Synthesis of novel, fluorescently tagged analogs of glycosylphosphatidylinositol (GPI) anchors

Article abstract of DOI:10.1080/07328303.2013.826671

Glycosylphosphatidylinositol (GPI) anchors are a group of complex glycolipids that attach extracellular proteins and glycoproteins to the eukaryotic cell outer membrane. To better understand GPI anchorage, it is necessary to have access to homogeneous, structurally defined, and functionalized GPIs and GPI analogs. In this regard, chemical synthesis is necessary, as GPI anchors are rather scarce and heterogeneous in natural sources. Three GPI analogs with phosphoglycerolipids linked to the pseudodisaccharide core and their fluorescein conjugates were prepared in this work as a small tool set useful for probing how the lipid composition and carbohydrate anomeric configuration may affect the properties of GPI anchors.

Full text of DOI:10.1080/07328303.2013.826671

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Synthesis of Novel, Fluorescently Tagged
Analogs of Glycosylphosphatidylinositol
(GPI) Anchors
Charles L. Johnson a & Zhongwu Guo a
a Department of Chemistry , Wayne State University , Detroit , MI ,
48202 , USA
Published online: 02 Sep 2013.
To cite this article: Charles L. Johnson & Zhongwu Guo (2013) Synthesis of Novel, Fluorescently
Tagged Analogs of Glycosylphosphatidylinositol (GPI) Anchors, Journal of Carbohydrate Chemistry,
32:5-6, 301-323, DOI: 10.1080/07328303.2013.826671
To link to this article: http://dx.doi.org/10.1080/07328303.2013.826671
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Journal of Carbohydrate Chemistry, 32:301–323, 2013
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2013.826671
Synthesis of Novel,
Fluorescently Tagged Analogs
of Glycosylphosphatidylinositol
(GPI) Anchors
Charles L. Johnson and Zhongwu Guo
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Glycosylphosphatidylinositol (GPI) anchors are a group of complex glycolipids that at-
tach extracellular proteins and glycoproteins to the eukaryotic cell outer membrane.
To better understand GPI anchorage, it is necessary to have access to homogeneous,
structurally defined, and functionalized GPIs and GPI analogs. In this regard, chemical
synthesis is necessary, as GPI anchors are rather scarce and heterogeneous in natural
sources. Three GPI analogs with phosphoglycerolipids linked to the pseudodisaccharide
core and their fluorescein conjugates were prepared in this work as a small tool set use-
ful for probing how the lipid composition and carbohydrate anomeric configuration may
affect the properties of GPI anchors.
Keywords Glycosylphosphatidylinositol; GPI anchor; Phospholipid; Glycolipid; Fluo-
rescein; Fluorescent tag
INTRODUCTION
Glycosylphosphatidylinositol (GPI) anchorage of extracellular membrane pro-
teins and glycoproteins to the cell surface exists in all eukaryotic species.[1–3] It
is generally acknowledged that GPIs take part in a number of biological roles
such as cell adhesion and recognition,[4] pathogenic infections,[5] signal trans-
duction,[6] and enzymatic reactions on the cell surface.[7] However, relatively
little is known about how the GPI structure might affect their biological func-
tions.[8] While all eukaryotic GPI anchors share the same core structure shown
in Figure 1,[1–3] they have a great deal of microheterogeneity including vari-
able lipid compositions and additional sugar chains and other functionalities
attached to the GPI core glycan. Due to this property, as well as the natural
Received May 16, 2013; accepted July 15, 2013.
Address correspondence to Zhongwu Guo, Department of Chemistry, Wayne State Uni-
versity, 5101 Cass Avenue, Detroit, MI 48202, USA. E-mail: zwguo@chem.wayne.edu
301

C. L. Johnson and Z. Guo
302
Figure 1: The generic core structure of GPI anchors found on all eukaryotic cells.
scarcity of GPIs in living systems, isolation of pure GPIs from nature for their
structural study would be impractical. Chemical synthesis has become the crit-
ical means to access homogeneous and structurally defined GPI anchors[9,10]
for the better understanding of GPI anchorage. In addition to natural GPI an-
chors, the synthesis of GPI analogs is also a necessary step for determining the
roles, structure–activity relationships, and many other studies of GPI anchors.
In this work, synthetic GPI analogs carrying a fluorescent tag were
prepared as a tool set that can be utilized to probe GPI anchorage. The
designed target molecules (Fig. 2), which had fluorescein incorporated at the
glucosamine 4-O-position of the GPI core pseudosaccharide, carried both un-
saturated and saturated lipids, 1 and 2, and with both α- and β-glycosidic link-
ages, 2 and 3. We planned to prepare GPI analogs that contained only the
core pseudosaccharide as studies have demonstrated that this structural mo-
tif was critical for some biological activities of GPI anchors,[11–13] GPI anchor
microdomain formation in membranes,[12,14] and GPI biosynthesis.[2] Moreover,
Figure 2: Designed GPI analogs carrying the fluorescein tag.

Synthetic GPI analogs
303
these GPI analogs can be used to compare with intact GPIs to provide more in-
sight into how variation in the lipid composition and anomeric configuration of
the GPI anchor may affect its distribution in lipid bilayers and other studies.
RESULTS AND DISCUSSION
The retrosynthesis of GPI analogs 1, 2, and 3 is outlined in Scheme 1. Because
some of these synthetic targets contained unsaturated lipids that are incom-
patible with catalytic hydrogenolysis employed to deprotect benzyl ethers, we
planned to use the para-methoxybenzyl (PMB) group for global hydroxyl pro-
tection.[15,16] Fluorescein would be linked to the GPI pseudodisaccharide via
a flexible diethylene glycol analog. Here we design to have an ether linkage,
instead of the more easily formed ester or other similar linkages, between the
linker and the carbohydrate chain to take advantage of the excellent stabil-
ity of the ether bond under physiological conditions. The phosphoglycerolipid
would be introduced by the 1H-phosphonate approach.[17] Finally, the Schmidt
glycosylation[18] would be used to couple inositol and glucosamine using imi-
date 11 as the glycosyl donor. In 11, the amino group was protected as an azide
to facilitate the formation of both α- and β-anomers during the glycosylation
reaction.
Scheme 1: Retrosynthesis of the target molecules.
The synthesis of inositol derivative 12 was a significant challenge, achiev-
able via combining two previously developed protocols (Sch. 2).[15,19] First, the
optically pure dicyclohexylidene ketal derivative of inositol (-)-13 was prepared
by regioselective protection of myo-inositol[20] and then enzymatic resolution of

C. L. Johnson and Z. Guo
304
the resulting enantiomers.[19] Thereafter, the 1,6-O-positions of 13 were tem-
porarily protected with allyl groups to differentiate them from the remain-
ing inositol positions. The ketal protecting groups of 14 were subsequently re-
moved by acidic methanolysis, which was followed by hydroxyl protection with
PMB to afford 15. To differentiate the inositol 1,6-O-positions, the two allyl
groups were removed by Cha’s method,[21] and the resulting diol 16 was sub-
jected to tin-directed regioselective allylation of the 6-O-position to furnish 12.
The final step provided a 3:1 selectivity for the desired regioisomer.
Scheme 2: Synthesis of the inositol derivative 12 (note: bold).
The synthesis of glucosamine derivative 11 (Sch. 3) as a glycosyl donor
started from peracetylated azido sugar 17.[22] Selective removal of the 1-O-
acetyl group with benzylamine was followed by tert-butyldiphenylsilyl (TB-
DPS) protection of the exposed anomeric position to give 18 as a β-anomer
exclusively. The remaining acetyl groups were subsequently removed with
sodium methoxide in methanol, which was followed by regioselective pro-
tection of the 4,6-O-positions with para-methoxybenzylidine to provide 19.
Ideally, the remaining free 3-O-position in 19 should be protected with a
PMB group, but we found that the conventional method to introduce PMB
utilizing NaH and PMBCl resulted in the decomposition of 19, presumably
due to the sensitivity of the 1-O-TBDPS group to strong basic conditions.
To address this issue, the 3-O-position was first protected with an acetyl
group that was replaced with a PMB group after the glycosylation reaction.
Hence, 19 was acetylated with acetic anhydride and pyridine, which was fol-
lowed by 1-O-position deprotection using buffered tetrabutylammonium flu-
oride (TBAF) to give hemiacetal 20. Finally, 20 was converted to the corre-
sponding trichloroacetimidate 11 by reaction with trichloroacetonitrile in the
presence of 1,8-diazabicycloundec-7-ene (DBU), which was ready to couple with
inositol derivative 12.
Glycosylation of 12 by 11 with trimethylsilyl triflate (TMSOTf) as the pro-
moter afforded a mixture of α- and β- (1.3:1.0) anomers 21 in a very good yield
(82%, Sch. 3). However, the two anomers were difficult to separate; therefore,

Synthetic GPI analogs
305
Scheme 3: Synthesis of the pseudodisaccharides.
the purification process was delayed until a later step to facilitate more con-
venient isomer separation. Before further transformations, the acetyl group at
the glucosamine 3-O-position in 21 was replaced with a PMB group upon basic
deacetylation and PMB-ation to give 22. Thereafter, regioselective benzylidine
acetal ring opening with sodium cyanoborohydride and HCl provided 6a and
6b that were readily separable by flash silica gel column chromatography. The
anomeric configurations of 6a and 6b were confirmed by 1H NMR: the coupling
constants of the anomeric protons were 3.6 Hz for 6a (α-anomer) and 7.9 Hz
for 6b (β-anomer).
The designed linker 8 for attaching fluorescein to the glucosamine residue
was prepared from commercially available diethylene glycol 23 in three sepa-
rate steps (Sch. 4), including (1) mono-tosylation using 1 equiv. of tosyl chlo-
ride, (2) azido substitution of the tosylate using sodium azide, and (3) tri-
flation of the remaining free hydroxyl group. In the meantime, synthesis of
1H-phosphonates 9 and 10 began with protecting the free hydroxyl group of
commercially available (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol 26 with
TBDPS, followed by removal of 2,3-O-acetonide under acidic conditions to af-
ford 27 (Sch. 4). Incorporation of oleic acid and stearic acid in 27 was achieved
with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensa-
tion reagent to give 28a and 28b in 81% and 67% yields, respectively. Triethy-
lamine trihydrofluoride (Et3N•3HF), instead of more basic TBAF, was used for
TBDPS deprotection in an effort to prevent acyl group migration from the sec-
ondary alcohol to the less hindered primary alcohol. Finally, a phosphonate
group was attached to the free hydroxyl group in 29a and 29b by reacting with
phosphonic acid and pivaloyl chloride to produce 9 and 10 in 76% and 71%
yields, respectively.
To provide orthogonality to the azido group introduced with linker 8, the 2-
azido group of the glucosamine residue in 6a and 6b was reduced using Zn and
acetic acid (Sch. 5), and the resulting free amino group was then protected with

