n-Pentyl glycoside methodology in the stereoselective construction of the tetrasaccharyl cap portion of Leishmania lipophosphoglycan

Article abstract of DOI:10.1021/jo9520102

Efficient and high yielding stereoselective assembly of the tetrasaccharyl cap region of the lipophosphoglycan from the protozoan parasite Leishmania using the n-pentenyl glycoside protocol is described in this paper. Both convergent and linear syntheses lead to the protected tetrasaccharide 14; however, the convergent assembly is more efficient in terms of product recovery. Regioselective reductive cleavage of benzylidene acetal 4 with triethylsilane-trifluoroacetic acid system liberates the required C-4 OH in excellent yield, without affecting the resident chloroacetate functionality.

Full text of DOI:10.1021/jo9520102

J . Org. Chem. 1996, 61, 2401-2406
2401
n -P en ten yl Glycosid e Meth od ology in th e Ster eoselective
Con str u ction of th e Tetr a sa cch a r yl Ca p P or tion of Leish m a n ia
Lip op h osp h oglyca n
Ashok Arasappan and Bert Fraser-Reid*
Paul M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708
Received November 14, 1995X
Efficient and high yielding stereoselective assembly of the tetrasaccharyl cap region of the
lipophosphoglycan from the protozoan parasite Leishmania using the n-pentenyl glycoside protocol
is described in this paper. Both convergent and linear syntheses lead to the protected tetrasac-
charide 14; however, the convergent assembly is more efficient in terms of product recovery.
Regioselective reductive cleavage of benzylidene acetal 4 with triethylsilane-trifluoroacetic acid
system liberates the required C-4 OH in excellent yield, without affecting the resident chloroacetate
functionality.
In tr od u ction
Synthetic work in the area of membrane-bound glyco-
conjugates is gaining importance as is evident from
contributions from this5 and other laboratories.6 How-
ever, specific work in the area of Leishmania LPG is
minimal, including the syntheses of phosphoglycan frag-
ments of L. donovani.7 Herein, we report our work
involving the synthesis of the tetrasaccharyl cap moiety
of Leishmania LPG.
The human disease Leishmaniasis is an ancient,
widespread, and poorly understood affliction prevalent
throughout the tropical and subtropical regions of the
world. Protozoan parasites belonging to the genus Leish-
mania, which are the causative agents for this disease,
live within the digestive tract of the sandfly and are
transferred to the host mammal’s bloodstream during
feeding. They then cleverly survive the very mechanism
which is supposed to destroy them and infection of the
host follows.1 There is evidence that cell surface glyco-
conjugates play a key role in mediating the interactions
which protect the parasite in the host’s hydrolytic
environment.2 Insights into the structural assignments
of these and related glycoconjugates have poured forth
in recent years,3 and on the basis of reports in the
literature, a generic structure of the cell surface lipo-
phosphoglycan (LPG) of Leishmania is shown below
(Figure 1).2
Since their advent,8 the n-pentenyl glycosides (NPGs)
have been effectively utilized in synthetic9 and mecha-
nistic10 carbohydrate chemistry. The mild conditions
required for NPG activation tolerate a wide array of
commonly employed protecting groups. The compat-
ibility of the n-pentenyl group with a variety of synthetic
manipulations enables its installation early on in the
synthesis. Coupling reactions are frequently complete
even before their progress can be monitored by TLC. On
the other hand, the reactivity of the pentenyl group can
be erased by dibromination of the olefinic residue.
Restoration of the double bond is readily accomplished
The complex oligosaccharide array may be divided into
three components, namely cap, repeating unit, and
glycosylphosphatidylinositol (GPI) anchor, interlinked by
phosphate residues. Tentative structure-activity rela-
tionships have been assigned for each of the three
components2,3 as follows: the cap is thought to attach
the parasite to the digestive tract of the sandfly and may
also contain the epitope responsible for recognition by the
mammalian host macrophage; the repeating unit is
presumed to form a macromolecular diffusion barrier,
which prevents the binding of host’s antibodies to the
LPG epitopes; the GPI moiety is implicated in many
functions, the most fundamental of which is to anchor
the oligosaccharide (or protein) to the plasma membrane.4
(4) (a) Pimento, P. F. P.; Saraiva, E. M. B.; Sacks, D. L. Exp.
Parasitol. 1991, 72, 191. (b) Tolsen, D. L.; Turco, S. J .; Beecroft, R. P.;
Pearson, T. W. Mol. Biochem. Parasitol. 1989, 35, 109. (c) Chan, B.
L.; Chao, M. V.; Saltiel, A. R. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
1756. (d) Eardley, D. D.; Koshland, M. E. Science 1991, 251, 78. (e)
Saltiel, A. R.; Fox, J . A.; Sherline, P.; Cuatrecasas, P. Science 1986,
233, 967.
(5) (a) Madsen, R.; Udodong, U. E.; Roberts, C.; Mootoo, D. R.;
Konradsson, P.; Fraser-Reid, B. J . Am. Chem. Soc. 1995, 117, 1554
and references cited therein. (b) Campbell, A. S.; Fraser-Reid, B.
BioMed. Chem. 1994, 2, 1209. (c) Campbell, A. S.; Fraser-Reid, B. J .
Am. Chem. Soc. 1995, 117, 10387.
(6) See, for example: (a) Cottaz, S.; Brimacombe, J . S.; Ferguson,
M. A. J . J . Chem. Soc., Perkin Trans. 1 1995, 1673. (b) Cottaz, S.;
Brimacombe, J . S.; Ferguson, M. A. J . Carbohydr. Res. 1995, 270, 85.
