Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphocyte CD52 antigen

Article abstract of DOI:10.1002/chem.201102545

The first total synthesis of a glycosylphosphatidylinositol (GPI) anchor bearing a polyunsaturated arachidonoyl fatty acid is reported. This lipid is found in mammalian GPIs that do not undergo lipid remodeling, a process that has important implications in the localization and function of GPI-anchored proteins. Incorporation of the oxidation- and reduction-sensitive arachidonoyl lipid in the target GPI was accomplished by using the para-methoxybenzyl (PMB) group for permanent hydroxyl group protection, which featured a selective, rapid, and efficient global deprotection protocol. The flexibility of this synthetic strategy was further highlighted by the inclusion of two additional GPI core structural modifications present in the GPI anchor of the human lymphocyte CD52 antigen. Lipid diversity in GPI synthesis: The convergent synthesis of an arachidonoyl-containing glycosylphosphatidylinositol (GPI) anchor of the human lymphocyte CD52 antigen is reported. Incorporation of the oxidation- and reduction-sensitive polyunsaturated arachidonoyl lipid, as well as two additional GPI core modifications (see structure), was enabled by using a para-methoxybenzyl (PMB)-based global protection strategy. Copyright

Full text of DOI:10.1002/chem.201102545

DOI: 10.1002/chem.201102545
Total Synthesis of a Glycosylphosphatidylinositol Anchor of the Human
Lymphocyte CD52 Antigen
Srinivas Burgula, Benjamin M. Swarts, and Zhongwu Guo*[a]
Abstract: The first total synthesis of a
glycosylphosphatidylinositol (GPI)
anchor bearing polyunsaturated
arachidonoyl fatty acid is reported.
This lipid is found in mammalian GPIs
that do not undergo lipid remodeling, a
process that has important implications
in the localization and function of GPI-
anchored proteins. Incorporation of the
oxidation-
and
reduction-sensitive
manent hydroxyl group protection,
which featured a selective, rapid, and
efficient global deprotection protocol.
The flexibility of this synthetic strategy
was further highlighted by the inclusion
of two additional GPI core structural
modifications present in the GPI
anchor of the human lymphocyte
CD52 antigen.
arachidonoyl lipid in the target GPI
was accomplished by using the para-
methoxybenzyl (PMB) group for per-
a
Keywords: antigens · glycolipids ·
GPI anchor · lipid remodeling · to-
tal synthesis
Introduction
tified, including a major form of the human lymphocyte
CD52 antigen.[7] The effects of unremodeled lipids on the
function of lymphocyte CD52 and other GPI-APs have not
been thoroughly explored, though in general it is believed
that the structure of GPI lipids has important implications
in the localization and function of GPI-APs. For example, a
recent study demonstrated that saturated fatty acids result-
ing from lipid remodeling are required for association of
mammalian GPI-APs with lipid rafts.[8] On the other hand,
unsaturated lipids found in GPIs from the protozoan para-
site Trypanosoma cruzi are thought to be responsible for
potent proinflammatory activity.[9]
Numerous eukaryotic cell surface proteins are anchored to
the plasma membrane by glycosylphosphatidylinositols
(GPIs), a family of glycolipids that are post-translationally
attached to proteins at their C termini.[1] GPI-anchored pro-
teins (GPI-APs) play important roles in many biological and
pathological events, such as cell recognition and adhesion,[2]
signal transduction,[3] host defense,[1c,4] and acting as recep-
tors for viruses and toxins.[4] In the 1980s, Ferguson and co-
workers elucidated the GPI anchor core structure conserved
among all species: H2NEt-(PO4)-6-Mana
G
U
6)Mana
(1!4)GlcNH2a
E
The CD52 antigen is a GPI-anchored glycopeptide ex-
pressed by human lymphocyte and sperm cells.[7b,10] Al-
though lymphocyte and sperm CD52 share an identical do-
decapeptide sequence, structural variations in their N-glycan
and GPI anchor lead to distinct biological activities.[11]
Whereas lymphocyte CD52 is a target for monoclonal anti-
body-based therapy of immune system related diseases, such
as leukemia,[12] antibodies specific to sperm CD52 have been
identified in infertile women, suggesting potential for immu-
nocontraceptive development.[13] Intriguingly, there are two
major forms of lymphocyte CD52, which exhibit two ex-
tremes of GPI lipid remodeling, one carrying exclusively dis-
tearoyl-PI and the other carrying inositol-palmitoylated
stearoyl-arachidonoyl PI.[7b] Evaluating the consequences of
lipid structural variations on GPI-AP trafficking, surface lo-
calization, and antibody binding can be aided by chemical
synthesis, which is an invaluable approach to obtaining
structurally defined GPI anchors for biological study.[14]
To develop a strategy for accessing GPIs relevant to
studying the effects of lipid remodeling and to continue our
synthetic studies of GPI anchors[15] and the human CD52 an-
tigen,[16] we targeted lymphocyte CD52 GPI anchor 1 for
total synthesis. The target molecule contained modifications
of the GPI core glycan present in lymphocyte CD52, includ-
lipid.[5] Modifications of the core structure are frequent and
primarily include additional glycans and phosphoethanola-
mine groups linked to Man residues, while the phosphatidy-
linositol (PI) fatty acids can differ in chain length and unsa-
turation.[1c,d]
The structural diversity of GPI lipids is generated by the
“lipid remodeling” process of GPI-APs.[6] GPI biosynthesis
begins from cellular PI, which in mammalian cells typically
contains a polyunsaturated arachidonoyl lipid (20:4) at the
sn-2 position. The arachidonoyl group can be replaced by
other lipids through a series of transformations in the Golgi
apparatus, and as a result most mature mammalian GPI-
APs contain only saturated fatty acids. However, several
mature GPI-APs bearing 2-arachidonoyl PI have been iden-
[a] Dr. S. Burgula,+ Dr. B. M. Swarts,+ Prof. Dr. Z. Guo
Department of Chemistry, Wayne State University
5101 Cass Avenue, Detroit, MI 48202 (USA)
Fax : (+1)313-577-8822
E-mail: zwguo@chem.wayne.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102545.
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Chem. Eur. J. 2012, 18, 1194 – 1201

FULL PAPER
ing additional phosphoethanolamine and mannose groups
linked to the 2-O-positions of Man-I and Man-III, respec-
tively. Most importantly, compound 1 contained a stearoyl-
arachidonoyl PI moiety, which is present in lymphocyte
CD52 and other GPI-APs that do not undergo lipid remod-
eling. To achieve the synthetically challenging incorporation
of a polyunsaturated arachidonoyl lipid in GPI 1, we em-
ployed a strategy based on para-methoxybenzyl (PMB) hy-
droxyl protection, which was recently developed in our labo-
ratory to enable the synthesis of GPIs carrying sensitive
functionalities that are not compatible with traditional car-
bohydrate protection chemistries.[15d,e]
tive to: 1) heat, base, and acid, which promote rearrange-
ment to the more stable 1-arachidonoyl isomer, and 2) air
and light, which result in oxidation of the lipid alkenes.[18]
Care was taken in the synthesis to minimize exposure to
these conditions. The preparation of phosphoramidite 6
commenced with stearoylation of optically pure glycerol de-
rivative 10, which was carried out with EDCI and DMAP
Results and Discussion
A convergent approach was used for the construction of
GPI 1 that relied on a key glycosylation between PMB-pro-
tected coupling partners pseudodisaccharide acceptor 2 and
tetramannosyl donor 3 (Scheme 1). Silyl ether protecting
groups at the Man-I 2-O- and Man-III 6-O-positions would
be selectively removed at a late stage to enable double
phosphorylation of these sites with phosphoramidite 4. Ac-
ceptor 2 could be accessed through phospholipidation of 5
with phosphoramidite 6, which would attach the arachidono-
yl-containing glycerolipid. Glycosyl donor 3 would arise
from sequential a-mannosylations of disaccharide 7. Guided
by previous success, we used the Schmidt method[17] to ste-
reoselectively couple all PMB-protected glycosylation part-
ners.
Scheme 2. Synthesis of pseudodisaccharide 2. All reactions involving
arachidonoyl lipid were carried out with exclusion of light and air. Re-
agents and conditions: a) stearic acid, EDCI, DMAP, CH2Cl2/DMF (4:1),
70%; b) AcOH, CH2Cl2, H2O, 69%; c) TBSCl, imidazole, DMF, 77%;
d) arachidonic acid, EDCI, DMAP, CH2Cl2/DMF (9:1), 99%;
e) Et3N·3HF, THF, CH3CN, 98%; f) diisopropylammonium tetrazolide,
CH2Cl2, CH3CN, 88%; g) 5, 1H-tetrazole, CH2Cl2; then tBuOOH,
ꢀ408C; h) Et3N·3HF, THF, CH3CN, 55% (two steps). DMAP: 4-dime-
thylamino pyridine, DMF: N,N-dimethylformamide, EDCI: 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide, TBS: tert-butyldimethylsilyl.
