First total synthesis of a GPI-anchored peptide

Article abstract of DOI:10.1021/jo034213t

A GPI-anchored dipeptide of sperm CD52 antigen was prepared through a convergent synthesis. First, the dipeptide with its C-terminus free and the GPI with its nonreducing end phosphoeth-anolamine bearing a free amino group were synthesized separately. Then, the two building blocks were coupled with use of EDC/HOBt as the condensation reagent. Finally, the GPI-anchored peptide was deprotected to give the target molecule 1.

Full text of DOI:10.1021/jo034213t

First Total Synthesis of a GPI-Anchored Peptide
Jie Xue, Ning Shao, and Zhongwu Guo*
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106-7078
zxg5@po.cwru.edu
Received February 18, 2003
A GPI-anchored dipeptide of sperm CD52 antigen was prepared through a convergent synthesis.
First, the dipeptide with its C-terminus free and the GPI with its nonreducing end phosphoeth-
anolamine bearing a free amino group were synthesized separately. Then, the two building blocks
were coupled with use of EDC/HOBt as the condensation reagent. Finally, the GPI-anchored peptide
was deprotected to give the target molecule 1.
Introduction
Many proteins and glycoproteins are anchored to
eukaryotic cells by glycosylphosphatidyl inositols (GPIs),1
and the GPI-anchored proteins/glycoproteins, as well as
the GPIs, play a pivotal role in numerous biological
functions.2-4 To study these functions, it is essential to
access homogeneous glycoforms of GPIs and GPI-an-
chored structures. The problem is that it is difficult, if
not impossible, to prepare homogeneous glycoconjugates
biochemically. Therefore, chemical synthesis becomes an
important tool for obtaining homogeneous, well-defined
samples for these investigations.
Figure 1 highlights the typical structure of a GPI-
anchored protein or glycoprotein.1 All GPIs share a
conserved common core with the tetrasaccharide linked
to the 6-O-position of inositol. The variable lipid chains
are attached to the 1-O-position of inositol through a
phosphate group. The GPI may have other carbohydrate
residues as branches, and sometimes the inositol is
modified by an acyl group. The peptides/glycopeptides are
attached to the nonreducing end of the GPI glycans via
the C-termini and a phosphoethanolamine group.
FIGURE 1. Typical structure of GPI-anchored proteins/
glycoproteins.
Ley,11,12 Martin-Lomas,13 Ogawa,14-16 Schmidt,17-21 See-
berger,22,23 and us.24 The P. faclciparum GPI was further
conjugated to a carrier protein through an artificial linker
to form a malaria vaccine that could induce protective
immune reactions in animals.23 However, there has been
no report yet describing the synthesis of a natural GPI-
peptide/glycopeptide.
This paper describes the first total synthesis of a
naturally linked GPI-peptide conjugate 1 (Figure 2). The
synthetic target is a fragment of sperm CD52. CD52
antigens play an important role in the human immune
system25-27 and the human reproduction process.28-30
Since the first characterization of a GPI by Ferguson
in 1988,5,6 the structural complexity of GPIs has attracted
significant interest in their total synthesis. Several GPIs
were therefore prepared by Ferguson,7,8 Fraser-Reid,9,10
(11) Baeschlin, D. K.; Chaperon, A. R.; Charbonneau, V.; Green, L.
G.; Ley, S. V.; Lu¨cking, U.; Walther, E. Angew. Chem., Int. Ed. 1998,
37, 3423-3428.
(12) Baeschlin, D. K.; Chaperon, L. R.; Green, L. G.; Hahn, M. G.;
Ince, S. J.; Ley, S. V. Chem. Eur. J. 2000, 6, 172-186.
(13) Martin-Lomas, M.; Khiar, N.; Garcia, S.; Koessler, J.; Nieto,
P. M.; Rademache, W. Chem. Eur. J. 2000, 6, 3608-3621.
(14) Murakata, C.; Ogawa, T. Carbohydr. Res. 1992, 235, 95-114.
(15) Murakata, C.; Ogawa, T. Tetrahedron Lett. 1991, 32, 671-674.
(16) Murakata, C.; Ogawa, T. Tetrahedron Lett. 1990, 31, 2439-
2442.
(1) Ferguson, M. A. J.; Williams, A. F. Annu. Rev. Biochem. 1988,
57, 285-320.
(2) Robinson, P. J. Cell Biol. Intern. Rep. 1991, 15, 761-767.
(3) Robinson, P. J.; Millrain, M.; Antoniou, J.; Simpson, E.; Mellor,
A. L. Nature 1989, 342, 85-87.
(4) Thomas, J. R.; Dwek, R. A.; Radmacher, T. W. Biochemistry 1990,
29, 5413-5422.
(17) Mayer, T. G.; Kratzer, B.; Schmidt, R. R. Angew. Chem., Intl.
Ed. Engl. 1994, 33, 2177-2181.
(5) Ferguson, M. A. J.; Homans, S. W.; Dwek, R. A.; Rademacher,
T. W. Science 1988, 239, 753-759.
(18) Mayer, T. G.; Weingart, R.; Mu¨nstermann, F.; Kawada, T.;
Kurzchalia, T.; Schmidt, R. R. Eur. J. Org. Chem. 1999, 2563-2571.
(19) Kratzer, B.; Mayer, T. G.; Schmidt, R. R. Eur. J. Org. Chem.
1998, 291-298.
(6) Homans, S. W.; Ferguson, M. A. J.; Dwek, R. A.; Rademacher,
T. W.; Anand, R.; Williams, A. F. Nature 1988, 333, 269-272.
(7) Crossman, A., Jr.; Brimacombe, J. B.; Ferguson, M. A. J.; Smith,
T. K. Carbohydr. Res. 1999, 321, 42-51.
(20) Mayer, T. G.; Schmidt, R. R. Eur. J. Org. Chem. 1999, 1153-
1165.