(c) Murakata, C.; Ogawa, T. Carbohydr. Res. 1992, 235, 95. (d) Boons,
G.-J .; Grice, P.; Leslie, R.; Ley, S. V.; Yeung, L. L. Tetrahedron Lett.
1993, 34, 9525. (e) Verduyn, R.; Belien, J . J . A.; Dreef-Tromp, C. M.;
van der Marel, G. A.; van Boom, J . H. Tetrahedron Lett. 1991, 32, 6637.
(f) Verduyn, R.; Elie, C. J . J .; Dreef, C. E.; van der Marel, G. A.; van
Boom, J . H. Recl. Trav. Chim. Pays-Bas 1990, 109, 591.
(7) (a) Nikolaev, A. V.; Chudek, J . A.; Ferguson, M. A. J . Carbohydr.
Res. 1995, 272, 179. (b) Nikolaev, A. V.; Rutherford, T. J .; Ferguson,
M. A. J .; Brimacombe, J . S. Bioorg. Med. Chem. Lett. 1994, 4, 785.
(8) Fraser-Reid, B.; Udodong, U. E.; Wu, Z.; Ottosson, H.; Merritt,
J . R.; Rao, C. S.; Roberts, C.; Madsen, R. Synlett 1992, 927 and
references cited therein.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
(1) (a) Turco, S. J . Biochem. Soc., Trans. 1988, 16, 259. (b) Puentes,
S. M.; Sacks, D. L.; da Silva, R. P.; J oiner, K. A. J . Exp. Med. 1988,
167, 887. (c) Gernmaro, R.; Florio, C.; Romeo, D. FEBS Lett. 1985,
180, 185.
(2) McConville, M. J . Cell Bio. Intl. Rep. 1991, 15, 779.
(3) (a) McConville, M. J .; Ferguson, M. A. J . Biochem. J . 1993, 294,
305. (b) Englund, P. T. Annu. Rev. Biochem. 1993, 62, 121. (c) Thomas,
J . R.; Dwek, R. A.; Rademacher, T. W. Biochemistry 1990, 29, 5413.
(d) Cross, G. A. M. Annu. Rev. Cell. Bio. 1990, 6, 1. (e) Low, M. G.
Biochim. Biophys. Acta 1989, 988, 427. (f) Ferguson, M. A. J .; Williams,
A. F. Annu. Rev. Biochem. 1988, 57, 2985. (g) Homans, S. W.; Ferguson,
M. A. J .; Dwek, R. A.; Rademacher, T. W.; Anand, R.; Williams, A. F.
Nature 1988, 333, 269. (h) Ferguson, M. A. J .; Homans, S. W.; Dwek,
R. A.; Rademacher, T. W. Science 1988, 239, 753.
(9) Madsen, R.; Fraser-Reid, B. in Modern Methods in Carbohydrate
Synthesis; Khan, S. H., O’Neill, R. A. Eds.; Harwood Academic
Publishers: Switzerland, 1995; Chapter 4.
(10) (a) Wilson, B. G.; Fraser-Reid, B. J . Org. Chem. 1995, 60, 317.
(b) Rodebaugh, R.; Fraser-Reid, B. J . Am. Chem. Soc. 1994, 116, 3155.
(c) Ratcliffe, A. J .; Mootoo, D. R.; Andrews, C. W.; Fraser-Reid, B. J .
Am. Chem. Soc. 1989, 111, 7661.
0022-3263/96/1961-2401$12.00/0 © 1996 American Chemical Society

2402 J . Org. Chem., Vol. 61, No. 7, 1996
Arasappan and Fraser-Reid
F igu r e 1. Structure of Leishmania lipophosphoglycan.
at the appointed time by reductive debromination using
zinc dust, sodium iodide, or samarium(II) iodide.11 Thus,
a specific n-pentenyl glycoside can be tailored to act as
acceptor or donor by employing the bromination-debro-
mination sequence. Finally, arming or disarming the
sugar entity for reactivity purposes can be achieved easily
by suitable choice of protecting groups. Due to these
properties, the n-pentenyl methodology has found good
use in complex oligosaccharide synthesis.12
3-hydroxyl group via the stannylene acetal13 intermediate
followed by chloroacetylation provided the NPG 4 in 84%
yield (Scheme 2). Although cyanoborohydride-HCl com-
bination14 is widely used for regioselective reductive ring
opening of benzylidene acetals, we decided to test the
triethylsilane-trifluroacetic acid combination described
in a recent report.15 Thus, treatment of the benzylidene
acetal 4 with 5 equiv of triethylsilane-trifluroacetic acid
at 0 °C followed by slow warming to room temperature
afforded the desired C-4 alcohol 5 in excellent yield. The
survival of the C-2 chloroacetate functionality under these
conditions is worthy of special note. In the final step
toward the key acceptor 6, the reactivity of the pentenyl
double bond was obliterated by dibromination using
standard conditions.8
Initial attempts to obtain disaccharide 9 via Koenigs-
Knorr coupling of the acceptor 6 and acetobromogalactose
7 under the agency of silver triflate16 were unsatisfactory.
However upon changing the donor to n-pentenyl galac-
toside 8 and employing the standard NPG conditions (1.3
equiv of NIS and 0.3 equiv of TESOTf), the coupling went
smoothly and rapidly at room temperature in dichlo-
romethane to provide the â-linked disaccharide 9 in a
very high yield (Scheme 3). Removal of the chloroacetyl
group using thiourea in refluxing ethanol resulted in the
acceptor 10, the common intermediate for the convergent
and linear syntheses.
