Inositolphosphoglycan mediators: An effective synthesis of the conserved linear GPI anchor structure

Article abstract of DOI:10.1081/CAR-200066956

An effective new preparative synthesis of the conserved linear pseudopentasaccharide structure of the GPI anchors and of the full GPI structure has been carried out that has permitted obtaining both molecules in sufficient quantities as to perform further

Full text of DOI:10.1081/CAR-200066956

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Inositolphosphoglycan Mediators: An
Effective Synthesis of the Conserved
Linear GPI Anchor Structure
Javier López‐Prados a & Manuel Martín‐Lomas a
a Grupo de Carbohidratos , Instituto de Investigaciones Químicas,
CSIC, Américo Vespucio, s/n , Sevilla, Spain
Published online: 16 Aug 2006.
To cite this article: Javier López‐Prados & Manuel Martín‐Lomas (2005) Inositolphosphoglycan
Mediators: An Effective Synthesis of the Conserved Linear GPI Anchor Structure , Journal of
Carbohydrate Chemistry, 24:4-6, 393-414
To link to this article: http://dx.doi.org/10.1081/CAR-200066956
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Journal of Carbohydrate Chemistry, 24:393–414, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200066956
Inositolphosphoglycan
Mediators: An Effective
Synthesis of the Conserved
Linear GPI Anchor Structure
´
Javier Lopez-Prados and Manuel Mart´ın-Lomas
´
´
Grupo de Carbohidratos, Instituto de Investigaciones Quımicas, CSIC, Americo
Vespucio, s/n, Sevilla, Spain
An effective new preparative synthesis of the conserved linear pseudopentasaccharide
structure of the GPI anchors and of the full GPI structure has been carried out
that has permitted obtaining both molecules in sufficient quantities as to
perform further structural and biologic studies. The synthesis involves a 3 þ 2
block synthesis strategy in which a conveniently protected Man a(1 ! 4) GlcN3
a(1 ! 6) myo-Ins building block, previously used in the synthesis of inositolphospho-
glycan (IPG) mediators, is glycosylated with
trichloroacetimidate.
a
protected Man a(1 ! 2) Man
Keywords Insositolphosphoglycan mediators, Glycosylphosphatidyl inositol, Oligo-
saccharides synthesis
INTRODUCTION
Inositolphosphoglycans (IPGs) are partially characterized intracellular
mediators generated by enzymatic cleavage of glycosylphosphatidylinositols
(GPIs).[1,2] As a part of an ongoing program we have reported on the synthesis,
the three-dimensional structure, and the biologic activity of myo-inositol
Received January 27, 2005; accepted March 4, 2005.
Dedicated to the memory of Professor Jacques H. van Boom.
´
Address correspondence to Manuel Martın-Lomas, Grupo de Carbohidratos, Instituto
´
´
de Investigaciones Quımicas, CSIC, Americo Vespucio, s/n, Isla de La Cartuja, 41092
Sevilla, Spain. E-mail: mauel.martin-lomas@iiq.csic.es
393

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´
J. Lopez-Prados and M. Martın-Lomas
394
containing pseudooligosaccharides bearing some of the structural motifs that
have been postulated for natural IPGs.[3–6]
These studies have included the synthesis of pseudopentasaccharide 1 as
the product to be expected from a GPI-specific phospholipase C (GPI-PLC)-
mediated cleavage of a GPI anchor (2).[4] A further step in this investigation
required the synthesis of pseudopentasaccharide 3—the structure to be
expected from a GPI-specific phospholipase D (GPI-PLD)-mediated cleavage
of 2—and the synthesis of 2—the basic GPI anchor structure—as a model sub-
strate to investigate the structural requirements involved in the regulation of
GPI enzymatic cleavage. Because of their biochemical importance and as a
result of the challenges involved in their total synthesis, GPIs have received
a great deal of attention and have been chosen as a target to test methods
and strategies in complex oligosaccharide synthesis.[7] As a further contri-
bution, we describe herein an effective synthesis of 2 and 3 using the chemistry
and the intermediates developed in our laboratory in the course of this
program.
RESULTS AND DISCUSSION
The synthesis of 2 was first envisaged from 4, an advanced intermediate in the
synthesis of 1,[4] by regioselective phosphorylation using the phosphite-
phosphonium salt methodology.[8] We have previously shown[3] that the
equatorially oriented OH-1 in diols 5 and 6 regioselectively reacts with 1,2-
dimiristoyl-sn-glycero-3-yl dibenzyl phosphite (7)[3,9] leading to the phospholi-
pids 8 and 9 in 54% and 28% yield, respectively. The drastic effect of the
additional a-D-mannopyranosyl unit in 6 on the yield of this regioselective
reaction could be taken as an indication that the size of the glycan chain at
C-6 of the myo-inositol unit may play a role in this regioselective phosphoryl-
ation. Indeed, pseudopentasaccharide 4 did not react with phosphite 7 even
when using a large excess of 7 in a wide range of temperatures. It may be
expected that the use of a more reactive phosphorylating agent would result

GPI Synthesis
395
in a mixture of products since no regioselectivity had been observed[10] in
the reaction of phosphoramidite 10[11] with diol 5. It was therefore decided to
abandon this straightforward but rather costly route to 2 from 4 and to
develop a new synthetic strategy using some less elaborated intermediates
also prepared in our laboratory in the course of these studies.
Therefore, pseudotrisaccharide 16, with a convenient protecting group
pattern to construct the desired GPI structure, was chosen as its preparation
from 11, an intermediate in the synthesis of IPG- like molecules designed as
inhibitors of c-AMP dependent protein kinase (PKA),[6] seemed to be readily
feasible (Sch. 1). But also in this case the key step involved a regioselective
reaction on the myo-inositol 1,2 axial-equatorial diol system in which the
more reactive equatorially oriented hydroxyl group at position 1 may be steri-
cally hindered by the presence of the bulky disaccharide unit at position 6. The
free primary hydroxyl group in 11 was regioselectively silylated to give 12 in
almost quantitative yield. As could be expected, the dibutyltin mediated regio-
selective allylation of 12 gave the desired 1-O-allyl derivative 13 in 66% yield
accompanied by the 2-O-allyl derivative 14 that could be isolated in 24% yield.
The ratio 13 : 14 could not be significantly improved by changing the
reaction conditions. The influence of glycosylation at position 6 of myo-inositol

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J. Lopez-Prados and M. Martın-Lomas
396
Scheme 1: Reagents and conditions: a) TIPSCl, imidazol, DMAP, DMF nt 10 hr, 95%; b) Bn2SnO,
toluene, 1408C, 12 hr; c) All Br, TBAI, 908C, 2 hr; d) NaH, BnBr, DMF rt 2 hr, 96%; e) TBAF, THF, rt, 2 hr,
92%.
on the regioselectivity of this reaction is clearly evident when this result is
compared with that obtained by van Boom et al.[12] in the dibutyltin-
mediated regioselective allylation of the 1,2-diol system of 3,4,5,6-tetra-O-
benzyl-D-myo-inositol under similar experimental conditions. Conventional
benzylation of 13 gave 15, the silyl group of which was then removed to give
the desired pseudotrisaccharide 16 in excellent yield.
The glycosylation of 16 to complete the pseudopentasaccharide skeleton in
2 was performed with trichloroacetimidate 17 to afford the desired compound
18 in 80% yield accompanied by a small amount (11%) of the ß-anomer 19.
The allyl group in 18 was removed[13] to give 20 (Sch. 2). From 20, both the
IPG-like structure 3 and the GPI 2 were prepared. Removal of the benzyl
groups in 20 afforded 3 in quantitative yield (Sch. 3). Compound 3 has been pre-
viously synthesized by Fraser-Reid and coworkers following a different pro-
cedure.[14] For the synthesis of 2, compound 20 was phosphorylated using
phosphoramidite 10[11] to give the fully protected phospholipid 21 as a pair of
diastereomers in 86% yield (Sch. 3).
Since 21 is a considerably elaborated intermediately and a reasonable
amount of 2 was needed for the enzymatic studies, the final hydrogenation
step was carefully optimized. The best results were found using 10% Pd/C
and performing the hydrogenation in a 2 : 1 : 1 : 1 AcOEt : THF : EtOH : H2O

GPI Synthesis
397
Scheme 2: a) TMSOTf, Et2O, 2158C, 1 hr; b) Ir.H2, THF, rt, 30 min; c) NBS, H2O, rt, 10 min, 98%.
mixture[3] rather than in the usual 3 : 3 : 1 CHCl3 : MeOH : H2O mixture, which
has been used so far in the synthesis of GPI anchors.[7]
In summary, an effective new preparative synthesis of the conserved linear
pseudopentasaccharide structure of the GPI anchors (3) and of the full GPI
structure (2) has been carried out that has permitted obtaining both molecules
in sufficient quantities as to perform further structural and biological studies.
EXPERIMENTAL
General Procedures
Thin layer chromatography (TLC) analyses were performed on silica gel 60
F
254 precoated on aluminium plates (Merck), and the compounds were detected
by staining with cerium (IV) sulphate (13 g), phosphomolybdic acid (10 g), and
Scheme 3: Reagents and conditions: a) 10, tetrazol, nt, 30min; b) MCPBA, 2408C, 10min,
86%; c) H2 Pd/c, AcOEt/THF/EtOH/H2O, rt 3 hr, 92%; d) H2, Pd/c, MeOH/H2O, rt, 12hr, 100%.

