg, 90%) as an oil. [R]24D: +35.0° (c 0.22, CH2Cl2); IR (thin
n -P en ten yl 2-O-Ben zoyl-3,6-d i-O-ben zyl-4-O-ter t-bu ty-
d im eth ylsilyl-r-D-m a n n op yr a n osid e 19. To a solution of 16
(77 mg, 0.11 mmol) and 4-penten-1-ol (12 µL, 0.12 mmol) in
CH2Cl2(1 mL) was added TBSOTf (1.3 µL, 0.01 mmol). The
mixture was stirred at room temperature for 1 h, diluted with
CH2Cl2 (50 mL), washed with sat. NaHCO3 (2 × 50 mL) and
brine (1 × 50 mL), dried (Na2SO4), filtered, and concentrated.
Flash silica column chromatography (20 f 30% EtOAc/
hexanes) yielded 19 (18 mg, 25%; 76% based on recovered
desilylated product) as an oil. [R]24D: -38.5° (c 0.13, CH2Cl2);
1
film) 2871, 1740, 1454, 1366, 1067 cm-1; H NMR (CDCl3) δ
7.40-7.18 (m, 30H), 5.83-5.75 (m, 1H), 5.40-5.36 (m, 1H),
5.03-4.96 (m, 2H), 4.95-4.91 (m, 3H), 4.84 (d, J ) 1.2 Hz,
1H), 4.78 (d, J ) 12.2 Hz, 1H), 4.66 (d, J ) 11.9 Hz, 1H), 4.58
(d, J ) 11.0 Hz, 1H), 4.50-4.47 (m, 2H), 4.46-4.42 (m, 2H),
IR (thin film) 2926, 2360, 1724, 1267, 1109 cm-1 1H NMR
;

4242 J . Org. Chem., Vol. 66, No. 12, 2001
Hewitt and Seeberger
4.42 (d, J ) 11.9 Hz, 1H), 4.28 (d, J ) 11.9 Hz, 1H), 4.14 (t, J
) 9.5 Hz, 1H), 3.98-3.93 (m, 2H), 3.80-3.72 (m, 2H), 3.69-
3.64 (m, 3H), 3.61-3.57 (m, 1H), 3.48 (dd, J ) 5.5, 9.2 Hz,
1H), 3.44-3.41 (m, 2H), 3.33 (dd, J ) 2.8, 10.1 Hz, 1H), 2.18-
2.02 (m, 2H), 1.66-1.58 (m, 2H), 1.15 (s, 9H); 13C NMR (CDCl3)
δ 177.1, 138.8, 138.8, 138.2, 138.0, 128.6, 128.6, 128.4, 128.3,
128.3, 128.2, 128.0, 128.0, 128.0, 127.8, 127.8, 127.6, 127.5,
127.3, 100.4, 99.2, 81.2, 78.0, 77.6, 74.7, 73.7, 73.6, 73.3, 72.1,
71.0, 69.7, 67.2, 39.0, 30.5, 28.8, 27.5; FAB MS m/z (M+ + Na+)
calcd 967.4608, found 967.4594. A mixture of mannosyl
trichloroacetimidate 23 (50 mg, 0.078 mmol) and n-pentenyl
3,4,6-tri-O-benzyl-2-O-pivaloyl-â-D-galactopyranosyl-(1f4)-3,6-
di-O-benzyl-R-D-mannopyranoside acceptor (37 mg, 0.039 mmol)
was azeotroped with toluene (3 × 5 mL) and dried under
vacuum for 1 h. The mixture was dissolved in CH2Cl2 (1 mL),
and a 0.5 M solution of TMSOTf in CH2Cl2 (4 µL, 0.002 mmol)
was added. The solution was stirred at room temperature for
5 min and then diluted with CH2Cl2 (50 mL), washed with
sat. NaHCO3 (2 × 50 mL) and brine (1 × 50 mL), dried (Na2-
SO4), filtered, and concentrated. Flash silica column chroma-
tography (10 f 30% EtOAc/hexanes) yielded 25 (37 mg, 67%)
as an oil. [R]24D: +17.7° (c 1.0, CH2Cl2); IR (thin film) 2920,
ceptor (50 mg, 0.036 mmol) was azeotroped with toluene (3 ×
5 mL) and dried under vacuum for 1 h. The mixture was
dissolved in CH2Cl2 (1 mL), and a 0.5 M solution of TMSOTf
in CH2Cl2 (4 µL, 0.002 mmol) was added. The solution was
stirred at room temperature for 5 min and then diluted with
CH2Cl2 (50 mL), washed with sat. NaHCO3 (2 × 50 mL) and
brine (1 × 50 mL), dried (Na2SO4), filtered, and concentrated.
