Synthesis of an inositol phosphoglycan fragment found in Leishmania parasites

Article abstract of DOI:10.1016/S0040-4020(00)00239-8

Synthesis of 1 and 2a is described using a block synthetic strategy. Compound 4 was used as precursor for the two mannose derivatives which, coupled together, forms the dimannoside building block. Thioglycoside 7 was coupled to 8 yielding inositol phosphoglycan 9a, which was selectively deprotected and reacted with 2,3,4,6-tetra-O-benzyl-α-D-glucopyranos-1-yl H- phosphonate to form the protected target molecule 12. Deprotection of 12 by acidic deacetalisation/desilylation and subsequent catalytic hydrogenolysis resulted in cleavage of the anomeric phosphodiester to produce 1. Debenzylation with sodium in liquid ammonia followed by acidic deacetalisation/desilylation gave the target compound 2a. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00239-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3969±3975
Synthesis of an Inositol Phosphoglycan Fragment found in
Leishmania Parasites
Katinka Ruda,a,² Jan Lindberg,b Per J. Garegg,a Stefan Oscarsona and Peter Konradssonb,
*
aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
b
È È
Department of Chemistry/IFM, Linkoping University, S-581 83 Linkoping, Sweden
Received 25 October 1999; revised 3 March 2000; accepted 16 March 2000
AbstractÐSynthesis of 1 and 2a is described using a block synthetic strategy. Compound 4 was used as precursor for the two mannose
derivatives which, coupled together, forms the dimannoside building block. Thioglycoside 7 was coupled to 8 yielding inositol phospho-
glycan 9a, which was selectively deprotected and reacted with 2,3,4,6-tetra-O-benzyl-a-d-glucopyranos-1-yl H-phosphonate to form the
protected target molecule 12. Deprotection of 12 by acidic deacetalisation/desilylation and subsequent catalytic hydrogenolysis resulted in
cleavage of the anomeric phosphodiester to produce 1. Debenzylation with sodium in liquid ammonia followed by acidic deacetalisation/
desilylation gave the target compound 2a. q 2000 Published by Elsevier Science Ltd.
Introduction
These structures are supposedly involved in protection of
the parasite against hydrolytic enzymes in the insect midgut
(promastigote phase) and against digestion by lysosomal
enzymes in the mammalian host macrophages. It has also
been suggested that LPGs and GIPLs are involved in
mediating host±parasite interactions.3
In the different development stages in the lifecycle of the
Leishmania parasites, various myo-inositol containing
oligosaccharides are expressed on the cell surface. During
the promastigote stage the cell surface is covered with poly-
disperse lipophosphoglycans (LPGs) and low molecular-
mass glycoinositolphospholipids (GIPLs). In the amastigote
stage, the cell surface of the parasite is covered with GIPLs
and glycosphingolipids. The GIPLs are the major glyco-
lipids synthesized by Leishmania parasites, they coat a
signi®cant proportion of the plasma membrane and are
immunogenic.1
The synthesized lipid free GIPLs, inositolphosphoglycans
(IPGs), will be evaluated for cytokine responses in macro-
phages. In order to understand the biosynthetic pathways of
the GIPLs and their biological role, we have synthesized the
IPG 1 and 2a corresponding to parts of the complete LPG.
The enzymes that will be studied using these compounds are
the putative galactofuranosyltransferase and the alpha 1,3-
and alpha 1,6-galactopyranosyltransferases, which attach
the remaining three galactose residues to complete the
synthesis of the GIPL-core.4
Up to now several GPI-anchors have been synthesized.2 All
characterized GPI-anchors contain the identical ethanola-
mine-phosphate-6Mana1-2Mana1-6Mana1-4GlcNa1-6myo-
inositol backbone. The Leishmania LPGs, in contrast to
the GPI-anchors, all contain a Gala1-6Gala1-3Galfb1-
3Mana1-3Mana1-4GlcNa1-core hexasaccharide linked to
lysoalkylphosphatidylinositol, sharing the common Mana1-
4GlcNa1-6Ins-unit with the GPI-anchors.1 The C6 hydroxyl
of the mannose residue most distal to the glucosamine is
usually substituted with a glucosyl-a-1-phosphate. The
GIPLs of Leishmania closely resembles the LPG core oligo-
saccharide-PI part and some of the GIPLs have been
suggested as biosynthetic precursors to LPGs.
Results and Discussion
Compound 4 (Scheme 1) was obtained by benzylation of 35
and was used as a precursor for both mannose residues.
Reductive benzylidene opening of 4, followed by benzyl-
ation and removal of the silyl protecting group or mono-
chloroacetylation of position 6 gave derivatives 5 and 6a,
respectively. After transformation of 6a to 6b, by bromine
treatment, 6b was coupled to 5 in 80% yield. Previous
glycosylations6 with glycosyl bromide 6b had shown that
the amount of sym-collidine is crucial. Absence of sym-
collidine resulted in deprotection of the silyl group. More
than 0.65 mol equiv. of sym-collidine, relative to silver
tri¯ate, gave lower yields. Disaccharide 7 was activated
by dimethyl(methylthio)sulfonium tri¯uoromethanesulfonate
Keywords: Leishmania parasites; oligosaccharides; inositol phosphoglycan.