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J. Lopez-Prados and M. Martın-Lomas
398
sulphuric acid (60 mL) solution in water (1 L). Pseudopentasaccharide 3 was
visualized with sulphuric acid/ethanol solution (1 : 9) followed by heating at
over 1008C. Phospholipid 2 was visualized with H2SO4/MoO3 Dittmer
stainer[15] at rt. Column liquid chromatography was carried out on silica gel
60 (0.2–0.063 mm or 0.040–0.015 mm; Merck). Gel filtration chromatography
was performed on Sephadex G-25 Pharmacia H2O/MeOH 9/1 for 2 and
Sephadex LH-20 Pharmacia in MeOH 100% for 1. Ion-exchange chromato-
graphy was performed on Amberlite IRA-402 Cl2. Optical rotations were deter-
1
mined with a Perkin-Elmer 341 polarimeter. H and 13C NMR spectra were
acquired on a Brucker DRX-500 spectrometer and chemical shifts are given
in ppm (d) relative to tetramethylsilane as an internal reference or relative
to D2O. Elemental analyses were performed with a Leco CHNS-932 apparatus
after drying analytical samples under vacuum over phosphorous pentoxide for
24 hr. High-(HRMS) and low-resolution fast atom bombardment mass spectra
(F AB-MS) were carried out by the Mass Spectrometry Service, University of
Seville, with a Kratos MS-80 RFA spectrometer. MALDI-TOF mass spectra
were recorded with a MALDI-TOF GSG System spectrometer.
2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-a-D-mannopyranosyl-(1!4)-2-azido-
3,-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-3,4,5-tri-O-benzyl-
D-myo-inositol (12). TIPSCl (245 mL, 1.145 mmol) was added under argon to a
stirred solution of 11[6] (478 mg, 0.382 mmol), imidazol (156 mg, 2.92 mmol), and
DMAP (5 mg, 0.041 mmol) in DMF (7.6 mL); stirring was continued at rt for
10 hr. The reaction mixture was then diluted with AcOEt (50 mL) and washed
with a saturated NaHCO3 solution (50 mL), and the aqueous phase was extracted
with AcOEt (2 ꢀ 25 mL). The combined organic phases were washed with satu-
rated NaCl solution (3 ꢀ 100 mL), dried over MgSO4, and evaporated to dryness.
The residue was purified by column chromatography (hexane/AcOEt 9/1, 4/1,
1
2/1) to yield 12 (510 mg, 95%) as a white foam. [a]D 32.7 (c ¼ 1.0, CHCl3). H-
RMN (CDCl3,): d 7.36–7.19 (m, 37H, ArH), 7.18–7.13 (m, 3H, ArH), 5.43
(d, J ¼ 3.5 Hz, 1H, H1b), 5.22 (d, J ¼ 1.5 Hz, 1H, H1c), 4.93 (d, J ¼ 11.0 Hz, 1H,
CHbenzyl.), 4.89 (d, J ¼ 10.5 Hz, 1H, CHbenzyl.), 4.88 (d, J ¼ 10.5 Hz, 1H,
CHbenzyl.), 4.84 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.737 (d, J ¼ 10.5 Hz, 2H, 2 ꢀ
CHbenzyl.), 4.735 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.71 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.673 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.668 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.53 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.45 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.38 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.32 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.25 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.24 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.17 (t, J ¼ 2.5 Hz, 1H, H2a), 4.13 (t, J ¼ 9.5 Hz, 1H, H4c), 3.980
(t, J ¼ 9.5 Hz, 1H, H6a), 3.975 (t, J ¼ 9.5 Hz, 1H, H4a), 3.91 (m, 1H, H5b), 3.89
(dd, J1 ¼ 3.5 Hz, J2 ¼ 11.0 Hz, 1H, H6c), 3.87 (t, J ¼ 9.5 Hz, 1H, H4b), 3.820
(dd, J1 ¼ 3.0 Hz, J2 ¼ 9.5 Hz, 1H, H3c), 3.819 (t, J ¼ 9.5 Hz, 1H, H3b), 3.72
0
(t, J ¼ 2.5 Hz, 1H, H2c), 3.71 (dd, J1 ¼ 1.0 Hz, J2 ¼ 11.0 Hz, 1H, H6c ), 3.63

GPI Synthesis
399
(m, 1H, H1a), 3.62 (broad s, 1H, OHeq.), 3.53 (dd, J1 ¼ 3.0 Hz, J2 ¼ 11.0 Hz, 1H,
H6b), 3.52 (m, 1H, H5c), 3.50 (dd, J1 ¼ 4.0 Hz, J2 ¼ 10.0 Hz, 1H, H2b), 3.48 (dd,
J1 ¼ 2.5 Hz, J2 ¼ 9.5 Hz, 1H, H3a), 3.39 (t, J ¼ 9.5 Hz, 1H, H5a), 3.31 (dd,
0
J1 ¼ 1.0 Hz, J2 ¼ 11.0 Hz, 1H, H6b ), 2.51 (broad s, 1H, OHax.), 1.05–0.95
(m, 21H, 3 ꢀ (CH3)2CH TIPS þ 3 ꢀ (CH3)2CH TIPS). 13C-RMN (CDCl3): d
139.05, 138.63, 138.57 (ArC), 138.43 (2 ꢀ ArC), 138.29, 137.69, 137.54 (ArC),
128.54, 128.47, 128.35, 128.33, 128.24, 128.13, 127.97, 127.91, 127.83, 127.73,
127.60, 127.48, 127.42, 127.38, 127.28, 127.17, 127.12, 127.08, 126.95 (ArCH),
100.27 (C1c), 98.52 (C1b), 81.58 (C4a), 81.02 (C5a), 80.61 (C6a), 80.57 (C3b), 79.74
(C3a þ C3c), 76.66 (C4b), 75.99 (C2c), 75.85, 75.00, 74.89, 74.39 (CH2 benzyl.),
74.15 (C4c þ C5c), 73.04, 72.71 (CH2 benzyl.), 72.66 (C1a), 72.08, 72.02
(CH2 benzyl.), 71.14 (C5b), 69.46 (C2a), 68.56 (C6b), 64.43 (C2b), 62.54 (C6c), 17.99,
17.93 ((CH3)2CH TIPS), 11.97 ((CH3)2CH TIPS). FABþ calcd. for
C83H99O15N3Si: Mþ ¼ 1405.69, [M þ Na]þ ¼ 1428.68. Found: m/z 1428
[M þ Na]þ. Anal. calcd. for C83H99O15N3Si: C, 70.86; H, 7.09; N, 2.77; found: C,
70.76; H, 7.28; N, 2.77.
2,3,4-tri-O-benzyl-6-O-triisopropylsilyl-a-D-mannopyranosyl-(1 ! 4)-2-
azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-allyl-3,
4,6-tri-O-benzyl-D-myo-inositol (13) and 2,3,4 -tri-O-benzyl-6-O-triiso-
propyl-a-D-mannopyranosyl-(1!4)-2-azido-3,6-di-O-benzyl-2-deoxy-a-D-
glucopyranosyl-(1!6)-2-O-allyl-3,4,6-tri-O-benzyl-D-myo-inositol (14). Dibu-
tyltin oxide (100 mg, 0.402 mmol) was added to a solution of 12 (470 mg,
0.334 mmol) in dry toluene (16.7 mL), and the mixture was gently heated
under reflux in a dean-stark apparatus for 12 hr. The reaction mixture was
then evaporated to dryness and the residue was solved in allyl bromide
(3.2 mL). TBAI (148 mg, 0.401 mmol) was added and the mixture was gently
refluxed for 2 hr under argon. The mixture was then evaporated and the
residue was coevaporated with AcOEt (2 ꢀ 5 mL). The residue was fractionated
by column chromatography (hexane/AcOEt 9/1, 8/1, 7/1, 6/1, 5/1) to give 13
(319 mg, 66%) and 14 (116 mg, 24%) as white foams.
Data for 13: [a]D þ 41.5 (c ¼ 0.7, CHCl3). 1H-RMN (CDCl3): d 7.38–7.16
(m, 37H, ArH), 7.13 (m, 2H, ArH), 7.07 (m, 1H, ArH), 5.96 (ddt, J1 ¼ 6.0 Hz,
J2 ¼ 10.5 Hz, J3 ¼ 17.0 Hz, 1H, H2), 5.70 (d, J ¼ 3.5 Hz, 1H, H1b), 5.29 (broad
dd, J1 ¼ 1.5 Hz, J2 ¼ 17.0 Hz, 1H, H3), 5.21 (d, J ¼ 2.0 Hz, 1H, H1c),
0
5.20 (broad dd, J1 ¼ 1.0 Hz, J2 ¼ 10.0 Hz, 1H, H3 ), 4.92 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.896 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.895 (d, J ¼ 12.0 Hz,
1H, CHbenzyl.), 4.89 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.76 (d, J ¼ 11.0 Hz, 1H,
CHbenzyl.), 4.75 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.73 (d, J ¼ 11.5 Hz,
1H, CHbenzyl.), 4.72 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.69 (d, J ¼ 11.0 Hz, 1H,
CHbenzyl.), 4.61 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.55 (d, J ¼ 12.0 Hz,
1H, CHbenzyl.), 4.45 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.40 (d, J ¼ 12.0 Hz, 1H,