(8) Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. Carbohydr.
Res. 1995, 270, 85-91.
(21) Pekari, K.; Schmidt, R. R. J. Org. Chem. 2003, 68, 1295-1308.
(22) Hewitt, M. C.; Snyder, D. A.; Seeberger, P. H. J. Am. Chem.
Soc. 2002, 124, 13434-13436.
(9) Udodong, U. E.; Madsen, R.; Roberts, C.; Fraser-Reid, B. J. Am.
Chem. Soc. 1993, 115, 7886-7887.
(23) Schofield, L.; Hewitt, M. C.; Evans, K.; Siomos, M.-A.; See-
berger, P. H. Nature 2002, 418, 785-789.
(10) Campbell, A. S.; Fraser-Reid, B. J. Am. Chem. Soc. 1995, 117,
10387-10388.
(24) Xue, J.; Guo, Z. Bioorg. Med. Chem. Lett. 2002, 12, 2015-2018.
10.1021/jo034213t CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/19/2003
4020
J. Org. Chem. 2003, 68, 4020-4029

First Total Synthesis of a GPI-Anchored Peptide
FIGURE 2. The synthetic target.
SCHEME 1. Retrosynthetic Analysis
Additionally, the unique structure of sperm CD52 also
causes our attention. For example, the GPI of sperm
CD52 has a long acyl chain on the inositol 2-O-position.
Thus, the synthesis of sperm CD52 and its GPI is
challenging in regard to the planning of protecting tactics
and the preparation of the required inositol derivatives.
lead to a convergent synthetic procedure and avoid the
potential chemistry compatibility problems.
In the synthesis of GPI fragment 3, the benzyl group
was chosen as the permanent protection, since it can be
later deprotected globally under mild conditions. The
presence of an ester bond in the final synthetic target
prohibited acyl groups, such as acetyl and benzoyl, from
being used.
Results and Discussion
Fragment 3 was further disconnected into three smaller
pieces 4, 5, and 6. The primary amino group in 4 was
protected by an Fmoc that can be easily and selectively
removed. Fragment 5 was designed as a thioglycoside to
facilitate its direct use as a glycosyl donor, while the 6-O-
position at its nonreducing end had to be differentiated
from that of other hydroxyls. This design also enabled
the use of semiorthogonal glycosylations in the prepara-
tion of 5 from monosaccharides 7, 8, and 9.
For retrosynthesis of 1 (Scheme 1), the amido bond
between the peptide and GPI was disconnected first to
yield two key fragments 2 and 3. As mentioned above,
this amido linkage is shared by all GPI-anchored proteins
and glycoproteins (Figure 1). Then, a synthetic strategy
thus developed may be of general application. Moreover,
preparing the GPI and the peptide separately and
coupling the two fragments at the final stage could also
Meanwhile, 10 and 11 were designed as the building
blocks of 6. The azido group in 10 was stable to most
reactions involved, whereas it could be easily reduced to
form D-glucosamine. The 1-, 2-, and 6-O-positions of 11
had to be discriminated, as the 6-O-position is linked to
the GPI glycan and the 1-O- and 2-O-positions are
respectively linked to the phospholipid and acyl group
in the natural structure of sperm CD52. We planned to
protect its 1-O-position with a p-methoxylbenzyl (MBn),
which can be selectively removed by mild oxidants for
(25) Treumann, A.; Lifely, M. R.; Schneider, P.; Ferguson, M. A. J.
J. Biol. Chem. 1995, 270, 6088-6099.
(26) Xia, M.-Q.; Hale, G.; Lifely, M. R.; Ferguson, M. A. J.; Capmbell,
D.; Packman, L.; Waldmann, H. Biochem. J. 1993, 293, 633-640.
(27) Hale, G.; Xia, M.-Q.; Tighe, H. P.; Dyer, J. S.; Waldmann, H.
Tissue Antigens 1990, 35, 118-127.
(28) Schroter, S.; Derr, P.; Conradt, H. S.; Nimtz, M.; Hale, G.;
Kirchhoff, C. J. Biol. Chem. 1999, 274, 29862-29873.
(29) Kameda, K.; Tsuji, Y.; Koyama, K.; Isojima, S. Biol. Reprod.
1992, 46, 349-357.
(30) Eccleston, E. D.; White, T. W.; Howard, J. B.; Hamilton, D. W.
Mol. Reprod. Dev. 1994, 37, 110-119.
J. Org. Chem, Vol. 68, No. 10, 2003 4021

Xue et al.
SCHEME 2a
a Reagents and conditions: (a) Bu2SnO, toluene, reflux, 2.5 h; then AllBr, CsF, DMF, rt, 16 h; 71-80%; (b) MBnCl, NaH, DMF, rt, 3
h; 85%; (c) AcCl, MeOH-CH2Cl2, rt, 10 min; 60-68%; (d) BnBr, NaH, DMF, rt, 3 h; >99%; (e) AcCl, MeOH-CH2Cl2, rt, 2 h; 78-89%; (f)
Bu2SnO, toluene, reflux, 2.5 h; then BnBr, CsF, DMF, rt, 15 h; 98%; (g) Bu2SnO, toluene, reflux, 2.5 h; then MBnCl, CsF, KI, DMF, rt,
16 h; 94%; (h) C15H31COOH, DCC, DMAP, CH2Cl2, rt, overnight; >99%; (i) PdCl2, NaOAc, AcOH, H2O, rt, 18 h; 94%.