The preparation of pseudodisaccharide 2 is shown in
Scheme 2. 2-Arachidonoyl glycerides are known to be sensi-
Scheme 1. Retrosynthesis of lymphocyte CD52 GPI anchor 1. Ac: acetyl, All: allyl, Arach: arachidonoyl, Fmoc: 9-fluorenylmethoxycarbonyl, Stear:
stearoyl, TBDPS: tert-butyldiphenylsilyl, TES: triethylsilyl.
Chem. Eur. J. 2012, 18, 1194 – 1201
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Z. Guo et al.
(70%). After acid-catalyzed acetonide hydrolysis, the result-
ing diol 11 was treated with TBSCl and imidazole to selec-
tively protect its primary hydroxyl group. Next, with rigor-
ous exclusion of air, light, and moisture, the secondary alco-
hol was esterified with arachidonic acid in the presence of
EDCI and DMAP. The reaction proceeded in 99% yield,
and NMR spectroscopic analysis of the product confirmed
that the polyunsaturated lipid chain was intact. Previous re-
ports describing arachidonoyl migration during deprotection
of the adjacent hydroxyl group[18] were echoed in our work,
as desilylation with TBAF/AcOH was accompanied by un-
wanted transesterification. Fortunately, triethylamine trihy-
drofluoride proved mild enough to effect migration-free re-
moval of the TBS group, and gave stearoyl-arachidonoyl
glyceride 12 in 98% yield. Compound 12 was converted to
its corresponding phospholipid precursor 6 by coupling with
commercially available 2-cyanoethyl N,N,N’,N’-tetraisoprop-
yl phosphoramidite in 88% yield.
Scheme 3. Synthesis of mannosyl donors 8 and 9. a) NaOMe, MeOH;
b) TBDPSCl, imidazole, DMF, 68% (two steps); c) NaH, PMBCl, DMF,
71%; d) AcOH, CH2Cl2, H2O, 78%; e) Cl3CCN, DBU, CH2Cl2, 94%;
f) NaH, PMBCl, DMF, 85% (two steps); g) PdCl2, NaOAc, AcOH, H2O,
70%; h) Cl3CCN, DBU, CH2Cl2, 92%. DBU: 1,8-diazabicycloundec-7-
ene.
Phospholipidation of the PMB-protected pseudodisac-
charide 5[15d] was realized by treatment with freshly prepared
phosphoramidite 6 in the presence of 1H-tetrazole. Chemo-
selective oxidation of the resulting intermediate phosphite
to phosphate 13 with tBuOOH was performed in situ at
ꢀ408C without affecting the sensitive lipid alkenes. After
partial purification on silica gel, compound 13 was treated
with triethylamine trihydrofluoride to remove the sterically
hindered glucosamine 4-O-TBS group to afford pseudodi-
saccharide 2 in 55% yield over two steps. At this stage, the
approximately 1:1 diastereomeric mixture was separated by
semipreparative HPLC in order to simplify characterization
of downstream GPI intermediates. At each step in the syn-
thesis of 2, the structural integrity of the arachidonoyl lipid
was confirmed by NMR spectroscopy and mass spectrome-
try.
With pseudodisaccharide 2 in hand, we turned our atten-
tion to the synthesis of tetramannosyl donor 3. Previously,
we synthesized PMB-protected dimannose thioglycoside
7,[15d] which possessed the appropriate features to access 3
en route to the synthetic target 1: 1) orthogonal protection
at the Man-I 2-O-position to enable later phosphorylation;
2) anomeric thioether for facile conversion to a trichloroace-
timidate glycosyl donor; 3) free Man-II 2-O-position for ex-
tension of the glycan chain. For the latter purpose, PMB-
protected mannosyl donors 8 and 9 were prepared from
mannose penta-acetate 14 (Scheme 3). To obtain Man-III
donor 8, known orthoester 15[19] was first converted to 16 by
sequential deacetylation with NaOMe/MeOH, 6-O-silylation
with TBDPSCl/imidazole (68% over two steps), and para-
methoxybenzylation of the 3-O- and 4-O-positions (71%).
After acid-catalyzed regioselective opening of the orthoester
ring (78%), the resulting hemiacetal was converted to tri-
chloroacetimidate 8 by Cl3CCN and DBU. Man-IV donor 9
was prepared from allyl glycoside 17. Replacement of the
acetates by PMB groups gave 18 (85% over two steps),
which underwent palladium-catalyzed anomeric deallylation
followed by treatment with Cl3CCN and DBU to give tri-
chloroacetimidate 9.
Assembly of the tetramannose fragment from building
blocks 7–9 is depicted in Scheme 4. Coupling of dimannose
alcohol 7 and Man-III donor 8 was performed under stan-
dard Schmidt conditions, that is, catalytic TMSOTf and MS
4 ꢁ in CH2Cl2 at 08C. Trimannose 19 was produced in 72%
yield with complete a stereoselectivity imparted by neigh-
boring group participation from the donor 2-O-acetyl group,
which was subsequently removed by using NaOMe/MeOH
to afford 20 in 97% yield. To install the Man-IV unit, we
pursued a route initiated by glycosylation of 20 with known
mannosyl trichloroacetimidate 21,[15d] which carried a tempo-
rary stereodirecting acetyl group at its 2-O-position that
would be converted to a PMB group following a stereose-
lective glycosylation (Scheme 4A). Coupling of 20 and 21
under standard Schmidt conditions furnished the desired tet-
ramannoside in a moderate 44% yield with excellent stereo-
selectivity. However, subsequent attempts to replace the
Man-IV 2-O-acetate with a PMB group failed due to base-
promoted cleavage of the TBDPS group. To circumvent this
problem and improve reactivity with an armed glycosyl
donor,[20] trimannose 20 was instead coupled with fully
PMB-protected donor 9 by using Schmidt conditions to di-
rectly give 22 in an increased yield (Scheme 4B). However,
the stereoselectivity of this reaction was low (a/b, 3:2),
which was in contrast to previous results obtained in a
nearly identical system in which acceptor 7 was glycosylated
by a similar 2-O-PMB-protected mannosyl donor with com-
plete a stereoselectivity.[15d]
The a- and b-anomers of 22 were inseparable by HPLC
on large scale, so we explored conditions for removal of the
Man-I 2-O-allyl group for two purposes, namely, to improve
anomer separation and avoid late-stage deallylation, as the
latter is incompatible with the arachidonoyl lipid. Chaꢂs tita-
nium(IV)-mediated deallylation[21] was efficient for this pur-
pose (78% over two steps), whereas other methods were
low-yielding. After anomer separation by semipreparative
HPLC, the free hydroxyl group of 23 was protected with a
TES group (76%) to set up the Man-I 2-O- and Man-III 6-
O-positions for later double phosphorylation.
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Total Synthesis of CD52 GPI Anchor
FULL PAPER
Scheme 4. Synthesis of tetramannoside 24. Reagents and conditions: a) 8, TMSOTf (cat.), MS 4 ꢁ, CH2Cl2, 72%; b) NaOMe, MeOH, CH2Cl2, 97%;
c) 21, TMSOTf (cat.), MS 4 ꢁ, CH2Cl2, 44% (a only); d) NaOMe, MeOH, CH2Cl2, 78%; e) 9, TMSOTf (cat.), MS 4 ꢁ, CH2Cl2 (a/b, 3:2); f) Ti(OiPr)4,
ACHTUNGTRENNUNG
cyclopentylmagnesium chloride, THF, 78% (two steps); g) TESOTf, Et3N, CH2Cl2, 76%. Tf: trifluoromethanesulfonyl, THF: tetrahydrofuran, TMS: tri-
methylsilyl.
After pseudodisaccharide 2 and tetramannose thioglyco-
side 24 were obtained, they were used to construct the
target GPI (Scheme 5). Direct glycosylation of 2 by using
thioglycoside 24 was inefficient, so 24 was converted to tri-
chloroacetimidate 3 through NIS-promoted hydrolysis of the
anomeric thioether (92%) followed by treatment of the in-
termediate hemiacetal 25 with Cl3CCN and DBU (99%).