´
´
J. Lopez-Prados and M. Martın-Lomas
400
CHbenzyl.), 4.35 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.31 (d, J ¼ 12.0 Hz,
1H, CHbenzyl.), 4.22 (broad t, J ¼ 2.5 Hz, 1H, H2a), 4.18 (t, J ¼ 9.5 Hz, 1H,
H6a), 4.17 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.16 (t, J ¼ 9.5 Hz, 1H, H4c), 4.15
0
(m, 1H, H1), 4.07 (broad dd, J1 ¼ 6.0 Hz, J2 ¼ 12.5 Hz, 1H, H1 ), 4.05 (broad
d, J ¼ 9.5 Hz, 1H, H5b), 4.01 (t, J ¼ 9.5 Hz, 1H, H4a), 3.87 (dd, J1 ¼ 3.0 Hz,
J2 ¼ 11.0 Hz, 1H, H6c), 3.84 (m, 2H, H3bþ H4b), 3.76 (dd, J1 ¼ 2.5 Hz,
J2 ¼ 9.5 Hz, 1H, H3c), 3.66 (t, J ¼ 2.5 Hz, 1H, H2c), 3.65 (broad dd,
0
J1 ¼ 1.0 Hz, J2 ¼ 11.5 Hz, 1H, H6c ), 3.43 (m, 3H, H6b þ H1a þ H3a), 3.41
(t, J ¼ 9.5 Hz, 1H, H5a), 3.39 (broad d, J ¼ 10.0 Hz, 1H, H5c), 3.36 (dd,
0
J1 ¼ 3.5 Hz, J2 ¼ 11.5 Hz, 1H, H6b ), 3.28 (dd, J1 ¼ 3.5 Hz, J2 ¼ 10.0 Hz, 1H,
H2b), 2.39 (s, 1H, OHax.), 1.03–0.95 (m, 21H, 3 ꢀ (CH3)2CH TIPS þ 3 ꢀ (CH3)2-
CH TIPS). 13C-RMN (CDCl3,): d 139.34, 138.78, 138.74 (ArC), 138.47 (2 ꢀ ArC),
138.17 (ArC), 137.82 (2 ꢀ ArC), 133.98 (C2), 128.50, 128.47, 128.45, 128.30,
128.23, 128.19, 128.12, 127.94, 127.89, 127.71, 127.67, 127.59, 127.56, 127.42,
127.37, 127.32, 127.20, 127.11, 127.02, 126.94 (ArCH), 117.87 (C3), 100.36
(C1c), 97.34 (C1b), 81.35 (C4a), 81.13 (C1a), 80.88 (C5a), 79.87 (C3b), 79.65 (C3a),
79.59 (C3c), 76.40 (C2c), 76.38 (C4b), 75.90, 75.44, 74.74 (CH2 benzyl.), 74.66 (C6a),
74.08 (C4c), 73.90 (C5c), 73.58, 73.03, 72.77, 72.14, 71.94 (CH2 benzyl.), 70.88
(C1), 70.01 (C5b), 68.53 (C6b), 66.38 (C2a), 63.10 (C2b), 62.39 (C6c), 18.00, 17.94
((CH3)2CH TIPS), 11.98 ((CH3)2CH TIPS). FABþ calcd. for C86H103O15N3Si:
Mþ ¼ 1445.71; Found: 1468, [M þ Na]. Anal. calcd. for C86H103O15N3Si H2O:
.
C, 70.51; H, 7.23; N, 2.87. Found: C, 70.43; H, 7.18; N, 2.75.
Data for 14: [a]D þ 34.1 (c ¼ 0.6 CHCl3). 1H-RMN (CDCl3): d 7.35–7.18
(m, 37H, ArH), 7.16–7.10 (m, 3H, ArH), 5.94 (ddt, J1 ¼ 6.0 Hz, J2 ¼ 10.5 Hz,
J3 ¼ 17.0 Hz, 1H, H2), 5.55 (d, J ¼ 4.0 Hz, 1H, H1b), 5.29 (dq, J1 ¼ 1.5 Hz,
J2 ¼ 17.5 Hz, 1H, H3), 5.22 (d, J ¼ 2.0 Hz, 1H, H1c), 5.18 (broad dd,
0
J1 ¼ 1.5 Hz, J2 ¼ 10.0 Hz, 1H, H3 ), 4.95 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.),
4.90 (d, J ¼ 10.5 Hz, 2H, 2 ꢀ CHbenzyl.), 4.86 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.),
4.78 (d, J ¼ 10.5 Hz, 2H, 2 ꢀ CHbenzyl.), 4.70 (s, 2H, CH2 benzyl.), 4.68
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.65 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.),
4.54 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.46 (ddt, J1 ¼ 1.5 Hz, J2 ¼ 5.5 Hz,
J3 ¼ 13.0 Hz, 1H, H1), 4.44 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.35 (d, J ¼ 12.5 Hz,
1H, CHbenzyl.), 4.34 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.28 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.222 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.215 (ddt, J1 ¼ 1.0 Hz,
0
J2 ¼ 6.0 Hz, J3 ¼ 13.0 Hz, 1H, H1 ), 4.15 (t, J ¼ 9.5 Hz, 1H, H4c), 4.01
(t, J ¼ 9.5 Hz, 1H, H4a), 3.96 (t, J ¼ 9.5 Hz, 1H, H6a), 3.94 (broad d, J ¼ 9.0 Hz,
1H, H5b), 3.91 (t, J ¼ 2.5 Hz, 1H, H2a), 3.89 (dd, J1 ¼ 3.5 Hz, J2 ¼ 11.0 Hz, 1H,
H6c), 3.86 (t, J ¼ 9.0 Hz, 1H, H4b), 3.82 (t, J ¼ 10.0 Hz, 1H, H3b), 3.81 (dd,
J1 ¼ 3.0 Hz, J2 ¼ 9.5 Hz, 1H, H3c), 3.70 (t, J ¼ 3.0 Hz, 1H, H2c), 3.69 (dd,
0
J1 ¼ 1.0 Hz, J2 ¼ 11.0 Hz, 1H, H6c ), 3.61 (ddd, J1 ¼ 3.0 Hz, J2 ¼ 7.0 Hz,
J3 ¼ 10.0 Hz, 1H, H1a), 3.49 (m, 1H, H5c), 3.48 (dd, J1 ¼ 3.5 Hz, J2 ¼ 11.0 Hz,
1H, H6b), 3.45 (dd, J1 ¼ 4.0 Hz, J2 ¼ 10.0 Hz, 1H, H2b), 3.44 (dd, J1 ¼ 2.0 Hz,

GPI Synthesis
401
J2 ¼ 9.5 Hz, 1H, H3a), 3.39 (t, J ¼ 9.5 Hz, 1H, H5a), 3.33 (dd, J1 ¼ 1.5 Hz,
0
J2 ¼ 11.0 Hz, 1H, H6b ), 3.13 (d, J ¼ 7.0 Hz, 1H, OHeq.), 1.07–0.93 (m, 21H,
3 ꢀ (CH3)2CH TIPS þ 3 ꢀ (CH3)2CH TIPS).13C-RMN (CDCl3): d 139.16,
138.67, 138.63, 138.52, 138.47, 138.33, 138.03, 137.67 (ArC), 135.11 (C2),
128.46, 128.41, 128.30, 128.24, 128.13, 127.95, 127.93, 127.79, 127.72, 127.69,
127.53, 127.43, 127.41, 127.40, 127.38, 127.26, 127.18, 127.08, 127.04, 127.93
(ArCH), 117.15 (C3), 100.34 (C1c), 97.85 (C1b), 81.78 (C4a), 81.26 (C5a), 80.83
(C3a), 80.41 (C3b), 79.97 (C6a), 79.73 (C3c), 76.92 (C2a), 76.68 (C4b), 76.12 (C2c),
75.77, 75.11, 74.85, 74.22 (CH2 benzyl.), 74.12 (C5c), 74.10 (C4c), 73.86 (C1), 73.25
(C1a), 73.01, 72.96, 72.06, 72.05 (CH2 benzyl.), 70.71 (C5b), 68.56 (C6b), 64.07
(C2b), 62.50 (C6c), 18.00, 17.94 ((CH3)2CH TIPS), 11.98 ((CH3)2CH TIPS).
FABþ calcd. for C86H103O15N3Si: Mþ ¼ 1445.71, [M þ Na]þ ¼ 1468.70. Found:
m/z 1468 [M þ Na]þ.
2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-a-D-mannopyranosyl-(1 ! 4)-2-
azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-allyl-2,3,
4,5-tetra-O-benzyl-D-myo-inositol (15). A solution of 13 (280mg,
0.194 mmol) in dry DMF (3.9 ml) cooled to 08C was treated under argon with
NaH (24 mg of a 60% suspension in mineral oil, 0.600 mmol). Benzyl bromide
(70 mL, 0.589 mmol) was added and the mixture was stirred for 2 hr at rt.
After cooling in an ice bath, 30% NH3 (0.1 mL) was added and the solution
was stirred for 10 min. The reaction mixture was then diluted with AcOEt
(50 mL) and washed with 10% HCl (25 mL). The aqueous phase was extracted
with AcOEt (2 ꢀ 25) and the combined organic phases were washed with satu-
rated NaCl solution (3 ꢀ 100 mL), dried over MgSO4, and evaporated to
dryness. The residue was purified by column chromatography (hexane/AcOEt
19/1, 14/1, 9/1) to give 15 (286mg, 96%) as a white foam. [a]D þ 46.5 (c ¼ 0.8,
CHCl3). 1H-RMN (CDCl3): d 7.41 (m, 2H, ArH), 7.36–7.11 (m, 42H, ArH), 7.07
(m, 1H, ArH), 5.92 (ddt, J1 ¼ 5.5 Hz, J2 ¼ 10.5 Hz, J3 ¼ 17.5 Hz, 1H, H2), 5.76
(d, J ¼ 3.5 Hz, 1H, H1b), 5.27 (broad dd, J1 ¼ 1.5 Hz, J2 ¼ 17.0 Hz, 1H, H3),
5.21 (d, J ¼ 2.0 Hz, 1H, H1c), 5.17 (broad dd, J1 ¼ 1.0 Hz, J2 ¼ 10.5 Hz, 1H,
0
H3 ), 4.97 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.92 (d, J ¼ 10.5 Hz, 1H, CHbenzyl.),
4.90 (broad d, J ¼ 11.0 Hz, 2H, 2 ꢀ CHbenzyl.), 4.83 (s, 2H, CH2 benzyl.), 4.73 (d,
J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.72 (d, J ¼ 10.5 Hz, 1H, CHbenzyl.), 4.70 (d,
J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.66 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.614
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.612 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.57 (d,
J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.47 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.37 (d,
J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.34 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.31
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.27 (t, J ¼ 9.5 Hz, 1H, H6a), 4.18
(t, J ¼ 10.0 Hz, 1H, H4c), 4.16 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.11 (t,
J ¼ 9.5 Hz, 1H, H4a), 4.024 (t broad, J ¼ 2.0 Hz, 1H, H2a), 4.018 (broad d,
J ¼ 10.0 Hz, 1H, H5b), 4.01 (m, 1H, H1), 3.97 (broad dd, J1 ¼ 5.5 Hz,
0
J2 ¼ 12.0 Hz, 1H, H1 ), 3.87 (dd, J1 ¼ 2.5 Hz, J2 ¼ 11.0 Hz, 1H, H6c), 3.85