Retr osyn th etic An a lysis
Two pathways can be envisioned to the tetrasaccharyl
cap moiety of Leishmania LPG. Path a leads to disac-
charides I and II (Scheme 1), each of which in turn can
be obtained from the monosaccharides III-VI. By ap-
plying the bromination-debromination protocol, both III
and IV could arise from the same precursor 11 (see
below), while V and VI should be available by existing
methodologies. The alternative, path b, is linear and
leads to the monosaccharide III and the trisaccharide
VII. Further disconnection of the latter gives rise to the
previously described saccharides, II and III. The pro-
tected tetrasaccharide has been constructed via both
synthetic routes, paths a and b, as described in this
paper.
The dimannan donor required for the convergent
Resu lts a n d Discu ssion
(13) (a) David, S.; Hanessian, S. Tetrahedron 1985, 41, 643. (b)
Wagner, D.; Verheyden, J . P. H.; Moffatt, J . G. J . Org. Chem. 1974,
39, 24.
Starting from D-mannose, 1, the known diol 28 was
prepared in two steps. Regioselective benzylation of the
(14) (a) Garegg, P. J . Acc. Chem. Res. 1992, 25, 575. (b) Garegg, P.
J .; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108, 97.
(15) DeNinno, M. P.; Etienne, J . B.; Duplantier, K. C. Tetrahedron
Lett. 1995, 36, 669.
(16) (a) Hanessian, S.; Banoub, J . Carbohydr. Res. 1977, 53, C13.
(b) Arcamone, F.; Penco, S.; Redaelli, S.; Hanessian, S. J . Med. Chem.
1976, 19, 1424.
(11) Merritt, J . R.; Debenham, J .; Fraser-Reid, B. J . Carbohydr.
Chem., in press.
(12) (a) Merritt, J . R.; Naisang, E.; Fraser-Reid, B. J . Org. Chem.
1994, 59, 4443. (b) Udodong, U. E.; Rao, C. S.; Fraser-Reid, B.
Tetrahedron 1992, 48, 4713. See also ref 5a.

Tetrasaccharyl Cap Portion of Leishmania LPG
J . Org. Chem., Vol. 61, No. 7, 1996 2403
Sch em e 1
Sch em e 2a
Sch em e 3a
a
Key: (i) 7, AgOTf, CH2Cl2, 4 Å MS, 1 h (<10% of 9); (ii) 8,
NIS, TESOTf, CH2Cl2 (87% of 9); (iii) Thiourea, NaHCO3, EtOH/
EtOAc, reflux, 2 h.
after column chromatography. The structure was con-
firmed by NMR (1H and 13C), mass and combustion
analyses.
a
Key: (i) (a) Bu2SnO, MeOH, reflux, 1 h; (b) BnBr, DMF, 80-
110 °C, 2 h; (ii) (ClCH2CO)2O, pyr, CH2Cl2, 0-10 °C, 30 min; (iii)
Et3SiH, CF3CO2H, CH2Cl2, 0-20 °C, 2 h; (iv) Et4NBr, Br2, CH2Cl2,
0 °C.
For the linear synthesis, a mannan donor with a
suitable protecting group at C-2 was required. Chloro-
acetate was the obvious choice, and the required donor
15 was obtained from the corresponding acetate 1112a in
two simple steps, namely deacylation with potassium
carbonate/methanol and reprotection with chloroacetic
anhydride and pyridine (Scheme 6). Coupling of the
donor 15 to the disaccharide acceptor 10 was carried out
as described above. The reaction took place smoothly to
afford the trisaccharide 16 in 63% yield (81% based on
recovered acceptor) after purification. Deblocking of
chloroacetate was accomplished using thiourea and so-
dium bicarbonate in refluxing ethanol. The final coupling
was carried out with the trisaccharide acceptor 17 and
mannan donor 11 to provide the previously described
tetrasaccharide 14. However, in this case, TLC analysis
of the crude reaction mixture showed the product and
synthesis was prepared from the known dibromo precur-
sor 12.12a Reductive debromination of 12 was carried out
under both zinc dust and sodium iodide conditions
(Scheme 4).11 Though both methods afforded donor 13,
sonication of dibromo compound 12 with freshly activated
zinc dust seemed to work better in terms of product
recovery.
Coupling of the dimannan donor 13 to the disaccharide
acceptor 10 was next investigated under standard NPG
coupling conditions. Slow addition of the donor to a
mixture of the acceptor 10, NIS, and catalytic TESOTf
in dichloromethane at room temperature under argon
atmosphere resulted in instant formation of the tetrasac-
charide 14 (Scheme 5), which was isolated in a high yield

2404 J . Org. Chem., Vol. 61, No. 7, 1996
Arasappan and Fraser-Reid
Sch em e 4a
construction of the core GPI anchor17 and will be reported
in due course.
Exp er im en ta l Section
Gen er a l Met h od s. All reactions were performed under
argon in oven-dried glassware. Dichloromethane was distilled
from calcium hydride. NIS was recrystallized from CH2Cl2/
hexanes. All other solvents and reagents were used as
purchased. The carbohydrate derivatives were taken up in a
small quantity of toluene and placed under vacuum overnight
prior to use. Compounds were visualized on the TLC plate
by charring with H2SO4/EtOH/H2O (1/10/10). Flash column
chromatography was carried out as described by Still18 with
silica gel 60 (230-400 mesh, Merck). Unless stated otherwise,
1H and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, using CDCl3. Chemical shifts are reported in
ppm relative to residual solvent resonance.