Flash silica column chromatography (10 f 30% EtOAc/
hexanes) yielded 27 (50 mg, 75%) as an oil. [R]24D: +10.8° (c
0.48, CH2Cl2); IR (thin film) 2924, 2361, 1741, 1454, 1365 cm-1
;
1H NMR (CDCl3) δ 7.36-7.08 (m, 64H), 5.78-5.70 (m, 1H),
5.47-5.46 (m, 1H), 5.40 (dd, J ) 7.9, 10.1 Hz, 1H), 5.12 (d, J
) 1.5 Hz, 1H), 4.99-4.84 (m, 5H), 4.81-4.74 (m, 5H), 4.71-
4.59 (m, 6H), 4.57-4.32 (m, 14H), 4.30-4.17 (m, 4H), 4.13-
4.09 (m, 2H), 4.01 (t, J ) 2.1 Hz, 1H), 3.99-3.91 (m, 4H), 3.90-
3.52 (m, 18H), 3.48-3.35 (m, 5H), 3.26-3.21 (m, 1H), 2.09 (s,
3H), 2.01-1.99 (m, 3H), 1.57-1.53 (m, 8H), 1.12 (s, 9H); 13C
NMR (CDCl3) δ 177.6, 170.8, 139.9, 139.4, 139.3, 139.2, 139.1,
139.0, 138.7, 138.6, 138.5, 129.0, 129.0, 128.9, 128.9, 128.8,
128.8, 128.7, 128.7, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4,
128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.7,
115.5, 101.6. 100.9, 100.1, 99.3, 81.8, 80.4, 78.7, 78.2, 77.9, 75.8,
75.7, 75.3, 75.1, 74.9, 74.2, 74.0, 73.9, 73.9, 73.7, 73.3, 73.0,
72.8, 72.5, 72.1, 70.2, 69.7, 69.4, 68.9, 67.7, 39.5, 31.0, 30.4,
29.3, 28.0, 21.9; FAB MS m/z (M+ + Na+) calcd 1873.8588,
found 1873.8537.