´
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J. Lopez-Prados and M. Martın-Lomas
402
(t, J ¼ 9.0 Hz, 1H, H4b), 3.83 (t, J ¼ 9.0 Hz, 1H, H3b), 3.78 (dd, J1 ¼ 3.0 Hz,
J2 ¼ 9.5 Hz, 1H, H3c), 3.67 (t, J ¼ 2.5 Hz, 1H, H2c), 3.63 (broad dd,
0
J1 ¼ 1.0 Hz, J2 ¼ 11.5 Hz, 1H, H6c ), 3.43 (t, J ¼ 9.5 Hz, 1H, H5a), 3.39 (m, 2H,
H3a þ H6b), 3.37 (m, 2H, H5c þ H1a), 3.31 (dd, J1 ¼ 3.0 Hz, J2 ¼ 11.0 Hz, 1H,
0
H
6b ), 3.26 (dd, J1 ¼ 4.0 Hz, J2 ¼ 10.0 Hz, 1H, H2b), 1.02–0.93 (m, 21H,
3 ꢀ (CH3)2CH TIPS þ 3 ꢀ (CH3)2CH TIPS). 13C-RMN (CDCl3): d 139.37
(ArC), 138.76 (2 ꢀ ArC), 138.54, 138.43, 138.33, 138.23, 137.86 (ArC), 134.20
(C2), 128.44, 128.40, 128.26, 128.23, 128.21, 128.18, 128.08, 128.00, 127.86,
127.69, 127.63, 127.50, 127.46, 127.42, 127.39, 127.31, 127.16, 127.02, 126.99,
126.97, 126.91 (ArCH), 117.01 (C3), 100.38 (C1c), 97.55 (C1b), 81.90 (C1a),
81.87 (C4a), 81.38 (C5a), 80.85 (C3a), 79.93 (C3b), 79.57 (C3c), 76.52 (C4b), 76.41
(C2c), 75.78 (CH2 benzyl.), 75.36 (C6a), 75.24, 74.74 (CH2 benzyl.), 74.03 (CH2 benzyl.
-
C4c), 73.88 (C5c), 73.63, 73.02, 72.80 (CH2 benzyl.), 72.72 (C2a), 72.13, 71.92
(CH2 benzyl.), 70.76 (C1), 69.86 (C5b), 68.47 (C6b), 63.17 (C2b), 62.37 (C6c), 17.99,
17.93 ((CH3)2CH TIPS), 11.96 ((CH3)2CH TIPS). FABþ calcd. for
C93H109O15N3Si: Mþ ¼ 1535.77; [M þ Na]þ ¼ 1558.76. Found: m/z 1559
[M þ Na]þ. Anal. calcd. for C93H109O15N3Si: C, 72.68; H, 7.15; N, 2.73. Found:
C, 72.75; H, 7.48; N, 2.33.
2,3,4-Tri-O-benzyl-a-D-mannopyranosyl-(1 ! 4)-2-azido-3,6-di-O-benzyl-
2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-allyl-2,3,4,5-tetra-O-benzyl-D-
myo-inositol (16). TBAF (1.0 M in THF, 468 mL) was added to a solution of 15
(240 mg, 0.156 mmol) in dry THF (3.1 mL) under argon, and the mixture was
stirred for 2 hr at rt. The reaction mixture was diluted with AcOEt (25 mL)
and washed with saturated NaHCO3 solution (25 mL). The aqueous phase
was extracted with AcOEt (2 ꢀ 25 mL) and the combined organic phases
were washed with saturated NaCl solution (3 ꢀ 100 mL), dried over MgSO4,
and evaporated to dryness. The residue was purified by column chromato-
graphy (hexane/AcOEt 9/1, 4/1, 2/1) to give 16 (198 mg, 92%) as a white
foam. [a]D þ 54.2 (c ¼ 1.0, CHCl3). 1H-RMN (CDCl3): d 7.42 (m, 2H, ArH),
7.38–7.16 (m, 40H, ArH), 7.13–7.05 (m, 3H, ArH), 5.94 (ddt, J1 ¼ 5.5 Hz,
J2 ¼ 10.5 Hz, J3 ¼ 17.5 Hz, 1H, H2), 5.72 (d, J ¼ 3.5 Hz, 1H, H1b), 5.28 (broad
dd, J1 ¼ 1.5 Hz, J2 ¼ 17.0 Hz, 1H, H3), 5.21 (d, J ¼ 2.0 Hz, 1H, H1c), 5.19
0
(broad dd, J1 ¼ 1.5 Hz, J2 ¼ 10.5 Hz, 1H, H3 ), 5.02 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.94 (broad d, J ¼ 10.5 Hz, 2H, 2 ꢀ CHbenzyl.), 4.88 (d, J ¼ 11.0 Hz,
1H, CHbenzyl.), 4.85 (s, 2H, CH2 benzyl.), 4.75 (d, J ¼ 10.5 Hz, 1H, CHbenzyl.),
4.69 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.68 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.63
(broad d, J ¼ 12.0 Hz, 2H, 2 ꢀ C Hbenzyl.), 4.60 (d, J ¼ 11.5 Hz, 2H, 2 ꢀ
CHbenzyl.), 4.51 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.48 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.39 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.32 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.25 (t, J ¼ 10.0 Hz, 1H, H6a), 4.14 (t, J ¼ 9.5 Hz, 1H, H4a), 4.09
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.04 (t, J ¼ 2.5 Hz, 1H, H2a), 4.03 (broad dd,
J1 ¼ 5.5 Hz, J2 ¼ 12.0 Hz, 1H, H1), 3.99 (broad d, J ¼ 10.0 Hz, 1H, H5b), 3.98

GPI Synthesis
403
0
(broad dd, J1 ¼ 6.0 Hz, J2 ¼ 12.5 Hz, 1H, H1 ), 3.88 (t, J ¼ 9.0 Hz, 1H, H4b), 3.85
(t, J ¼ 9.5 Hz, 1H, H4c), 3.83 (t, J ¼ 9.5 Hz, 1H, H3b), 3.75 (dd, J1 ¼ 3.0 Hz,
0
J2 ¼ 9.0 Hz, 1H, H3c), 3.64 (t, J ¼ 2.5 Hz, 1H, H2c), 3.56 (m, 2H, H6c þ H6c ),
3.43 (t, J ¼ 9.5 Hz, 1H, H5a), 3.40 (m, 1H, H3a), 3.39 (m, 1H, H1a), 3.38
(m, 1H, H5c), 3.31 (dd, J1 ¼ 1.5 Hz, J2 ¼ 12.0 Hz, 1H, H6b), 3.26 (dd,
0
J1 ¼ 2.0 Hz, J2 ¼ 12.5 Hz, 1H, H6b ), 3.23 (dd, J1 ¼ 3.5 Hz, J2 ¼ 10.0 Hz, 1H,
H2b), 2.17 (broad s, 1H, OH). 13C-RMN (CDCl3): d 138.71, 138.65, 138.49
(ArC), 138.45 (2 ꢀ ArC), 138.31, 138.20, 138.16, 138.88 (ArC), 134.17 (C2),
128.48, 128.44, 128.39, 128.31, 128.26, 128.20, 128.17, 128.06, 128.00, 127.84,
127.76, 127.69, 127.64, 127.62, 127.58, 127.54, 127.52, 127.48, 127.43, 127.32,
127.24, 127.15, 126.90, 126.80 (ArCH), 117.09 (C3), 100.64 (C1c), 97.57 (C1b),
81.87 (C4a), 81.77 (C1a), 81.35 (C5a), 80.83 (C3a), 80.11 (C3b), 79.00 (C3c), 76.81
(C4b), 76.67 (C2c), 75.80 (CH2 benzyl.), 75.52 (C6a), 75.23 (CH2 benzyl.), 74.86
(C4c), 74.71, 74.06, 73.94, 73.56 (CH2 benzyl.), 73.23 (C5c), 72.81 (CH2 benzyl.),
72.73 (C2a), 72.37, 72.18 (CH2 benzyl.), 70.77 (C1), 70.05 (C5b), 67.94 (C6b),
63.36 (C2b), 62.24 (C6c). FABþ calcd. for C84H89O15N3: Mþ ¼ 1379.63;
[M þ Na]þ ¼ 1402.62. Found: m/z 1402 [M þ Na]þ. Anal. calcd. for
.
C84H89O15N3 H2O: C, 72.14; H, 6.56; N, 3.00. Found: C, 71.92; H, 6.71;
N, 2.81.
2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl-(1 ! 2)-3,4,6-tri-O-benzyl-a,
b-D-mannopyranosyl trichloroacetimidate (17). To a solution of the
corresponding lactol[16] (300 mg, 0.308 mmol) in anhydrous CH2Cl2 (3.1 mL),
trichloroacetonitrile (463 mL, 4.618 mmol) and DBU (0.1 M in CH2Cl2,
154 mL) were added and the solution was stirred at rt for 2 hr. The solution
was evaporated to dryness and the residue was redisolved in AcOEt and coeva-
porated (2 ꢀ 5 mL). The crude product was purified on a short silica gel column
(hexane/AcOEt/Et3N 4/0.9/0.1) to give 17 (324 mg, 94%) as a mixture of the a
and ß anomers. Data for the a anomer: 1H-RMN (CDCl3): d 8.52 (s, 1H,
OCNHCCl3), 7.36–7.14 (m, 35H, ArH), 6.32 (d, J ¼ 1.5 Hz, 1H, H1d), 5.24
(d, J ¼ 1.5 Hz, 1H, H1e), 4.85 (d, J ¼ 10.5 Hz, 2H, 2 ꢀ CHbenzyl.),
4.70 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.69 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.66
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.64 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.),
4.60 (broad d, J ¼ 12.5 Hz, 2H, 2 ꢀ CHbenzyl.), 4.56 (d, J ¼ 12.5 Hz, 1H,
CHbenzyl.), 4.51 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.504 (d, J ¼ 12.0 Hz,
1H, CHbenzyl.), 4.497 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.49 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.46 (d, J ¼ 12.0 Hz, 1H, CHbenzyl), 4.17 (t, J ¼ 2.0 Hz, 1H, H2d),
4.01 (t, J ¼ 9.5 Hz, 1H, H4e), 3.95 (m, 2H, H3d þ H5d), 3.945 (m, 1H, H5e),
3.938 (m, 1H, H4d), 3.89 (dd, J1 ¼ 3.0 Hz, J2 ¼ 9.0 Hz, 1H, H3e), 3.84
(t, J ¼ 3.0 Hz, 1H, H2e), 3.81 (m, 2H, H6e þ H6d), 3.75 (dd, J1 ¼ 1.5 Hz,
J2 ¼ 11.0 Hz, 1H, H6e ), 3.70 (broad d, J ¼ 10.5 Hz, 1H, H6d ). 13C-RMN
(CDCl3): d 159.94 (OCNHCCl3), 138.57, 138.52 (ArC), 138.41 (2 ꢀ ArC),
138.33, 138.15, 137.82 (ArC), 128.48, 128.40, 128.25, 128.22, 128.18, 128.09,
0
0