SCHEME 3a
the introduction of phospholipid, while the acyl group was
brought in early on.
Though many protocols for selective protection and
deprotection of inositols have been developed recently,31-38
preparing inositol derivatives that contain several uniquely
modified hydroxyl groups, e.g. 11, can be a major
undertaking, especially since these derivatives are usu-
ally used as the basic starting materials and demanded
in large quantities in GPI synthesis. To meet this
demand, we have developed a new synthetic procedure
(Scheme 2) for an inositol derivative 2324 having 1-O-,
2-O-, and 6-O-positions differently protected. These posi-
tions were also distinguished from other hydroxyl groups.
This new procedure is highlighted by repeated uses of
tin complex to facilitate the regioselective alkylations, as
well as the use of both enantiomers of 12, for synthesizing
the same target molecules 23 and 11. The synthetic
efficiency was thus significantly improved, and 11 was
prepared on multigram scales.
The optically pure intermediates (+)-12 and (-)-12
were prepared from myo-inositol according to a reported
procedure, using a selective enzymatic reaction to resolve
the enantiomers.31 After a series of regioselective protec-
tions and deprotections, both (+)-12 and (-)-12 were
transformed to 23 in 32% and 42% overall yields. Then,
a palmitoyl group was introduced to the only free 2-OH
in 23, using DCC as the coupling reagent to afford 24
(>99%). The allyl group in 24 was finally removed by
acetic acid and PdCl2, and the resulting 11 was ready as
a glycosyl acceptor to be coupled with 10.
a Reagents and conditions: (a) Ac2O-AcOH (1:1), H2SO4 (cat.),
0 °C, 15 min; 72%; (b) BnNH2, Et2O, rt, 2 h; >99%; (c) DAST,
CH2Cl2, 0 °C, 30 min; >99%; (d) NaOMe, MeOH, rt, 2 h; >99%;
(e) BnBr, NaH, DMF, rt, 3 h; 75%; (f) AgOTf, HfCp2Cl2, MS 4 Å,
Et2O; 40%; (g) PdCl2, NaOAc, AcOH, H2O, rt, 18 h, 99%.
to a literature procedure.39,40 The five-membered ring in
26 was opened via acetolysis in acetic anhydride and
acetic acid (1:1), using a small amount of concentrated
sulfuric acid as a catalyst, yielding 27 as an anomeric
mixture (R:â 3.5:1.0). The anomeric acetyl group was then
removed with benzylamine, and the resulting hemiacetal
28 was treated with DAST at low temperature to afford
the corresponding glycosyl fluoride 29 (R:â 1:6).
The reaction of 10 and 11 was effected by dichloro-
hafnocene and silver perchlorate41,42 in anhydrous ether
to afford a mixture containing both the R-pseudodisac-
charide (31, GlcN3: H-1 δ 5.65, J1,2 ) 3.8 Hz; C-1 δ 97.5)
and its â-anomer, slightly in favor of the former (4:3).
This result was different from most references that report
moderate yields of only R-anomer in similar reactions,
which might suggest that the long acyl chain on the 2-O-
position of inositol had a significant influence on the
reaction. It was also observed that solvent choice would
affect the stereochemical outcome, as the glycosylation
in DCM produced a mixture slightly in favor of the
The glycosyl fluoride 10 as a glycosyl donor was
prepared from commercial D-glucal (25, Scheme 3). The
transformation of 25 to 26 was accomplished according
(31) Gou, D.; Liu, Y.; Chen, C. Carbohydr. Res. 1992, 234, 51-64.
(32) Lu, P.; Gou, D.; Shieh, W.; Chen, C. Biochemistry 1994, 33,
11586-11587.
(33) Wang, D.; Hsu, A.; Song, X.; Chiou, C.; Chen, C. J. Org. Chem.
1998, 63, 5430-5437.
(34) Desai, T.; Fernandez-Mayoralas, A.; Gigg, J.; Gigg, R.; Payne,
S. Carbohydr. Res. 1990, 205, 105-123.
(35) Desai, T.; Gigg, J.; Gigg, R.; Payne, S. Carbohydr. Res. 1992,
225, 209-228.
(39) Tailler, D.; Jacquinet, J.-C.; Noirot, A.-M.; Beau, J.-M. J. Chem.,
Soc. Perkin Trans. 1 1992, 3163-3165.
(36) Leung, L. W.; Bittman, R. Carbohydr. Res. 1998, 305, 171-
179.
(40) Xue, J.; Guo, Z. Tetrahedron Lett. 2001, 42, 6487-6489.
(41) Suzuki, K.; Maeta, H.; Matsumoto, T.; Tsuchihashi, G. Tetra-
hedron Lett. 1988, 29, 3571-3574.
(37) Garegg, P. J.; Iversen, T.; Johansson, R.; Lindberg, B. Carbo-
hydr. Res. 1984, 130, 322-326.
(38) Garegg, P. J.; Konradsson, P.; Oscarson, S.; Ruda, K. Tetrahe-
dron 1997, 53, 17727-17734.
(42) Suzuki, K.; Maeta, H.; Matsumoto, T. Tetrahedron Lett. 1989,
30, 4853-4856.
4022 J. Org. Chem., Vol. 68, No. 10, 2003

First Total Synthesis of a GPI-Anchored Peptide
SCHEME 4a
a Reagents and conditions: (a) Ac2O, AcOH, pyr, rt, overnight; 95%; (b) a. HBr, AcOH, rt, 6 h; b. 2,6-lutidine, MeOH-CH2Cl2, rt, 12
h; 87%; (c) BnCl, KOH, reflux, 4 h; 66%; (d) TMSCl, CH2Cl2; >99%; (e) NaH, BnBr, DMF, rt, 16 h; >99%; (f) Ac2O, HOAc, H2SO4, 0 °C,
0.5 h; 70%; (g) BF3‚Et2O, EtSH, 0 °C, 0.5 h; 65%; (h) HBr in HOAc, CH2Cl2; (i) NaOMe, MeOH; 91%; (j) AgOTf, MS 4 Å, CH2Cl2; 53-82%;
(k) TBDMSCl, imidazole, DMAP, DMF; 92%.
