First synthesis of a malarial prototype: A fully lipidated and phosphorylated GPI membrane anchor

Article abstract of DOI:10.1016/j.tetlet.2003.10.179

A strategy is described for syntheses of a fully lipidated and phosphorylated prototype of the GPI of Plasmodium falciparum, the causative agent of lethal cerebral, drug-resistant malaria. Orthoesters, prepared in four steps from D-mannose, and methyl α-D

Full text of DOI:10.1016/j.tetlet.2003.10.179

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 879–882
First synthesis of a malarial prototype: a fully lipidated and
phosphorylated GPI membrane anchor
Jun Lu, K. N. Jayaprakash and Bert Fraser-Reid*
Natural Products and Glycotechnology Research Institute, Inc., (NPG), 4118 Swarthmore Road, Durham, NC 27706, USA
Received 15 September 2003; revised 17 October 2003; accepted 20 October 2003
Abstract—A strategy is described for syntheses of a fully lipidated and phosphorylated prototype of the GPI of Plasmodium fal-
ciparum, the causative agent of lethal cerebral, drug-resistant malaria. Orthoesters, prepared in four steps from D-mannose, and
methyl a- -glucopyranoside are the key starting materials. The latter furnishes the inositol moiety using Benderꢀs procedure, while
D
the former gives the other four units of the pseudo-pentasaccharide. The strategy for installing the three biologically important acyl
units of the phosphoinositide has been worked out. The critical, biosynthetically important C2-O-acyl group of the inositol is
exceptionally stable, showing no tendency to migrate to the cis-related C3-OH in several test substrates.
ꢀ 2003 Elsevier Ltd. All rights reserved.
The insurgence of cerebral malaria that traditionally
claims 2 to 3 million lives annually, mainly in tropical
ꢁThird Worldꢀ nations,1 now threatens the northern and
southern temperate zones.2 This development has
accelerated attention to the causative parasite, Plasmo-
dium falciparum, prompting determination of its gen-
ome,3 and development of various drugs and vaccine
candidates.4
variant surface glycoprotein (VSG) of Tyrpanosoma
€
brucei, Guther and Ferguson have established a parasite
biosynthetic pathway,9 which enables comparisons to be
drawn with the mammalian pathway deduced by
Kinoshita and Inoue.10 A crucial difference is that in
mammals inositol acylation necessarily precedes addi-
tion of the first mannose (i.e., unit I). However, in the
case of T. brucei, the mannosylation does not require
prior inositol acylation.11
Seminal 1992 publications by Playfair and co-workers
implicated phosphoinositide antigens,5 and subsequent
studies6 culminated in the assignment of the glycosyl-
phosphatidylinositol (GPI) candidate structure 1a by
Schwarz and co-workers.7 Subsequent refinement by
Gowda and co-workers has shown that the lipid residues
of 1a, exhibit wide structural variability, which pro-
foundly affect biological activity.8 In this manuscript, we
report the synthesis and properties of a malarial GPI
prototype, 1b, in which the crucially important lipid
residues of the phosphoinositol moiety have been
incorporated by a strategy that should be readily
extended to other such heavily lipidated GPIs.
Inositol acylation therefore enforces a subtle distinction
between mammalian and parasite biosynthetic pathways
that has provided an opportunity for therapeutic inter-
vention.12
Further attention to lipidation arises from the fact that
the inositol acyl group does not normally persist in
mature GPIs. However, reacylation is sometimes seen,
notably in human derived GPI-anchored proteins such
as CD52,13 and AchE.14 In these compounds, as in the
case of 1a, the inositolꢀs acyl group was assigned at C2
because a cyclic phosphate was not produced15 upon
treatment with phospholipase C (PLC). However, acyl
migration to the cis-related C3-OH of 1 is a possibility
and such an occurrence would compromise the above-
mentioned PLC diagnosis.
Although GPIs have a conserved glycan core comprising
units I fi V of 1, there are significant differences in how
they are assembled in nature. By using cell-free systems
to study the most readily available GPI, namely the
Several total syntheses of GPI have been reported,16
none of which has had to confront phospholipidation as
well as inositol acylation.17 Anticipating that the latter
feature would present new challenges, we carried out
* Corresponding author. Tel./fax: +1-919-493-6113; e-mail:
dglucose@aol.com
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.179