The key glycosylation between 3 and pseudodisaccharide 2
under Schmidt conditions gave the desired pseudohexasac-
charide, which, after workup but without purification, was
directly treated with triethylamine trihydrofluoride to effect
desilylation of the Man-I 2-O-TES and Man-III 6-O-TBS
groups. Although the resulting diol 26 was isolated in a
moderate yield (39% over two steps) the stereoselectivity of
the glycosylation step was excellent (only the a-isomer was
observed). Compound 26 was fully characterized, and the
stereochemistry of each mannosidic bond was established to
be a by using 13C NMR JCH coupling constants: d=101.9
(JCH =179.7 Hz, Man-III), 100.5 (JCH =173.4 Hz, Man-I),
99.3 (JCH =178.1 Hz, Man-IV), 99.2 ppm (JCH =178.1 Hz,
Man-II).[22]
To install both phosphoethanolamine units simultaneous-
ly, diol 26 was doubly phosphorylated by using phosphora-
midite 4 in the presence of 1H-tetrazole followed by chemo-
selective oxidation with tBuOOH at ꢀ408C. The reaction
proceeded in 37% yield to afford 27, which existed as an in-
consequential mixture of diastereomers originating at the
newly formed stereogenic phosphates. A monophosphorylat-
ed product was also obtained in 15% yield. Finally, com-
pound 27 was globally deprotected in under 3 h by using a
three-step, one-pot procedure involving zinc-mediated re-
duction of GlcN3 to GlcNH2, DBU-promoted cleavage of
the cyanoethoxyl and Fmoc groups, and TFA treatment to
remove all PMB groups. Following purification by using Se-
phadex-LH20 size exclusion chromatography, GPI 1 was ob-
tained in 90% yield over the final three steps. The structure
Scheme 5. Completion of GPI 1. Reagents and conditions: a) NIS, AgOTf, TTBP, wet CH2Cl2, 92%; b) Cl3CCN, DBU, CH2Cl2, 99%; c) 2, TMSOTf
(cat.), MS 4 ꢁ, Et2O; d) Et3N·3HF, THF, CH3CN, 39% (two steps); e) 4, 1H-tetrazole, CH2Cl2, CH3CN; then tBuOOH, ꢀ408C, 37%; f) Zn, AcOH,
CH2Cl2, 1 h; DBU, CH2Cl2, 1 h, CH2Cl2/TFA (9:1), 30 min, 90% (three steps). NIS: N-iodosuccinimide, TTBP: 2,4,6-tri-tert-butylpyrimidine.
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Z. Guo et al.
(d, J=3.7 Hz, 1H, 1-position), 5.25–5.20 (m, 1H), 4.94 (d, J=10.7 Hz,
1H), 4.92 (d, J=10.7 Hz, 1H), 4.86 (d, J=10.4 Hz, 1H), 4.81 (d, J=
11.0 Hz, 1H), 4.79 (d, J=10.7 Hz, 1H), 4.69 (d, J=10.4 Hz, 1H), 4.68 (d,
J=11.3 Hz, 1H), 4.67 (d, J=11.0 Hz, 1H), 4.63–4.59 (m, 2H), 4.42–4.40
(m, 2H), 4.37 (d, J=11.6 Hz, 1H), 4.36–4.32 (m, 2H), 4.29–4.21 (m, 4H),
4.16–4.12 (m, 1H), 4.12–4.08 (m, 1H), 4.04–4.00 (m, 2H), 3.82 (s, 3H),
3.80 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.77–3.76 (m, 1H),
3.75 (s, 3H), 3.70–3.65 (m, 2H), 3.51 (dd, J=9.8, 1.5 Hz, 1H), 3.40 (t, J=
9.5 Hz, 1H), 3.29 (ddd, J=14.6, 10.7, 4.0 Hz, 2H), 3.17 (dd, J=10.4,
3.7 Hz, 1H), 2.85–2.76 (m, 6H), 2.33–2.27 (m, 4H), 2.10–2.03 (m, 6H),
1.71–1.64 (m, 2H), 1.61–1.55 (m, 2H), 1.39–1.34 (m, 4H), 1.33–1.20 (m,
30H), 0.91–0.84 ppm (m, 6H); 13C NMR (CDCl3, 125 MHz): d=173.2,
172.6, 159.4, 159.2, 159.15, 159.10, 159.08, 159.03, 130.8, 130.7, 130.5,
130.3, 130.25, 130.23, 130.0, 129.8, 129.5, 129.29, 129.26, 129.04, 128.96,
128.7, 128.6, 128.3, 128.1, 127.8, 127.5, 116.4, 114.0, 113.8, 113.7, 113.65,
113.63, 97.5, 81.2, 80.6, 80.3, 78.3, 76.4, 75.5, 75.3, 74.9, 74.4, 73.0, 72.5,
72.1, 69.7, 69.4, 68.7, 66.0, 62.43, 62.40, 62.3, 61.4, 55.26, 55.25, 55.23, 33.9,
33.5, 31.9, 31.5, 29.72, 29.71, 29.67, 29.5, 29.4, 29.3, 29.1, 27.2, 26.4, 25.63,
25.60, 24.8, 24.7, 22.7, 22.6, 19.7, 19.6, 14.14, 14.09 ppm; 31P NMR
(CDCl3, 160 MHz): d=ꢀ1.40 ppm; [a]2D5 = +32.08 (c 0.5, CHCl3); HRMS
(ESI): calcd for C104H143N4NaO23P [M+Na]+ m/z: 1869.9778; found,
1869.9824. Isomer 2b was carried forward to complete the synthesis.
and homogeneity of the target compound were confirmed
by using 1H, 31P and 2D NMR spectroscopy and MALDI
mass spectrometry.
Conclusions
The construction of lymphocyte CD52 GPI anchor 1 repre-
sents the first total synthesis of a GPI anchor bearing unre-
modeled PI fatty acids, namely a polyunsaturated arachido-
noyl lipid at the sn-2 position. GPI 1 and related compounds
are useful for studying the impact of lipid structures on the
functions of GPIs and GPI-APs. Inclusion of the oxidation-
and reduction-sensitive arachidonoyl lipid was made possi-
ble by utilizing global PMB protection, which featured a
very mild and rapid three-step, one-pot deprotection proto-
col. Furthermore, two additional GPI core structural modifi-
cations present in lymphocyte CD52, specifically additional
phosphoethanolamine and mannose units, were incorporat-
ed in the target molecule, which further demonstrated the
applicability of the PMB protection strategy to the efficient
synthesis of highly complex and sensitively functionalized
GPI anchors.
Phenyl [2,3,4,6-tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[6-O-(tert-butyldiphenylsilyl)-3,4-di-O-(p-methoxybenzyl)-a-d-mannopyr-
anosyl]-(1!2)-[3,4,6-tri-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!
6)-2-O-allyl-3,4-di-O-(p-methoxybenzyl)-1-thio-a-d-mannopyranoside
(22): A mixture of trichloroacetimidate donor 9 (600 mg, 0.74 mmol), tri-
mannose acceptor 20 (850 mg, 0.49 mmol), and MS 4 ꢁ (300 mg) in anhy-
drous CH2Cl2 (10 mL) was stirred under an Ar atmosphere at room tem-
perature for 1 h. The reaction mixture was cooled to 08C, then TMSOTf
(13 mL, 0.07 mmol) was added and the reaction was stirred for 10 min.
Neutralization with triethylamine was followed by filtration through a
Celite pad to remove MS 4 ꢁ and then by concentration under vacuum.