â-anomer. Though difficult, the two anomers were sepa-
rable on a silica gel column. However, we found that after
the removal of the 4′-O-allyl group by acetic acid and
PdCl2, the resulting 6 was easily separated from its
â-anomer via flash column chromatography. Several
grams of the building block 6 was thus obtained.
Building block 5 was constructed from three monosac-
charide units 7, 8, and 9 as shown in Scheme 4. As
mentioned, we intended to have an alkylthio group linked
to the reducing end of 5 so that it can be directly used as
a glycosyl donor.43 Meanwhile, the application of orthogo-
nal glycosylations44-46 in the construction of 5 could save
several steps of anomeric deprotection and activation.
Monosaccharide 8 was readily prepared from D-man-
nose following a reported procedure,47 while 748 and 9
were both derived from R-methyl mannoside 36 (Scheme
4). Thus, after perbenzylation of 36, the resulting 37 was
treated with acetic acid, anhydride, and sulfuric acid to
acetolyze the protecting groups on the 1-O- and 6-O-
positions to afford 38. When 38 reacted with ethanethiol
using trifluoroborane etherate as the promoter, the
R-thioglycoside 39 (H-1: δ 5.33, J1,2 ) 1.3 Hz) was formed
in 65% yield. Deacetylation of 39 gave building block 7.
On the other hand, treating 38 with hydrogen bromide
in acetic acid afforded the glycosyl bromide 9.
The glycosylation of 7 by 8 with silver triflate as the
promoter was stereospecific and gave the R-disaccharide
40 (H-1′: δ 4.94, J1,2 ) 1.2 Hz; C-1′: δ 99.6) in 82% yield.
On treatment with sodium methoxide, 40 was trans-
formed to the glycosyl acceptor 41, which was then
glycosylated by 9, again with silver triflate as the
promoter, to give the trisaccharide 42 (53%). The stere-
ochemistry of the latter glycosyl linkage was supported
by the NMR data (H-1′′: δ 5.14, J1,2 ) 1.2 Hz). The 6′′-
acetyl group in 42 was subsequently replaced by a tert-
butyldimethylsilyl (TBDMS) group to afford the triman-
nose building block 5.
The coupling of 5 with 6 (Scheme 5) was accomplished
under the promotion of N-iodosuccinimide (NIS) and
triflic acid (TfOH) to yield the pseudopentasaccharide 44
(92%, H-1′: δ 5.21, J1,2 < 1 Hz). Its MALDI-TOF-MS also
supported the structural assignment. Then, the TBDMS
group was removed with tetrabutylammonium fluoride
(TBAF) to afford 45. This reaction was clean but unex-
pectedly slow, probably due to the large steric bulk of
the substrate.
Direct phosphorylation of 45 with 2 equiv of 4 led to a
slow and incomplete reaction, yielding only 15% of 46.
The use of 7 equiv of 4 could push the reaction to
completion but complications arose due to side reactions
involving the azido moiety. This result was a surprise to
us, as literature reported good results in similar reactions
with a large excess (up to 8 equiv) of the phosphorylating
reagents. The unusual reactivity of 45 might be caused
by the long acyl chain on the inositol. To avoid this
problem, the azide was thus reduced with triethylphos-
phine, and the benzyloxycarbonyl (CBz) group was used
to protect the resulting free amine. Both reactions were
clean, showing only one new spot on TLC, and the
MALDI-TOF-MS of the product offered a single molecular
peak that was consistent with 47. However, its room
temperature 1H NMR spectrum showed two sets of
signals (ca. 8:1). Recording the NMR at 50 °C coalesced
the signals into one set that agreed well with 47,
suggesting that 47 has two rotamers at room tempera-
ture. 47 was obtained from 45 in 70% yield.
Once the azido moiety was converted to an amide, the
phosphorylation reaction was rather smooth. Treating 47
with 4 and tetrazole, and then tert-butyl hydroperoxide,
gave an inseparable diastereoisomeric mixture (4.5:1) of
48 in 84% yield. The removal of Fmoc with a dimethyl-
amine/THF solution (1 M) yielded the GPI fragment 3
having a free amino group at its nonreducing end.
The dipeptide fragment 2 was easily prepared from the
reaction of commercial perfluorophenyl (Pfp) ester of
Fmoc-proline with free serine in DMF. After purification
with a silica gel column, 2 (4 equiv) was coupled with 3,
using HOBt/EDC, to give 49 in 60% isolated yield
(Scheme 6). The unprotected side chain of serine did not
affect the coupling reaction. Fmoc was then removed, and
the product 50 was carefully purified by column chro-
(43) Norburg, T. In Modern Methods in Carbohydrate Synthesis;
Khan, S. H., O’Nell, R. A., Eds.; Harwood Academic Publishers:
Amsterdam, The Netherlands, 1996; pp 82-106.
(44) Ito, Y.; Kanie, O.; Ogawa, T. Angew. Chem., Int. Ed. Engl. 1996,
35, 2510-2512.
(45) Raghavan, S.; Kahne, D. J. Am. Chem. Soc. 1993, 115, 158-
1581.
(46) Mootoo, D. R.; Konradsson, P.; Udodong, U.; Fraser-Reid, B. J.
Am. Chem. Soc. 1988, 110, 5583-5584.