´
´
J. Lopez-Prados and M. Martın-Lomas
404
128.01, 127.94, 127.84, 127.80, 127.75, 127.67, 127.47, 127.43, 127.40, 127.37
(ArCH), 99.59 (C1e), 96.83 (C1d), 90.94 (OCNHCCl3), 79.74 (C3e), 78.97 (C3d),
75.31, 74.92 (CH2 benzyl.), 74.88 (C2e), 74.84 (C4e), 74.78 (C4d), 74.12 (C5d),
73.34, 73.23, 72.72 (CH2 benzyl.), 72.69 (C2d), 72.29 (C5e), 72.26, 72.24
(CH2 benzyl.), 69.02 (C6e), 68.75 (C6d). Coupled 13C-RMN (CDCl3): d 99.59
(d, JC-H ¼ 172.3 Hz, C1e), 96.83 (d, JC-H ¼ 178.1 Hz, C1d).
2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl-(1 ! 2)-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl-(1 ! 6)-2,3,4-tri-O-benzyl-a-D-mannopyranosyl-(1 ! 4)-
2-azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-allyl-
2,3,4, 5-tetra-O-benzyl-D-myo-inositol (18) and 2,3,4,6-tetra-O-benzyl-a-
D-mannopyranosyl-(1 ! 2)-3,4,6-tri-O-benzyl-a-D-mannopyranosyl-(1!6)-
2,3,4-tri-O-benzyl-a-D-mannopyranosyl-(1 ! 4)-2-azido-3,6-di-O-benzyl-
2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-allyl-2,3,4,5-tetra-O-benzyl-D-
myo-inositol (19). To a solution of 16 (100 mg, 0.072 mmol) and 17 (97 mg,
˚
0.087 mmol) in anhydrous ether (1.5 mL), 4 A molecular sieves were added
and the mixture was cooled to 2158C under argon atmosphere. TMSOTf
(0.055 M in Et2O, 66 mL) was added and the reaction mixture stirred for 1 hr.
Then the mixture was neutralized with Et3N and evaporated to dryness. The
residue was purified by column chromatography (hexane/EtOAc 9/1) to give
18 (166 mg, 80%) and 19 (18 mg, 11%) as white foams.
Data for 18: [a]D þ 49.4 (c ¼ 1.1, CHCl3). 1H-RMN (C6D6): d 7.49–7.42 (m, 8H,
ArH), 7.40–7.26 (m, 25H, ArH), 7.24–6.97 (m, 47H, ArH), 5.974 (d, J ¼ 3.5 Hz,
1H, H1b), 5.968 (ddt, J1 ¼ 5.5 Hz, J2 ¼ 11.0 Hz, J3 ¼ 17.0 Hz, 1H, H2), 5.59
(d, J ¼ 2.0 Hz, 1H, H1c), 5.45 (d, J ¼ 2.0 Hz, 1H, H1d), 5.31 (d, J ¼ 2.0 Hz, 1H,
H1e), 5.28 (dq, J1 ¼ 2.0 Hz, J2 ¼ 17.5 Hz, 1H, H3), 5.18 (d, J ¼ 11.0 Hz, 1H,
CHbenzyl.), 5.13 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 5.12 (broad dd, J1 ¼ 1.5 Hz,
0
J2 ¼ 10.5 Hz, 1H, H3 ), 5.06 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.98
(d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.95 (broad d, J ¼ 12.0 Hz, 3H, 3 ꢀ CHbenzyl.),
4.89 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.84 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.77
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.72 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.71
(d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.68 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.664
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.656 (t, J ¼ 9.5 Hz, 1H, H6a), 4.62
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.60 (broad s, 2H, CH2 benzyl.), 4.59
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.55 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.54
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.49 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.46
(d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.45 (broad s, 2H, CH2 benzyl.), 4.44 (m, 1H,
H5b), 4.43 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.42 (d, J ¼ 11.5 Hz, 2H, 2 ꢀ
CHbenzyl.), 4.41 (d, J ¼ 12.5 Hz, 1H, CHbenzyl.), 4.38 (d, J ¼ 11.5 Hz, 1H,
CHbenzyl.), 4.37 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.36 (t, J ¼ 9.5 Hz, 1H, H4c),
4.33 (m, 3H, H2d þ 2 ꢀ CHbenzyl.), 4.32 (t, J ¼ 9.5 Hz, 1H, H4a), 4.31 (m, 2H,
H5e þ H4e), 4.27 (t, J ¼ 9.5 Hz, 1H, H4d), 4.22 (dd, J1 ¼ 3.0 Hz, J2 ¼ 9.0 Hz,

GPI Synthesis
405
1H, H3d), 4.21 (m, 1H, H6c), 4.20 (t, J ¼ 10.0 Hz, 1H, H3b), 4.19 (dd, J1 ¼ 3.0 Hz,
J2 ¼ 8.5 Hz, 1H, H3e), 4.11 (t, J ¼ 9.5 Hz, 1H, H4b), 4.09 (dd, J1 ¼ 3.0 Hz,
J2 ¼ 9.0 Hz, 1H, H3c), 4.08 (broad t, J ¼ 2.5 Hz, 1H, H2e), 4.06 (broad ddd,
J1 ¼ 1.0 Hz, J2 ¼ 4.0 Hz, J3 ¼ 9.5 Hz, 1H, H5d), 3.96 (broad dt, J1 ¼ 2.5 Hz,
J2 ¼ 9.5 Hz, 1H, H5c), 3.93 (t, J ¼ 2.5 Hz, 1H, H2a), 3.91 (t, J ¼ 2.5 Hz, 1H,
0
H2c), 3.90 (m, 1H, H1), 3.88 (m, 1H, H6e), 3.85 (m, 1H, H6d), 3.84 (m, 1H, H1 ),
0
3.78 (dd, J1 ¼ 1.5 Hz, J2 ¼ 11.0 Hz, 1H, H6c ), 3.76 (broad d, J ¼ 11.5 Hz, 1H,
0
0
0
H
6e ), 3.65 (dd, J1 ¼ 1.5 Hz, J2 ¼ 11.5 Hz, 1H, H6d ), 3.62 (m, 2H, H6b þ H6b ),
3.52 (t, J ¼ 9.5 Hz, 1H, H5a), 3.33 (dd, J1 ¼ 2.0 Hz, J2 ¼ 10.0 Hz, 1H, H1a),
3.20 (dd, J1 ¼ 2.5 Hz, J2 ¼ 10.0 Hz, 1H, H3a), 3.03 (dd, J1 ¼ 3.5 Hz,
J2 ¼ 10.0 Hz, 1H, H2b). 13C-RMN (C6D6): d 140.25 (2 ꢀ ArC), 140.22 (ArC),
140.15 (3 ꢀ ArC), 140.07 (2 ꢀ ArC), 139.95 (ArC), 139.88 (2 ꢀ ArC), 139.82,
139.55, 139.52, 139.47, 139.07 (ArC), 135.46 (C2), 129.56, 129.33, 129.29,
129.28, 129.26, 129.22, 129.15, 129.13, 129.10, 129.06, 129.04, 129.02, 128.96,
128.92, 128.86, 128.82, 128.76, 128.74, 128.72, 128.68, 128.62, 128.53, 128.48,
128.43, 128.20, 128.18, 128.13, 128.09, 128.05, 128.01, 127.98, 127.93
(ArCH), 117.62 (C3), 101.43 (C1c), 101.21 (C1e), 100.85 (C1d), 98.80 (C1b),
83.10 (C1a), 82.99 (C4a), 82.68 (C5a), 81.96 (C3a), 81.43 (C3e), 81.18 (C3c), 80.84
(C3b), 80.43 (C3d), 77.61 (C2e), 77.49 (C4b), 76.68 (C2c), 76.67, 76.39
(CH2 benzyl.), 76.33 (C6a), 76.26 (C2d), 75.97, 75.95, 75.91 (CH2 benzyl.), 75.84
(C4e), 75.80 (C4d), 75.75 (C4c), 75.07, 74.36, 74.21, 74.04 (CH2 benzyl.),
73.84 (C5e), 73.80 (CH2 benzyl.), 73.77 (C2a), 73.61 (C5c), 73.52 (CH2 benzyl.),
73.44 (C5d þ CH2 benzyl.), 73.11, 72.90, 72.73, 72.31 (CH2 benzyl.), 71.61
(C1), 71.30 (C5b), 70.68 (C6e), 70.34 (C6d), 70.03 (C6b), 68.03 (C6c), 63.61 (C2b).
Coupled 13C-RMN (C6D6): d 101.43 (d, JC-H ¼ 168.8 Hz, C1c), 101.21 (d, JC-H
¼
168.4 Hz, C1e), 100.85 (d, JC-H ¼ 170.5 Hz, C1d), 98.80 (d, JC-H ¼ 175.5 Hz, C1b).
FABþ calcd. for C145H151O25N3 Mþ ¼ 2333.89; [M þ Na]þ ¼ 2356.88. Found:
m/z 2357 [M þ Na]þ. Anal: calcd. for C141H151O25N3: C, 74.56: H, 5.62;
N, 1.80. Found: C, 74.17; H, 6.85; N, 1.52.
Data for 19: [a]D þ 26.8 (c ¼ 0.6, CHCl3. 1H-RMN (C6D6): d 7.46 (m, 6H, ArH),
7.41–7.23 (m, 26H, ArH), 7.20–6.96 (m, 48H, ArH), 6.03 (d, J ¼ 4.0 Hz, 1H,
H1b), 5.95 (ddt, J1 ¼ 6.5 Hz, J2 ¼ 10.5 Hz, J3 ¼ 17.0 Hz, 1H, H2), 5.62 (broad
s, 1H, H1e), 5.58 (broad d, J ¼ 1.5 Hz, 1H, H1c), 5.27 (broad dd, J1 ¼ 1.5 Hz,
J2 ¼ 17.5 Hz, 1H, H3), 5.13 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 5.11 (broad d,
0
J ¼ 10.5 Hz, 1H, H3 ), 4.99 (broad d, J ¼ 11.0 Hz, 4H, 4 ꢀ CHbenzyl.), 4.98
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.96 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.87
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.81 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.78
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.74 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.71
(m, 1H, H5e), 4.70 (d, J ¼ 11.0 Hz, 2H, 2 ꢀ CHbenzyl.), 4.69 (d, J ¼ 11.0 Hz,
1H, CHbenzyl.), 4.67 (t, J ¼ 10.0 Hz, 1H, H6a), 4.65 (broad s, 1H, H1d), 4.63
(d, J ¼ 12.0 Hz, 2H, 2 ꢀ CHbenzyl.), 4.57 (broad d, J ¼ 11.5 Hz, 2H, 2 ꢀ
CHbenzyl.), 4.56 (t, J ¼ 9.5 Hz, 1H, H4e), 4.53 (broad s, 2H, CH2 benzyl.), 4.520