The residue was purified by silica gel column chromatography (toluene/
EtOAc 10:1) to give an inseparable mixture of a,b-anomers 22. At this
point the isolated yield could not be obtained due to the presence of an
inseparable impurity, but a small amount of mixture 22 was subjected to
semipreparative HPLC to obtains a,b-anomers for characterization
(Waters Nova-Pak Silica 6 mm, 300ꢃ19 mm, eluent 33% acetone in hex-
anes, 10 mLminꢀ1, 22a tR =23.35 min, 22b tR =24.00 min, partially over-
lapping peaks). Characterization data (listed below for each anomer of
22) was obtained, but the poor separation made purification by semipre-
parative HPLC on a larger scale impossible, so we opted to purify after
the next step (deallylation) to obtain the isolated yield for the glycosyla-
tion–deallylation two-step sequence. Characterization data for 22a:
1H NMR (CDCl3, 500 MHz, a-isomer): d=7.71 (d, J=6.7 Hz, 2H), 7.67
(dd, J=7.3, 1.8 Hz, 2H), 7.38 (d, J=7.6 Hz, 2H), 7.33–7.26 (m, 10H),
7.25–7.20 (m, 6H), 7.18–7.13 (m, 6H), 7.11–6.99 (m, 9H), 6.89 (d, J=
8.5 Hz, 2H), 6.82–6.75 (m, 12H), 6.74–6.72 (m, 6H), 6.70–6.67 (m, 2H),
5.87–5.79 (m, 1H), 5.44 (d, J=1.2 Hz, 1H, Man-I 1-position), 5.37 (d, J=
1.5 Hz, 1H, Man-III 1-position), 5.30 (d, J=1.2 Hz, 1H, Man-IV 1-posi-
tion), 5.24 (dd, J=17.1, 1.2 Hz, 1H), 5.12 (dd, J=10.4, 1.2 Hz, 1H), 4.82
(d, J=1.2 Hz, 1H, Man-II 1-position), 4.81 (d, J=10.4 Hz, 1H), 4.78 (d,
J=10.4 Hz, 1H), 4.78 (d, J=10.4 Hz, 1H), 4.75 (d, J=11.0 Hz, 1H),
4.64–4.56 (m, 5H), 4.53 (d, J=10.7 Hz, 1H), 4.51–4.48 (m, 2H), 4.47 (d,
J=11.0 Hz, 1H), 4.42 (d, J=10.4 Hz, 1H), 4.39 (d, J=11.9 Hz, 1H), 4.38
(d, J=10.1 Hz, 1H), 4.37 (d, J=10.4 Hz, 1H), 4.37–4.31 (m, 4H), 4.27–
4.25 (m, 2H), 4.16–4.13 (m, 2H), 4.09–4.05 (m, 1H), 4.04–3.98 (m, 2H),
3.96–3.92 (m, 3H), 3.90–3.86 (m, 5H), 3.85–3.83 (m, 2H), 3.83 (s, 3H),
3.83–3.80 (m, 2H), 3.80 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H), 3.73 (s, 3H),
3.72 (s, 3H), 3.72 (s, 3H), 3.71 (s, 3H), 3.71–3.70 (m, 2H), 3.70 (s, 3H),
3.68–3.64 (m, 2H), 3.66 (s, 3H), 3.60–3.56 (m, 2H), 3.56 (s, 3H), 3.54–
3.50 (m, 2H), 0.99 ppm (s, 9H); 13C NMR (CDCl3, 125 MHz, a-isomer):
d=159.5, 159.4, 159.35, 159.30, 159.2, 159.10, 159.08, 136.1, 135.9, 135.2,
134.9, 134.0, 133.4, 131.5, 131.2, 131.1, 130.9, 130.85, 130.77, 130.7, 130.6,
130.5, 130.4, 130.0, 129.9, 129.8, 129.65, 129.56, 129.5, 129.4, 129.2, 127.93,
127.89, 127.2, 118.0, 114.1, 114.0, 113.94, 113.88, 113.8, 113.74, 113.71,
100.2 (Man-III), 99.5 (Man-IV), 99.4 (Man-II), 86.1 (Man-I), 80.4, 80.1,
79.6, 76.6, 75.0, 74.9, 74.8, 74.7, 74.6, 74.5, 74.4, 74.2, 73.6, 73.2, 72.9, 72.5,
Experimental Section
This section contains experimental procedures as well as MS and NMR
spectroscopy data for selected key intermediates and the target com-
pound. For all other procedures and characterization data, refer to the
Supporting Information.
6-O-[2-Azido-2-deoxy-3,6-di-O-(p-methoxybenzyl)-a-d-glucopyranosyl]-
1-O-[(2-cyanoethoxy)-(2-O-arachidonoyl-3-O-stearoyl-sn-glycerol)-phos-
phono]-2,3,4,5-tetra-O-(p-methoxybenzyl)-myo-inositol (2): A solution of
freshly prepared phosphoramidite
0.10 mmol) was slowly added to
6
a
(84 mg in 1.0 mL dry CH2Cl2,
stirring solution of (40 mg,
5
0.030 mmol) and 1H-tetrazole (0.45m solution in acetonitrile, 0.44 mL,
0.20 mmol) in anhydrous CH2Cl2/CH3CN (3:1, 6 mL) in the dark under
Ar at room temperature. After being stirred at room temperature under
Ar for 1 h, the reaction mixture was cooled to ꢀ408C and treated with
tert-butyl hydroperoxide (5.5m solution in decane, 60 mL, 0.33 mmol).
The solution was further stirred for 3 h at ꢀ208C, then Me2S (49 mL,
0.66 mmol) was added and stirring was continued for an additional 1 h at
ꢀ208C. The mixture was poured into saturated aqueous NaHCO3 and ex-
tracted three times with CH2Cl2. The combined organic layer was dried
over Na2SO4 and concentrated under vacuum. Purification by silica gel
column chromatography gave the desired intermediate 13 (briefly charac-
terized by MALDI-MS), which was then dissolved in anhydrous THF/
CH3CN (1:1, 2.5 mL) and treated with Et3N·3HF (0.5 mL) under Ar at
room temperature. After being stirred for 13 days at room temperature,
the reaction was quenched by dropwise addition of saturated aqueous
NaHCO3. The mixture was extracted three times with CH2Cl2, and the
combined organic layer was dried over Na2SO4, concentrated in vacuum,
and purified by silica gel column chromatography (hexanes/acetone 2:1)
to afford compound 2 (34 mg, 55% over two steps) as an approximately
1:1 diastereomeric mixture originating at phosphorus atom. Semiprepara-
tive HPLC (Waters Nova-Pak Silica 6 mm, 300ꢃ19 mm, eluent 30% ace-
tone in hexane, 10 mLminꢀ1, isomer 2a tR =13.4 min, isomer 2b tR =
13.9 min) was used to separate the mixture. Characterization data for 2a:
1H NMR (CDCl3, 500 MHz): d=7.35 (d, J=8.5 Hz, 2H), 7.32 (d, J=
8.5 Hz, 2H), 7.28 (d, J=8.5 Hz, 2H), 7.21 (d, J=8.5 Hz, 2H), 7.16–7.12
(m, 4H), 6.90–6.86 (m, 6H), 6.84 (dd, J=8.5, 1.8 Hz, 2H), 6.79 (dd, J=
8.5, 1.8 Hz, 2H), 6.77 (dd, J=8.8, 1.8 Hz, 2H), 5.43–5.32 (m, 8H), 5.39
1198
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1194 – 1201

Total Synthesis of CD52 GPI Anchor
FULL PAPER
72.4, 72.1, 71.9, 71.7, 71.6, 71.2, 69.0, 68.6, 66.7, 66.0, 63.4, 55.52, 55.48,
55.46, 55.4, 55.34, 55.31, 55.2, 27.1, 19.5 ppm; 13C NMR JCH values
(125 MHz): d=100.2 (JCH =176.0 Hz, Man-III), 99.5 (JCH =176.0 Hz,
Man-IV), 99.4 (JCH =174.0 Hz, Man-II), 86.1 ppm (JCH =169.0 Hz, Man-
I); HRMS (ESI): calcd for C137H156NaO31SSi [M+Na]+ m/z: 2380.0018;
found, 2380.0103. Characterization data for 22b: 1H NMR (CDCl3,
500 MHz, b-isomer): d=7.70 (d, J=6.7 Hz, 2H), 7.64 (d, J=6.7 Hz, 2H),
7.41 (d, J=7.3 Hz, 2H), 7.38–7.33 (m, 2H), 7.33–7.29 (m, 8H), 7.26–7.23
(m, 6H), 7.22–7.16 (m, 4H), 7.14–7.07 (m, 8H), 7.05–7.00 (m, 2H), 6.97
(d, J=8.5 Hz, 2H), 6.88 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H),
6.82–6.71 (m, 11H), 6.69–6.67 (m, 4H), 6.59 (d, J=8.5 Hz, 2H), 5.89–5.80