(47) Garegg, P. J.; Maron, L. Acta Chem. Scand., Ser. B 1979, B33,
39-41.
(48) Tennant-Eyles, R. J.; Davis, B. G.; Fairbanks, A. J. Tetrahe-
dron: Asymmetry 2000, 11, 231-243.
J. Org. Chem, Vol. 68, No. 10, 2003 4023

Xue et al.
SCHEME 5a
a Reagents and conditions: (a) NIS, TfOH, DCM-ether (1:1), 0 °C, 30 min; 90%; (b) TBAF, HOAc, DCM, 35-40 °C, 3 d; 95%; (c) 4 (2
equiv), tetrazole, DCM-MeCN (3:1), rt, 6 h; then t-BuOOH; 15%; (d) PEt3, CBz2O, MeOH-DCM (1:2), rt, overnight; 70%; (e) 4 (7 equiv),
tetrazole, DCM-MeCN (3:1), rt, 6 h; then t-BuOOH; 84%; (f) Me2NH, THF, rt, 1 h, 95%.
SCHEME 6a
sides and those smaller than 170 Hz are observed in
1
â-glycosides. The H and 13C signals of the 2-O-position
of inositol shifted downfield to δ 5.41 and 75.6, respec-
tively, indicating that this position is linked to the acyl
group. The R-proton signals (δ 4.45 and 4.38) of the amino
acid residues were also distinctive. Both were triplets
(Figure 3a in D2O) and had 3JH,H coupling of 5.4 and 7.2
Hz, respectively. The chemical shift (δ 1.72) of the 31P
signal showed the phosphate group.
In summary, this paper described the first total
synthesis of a GPI-anchored peptide through a highly
convergent procedure, in which a key synthetic design
was to separately prepare the properly protected peptide
and GPI. The two fragments were effectively coupled
between the free peptide C-terminus and the free amino
group of phosphoethanolamine at the nonreducing end
of GPI, using EDC/HOBt as the condensation reagent.
To facilitate the preparation of the key building blocks,
e.g. the core trimannose 5 and the specifically protected
inositol 23, new synthetic routes were developed that
could lead to the expected structures on large scales. The
results may be applied to the synthesis of other GPI-
anchored structures. On the other hand, unusual reac-
tions were observed with the 2-O-palmitoyl derivatives
of inositol, suggesting the significant influence of steric
hindrance caused by the large acyl group.
a Reagents and conditions: (a) EDC, HOBt, DCM-DMF (10:1),
rt, overnight; 60%; (b) Me2NH, THF, rt, 1 h; 85%; (c) 10% Pd/C,
H2, CH3Cl-MeOH-H2O (10:10:3), 1 d; 91%.
matography. Global deprotection of 50 in hydrogen
atmosphere with 10% Pd/C as a catalyst offered the final
synthetic target 1 as a white solid (91%).
1
1
1
The 1D H, 31P NMR, 2D H-1H, H-13C NMR, and
MS of the final product agreed well with the expected
structure of 1. For instance, the chemical shifts and J
3
coupling constants of the anomeric protons (δ 5.34, 5.20,
5.13, 4.98; all J1,2 < 1 Hz; Figure 3a,b) clearly indicated
the R-glycosyl linkages of sugar units. The R-configura-
tions were also supported by the chemical shifts of
anomeric carbons (δ: 97.9, Glc; 100.0, Man; 102.8, Man;
104.2, Man) and the 1JC,H coupling constants between the
anomeric H and C (1JC,H ) 174 (Glc), 174 (Man), 174
(Man), 174 Hz (Man)) derived from its heteronuclear
multiple quantum coherence (HMQC) spectrum (Figure
Experimental Section
(+)-6-O-A1lyl-2,3:4,5-di-O-cyclohexylidene-myo-inosi-
tol (13).33 A mixture of (+)-12 (1.8 g, 5.3 mmol) and Bu2SnO
(1.37 g, 5.5 mmol) in 60 mL of toluene was refluxed with
azeotropic removal of water for 2.5 h. After concentration to
dryness under reduced pressure, to the residue were added
DMF (30 mL), CsF (2.4 g, 15 mmol), and allyl bromide (0.58
mL, 6.4 mmol) at -10 °C. The mixture was stirred at room
1
3c). According to Bock and Petersen,49 JC,H coupling
constants greater than 170 Hz are observed in R-glyco-
(49) Bock, K.; Petersen, C. J. Chem. Soc., Perkin Trans. 2 1974,
293-297.
4024 J. Org. Chem., Vol. 68, No. 10, 2003

First Total Synthesis of a GPI-Anchored Peptide
FIGURE 3. 1H NMR spectra of the final synthetic target 1 in D2O (a) and CD3OD (b): the anomeric region of sugar units, R-Hs
of amino acids, and 2-H of inositol. 1H-13C HQMC spectrum (coupled) of 1 in CD3OD (c): the anomeric region. MALDI-TOF-MS
spectrum of 1 (d).
temperature for 16 h, and then diluted with ethyl acetate. The
organic solution was washed with brine, dried with Na2SO4,
and then concentrated under vacuum. Column chromatogra-
phy of the residue gave 13 as a colorless syrup (1.62 g, 80%).