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J. Lopez-Prados and M. Martın-Lomas
406
(m, 1H, H5b), 4.518 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.51 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.49 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.48 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.47 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.444 (d, J ¼ 12.0 Hz, 1H,
CHbenzyl.), 4.443 (broad t, J ¼ 2.5 Hz, 1H, H2d), 4.41 (d, J ¼ 12.0 Hz, 2H, 2 ꢀ
CHbenzyl.), 4.40 (broad s, 2H, CH2 benzyl.), 4.398 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.),
4.35 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.31 (dd, J1 ¼ 3.5 Hz, J2 ¼ 9.5 Hz, 1H,
H3e), 4.30 (t, J ¼ 10.0 Hz, 1H, H4a), 4.21 (broad t, J ¼ 10.0 Hz, 2H, H3bþ H4c),
0
4.15 (t, J ¼ 9.5 Hz, 1H, H4b), 4.13 (m, 4H, H6e þ H6e þ H6c þ H2e), 4.12
(m, 1H, H5c), 4.10 (t, J ¼ 9.5 Hz, 1H, H4d), 4.05 (broad d, J ¼ 11.5 Hz, 1H,
0
H
6c ), 4.03 (dd, J1 ¼ 3.0 Hz, J2 ¼ 9.5 Hz, 1H, H3c), 3.92 (broad t, J ¼ 2.0 Hz,
1H, H2a), 3.89 (broad t, J ¼ 3.0 Hz, 1H, H2c), 3.88 (broad dd, J1 ¼ 6.0 Hz,
0
J2 ¼ 12.0 Hz, 1H, H1), 3.83 (broad dd, J1 ¼ 5.5 Hz, J2 ¼ 12.0 Hz, 1H, H1 ),
3.79 (dd, J1 ¼ 5.0 Hz, J2 ¼ 11.5 Hz, 1H, H6d), 3.71 (broad d, J ¼ 12.0 Hz, 1H,
0
0
H
6d ), 3.70 (broad s, 2H, H6b þ H6b ), 3.51 (t, J ¼ 9.5 Hz, 1H, H5a), 3.42 (dd,
J1 ¼ 2.5 Hz, J2 ¼ 9.5 Hz, 1H, H3d), 3.38 (broad dd, J1 ¼ 3.0 Hz, J2 ¼ 9.0 Hz,
1H, H5d), 3.32 (dd, J1 ¼ 2.0 Hz, J2 ¼ 9.5 Hz, 1H, H1a), 3.19 (dd, J1 ¼ 2.0 Hz,
J2 ¼ 9.5 Hz, 1H, H3a), 3.16 (dd, J1 ¼ 4.0 Hz, J2 ¼ 10.0 Hz, 1H, H2b). 13C-RMN
(C6D6): d 140.62, 140.50, 140.46, 140.25, 140.21, 140.20, 140.16, 140.13,
139.98 (ArC), 139.86 (3 ꢀ ArC), 139.53, 139.45, 139.19, 139.12 (ArC), 135.40
(C2), 129.49, 129.37, 129.34, 129.28, 129.22, 129.14, 129.09, 129.06, 129.04,
128.96, 128.91, 128.86, 128.81, 128.72, 128.68, 128.62, 128.54, 128.51, 128.43,
128.38, 128.26, 128.20, 128.16, 128.14, 128.09, 127.93, 127.88, 127.86, 127.82
(ArCH), 117.70 (C3), 101.56 (C1d), 101.00 (C1c), 99.79 (C1e), 98.67 (C1b), 84.27
(C3d), 83.10 (C1a), 82.95 (C4a), 82.69 (C5a), 81.92 (C3a), 81.56 (C3e), 81.02
(C3b), 80.72 (C3c), 77.50 (C2c), 77.15 (C5d), 77.08 (C2e), 77.04 (C4b), 76.78
(CH2 benzyl.), 76.47 (C4c), 76.33 (CH2 benzyl.), 76.17 (C4e), 76.06 (C6a), 75.81 (C4d
-
2 ꢀ CH2 benzyl.), 75.62, 75.03 (CH2 benzyl.), 74.51 (C5c), 74.33, 74.30, 74.21, 74.05
(CH2 benzyl.), 73.81 (C2a), 73.76 (C5e), 73.51 (CH2 benzyl.), 73.46 (C2d), 73.41
(CH2 benzyl.), 73.12 (CH2 benzyl.), 73.09 (2 ꢀ CH2 benzyl.), 72.77 (CH2 benzyl.), 71.62
(C1), 71.18 (C5b), 70.93 (C6c), 70.43 (C6d), 70.05 (C6b), 69.63 (C6e), 64.07 (C2b).
Coupled 13C-RMN (C6D6): d 101.56 (d, JC-H ¼ 156.0 Hz, C1d), 101.00 (d, JC-
¼ 167.8 Hz, C1c), 99.79 (d, JC-H ¼ 168.9 Hz, C1e), 98.67 (d, JC-H ¼ 174.8 Hz,
H
C1b). FABþ calcd. for C145H151O25N3; Mþ ¼ 2333.89; [M þ Na]þ ¼ 2356.88.
Found: m/z 2359 [M þ Na þ 2]þ.
2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl-(1 ! 2)-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl-(1 ! 6)-2,3,4-tri-O-benzyl-a-D-mannopyranosyl-(1!4)-
2-azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-2,3,4,5-tetra-
O-benzyl-D-myo-inositol (20). To a solution of 18 (120 mg, 0.051 mmol) in
anhydrous THF (1 mL) under argon atmosphere (230 mL) of a solution of
[(1,5-cyclooctadiene)-bis-(methyl-diphenylphosphine)-iridium], hexafluoropho-
sphate (4 mg, 4.7 ꢀ 1023 mmol) in THF (390 mL), previously treated with H2,
was added and the mixture was stirred for 30 min. Then, the reaction

GPI Synthesis
407
mixture was treated in the dark with NBS (14 mg, 0.079 mmol) and water
(184 mL, 10.214 mmol) and stirred for 10 min at rt. The mixture was then
diluted with AcOEt (25 mL), washed with saturated NaHCO3 solution
(2 ꢀ 25 mL) and NaCl (3 ꢀ 25 mL), dried over MgSO4, and evaporated to
dryness. The residue was purified by column chromatography to give pure 20
1
(115 mg, 98%) as a white foam. [a]D þ 41.9 (c ¼ 1.1 CHCl3). H-RMN (C6D6): d
7.49–6.97 (m, 80H, ArH), 5.59 (d, J ¼ 2.0 Hz, 1H, H1c), 5.48 (d, J ¼ 3.5 Hz, 1H,
H1b), 5.47 (d, J ¼ 1.5 Hz, 1H, H1d), 5.31 (d, J ¼ 1.5 Hz, 1H, H1e), 5.13
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 5.09 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.),
5.05 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.99 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.97
(d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.95 (d, J ¼ 12.0 Hz, 1H, CHbenzyl.),
4.94 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.84 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.82
(d, J ¼ 12.0 Hz, 1H, CHbenzyl.), 4.78 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.),
4.71 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.68 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.67
(d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.65 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.),
4.60 (d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.59 (broad s, 2H, CH2 benzyl.), 4.56
(d, J ¼ 11.0 Hz, 1H, CHbenzyl.), 4.54 (d, J ¼ 10.5 Hz, 1H, CHbenzyl.),
4.51 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.48 (d, J ¼ 11.5 Hz, 1H, CHbenzyl.), 4.45–
4.30 (m, 11H, 11 ꢀ CHbenzyl.), 4.33 (m, 1H, H2d), 4.32 (m, 2H, H4c þ H5e), 4.30
(m, 1H, H5b), 4.293 (t, J ¼ 9.5 Hz, 1H, H4e), 4.285 (t, J ¼ 9.5 Hz, 1H, H4d), 4.27
(t, J ¼ 9.5 Hz, 1H, H6a), 4.25 (t, J ¼ 10.0 Hz, 1H, H4a), 4.22 (m, 2H, H3d þ H6c),
4.19 (dd, J1 ¼ 3.5 Hz, J2 ¼ 9.0 Hz, 1H, H3e), 4.09 (t, J ¼ 9.5 Hz, 1H, H3b), 4.08
(m, 3H, H3c þ H2e þ H5d), 4.05 (m, 1H, H5c), 4.04 (t, J ¼ 9.5 Hz, 1H, H4b), 3.90
(broad t, J ¼ 2.5 Hz, 1H, H2c), 3.88 (broad s, 1H, H2a), 3.87 (dd, J1 ¼ 5.5 Hz,
J2 ¼ 10.5 Hz, 1H, H6e), 3.85 (dd, J1 ¼ 3.5 Hz, J2 ¼ 11.5 Hz, 1H, H6d), 3.79
0
0
(broad d, J ¼ 10.5 Hz, 1H, H6c ), 3.77 (broad d, J ¼ 11.0 Hz, 1H, H6e ), 3.71 (dd,
0
J1 ¼ 3.5 Hz, J2 ¼ 11.0 Hz, 1H, H6b), 3.64 (broad d, J ¼ 11.5 Hz, 1H, H6d ), 3.62
0
(broad d, J ¼ 11.0 Hz, 1H,
H
6b ), 3.59 (ddd, J1 ¼ 2.5 Hz, J2 ¼ 6.0 Hz,
J3 ¼ 9.0 Hz, 1H, H1a), 3.43 (t, J ¼ 9.5 Hz, 1H, H5a), 3.23 (dd, J1 ¼ 2.5 Hz,
J2 ¼ 10.0 Hz, 1H, H3a), 3.15 (d, J ¼ 6.0 Hz, 1H, OHeq.), 3.07 (dd, J1 ¼ 4.0 Hz,
J2 ¼ 10.5 Hz, 1H, H2b). 13C-RMN (C6D6, 125 MHz): d 139.54 (2 ꢀ ArC), 139.49
(3 ꢀ ArC), 139.47, 139.40 (ArC), 139.27 (2 ꢀ ArC), 139.23, 139.20, 139.09,
139.04, 138.90, 138.82, 138.25 (ArC), 128.73, 128.68, 128.66, 128.64,
128.61, 128.55, 128.53, 128.51, 128.47, 128.45, 128.44, 128.40, 128.35, 128.24,
128.19, 128.12, 128.09, 128.05, 128.00, 127.92, 127.91, 127.88, 127.86, 127.84,
127.80, 127.61, 127.59, 127.57, 127.56, 127.54, 127.50, 127.45, 127.41, 127.38,
127.26 (ArCH), 100.59 (C1c þ C1e), 100.15 (C1d), 98.33 (C1b), 82.31 (C4a), 82.10
(C5a), 81.29 (C3a), 81.02 (C3b), 80.78 (C3e), 80.65 (C6a), 80.58 (C3c), 79.95 (C3d),
77.41 (C2a), 76.86 (C2c), 76.80 (C4b), 76.01 (C2e), 75.73 (2 ꢀ CH2 benzyl.), 75.64
(C4e), 75.40, 75.34 (CH2 benzyl.), 75.20 (C2d), 75.17 (C4c þ 2 ꢀ CH2 benzyl.), 75.15
(C4d), 74.13, 73.76 (CH2 benzyl.), 73.70 (C1a), 73.59, 73.40 (CH2 benzyl.), 73.17
(C5c), 73.15 (C5e), 72.82 (2 ꢀ CH2 benzyl.), 72.77 (C5d), 72.68, 72.29, 72.11, 71.79
(CH2 benzyl.), 71.42 (C5b), 70.14 (C6e), 69.70 (C6d), 69.47 (C6b), 67.38 (C6c), 64.24