(m, 1H), 5.47 (d, J=1.2 Hz, 1H, Man-I 1-position), 5.41 (d, J=1.5 Hz,
1H, Man-III 1-position), 5.25 (dd, J=17.1, 1.5 Hz, 1H), 5.13 (dd, J=10.4,
1.2 Hz, 1H), 4.96 (d, J=11.0 Hz, 1H), 4.84 (d, J=1.2 Hz, 1H, Man-II 1-
position), 4.82 (d, J=1.2 Hz, 1H, Man-IV 1-position), 4.81 (d, J=
10.1 Hz, 1H), 4.80 (d, J=10.4 Hz, 1H), 4.75 (d, J=10.4 Hz, 1H), 4.71 (d,
J=11.0 Hz, 1H), 4.62–4.58 (m, 4H), 4.56–4.52 (m, 1H), 4.50–4.46 (m,
4H), 4.44–4.33 (m, 7H), 4.31–4.23 (m, 3H), 4.19 (dd, J=9.5, 4.6 Hz, 1H),
4.13–4.09 (m, 1H), 4.08–3.99 (m, 3H), 3.94–3.86 (m, 7H), 3.84–3.82 (m,
2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H), 3.80–3.78 (m,
2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.73–3.71 (m, 1H), 3.70 (s, 3H), 3.70 (s,
3H), 3.68–3.66 (m, 1H), 3.65 (s, 3H), 3.65 (s, 3H), 3.60–3.52 (m, 4H),
3.50 (s, 3H), 3.25 (dd, J=9.2, 2.7 Hz, 1H), 3.13 (dd, J=9.2, 2.7 Hz, 1H),
0.99 ppm (s, 9H); 13C NMR (CDCl3, 125 MHz, b-isomer): d=159.5,
159.4, 159.28, 159.27, 159.24, 159.22, 159.18, 159.1, 159.0, 136.1, 135.8,
135.3, 134.9, 134.1, 133.6, 131.5, 131.4, 131.3, 131.12, 131.09, 131.0, 130.9,
130.8, 130.72, 130.68, 130.5, 130.4, 130.3, 130.2, 129.88, 129.86, 129.83,
129.81, 129.78, 129.73, 129.66, 129.63, 129.56, 129.5, 129.4, 129.3, 127.84,
127.82, 127.2, 118.0, 114.2, 114.1, 113.9, 113.82, 113.79, 113.78, 113.7,
113.6, 113.5, 99.4 (Man-II), 99.2 (Man-III), 86.0 (Man-I), 82.2 (Man-IV),
80.6, 80.3, 78.0, 77.5, 76.6, 76.0, 75.1, 74.91, 74.87, 74.8, 74.7, 74.6, 74.5,
74.3, 74.2, 73.8, 73.4, 73.2, 73.0, 72.4, 72.1, 71.6, 70.8, 70.1, 69.5, 69.0, 66.7,
63.8, 55.52, 55.47, 55.4, 55.34, 55.31, 55.29, 55.21, 55.17, 27.0, 19.5 ppm;
13C NMR JCH values (125 MHz): d=99.4 (JCH =163.0 Hz, Man-II), 99.2
(s, 3H), 3.70 (s, 3H), 3.67 (s, 3H), 3.67–3.65 (m, 2H), 3.61–3.59 (m, 2H),
3.57 (s, 3H), 3.55–3.50 (m, 2H), 2.64 (brs, 1H), 1.00 ppm (s, 9H);
13C NMR (CDCl3, 125 MHz): d=159.7, 159.4, 159.30, 159.28, 159.22,
159.17, 159.12, 159.08, 136.1, 135.9, 134.6, 134.1, 133.5, 131.4, 131.23,
131.20, 131.16, 130.9, 130.82, 130.76, 130.62, 130.60, 130.0, 129.92, 129.87,
129.8, 129.65, 129.62, 129.58, 129.54, 129.52, 129.3, 129.2, 127.92, 127.86,
127.3, 114.2, 114.1, 114.0, 113.95, 113.90, 113.85, 113.80, 113.76, 113.7,
100.2 (Man-III), 99.58 (Man-IV), 99.58 (Man-II), 87.5 (Man-I), 80.4, 80.2,
80.1, 79.3, 75.0, 74.93, 74.87, 74.7, 74.6, 74.54, 74.47, 74.3, 73.9, 73.5, 73.2,
73.0, 72.6, 72.5, 72.4, 72.24, 72.19, 71.9, 71.83, 71.76, 71.3, 69.9, 69.0, 68.8,
66.4, 63.4, 55.53, 55.52, 55.49, 55.46, 55.41, 55.40, 55.38, 55.36, 55.32,
55.24, 27.2, 19.5 ppm; 13C NMR JCH values (125 MHz): d=100.2 (JCH
172.5 Hz, Man-III), 99.58 (JCH =172.5 Hz, Man-IV), 99.58 (JCH
=
=
172.5 Hz, Man-II), 87.5 ppm (JCH =166.0 Hz, Man-I); [a]2D5 = +49.88 (c
0.5, CHCl3); HRMS (ESI): calcd for C134H152NaO31SSi [M+Na]+ m/z:
2339.9705; found, 2339.9761.
Phenyl [2,3,4,6-tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[6-O-(tert-butyldiphenylsilyl)-3,4-di-O-(p-methoxybenzyl)-a-d-mannopyr-
anosyl]-(1!2)-[3,4,6-tri-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!
6)-3,4-di-O-(p-methoxybenzyl)-2-O-triethylsilyl-1-thio-a-d-mannopyrano-
side (24): TESOTf (29 mL, 0.13 mmol) was added slowly to a solution of
tetramannose 23 (20 mg, 8.6 mmol) and triethylamine (36 mL, 0.26 mmol)
in anhydrous CH2Cl2 (1.5 mL) and stirred under Ar at 08C. After being
stirred for 2 h while being warmed to room temperature, the reaction
mixture was poured into saturated aqueous NaHCO3, which was then ex-
tracted three times with CH2Cl2. The combined organic layer was dried
over Na2SO4, concentrated under vacuum, and purified by silica gel
column chromatography (toluene/EtOAc 8:1) to afford 24 (16 mg, 76%)
as syrup. 1H NMR (CDCl3, 500 MHz): d=7.77 (dd, J=6.4, 1.5 Hz, 2H),
7.73 (dd, J=7.6, 1.5 Hz, 2H), 7.45–7.42 (m, 3H), 7.40–7.30 (m, 9H),
7.28–7.21 (m, 6H), 7.22–7.15 (m, 7H), 7.16–7.12 (m, 5H), 7.10–7.02 (m,
5H), 6.91 (d, J=8.5 Hz, 2H), 6.87–6.81 (m, 7H), 6.81–6.76 (m, 8H),
6.76–6.74 (m, 3H), 5.42 (brs, 1H, Man-III 1-position), 5.34 (d, J=1.2 Hz,
1H, Man-IV 1-position), 5.31 (d, J=1.2 Hz, 1H, Man-I 1-position), 4.85
(d, J=1.2 Hz, 1H, Man-II 1-position), 4.84 (d, J=10.4 Hz, 1H), 4.81 (d,
J=10.4 Hz, 1H), 4.79 (d, J=11.0 Hz, 1H), 4.78 (d, J=11.0 Hz, 1H), 4.69
(d, J=11.3 Hz, 1H), 4.66 (d, J=11.9 Hz, 1H), 4.64 (d, J=11.6 Hz, 1H),
4.62 (d, J=11.6 Hz, 1H), 4.60 (d, J=11.6 Hz, 1H), 4.58 (d, J=11.0 Hz,
1H), 4.56 (d, J=10.4 Hz, 1H), 4.55 (d, J=11.3 Hz, 1H), 4.54 (d, J=
11.9 Hz, 1H), 4.52 (d, J=11.0 Hz, 1H), 4.46 (d, J=10.7 Hz, 1H), 4.43 (d,
J=10.4 Hz, 1H), 4.42 (d, J=10.4 Hz, 1H), 4.42 (d, J=11.0 Hz, 1H), 4.41
(d, J=10.4 Hz, 1H), 4.40 (d, J=10.4 Hz, 1H), 4.38 (d, J=10.7 Hz, 1H),
4.37 (d, J=11.0 Hz, 1H), 4.29 (d, J=2.0 Hz, 2H), 4.27 (brs, 1H), 4.20 (t,
J=7.6 Hz, 1H), 4.15 (dd, J=2.0, 1.5 Hz, 1H), 4.06 (t, J=9.5 Hz, 1H),
4.01–3.95 (m, 3H), 3.95–3.91 (m, 3H), 3.91–3.86 (m, 5H), 3.84 (s, 3H),
3.83 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.81–3.77 (m, 3H), 3.76 (s, 3H),
3.75 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.73 (s, 3H), 3.69 (s, 3H), 3.69–
3.65 (m, 2H), 3.60 (s, 3H), 3.60–3.53 (m, 2H), 1.03 (s, 9H), 0.95 (t, J=
7.9 Hz, 9H), 0.60 ppm (distorted q, J=7.9 Hz, 6H); 13C NMR (CDCl3,
125 MHz): d=159.4, 159.35, 159.33, 159.24, 159.21, 159.18, 159.13, 159.12,
136.1, 135.9, 135.3, 134.1, 133.4, 131.5, 131.32, 131.27, 131.15, 131.12,
130.9, 130.80, 130.78, 130.7, 130.62, 130.59, 129.9, 129.80, 129.78, 129.7,
129.64, 129.58, 129.55, 129.54, 129.4, 129.31, 129.26, 128.0, 127.9, 127.3,
114.1, 114.02, 113.99, 113.96, 113.91, 113.88, 113.86, 113.79, 113.75, 100.2
(Man-III), 99.6 (Man-IV), 99.2 (Man-II), 89.7 (Man-I), 80.7, 80.15, 80.14,
80.10, 75.03, 74.97, 74.7, 74.6, 74.5, 74.4, 74.3, 73.7, 73.2, 72.9, 72.55, 72.49,
72.4, 72.2, 72.1, 72.0, 71.7, 71.5, 71.4, 68.8, 68.6, 67.0, 63.4, 55.52, 55.51,
55.49, 55.46, 55.42, 55.40, 55.39, 55.33, 55.2, 27.2, 19.5, 7.2, 5.3 ppm;
[a]2D5 = +40.08 (c 0.5, CHCl3); HRMS (ESI): calcd for C140H166NaO31SSi2
[M+Na]+ m/z: 2454.0570; found, 2454.0669.