* Corresponding author. Tel.: 146-13-28-1728; fax: 146-13-28-1399;
e-mail: petko@ifm.liu.se
²
Current address: Labwell AB, Hamnesplanaden 5, S-753 19 Uppsala,
Sweden.
0040±4020/00/$ - see front matter q 2000 Published by Elsevier Science Ltd.
PII: S0040-4020(00)00239-8

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K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
Figure 1.
Scheme 1.

K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
3971
Scheme 2.
(DMTST) in dichloromethane and coupled with myo-inositol
derivative 87 in 78% yield (!9a). Building blocks 7 and 8
can be used in the synthesis of other core structures found in
Leishmania.1 Inositol phosphoglycan 9a carries the possi-
bility to introduce the glucose phosphate at the 6 position, as
well as the trigalactose unit at the 3 position of the terminal
mannose residue (compare structure 2b, Fig. 1).
The anomerically pure a-d-glucopyranosyl H-phosphonate
derivative8 11 (Scheme 2) was prepared by phosphonyl-
ation9 of 2,3,4,6-tetra-O-benzyl-a-d-glucopyranose 10,
obtained by recrystallization of commercially available
2,3,4,6-tetra-O-benzyl-a/b-d-glucopyranose from ethyl
acetate. The base catalyzed anomerization has been shown
to be slow enough to permit phosponylation without
Scheme 3.

3972
K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
affecting the anomeric con®guration.10 Coupling of 11 and
9b followed by in situ oxidation with I2 in pyridine/water
produced compound 12 in 80% yield. The compound 9b
was synthesized from 9a by removal of the chloroacetyl
group with sodium methoxide in a mixture of methanol
and dichloromethane. In a ®rst attempt to deprotect 12 it
was decided to start the deprotection with the removal of the
acetal and silyl protecting groups with tri¯uoroacetic acid in
chloroform followed by catalytic hydrogenolysis with
Pd(OH)2/C. However, only 1 could be isolated in 72% yield.
(8.55 g, 20 mmol) and benzyl bromide (4.76 mL,
40 mmol) in DMF (80 mL) was added dropwise to a suspen-
sion of NaH (60%, 2.4 mg, 0.06 mmol) in DMF (100 mL).
After 15 min the excess NaH was destroyed with MeOH,
and the reaction mixture was diluted with toluene and
washed with H2O followed by normal workup. FC (light
petroleum (45±65)/EtOAc 19:1) afforded
15 mmol, 75%) as a colorless syrup. Rf 0.38 (light petro-
4 (7.79 g,
leum (45±65)/EtOAc 6:1); [a]Dˆ175 (c 1.2, CHCl3); IR
n
max cm21 2896, 2953, 2927, 2856, 1455, 1382, 1255, 1212,
1098, 1058, 1014; NMR (CDCl3) 1H, d 0.04 (s, 3H), 0.08 (s,
3H), 0.89 (s, 9H), 1.23 (t, 3H, Jˆ7.2 Hz), 2.56 (m, 2H),
3.78±3.84 (m, 2H), 4.09±4.20 (m, 4H), 4.69 (benzylic d,
1H, Jˆ12.1 Hz), 4.86 (benzylic d, 1H, Jˆ11.9 Hz), 5.28 (s,
1H), 5.56 (s, 1H), 7.31±7.47 (m, 10H); 13C, d 24.8, 24.4,
15.0, 18.4, 25.4, 25.9, 64.9, 68.7, 71.0, 74.0, 79.4, 81.2,
84.1, 101.9, 126.3, 127.8, 127.9, 128.0, 128.4, 128.8,
137.7, 138.3; HRMS Calcd for C28H40O5SiS: [M1Na]1
539.2263. Found [M1Na]1 539.2282.
In order to optimize the conditions for the ®nal deprotection,
model compounds 14 and 15 were synthesized. Compound
14 (Scheme 3) was dissolved in chloroform/tri¯uoroacetic
acid 10:1, conditions required for the deprotection of the
silyl and acetal protecting groups.7 After 2 h TLC and
NMR-analysis showed complete solvolysis of the anomeric
phosphate. The rate of hydrolysis for the deprotected
compound 15 was determined in 0.1 M HCl. After 96 h
50% of the disaccharide had been hydrolysed. Deprotection
of compound 8 with sodium in liquid ammonia followed by
acidic hydrolysis of the acetals in 0.1 M HCl, showed after
6 h complete cleavage of the acetals. This pronounced
difference in stability of the protected and the deprotected
model compounds were also found in compounds 12 and 2a.
Reversal of the deprotection order of compound 12
proceeded as expected without any signi®cant hydrolysis
of the anomeric phosphate. Debenzylation with sodium in
liquid ammonia followed by acidic hydrolysis of the silyl
group and the acetals produced 2a in 74% yield. The now
completed synthesis of fragments 1 and 2a will be followed
up with attempts to synthesize the complete Leishmania
core octasaccharide 2b, using a newly constructed galactose
trisaccharide unit and 7 and 8 as key building blocks.