C. L. Johnson and Z. Guo
306
Scheme 4: Synthesis of the linker and the phosphorylating reagents.
a tert-butoxycarbonyl (Boc) group. It was envisioned that the Boc group could
be easily removed under mild acidic conditions along with the PMB groups in
the final deprotection step. The addition of linker 8 to the glucosamine 4-O-
position was realized by reacting 8 with 30a and 30b under the influence of
sodium hydride, which was followed by removal of the inositol 1-O-allyl pro-
tection to obtain 32a and 32b ready for the attachment of the phospholipid
moiety. Phospholipidation of 32a and 32b was achieved with excessive 9 or 10
(2.5 equiv.) in the presence of pivaloyl chloride, followed by oxidation in situ
with iodine to afford 33a (75%), 33b (53%), and 34 (36%, but 76% based on re-
covered 32a). The products were rather difficult to separate from the excessive
reagent and other side products, which affected the isolated yields.
Scheme 5: Final stage assembly of the target molecules.
The installation of the fluorescent tag was achieved in two steps, including
reduction of the linker azide by Zn and acetic acid and reaction of the result-
ing primary amine with commercial activated ester of fluorescein 7 to give

Synthetic GPI analogs
307
compounds 4 and 5 in excellent yields. Finally, all of the Boc and PMB pro-
tecting groups were smoothly removed using 10% trifluoroacetic acid (TFA) in
dichloromethane in the presence of a catalytic amount of DBU to afford the de-
sired target molecules in good yields (60%–78%). It is worth mentioning that
the catalytic amount of DBU was necessary for the rapid and complete PMB
deprotection, as initial attempts for PMB deprotection using 10%–40% TFA
in DCM could not be completed after 4 h of reaction. We believe this may be
because of the influence of DBU on the equilibrium of the PMB deprotection
reaction that is reversible under anhydrous conditions in an aprotic solvent,
and DBU may serve as a base to buffer the reaction and shift it to completion.
CONCLUSIONS
An efficient synthesis was developed for the preparation of fluorescent-tagged
GPI analogs. The installed fluorescent tag makes it possible to visualize the
GPI analogs, which would facilitate many studies on GPI anchorage, such as
the implications of GPI structure variations, including the lipid composition
and the anomeric configuration, on the distribution, mobility, and other prop-
erties of GPIs in lipids and similar biological systems. The research results
should be of particular interest as GPI analogs 1–3 contain the GPI compo-
nents that are the most proximal to the lipid membrane. Moreover, the GPI
core pseudodisaccharide is believed to play a pivotal role in the biological func-
tion of GPI anchors.
EXPERIMENTAL SECTION
General Methods
Reagents were obtained from commercial sources and used without further
purification unless otherwise noted. Anhydrous solvents were obtained from
commercial sources or from an alumina column solvent purification system.
Molecular sieves were flame dried and used immediately after cooling under
dry conditions. Analytical TLC was preformed using glass-backed silica gel 60
˚
A plates (250 μm thickness). TLC plate detection was done using a UV lamp, by
charring with phosphomolybdic acid in EtOH, or by charring with 5% H2SO4
in EtOH. Column chromatography was carried out using standard-grade silica
1
gel (40–63 μm particle size). H NMR spectra were recorded at 400, 500, or
600 MHz with chemical shifts reported in ppm (δ) relative to CHCl3 (7.26 ppm)
or tetramethylsilane (0.00 ppm). 13C NMR spectra were recorded at 100 or
125 MHz relative to the 13C signal of CDCl3 (77.23 ppm). Coupling constants
(J) are reported in hertz (Hz). Mass spectrometry was performed using either a
Bruker Daltonics Ultraflex MALDI TOF MS or Waters LCT Premier XE high-
resolution ESI MS.

C. L. Johnson and Z. Guo
308
tert-Butyldiphenylsilyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
D-glucopyranoside (18)
To 17 (4.84 g, 13.0 mmol) stirring in anhydrous Et2O (80 mL) under an
Ar atmosphere was added benzylamine (21.3 mL, 194.7 mmol). After stir-
ring for 2 h, the reaction was quenched with 1 M aq. HCl solution and then
slowly poured into saturated aq. NaHCO3 solution. The aq. layer was extracted
three times with Et2O. Combined organic portions were washed with brine
and dried over MgSO4 before being concentrated under reduced pressure. The
residue was purified by silica gel column chromatography to give the corre-
sponding hemiacetal intermediate (3.56 g, 83%) as colorless syrup. To a solu-
tion of the intermediate (3.56 g, 10.8 mmol), imidazole (1.32 g, 19.4 mmol),
and catalytic 4-(dimethylamino)pyridine (DMAP, 16 mg, 0.13 mmol) stir-
ring in anhydrous DMF (70 mL) under an Ar atmosphere was added tert-
butylchlorodiphenylsilane (5.03 mL, 19.35 mmol). After stirring overnight,
DMF was removed under reduced pressure and the remaining residue was
diluted with EtOAc before pouring into distilled water. The aq. layer was
extracted three times with EtOAc and the combined organic portions were
washed with brine, dried over MgSO4, and concentrated under reduced pres-
sure. The remaining residue was purified by silica gel column chromatogra-
1
phy to give 18 (4.32 g, 71%) as colorless syrup. H NMR (400 MHz, CDCl3): δ
7.71–7.68 (m, 4H), 7.50–7.26 (m, 6H), 4.98–4.95 (t, J = 9.3, Hz, 1H), 4.90–4.85
(t, J = 9.3, Hz, 1H), 4.47–4.45 (d, J = 7.8 Hz, 1H, 1 position), 4.04–4.00 (dd, J
= 5.2, 12.4 Hz, 1H), 3.85–3.82 (dd, J = 2.4, 12.4Hz, 1H), 3.59–3.55 (dd, 7.8Hz,
J = 10.3, 1H), 3.29–3.25 (m, 1H), 2.07 (s, 3H), 1.95 (s, 6H), 1.12 (s, 9H). 13C
NMR (CDCl3, 100 MHz): δ 170.46, 169.99, 169.60, 135.85, 135.79, 132.12,
130.11, 129.89, 127.68, 127.39, 96.64, 72.65, 71.43, 68.58, 66.23, 61.89, 26.70,
20.68, 20.56, 20.55, 19.09.
tert-Butyldiphenylsilyl2-azido-2-deoxy-4,6-O-(p-methoxybenzy
lidene)-D-glucopyranoside, (19)
A solution of 18 (4.32 g, 7.59 mmol) in 0.05 M NaOMe in MeOH (40 mL)
was stirred at 0◦C for 40 min while being monitored by TLC. The reaction was
then neutralized to pH 6–7 with amberlyst acidic resin. The resin was removed
by filtration and the solution was condensed under reduced pressure to give
the triol as a white solid (3.27 g, 97%) that was used directly for the next step.
The crude triol was dissolved in anhydrous DMF under an Ar atmosphere be-
fore adding camphor sulfonic acid (176 mg, 0.76 mmol) and para-anisaldehyde
dimethyl acetal (1.70 mL, 9.87 mmol). The reaction was stirred overnight at
rt before dilution with EtOAc and poured into a saturated aq. NaHCO3 solu-
tion. The aq. layer was extracted three times with EtOAc and the combined
organic portions were washed with brine, dried over MgSO4, and concentrated

Synthetic GPI analogs
309
under reduced pressure. The remaining residue was purified by silica gel col-
1
umn chromatography to afford 19 (3.59 g, 84%) as colorless syrup. H NMR
(400 Hz, CDCl3): δ 7.71–7.69 (m, 4H), 7.46–7.26 (m, 6H), 5.42 (s, 1H), 4.53–4.51
(d, J = 7.6 Hz, 1H, 1 position), 3.98–3.90 (dd, J = 4.8, 10.8 Hz, 1H), 3.78 (s,
3H), 3.61–3.54 (m, 2H), 3.51–3.42 (m, 2H), 3.03–2.99 (m, 1H), 2.56–2.55 (d, J =
2.8 Hz, 1H), 1.12 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 135.86, 133.03, 132.35,
130.06, 129.87, 129.30, 129.03, 128.22, 127.62, 127.55, 127.41, 114.31, 113.70,
101.85, 97.23, 80.67, 72.16, 69.17, 68.23, 65.96, 55.29, 26.79, 19.11. HR ESI
MS: calcd. for C30H36N3O6 [M + Na]+ m/z, 562.2373; found, 562.2372.
2-Azido-2-deoxy-3-O-acetyl-4,6-O-(p-methoxybenzylidene)
-D-glucopyranose (20)
To 19 (2.615 g, 4.66 mmol) stirring in pyridine (20 mL) at 0◦C was added
acetic anhydride (4.4 mL, 46.6 mmol). The reaction was stirred for 1 h be-
fore pyridine and acetic anhydride were removed under reduced pressure. The
remaining residue was diluted with EtOAc before it was poured into a sat-
urated aq. NaHCO3 solution. The aq. layer was extracted three times with
EtOAc and the combined organic portions were washed with brine, dried over
MgSO4, and concentrated under reduced pressure. Purification of the remain-
ing residue by silica gel column chromatography gave the acetylated interme-
diate (2.50 g, 89%) as syrup. To facilitate TBDPS removal, a stock solution was
prepared containing 11.9 mL of 1.0 M TBAF in THF and 0.70 mL glacial acetic
acid. To the acetylated intermediate (2.49 g, 4.12 mmol) stirring in anhydrous
THF at 0◦C under an Ar atmosphere was added the freshly prepared, buffered
TBAF solution (5.8 mL, 5.48 mmol TBAF). After stirring for another 15 min,
the solvent was removed under reduced pressure and the remaining residue
was diluted with EtOAc before being poured into distilled water. The aq. layer
was extracted three times with EtOAc and the combined organic portions were
washed with brine, dried over MgSO4, and concentrated under reduced pres-
sure. The remaining residue was purified by silica gel column chromatogra-
1
phy to give 20 as a white solid (1.20 g, 80%). H NMR (400 MHz, CDCl3): δ
7.45–7.30 (m, 2H), 6.94–6.82 (m, 2H), 5.66–5.60 (t, J = 9.8 Hz, 1H), 5.48–5.42
(d, J = 5.4 Hz, 1H), 5.20–5.31 (t, J = 9.8 Hz, 1H), 4.84–4.78 (d, J = 7.8 Hz,
1H), 4.41–4.14 (m, 2H), 3.83–3.77 (s, 3H), 3.77–3.67 (m, 1H), 3.65–3.57 (m,
1H), 3.57–3.49 (m, 1H), 3.49–3.42 (dd, 7.8, 9.8 Hz, 1H), 3.37–3.31 (dd, J = 3.4,
10.2 Hz, 1H), 3.07–3.01 (s, 1H), 2.15–2.11 (s, 3H). 13C NMR (100 MHz, CDCl3):
of α:β mixture, δ 160.13, 132.02, 127.50, 127.42, 114.31, 113.59, 101.65, 101.49,
96.71, 83.18, 79.34, 78.48, 71.13, 68.99, 68.72, 68.28, 66.65, 65.80, 62.26, 55.59,
55.29, 20.87. HR ESI MS: calcd. for C16H19N3O7 [M+Na]+ m/z, 388.1121;
found, 388.1125.