(JCH =164.0 Hz, Man-III), 86.0 (JCH =168.0 Hz, Man-I), 82.2 ppm (JCH
=
139.0 Hz, Man-IV); HRMS (ESI) data for b-isomer were consistent with
the a-isomer.
Phenyl [2,3,4,6-tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[6-O-(tert-butyldiphenylsilyl)-3,4-di-O-(p-methoxybenzyl)-a-d-mannopyr-
anosyl]-(1!2)-[3,4,6-tri-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!
6)-3,4-di-O-(p-methoxybenzyl)-1-thio-a-d-mannopyranoside (23): Tita-
nium(IV) isopropoxide (0.76 g in 5.0 mL hexanes, 2.7 mmol) was added
to a solution of a,b-mixture 22 (1.3 g, which also contained some insepa-
rable impurity from the previous glycosylation) in anhydrous THF
(30 mL). Cyclopentylmagnesium chloride (2.87m solution in Et2O,
1.86 mL, 5.36 mmol) was added to the reaction mixture, dropwise under
an Ar atmosphere at room temperature over a period of 1 h. After being
stirred for an additional 2 h, the reaction mixture was poured into water.
The mixture was extracted three times with CH2Cl2, and then the com-
bined organic layer was washed with brine, dried over Na2SO4, and con-
centrated under vacuum. The resulting residue was purified by silica gel
column chromatography (toluene/EtOAc 5:1) to give 23 (0.895 g, 78%
over two steps). At this stage, the a,b-mixture was purified by semipre-
parative HPLC (Waters Nova-Pak Silica 6 mm, 300ꢃ19 mm, eluent 35%
acetone in hexanes, 10 mLminꢀ1, 23 tR =36.19 min, b-isomer of 23 tR =
38.78 min), which showed an anomeric ratio of 3:2. Characterization data
for 23: 1H NMR (CDCl3, 500 MHz): d=7.74 (dd, J=6.4, 1.8 Hz, 2H),
7.69 (dd, J=7.6, 1.8 Hz, 2H), 7.33–7.26 (m, 11H), 7.24–7.22 (m, 6H),
7.19–7.15 (m, 5H), 7.13–7.11 (m, 7H), 7.08 (d, J=8.5 Hz, 2H), 7.04 (d,
J=8.5 Hz, 2H), 7.02 (d, J=7.32 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 6.83–
6.78 (m, 9H), 6.76–6.73 (m, 10H), 5.39 (brs, 1H, Man-I 1-position), 5.37
(brs, 1H, Man-III 1-position), 5.30 (brs, 1H, Man-IV 1-position), 4.86 (d,
J=1.2 Hz, 1H, Man-II 1-position), 4.83 (d, J=10.4 Hz, 1H), 4.78 (d, J=
9.5 Hz, 1H), 4.76 (d, J=10.4 Hz, 1H), 4.72 (d, J=10.7 Hz, 1H), 4.62–4.57
(m, 5H), 4.56 (d, J=10.4 Hz, 1H), 4.52 (d, J=10.4 Hz, 1H), 4.50 (d, J=
10.4 Hz, 1H), 4.49 (d, J=10.7 Hz, 1H), 4.43–4.34 (m, 8H), 4.29–4.26 (m,
2H), 4.15–4.08 (m, 3H), 4.02–3.93 (m, 4H), 3.90–3.86 (m, 4H), 3.85–3.83
(m, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.79–3.76 (m, 2H), 3.78
(s, 3H), 3.76–3.74 (m, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.73 (s, 3H), 3.72
[2,3,4,6-Tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-[6-O-
(tert-butyldiphenylsilyl)-3,4-di-O-(p-methoxybenzyl)-a-d-mannopyrano-
syl]-(1!2)-[3,4,6-tri-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!6)-
3,4-di-O-(p-methoxybenzyl)-2-O-triethylsilyl-d-mannopyranose
(25):
2,4,6-Tri-tert-butylpyrimidine (26 mg, 0.11 mmol), N-iodosuccinimide
(16 mg, 0.07 mmol), and silver triflate (18 mg, 0.07 mmol) were added to
a solution of tetramannose thioglycoside 24 (88 mg, 0.036 mmol) in wet
CH2Cl2 (5 mL) at 08C. After being stirred for 2 h at room temperature,
saturated aqueous Na2S2O3 was added at 08C and the reaction mixture
Chem. Eur. J. 2012, 18, 1194 – 1201
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1199

Z. Guo et al.
was stirred for an additional 30 min while being warmed to room temper-
ature. The reaction mixture was extracted three times with CH2Cl2, and
the combined organic layer was washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (toluene/EtOAc 5:1) to afford tetra-
mannose hemiacetal 25 (78 mg, 92%) as syrup. 1H NMR (CDCl3,
500 MHz): d=7.78 (dd, J=7.3, 1.5 Hz, 2H), 7.75 (dd, J=7.0, 1.5 Hz,
2H), 7.39–7.32 (m, 8H), 7.31–7.24 (m, 8H), 7.22–7.19 (m, 5H), 7.18–7.12
(m, 5H), 7.06 (dd, J=8.5, 2.7 Hz, 4H), 6.87 (d, J=8.8 Hz, 2H), 6.85 (d,
J=7.9 Hz, 2H), 6.84 (d, J=7.9 Hz, 2H), 6.83 (d, J=7.0 Hz, 2H), 6.82 (d,
J=7.0 Hz, 2H), 6.81–6.75 (m, 10H), 5.33 (brs, 1H, Man-III 1-position),
5.28 (brs, 1H, Man-IV 1-position), 4.85 (d, J=2.1 Hz, 1H, Man-II 1-posi-
tion), 4.84 (d, J=10.4 Hz, 1H), 4.83 (brs, 1H, Man-I 1-position), 4.79 (d,
J=10.7 Hz, 1H), 4.75 (d, J=10.7 Hz, 1H), 4.68 (d, J=10.7 Hz, 1H), 4.65
(d, J=11.3 Hz, 1H), 4.62 (d, J=11.6 Hz, 1H), 4.60 (d, J=11.0 Hz, 1H),
4.58–4.51 (m, 6H), 4.50–4.38 (m, 6H), 4.33 (d, J=10.4 Hz, 1H), 4.31 (d,
J=11.3 Hz, 1H), 4.29 (d, J=11.9 Hz, 1H), 4.25 (d, J=10.4 Hz, 1H), 4.21
(dd, J=2.0, 1.2 Hz, 1H), 4.12 (dd, J=2.5, 1.5 Hz, 1H), 4.00–3.94 (m,
5H), 3.93–3.85 (m, 6H), 3.82 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.80 (s,
3H), 3.79–3.77 (m, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.74 (s,
3H), 3.73 (s, 3H), 3.72 (s, 3H), 3.68 (s, 3H), 3.67–3.60 (m, 5H), 3.59–3.51
(m, 2H), 2.39 (brs, 1H), 1.05 (s, 9H), 0.95 (t, J=7.9 Hz, 9H), 0.58 ppm
(distorted q, J=7.6 Hz, 6H); 13C NMR (CDCl3, 125 MHz): d=159.43,
159.40, 159.36, 159.32, 159.26, 159.22, 159.19, 159.17, 159.14, 136.2, 135.9,
134.1, 133.4, 131.3, 131.2, 131.1, 130.9, 130.81, 130.76, 130.5, 129.8, 129.75,
129.70, 129.67, 129.64, 129.5, 129.3, 128.0, 127.9, 114.05, 114.01, 113.95,
113.93, 113.90, 113.82, 113.79, 113.77, 99.70 (Man-III), 99.70 (Man-IV),
99.5 (Man-II), 95.1 (Man-I), 80.1, 80.0, 74.9, 74.85, 74.77, 74.7, 74.5, 73.4,
73.2, 73.0, 72.5, 72.4, 72.2, 72.0, 71.9, 71.85, 71.80, 70.4, 69.6, 69.1, 68.8,
63.3, 55.49, 55.48, 55.46, 55.43, 55.40, 55.37, 55.35, 27.2, 19.5, 7.2, 5.2 ppm;
HRMS (ESI): calcd for C134H162NaO32Si2 [M+Na]+ m/z: 2362.0485;
found, 2362.0591.