Ethyl 2,4-di-O-benzyl-3-O-t-butyldimethylsilyl-6-O-chloro-
acetyl-1-thio-a-d-mannopyranoside (6a). A mixture of 4
(392 mg, 0.76 mmol), borane trimethylamine complex
Ê
(1.19 g, 15.2 mmol) and 4 A MS (2 g) in CH2Cl2 (25 mL)
and Et2O (5 mL) was cooled to 08C. AlCl3 (405 mg,
3.03 mmol) was added and, after 10 min, the reaction
mixture was ®ltered through a pad of Celite. 1 M HCl
(30 mL) was added to the ®ltrate and the mixture was stirred
for 30 min. The organic phase was then separated and
washed with NaHCO3 (aq). Normal workup and FC
(toluene/EtOAc 9:1) yielded ethyl 2,4-di-O-benzyl-3-O-t-
butyldimethylsilyl-1-thio-a-d-mannopyranoside (338 mg,
0.65 mmol, 86%). Rf 0.24 (toluene/EtOAc 9:1). To a solu-
tion of this compound (3.74 g, 7.20 mmol) in CH2Cl2
(45 mL) and pyridine (3 mL) was added chloroacetyl
chloride (0.86 mL, 11 mmol). After 5 min, the reaction
mixture was washed with 10% CuSO4 (aq) (300 mL) and
NaHCO3 (aq). 6a was quantitatively isolated (4.23 g,
7.20 mmol) as a colorless syrup, after normal workup and
FC (light petroleum (45±65)/EtOAc 18:1!6:1). Rf 0.80
(toluene/EtOAc 9:1); [a]Dˆ1103 (c 1.2, CHCl3); IR
Experimental
General methods
Normal workup means drying the organic phase (Na2SO4),
®ltration, and evaporation of the solvent in vacuo at or
below 308C except for N,N-dimethylformamide when
508C were used. TLC: 0.25 mm precoated silica-gel plates
(MERCK silica-gel 60F254); detection by spraying the plates
with 8% aq. H2SO4 solution followed by heating at ca
2508C. Optical rotations were recorded at room temperature
with a Perkin±Elmer 241 polarimeter. Flash Chromato-
graphy (FC): Silica gel MERCK 60 (0.040±0.063 mm).
1H and 13C NMR spectra were performed on a JEOL
JNM-GSX 270, temperature 308C unless otherwise stated.
Chemical shifts are given in ppm relative to TMS in CDCl3
(dˆ0.00) or acetone in D2O (13C: dˆ31.00, 1H: dˆ2.22) as
internal standards; 31P, phosphoric acid (dˆ0.00) was used
as external standard. pHp in D2O is given as an uncorrected
value calibrated against H2O-buffer solutions. Mass spectra
were recorded on a JEOL SX 102 Mass Spectrometer. IR
spectra were recorded as ®lms on CaF2 plates (syrups) or as
KBr pellets (solids) on a Perkin±Elmer SPECTRUM 1000
FT-IR Spectrometer.
n
max cm21 2955, 2928, 2884, 2857, 1764, 1738, 1454,
1
1259, 1101; NMR (CDCl3) H, d 0.11 (s, 3H), 0.12 (s,
3H), 0.95 (s, 9H), 1.23 (t, 3H, Jˆ7.3 Hz), 2.57 (m, 2H),
3.73 (s, 1H), 3.85±3.90 (m, 3H), 4.06±4.20 (m, 2H),
4.29±4.40 (m, 2H), 4.55, (d, 1H, Jˆ11.4 Hz), 4.65 (d, 1H,
Jˆ11.9 Hz), 4.77 (d, 1H, Jˆ11.9 Hz), 4.89 (d, 1H,
Jˆ11.4 Hz), 5.28 (s, 1H), 7.30±7.37 (m, 10H); 13C, d
24.7, 24.4, 15.0, 18.0, 25.4, 25.9, 40.6, 64.9, 70.4, 72.7,
74.0, 75.0, 75.3, 80.7, 82.4, 127.5, 127.6, 127.7, 127.8,
128.2, 128.3, 138.0, 138.3, 166.8; HRMS Calcd for
C30H43O6ClSiS: [M1Na]1 617.2136. Found [M1Na]1
617.2149.
Ethyl 2,4,6-tri-O-benzyl-1-thio-a-d-mannopyranoside (5).
Benzyl bromide (1.2 mL, 0.010 mmol) and ethyl 2,4-di-O-
benzyl-3-O-t-butyldimethylsilyl-1-thio-a-d-mannopyrano-
side (2.63 g, 5.06 mmol) were dissolved in DMF (50 mL).