J values are
reported in Hz. Spectral assignments were done using COSY
and HETCOR experiments. FAB mass spectral data were
obtained on a J eol J MS-SX-102 high-resolution mass spec-
trometer. Elemental analyses were performed by Atlantic
Microlab, Inc.
a
Key: (i) NaI, MEK, reflux, 4 h (73%); (ii) n-Bu4NI, Zn dust,
EtOH/EtOAc, sonication, 17 h (86%).
Sch em e 5
Gen er a l Cou p lin g P r oced u r e. To a solution (0.1 M) of
the glycosyl acceptor (1.0 equiv) in dry dichloromethane was
added N-iodosuccinimide (NIS, 1.3 equiv) followed by trieth-
ylsilyl trifluoromethanesulfonate (TESOTf, 0.3 equiv). The
glycosyl donor was dissolved in dichloromethane (0.4 M
solution) which was added dropwise to the reaction mixture
at room temperature. The mixture was stirred until it turned
into a pale pink homogeneous solution (2-3 min). The
reaction was quenched with 10% aqueous sodium thiosulfate
and saturated aqueous sodium bicarbonate solutions. The
organic layer was separated and the aqueous layer washed
with CH2Cl2 once. The combined organic layer was dried over
sodium sulfate, and solvent was stripped off in vacuo. The
crude residue was then subjected to flash column chromatog-
raphy.
Sch em e 6a
P en ten yl 3-O-Ben zyl-4,6-O-ben zylid en e-r-D-m a n n op y-
r a n osid e (3). A mixture of the n-pentenyl mannoside 2 (2 g,
5.95 mmol, 1.0 equiv) and dibutyltin oxide (1.5 g, 5.95 mmol,
1.0 equiv) was refluxed in methanol (100 mL) until the solution
turned completely homogeneous (∼1 h). The solvent was then
removed by rotary evaporation. The oily residue was taken
up in DMF (20 mL), and benzyl bromide (1.0 mL, 8.33 mmol,
1.4 equiv) was added. The reaction mixture was heated to 80-
110 °C for 2 h when TLC analysis indicated complete disap-
pearance of the starting material. DMF was removed in vacuo,
and the residue was directly subjected to column chromatog-
raphy using 25/75 EtOAc/petroleum ether. The product 3 (1.5
g, 59%) was isolated as a colorless oil: 1H NMR δ 1.79-1.88
(m, 2H), 2.24-2.31 (m, 2H), 3.11 (br s, 1H, OH), 3.52-3.59
(m, 1H, OCH2), 3.80-3.88 (m, 1H, OCH2), 3.94-4.00 (m, 2H,
H-5, H-6), 4.07 (dd, 1H, H-3), 4.14-4.15 (m, 1H, H-2), 4.27
(ap t, 1H, H-4), 4.38-4.45 (m, 1H, H-6), 4.94 (ABq, 2H, ArCH2),
4.96 (s, 1H, H-1), 5.13-5.23 (m, 2H, dCH2), 5.77 (s, 1H, ArCH),
5.89-6.03 (m, 1H, CHd), 7.37-7.68 (m, 10H, ArH); 13C NMR
δ 28.40, 30.11, 63.24 (C-5), 66.97 (OCH2), 68.75 (C-6), 69.86
(C-2), 72.99 (ArCH2), 75.72 (C-3), 78.81 (C-4), 99.95 (C-1),
101.37 (ArCH), 114.95 (dCH2), 125.92-128.32 (ArC’s), 137.45
(ipso C), 137.74 (CHd), 137.92 (ipso C); HRMS (FAB) Calcd
for C25H30O6 427.2121 (M + H)+, found 427.2134.
P en ten yl 2-O-(Ch lor oa cetyl)-3-O-ben zyl-4,6-O-ben zyl-
id en e-r-D-m a n n op yr a n osid e (4). To the mannoside 3 (1.175
g, 2.76 mmol, 1.0 equiv) in dichloromethane (14 mL) at 0 °C
was added pyridine (0.45 mL, 5.52 mmol, 2.0 equiv) followed
by chloroacetic anhydride (0.472 g, 2.76 mmol, 1.0 equiv) in
one bulk. The reaction mixture was slowly warmed to 10 °C
over 30 min at which time TLC indicated reaction completion.
The reaction was quenched by addition of crushed ice and
water. The organic layer was separated and dried over sodium
sulfate. The solvent was removed, and the residue was
a
Key: (i) (a) K2CO3, MeOH, 4 h; (b) (ClCH2CO)2O, pyr, CH2Cl2,
0 °C to RT, 30 min; (ii) NIS, TESOTf, CH2Cl2; (iii) Thiourea,
NaHCO3, EtOH/EtOAc, reflux, 7 h.
several other unidentified compounds. After repeated
column chromatography the tetrasaccharide was isolated
in a modest yield of 35%.
In conclusion, the tetrasaccharyl cap portion of Leish-
mania LPG was assembled in an efficient manner using
the NPG protocol. Although both the convergent and
linear synthetic sequences resulted in the tetrasaccharide
14, the former is the method of choice in terms of product
recovery. The triethylsilane-trifluroacetic acid system
provided the required regioselective reductive ben-
zylidene acetal cleavage in an excellent yield, the C-2
chloroacetate functionality being tolerated by the reaction
conditions. Work is already in progress towards the
(17) Arasappan, A.; Fraser-Reid, B. Tetrahedron Lett. 1995, 36,
7967.