1741, 1453, 1368, 1235 cm-1 1H NMR (CDCl3) δ 7.37-7.12
;
(m, 45H), 5.81-5.74 (m, 1H), 5.56 (dd, J ) 1.8, 3.1 Hz, 1H),
5.39 (dd, J ) 8.2, 10.1 Hz, 1H), 5.14 (d, J ) 1.5 Hz, 1H), 5.03-
4.99 (m, 1H), 4.96-4.94 (m, 2H), 4.85 (d, J ) 11.6 Hz, 1H),
4.82-4.78 (m, 3H), 4.69-4.56 (m, 5H), 4.50-4.42 (m, 6H), 4.37
(d, J ) 11.9 Hz, 1H), 4.28 (d, J ) 11.6 Hz, 1H), 4.24 (d, J )
10.7 Hz, 1H), 4.18-4.14 (m, 1H), 4.04-4.03 (m, 1H), 3.95-
3.91 (m, 3H), 3.87 (dd, J ) 3.1, 9.2 Hz, 1H), 3.83-3.76 (m,
3H), 3.72-3.69 (m, 3H), 3.64-3.58 (m, 3H), 3.53 (dd, J ) 4.9,
8.9 Hz, 1H), 3.45-3.41 (m, 2H), 3.32 (dt, J ) 6.4, 9.8 Hz, 1H),
2.09-2.03 (m, 5H), 1.65-1.61 (m, 3H), 1.14 (s, 9H); 13C NMR
(CDCl3) δ 177.6, 170.5, 139.5, 139.4, 139.2, 138.9, 138.8, 138.7,
138.7, 138.6, 129.1, 129.0, 129.0, 129.0, 129.0, 128.9, 128.8,
128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2,
128.2, 128.0, 127.7, 127.7, 127.6, 115.6, 101.2, 100.1, 99.4, 81.8,
79.3, 78.6, 78.0, 75.7, 75.6, 75.0, 74.9, 74.5, 74.1, 74.0, 74.0,
73.7, 73.3, 72.9, 72.6, 72.5, 72.1, 69.8, 69.6, 69.0, 68.8, 67.7,
39.5, 31.0, 29.3, 28.0, 21.8; FAB MS m/z (M+ + Na+) calcd
1441.6645, found 1441.6684.
n -P en ten yl (2-O-Acetyl-3,4,6-tr i-O-ben zyl-r-D-m an n opy-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f2)-[(3,4,6-t r i-O-b en zyl-2-O-p iva loyl-â-D-ga la ct op yr a -
n osyl)-(1f4)]-3,6-d i-O-ben zyl-r-D-m a n n op yr a n osid e 27.
To a solution of 25 (37 mg, 0.026 mmol) in CH2Cl2 (1 mL) was
added a 0.5 M solution of NaOMe in MeOH (78 µL, 0.039
mmol). After stirring at room temperature for 30 min, the
solution was diluted with CH2Cl2 (50 mL), washed with sat.
NaHCO3 (2 × 50 mL) and brine (1 × 50 mL), dried (Na2SO4),
filtered, and concentrated. Flash silica column chromatography
(10 f 30% EtOAc/hexanes) yielded n-pentenyl (3,4,6-tri-O-
benzyl-R-D-mannopyranosyl)-(1f2)-[(3,4,6-tri-O-benzyl-2-O-
pivaloyl-â-D-galactopyranosyl)-(1f4)]-3,6-di-O-benzyl-R-D-man-
nopyranoside (27 mg, 76%) as an oil. [R]24D: +28.2° (c 0.29,
CH2Cl2); IR (thin film) 2918, 2362, 1740, 1454, 1364 cm-1; 1H
NMR (CDCl3) δ 7.36-7.13 (m, 40H), 5.80-5.75 (m, 1H), 5.46-
5.41 (m, 1H), 5.19 (d, J ) 1.8 Hz, 1H), 5.03-4.98 (m, 1H),
4.97-4.92 (m, 3H), 4.85-4.82 (m, 2H), 4.75 (dd, J ) 3.1, 11.0
Hz, 1H), 4.67-4.62 (m, 5H), 4.57-4.43 (m, 6H), 4.40-4.38 (m,
2H), 4.35-4.30 (m, 2H), 4.26 (dd, J ) 3.1, 11.9 Hz, 1H), 4.23-
4.19 (m, 1H), 4.06-4.00 (m, 2H), 3.92-3.79 (m, 5H), 3.77-
3.66 (m, 7H), 3.61-3.50 (m, 2H), 3.48-3.43 (m, 3H), 3.32-
3.27 (m, 1H), 2.20 (s, 1H), 2.07-2.02 (m, 2H), 1.64-1.57 (m,
2H), 1.16 (s, 9H); 13C NMR (CDCl3) δ 177.6, 139.8, 139.4, 139.3,
139.1, 139.0, 138.7, 138.7, 138.5, 129.1, 129.1, 129.0, 128.9,
128.8, 128.7, 128.6, 128.6, 128.5, 128.4, 128.4, 128.4, 128.3,
128.3, 128.2, 128.2, 128.1, 127.8, 115.6, 101.6, 100.9, 99.5, 81.8,
80.8, 78.5, 78.0, 76.1, 75.6, 75.1, 75.1, 74.3, 74.2, 74.2, 74.1,
74.1, 74.0, 73.4, 72.7, 72.5, 72.5, 72.4, 72.3, 69.8, 69.6, 69.4,
68.9, 67.7, 39.5, 31.0, 29.3, 28.0; FAB MS m/z (M+ + Na+) calcd
1399.6540, found 1399.6492. A mixture of 23 (45 mg, 0.072