The solution was added dropwise to a slurry of NaH (60%,
405 mg, 0.010 mmol) in DMF (50 mL). MeOH was added
after 15 min to destroy the excess of NaH. The mixture was
concentrated and the residue was dissolved in toluene and
washed with H2O. The organic phase was concentrated and
Ethyl 2-O-benzyl-4,6-O-benzylidene-3-O-t-butyldimethyl-
silyl-1-thio-a-d-mannopyranoside (4). A solution of 35

K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
3973
the resulting syrup was dissolved in CHCl3/BF3´Et2O (50:1,
51 mL). The mixture was heated at 458C for 1.5 h and then
neutralised with NaHCO3 (aq). Normal workup and FC (tol-
uene/EtOAc 20:1) afforded 5 (1.896 g, 3.833 mmol, 76%)
as a colorless syrup. Rf 0.44 (toluene/EtOAc 9:1);
[a]Dˆ185 (c 0.99, CHCl3); IR nmax cm21 3546, 3470,
3062, 3029, 2926, 2869, 1496, 1453, 1397, 1351, 1267,
colorless syrup. Rf 0.53 (toluene/EtOAc 4:1); [a]Dˆ149 (c
0.98, CHCl3); IR nmax cm21 2929, 2107, 1762, 1497, 1454,
1372, 1253, 1111, 1050, 1006; NMR (CDCl3) 1H, d 0.05 (s,
3H), 0.06 (s, 3H), 0.83 (s, 3H), 0.89 (s, 3H), 0.91 (s, 9H),
0.98 (s, 3H), 1.10±1.41 (m, 9H), 1.60±2.10 (m, 4H), 3.26
(dd, 1H, Jˆ9.3, 3.7 Hz), 3.43±5.20 (m, 45H), 5.49 (s, 1H),
7.10±7.40 (m, 45H); 13C, d 24.7, 24.3, 10.0, 18.0, 20.3,
20.5, 26.0, 27.0±27.1 (3C), 29.9 40.7, 43.9, 45.2, 48.0, 51.6,
62.9±80.3, 97.3 (JC,Hˆ174.1 Hz), 98.6 (JC,Hˆ170.5 Hz),
100.5 (JC,Hˆ170.5 Hz), 112.5, 119.1, 126.9±129.1, 135.7,
137.8±138.6, 166.9; HRMS Calcd for C108H129O24ClN3PSi:
[M1Cs]1 2078.7216. Found: [M1Cs]1 2078.7258.
1
1207, 1097; NMR (CDCl3) H, d 1.26 (t, 3H, Jˆ7.5 Hz),
2.50±2.72 (m, 2H), 3.68±3.85 (m, 4H), 3.95 (dd, 1H, Jˆ9.1,
3.6 Hz), 4.13 (ddd, 1H, Jˆ9.5, 4.4, 1.8 Hz), 4.48±4.55 (m,
3H), 4.68 (d, 1H, Jˆ12.1 Hz), 4.77 (d, 1H, Jˆ11.7 Hz), 4.85
(d, 1H, Jˆ10.9 Hz), 5.49 (s, 1H), 7.20±7.40 (m, 15H); 13C,
d 14.9, 25.0, 69.1, 71.3, 72.1, 72.3, 73.3, 74.7, 76.8, 79.9,
81.0, 127.4±128.5, 137.6, 138.3, 138.5; HRMS Calcd for
C29H34O5S: [M1Na]1 517.2025. Found [M1Na]1
517.2037.
(2,4-Di-O-benzyl-3-O-t-butyldimethylsilyl-a-d-manno-
pyranosyl)-(1!3)-(2,4,6-tri-O-benzyl-a-d-mannopyra-
nosyl)-(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-a-d-
glucopyranosyl)-(1!6)-1-O-dibenzyloxyphosphoryl-4,5-
O-isopropylidene-2,3-O-(d-1,7,7-trimethyl[2.2.1]bicyclo-
hept-6-ylidene)-d-myo-inositol (9b). 9a (226 mg, 0.116
mmol) was dissolved in CH2Cl2/MeOH (2:1, 30 mL) and
methanolic sodium methoxide (1 M, 0.2 mL) was added.
The reaction mixture was concentrated after 5 min, where-
after FC (toluene/EtOAc 6:1, 0.25% Et3N) afforded 9b
(186 mg, 0.100 mmol, 86%) as a colorless syrup. Rf 0.47
(toluene/EtOAc 4:1); [a]Dˆ140 (c 1.1, CHCl3); IR
Ethyl (2,4-di-O-benzyl-3-O-t-butyldimethylsilyl-6-O-chloro-
acetyl-a-d-mannopyranosyl)-(1!3)-2,4,6-tri-O-benzyl-1-
thio-a-d-mannopyranoside (7). Br2 (20 mL, 0.392 mmol)
was added to a solution of 6a (224 mg, 0.377 mmol) in
Ê
CH2Cl2 (10 mL) containing 4 A MS (1 g). After 10 min
the reaction mixture was concentrated and co-concentrated
twice with toluene (Na-dried). This bromosugar 6b, 5
(149 mg, 0.301 mmol) and collidine (65 mL, 0.489 mmol)
were dissolved in CH2Cl2 (20 mL) and an additional 0.5 g
n
max cm21 2928, 2107, 1497, 1454, 1372, 1256, 1111,
1
Ê
4 A MS was added. The mixture was cooled to 2308C and
1050, 1006; NMR (CDCl3) H, d 0.03 (s, 6H), 0.83 (s,
3H), 0.89 (s, 3H), 0.90 (s, 9H), 0.97 (s, 3H), 1.10±1.42
(m, 9H), 1.60±2.18 (m, 4H), 3.24 (dd, 1H, Jˆ9.6, 3.5 Hz),
3.43±5.20 (m, 43H), 5.39 (s, 1H), 7.10±7.40 (m, 45H); 13C,
d 24.7, 24.3, 10.0, 18.0, 20.3, 20.5, 26.0, 26.9±27.1 (3C),
29.8, 43.9, 45.1, 48.0, 51.5, 62.5±80.2, 97.2, 99.3, 100.4,
112.5, 119.0, 126.6±128.6, 135.7, 138.0±138.5; HRMS
Calcd for C106H128O23N3PSi: [M1Na]1 1892.8343.