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J. Lopez-Prados and M. Martın-Lomas
408
(C2b). FABþ calcd. for C142H147O25N3: Mþ ¼ 2294.03, [M þ Na]þ ¼ 2317.02.
Found: m/z 2317 [M þ Na]þ.
2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl-(1 ! 2)-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl-(1 ! 6)-2,3,4-tri-O-benzyl-a-D-mannopyranosyl-(1!4)-
2-azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1!6)-1-O-(10,20-di-O-
miristoyl-sn-glycero-30-R,S-benzyl-phosphatidyl)-2,3,4,5-tetra-O-benzyl-
D-myo-inositol (21). To a solution of 20 (45 mg, 0.020 mmol) and 1H-tetrazol
(3 mg, 0.043 mmol) in anhydrous CH2Cl2 (200 mL), phosphoramidite 10[11]
(0.2 M in CH2Cl2, 200 mL) was added and the mixture was stirred under argon
for 30 min. The reaction mixture was cooled to 2408C, MCPBA (70%, 5 mg,
0.020 mmol) was added, and the mixture was stirred for 10 min. The reaction
mixture was neutralized with Et3N (10% solution in CH2Cl2) and fractionated
using three preparative TLC plates previously treated with Et3N (hexane/
AcOEt 3/1) to obtain 21 (51 mg, 86%) as a mixture of two diastereomers I and
II 1 : 0.2. 1H-RMN (C6D6): d 7.60–7.01 (m, 153H, 85 ꢀ ArHI þ 85 ꢀ ArHII),
5.80 (d, J ¼ 3.5 Hz , 1H, H1bI), 5.75 (d, J ¼ 3.5 Hz, 0.8H, H1bII), 5.57
(q, J ¼ 5.0 Hz, 1H, H2II), 5.54 (broad s, 0.8H, H1cII), 5.53 (broad s, 1H, H1cI),
5.45 (s, 1.8H, H1dI þ H1dII), 5.35 (q, J ¼ 5.0 Hz, 1H, H2I), 5.30 (broad s, 1.8H,
H1eI þ H1eII), 5.29 (dd, J1 ¼ 9.0 Hz, J2 ¼ 11.5 Hz, 1H, CHbenzyl.I phosphate),
5.24–4.92 (m, 18.8H, 10 ꢀ Hbenzyl.I þ 10 ꢀ CHbenzyl.II þ CHbenzyl. II phosphate),
4.88 (broad s, 0.8H, H2aII), 4.81–4.62 (m, 10.8H, 6 ꢀ CHbenzyl. I þ 6 ꢀ CHbenzyl.
II), 4.79 (m, 1.6H, H1aII þ H6aII), 4.77 (t, J ¼ 10.0 Hz, 1H, H6aI), 4.75 (broad s,
1H, H2aI), 4.64 (m, 1H, H1aI), 4.61–4.50 (m, 11.8H, 7 ꢀ CHbenzyl. I þ 6 ꢀ CHbenzyl.
II), 4.47 (m, 1H, H5bI), 4.46 (m, 0.8H, H5bII), 4.46–4.30 (m, 18.8H, 10 ꢀ CHbenzyl.
I þ 11 ꢀ CHbenzyl. II), 4.38 (m, 0.8H, H3II), 4.35 (m, 1.8H, H4cI þ H4cII), 4.33
(m, 2.6H, H4aII þ H2dIþ H2dII), 4.31 (m, 4.6H, H1I þ H5eI þ H5eII þ H4eI þ H4eII),
0
4.30 (m, 1.6H, H1II þ H1 II), 4.29 (t, J ¼ 10.0 Hz, 1H, H4aI), 4.24 (t, J ¼ 10.0 Hz,
0
1H, H3bI), 4.23 (m, 2.6H, H3bII þ H4dI þ H4dII), 4.21 (m, 2.6H, H3 II þ H3dI
þ
H
3dII), 4.20 (m, 1.8H, H6cI þ H6cII), 4.18 (m, 1.8H, H3eI þ H3eII), 4.16 (m, 2H,
0
0
H3I þ H3 I), 4.10 (dd, J1 ¼ 4.5 Hz, J2 ¼ 11.0 Hz, 1H, H1 I), 4.08 (m, 1.8H,
H3cI þ H3cII), 4.07 (m, 2.8H, H4bI þ H2eI þ H2eII), 4.06 (t, J ¼ 10.0 Hz, 0.8H,
H
4bII), 4.03 (broad dd, J1 ¼ 3.5 Hz, J2 ¼ 10.0 Hz, 1.8H, H5dI þ H5dII), 3.95
(broad d, J ¼ 9.5 Hz, 1.8H, H5cI þ H5cII), 3.91 (broad t, J ¼ 2.0 Hz, 0.8H, H2cII),
3.90 (broad t, J ¼ 2.0 Hz, 1H, H2cI), 3.87 (broad dd, J1 ¼ 3.5 Hz, J2 ¼ 10.5 Hz,
1.8H, H6eI þ H6eII), 3.83 (dd, J1 ¼ 4.0 Hz, J2 ¼ 11.5 Hz, 1.8H, H6dI þ H6dII),
0
0
0
0
3.75 (broad d, J ¼ 11.0 Hz, 3.6H, H6c I þ H6c IIþ H6e I þ H6e II), 3.65 (broad s,
0
0
0
3.6H, H6bI þ H6bII þ H6b I þ H6b II), 3.64 (broad d, J ¼ 11.0 Hz, 1.8H, H6d I
þ
0
H
6d II), 3.63 (t, J ¼ 10.0 Hz, 0.8H, H5aII), 3.56 (dd, J1 ¼ 2.0 Hz, J2 ¼ 10.0 Hz,
0.8H, H3aII), 3.49 (t, J ¼ 9.5 Hz, 1H, H5aI), 3.36 (dd, J1 ¼ 1.5 Hz, J2 ¼ 10.0 Hz,
1H,
H
3aI), 3.11 (dd, J1 ¼ 3.5 Hz, J2 ¼ 10.5 Hz, 0.8H, H2bII), 3.07 (dd,
J1 ¼ 3.5 Hz, J2 ¼ 10.5 Hz, 1H, H2bI), 2.27 (m, 3.2H, 2 ꢀ OCOCH2 aII), 2.17
(m, 4H, 2 ꢀ OCOCH2 aI), 1.58 (m, 7.2H, 2 ꢀ CH2 bI þ 2 ꢀ CH2 bII), 1.37–1.16

GPI Synthesis
409
(m, 72H, 2 ꢀ 10 ꢀ CH2 I þ 2 ꢀ 10 ꢀ CH2 II), 0.91 (t, J ¼ 7.0 Hz, 10.8H, 2 ꢀ CH3
I þ 2 ꢀ CH3 II). 13C-RMN (C6D6): d 173.45, 173.42, 173.34, 173.33 (OCOC13H27),
140.23, 140.20, 140.12, 140.07, 140.05, 140.03, 139.99, 139.92, 139.81, 139.78,
139.50, 139.39, 139.36, 139.05, 139.03 (ArC), 137.06 (d, JC-P ¼ 5.5 Hz, ArCI phos-
phate), 136.89 (d, JC-P ¼ 6.4 Hz, ArCII phosphate), 129.59, 129.56, 129.55,
129.51, 129.31, 129.29, 129.26, 129.23, 129.22, 129.20, 129.18, 129.16, 129.15,
129.12, 129.09, 129.07, 129.04, 129.02, 128.96, 128.91, 128.81, 128.74, 128.72,
128.67, 128.62, 128.58, 128.55, 128.53, 128.43, 128.38, 128.19, 128.11, 128.06,
128.01, 127.99, 127.89 (ArCH), 101.81 (C1cII), 101.73 (C1cI), 101.21
(C1eI þ C1eII), 100.88 (C1dI þ C1dII), 98.80 (C1bI), 98.73 (C1bII), 82.62 (C4aII),
82.57 (C4aI), 82.33 (C5aII), 82.24 (C5aI), 81.72 (C3aII), 81.53 (C3aI), 81.40
(C3eI þ C3eII), 81.14 (m, C1aII þ C3cI þ C3cII), 81.00 (m, C1aI), 80.59 (C3bII), 80.56
(C3bI), 80.38 (C3dI þ C3dII), 78.11 (C4bII), 78.01 (C4bI), 77.94 (C2aI), 77.76 (C2aII),
77.58 (C2cI þ C2cII), 76.98, 76.90 (CH2 benc), 76.63 (C2eI þ C2eII þ CH2 benzyl.),
76.48, 76.42, 76.35 (CH2 benzyl.), 76.32 (C2dI þ C2dII þ 2 ꢀ CH2 benzyl.), 75.94 (3 ꢀ
CH2 benzyl.), 75.89 (CH2 benzyl.), 75.83 (C6aI þ C6aII þ 2 ꢀ CH2 benzyl.), 75.77 (C4dI
þ
C4dII þ C4eI þ C4eII), 75.70 (C4cI þ C4cII), 74.29, 74.27 (CH2 benzyl.), 74.20 (2 ꢀ
CH2 benzyl.), 74.04 (2 ꢀ CH2 benzyl.), 73.97 (CH2 benzyl.), 73.79 (C5eI þ C5eII), 73.62
(C5cI þ C5cII), 73.53, 73.46 (CH2 benzyl.), 73.43 (C5dI þ C5dII þ 2 ꢀ CH2 benzyl.),
73.17, 73.15 (CH2 benzyl.), 72.85 (2 ꢀ CH2 benc), 72.72 (2 ꢀ CH2 benzyl.), 72.29 (2 ꢀ
CH2 benzyl.), 71.59 (C5bI), 71.57 (C5bII), 70.90 (d, JC-P ¼ 6.0 Hz, C2II), 70.86 (d, JC-
¼ 5.0 Hz, CH2 benzyl.I phosphate), 70.71 (C6eI þ C6eII), 70.62 (d, JC-P ¼ 7.1 Hz,
P
C2I), 70.55 (d, JC-P ¼ 5.6 Hz, CH2benzyl.II phosphate), 70.32 (C6dI þ C6dII), 70.07
(C6bII), 70.04 (C6bI), 68.05 (C6cI þ C6cII), 66.65 (d, JC-P ¼ 6.3 Hz, C3II), 66.56
(d, JC-P ¼ 5.4 Hz, C3I), 63.81 (C2bII), 63.69 (C2bI), 62.50 (C1II), 62.45 (C1I), 35.02,
34.96, 34.76, 34.71 (OCOCH2 a), 32.92, 30.77, 30.75, 30.74, 30.72, 30.70, 30.57,
30.55, 30.42, 30.39, 30.35, 30.33, 30.13, 30.08 (CH2), 25.85, 25.82 (CH2 b), 25.80
(2 ꢀ CH2 b), 23.70 (4 ꢀ CH2), 14.96 (4 ꢀ CH3). 31P-RMN (C6D6, 202 MHz): d–
1.15 (PꢁI),–1.50 (PꢁII). FABþ calcd. for C180H212O32N3P: Mþ ¼ 2958.47;
[M þ Na]þ ¼ 2981.46. Found: m/z 2982 [M þ Na]þ.
a-D-Mannopyranosyl-(1 ! 2)-a-D-mannopyranosyl-(1 ! 6)-a-D-mannopyr-
anosyl-(1 ! 4)-2-ammonio-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-1-O-(10,20-
O-miristoyl-sn-glycero-30-phosphatidyl)-D-myo-inositol (2). A suspen-
sion of 21 (25 mg, 8.4 ꢀ 1023 mmol) in a 1/1/1/0.1 mixture of AcOEt/THF/
EtOH/H2O (4.3 mL) was hydrogenated for 3 hr with stirring using 10% Pd
over charcoal (45 mg, 0.042 mol) as catalyst. The reaction mixture was
filtered over Celite, washed with MeOH (10 mL), and evaporated to dryness
1
to afford 2 as a white solid. [a]D þ 29.1 (c ¼ 0.2, MeOH). H-RMN (CD3OD): d
5.54 (d, J ¼ 4.0 Hz, 1H, H1b), 5.29 (m, 1H, H2), 5.27 (d, J ¼ 1.5 Hz, 1H, H1c),
5.16 (d, J ¼ 1.5 Hz, 1H, H1d), 5.02 (d, J ¼ 2.0 Hz, 1H, H1e), 4.48 (dd,
J1 ¼ 3.0 Hz, J2 ¼ 12.0 Hz, 1H, H1), 4.30 (broad ddd, J1 ¼ 2.0 Hz, J2 ¼ 4.0 Hz,
0
J3 ¼ 10.0 Hz, 1H, H5b), 4.23 (dd, J1 ¼ 7.0 Hz, J2 ¼ 12.0 Hz, 1H, H1 ), 4.20