880
J. Lu et al. / Tetrahedron Letters 45 (2004) 879–882
featuring tin-mediated chemistry,20 were applied, which
O
X
O
RO
N
H
P
HO
2
O
paved the way to the O2-acylated inositols 6a and 6b. As
shown in Scheme 2 this strategy, which has also been
reported by Guo and co-workers,17 was unreliable,
indicating that a different approach would be needed. A
more advanced substrate was chosen for testing, and
this was developed in Scheme 3.
R'
H
Bn
R" R'''
O
i
HO
HO
HO
a
b
H
H
V
iii
iv
PMB Bn
c TBDMS Bn Bn
O
ii
O
R'O
Ph
IV
O
O
O
d
Bn
e Tr
Bn Bn
Ac Ac
HO
R" O
RO'''
O
HO
2a
O
OH
O
4 steps from
D-mannosa
HO
HO
IIIOH
OH
O
O
O
O
II
HO
HO
HO
In keeping with the retrosynthetic plan in Scheme 1, the
inositol moiety was obtained from methyl a-D-gluco-
R' I
1
O
HO
3 HO
H2N
OMe
a P. falciparum candidate
structures: R=H or α Man;
R,'R",R'''=various fatty
acyl groups; X=protein
OH
O
OH
O
2
O
P
1
pyranoside 3,21 while the ꢁrestꢀ of 1, came from the
n-pentenyl orthoester (NPOE) 2a.22 Coupling of 2b with
423 (Scheme 3) under the agency of ytterbium(III)triflate
[Yb(OTf)3]24 and NIS gave a pseudo-disaccharide, 7a,
the lone benzoate of which was replaced by triflate, 7b,
so as to employ Deshongꢀs novel azide displacement to
give 8a.25
3
O
refs.21,23
OH
BnO
O
OBn
OBn
3
HO
O
2
1
O
b. X=H;R=H; R'=C4H9
4
O
R'''
R"
O
R"=R'''=C5H11
O
Scheme 1. Reagents and conditions: (i) (a) (n-Bu3Sn)2O, benzene/
PhCH2Br/Bu4NI; (b) PMBCl/NaH/DMF; (ii) PhCH2Br/NaH/Bu4NI;
(iii) TBDMSCl/THF/imidazole; (iv) Ph3CCl/pyridine/Ac2O.
Removal of PMB to give 8b, followed by efficient (79%)
reaction with NPOE 2c, paved the way to the pseudo-
trisaccharide 9a and thence 9b. Tin-mediated chemis-
try20 was applied to effect O1-allylation and subsequent
O2-acylation gave 10a and b. However, attempts to
remove the 1-O-allyl group under standard conditions
gave 11a in poor yield, and in the case of 11b, there was
gross decomposition.
O
R
OR'
2
O
2
BnO
HO
OBn
OBn
OH
OH
ref. 18
BnO
BnO
O
RO
1
1
5
O
P
O
R
R'
O
R"
O
OH
a
b
H
H
6
O
H
Alk
H
R'
R
R' R"
c
Acyl
a Oct Hex Hex
b PalmHex Hex
O
A different approach was therefore needed for substrates
more elaborate than 6. The pseudo-trisaccharide 9c
(Scheme 3) obtained from 8a by use of NPOE 2d, led to
9d, and the related acceptor 9e was coupled to the third
NPOE, 2d (Scheme 4) followed by debenzoylation gave
pseudo-tetrasaccharide acceptor 12.
Scheme 2.
model studies on the inositol moiety by itself.18 Nearly
40 years ago, Angyal and Tate had shown that the vic-
inal 1, 2-OH groups of 5a, could be differentiated by
chemoselective reactions with alkylating and acylating
agents giving rise to 5b and 5c, respectively, as major
products.19 The desired acylation therefore could not be
achieved directly, and so protection/deprotection steps,
The next step was very problematic. We had hoped to
use the 3,4-di-O-benzyl NPOE 2c again, but it failed to
couple with acceptor 12. Several probing experiments
revealed that acylated NPOEs worked better. Accord-
BnO
BnO
2c/vi
or
2d/vi
iv
OR
O
Ph
RO
BnO
O
N
o
O
BnO
PMBO
O
O
O
BnO
BnO
3
4
PMBO
BnO
OBn
OBn
O
O
BnO
O
OBn
OBn
O
7
i
O
2b
a R8=PMB
b R=H
a R=Bz
b R=Tf
ii,iii
v
BnO
R'O
BnO
OR
O
OBn
OBn
BnO
BnO
O
Bn
O
O
BnO
BnO
O
BnO
OBn
O
OBn
O
OBn
O
O
ix,x
xi
r.t.
O
O
O
R
BnO
R
BnO
O
BnO
BnO
O
N
BnO
3
N
O
3
BnO
N
3
O
OBn
OBn
O
OBn
OBn
OBn
OBn
O
HO
10AllylO
9
11
O
a R=Bz;R'=Bn
b R=R'=Bn
or
c R=Bz;R'=TBDMS
d R=Bn;R'=TBDMS
e R=Bn; R'=H
ii,vii
ii,vii
viii
Scheme 3. Reagents and conditions: (i) NIS/Yb(OTf)3/0.3 equiv/CH2Cl2/0 ꢁC (99%); (ii) NaOMe/MeOH/CH2Cl2 (87%); (iii) Tf2O/PyDMAP/CH2Cl2
ꢀ
(93%); (iv) TMSN3/TBAF/THF (39%); (v) BF3ÆEt2O/CH2Cl2 (86%); (vi) NIS/BF3ÆEt2O/CH2Cl2/0 ꢁC; (vii) BnBr/NaH/Bu4NI; (viii) TBAF/MS(4 A)/
THF (83%); (ix) CSA/ethylene glycol (68%); (x) (a) Bu2SnO, toluene, then DMF, )10 ꢁC allyl bromide, CsF, 18 h (54%); (b) RCOCl, pyridine; (xi)
PdCl2, HOAc, H2O, NaOAc.