Found: [M1Na]1 1892.8344.
silver tri¯uoromethanesulfonate (166 mg, 0.753 mmol) in
Na-dried toluene (5 mL) was added dropwise. After
20 min the reaction was quenched by the addition of Et3N
(1 mL). The mixture was ®ltered through Celite, washed
with NaHCO3 (aq), and subjected to normal workup [FC
(light petroleum (45±65)/EtOAc 18:1!9:1)] to yield 7
(245 mg, 0.240 mmol, 79%) as a colorless syrup. Rf 0.53
(light petroleum (45±65)/EtOAc 4:1); [a]Dˆ176 (c 1.1,
CHCl3); IR nmax cm21 2954, 2928, 2857, 1763, 1497,
1
1455, 1253, 1092; NMR (CDCl3) H, d 0.09 (s, 3H), 0.11
2,3,4,6-Tetra-O-benzyl-a-d-glucopyranos-1-yl H-phos-
phonate, triethylammonium salt (11). Diphenyl phosphite
(0.50 mL, 2.6 mmol) and 2,3,4,6-tetra-O-benzyl-a-d-gluco-
pyranose 10 (202 mg, 0.374 mmol) were dissolved in pyri-
dine. After 20 min Et3N/H2O (1:1, 2 mL) was added and the
mixture was stirred for an additional 15 min. The solvents
were removed and the residue was dissolved in CH2Cl2 and
washed twice with 5% NaHCO3 (aq). Normal workup and
FC (CHCl3/MeOH 20:1 with 0.5% Et3N) then yielded 11
(240 mg, 0.340 mmol, 91%) as a colorless syrup. Rf 0.60
(CHCl3/MeOH 5:1); [a]Dˆ161 (c 1.1, CHCl3); IR
(s, 3H), 0.94 (s, 9H), 1.27 (t, 3H, Jˆ7.3 Hz), 2.62 (m, 2H),
3.58±4.30 (m, 16H), 4.45±4.66 (m, 6H), 4.78 (d, 1H,
Jˆ11.7 Hz), 4.89 (d, 1H, Jˆ11.7 Hz), 5.13 (s, 1H), 5.49
(s, 1H), 7.15±7.45 (m, 25H); 13C, d 24.7, 24.3, 15.0,
18.0, 25.3, 26.0, 40.9, 65.5, 69.1, 70.6, 71.7, 72.2, 73.1,
73.3, 73.4, 74.6, 74.9, 75.4, 75.6, 79.3, 79.5, 79.6, 81.5,
100.4 (JC,Hˆ170.5 Hz), 126.9±128.5, 137.9, 138.2, 138.3,
138.4, 138.5, 167.1; HRMS Calcd for C57H71O11ClSiS:
[M1Na]1 1049.4073. Found [M1Na]1 1049.4093.
(2,4-Di-O-benzyl-3-O-t-butyldimethylsilyl-6-O-chloro-
acetyl-a-d-mannopyranosyl)-(1!3)-(2,4,6-tri-O-benzyl-
a-d-mannopyranosyl)-(1!4)-(2-azido-3,6-di-O-benzyl-
2-deoxy-a-d-glucopyranosyl)-(1!6)-1-O-dibenzyloxy-
phosphoryl-4,5-O-isopropylidene-2,3-O-(d-1,7,7-
trimethyl[2.2.1]bicyclohept-6-ylidene)-d-myo-inositol
(9a). To a mixture of 6-O-(2-azido-3,6-di-O-benzyl-2-
deoxy-a-d-glucopyranosyl)-1-O-dibenzyloxyphosphoryl-4,5-
O-isopropylidene-2,3-O-(d-1,7,7-trimethyl[2.2.1]bicyclohept-
6-ylidene)-d-myo-inositol 87 (150 mg, 0.153 mmol), 7
n
max cm21 3029, 2914, 2866, 2362, 1496, 1453, 1360,
1
1224, 1092, 1071, 929; NMR (CDCl3) H, d 1.18 (t, 9H,
Jˆ7.2 Hz), 2.89 (m, 6H), 3.66±3.73 (m, 4H), 4.02±4.06 (m,
2H), 4.43±4.77 (m, 4H), 4.82±4.96 (m, 4H), 5.89 (dd, 1H,
Jˆ8.8, 2.9 Hz), 7.03 (d, 1H, Jˆ646 Hz), 7.18±7.37 (m,
20H); 13C, d 8.4, 45.3, 68.5, 71.5, 72.3, 73.4, 74.8, 75.5,
77.5, 79.8 (d, Jˆ5.5 Hz), 81.6, 92.4 (d, Jˆ5.5 Hz, JC±
ˆ176 Hz), 127.5±128.3, 138.1, 138.3, 138.6, 138.8; 31P,
H
d 1.0 (d, Jˆ660 Hz); HRMS Calcd for C34H37O8P:
[M1Na]1 627.2124. Found [M1Na]1 627.2109.
Ê
(203 mg, 0.198 mmol) and 4 A MS (1.5 g) in CH2Cl2
(15 mL) under an Ar-atmosphere was added DMTST
(59 mg, 0.229 mmol). After 1 h, Et3N (1 mL) was added.