C. L. Johnson and Z. Guo
310
6-O-[2-Azido-2-deoxy-3-O-acetyl-4,6-O-(p-methoxybenzylidene)-
α,β-glucopyranosyl]-1-O-allyl-2,3,4,5-tetra-O-(p-methoxy
benzyl)-myo-inositol (21)
To a solution of 20 (440 mg, 1.20 mmol) and trichloroacetonitrile (1.05 mL,
10.5 mmol) stirring in anhydrous CH2Cl2 (8 mL) was added 1 drop of DBU.
After stirring for 50 min, the solvent was removed under reduced pressure
and the remaining residue was purified with a Et3N-neutralized silica gel col-
umn (1% Et3N in mobile phase) to give trichloroacetimidate 2-azido-2-deoxy-
3-O-acetyl-4,6-O-(p-methoxybenzylidene)-D-glucopyranose (11) (503 mg, 82%)
as syrup, which was immediately used for the next step of reaction. After a
mixture of freshly prepared 11 (503 mg, 0.99 mmol), 12 (400 mg, 0.58 mmol),
˚
and flame-dried MS 4 A (200 mg) in anhydrous toluene and dioxane (3:2,
7 mL) was stirred for 1 h under an Ar atmosphere at rt, it was cooled to
◦
−10 C, and TMSOTf (24 μL, 0.1 mmol) was added while the reaction was
monitored by TLC. After stirring for another 25 min, the reaction mixture
˚
was neutralized with Et3N, filtered through celite to remove MS 4 A, concen-
trated under reduced pressure, and then purified by silica gel column chro-
matography to give 21 as an α:β mixture (497 mg, 81%). 1H NMR of α-anomer
(400 MHz, CDCl3): δ 7.37–7.27 (m, 6H), 7.27–7.19 (m, 4H), 6.93–6.80 (m, 10H),
5.94–5.83 (m, 1H), 5.43–5.40 (s, 1H), 5.35–5.27 (dd, J = 2.0, 17.6 Hz, 1H),
5.19–5.13 (dd J = 1.5, 10.8 Hz, 1H), 5.13–5.02 (m, 2H), 4.97–4.86 (m, 2H),
4.77–4.74 (d, J = 4.4 Hz, 1H, 1 position), 4.74–4.65 (m, 2H), 4.58–4.50 (m,
2H), 4.37–4.31 (t, J = 9.3 Hz, 1H), 4.28–4.21 (m, 1H), 4.22–4.15 (dd, J = 4.9,
13.2 Hz, 1H), 4.08–3.93 (m, 3H), 3.85–3.75 (m, 12H), 3.76–3.68 (t, J = 10.3 Hz,
1H), 3.59–3.47 (m, 2H), 3.45–3.39 (dd, J = 8.3, 9.8 Hz, 1H), 3.37–3.31 (2.0,
9.8 Hz, 1H), 3.26–3.20 (m, 1H), 3.20–3.14 (dd, J = 2.0, 9.8 Hz, 1H), 2.14–2.10 (s,
3H). 13C NMR of α-anomer (100 MHz, CDCl3): δ 159.27, 159.19, 159.11, 135.19,
130.89, 130.81, 129.90, 129.84, 129.49, 129.42, 129.20, 116.20, 113.94, 113.78,
113.57, 100.82, 83.33, 81.64, 80.54, 78.33, 75.40, 74.08, 72.32, 72.20, 64.44,
55.26, 29.69, 21.05, 20.97, 14.19. MALDI-TOF MS: calcd. for C57H65N3NaO16
[M+Na]+ m/z, 1070.4; found, 1070.1.
6-O-[2-Azido-2-deoxy-3-O-(p-methoxybenzyl)-4,6-O-(p-methoxy
benzylidene)-α,β-D-glucopyranosyl]-1-O-allyl-2,3,4,5-tetra-
O-(p-methoxybenzyl)-myo-inositol (22)
To 21 (497 mg, 0.47 mmol) stirring in MeOH and CH2Cl2 (8:1, 9 mL) at
rt was added freshly prepared 2 M NaOMe in MeOH (300 μL, 0.60 mmol).
After stirring for 3.5 h, the solvent was removed under reduced pressure and
the remaining residue was purified by silica gel column chromatography to
give the corresponding deacetylated intermediate (428 mg, 90%). To the inter-
mediate (103 mg, 0.102 mmol) stirring in anhydrous DMF (1.5 mL) under an

Synthetic GPI analogs
311
Ar atmosphere at 0◦C was added NaH (60% dispersion in mineral oil, 8 mg,
0.20 mmol). After stirring for 30 min at 0◦C, p-methoxybenzyl chloride (27 μL,
0.20 mmol) was added, and the mixture was stirred overnight at rt. DMF was
then removed under reduced pressure, and the remaining residue was diluted
with EtOAc and poured into distilled water. The aq. layer was extracted three
times with EtOAc and the combined organic portions were washed with brine,
dried over MgSO4, and concentrated under reduced pressure. The remaining
residue was purified by silica gel column chromatography to give 22 (108 mg,
94%). 1H NMR of α-anomer (400 MHz, CDCl3): δ 7.46–7.17 (m, 12H), 7.00–6.76
(m, 12H), 5.94–5.79 (m, 1H), 5.54–5.48 (s, 1H), 5.35–5.25 (dd, 1.5, 17.1 Hz,
1H), 5.18–5.10 (dd, J = 1.0, 10.3 Hz, 1H), 4.96–4.66 (m, 9H), 4.57–4.50 (m,
2H), 4.42–4.13 (m, 3H), 4.10–3.92 (m, 3H), 3.87–3.71 (m, 18H), 3.67–3.59 (t, J
= 9.3 Hz, 2H), 3.52–3.44 (m, 2H), 3.41–3.29 (m, 3H), 3.23–3.13 (m, 3H). 13C
NMR of α-anomer (100 MHz, CDCl3): δ 159.56, 159.18, 159.14, 159.04, 135.30,
131.99, 130.86, 129.82, 129.59, 129.50, 129.44, 129.19, 116.00, 114.30, 113.76,
113.55, 113.48, 101.21, 83.45, 82.12, 81.67, 80.50, 78.43, 75.26, 75.22, 74.72,
74.07, 74.06, 72.32, 72.25, 70.30, 66.62, 61.79, 55.59, 55,26, 29.69. HR ESI MS:
calcd. for C63H71N3O16 [M+Na]+ m/z, 1148.4732; found, 1148.4749.
6-O-[2-Azido-2-deoxy-3,6-di-O-(p-methoxybenzyl)-α,β-D-gluco
pyranosyl]-1-O-allyl-2,3,4,5-tetra-O-(p-methoxybenzyl)-myo-
inositol (6a and 6b)
To a mixture of 22 (209 mg, 0.19 mmol), NaBH3CN (117 mg, 1.86 mmol),
˚
MS 4A (150 mg), and methyl orange indicator stirring in anhydrous THF
(8 mL) at 0◦C under an Ar atmosphere was added an HCl solution (1 M in
Et2O) dropwise until pH ≈1 was reached (the reaction mixture became a red
color as indicated by methyl orange). After stirring for 45 min, the reaction
was quenched by adding ice cold saturated aq. NaHCO3 solution. The aq.
layer was extracted thre times with EtOAc and the combined organic por-
tions were washed with brine, dried over MgSO4, and concentrated under re-
duced pressure. The remaining residue was purified by silica gel chromatog-
raphy to give 6a and 6b (95.8 mg α-anomer, 72.2 mg β-anomer, 80% overall
yield). 1H NMR of 6a (400 MHz, CDCl3): 7.38–7.29 (m, 4H), 7.29–7.19 (m, 4H),
7.19–7.10 (m, 4H), 6.92–6.81 (m, 8H), 6.81–6.73 (m, 4H), 5.99–5.87 (m, 1H),
5.71–5.68 (d, J = 3.4 Hz, 1H, 1 position), 5.30–5.23 (dd, J = 1.5, 17.1 Hz,
1H), 5.20–5.15 (J = 1.5, 11.7 Hz, 1H), 4.96–4.69 (m, 7H), 4.64–4.51 (m, 3H),
4.39–4.33 (d, J = 11.7 Hz, 1H), 4.24–4.15 (m, 2H), 4.11–3.93 (m, 6H), 3.84–3.72
(m, 18H), 3.72–3.63 (m, 2H), 3.63–3.59 (dd, J = 3.4, 4.9 Hz, 1H), 3.41–3.30 (m,
3H), 3.29–3.25 (m, 2H), 3.23–3.17 (dd, J = 3.4, 10.3 Hz, 1H). 13C NMR of 6a
(100 MHz, CDCl3): δ 159.19, 159.10, 159.04, 158.91, 134.34, 130.42, 129.81,
129.26, 129.15, 127.94, 116.95, 113.93, 113.77, 113.67, 113.57, 97.43, 81.94,
81.72, 81.30, 80.64, 75.30, 75.21, 74.94, 74.45, 73.63, 73.00, 72.38, 72.27, 70.85,