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J. Lopez-Prados and M. Martın-Lomas
410
(broad td, J1 ¼ 2.5 Hz, J2 ¼ 9.5 Hz, 1H, H1a), 4.12 (broad t, J ¼ 2.5 Hz, 1H, H2a),
0
4.08 (m, 2H, H3 þ H3 ), 4.03 (dd, J1 ¼ 9.5 Hz, J2 ¼ 10.0 Hz, 1H, H3b), 4.02 (dd,
J1 ¼ 2.0 Hz, J2 ¼ 3.0 Hz, 1H, H2e), 4.00 (t, J ¼ 9.5 Hz, 1H, H6a), 3.99 (dd,
J1 ¼ 2.0 Hz, J2 ¼ 3.0 Hz, 1H, H2c), 3.95 (dd, J1 ¼ 1.5 Hz, J2 ¼ 3.5 Hz, 1H,
H2d), 3.93 (m, 1H, H3d), 3.90 (m, 1H, H6c), 3.89 (m, 1H, H6b), 3.88 (m, 1H,
0
0
H6e), 3.87 (m, 1H, H6d), 3.83 (m, 2H, H6b þ H6c ), 3.80 (m, 1H, H5c), 3.76
0
0
(m, 1H, H5e), 3.74 (m, 2H, H3e þ H6e ), 3.72 (m, 1H, H6d ), 3.697 (broad t,
J ¼ 9.5 Hz, 2H, H4b þ H4a), 3.696 (dd, J1 ¼ 3.0 Hz, J2 ¼ 9.5 Hz, 1H, H3c),
3.66 (m, 2H, H4d þ H5d), 3.65 (t, J ¼ 9.5 Hz, 1H, H4c), 3.62 (t, J ¼ 9.5 Hz,
1H, H4e), 3.41 (dd, J1 ¼ 3.0 Hz, J2 ¼ 10.0 Hz, 1H, H3a), 3.33
(t, J ¼ 9.0 Hz, 1H, H5a), 3.20 (dd, J1 ¼ 4.0 Hz, J2 ¼ 10.5 Hz, 1H, H2b), 2.39
(t, J ¼ 7.5 Hz, 2H, OCOCH2 a), 2.34 (t, J ¼ 7.5 Hz, 2H, OCOCH2 a), 1.64
(m, 4H, 2 ꢀ CH2 b), 1.32 (m, 40H, 2 ꢀ 10 ꢀ CH2), 0.93 (t, J ¼ 7.0 Hz, 6H,
2 ꢀ CH3). 13C-RMN (CD3OD, 125 MHz): d 175.00, 174.72 (OCOC13H27),
104.15 (C1e), 102.81 (C1c), 100.01 (C1d), 96.98 (C1b), 80.47 (C2d), 79.33 (C6a),
78.22 (d, JC-P ¼ 6.1 Hz, C1a), 78.01 (C4b), 74.92 (C5e), 74.90 (C5a), 74.49
(C5d), 74.34 (C4a), 74.30 (C5c), 73.35 (C2a), 72.63 (C3a), 72.48 (C3c), 72.44
(C3e þ C5b), 72.09 (C2c), 72.01 (C3d), 71.92 (C2e), 71.79 (C2), 71.72 (C3b),
69.05 (C4d), 68.76 (C4e), 68.56 (C4c), 68.06 (C6c), 65.02 (C3), 63.70 (C1),
63.02 (C6d þ C6e), 62.06 (C6b), 56.04 (C2b), 35.12, 34.95 (OCOCH2 a), 33.09,
30.83, 30.79, 30.69, 30.67, 30.50, 30.24, 30.21 (CH2), 26.05, 26.02
(CH2 b), 23.74 (2 ꢀ CH2), 14.44 (2 ꢀ CH3). 31P-RMN (CD3OD, 202 MHz):
d–0.81. FABþ HRMS calcd. for C61H112O32NP: Mþ ¼ 1401.6873;
[M þ H]þ ¼ 1402.7143; [M þ Na]þ ¼ 1424.6961. Found: m/z 1402.6983
[M þ H]þ; 1424.6803 [M þ Na]þ.
a-D-Mannopyranosyl-(1 ! 2)-a-D-mannopyranosyl-(1 ! 6)-a-D-mannopyr-
anosyl-(1 ! 4)-2-ammonio-2-deoxy-a-D-glucopyranosyl-(1 ! 6)-D-myo-
inositol (3). A suspension of 20 (30 mg, 0.013 mmol) in a 9/1 MeOH/H2O
mixture (6.5 mL) was hydrogenated with stirring for 12 hr in the presence of
10% Pd on charcoal (69 mg, 0.065 mmol). The mixture was filtered over
Celite and evaporated to dryness. The residue was dissolved in H2O and
passed through an Amberlite IRA-408 (Cl2 form) column and lyophilized to
give 3 (12 mg, 100%) as a white solid. [a]D þ 90.4 (c ¼ 0.3, H2O). 1H-RMN
(D2O): d 5.39 (d, J ¼ 4.0 Hz, 1H, H1b), 5.21 (d, J ¼ 1.5 Hz, 1H, H1c), 5.12
(d, J ¼ 1.5 Hz, 1H, H1d), 5.00 (d, J ¼ 1.5 Hz, 1H, H1e), 4.13 (dt, J1 ¼ 3.0 Hz,
J2 ¼ 10.0 Hz, 1H, H5b), 4.05 (broad dd, J1 ¼ 2.0 Hz, J2 ¼ 3.0 Hz, 2H,
H2c þ H2e), 4.02 (t, J ¼ 9.0 Hz, 1H, H3b), 4.00 (m, 2H, H2a þ H2d), 3.95 (dd, 1H,
J1 ¼ 3.5 Hz, J2 ¼ 9.5 Hz, 1H, H3d), 3.93 (dd, J1 ¼ 5.0 Hz, J2 ¼ 11.0 Hz, 1H,
H6c), 3.87 (broad dd, 2H, J1 ¼ 1.5 Hz, J2 ¼ 12.0 Hz, 1H, H6d þ H6e), 3.82
0
(m, 4H, H6b þ H6b þ H3e þ H5c), 3.78 (dd, J1 ¼ 3.0 Hz, J2 ¼ 9.5 Hz, 1H, H3c),
0
0
0
3.76 (m, 1H, H5e), 3.74 (m, 1H, H6d ), 3.72 (m, 2H, H4b þ H6c ), 3.71 (m, 4H, H6e
-
H4c þ H1a þ H6a), 3.67 (m, 2H, H4d þ H5d), 3.62 (t, J ¼ 10.0 Hz, 1H, H4a), 3.60

GPI Synthesis
411
(t, J ¼ 9.5 Hz, 1H, H4e), 3.49 (dd, J1 ¼ 2.5 Hz, J2 ¼ 10.0 Hz, 1H, H3a), 3.35
(m, 1H, H5a), 3.27 (dd, J1 ¼ 3.5 Hz, J2 ¼ 10.5 Hz, 1H, H2b). 13C-RMN (C6D6): d
101.91 (C1e), 101.50 (C1c), 97.95 (C1d), 96.59 (C1b), 79.92 (C6a), 78.24 (C2d),
76.30 (C4b), 72.83 (C5e), 72.32 (C5d), 72.31 (C5a), 72.13 (C4a), 72.01 (C2a), 71.84
(C5c), 71.18 (C1a), 70.58 (C5b), 70.49 (C3a), 70.01 (C3c), 69.90 (C3b þ C3e), 69.79
(C3d þ C2c), 69.56 (C2e), 66.53 (C4d), 66.47 (C4e), 66.04 (C4c), 65.88 (C6c), 60.72
(C6e), 60.53 (C6d), 59.80 (C6b), 54.21 (C2b). FABþ HRMS calcd. for C30H53O25N:
Mþ ¼ 827.2890; [M þ Na]þ ¼ 850.2788. Found: m/z 850.2796 [M þ Na]þ.
SUPPORTING INFORMATION
RMN Spectra
1H-RMN and HSQC spectra of GPI 2 and pseudopentasaccharide 3
acquired on a Bruker DRX-500 spectrometer in CD3OD at pH ¼ 6.4 and deu-
terium oxide at pH ¼ 6.9, respectively, are shown:

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J. Lopez-Prados and M. Martın-Lomas
412

GPI Synthesis
413
ACKNOWLEDGMENTS
We thank the Ministry of Science and Technology (Grant BQU 2002-03734) and
Rodaris Pharmaceuticals for financial support.
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