[R]D +8.2 (c 0.8, CH2Cl2) (lit.33 [R]D +4.3 (c 2.8, CHCl3)). 1H
NMR (CDCl3, 300 MHz): δ 5.87-6.00 (m, 1 H), 5.32 (dd, J )
17.2, 2.4 Hz, 1 H), 5.20 (d, J ) 10.3 Hz, 1 H), 4.42 (dd, J )
7.4, 3.6 Hz, 1 H), 4.31 (t, J ) 7.2 Hz, 1 H), 4.10-4.27 (m, 3 H),
3.98 (m, 1 H), 3.82 (dd, J ) 7.9, 1.7 Hz, 1 H), 3.48 (dd, J )
10.5, 7.9 Hz, 1 H), 2.60 (d, J ) 1.4 Hz, 1 H), 1.34-1.77 (m, 20
H). 13C NMR (CDCl3, 50 MHz): 134.4, 117.4, 113.0, 111.3, 79.8,
78.7, 76.7, 76.5, 75.3, 72.2, 70.6, 36.7, 36.6, 36.3, 33.5, 25.2,
25.1, 23.9, 23.8, 23.7, 23.4.
6-O-Allyl-1-O-(p-methoxybenzyl)-2,3:4,5-di-O-cyclohex-
ylidene-myo-inositol (14).33,50 To a solution of 13 (1.8 g, 4.73
mmol) and 4-methoxybenzyl chloride (0.77 mL, 5.7 mmol) in
DMF (30 mL) was added NaH (284 mg, 60%, 7.1 mmol) at 0
°C. The mixture was stirred at room temperature for 3 h. The
excess NaH was neutralized by MeOH, and the mixture was
diluted with ethyl acetate. After workup as mentioned above,
column chromatography of the residue afforded 13 as a
colorless syrup (2.0 g, 85%). 1H NMR (CDCl3, 300 MHz): δ
7.30 (d, J ) 8.6 Hz, 2 H), 6.81 (d, J ) 8.6 Hz, 2 H), 5.81-5.93
(m, 1 H), 5.27 (dd, J ) 17.2, 1.7 Hz, 1 H), 5.17 (dd, J ) 10.4,
1.6 Hz, 1 H), 4.68 (d, J ) 11.8 Hz, 1 H), 4.62 (d, J ) 11.8 Hz,
1 H), 4.26-4.34 (m, 2 H), 3.96-4.13 (m, 3 H), 3.81 (s, 3 H),
3.77 (dd, J ) 8.0, 3.0 Hz, 1 H), 3.69 (t, J ) 3.1 Hz, 1 H), 3.42
(dd, J ) 10.6, 7.8 Hz, 1 H), 1.35-1.79 (m, 20 H). 13C NMR
(CDCl3, 50 MHz): δ 159.3, 134.6, 130.3, 129.5, 117.0, 113.8,
112.8, 111.6, 79.6, 78.8, 78.5, 77.0, 76.5, 75.1, 73.0, 70.7, 55.3,
36.7, 36.5, 34.7, 25.2, 25.1, 24.0, 23.8, 23.7.
was stirred at room temperature for 3 h. The excess NaH was
neutralized by MeOH, and the solution was diluted with ethyl
acetate. The organic layer was washed with water and brine,
dried, and condensed to give crude (-)-6-O-allyl-1-O-(p-meth-
oxybenzyl)-2,3-O-cyclohexylidene-myo-inositol (16) as a color-
less syrup (>99%). To the solution of crude 16 in MeOH-
CH2Cl2 (1:3, 88 mL) was added acetyl chloride (0.l9 mL), and
the mixture was stirred at room temperature for 2 h. Then,
triethylamine (0.73 mL) was added. Following concentration,
column chromatography of the residue gave 17 (0.97 g, 78%).
[R]D +13.5 (c 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz): δ 7.29-
7.36 (m, 12 H), 6.89 (d, J ) 8.7 Hz, 2 H), 5.94-6.06 (m, 1 H),
5.30 (dd, J ) 17.2, 1.8 Hz, 1 H), 5.18 (d, J ) 8.5 Hz, 1 H), 4.95
(d, J ) 11.2 Hz, 1 H), 4.92 (d, J ) 10.7 Hz, 1 H), 4.81 (d, J )
10.7 Hz, 1 H), 4.74 (d, J ) 11.2 Hz, 1 H), 4.67 (d, J ) 11.4 Hz,
1 H), 4.62 (d, J ) 11.4 Hz, 1 H), 4.30-4.44 (m, 2 H), 4.14 (m,
1 H), 3.73-3.84 (m, 5 H), 3.36-3.48 (m, 3 H), 2.53 (br, 1 H),
2.47 (d, J ) 2.8 Hz, 1 H). 13C NMR (CDCl3, 50 MHz): δ 159.5,
138.6, 135.3, 130.0, 129.6, 128.6, 128.5, 128.1, 128.0, 127.9,
127.7, 116.7, 114.0, 83.3, 81.4, 81.3, 79.5, 75.8, 75.6, 74.6, 72.5,
71.8, 69.4, 55.3. HRFABMS: Calcd for C31H37O7 519.2364,
found 519.2382.