C. L. Johnson and Z. Guo
312
69.28, 68.97, 68.87, 68.75, 62.87, 55.28, 55.26, 55.24, 55.21. HR ESI MS: calcd.
1
for C63H73N3O16 [M+Na]+ m/z, 1150.4889; found, 1150.4911. H NMR of 6b
(500 MHz, CDCl3): δ 7.39–7.23 (m, 12H), 6.96–6.78 (m, 12H), 5.92–5.82 (m,
1H), 5.31–5.25 (dd, J = 1.5, 17.4 Hz, 1H), 5.13–5.07 (d, J = 10.7 Hz, 1H),
5.00–4.73 (m, 7H), 4.61–4.47 (m, 4H), 4.41–4.35 (t, J = 9.5 Hz, 1H), 4.30–4.23
(dd, J = 4.9, 13.4 Hz, 1H), 4.09–3.95 (m, 4H), 3.87–3.76 (m, 18H), 3.75–3.70
(dd, J = 4.9, 10.7 Hz, 1H), 3.69–3.64 (m, 2H), 3.57–3.52 (t, J = 9.2 Hz, 1H),
3.40–3.33 (m, 2H), 3.31–3.26 (m, 1H), 3.24–3.18 (m, 2H), 2.89 (s, 1H). 13C
NMR of 6b (125 MHz, CDCl3): δ 159.43, 159.25, 159.18, 159.16, 159.10, 158.90,
135.53, 131.09, 130.88, 130.48, 130.25, 130.00, 129.90, 129.76, 129.52, 129.50,
129.42, 129.23, 115.81, 113.96, 113.81, 113.80, 113.78, 113.76, 113.42, 101.07,
83.76, 82.05, 81.77, 80.45, 78.78, 78.04, 75.29, 75.05, 74.91, 74.65, 73.70, 73.67,
73.51, 72.94, 72.30, 72.13, 69.94, 66.41, 55.28, 55.24. HR ESI MS: calcd. for
C63H73N3O16 [M+Na]+ m/z, 1150.4889; found, 1150.4844.
2-(2-Hydroxyethoxy)ethyl-p-methylbenzenesulfonate (24)
To a solution of commercially available diethyleneglycol 23 (4.0 g,
37.7 mmol) and p-toluenesulfonyl chloride (7.55 g, 39.6 mmol) stirring in anhy-
drous CH2Cl2 under an Ar atmosphere at 0◦C was slowly added Et3N (15.8 mL,
113.1 mmol). After stirring overnight at rt, the reaction was diluted with
CH2Cl2 and poured into a saturated aq. NaHCO3 solution. The aq. layer was
extracted three times with CH2Cl2, and the combined organic portions were
washed with brine, dried over MgSO4, and concentrated under reduced pres-
sure. The product mixture was purified by silica gel column chromatography to
give 24 (3.56 g, 36%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.83–7.76 (d,
J = 8.31 Hz, 2H), 7.37–7.30 (d, J = 7.34 Hz, 2H), 4.21–4.15 (m, 2H), 3.70–3.63
(m, 4H), 3.54–3.49 (m, 2H), 2.46–2.40 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
144.95, 129.84, 127.92, 72.46, 68.54, 61.61, 21.63.
2-(2-Azidoethoxy)ethanol (25)
A solution of 24 (738 mg, 2.84 mmol) and NaN3 (239 mg, 3.60 mmol) in
CH3CN was stirred for 36 h at 80◦C. A white precipitate was formed and re-
moved by filtration. The precipitate was washed three times with cold CH3CN.
The filtrate was evaporated under reduced pressure, diluted with CH2Cl2,
poured into a saturated aq. NaHCO3 solution, and extracted three times with
CH2Cl2. The combined organic portions were washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The remaining residue was
purified by silica gel column chromatography to give 25 (239 mg, 64%) as col-
orless oil. 1H NMR (400 MHz, CDCl3): δ 3.75–3.69 (m, 2H), 3.69–3.64 (m, 2H),
3.61–3.56 (m, 2H), 3.41–3.36 (m, 2H), 2.59–2.00 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 72.42, 69.99, 61.69, 50.69.

Synthetic GPI analogs
313
2-(2-Azidoethoxy)ethyl triflouromethanesulfonate (8)
To a solution of 25 (19.5 mg, 148.7 μmol) and Et3N (3 drops) stirring in an-
hydrous CH2Cl2 (1 mL) at 0◦C under an Ar atmosphere was slowly added tri-
flouromethanesulfonic anhydride (60 μL, 354 μmol). After stirring for 25 min,
the reaction mixture was diluted with CH2Cl2 and poured into saturated aq.
NaHCO3 solution. The aq. layer was extracted three times with CH2Cl2 and
the combined organic portions were washed with brine, dried over MgSO4,
and concentrated under reduced pressure. Compound 8 was obtained (32.6 mg,
83%) as colorless oil and used directly for the next step without further purifi-
cation. 1H NMR (400 MHz, CDCl3): δ 4.66–4.62 (t, J = 4.40 Hz, 2H), 3.85–3.81
(t, J = 4.40 Hz, 2H), 3.72–3.68 (t, J = 4.89 Hz, 2H), 3.44–3.38 (t, J = 4.89 Hz,
2H).
1-O-(tert-Butyldiphenylsilyl)-sn-glycerol (27)
To a solution of commercially available 26 (4.19 g, 31.60 mmol) and imi-
dazole (8.60 g, 126.0 mmol) stirring in anhydrous DMF (20 mL) under an Ar
atmosphere at rt was slowly added tert-butylchlorodiphenylsilane (16.4 mL,
63.2 mmol). After stirring overnight, the solvent was removed under reduced
pressure and the remaining residue was diluted with EtOAc and poured into
distilled water. The aq. layer was extracted three times with EtOAc and the
combined organic portions were washed with brine, dried over MgSO4, and
concentrated under reduced pressure. To a solution of the crude TBDPS-
protected intermediate stirring in THF and H2O (4:1, 20 mL) at 0◦C was added
TFA (1 mL). After stirring for 3 days at rt, the reaction was cooled to 0◦C
and saturated aq. NaHCO3 solution (100 mL) was added slowly. After stirring
for 15 min at 0◦C, the aq. layer was extracted three times with EtOAc. The
combined organic portions were washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The remaining residue was purified by
silica gel column chromatography to give 27 (5.77 g, 55%). 1H NMR (400 MHz,
CDCl3): δ 7.71–7.65 (d, J = 7.3 Hz, 4H), 7.48–7.37 (m, 6H), 3.85–3.78 (m, 1H),
3.76–3.66 (m, 2H), 3.66–3.60 (m, 1H), 2.65–2.59 (s, 2H), 1.12–1.04 (s, 9H). 13
C
NMR (100 MHz, CDCl3): δ 135.52, 132.93, 129.93, 127.85, 71.97, 65.20, 63.84,
26.86, 19.23.
1-O-(tert-Butyldiphenylsilyl)-2,3-di-O-oleoyl-sn-glycerol (28a)
To a solution of 27 (532 mg, 1.61 mmol), oleic acid (1.0 g, 3.54 mmol),
and catalytic DMAP (19.5 mg 0.16 mmol) stirring in CH2Cl2 and DMF (4:1,
16 mL) under an Ar atmosphere at rt was added EDC hydrochloride (679 mg,
3.54 mmol). After stirring overnight, the solvent was removed under reduced