(18) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

Tetrasaccharyl Cap Portion of Leishmania LPG
J . Org. Chem., Vol. 61, No. 7, 1996 2405
chromatographed on silica gel using 15/85 EtOAc/petroleum
ether. The product 4 (1.164 g, 84%) was isolated as a colorless
oil: 1H NMR δ 1.79-1.88 (m, 2H), 2.24-2.30 (m, 2H), 3.53-
3.60 (m, 1H, OCH2), 3.80-3.87 (m, 1H, OCH2), 3.94-4.02 (m,
2H, H-5, H-6), 4.13-4.22 (m, 2H, H-3, H-4), 4.31 (s, 2H, CH2-
Cl), 4.39-4.43 (m, 1H, H-6), 4.84 (ABq, 2H, ArCH2), 4.94 (d,
1H, J ) 1.5, H-1), 5.11-5.22 (m, 2H, dCH2), 5.58 (dd, 1H, H-2),
5.77 (s, 1H, ArCH), 5.87-5.98 (m, 1H, CHd), 7.37-7.67 (m,
10H, ArH); 13C NMR δ 28.30, 30.06, 40.75 (CH2Cl), 63.71 (C-
5), 67.39 (OCH2), 68.57 (C-6), 71.57 (C-2), 72.30 (ArCH2), 73.71
(C-3), 78.19 (C-4), 98.23 (C-1), 101.46 (ArCH), 115.13 (dCH2),
125.93-128.86 (ArC’s), 137.24 (ipso C), 137.59 (CHd), 137.67
(ipso C), 166.64. Anal. Calcd for C27H31O7Cl: C, 64.47; H,
6.21. Found: C, 64.41; H, 6.29.
P en ten yl 2-O-(Ch lor oa cetyl)-3,6-d i-O-ben zyl-r-D-m a n -
n op yr a n osid e (5). To the mannoside 4 (0.13 g, 0.26 mmol,
1.0 equiv) in dichloromethane (2 mL) at 0 °C was added
triethylsilane (0.21 mL, 1.3 mmol, 5.0 equiv). Trifluoroacetic
acid (0.1 mL, 1.3 mmol, 5.0 equiv) was then added dropwise
over a period of 2 min. The reaction mixture was slowly
brought to 20 °C over 2 h. Careful monitoring by TLC
indicated complete disappearance of the starting material at
this time. The reaction was diluted with CH2Cl2 (10 mL) and
quenched with saturated aqueous bicarbonate solution. The
organic layer was separated and dried over sodium sulfate and
the solvent removed in vacuo. Flash chromatography of the
crude residue using 20/80 EtOAc/hexanes afforded the product
5 (0.11 g, 84%) as a colorless thick oil: 1H NMR δ 1.76-1.86
(m, 2H), 2.20-2.27 (m, 2H), 2.62 (br s, 1H, OH), 3.52-3.59
(m, 1H, OCH2), 3.80-3.87 (m, 4H, H-5, H-6, OCH2), 3.93 (dd,
1H, H-3), 4.01-4.07 (m, 1H, H-4), 4.23 (s, 2H, CH2Cl), 4.72 (2
× ABq, 4H, ArCH2), 4.97 (d, 1H, J ) 1.5, H-1), 5.08-5.18 (m,
2H, dCH2), 5.51 (dd, 1H, H-2), 5.85-5.99 (m, 1H, CHd), 7.37-
7.46 (m, 10, ArH); 13C NMR δ 28.37, 30.12, 40.73 (CH2Cl),
67.04 (C-4), 67.24 (OCH2), 69.51 (C-6), 69.85 (C-2), 71.12 (C-
5), 71.76 (ArCH2), 73.42 (ArCH2), 77.42 (C-3), 97.40 (C-1),
115.01 (dCH2), 127.40-128.45 (ArC’s), 137.34 (ipso C), 137.71
(CHd), 138.00 (ipso C), 166.79. Anal. Calcd for C27H33O7Cl:
C, 64.21; H, 6.59. Found: C, 64.09; H, 6.63.
73.60 (ArCH2), 74.25, 75.81, 97.20 (C-1a), 100.53 (C-1b),
127.05-128.53 (ArC’s), 137.96 (ipso C), 138.01 (ipso C), 166.61,
169.25, 169.95, 170.00, 170.06. Anal. Calcd for C41H51O16Br2-
Cl: C, 49.49; H, 5.17. Found: C, 49.57; H, 5.24.
Dibr om op en ta n yl O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la c-
t op yr a n osyl)-(1f4)-3,6-d i-O-b en zyl-r-D-m a n n op yr a n o-
sid e (10). The disaccharide 9 (1.012 g, 1.02 mmol, 1.0 equiv)
was taken up in EtOH (22 mL), and a minimum amount of
EtOAc (3 mL) was added to effect dissolution. Sodium
bicarbonate (0.171 g, 2.04 mmol, 2.0 equiv) and thiourea (0.093
g, 1.22 mmol, 1.2 equiv) were added, and the mixture was
refluxed for 2 h. The solvent was then removed by rotary
evaporation and the residue taken in dichloromethane. The
dichloromethane layer was washed with water once and dried
over sodium sulfate, and solvent was removed. Flash column
chromatography of the crude product using 40/60 EtOAc/
petroleum ether resulted in 0.802 g (86%) of the disaccharide
acceptor 10: 1H NMR δ 1.79-2.02 (m, 3H), 2.04 (s, 3H), 2.06
(s, 3H), 2.09 (s, 3H), 2.19 (s, 3H), 2.22-2.35 (m, 1H), 2.73 (br,
1H, OH), 3.54-3.59 (m, 1H, OCH2), 3.68-3.87 (m, 7H), 3.92-
3.98 (ddd, 1H, H-2a), 4.01-4.30 (m, 5H), 4.55-4.92 (m, 6H,
H-1b, H-3b, 2 × ArCH2), 4.95 (s, 1H, H-1a), 5.21 (dd, 1H, H-2b),
5.39 (d, 1H, H-4b), 7.46-7.49 (m, 10 H, ArH); 13C NMR δ 20.50,
20.53, 20.58, 20.65, 26.67, 32.78, 36.05 (CH2Br), 52.30 (CHBr),
60.72, 66.63 (OCH2), 66.77 (C-4b), 67.91, 68.81 (C-2a), 69.47
(C-2b), 70.30, 70.77 (C-3b), 70.94, 72.42 (ArCH2), 73.56 (ArCH2),
74.12, 77.76, 99.07 (C-1a), 100.48 (C-1b), 127.18-128.38 (ArC’s),
137.96 (ipso C), 138.17 (ipso C), 169.23, 169.95, 170.09. Anal.