(-)-6-O-Allyl-1-O-(p-methoxybenzyl)-3,4,5-tri-O-benzyl-
myo-inositol (23).51 A mixture of 17 (0.97 g, 1.86 mmol) and
Bu2SnO (0.56 g, 2.23 mmol) in toluene (60 mL) was refluxed
under N2 with azeotropic removal of water for 2.5 h and was
then concentrated to dryness. To the residue were added DMF
(20 mL), CsF (0.71 g, 4.7 mmol), and benzyl bromide (0.28 mL,
2.38 mmol) at -10 °C. After being stirred at -10 °C for 1 h,
the reaction mixture was allowed to warm to room tempera-
ture and stirred for 16 h. The reaction was diluted with ethyl
acetate, washed with water, dried with Na2SO4, and finally
concentrated to dryness. Column chromatography of the
residue afforded 23 as a colorless syrup (1.11 g, 97.5%). [R]D
-8 (c 1.0, CHCl3) (lit.51 [R]D -10 (c 1.2, CHCl3)). 1H NMR
(CDCl3, 300 MHz): δ 7.31-7.36 (m, 17 H), 6.92 (d, J ) 8.5,
Hz 2 H), 5.96-6.08 (m, 1 H), 5.32 (dd, J ) 17.3, 1.6 Hz, 1 H),
5.19 (d, J ) 10.4 Hz, 1 H), 4.85-4.94 (m, 4 H), 4.73 (s, 2 H),
4.68 (d, J ) 11.4 Hz, 1 H), 4.64 (d, J ) 11.4 Hz, 1 H), 4.42 (m,
2 H), 4.19 (br, 1 H), 3.98 (t, J ) 9.5 Hz, 1 H), 3.85 (m, 1 H),
3.84 (s, 3 H), 3.42 (t, J ) 9.3 Hz, 1 H), 3.39 (dd, J ) 9.6, 2.5
Hz, 1 H), 3.33 (dd, J ) 9.6, 2.5 Hz, 1 H), 2.48 (s, 1 H). 13C
NMR (CDCl3, 50 MHz): δ 159.4, 138.9, 138.8, 138.1, 135.4,
130.2, 129.5, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 127.6,
116.7, 114.0, 83.2, 81.2, 81.0, 79.8, 79.3, 76.0, 75.9, 74.6, 72.8,
72.5, 67.8, 55.4.
(-)-6-O-Allyl-1-O-(p-methoxybenzyl)-2,3-O-cyclohexyl-
idene-myo-inositol (15).33 A solution of 14 (2.0 g, 4.0 mmol)
in MeOH-CH2Cl2 (1:3, 68 mL) was stirred with acetyl chloride
(87 µL) at room temperature for 10 min. After the reaction
was quenched with triethylamine (0.4 mL), the mixture was
concentrated. Column chromatography of the residue yielded
15 as a colorless syrup (1.0 g, 60%). [R]D -8.3 (c 3.0, CH2Cl2)
1
(lit.33 [R]D +6.5 (c 3, CHCl3)). H NMR (CDCl3, 300 MHz): δ
7.30 (d, J ) 8.8 Hz, 2 H), 6.87 (d, J ) 8.8 Hz, 2 H), 5.87-6.04
(m, 1 H), 5.16-5.35 (m, 2 H), 4.70, 4.65 (2d, J ) 12 Hz, 2 H),
4.44 (ddt, J ) 5.6, 12.6, 1.4 Hz, 1 H), 4.16-4.25 (m, 2 H), 3.89
(dd, J ) 7.5, 5.2 Hz, 1 H), 3.81 (s, 3 H), 3.71 (dt, J ) 2.2, 10.0
Hz, 1 H), 3.59-3.62 (m, 2 H), 3.20-3.32 (m, 1 H), 2.88 (t, J )
2.6 Hz, 2 H), 1.33-1.77 (m, 10 H). 13C NMR (CDCl3, 50 MHz):
δ 134.9, 130.1, 129.7, 117.2, 113.9, 110.7, 80.0, 78.2, 77.4, 75.2,
73.9, 73.7, 73.1, 72.4, 55.3, 37.8, 35.2, 25.0, 23.9, 23.6.
(-)-3,4-Di-O-benzyl-1,2-O-cyclohexylidene-myo-inosi-
tol (19).32 The benzylation of (-)-12 (2.27 g, 6.67 mmol)
followed the same procedure described for 16, which offered
(-)-3,4-di-O-benzyl-1,2;5,6-di-O-cyclohexylidene-myo-inositol
(18) as a colorless syrup in >99% yield. It was directly used
in the next step. Thus, to the solution of 18 in MeOH-CH2Cl2
(+)-6-O-Allyl-1-O-(p-methoxybenzyl)-4,5-di-O-benzyl-
myo-inositol (17). To the solution of 15 (1.0 g, 2.38 mmol)
and benzyl bromide (1.0 mL, 6.5 mmol) in DMF (25 mL) was
added NaH (560 mg, 60%, 14.2 mmol) at 0 °C. The mixture
(50) Madsen, R.; Udodong, U. E.; Roberts, C.; Mootoo, D. R.;
Konradsson, P.; Fraser-Reid, B. J. Am. Chem. Soc. 1995, 117, 1554-
1556.
(51) Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. J. Chem. Soc.,
Perkin Trans. 1 1993, 2945-2951.
J. Org. Chem, Vol. 68, No. 10, 2003 4025

Xue et al.
(1:3, 112 mL) was added acetyl chloride (145 µL). The mixture
was stirred at room temperature for 10 min, and then
triethylamine (0.67 mL) was added. After this mixture was
concentrated, column chromatography of the residue afforded
19 as a colorless syrup (1.98 g, 68%). [R]D -5.8 (c 1.0, CHCl3)
11.2 Hz, 1 H), 4.66 (d, J ) 10.8 Hz, 1 H), 4.53 (d, J ) 11.2 Hz,
1 H), 4.47 (d, J ) 10.8 Hz, 1 H), 4.39 (dd, J ) 12.1, 5.8 Hz, 1
H), 4.28 (dd, J ) 12.1, 5.8 Hz, 1 H), 3.84 (t, J ) 9.4 Hz, 1 H),
3.82 (s, 3 H), 3.72 (t, J ) 9.4 Hz, 1 H), 3.37-3.48 (m, 3 H),
2.38 (t, J ) 7.3 Hz, 2 H), 1.64 (m, 2 H), 1.12-1.37 (m, 24 H),
0.90 (t, J ) 7.0 Hz, 3 H). 13C NMR (CDCl3, 50 MHz): δ 173.3,
159.3, 138.8, 138.6, 137.8, 135.4, 130.0, 129.7, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.8, 127.6, 116.6, 113.8, 113.7,
83.0, 81.5, 81.2, 78.4, 78.0, 76.3, 76.0, 74.7, 72.2, 71.9, 66.5,
55.3, 34.6, 32.0, 29.8, 29.6, 29.5, 29.4, 29.1, 25.4, 22.8, 14.2.