The reaction mixture was ®ltered through a pad of Celite
and concentrated. FC (toluene/EtOAc 12:1, 0.25% Et3N) of
the residue yielded 78% (232 mg, 0.119 mmol) of 9a as a
[(2,3,4,6-Tetra-O-benzyl-a-d-glucopyranos-1-yl) (2,4-di-
O-benzyl-3-O-t-butyldimethylsilyl-a-d-mannopyranos-
6-yl triethylammonium phosphate)]-(1!3)-(2,4,6-tri-O-
benzyl-a-d-mannopyranosyl)-(1!4)-(2-azido-3,6-di-O-
benzyl-2-deoxy-a-d-glucopyranosyl)-(1!6)-1-O-dibenzyl-

3974
K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
oxyphosphoryl-4,5-O-isopropylidene-2,3-O-(d-1,7,7-
trimethyl[2.2.1]bicyclohept-6-ylidene)-d-myo-inositol
(12). 11 (58 mg, 0.082 mmol) and 9b (102 mg, 0.055 mmol)
were dissolved in CH3CN/pyridine (3:1, 16 mL) and
pivaloyl chloride (33 mL, 0.269 mmol) was added. The
coupling reaction was ready within 5 min according to
TLC, when I2 (28 mg, 0.109 mmol) in pyridine/water
(49:1, 10 mL) was added. After an additional 1 h 15 min,
the reaction was diluted with CHCl3 and washed with 10%
Na2S2O3 (aq). Normal workup and FC (CHCl3!CHCl3/
MeOH 20:1 with 0.25% Et3N) gave 12 (114 mg,
0.044 mmol, 81%) as a colorless syrup. Rf 0.27 (CHCl3/
MeOH 20:1); [a]Dˆ137 (c 1.2, CHCl3); IR nmax cm21
2925, 2107, 1497, 1453, 1351, 1252, 1174, 1110, 1009;
Sephadex G-15 column eluted with H2O containing 1%
n-butanol afforded 2a (11 mg, 0.011 mmol, 74%) as a
white solid. [a]Dˆ1100 (c 1.0, H2O); IR nmax cm21 3418,
2931, 1635, 1402, 1384, 1224, 1090, 1048; NMR (D2O)
1
pHpˆ5.6 H, d 3.32 (dd, 1H, Jˆ10.6, 4.0 Hz), 3.43 (t, 1H,
Jˆ9.3 Hz), 3.49 (t, 1H, Jˆ9.3 Hz), 3.55±4.27 (m, 26H),
4.29 (t, 1H, Jˆ2.2), 5.07 (d, 1H, Jˆ0.7 Hz), 5.30 (d, 1H,
Jˆ1.1 Hz), 5.52 (dd, 1H, Jˆ7.3, 3.6 Hz), 5.71 (d, 1H,
Jˆ4.0 Hz); 13C, d 54.7, 60.8, 61.1, 61.6, 66.1 (d,
Jˆ5.5 Hz), 66.3, 67.4, 69.9, 70.6, 70.7, 71.0, 71.3, 71.4,
72.0, 72.1, 72.6, 73.1 (2C), 73.2, 73.3, 73.6, 74.6, 76.0,
76.4 (d, Jˆ5.6 Hz), 77.5 (d, Jˆ5.6 Hz), 80.0, 95.7, 96.1
(d, Jˆ7.4 Hz), 102.3, 103.6; 31P (decoupled), d 21.11,
1.44; HRMS Calcd for C30H55NO31P2: [M2H]2 986.2155.
Found [M2H]2 986.2200.
1
NMR (CDCl3) H, d 20.07 (s, 3H,), 20.02 (s, 3H,) 0.83
(s, 3H), 0.85 (s, 9H), 0.91 (s, 3H), 0.98 (s, 3H), 1.02 (t, 9H,
Jˆ7.3 Hz), 1.10±1.46 (m, 9H), 1.50±1.75 (m, 2H), 1.90±
2.08 (m, 2H), 2.62 (m, 6H), 3.22±5.18 (m, 58H), 5.50 (s,
1H), 5.94 (dd, 1H, Jˆ7.8, 3.1 Hz), 7.05±7.45 (m, 65H); 13C,
d 24.7, 24.3, 8.4, 10.0, 17.9, 20.2, 20.5, 26.0, 27.0, 27.3
(2C), 29.9, 43.6, 45.2, 47.9, 51.5, 63.6±81.6, 93.1, 97.6,
98.9, 100.7, 112.4, 119.0, 126.9±128.5, 135.6, 135.7,
138.0±139.2; 31P (decoupled), d 21.1, 20.6; HRMS
Calcd for C140H163O31N3P2Si: [M2H12Cs]1 2736.8546.
Found [M2H12Cs]1 2736.8562.
(2,3,4,6-Tetra-O-benzyl-a-d-glucopyranos-1-yl) (methyl
2,3,4-tri-O-benzyl-a-d-mannopyranosid-6-yl) triethyl-
ammonium phosphate (14). 11 (170 mg, 0.24mmol) and
1311 (82 mg, 0.18 mmol) were dissolved in CH3CN/pyridine
(3:1, 12 mL) and pivaloyl chloride (0.15 mL, 1.22 mmol)
was added. A 2% solution of I2 in pyridine/water, 49:1
(7.0 mL, 0.55 mmol) was added to the reaction mixture
after 40 min. After an additional 20 min, the reaction was
diluted with CHCl3 and washed with 10% Na2S2O3 (aq).