(brs, 1H, Man-I 1-position), 4.97 (d, J=11.0 Hz, 1H), 4.96 (brs, 1H,
Man-II 1-position), 4.92 (dd, J=10.4, 2.5 Hz, 1H), 4.85 (d, J=10.4 Hz,
1H), 4.84 (d, J=9.2 Hz, 1H), 4.81–4.71 (m, 5H), 4.69–4.62 (m, 4H),
4.60–4.56 (m, 4H), 4.56–4.50 (m, 4H), 4.49–4.45 (m, 5H), 4.44–4.39 (m,
4H), 4.39–4.34 (m, 4H), 4.33–4.30 (m, 2H), 4.29–4.27 (m, 2H), 4.25–4.15
(m, 8H), 4.04–3.96 (m, 4H), 3.86–3.81 (m, 7H), 3.82 (s, 3H), 3.80 (s, 3H),
3.79 (s, 3H), 3.77 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.76 (s, 3H), 3.75 (s,
3H), 3.73 (s, 3H), 3.72 (s, 3H), 3.70 (s, 3H), 3.70 (s, 3H), 3.70–3.67 (m,
2H), 3.67 (s, 3H), 3.66–3.64 (m, 3H), 3.63 (s, 3H), 3.61 (s, 3H), 3.62–3.56
(m, 2H), 3.57 (s, 3H), 3.57–3.52 (m, 3H), 3.51–3.43 (m, 4H), 3.40–3.36
(m, 2H), 3.36 (s, 3H), 3.22 (dd, J=10.7, 2.1 Hz, 1H), 3.16 (dd, J=9.8,
3.7 Hz, 1H), 3.09 (ddd, J=10.7, 2.1, 0.9 Hz, 1H), 2.84–2.77 (m, 6H),
2.68–2.63 (m, 2H), 2.59 (brs, 1H), 2.37–2.32 (m, 2H), 2.30–2.26 (m, 2H),
2.12–2.08 (m, 2H), 2.08–2.03 (m, 2H), 1.72–1.65 (m, 3H), 1.61–1.55 (m,
2H), 1.39–1.33 (m, 4H), 1.32–1.22 (m, 30H), 0.91–0.87 ppm (m, 6H);
13C NMR (CDCl3, 125 MHz): d=173.5, 172.9, 159.6, 159.4, 159.35,
159.32, 159.26, 159.22, 159.17, 159.15, 159.14, 131.09, 131.07, 131.02,
130.92, 130.89, 130.84, 130.7, 130.6, 130.49, 130.47, 130.3, 129.87, 129.83,
129.81, 129.80, 129.77, 129.75, 129.74, 129.72, 129.6, 129.5, 129.3, 129.25,
129.22, 129.1, 129.0, 128.8, 128.6, 128.5, 128.3, 128.1, 127.7, 116.6, 114.12,
114.10, 114.05, 114.04, 114.01, 113.96, 113.93, 113.90, 113.84, 113.83,
113.79, 113.70, 101.9 (Man-III), 100.5 (Man-I), 99.3 (Man-IV), 99.2 (Man-
II), 98.0 (GlcN3), 81.4, 80.7, 80.6, 79.8, 79.7, 79.5, 79.4, 78.5, 76.42, 76.41,
75.65, 75.56, 75.3, 75.0, 74.9, 74.7, 74.65, 74.59, 74.5, 74.3, 74.2, 73.90,
73.88, 73.6, 73.19, 73.15, 73.13, 73.0, 72.8, 72.58, 72.53, 72.48, 72.40, 71.8,
71.64, 71.59, 71.4, 70.05, 70.01, 69.7, 69.0, 68.8, 68.7, 68.2, 66.5, 66.0, 64.9,
63.25, 63.23, 62.51, 62.48, 62.45, 62.40, 62.37, 61.7, 55.51, 55.48, 55.46,
55.44, 55.42, 55.40, 55.37, 55.35, 55.33, 55.27, 55.23, 55.20, 41.3, 34.2, 33.8,
32.2, 31.7, 30.0, 29.9, 29.8, 29.62, 29.58, 29.4, 27.5, 26.70, 26.67, 26.65, 25.9,
25.8, 25.1, 24.9, 22.9, 22.8, 19.85, 19.78, 14.4, 14.3 ppm. Configurations of
anomeric positions were established as a by coupled 13C NMR JCH values
(125 MHz): d=101.9 (JCH =179.7 Hz, Man-III), 100.5 (JCH =173.4 Hz,
Man-I), 99.3 (JCH =178.1 Hz, Man-IV), 99.2 ppm (JCH =178.1 Hz, Man-
II); 31P NMR (CDCl3, 160 MHz): d=ꢀ1.79 ppm; [a]2D5 = +43.08 (c 0.4,
CHCl3); HRMS (ESI): calcd for C216H271N4NaO54P [M+Na]+ m/z:
3838.8218; found, 3838.8269.
6-O-{[2,3,4,6-Tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[3,4-di-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-[3,4,6-tri-O-
(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!6)-[3,4-di-O-(p-methoxy-
benzyl)-a-d-mannopyranosyl]-(1!4)-[2-azido-2-deoxy-3,6-di-O-(p-me-
thoxybenzyl)-a-d-glucopyranosyl]}-1-O-[(2-cyanoethoxy)-(2-O-arachido-
noyl-3-O-stearoyl-sn-glycerol)-phosphono]-2,3,4,5-tetra-O-(p-methoxy-
benzyl)-myo-inositol (26): DBU (1 drop) and trichloroacetonitrile
(0.3 mL) were added to a solution of tetramannose hemiacetal 25 (78 mg,
0.03 mmol) in anhydrous CH2Cl2 (5 mL). After being stirred under an Ar
atmosphere at 08C for 30 min, the reaction mixture was concentrated
under vacuum and purified with a triethylamine-treated neutral silica gel
column to furnish tetramannose donor 3 (82 mg, 99%). A mixture of the
resulting trichloroacetimidate 3 (44 mg, 0.02 mmol), acceptor 2 (10 mg,
5.95 mmol), and MS 4 ꢁ (10 mg) in anhydrous Et2O (4 mL) was stirred
under an Ar atmosphere at room temperature for 1 h. The reaction mix-
ture was cooled to 08C, TMSOTf (0.05m solution in anhydrous Et2O,
40 mL) was added, and the reaction was stirred, overnight, while being
warmed to room temperature. The reaction was neutralized by addition
of saturated aqueous NaHCO3. After filtration through a Celite pad to
remove MS 4 ꢁ, the mixture was extracted three times with CH2Cl2, and
the combined organic layer was dried over Na2SO4 and concentrated
under vacuum. The residue was partially purified by silica gel column
chromatography to give the intermediate a-pseudohexasaccharide, which
was taken directly to the next step. Et3N·3HF (0.5 mL) was added to a
solution of the crude material (13.7 mg) in anhydrous THF/CH3CN (1:1,
1 mL) under an Ar atmosphere at room temperature. After being stirred
for 7 days at room temperature, the reaction was quenched by dropwise
addition of saturated aqueous NaHCO3 at 08C. The aqueous layer was
extracted three times with CH2Cl2, and the combined organic layer was
washed with brine, dried over Na2SO4, and concentrated under vacuum.
The residue was purified by silica gel column chromatography (toluene/
EtOAc 2.5:1) to give diol 26 (8.1 mg, 39% for two steps) as syrup.