C. L. Johnson and Z. Guo
314
pressure and the remaining residue was diluted with CH2Cl2, poured into dis-
tilled water, and extracted three times with CH2Cl2. The combined organic por-
tions were washed with brine, dried over MgSO4, and concentrated under re-
duced pressure. The remaining residue was purified by silica gel column chro-
matography to give 28a (1.13 g, 81%). 1H NMR (400 MHz, CDCl3): δ 7.69–7.63
(m, 4H), 7.46–7.35 (m, 6H) 5.39–5.29 (m, 4H), 5.20–5.14 (m, 1H), 4.43–4.38
(dd, J = 4.0, 11.9, 1H), 4.24–4.19 (dd, J = 6.1, 11.6, 1H), 3.80–3.72 (m, 2H),
2.33–2.21 (m, 4H), 2.04–1.97 (m, 8H), 1.63–1.56 (m, 4H), 1.36–1.22 (m, 40H),
1.08–1.03 (s, 9H), 0.90–0.86 (t, 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ
173.33, 172.94, 135.51, 133.04, 133.00, 129.98, 129.80, 129.70, 127.74, 127.72,
71.49, 62.37, 62.31, 34.30, 34.11, 31.92, 29.78, 29.72, 29.53, 29.33, 29.22, 29.20,
29.12, 27.23, 27.18, 26.71, 24.91, 24.87, 22.69, 19.22, 14.12.
1-O-(tert-Butyldiphenylsilyl)-2,3-di-O-stearyl-sn-glycerol (28b)
To a solution of 27 (1.258 g, 3.81 mmol), stearic acid (2.40 g, 8.37 mmol),
and catalytic DMAP (92.8 mg 0.76 mmol) stirring in CH2Cl2 and DMF (4:1,
40 mL) under an Ar atmosphere at rt was added EDC hydrochloride (1.60 g,
8.37 mmol). After stirring overnight, the solvent was removed under reduced
pressure. The remaining residue was diluted with CH2Cl2, poured into dis-
tilled water, and extracted three times with CH2Cl2. The combined organic
portions were washed with brine, dried over MgSO4, and concentrated un-
der reduced pressure. The remaining residue was purified by silica gel col-
1
umn chromatography to give 28b (2.19g, 67%) H NMR (400 MHz, CDCl3): δ
7.71–7.63 (m, 4H), 7.47–7.36 (m, 6H), 5.22–5.15 (m, 1H), 4.45–4.39 (dd, J =
3.91, 11.74 Hz, 1H), 4.26–4.20 (dd, J = 6.36, 11.74 Hz, 1H), 3.83–3.73 (m, 2H),
2.32–2.24 (m, 4H), 1.65–1.55 (m, 4H), 1.38–1.20 (s, 56H), 1.08–1.05 (s, 9H),
0.92–0.87 (t, J = 6.85 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 173.38, 172.99,
135.57, 135.51, 133.04, 133.00, 129.98, 129.80, 129.70, 127.74, 127.72, 71.49,
62.37, 62.30, 34.32, 34.14, 31.94, 29.72, 29.70, 29.68, 29.65, 29.50, 29.38, 29.31,
29.30, 29.15, 29.13, 26.71, 24.93, 24.89, 22.70, 19.22, 14.12.
2,3-di-O-Oleoyl-sn-glycerol (29a)
To a solution of 28a (441 mg, 0.513 mmol) in CH3CN, THF, and CH2Cl2
(1:1:1, 6 mL) stirring under an Ar atmosphere at rt was slowly added
Et3N•3HF (1.0 mL). After stirring for 24 h, the reaction mixture was cooled
to 0◦C and quenched with saturated aq. NaHCO3 solution. After stirring for
30 min at 0◦C, the quenched reaction mixture was poured into saturated aq.
NaHCO3 solution and extracted three times with CH2Cl2. The combined or-
ganic portions were dried over MgSO4, concentrated under reduced pressure,
and purified by silica gel column chromatography to give 28a (233 mg, 73%).
1H NMR (400 MHz, CDCl3): δ 5.40–5.28 (m, 4H), 5.11–5.04 (pent., J = 4.9 Hz,

Synthetic GPI analogs
315
1H), 4.35–4.28 (dd, J = 4.9, 12.2 Hz, 1H), 4.26–4.20 (dd, J = 5.9, 12.2 Hz, 1H),
3.74–3.70 (d, 4.89 Hz, 2H), 2.37–2.28 (q, J = 8.3 Hz, 4H), 2.06–1.95 (m, 8H),
1.67–1.57 (m, 4H), 1.37–1.20 (m, 40H), 0.91–0.85 (t, J = 6.4 Hz, 6H).
1-O-(Phosphonooxyl)-2,3-di-O-oleoyl-sn-glycerol (9)
A mixture of 29a and phosphonic acid were coevaporated with anhydrous
pyridine three times and then dried under reduced pressure for over 2 h. The
resulting mixture was dissolved in 5 mL of anhydrous pyridine under an Ar
atmosphere before the addition of pivaloyl chloride (20 μL, 0.161 mmol). The
reaction mixture was stirred overnight at rt before the solvent was removed
under reduced pressure. The residue was purified by silica gel column chro-
matography (1% Et3N in mobile phase) to give 9 as a Et3N salt (64.5 mg, 76%).
1H NMR (400 MHz, CDCl3): δ 11.67–11.34 (s, broad, 3H, Et3NH+), 7.57–7.53
(s, 0.5H), 5.94–5.90 (s, 0.5H), 5.30–5.20 (m, 4H), 5.15–5.07 (m, 1H), 4.30–4.23
(dd, J = 4.1, 15.4 Hz, 1H), 4.11–4.03 (dd, J = 6.5, 12.2 Hz, 1H), 4.00–3.92
(m, 2H), 3.09–2.99 (m, 25.5H, Et3N), 2.25–2.17 (m, 4H), 1.95–1.87 (m, 8H),
1.55–1.45 (m, 4H), 1.35–1.27 (t, J = 7.3 Hz, 38.2 Hz, Et3N), 1.26–1.11 (m,
40H), 0.82–0.75 (t, J = 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 173.23,
172.83, 129.89, 129.62, 69.98, 69.90, 62.26, 62.13, 52.83, 45.89 (Et3N), 34.14,
33.95, 31.81, 29.67, 29.64, 29.44, 29.23, 29.12, 29.05, 28.99, 27.09, 24.76, 22.60,
14.05, 8.62 (Et3N). 31P NMR (160 MHz, CDCl3): δ 4.30.
2,3-di-O-Stearyl-sn-glycerol (29b)
To a solution of 28b (359 mg, 0.42 mmol) in CH3CN, THF, and CH2Cl2
(1:1:1, 6 mL) stirring under an Ar atmosphere at rt was added Et3N•3HF
(1.0 mL). After stirring for 24 h, the reaction mixture was cooled to 0◦C and
quenched with saturated aq. NaHCO3 solution. After stirring for 30 min at
0◦C, the quenched reaction mixture was poured into a saturated aq. NaHCO3
solution and extracted three times with CH2Cl2. The combined organic por-
tions were dried over MgSO4, concentrated under reduced pressure, and puri-
1
fied by silica gel column chromatography to give 29b (206 mg, 78%). H NMR
(400 MHz, CDCl3): δ 5.12–5.05 (m, 1H), 4.35–4.29 (dd, J = 4.9, 12.2 Hz, 1H),
4.27–4.21 (dd, J = 5.7, 12.2 Hz, 1H), 3.75–3.71 (d, J = 5.7 Hz, 2H), 2.38–2.29
(q, J = 7.30 Hz, 4H), 1.66–1.57 (m, 4H), 1.36–1.18 (broad s, 56H), 0.90–0.85 (t,
J = 6.5 Hz, 6H).
1-O-(Phosphonooxyl)-2,3-di-O-stearyl-sn-glycerol (10)
A mixture of 29b and phosphonic acid were coevaporated with anhydrous
pyridine three times and dried under reduced pressure for over 2 h. The
resulting mixture was dissolved in 5 mL of anhydrous pyridine under an Ar
atmosphere before the addition of pivaloyl chloride (62 μL, 0.50 mmol). The
reaction mixture was stirred overnight at rt. The solvent was removed under

C. L. Johnson and Z. Guo
316
reduced pressure and the residue was purified by silica gel column chromatog-
raphy (1% Et3N in mobile phase) to give 10 as an Et3N salt (161.4 mg, 71%).
1H NMR (400 MHz, CDCl3): δ 11.81–11.65 (s, broad, 4H), 7.50–7.47 (s, 0.5H),
5.95–5.91 (s, 0.5H), 5.13–5.05 (m, 1H), 4.28–4.21 (dd, J = 3.2, 11.35 Hz, 1H),
4.08–4.01 (dd, J = 6.49, 12.16 Hz, 1H), 3.89–3.83 (dd, J = 5.7, 8.1 Hz, 2H),
3.05–2.97 (m, 34H, Et3N), 2.21–2.13 (q, J = 7.3 Hz, 4H), 1.52–1.41 (m, 4H),
1.31–1.26 (t, J = 7.3 Hz, 50H, Et3N), 1.17–1.11 (s, broad, 56H), 0.78–0.73 (t,
J = 7.3 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 173.29, 172.91, 62.37, 45.78
(Et3N), 34.20, 33.99, 31.81, 29.60, 29.41, 29.26, 29.02, 24.78, 22.78, 22.58,
14.02, 8.62 (Et3N). 31P NMR (160 MHz, CDCl3): δ 4.08
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-3,6-di-O-(p-methoxy
benzyl)-α-D-glucopyranosyl]-1-O-allyl-2,3,4,5-tetra-O-(p-
methoxybenzyl)-myo-inositol (30a) and 6-O-[2-N-(tert-butyl
carbamate)-2-deoxy-3,6-di-O-(p-methoxybenzyl)-β-D-
glucopyranosyl]-1-O-allyl-2,3,4,5-tetra-O-(p-methoxybenzyl)-
myo-inositol (30b)
To a mixture of 6a (138 mg, 0.122 mmol) and zinc powder (200 mg,
3.05 mmol) stirring in anhydrous CH2Cl2 (7 mL) under an Ar atmosphere at
rt was added acetic acid (∼100 μL). After stirring for 1 h, the reaction was
diluted with CH2Cl2, poured into saturated aq. NaHCO3, and extracted four
times with CH2Cl2. The combined organic portions were dried over MgSO4 and
concentrated under reduced pressure. The remaining residue was dissolved in
MeOH and CH2Cl2 (1:1, 10 mL) before the addition of di-tert-butyl dicarbonate
(56 μL, 0.244 mmol) and Et3N (100 μL). After stirring overnight, the solvent
was removed under reduced pressure and the remaining residue was purified
1
by silica gel column chromatography to give 30a (115 mg, 78% two steps). H
NMR for 30a (500 MHz, CDCl3): δ 7.37–7.10 (m, 12H), 6.93–6.77 (m, 12H),
5.96–5.85 (m, 1H), 5.66–5.60 (d, J = 9.8 Hz, 1H), 5.40–5.35 (d, J = 3.1 Hz,
1H, 1 position), 5.33–5.25 (d, J = 17.4 Hz, 1H), 5.22–5.16 (d, J = 10.7 Hz, 1H),
4.91–4.85 (d, J = 10.4 Hz, 1H), 4.85–4.79 (d, J = 9.8 Hz, 1H), 4.79–4.71 (m, 5H),
4.69–4.63 (d, J = 11.0 Hz, 1H), 4.61–4.51 (dd, J = 11.3, 23.2 Hz, 2H), 4.42–4.37
(d, J = 11.3 Hz, 1H), 4.28–4.23 (d, J = 11.6 Hz, 1H), 4.17–4.12 (t, J = 9.8 Hz,
1H), 4.07–3.99 (m, 3H), 3.99–3.88 (m, 3H), 3.87–3.75 (m, 18H), 3.75–3.67 (m,
2H), 3.56–3.43 (m, 3H), 3.38–3.26 (m, 3H), 3.24–3.18 (d, J = 9.5 Hz, 1H),
2.54–2.49 (s, 1H), 1.50–1.40 (s, 9H); 13C NMR for 30a (125 MHz, CDCl3): δ
155.41, 134.22, 131.04, 131.01, 130.80, 130.41, 130.19, 129.57, 129.50, 129.48,
129.46, 129.25, 129.21, 117.82, 113.79, 113.75, 113.66, 113.60, 99.70, 82.45,
81.83, 80.64, 80.50, 80.43, 79.04, 77.72, 75.33, 75.10, 73.56, 74.36, 73.12, 72.36,
71.82, 71.75, 70.91, 70.66, 69.90, 55.30, 55.26, 55.22, 54.21, 28.55. HR ESI MS:
calcd. for C68H83NO18 [M+Na]+ m/z, 1224.5508; found, 1224.5511. Compound