J. Lu et al. / Tetrahedron Letters 45 (2004) 879–882
881
O
OBn
RO
OBn
NHCBz
OBn
O
P
O
BnO
O
BnO
BnO
BnO
BnO
BnO
OH
O
BnO
BnO
O
O
O
iv
2e
BnO
O
BnO
BnO
BnO
2d
vi
O
O
OR
O
O
BnO
OBn
O
i,ii
9e
BnO
BnO
i,ii
O
BnO
BnO
OBn
O
OBn
BnO
BnO
OBn
O
O
O
O
OBn
BnO
BnO
O
O
N
O
BnO
N3
O
BnO
BnO
N3
OBn
OR
O
BnO
OBn
OBn
3
O
OBn
OBn
OBn
O
O
13
O
14
RO
12
a R=Tr
b R=H
a R,R=C6H10
b R=H
iii
v
O
OBn
O
O
OBn
P
O
NHCBz
O
OBn
O
O
OBn
BnO
BnO
NHCBz
P
O
BnO
BnO
O
O
O
OBn
O
O
OBn
P
BnO
BnO
BnO
BnO
O
NHCBz
BnO
BnO
BnO
O
O
O
ix
OBn
O
O
O
1b
BnO
BnO
BnO
BnO
BnO
BnO
viii
OBn
O
O
O
vii
BnO
O
O
OBn
O
OBn
O
Bn
O
O
BnO
BnO
N3
BnO
OBn
OBn
Bn
O
O
OBn
OBn
O
O
O
O
BnO
O
O
O
O
BnO
BnO
N3
P
OBn
O
OBn
O
O
O
BnO
N3
O
OBn
O
OBn
OBn
O
O
HO
OMe
C3H7
15
16
17
O
Scheme 4. Reagents and conditions: (i) NIS/BF3ÆEt2O/CH2Cl2/0 ꢁC; (ii) (a) NaOMe/MeOH/CH2Cl2; (b) BnBr/NaH/Bu4NI; (iii) HCOOH/Et2O
(60%); (iv) CbzNH(CH2)2OP(OBn)N(i-Pr)2/1H-tetrazole/CH2Cl2; (v) CSA/ethylene glycol; (vi) C4H9C(OMe)3, CSA, CH3CN, 1 h (90%); (vii)
Yb(OTf)3, CH2Cl2, 0 ꢁC, 1 h (50%); (viii) C5H11COOCH2CH(OCOC5H11)CH2OP(OBn)N(i-Pr)2/1H-tetrazole, )40 ꢁC/mcpba (66%); (ix) Pearlmanꢀs
catalyst, atm pressure.
ingly, the tritylated diester 2e afforded the pseudo-penta-
saccharide 13 (75%). Protecting group adjustments
preceded installation of the phosphoethanolamine
complex26 and liberation of the inositolꢀs cis-diol 14b.
Acknowledgements
We thank the World Health Organization, the Human
Frontier Science Program Organization, and the Mizu-
tani Foundation for support.
Given the poor yields in the conversion of 10 into 11, the
stereoelectronic decomposition of cyclic orthoesters
pioneered by King and Allbutt27 was an attractive
option. Indeed, treatment of diol 14b with trimethyl
orthovalerate afforded the cyclic orthoester 15, which
was usually not isolated, but treated directly with
ytterbium(III)triflate24 to give the desired b-hydroxyes-
ter 16,28 as the major regioisomer in keeping with
expectations.27 Reaction with the readily prepared26 di-
acylatedglycerylphosphoamidite gave the fully lipidated
material 17.28 Debenzylation by atmospheric hydro-
genolysis over Pearlmanꢀs catalyst finally gave 1b.28
References and notes
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M.; Mungall, C.; Venter, J. C.; Carucci, D. J.; Hoffmann,
With respect to the crucial question of migration of the
inositolꢀs acyl group, we have examined three substrates:
(1) 2-O-pentanoyl myoinositol, (2) the fully lipidated
pseudo-disaccharide corresponding to units I and II of
17, and (3) the synthetic material 1b. In the first,
1H NMR easily gave no evidence of any acyl migration
over several months. The same conclusions were reached
for the other two substrates, based on the signal at
5.43 ppm that is assignable to the equatorial proton a to
the acyl group. (As a point of interest, we have also
synthesized the regioisomer of 1b with the inositolꢀs C1
and C2 functionalities reversed. The axial proton a to
the acyl group comes to much higher field ꢀ4.7 ppm.)