mmol) and n-pentenyl (3,4,6-tri-O-benzyl-R-D-mannopyranosly)-
(1f2)-[(3,4,6-tri-O-benzyl-2-O-pivaloyl-â-D-galactopyranosyl)-
(1f4)]-3,6-di-O-benzyl-R-D-mannopyranoside trisaccharide ac-
Solid -P h a se Syn th esis of n -P en ten yl (2-O-Acetyl-3,4,6-
tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben -
zyl-r-D-m a n n op yr a n osyl)-(1f2)-[(3,4,6-tr i-O-ben zyl-2-O-
p iva loyl-â-D-ga la ctop yr a n osyl)-(1f4)]-3,6-d i-O-ben zyl-r-
D-m a n n op yr a n osid e 27. Note: Intermediates were analyzed
by removing ca. 2 mg of resin and cleaving from resin using
Grubbs catalyst in an atmosphere of ethylene, followed by TLC.
Compounds made in solution were used as a reference. Our
functionalized resin (17) was prepared in the following man-
ner: Mono-DMT protected octenediol was reacted with Mer-
rifield’s resin (chloromethylated polystyrene cross-linked with
1% divinylbenzene), followed by removal of the DMT group
by dichloroacetic acid. The resin loading could be determined
upon cleavage of the DMT group by a colorimetric assay.38
Octenediol functionalized Merrifield resin32 17 (417 mg, 0.6
mmol/g, 0.25 mmol) was swollen in CH2Cl2 (4 mL) and shaken
for 15 min. Following addition of donor 16 (542 mg, 0.75 mmol,
3 equiv) in CH2Cl2 (4 mL), the flask was shaken for 15 min. A
0.5 M solution of TMSOTf in CH2Cl2 (150 µL, 0.075 mmol) was
added and shaking continued. After 1 h at room temperature,
10 mL of methanol was added, and the resin was washed with
3 × 5 mL of MeOH, 5 mL of 1:1 MeOH:THF, 3 × 5 mL each;
THF and CH2Cl2. The glycosylation procedure was repeated,
followed by identical washing (double glycosylation). The resin
was then dried under vacuum for 12 h. Resin-bound 18 (514
mg, 0.25 mmol) was swollen in THF (8 mL) and shaken for 15
min. Following addition of a 1.0 M solution of TBAF in THF
(2.52 mL, 2.52 mmol), the reaction mixture was shaken for 12
h at room temperature. The resin was then washed with 3 ×
5 mL of MeOH, 5 mL of MeOH/THF (1:1), 3 × 5 mL of THF,
3 × 5 mL of CH2Cl2, and dried under vacuum over P2O5 for 12
h prior to glycosylation. Acceptor functionalized resin (233 mg,
0.11 mmol) was swollen in CH2Cl2 (3 mL) and shaken for 15
min. Following addition of donor 20 (257 mg, 0.34 mmol, 3
equiv) in CH2Cl2 (2 mL), the flask was shaken for 15 min and
cooled to -78 °C. A 0.5 M solution of TMSOTf in CH2Cl2 (686
µL, 0.34 mmol) was added and shaking continued for 1 h. Upon
warming to room temperature, 10 mL of methanol were added,
and the resin was washed with 3 × 5 mL of MeOH, 5 mL of
MeOH/THF (1:1), 3 × 5 mL each; THF and CH2Cl2. The
glycosylation procedure was repeated, followed by identical
washing (double glycosylation). Benzoyl ester protected car-
bohydrate bound resin 21 (282 mg, 0.11 mmol) was swollen
in CH2Cl2 (10 mL) and shaken for 15 min. A 0.5 M solution of
NaOMe in MeOH (1.35 mL, 0.68 mmol) was added and
(38) Pon, R. T. In: Methods in Molecular Biology 20: Protocols for
Oligonucleotides and Analogues; Agrawal, S., Ed.; Humana Press:
Totowa, 1993; p 467.