Synthetic GPI analogs
317
6b underwent an analogous procedure to give 30b (64 mg, 65% two steps). 1H
NMR for 30b (500 MHz, CDCl3): δ 7.36–7.17 (m, 12H), 6.94–6.77 (m, 12H),
5.90–5.81 (m, 1H), 5.28–5.21 (dd, J = 1.5, 18.9 Hz, 1H), 5.09–5.01 (t, J =
9.2 Hz, 2H), 4.89–4.82 (m, 2H, includes 1-pos), 4.82–4.73 (dd, J = 11.6, 20.1 Hz,
2H), 4.71–4.69 (d, J = 10.1 Hz, 1H), 4.65–4.60 (d, J = 11.0 Hz, 1H), 4.58–4.46
(m, 6H), 4.33–4.24 (m, 2H), 4.21–4.15 (t, J = 9.2 Hz, 1H), 4.04–3.93 (m, 3H),
3.83–3.76 (m, 18H), 3.71–3.68 (d, J = 4.6 Hz, 2H), 3.58–3.52 (t, J = 8.9 Hz,
1H), 3.41–3.30 (m, 4H), 3.26–3.19 (m, 1H), 3.19–3.13 (d, J = 9.5 Hz, 1H), 2.69
(s, 1H), 1.44 (s, 9H). 13C NMR for 30b (125 MHz, CDCl3): δ 159.21, 159.17,
159.13, 158.94, 158.92, 135.65, 131.21, 130.84, 130.52, 130.15, 129.58, 129.55,
129.44, 129.16, 128.17, 115.61, 113.90, 113.83, 113.75, 113.44, 101.35, 81.49,
80.48, 79.36, 79.18, 75.28, 74.61, 73.82, 73.70, 73.41, 72.65, 72.36, 72.12, 70.42,
55.24, 28.47. HR ESI MS: calcd. for C68H83NO18 [M+Na]+ m/z, 1224.5508;
found 1224.5465.
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)
ethyl)-3,6-di-O-(p-methoxybenzyl)-α-D-glucopyranosyl]-1-O-
allyl-2,3,4,5-tetra-O-(p-methoxybenzyl)-myo-inositol (31a)
and 6-O-[2-N-(tert-butylcarbamate)-2-deoxy-4-O-(2-(2-azido
ethoxy)ethyl)-3,6-di-O-(p-methoxybenzyl)-β-D-glucopyran
osyl]-1-O-allyl-2,3,4,5-tetra-O-(p-methoxybenzyl)-myo-
inositol (31b)
To 30a (114 mg, 94.8 μmol) stirring in anhydrous THF at 0◦C was added
NaH (23 mg of 60% dispersion in mineral oil, ∼570 μmol). After stirring for
30 min, 8 was added from a stock solution containing 103 mg of 8 in anhy-
drous THF (initially 16.8 mg, 64 μmol) and the reaction was monitored by
TLC. Additional 30- to 50-μL portions from the stock solution were added
(estimated ∼2 eq. with respect to 30a) until TLC showed a nearly complete
reaction (alkylation of Boc-protected amine was observed by HR ESI-MS in
initial experiments upon using a larger excess of 8). The reaction was then di-
luted with EtOAc, poured into distilled water, and extracted four times with
EtOAc. The combined organic portions were washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The remaining residue was
1
purified by silica gel chromatography to give 31a (104 mg, 83%). H NMR
(500 MHz, CDCl3): δ 7.37–7.11 (m, 12H), 6.91–6.77 (m, 12H), 5.92–5.82 (m,
1H), 5.62–5.56 (d, J = 10.1 Hz, 1H), 5.37–5.33 (d, J = 2.8 Hz, 1 position, 1HH),
5.30–5.23 (d, J = 17.4 Hz, 1H), 5.19–5.13 (d, J = 10.1 Hz, 1H), 4.91–4.85 (d,
J = 10.1 Hz, 1H), 4.84–4.80 (d, J = 9.8 Hz, 1H), 4.80–4.70 (m, 5H), 4.70–4.66
(d, J = 11.0 Hz, 1H), 4.61–4.51 (dd, J = 11.6, 23.2 Hz, 2H), 4.50–4.44 (d, J =
11.6 Hz, 1H), 4.23–4.17 (d, J = 11.6 Hz, 1H), 4.16–4.09 (t, J = 9.5 Hz, 1H),
4.06–4.00 (t, J = 9.5, 1H), 4.00–3.93 (m, 3H), 3.93–3.85 (m, 3H), 3.85–3.74 (m,

C. L. Johnson and Z. Guo
318
18H), 3.62–3.52 (m, 5H), 3.52–3.43 (m, 3H), 3.42–3.36 (d, J = 10.7 Hz, 1H),
3.36–3.23 (m, 5H), 3.20–3.13 (d, J = 9.8 Hz, 1H), 1.49–1.38 (s, 9H). 13C NMR
(125 MHz, CDCl3): δ 159.20, 159.08, 159.05, 159.01, 158.95, 155.41, 134.21,
131.13, 131.04, 130.85, 130.81, 130.53, 130.41, 129.56, 129.48, 129.44, 129.25,
129.23, 117.77, 113.77, 113.74, 113,57, 113.54, 99.83, 82.46, 81.85, 80.74,
80.49, 80.43, 78.86, 78.56, 77.91, 75.33, 75.01, 74.48, 69.76, 68.09, 55.29, 55.26,
55.22, 54.85, 50.70, 28.55. HR ESI-MS: calcd. for C72H90N4O19 [M+Na]+ m/z,
1337.6097; found, 1337.6078. Compound 30b underwent an analogous pro-
1
cedure to give 31b (51.1 mg, 73%). H NMR for 31b (500 MHz, CDCl3): δ
7.36–7.16 (m, 12H), 6.95–6.73 (m, 12H), 5.89–5.80 (m, 1H), 5.27–5.19 (dd, J
= 1.8, 17.4 Hz, 1H), 5.07–4.99 (m, 2H), 4.89–4.44 (m, 12H, includes 1 position),
4.38–4.30 (m, 1H), 4.26–4.19 (t, J = 9.2, 1H), 4.19–4.12 (broad s, 1H), 4.04–3.94
(m, 4H), 3.92–3.86 (m, 2H), 3.85–3.74 (m, 18H), 3.73–3.66 (m, 3H), 3.60–3.54
(m, 2H), 3.54–3.49 (m, 2H), 3.42–3.27 (m, 8H), 3.19–3.13 (d, J = 9.46, 1H), 1.43
(s, 9H). 13C NMR (125 MHz, CDCl3): δ 159.12, 158.89, 155.46, 135.80, 131.33,
131.24, 130.91, 130.87, 130.74, 130.54, 129.81, 129.58, 129.52, 129.31, 129.16,
128.11, 115.59, 113.91, 113.74, 113.73, 113.68, 113.59, 113.42, 101.39, 83.90,
81.84, 81.56, 80.50, 79.23, 79.02, 78.79, 75.27, 75.21, 74.92, 74.54, 74.36, 73.70,
73.19, 72.57, 72.08, 71.73, 70.79, 69.83, 68.37, 57.68, 55.28, 55.25, 55.23, 55.21,
50.68, 28.47. HR ESI MS: calcd. for C72H90N4O19 [M+Na]+ m/z, 1337.6097;
found, 1337.6099.
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)
ethyl)-3,6-di-O-(p-methoxybenzyl)-α-D-glucopyranosyl]-2,3,4,
5-tetra-O-(p-methoxybenzyl)-myo-inositol (32a) and 6-O-[2-N-
(tert-butylcarbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)ethyl)
-3,6-di-O-(p-methoxybenzyl)-β-D-glucopyranosyl]-2,3,4,5-
tetra-O-(p-methoxybenzyl)-myo-inositol (32b)
A solution of [Ir(CODPMe2Ph2)2]PF6 (6.0 mg, 7 μmol) in anhydrous THF
(1.5 mL) was stirred under an H2 atmosphere until the color of the solu-
tion turned from red to pale yellow (∼10 min). The H2 atmosphere was ex-
changed for Ar and a solution of 31a in anhydrous THF (1 mL) was added
to the catalyst solution. After stirring for 1.5 h, the solvent was removed un-
der reduced pressure and the remaining residue was diluted with acetone and
H2O (9:1, 1.5 mL) followed by the addition of HgCl2 and HgO. After stirring
for 10 min, the solvent was removed and the remaining residue was purified
1
by silica gel chromatography to give 32a (89.5 mg, 90%). H NMR for 32a
(500 MHz, CDCl3): δ 7.33–7.18 (m, 12H), 6.19–6.77 (m, 12H), 5.37–5.33 (m,
1H), 5.33–5.29 (d, J = 2.4 Hz, 1H), 5.00–4.95 (d, J = 11.3Hz, 1H), 4.88–4.84 (d,
J = 10.4 Hz, 1H), 4.82–4.77 (d, J = 9.8 Hz, 1H), 4.75–4.72 (d, J = 6.1 Hz, 1H),
4.72–4.70 (d, J = 6.4 Hz, 1H), 4.69–4.61 (m, 4H), 4.58–4.53 (d, J = 11.0 Hz,