Normal workup and FC (CHCl3/MeOH 20:1!10:1,
0.25% Et3N) gave 14 (169 mg, 0.14 mmol, 82%) as a
colorless syrup. Rf 0.32 (CHCl3/MeOH 9:1); [a]Dˆ177
(c 0.76, CHCl3); IR nmax cm21 3030, 2925, 1496, 1453,
1363, 1237, 1098; NMR (CDCl3) 1H, d 1.11 (t, 9H,
Jˆ7.3 Hz), 2.77 (m, 6H), 3.25 (s, 3H), 3.50±92 (m, 8H),
3.95±4.11 (m, 2H), 4.20±4.35 (m, 3H), 4.42±4.94 (m
14H), 5.93 (dd, 1H, Jˆ7.7, 2.9 Hz), 7.05±7.41 (m, 35H);
13C, d 8.4, 45.1, 54.7, 64.6 (d, Jˆ4.9 Hz), 68.5, 71.2, 71.3,
71.5, 71.8, 72.2, 73.0, 73.3, 74.7, 74.9, 75.0, 75.3 (2C), 77.4,
80.3, 81.6, 93.0 (d, Jˆ5.6 Hz), 98.9, 127.3±128.3, 138.3±
139.0; 31P (decoupled), d 21.01; HRMS Calcd for
C62H67O14P: [M1Na]1 1089.4166. Found: [M1Na]1
1089.4164.
(a-d-Mannopyranosyl 6-phosphate)-(1!3)-a-d-manno-
pyranosyl-(1!4)-2-amino-2-deoxy-a-d-glucopyranosyl-
(1!6)-d-myo-inositol 1-phosphate, pyridinium salt (1).
To a stirred solution of 12 (129 mg, 0.050 mmol) in
CHCl3 (10 mL) was added CF3COOH (1 mL). After 4 h
the reaction mixture was neutralized with NaHCO3 (aq).
The organic phase was concentrated and the residue
dissolved in EtOAc/MeOH/H2O (8:2:1, 11 mL). A catalytic
amount of Pd(OH)2 on carbon was added and the mixture
was hydrogenolysed at 120 psi for 48 h (after 24 h H2O
(1 mL) was added). The mixture was ®ltered through Celite
and concentrated. Gel ®ltration of the residue on a Bio-Gel
P2 column eluted with a pyridinium acetate buffer (pH 5)
afforded 1 (30 mg, 0.036 mmol, 72%) as a white solid.
[a]Dˆ188 (c 1.4, H2O); IR nmax cm21 3428, 2926, 1635,
(a-d-Glucopyranos-1-yl) (methyl a-d-mannopyranosid-
6-yl) ammonium phosphate (15). To NH3 (l) (ca. 20 mL) at
2338C was added 14 (54 mg, 0.046 mmol) in THF (2 mL).
To the stirred mixture was added a minimum amount of
sodium for the mixture to turn deep blue. After 1 min
NH4Cl was added until the color disappeared. The mixture
was concentrated and gel ®ltration of the residue on a
Pharmacia Sephadex G-15 column eluted with H2O contain-
ing 1% n-butanol afforded 15 (16 mg, 0.076 mmol, 78%) as
a white solid. [a]Dˆ136 (c 1.4, H2O); IR nmax cm21 3404,
2936, 1634, 1401, 1225, 1135, 1093, 1048, 943, 876; NMR
(D2O) pHpˆ6.3 1H, d 3.41 (s, 3H), 3.48 (t, 1H, Jˆ9.3 Hz),
3.57 (dt, 1H, Jˆ9.9, 2.6 Hz), 3.70±3.93 (m, 7H), 4.08±4.21
(m, 2H), 4.76 (d, 1H, Jˆ1.8 Hz), 5.52 (dd, 1H, Jˆ6.9,
3.6 Hz); 13C, d 55.6, 61.1, 65.3 (d, Jˆ3.7 Hz), 67.0, 69.9,
70.6, 71.2, 72.1, 72.2, 73.3, 73.6, 96.1 (d, Jˆ5.5 Hz), 101.7;
31P (decoupled), d 21.11; HRMS Calcd for C13H25O14P:
[M2H]2 435.0904. Found: [M2H]2 435.0903.