1H NMR (CDCl3, 500 MHz): d=7.34–7.26 (m, 10H), 7.25–7.15 (m, 15H),
7.13–7.07 (m, 9H), 7.02 (d, J=8.5 Hz, 2H), 6.89–6.84 (m, 8H), 6.81–6.78
(m, 8H), 6.76–6.72 (m, 16H), 5.43–5.30 (m, 8H), 5.36 (d, J=3.7 Hz, 1H,
GlcN3 1-position), 5.30–5.26 (m, 1H, glyceride CH), 5.21 (d, J=2.4 Hz,
1H, Man-III 1-position), 5.21 (d, J=2.4 Hz, 1H, Man-IV 1-position), 5.13
6-O-{[2,3,4,6-Tetra-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[6-O-[(2-cyanoethoxy)-[2-(9-fluorenylmethoxycarbonylamino)ethyl]-
phosphono]]-3,4-di-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!2)-
[3,4,6-tri-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!6)-[2-O-[(2-cy-
anoethoxy)-[2-(9-fluorenylmethoxycarbonylamino)ethyl]-phosphono]]-
3,4-di-O-(p-methoxybenzyl)-a-d-mannopyranosyl]-(1!4)-[2-azido-2-
deoxy-3,6-di-O-(p-methoxybenzyl)-a-d-glucopyranosyl]}-1-O-[(2-cyanoe-
thoxy)-(2-O-arachidonoyl-3-O-stearoyl-sn-glycerol)-phosphono]-2,3,4,5-
tetra-O-(p-methoxybenzyl)-myo-inositol (27): Freshly prepared phos-
phoramidite 4 (15.5 mg in 500 mL dry CH2Cl2, 0.03 mmol) was added to a
stirring solution of pseudohexasaccharide 26 (4.1 mg, 1.0 mmol) and MS
4 ꢁ (5 mg) in anhydrous CH2Cl2/CH3CN (3:1, 1.5 mL). Then, 1H-tetra-
zole (0.45m solution in acetonitrile, 143 mL, 0.06 mmol) was added under
Ar at room temperature. After being stirred at room temperature under
Ar, overnight, the reaction mixture was cooled to ꢀ408C and treated
with tert-butyl hydroperoxide (5.5m solution in decane 23 mL,
0.13 mmol). The solution was further stirred for 1 h at ꢀ408C, after
which Me2S (19 mL, 0.26 mmol) was added and stirring was continued for
an additional 1 h at ꢀ408C. After passage through a Celite pad to
remove MS 4 ꢁ, the mixture was poured into saturated aqueous
NaHCO3 and extracted three times with CH2Cl2. The combined organic
layer was dried over Na2SO4 and concentrated under vacuum. Purifica-
tion of the crude material by silica gel column chromatography afforded
diphosphorylated GPI 27 and also the Man-III monophosphorylated
GPI. Both compounds were further purified by using Sephadex LH-20
size exclusion chromatography (CHCl3/MeOH, 3:1) to provide 27
(1.85 mg, 37%) and a monophosphorylated compound (0.7 mg, 15%),
both of which existed as complex diastereomeric mixtures resulting from
the newly formed stereogenic phosphates. This was inconsequential as
the following global deprotection step rendered the phosphorus atoms
nonstereogenic. Compound 27: 1H NMR (CDCl3, 500 MHz, resolved sig-
nals): d=7.73–7.70 (m, 4H), 7.55–7.48 (m, 4H), 7.40–7.28 (m, 10H),
7.26–7.19 (m, 12H), 7.18–7.10 (m, 12H), 7.09–7.04 (m, 4H), 7.01–6.95 (m,
1200
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1194 – 1201

Total Synthesis of CD52 GPI Anchor
FULL PAPER
4H), 6.87–6.80 (m, 10H), 6.80–6.69 (m, 24H), 5.55 (brs, 1H, Man-I 1-po-
sition), 5.41–5.31 (m, 8H), 5.35 (brs, 1H, GlcN3 1-position), 5.30–5.26 (m,
1H, glyceride CH), 5.10 (s, 1H, Man-III 1-position), 4.88 (s, 1H, Man-IV
1-position), 4.84 (s, 1H, Man-II 1-position), 2.84–2.76 (m, 6H), 2.64–2.58
(m, 2H), 2.33–2.20 (m, 8H), 2.08–2.00 (m, 4H), 1.72–1.54 (m, 4H), 1.38–
1.20 (m, 34H), 0.90–0.87 ppm (m, 6H); 31P NMR (CDCl3, 160 MHz): d=
ꢀ0.78, ꢀ0.82, ꢀ1.26, ꢀ1.29, ꢀ1.64, ꢀ1.65, ꢀ1.85, ꢀ2.25, ꢀ2.26, ꢀ2.69,
ꢀ2.71, ꢀ3.00, ꢀ3.05 ppm; MALDI-ToF MS (positive mode): calcd For
C256H310N8NaO64P3 [M+H+Na]+ m/z: 4636.0; found, 4636.5. Monophos-
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2000, 19, 1476–1485.
1
phorylated compound: H NMR (CDCl3, 500 MHz, resolved signals): d=
7.73–7.70 (m, 2H), 7.53–7.51 (m, 2H), 7.36–7.28 (m, 10H), 7.26–7.21 (m,
6H), 7.20–7.13 (m, 10H), 7.12–7.05 (m, 8H), 7.04–6.98 (m, 4H), 6.90–
6.82 (m, 10H), 6.81–6.70 (m, 24H), 5.40–5.30 (m, 9H, includes glyceride
CH), 5.24 (brs, 2H), 5.15 (brs, 2H), 4.95 (brs, 1H), 2.83–2.77 (m, 6H),
2.65–2.60 (m, 2H), 2.35–2.21 (m, 6H), 2.07–2.00 (m, 4H), 1.70–1.55 (m,
4H), 1.37–1.20 (m, 34H), 0.90–0.85 ppm (m, 6H); 31P NMR (CDCl3,
160 MHz): d=ꢀ0.72, ꢀ1.29, ꢀ2.14, ꢀ2.30, ꢀ2.62, ꢀ2.64, ꢀ2.91,
ꢀ2.99 ppm.
MALDI-ToF
MS
(positive
mode):
calcd
For
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C236H292N6NaO59P2 [M+2H+Na]+ m/z: 4238.9; found, 4238.4.
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6-O-{[a-d-Mannopyranosyl]-(1!2)-[6-O-[(2-aminoethyl)-phosphono]-a-
d-mannopyranosyl]-(1!2)-[a-d-mannopyranosyl]-(1!6)-[2-O-[(2-ami-
noethyl)-phosphono]-a-d-mannopyranosyl]-(1!4)-[2-amino-2-deoxy-a-
d-glucopyranosyl]}-1-O-[(2-O-arachidonoyl-3-O-stearoyl-sn-glycerol)-
phosphono]-myo-inositol (1): Acetic acid (1 drop) and zinc powder
(10 mg) were added to a solution of pseudohexasaccharide 27 (1.85 mg,
0.40 mmol) in CH2Cl2 (500 mL). After being vigorously stirred for 1 h at
room temperature, MALDI-ToF MS showed complete reduction of the
azide; MALDI-ToF MS (positive mode): calcd For C256H311N6NaO64P3
[M+Na]+
m/z: 4609.0; found, 4609.0. The mixture was filtered through a Celite
plug and condensed in vacuum to remove acetic acid, and the resulting
residue was redissolved in CH2Cl2 (500 mL) and treated with DBU
(2 mL). The solution stirred for 1 h, after which MALDI-ToF MS con-
firmed removal of the Fmoc and cyanoethoxyl protecting groups;
MALDI-ToF MS (negative mode): calcd for C217H281N3O60P3 [MꢀH]ꢀ m/
z: 3981.8; found, 3981.6. Then, 20% TFA in CH2Cl2 (500 mL) was added
directly to the reaction mixture at 08C, to give a final concentration of
approximately 10% TFA. After being stirred for 30 min at room temper-
ature, the reaction was co-evaporated with toluene 5 times. Purification
of the crude product by Sephadex LH-20 size exclusion chromatography
(CHCl3/MeOH/H2O, 3:3:1) afforded GPI 1 (0.70 mg, 90%). 1H NMR
(CDCl3/CD3OD/D2O 3:3:1, 500 MHz, anomeric region): d=5.46 (1 H
Man-I), 5.42–5.32 (8H, arachidonoyl lipid sp2-CH), 5.25 (1H, glyceride
CH), 5.24 (1H, GlcNH2-1), 5.04 (1H, Man-III), 4.98 (1H, Man-IV),
4.98 ppm (1H, Man-II); 13C NMR (CDCl3/CD3OD/D2O 3:3:1, 125 MHz,
anomeric region): d=100.3 (Man-IV), 100.3 (Man-II), 101.5 (GlcNH2-1),
99.5 (Man-III), 97.5 ppm (Man-I); 31P NMR (CDCl3, 160 MHz): d=4.37,
3.59, 3.51 ppm; MALDI-ToF MS (negative mode): calcd for
C81H145N3O43P3 [MꢀH]ꢀ m/z: 1940.8; found, 1940.7.
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Received: August 16, 2011
Published online: December 21, 2011
Chem. Eur. J. 2012, 18, 1194 – 1201
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1201