Synthetic GPI analogs
319
1H), 4.46–4.41 (d, J = 11.9 Hz, 1H), 4.20–4.15 (d, J = 11.6 Hz, 1H), 4.00–3.95
(t, J = 9.5 Hz, 1H), 3.95–3.91 (m, 1H), 3.90–3.84 (m, 3H), 3.84–3.77 (m, 18H),
3.77–3.75 (m, 1H), 3.62–3.54 (m, 2H), 3.54–3.51 (t, J = 5.5 Hz, 2H), 3.51–3.31
(m, 6H), 3.39–3.33 (m, 1H), 3.31–3.27 (t, J = 5.5 Hz, 2H), 3.27–3.22 (d, J =
8.85 Hz, 1H), 2.95–2.92 (s, 1H), 1.41–1.35 (s, 9H). 13C NMR (125 MHz, CDCl3):
δ 158.97, 130.87, 130.77, 130.36, 130.19, 129.70, 129.61, 129.54, 129.45, 129.32,
113.92, 113.86, 113.73, 113.59, 113.54, 98.96, 81.45, 81.36, 80.80, 80.24, 80.06,
78.37, 75.33, 74.33, 72.90, 72.85, 71.59, 70.79, 69.75, 55.28, 55.26, 55.24, 55.22,
50.68, 42.57, 31.92, 30.85, 29.70, 28.40. HR ESI-MS: calcd. for C69H86N4O19
[M+Na]+ m/z, 1297.5784; found, 1297.5781. Compound 31b underwent an
1
analogous procedure to give 32b (59.8 mg, 94%). H NMR for 32b (500 MHz,
CDCl3): δ 7.33–7.14 (m, 12H), 6.19–6.76 (m, 12H), 4.94–4.88 (d, J = 10.9 Hz,
1H), 4.88–4.84 (d, J = 10.38, 1H), 4.82–4.75 (dd, J = 11.3, 14.7 Hz, 2H),
4.70–4.59 (m, 3H), 4.56–4.48 (m, 4H), 4.47–4.42 (d, J = 11.6, 1H), 4.40–4.34
(d, J = 7.9 Hz, 1 position), 4.06–4.02 (m, 1H), 4.01–3.86 (m, 4H), 3.81 (s,
6H), 3.79 (s, 3H), 3.78 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 3.73–3.60 (m, 5H),
3.60–3.55 (t, J = 4.9, 2H), 3.55–3.44 (m, 4H), 3.42–3.30 (m, 5H), 3.24–3.17
(m, 3H), 1.41 (s, 1H). 13C NMR (125 MHz, CDCl3): δ 159.24, 159.12, 159.09,
159.08, 158.92, 158.87, 155.38, 131.47, 131.27, 130.93, 130.46, 130.25, 129.84,
129.70, 129.37, 129.24, 128.33, 113.80, 113.74, 113.70, 113.41, 102.39, 82.69,
82.27, 81.73, 81.33, 80.28, 79.65, 78.48, 75.97, 75.30, 74.76, 74.58, 74.46, 73.96,
73.18, 71.93, 71.76, 71.22, 70.70, 69.88, 68.07, 55.25, 55.23, 55.22, 55.20, 50.66,
28.43. HR ESI-MS: calcd. for C69H86N4O19 [M+Na]+ m/z, 1297.5784; found,
1297.5776.
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)
ethyl)-3,6-di-O-(p-methoxybenzyl)-α-D-glucopyranosyl]-1-O-
[(2,3-di-O-oleoyl-sn-glycerol)-phosphono]-2,3,4,5-tetra-O-(p-
methoxybenzyl)-myo-inositol (33a) and 6-O-[2-N-(tert-butyl
carbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)ethyl)-3,6-di-O-
(p-methoxybenzyl)-β-D-glucopyranosyl]-1-O-[(2,3-di-O-
oleoyl-sn-glycerol)-phosphono]-2,3,4,5-tetra-O-(p-
methoxybenzyl)-myo-inositol (33b)
A mixture of 32a and 9 was coevaporated with anhydrous pyridine three
times and then dried under reduced pressure for >2 h. Under an Ar atmo-
sphere at rt, the mixture was dissolved in anhydrous pyridine followed by the
addition of pivaloyl chloride. The reaction mixture stirred overnight and was
monitored by TLC. The solvent was then removed and the remaining residue
was redissolved in a solution of pyridine and water (9:1) containing 22 mg
of iodine. After stirring for 5 h, the reaction was quenched with Na2S2O3.
The solvent was then removed under reduced pressure and the residue was

C. L. Johnson and Z. Guo
320
directly applied to silica gel chromatography to give 33a (47.6 mg, 75% as
an Et3N salt). 1H NMR for 33a (500 MHz, CDCl3), δ 7.37–6.96 (m, 12H),
6.92–6.52 (m, 12H), 5.41–5.30 (m, 5H), 5.27–3.91 (complex, 28H), 3.87–3.69
(m, 18H), 3.66–2.85 (complex, 17H), 2.37–2.07 (complex, 5H), 2.05–1.90 (m,
7H), 1.67–1.56 (m, 4H), 1.36–1.18 (s, broad, 48H), 0.90–0.84 (m, 6H). 13C NMR
(125 MHz, CDCl3, resolved signals): δ 167.76, 156.22, 132.42, 130.88, 129.95,
129.69, 129.31, 129.10, 128.78, 127.77, 72.89, 70.52, 69.62, 68.59, 68.14, 55.19,
54.92, 53.42, 50.58, 38.71, 31.92, 31.90, 31.82, 31.00, 30.34, 29.77, 29.70, 29.54,
29.40, 29.31, 29.20, 28.91, 27.22, 24.80, 23.72, 22.98, 22.68, 14.12, 10.96. 31P
NMR (160 MHz, CDCl3): δ −2.74. HR ESI-MS: calcd. for C108H157N4O26P [M-
H]- m/z, 1956.0751; found, 1956.0769. Compound 32b underwent an anal-
1
ogous procedure to give 33b (17.3 mg, 53% as an Et3N salt). H NMR for
33b (500 MHz, CDCl3): δ 11.77 (broad s, 2.4 H, Et3N+H), 7.36–7.10 (m, 12H),
6.91–6.72 (m, 12H), 5.42–5.28 (m, 5H), 5.28–5.22 (m, 1H), 5.09–5.01 (d, J =
9.8 Hz, 1H), 4.88–4.79 (m, 3H), 4.79–4.73 (d, J = 7.3 Hz, 1-pos), 4.66–4.44
(m, 8H), 4.42–4.34 (m, 3H), 4.31–4.26 (m, 1H), 4.25–4.18 (m, 2H), 4.17–4.02
(m, 4H), 4.01–3.94 (t, J = 8.6 Hz, 1H), 3.88–3.82 (m, 2H), 3.81–3.71 (m, 18H),
3.69–3.59 (m, 4H), 3.58–3.49 (m, 4H), 3.49–3.42 (m, 3H), 3.42–3.35 (m, 2H),
3.35–3.23 (m, 6H), 3.08–3.01 (m, 12.7 H, Et3N), 2.26–2.17 (t, J = 7.3 Hz, 4H),
2.04–1.94 (m, 8H), 1.58–1.47 (m, 4H), 1.40 (s, 9H), 1.35–1.29 (t, J = 7.3 Hz,
26.5H, Et3N), 1.25 (broad s, 39H, overlaped with Et3N), 0.90–0.84 (t, J =
7.0 Hz, 6H). 13C NMR (125 MHz, CDCl3, resolved signals): δ 173.33, 173.01,
159.05, 158.98, 158.88, 158.73, 130.64, 129.95, 129.77, 129.72, 129.59, 129.49,
129.08, 128.26, 113.88, 113.67, 113.65, 113.59, 113.38, 101.11, 81.50, 80.56,
78.99, 78.71, 75.13, 74.60, 74.39, 74.25, 73.16, 71.88, 71.73, 70.73, 70.54, 69.83,
63.02, 55.23, 55.21, 55.18, 55.16, 52.75, 50.64, 45.78 (Et3N CH2), 34.21, 34.02,
31.89, 29.78, 29.69, 29.52, 29.31, 29.25, 29.22, 29.17, 29.13, 29.10, 28.48, 27.30,
27.21, 24.85, 24.82, 22.67, 14.12, 8.58 (Et3N CH3), 7.80. 31P NMR (160 MHz,
CDCl3): δ −1.04. MALDI-TOF MS (negative mode) calcd. for C108H157N4O26P
[M-H]- m/z, 1956.1; found, 1955.8.
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-4-O-(2-(2-azidoethoxy)
ethyl)-3,6-di-O-(p-methoxybenzyl)-α-D-glucopyranosyl]-1-O-
[(2,3-di-O-stearyl-sn-glycerol)-phosphono]-2,3,4,5-tetra-O-(p-
methoxybenzyl)-myo-inositol (34)
A mixture of 32a and 10 was coevaporated with anhydrous pyridine three
times and then dried under reduced pressure for >2 h. Under an Ar atmo-
sphere at rt, the mixture was dissolved in anhydrous pyridine followed by
the addition of pivaloyl chloride. The reaction mixture stirred overnight and
was monitored by TLC. The solvent was removed and the remaining residue
was redissolved in a solution of pyridine and water (9:1) containing 22 mg of