Calcd for C39H50O15Br2: C, 50.99; H, 5.49. Found: C, 51.10;
H, 5.44.
P en ten yl O-(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 osid e
(13) via Zn Du st Meth od . To the dibromide 12 (0.077 g,
0.067 mmol, 1.0 equiv) was added EtOH (2 mL) and drops of
EtOAc to effect dissolution. Tetra-n-butylammonium iodide
(0.025 g, 0.067 mmol, 1.0 equiv) and freshly activated Zn dust
(0.022 g, 0.33 mmol, 5.0 equiv) were added, and the reaction
mixture was sonicated overnight (17 h) under argon atmo-
sphere. The reaction mixture was diluted with EtOAc and
filtered through a pad of Celite. The solvent was removed in
vacuo, and the residue was subjected to flash chromatography
using 25/75 EtOAc/petroleum ether to afford 0.57 g (86%) of
the donor 13. Via Na I m eth od . To a solution of the
dibromide 12 (0.05 g, 0.043 mmol, 1.0 equiv) in methyl ethyl
ketone (MEK, 2 mL) was added dry sodium iodide (0.007 g,
0.43 mmol, 10.0 equiv), and the mixture was refluxed for 4 h.
The solvent was removed by rotary evaporation, and the
residue was dissolved in dichloromethane and washed with
10% aqueous sodium thiosulfate solution. The organic phase
was dried over sodium sulfate and the solvent stripped off in
vacuo. Flash column chromatography using 30/70 EtOAc/
petroleum ether provided 0.31 g (73%) of the donor 13: 1H
NMR δ 1.69-1.79 (m, 2H), 2.15-2.22 (m, 2H), 2.27 (s, 3H),
3.39-3.47 (m, 1H, OCH2), 3.72-3.80 (m, 1H, OCH2), 3.83-
4.16 (m, 11H), 4.53-4.88 (m, 10H, 5 × ArCH2), 4.98-5.02 (m,
3H, H-1c, ArCH2), 5.06-5.17 (m, 2H, dCH2), 5.24 (d, 1H, J )
1.4, H-1d), 5.70 (dd, 1H, H-2d), 5.84-6.15 (m, 1H, CHd), 7.41-
7.49 (m, 30H, ArH); 13C NMR δ 21.12, 28.56, 30.21, 66.92
(OCH2), 68.63 (C-2d), 98.61 (C-1c), 99.49 (C-1d), 114.82 (dCH2),
170.10; HRMS (FAB) calcd for C61H68O12 991.4633 (M - H)+,
found 991.4584. Anal. Calcd: C, 73.77; H, 6.90. Found: C,
73.58; H, 6.92.
Dibr om op en ta n yl 2-O-(Ch lor oa cetyl)-3,6-d i-O-ben zyl-
r-D-m a n n op yr a n osid e (6). The pentenyl mannoside 5 (0.71
g, 1.41 mmol, 1.0 equiv) was dissolved in dichloromethane (8
mL), and tetraethylammonium bromide (0.15 g, 0.71 mmol,
0.5 equiv) was added in one portion. Bromine (73 µL, 1.41
mmol, 1.0 equiv) was then added dropwise, and when the
brown color persisted, the reaction was quenched with 10%
aqueous sodium thiosulfate solution. The organic phase was
separated and dried over sodium sulfate and the solvent
removed by rotary evaporation. Silica gel chromatography
provided the dibromide (0.8 g, 86%) as a thick oil: 1H NMR δ
1.82-2.04 (m, 3H), 2.32-2.41 (m, 1H), 2.60 (br s, 1H, OH),
3.58-3.63 (m, 1H, OCH2), 3.72 (ap t, 1H, CH2Br), 3.84-4.03
(m, 7H, H-3 to H-6, OCH2, CH2Br), 4.23 (s, 2H, CH2Cl), 4.24-
4.30 (m, 1H, CHBr), 4.72 (2 × ABq, 4H, ArCH2), 4.98 (d, 1H,
J ) 1.6, H-1), 5.50 (ap d, 1H, H-2), 7.37-7.46 (m, 10H, ArH);
13C NMR δ 26.52, 32.63, 35.93 (CH2Br), 40.67 (CH2Cl), 52.13
(CHBr), 66.72 (OCH2), 66.87, 69.43 (C-6), 69.72, 71.30, 71.67
(ArCH2), 73.38 (ArCH2), 77.17, 97.38 (C-1), 127.39-128.43
(ArC’s), 137.23 (ipso C), 137.91 (ipso C), 166.70; HRMS (FAB)
calcd for C27H33O7Br2Cl 661.0203 (M - H)+, found 661.0228.