1
1384, 1046; NMR (D2O) pHpˆ5.1 H, d 3.33 (dd, 1H,
Jˆ10.6, 3.7 Hz), 3.44 (t, 1H, Jˆ9.1 Hz), 3.55±4.28 (m,
21H), 4.35 (s, 1H), 5.06 (s, 1H), 5.28 (s, 1H), 5.65 (d, 1H,
Jˆ3.6 Hz); 13C, d 54.8, 60.9, 61.6, 65.5 (d, Jˆ3.7 Hz), 66.3,
67.6, 70.5, 70.7, 71.0, 71.1, 71.3, 71.5, 72.4, 73.0, 73.2,
73.3, 74.7, 76.2, 76.7, (d, 1H, Jˆ7.4 Hz), 77.5 (d, 1H,
Jˆ7.4 Hz), 80.1, 95.8, 102.4, 103.7; 31P (decoupled), d
0.46 (br, 2P); HRMS Calcd for C24H45O26NP2: [M1H]1
826.1784. Found [M1H]1 826.1794.
[(a-d-Glucopyranos-1-yl) (a-d-mannopyranos-6-yl) phos-
phate]-(1!3)-a-d-mannopyranosyl-(1!4)-2-amino-2-
deoxy-a-d-glucopyranosyl-(1!6)-d-myo-inositol 1-phos-
phate, ammonium salt (2a). To NH3 (l) (ca. 20 mL) at
2338C was added 12 (39 mg, 0.015 mmol) in THF
(2 mL). To the stirred mixture was added a minimum
amount of sodium for the mixture to turn deep blue. After
1 min NH4Cl was added until the color disappeared. The
mixture was concentrated and the residue was dissolved in
0.1 M HCl (10 mL). After 6 h the mixture was neutralized
with NH3 (25%, 0.1 mL), washed with ether (10 mL) and
concentrated. Gel ®ltration of the residue on a Pharmacia
Hydrolytic studies of compounds 14 and 15. To 14
(10 mg) in CHCl3 (3 mL) was added TFA (0.3 mL). After
2 h, when TLC-analysis showed only traces of 14, pyridine
(2 mL) was added and the mixture was concentrated. In the

K. Ruda et al. / Tetrahedron 56 (2000) 3969±3975
3975
65. (b) McConville, M. J.; Ferguson, M. A. J. Biochem. J. 1993,
294, 305 and references cited therein.
NMR-spectrum of the residue only traces of the glucose-
phosphate anomeric proton could be detected.
2. (a) Mootoo, D. R.; Konradsson, P.; Fraser-Reid, B. J. Am.
Chem. Soc. 1989, 111, 8540. (b) Verduyn, G. R.; Belien, J. J. A.;
Dreef-Tromp, C. M.; van Marel, G. A.; van Boom, J. H.
Tetrahedron Lett. 1991, 32, 6637. (c) Murakata, C.; Ogawa, T.
Carbohydr. Res. 1992, 235, 95. (d) Cottaz, S.; Brimacombe J. S.;
Ferguson, M. A. J. Chem. Soc., Perkin Trans. 1 1993, 2949.
(e) Udodong, U. E.; Madsen, R.; Roberts, C.; Fraser-Reid, B.
J. Am. Chem. Soc. 1993, 115, 7886. (f) Madsen, R.; Udodong,
U. E.; Roberts, C.; Mootoo, D. R.; Konradsson, P.; Fraser-Reid,
B. J. Am. Chem. Soc. 1995, 117, 1554. (g) Mayer, T. G.; Schmidt,
R. R. Eur. J. Org. Chem. 1999, 1153.
15 (4 mg) was dissolved in 0.1 M HCl (10 mL). After 24 h
half of the material was neutralized. In order to decrease the
salt concentration, Ag2CO3 (100 mg) was used for the
neutralization to precipitate chloride ions. After stirring
for 10 min the mixture was centrifuged and the supernatant
was passed through a DOWEX cation exchange resin
(NH14 -form) to remove traces of Ag1. After lyophilization
the product mixture was quanti®ed by the integrated
intensities of the glucose anomeric protons. After 96 h the
rest of the material was subjected to the same workup pro-
cedures. After 24 h and 96 h 16% and 50% of the anomeric
phosphate had been hydrolyzed.
3. (a) Hatzigeorgiou, D. E.; Geng, J; Zhu, B.; Zhang, Y.; Liu, K.;
Rom, W. N.; Fenton, M. J.; Turco, S. J.; Ho, J. L. Proc. Natl. Acad.
Sci., USA 1996, 93, 14708. (b) Lo, S. K.; Bovis, L.; Matura, R.;
Zhu, B.; He, S.; Lum, H.; Turco, S. J.; Ho, J. L. J. Immunol. 1998,
160, 1857.
Acknowledgements
4. Salvatore Turco. Personal communication.
5. Garegg, P. J.; Olsson, L.; Oscarson, S. J. Carbohydr. Chem.
1993, 12, 955.
This work, which is part of a collaboration with Dr
Salvatore Turco, Department of Biochemistry, University
of Kentucky, Lexington, USA and Dr Hannah Akuffo,
Microbiology and Tumor Biology Centre, Karolinska
Institute, Stockholm, Sweden, was supported by the
Swedish Natural Science Research Council. We also
6. Ruda, K. Doctoral Thesis, Stockholm University, 1998.
7. Garegg, P. J.; Konradsson, P.; Oscarson, S.; Ruda, K.
Tetrahedron 1997, 53, 17727.
8. Hermans, J. P. G.; de Vroom, E.; Elie, C. J. J.; van der Marel,
G. A.; van Boom, J. H. Recl. Trav. Chim. Pays-Bas 1986, 105, 510.
9. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A.
Tetrahedron Lett. 1994, 35, 3355.
È
thank Lars Swanson at AstraZeneca R & D, Molndal,
Sweden for the help with high resolution mass-spectrometry.
10. Bols, M.; Hansen, H. C.; Smith, B. I. Acta Chem. Scand. 1993,
47, 532.
11. Boren, H. B.; Eklind, K.; Garegg, P. J.; Lindberg, B.; Pilotti,
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