882
J. Lu et al. / Tetrahedron Letters 45 (2004) 879–882
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28. Supporting spectroscopic data for key compounds: For 16:
1H NMR (300 MHz, CDCl3) d 0.88 (t, 3H, –CH3), 1.34 (m,
2H, –CH2), 1.63 (m, 2H, –CH2), 2.40 (t, 2H, –CCH2–),
3.18–5.06 (m, 66H), 5.266 (d, 1H), 5.31 (d, 1H), 5.70 (t, 1H,
H-2, inositol), 7.06–7.31 (m, 70H, Ar-H). 13C NMR
(75 MHz, CDCl3) d 13.82, 22.18, 27.26, 29.77 (pentyl),
46.12, 65.19, 66.55, 66.79, 68.83, 69.69, 71.96, 72.80, 73.10,
73.69, 74.15, 74.48, 74.87, 75.21, 76.87, 77.37, 78.358,
79.57, 79.99, 80.68, 80.91, 95.13, 98.89, 99.10, 100.06,
110.81, 126.83, 127.04, 127.14, 127.27, 127.33, 127.42,
127.49, 127.62, 127.69, 127.76, 127.86, 127.94, 128.05,
128.12, 128.15, 128.20, 128.25, 128.30, 128.37, 137.59,
€
9. Guther, M. L. S.; Ferguson, M. A. J. EMBO J. 1995, 14,
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1
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138.01, 138.18, 138.29, 138.44, 138.67. For 17: H NMR
(300 MHz, CDCl3) d 0.87 (m, 9H, 2· –CH3), 1.26 (m, 10H,
–CH2), 1.57 (m, 6H, –CH2), 2.26 (m, 6H, 3-COCH2–),
3.17–5.21 (m, 72H), 5.47 (m, 1H, anomeric H), 5.89 (t, 1H,
H-2, inositol). 13C NMR (75 MHz, CDCl3) d 13.80, 13.99,
22.11, 22.36, 24.56, 39.77, 31.24, 31.295, 33.99, 34.01,
61.63, 66.59, 66.82, 68.89, 69.47, 69.98, 71.98, 72.23,
73.156, 74.15, 74.48, 74.88, 75.03, 76.59, 77.20, 79.62,
97.11, 98.67, 98.99, 99.28, 126.98, 127.08, 127.20, 127.30,
127.36, 127.42, 127.53, 127.55, 127.62, 127.71, 127.78,
127.86, 127.93, 127.98, 128.07, 128.13, 128.17, 128.27,
128.34, 128.44, 128.49, 128.56, 136.44, 137.62, 137.73,
137.99, 138.14, 138.31, 138.44, 138.58, 138.81, 172.31,
172.58, 172.99 (C@O). 31P NMR (126 MHz, CDCl3) d
)0.49, )0.469, 0.21, 0.37, 7.48, 7.56, 7.83, 7.87. MS calcd
2985.28, found 3010.4 (M+Na).
For 1b: 1H NMR (300 MHz, D2O): d 0.89 (m, 9H, 3·
–CH3), 1.31 (m, 10H, –CH2–),1.64 (m, 6H, –CH2–), 2.43–
2.53 (m, 6H, –CO–CH2–), 3.30–4.38 (m, 38H, mannose,
inositol), 5.03 (1H), 5.12 (1H), 5.22 (1H, anomeric H), 5.54
(1H, H-2, inositol). MS (FAB+, 1385.7 M+H; 1407.9
M+Na).
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