Anal. Calcd: C, 48.78; H, 5.00. Found: C, 48.84; H, 5.07.
Dibr om op en ta n yl O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la c-
top yr a n osyl)-(1f4)-2-O-(ch lor oa cetyl)-3,6-d i-O-ben zyl-r-
D-m a n n op yr a n osid e (9). The acceptor 6 (0.86 g, 1.30 mmol,
1.0 equiv) and donor 8 (0.704 g, 1.69 mmol, 1.3 equiv) were
coupled using the general procedure described above. The
crude product was chromatographed using 25/75 EtOAc/
petroleum ether to provide 1.122 g (87%) of the disaccharide
9: 1H NMR δ 1.75-2.02 (m, 3H), 2.02 (s, 3H), 2.06 (s, 6H),
2.21 (3, 3H), 2.25-2.40 (m, 1H), 3.54-4.28 (m, 15 H), 4.57-
4.90 (5H, H-1b, 2 × ArCH2), 4.91-4.95 (m, 2H, H-1a, H-3b),
5.21 (dd, 1H, H-2b), 5.36 (d, 1H, H-4b), 5.43 (ap t, 1H, H-2a),
7.37-7.46 (m, 10H, ArH); 13C NMR δ 20.53, 20.58, 20.64,
20.75, 26.59, 32.65, 35.94 (CH2Br), 40.70 (CH2Cl), 52.21
(CHBr), 66.67 (C-4b), 67.11 (C-6a b), 67.86 (C-6a b), 69.47
P en ten yl 2-O-(Ch lor oacetyl)-3,4,6-tr i-O-ben zyl-r-D-m an -
n op yr a n osid e (15). The pentenyl mannoside 11 (0.6 g, 1.07
mmol, 1.0 equiv) was dissolved in MeOH (12 mL), and
anhydrous potassium carbonate (0.06 g) was added. The
mixture was stirred at room temperature for 4 h. The solvent
was removed, and dichloromethane was added to the oily
residue. The organic phase was washed with water once and
dried over sodium sulfate and the solvent removed by rotary
evaporation. The residue was dissolved in dichloromethane
(5 mL) and cooled to 0 °C. Pyridine (0.34 mL, 4.28 mmol, 4.0
equiv) was added followed by chloroacetic anhydride (0.366 g,
2.14 mmol, 2.0 equiv) in two portions. The reaction was
warmed to 20 °C over 30 min, diluted with dichloromethane,
and quenched with crushed ice and water. The organic layer
or
or
(C-2b), 70.68 (C-2a), 70.73, 70.92 (C-3b), 71.14, 71.77 (ArCH2),

2406 J . Org. Chem., Vol. 61, No. 7, 1996
Arasappan and Fraser-Reid
was separated and dried over sodium sulfate, and the solvent
was removed in vacuo. Column chromatography using 15/85
EtOAc/petroleum ether resulted in 0.515 g (81%) of 15 as a
colorless thick oil: 1H NMR δ 1.78-1.87 (m, 2H), 2.22-2.29
(m, 2H), 3.55-3.62 (m, 1H, OCH2), 3.82-4.06 (m, 5H, H-4, H-5,
H-6, OCH2), 4.17 (dd, 1H, H-3), 4.31 (s, 2H, CH2Cl), 4.61-
4.98 (m, 6H, 3 × ArCH2), 5.02 (d, 1H, J ) 1.9, H-1), 5.10-
5.22 (m, 2H, dCH2), 5,80 (dd, 1H, H-2), 5.88-5.99 (m, 1H,
CHd), 7.30-7.52 (m, 15H, ArH); 13C NMR δ 28.39, 30.10, 40.87
(CH2Cl), 67.22 (OCH2), 68.59 (C-6), 70.59 (C-2), 71.26 (C-5),
71.93 (ArCH2), 73.32 (ArCH2), 74.03 (C-4), 75.15 (ArCH2), 78.02
(C-3), 97.26 (C-1), 114.98 (dCH2), 127.50-128.27 (ArC’s),
137.60 (ipso C), 137.70 (CHd), 138.03 (ipso C), 138.10 (ipso
C), 166.86; HRMS (FAB) calcd for C34H39O7Cl 593.2306 (M -
H)+, found: 593.2307. Anal. Calcd: C, 68.62; H, 6.60.
Found: C, 68.39; H, 6.64.
Dib r om op en t a n yl O-[2-O-(Ch lor oa cet yl)-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-
a cetyl-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 (16). The acceptor 10 (0.244 g, 0.266
mmol, 1.0 equiv) and donor 15 (0.206 g, 0.346 mmol, 1.3 equiv)
were coupled using the general procedure described above.