Synthesis of Potential Leishmaniasis Carbohydrate Vaccine
J . Org. Chem., Vol. 66, No. 12, 2001 4243
shaking continued for 2 h. The resin was then washed with 3
× 5 mL of 20% MeOH/CH2Cl2, 1 × 5 mL of MeOH, 3 × 5 mL
each: 20% MeOH/THF, THF and CH2Cl2 and dried under
vacuum for 12 h. Acceptor-bound resin (239 mg, 0.095 mmol)
was swollen in a solution of donor 23 (182 mg, 0.29 mmol, 3
equiv) in CH2Cl2 (6 mL) and shaken for 15 min. A solution of
0.5 M TMSOTf in CH2Cl2 (58 µL, 0.029 mmol) was added and
the reaction mixture was shaken for 1 h at room temperature.
The resin was washed with 3 × 5 mL of 20% MeOH/CH2Cl2,
1 × 5 mL of MeOH, 3 × 5 mL each: 20% MeOH/THF, THF
and CH2Cl2. The glycosylation and washing were repeated, and
the resin was dried under vacuum for 12 h. Acetate-protected
carbohydrate-bound resin 24 (262 mg, 0.094 mmol) was
swollen in CH2Cl2 (10 mL) and shaken for 15 min. A 0.5 M
solution of NaOMe in MeOH (1.13 mL, 0.57 mmol) was added
and shaking continued for 2 h. The resin was washed with 3
× 5 mL of 20% MeOH/CH2Cl2, 1 × 5 mL of MeOH, 3 × 5 mL
each: 20% MeOH/THF, THF and CH2Cl2 and dried under
vacuum for 12 h. Acceptor bound resin (250 mg, 0.09 mmol)
was swollen in a solution of donor 23 (172 mg, 0.27 mmol, 3
equiv) in CH2Cl2 (6 mL) and shaken for 15 min. A solution of
0.5 M TMSOTf in CH2Cl2 (54 µL, 0.027 mmol) was added, and
the reaction mixture was shaken for 1 h at room temperature.
The resin was washed with 3 × 5 mL of 20% MeOH/CH2Cl2,
1 × 5 mL of MeOH, 3 × 5 mL each: 20% MeOH/THF, THF
and CH2Cl2. The glycosylation and washing was repeated, and
the resin was dried under vacuum for 12 h. Resin-bound
tetrasaccharide 26 (281 mg, 0.09 mmol) was swollen in CH2-
Cl2 (6 mL). Grubbs catalyst (15 mg, 0.018 mmol) was added,
and the reaction mixture was purged with ethylene and stirred
at room temperature for 18 h. Another 10 mol % of the catalyst
(15 mg, 0.018 mmol) was added, and the solution was stirred
for an additional 18 h. The mixture was diluted with CH2Cl2
(100 mL) and passed through a plug of Celite. Following
concentration, the crude material was purified by flash silica
column chromatography (30 f 40% EtOAc/hexanes) to afford
n-pentenyl glycoside 27 (29 mg, 18% overall yield).
Ack n ow led gm en t. Financial support from: the
donors of the Petroleum Research Fund, administered
by the ACS (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.
Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
new compounds is included. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O015521Z