Synthetic GPI analogs
321
iodine. After stirring for 5 h, the reaction was quenched with Na2S2O3. The
solvent was removed under reduced pressure and the residue was directly ap-
plied to silica gel chromatography to give 34 (15.2 mg, 36% but 76% based on
1
recovered 32a). H NMR (500 MHz, CDCl3): δ 7.39–7.03 (m, 12H), 6.91–6.62
(m, 12H), 5.43–3.89 (complex, 30H), 3.89–3.62 (m, 18H), 3.60–2.77 (complex,
15H), 2.37–2.03 (complex, 5H), 1.65–1.58 (s, broad, 4H), 1.36–1.09 (s, broad,
56H), 0.93–0.83 (m, 8H). 31P NMR (160 MHz, CDCl3): δ −2.74. HR ESI-MS:
calcd. for C108H161N4O26P [M-H]- m/z, 1960.1082; found, 1960.1058.
6-O-[2-N-(tert-Butylcarbamate)-2-deoxy-4-O-[2-(2-amidofluori
seneylethoxy)ethyl)-3,6-di-O-(p-methoxybenzyl)-α-D-gluco
pyranosyl]-1-O-[(2,3-di-O-oleoyl-sn-glycerol)-phosphono]2,3,
4,5-tetra-O-(p-methoxybenzyl)-myo-inositol (4a), 6-O-[2-N-
(tert-butylcarbamate)-2-deoxy-4-O-[2-(2-amidofluoriseney
lethoxy)ethyl)-3,6-di-O-(p-methoxybenzyl)-β-D-glucopyra
nosyl]-1-O-[(2,3-di-O-oleoyl-sn-glycerol)-phosphono]2,3,4,5-
tetra-O-(p-methoxybenzyl)-myo-inositol (4b), and 6-O-[2-N-
(tert-butylcarbamate)-2-deoxy-4-O-(2-(2-aminoethoxy)ethyl)-
3,6-di-O-(p-methoxybenzyl)-α-D-glucopyranosyl]-1-O-[(2,3-di-
O-stearyl-sn-glycerol)-phosphono]-2,3,4,5-tetra-O-(p-
methoxybenzyl)-myo-inositol (5)
To 33a (18.5 mg, 9.4 μmol) stirring in anhydrous CH2Cl2 (1.5 mL) was
added zinc powder (61.4 mg, 0.94 mmol) and acetic acid (100 μL). The reaction
was monitored by MALDI-TOF MS and showed completion after about 6 h.
The reaction mixture was filtered and poured into a saturated aq. NaHCO3 so-
lution. The aq. layer was extracted four times with CH2Cl2 and the combined
organic portions were dried over MgSO4 before being concentrated under re-
duced pressure. The intermediate (15.5 mg, 8.0 μmol) was used directly for the
next step without further purification. The product was dissolved in anhydrous
CH2Cl2 (800 μL) followed by the addition of 7 (4.2 mg in 400 μL DMF) and one
drop of Et3N. The reaction stirred for 1 h before showing a complete reaction
(MALDI-TOF MS). The solvent was removed under reduced pressure before
purification by size exclusion chromatography to give 4a (16.3 mg, 76% for
two steps). MALDI-TOF MS (negative mode) for 4a: calcd. for C129H169N2O32P
[M−H]− m/z, 2288.1; found, 2287.6. Compounds 33b and 34 underwent an
analogous procedure to provided 4b (5.0 mg, 62% for two steps) and 5 (6.3 mg,
71% for two steps), respectfully. MALDI-TOF MS (negative mode) for 4b:
calcd. for C129H169N2O32P [M-H]− m/z, 2288.1; found, 2287.6. MALDI-TOF MS
(negative mode) for 5: calcd. for C129H173N2O32P [M-H]− m/z, 2292.2; found,
2291.5.

C. L. Johnson and Z. Guo
322
6-O-[2-Amino-2-deoxy-4-O-[2-(2-amidofluoriseneylethoxy)ethyl)-
α-D-glucopyranosyl]-1-O-[(2,3-di-O-oleoyl-sn-glycerol)-phos
phono]-myo-inositol (1), 6-O-[2-amino-2-deoxy-4-O-[2-(2-
amidofluoriseneylethoxy)ethyl)-β-D-glucopyranosyl]-1-O-
[(2,3-di-O-oleoyl-sn-glycerol)-phosphono]-myo-inositol (2),
and 6-O-[2-amino-2-deoxy-4-O-[2-(2-amidofluoriseney
lethoxy)ethyl)-α-D-glucopyranosyl]-1-O-[(2,3-di-O-stearyl-sn-
glycerol)-phosphono]-myo-inositol (3)
To 4a (4.4 mg, 1.9 μmol) stirring in anhydrous CH2Cl2 (2 mL) at 0◦C
was added 1 drop of DBU. After stirring for 1 min, a solution of TFA
(0.3 mL) and CH2Cl2 (2mL) was slowly added and the reaction was moni-
tored by MALDI-TOF MS. After stirring for 1 h, the solvent and TFA were
removed by coevaporation with toluene. The remaining residue was purified
by size exclusion chromatography to give 1 (2.2 mg, 78%). 1H NMR (600 MHz,
CDCl3:CD3OD:D2O [3:3:1], mixture of regioisomers): see supporting infor-
mation. 31P NMR (160 MHz, CDCl3): δ −0.098. MALDI-TOF MS (negative
mode): calcd. for C76H113N2O24P [M−H]− m/z, 1467.7; found, 1467.2. Com-
pounds 4b and 5 underwent an analogous procedure to provide 2 (3.0 mg,
74%) and 3 (2.0 mg, 60%), respectfully. 1H NMR of 2 and 3 (600 MHz,
CDCl3:CD3OD:D2O [3:3:1], mixture of regioisomers): see supporting informa-
tion. 31P NMR of 2 (160 MHz, CDCl3): δ −0.050. MALDI-TOF MS (negative
mode): calcd. for 2 C76H113N2O24P [M−H]− m/z, 1467.7; found, 1467.7. 31P
NMR of 3 (160 MHz, CDCl3): δ −0.043. MALDI-TOF MS (negative mode):
calcd. for 3 C76H117N2O24P [M−H]− m/z, 1471.7; found, 1471.3.
ACKNOWLEDGMENT
The authors thank the National Science Foundation (NSF CHE-1053848) for
financial support.
REFERENCES
1. Ferguson, M.A.J.; Williams, A.F. Cell-surface anchoring of proteins via glyco-
sylphosphatidylinositol structure. Ann. Rev. Biochem. 1988, 57, 285–320.
2. Englund, P.T. The structure and biosynthesis of glycosyl phosphatidylinositol pro-
tein anchors. Ann. Rev. Biochem. 1993, 62, 121–138.
3. Udenfriend, S.; Kodukula, K. How glycosylphosphatidylinositol-anchored mem-
brane protein are made. Ann. Rev. Biochem. 1995, 64, 563–591.
4. Trotter, J.; Klein, C.; Kramer, E. GPI-anchored proteins and glycosphingolipid-
rich rafts: platforms for adhesion and signaling. Neuroscientist 2000, 6, 271–284.
5. Cross, G.A.M. Cellular and genetic aspects of antigenic variation in trypanosomes.
Ann. Rev. Immunol. 1990, 8, 83–110.

Synthetic GPI analogs
323
6. Robinson, P.J. Signal transduction by GPI-anchored membrane proteins. Cell Biol.
Intern. Rep. 1991, 15, 761–767.
7. Hwa, K. Glycosyl phosphatidylinositol-linked glycoconjugates: structure, biosyn-
thesis and function. Adv. Exp. Med. Biol. 2001, 491, 207–214.
8. Paulick, M.G.; Bertozzi, C.R. The glycosylphosphatidylinositol anchor: a complex
membrane-anchoring structure for proteins. Biochemistry 2008, 47, 6991–7000.
9. Nikolaev, A.V.; Al-Maharik, N. Synthetic glycosylphosphatidylinositol (GPI) an-
chors: how these complex molecules have been made. Nat. Prod. Rep. 2011, 28,
970–1020, and references therein.
10. Swarts, B.M.; Guo, Z. Chemical synthesis of glycosylphosphatidylinositol anchors.
Adv. Carbohydr. Chem. Biochem. 2012, 67, 137–219, and references therein.
11. Martin-Lomas, M.; Khiar, N.; Garcia, S.; Koessler, J.-L.; Nieto, P.M.; Rademacher,
T.W. Inositolphosphoglycan mediators structurally related to glycosyl phosphatidyli-
nositol anchors: synthesis, structure and biological activity. Chem. Eur. J. 2000, 6,
3608–3621.
12. Stefaniu, C.; Vilotijevic, I.; Santer, M.; Silva, D.V.; Brezesinski, G.; Seeberger,
P.H. Subgel phase structure in monolayers of glycosylphosphatidylinositol glycolipids.
Angew. Chem. Int. Ed. 2012, 51, 12874–12878.
13. Bradley, J.E.; Chan, J.M.; Hagood, J.S. Effect of the GPI anchor of human Thy-1
on antibody recognition and function. Lab. Invest. 2013, 93, 365–374.
14. Seong, J.; Wang, Y.; Kinoshita, T.; Maeda, Y. Implications of lipid moiety in
oligomerization and immunoreactivities of GPI-anchored proteins. J. Lipid Res. 2013,
54, 1077–1091.
15. Swarts, B.M.; Guo, Z. Synthesis of a glycosylphosphatidylinositol (GPI) anchor
carrying unsturated lipid chains. J. Am. Chem. Soc. 2010, 6648–6650.
16. Burgula, S.; Swarts, B.M.; Guo, Z. Total synthesis of a glycosylphosphatidylinositol
anchor of the human lymphoctye CD52 antigen. Chem. Eur. J. 2012, 18, 1194–1201.
17. Lindh, I.; Stawinski, J. A general method for the synthesis of glycerophospholipids
and their analogs via H-phosphonate intermediates. J. Org. Chem. 1989, 54, 1338–1342.
18. Schmidt, R.R.; Michel, J. Simple syntheses of α- and β-O-glycosyl imidates; prepa-
ration of glycosides and disaccharides. Angew. Chem. Int. Ed. 1980, 19, 731–733.
19. Gou, D.; Liu, Y.; Chen, C. An efficient chemoenzymic access to optically active
myo-inositol polyphosphates. Carbohydr. Res. 1992, 234, 51–64.
20. Garegg, P.J.; Iversen, T.; Johansson, R.; Lindberg, B. Synthesis of some mono-O-
benzyl- and penta-O-methyl-myo-inositols. Carbohydr. Res. 1984, 130, 322–326.
21. Lee, J.; Cha, J.K. Selective cleavage of allyl ethers. Tetrahedron Lett. 1996, 37,
3663–3666.
22. Alper, P.B.; Hung, S.-C.; Wong, C.-H. Metal catalyzed diazo transfer for the syn-
thesis of azides from amines. Tetrahedron Lett. 1996, 37, 6029–6032.