Flash chromatography using 20/70 to 50/50 EtOAc/petroleum
ether provided 0.237 g (63%) of the product 16 and 0.056 g of
unreacted acceptor 10. Thus, the yield based on recovered
acceptor was 81%: 1H NMR δ 2.05 (s, 6H), 2.07 (s, 3H), 2.19
(s, 3H), 4.87-4.91 (m, 3H, H-1b, ArCH2), 4.95 (s, 1H, H-1a),
5.03 (dd, 1H, H-3b), 5.08 (s, 1H, H-1c), 5.26 (dd, 1H, H-2b),
5.39 (d, 1H, H-4b), 5.67 (dd, 1H, H-2c); 13C NMR δ 20.56, 20.80,
26.69, 32.76, 36.08 (CH2Br), 40.84 (CH2Cl), 52.34 (CHBr),
60.67, 66.66 (OCH2), 66.73 (C-4b), 68.26, 68.83, 69.59 (C-2b),
70.21 (C-2c), 70.41, 71.06, 71.34, 71.76, 71.97, 72.09, 73.40,
73.58, 74.04, 74.33, 75.04, 75.14, 77.98, 78.16, 98.43 (C-1a),
98.93 (C-1c), 101.06 (C-1b), 126.62-128.30 (ArC’s), 137.62 (ipso
C), 137.94 (ipso C), 138.08 (ipso C), 138.31 (ipso C), 138.40
(ipso C), 166.48, 169.55, 170.03, 170.19; HRMS (FAB) calcd
for C68H79O21Br2Cl 1423.3091 (M - H)+, found 1423.3082.
Anal. Calcd: C, 57.21; H, 5.58. Found: C, 57.18; H, 5.66.
Dibr om op en ta n yl O-(3,4,6-Tr i-O-ben zyl-r-D-m a n n op y-
r a n osyl)-(1f2)-O-[(2,3,4,6-tetr a -O-a cetyl-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 (17).
Dechloroacylation of the trisaccharide 16 (0.192 g, 0.134 mmol)
was carried out using the procedure described for 10. Column
chromatography of the crude residue afforded 0.129 g (71%)
of the trisaccharide acceptor 17 as a foam: 1H NMR δ 2.05 (s,
3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.23 (s, 3H), 4.56-4.93 (m, 11H,
H-1b, 5 × ArCH2), 5.02-5.05 (m, 2H, H-1a, H-3b), 5.14 (s,
1H, H-1c), 5.27 (dd, 1H, H-2b), 5.40 (d, 1H, H-4b); 13C NMR δ
20.54, 20.61, 20.79, 26.72, 32.75, 36.10, 52.41 (CHBr), 60.74,
66.67 (OCH2), 66.77 (C-4b), 68.29, 68.38, 69.20, 69.57 (C-2b),
70.32, 71.04 (C-3b), 71.37, 71.48, 72.03, 73.35, 73.55, 74.35,
74.96, 77.81, 79.96, 98.61 (C-1a), 100.92 (C-1c), 101.01 (C-1b),
126.82-127.79 (ArC’s), 137.86 (ipso C), 138.07 (ipso C), 138.12
(ipso C), 138.45 (ipso C), 138.50 (ipso C), 169.47, 170.06, 170.09,
170.18; HRMS (FAB) calcd for C66H78O20Br2 1349.3531 (M -
H)+, found 1349.3473. Anal. Calcd: C, 58.67; H, 5.82.
Found: C, 58.57; H, 5.78.
Dibr om op en ta n yl O-(2-O-Acetyl-3,4,6-tr i-O-ben 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-tetr a -O-a cetyl-â-D-ga la c-
t o p y r a n o s y l )-(1 f4 )]-3 ,6 -d i -O -b e n z y l -r-D -m a n n o -
p yr a n osid e (14) via Con ver gen t Syn th esis. Coupling of
the disaccharide acceptor 10 (0.235 g, 0.255 mmol, 1.0 equiv)
to the disaccharide donor 13 (0.33 g, 0.332 mmol, 1.3 equiv)
was performed using the general procedure described above.
Standard chromatography of the crude residue using 20/80 to
30/70 EtOAc/petroleum ether provided 0.319 g (69%) of the
tetrasaccharide 14. Via Lin ea r Syn th esis. The trisaccha-
ride acceptor 17 (0.074 g, 0.055 mmol, 1.0 equiv) and mannan
donor 15 (0.04 g, 0.072 mmol, 1.3 equiv) were coupled by the
procedure described above. The crude product was subjected
to flash column chromatography to provide 0.035 g (35%) of
the tetrasaccharide 14: 1H NMR (500 MHz) δ 4.76-4.84 (m,
3H, H-1b), 4.90 (dd, 1H, H-3b), 4.97 (s, 1H, H-1a or c), 4.99 (d,
1H, J ) 1, H-1d), 5.03 (s, 1H, H-1a or c), 5.15 (ap d, 1H, H-2b),
5.26 (d, 1H, H-4b), 5.50 (dd, 1H, H-2d); 13C NMR δ 20.47, 20.54,
20.75, 21.04, 26.69, 32.67, 36.04 (CH2Br), 52.36 (CHBr), 60.30,
66.56, 66.60 (C-4b), 66.67, 68.22, 68.45 (C-2d), 68.57, 69.53 (C-
2b), 70.15, 71.00 (C-3b), 71.10, 71.69, 71.79, 71.92, 72.05, 73.07,
73.15, 73.52, 74.01, 74.58, 74.68, 74.82, 74.94, 75.01, 75.78,
77.82, 79.42, 98.39 (C-1a or c), 99.17 (C-1d), 100.76 (C-1b), 101.03
(C-1a or c), 126.75-128.18 (ArC’s), 137.83, 138.14, 138.20, 138.26,
138.33, 138.39, 138.47, 169.44, 169.83, 169.99, 170.04, 170.11;
HRMS (FAB) calcd for C95H108O26Br2 1821.5417 (M - H)+,
found 1821.5505. Anal. Calcd: C, 62.50; H, 5.96. Found: C,
63.17; H, 5.94.
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (GM 51237) for support of this work.
We thank Tony Ribero for recording the 500 MHz NMR
spectra.
J O9520102