FABMS: calcd for C54H72O8 791.5, found 791.5.
(-)-2-O-Hexadecanoyl-1-O-(p-methoxybenzyl)-3,4,5-tri-
O-benzyl-myo-inositol (11). To a solution of 24 (2.2 g, 2.6
mmol) in acetic acid (12 mL) and water (13 drops) were added
PdCl2 (0.75 g, 4.23 mmol) and NaOAc (0.75 g) at room
temperature. After 18 h of stirring, the mixture was concen-
trated in a vacuum. The residue was dissolved in ethyl acetate,
and the solution was washed with aq NaHCO3, brine, and
water. The organic solution was dried over Na2SO4 and
concentrated. Flash chromatography of the residue gave 11
as a colorless syrup (1.98 g, 94%). [R]D -4.6 (c 1.0, CHCl3). 1H
NMR (CDCl3, 300 MHz): δ 7.29-7.40 (m, 17 H), 6.90 (d, J )
8.3 Hz, 2 H), 5.90 (bs, 1 H), 4.71-4.93 (m, 6 H), 4.57 (d, J )
11.3 Hz, 1 H), 4.42 (d, J ) 10.7 Hz, 1 H), 3.98 (t, J ) 9.5 Hz,
1 H), 3.87 (t, J ) 9.5 Hz, 1 H), 3.82 (s, 3 H), 3.52 (dd, J ) 9.5,
2.3 Hz, 1 H), 3.43 (t, J ) 9.2 Hz, 1 H), 3.29 (dd, J ) 9.8, 2.0
Hz, 1 H), 2.55 (s, 1 H), 2.40 (t, J ) 7.3 Hz, 2 H), 1.65 (m, 2 H),
1.20-1.40 (m, 24 H), 0.91 (t, J ) 6.0 Hz, 3 H). 13C NMR (CDCl3,
300 MHz): δ 173.4, 159.6, 138.8, 138.7, 137.8, 129.9, 129.5,
128.5, 128.4, 128.2, 128.1, 127.9, 127.8, 127.7, 114.0, 82.8, 81.3,
78.5, 77.2, 76.0, 75.8, 72.9, 72.3, 71.5, 65.7, 55.3, 34.5, 32.0,
29.8, 29.6, 29.4, 29.1, 25.3, 22.8, 14.2. HRFABMS: calcd for
C51H68O8Na+ 831.4811, found 831.4786.
1,6-Di-O-acetate-4-O-allyl-2-azido-3-O-benzyl-2-deoxy-
D-glucopyranose (27).53 To the solution of 26 (317 mg, 1.0
mmol) in acetic anhydride (5 mL) was added a solution of H2-
SO4 in HOAc (2%, 5 mL) with stirring at 0 °C. Fifteen minutes
later, NaOAc‚3H2O (500 mg) was introduced to quench the
reaction. The reaction mixture was concentrated, and the
residue was dispensed in ethyl acetate. The organic solution
was washed with aqueous NaHCO3 and, brine, and then dried
and concentrated. Column chromatography of the residue gave
27 as a colorless syrup (302 mg, 72%, R:â 3.5:1.0). 1H NMR
(CDCl3, 300 MHz) of the R-isomer: δ 7.38 (m, 5 H), 6.22 (d, J
) 3.6 Hz, 1 H), 5.94 (m, 1 H), 5.16-5.30 (m, 2 H), 4.89 (s, 2
H), 4.27-4.36 (m, 3 H), 4.09 (m, 1 H), 3.89 (m, 2 H), 3.47-
3.59 (m, 3 H), 2.17 (s, 3 H), 2.08 (s, 3 H).
6-Acetate-4-O-allyl-2-azido-3-O-benzyl-2-deoxy-D-glu-
copyranose (28).53 To the solution of 27 (2.25 g, 5.4 mmol)
in ether (50 mL) was added benzylamine (8.8 mL, 81 mmol).
The reaction mixture was stirred at room temperature for 2
h. After the reaction mixture was washed with dilute aq HCl
solution and water, the organic layer was dried and concen-
trated. Column chromatography of the crude product afforded
28 (>99%). 1H NMR (CDCl3, 300 MHz): δ 7.38 (m, 5 H), 5.90
(m, 1 H), 5.17-5.30 (m, 3 H), 4.89 (s, 2 H), 3.95-4.44 (m, 6
H), 3.38-3.54 (m, 3 H), 2.09 (s, 3 H).
1
(lit.32 [R]D -16.5 (c 1, CHCl3)). H NMR (CDCl3, 300 MHz): δ
7.31-7.41 (m, 10 H), 5.00 (d, J ) 11.3 Hz, 1 H), 4.77 (s, 2 H),
4.71 (d, J ) 11.3 Hz, 1 H), 4.32 (dd, J ) 5.1, 3.8 Hz, 1 H), 3.93
(dd, J ) 7.6, 5.3 Hz, 1 H), 3.68-3.78 (m, 3 H), 3.31 (dd, J )
10.0, 8.5 Hz, 1 H), 2.64 (br, 2 H), 1.41-1.80 (m, 10 H).
(-)-6-O-Allyl-3,4-di-O-benzyl-1,2-O-cyclohexylidene-
myo-inositol (20).52 Regioselective allylation of 19 (1.95 g, 4.43
mmol) followed the same procedure described for 13. The
reaction afforded 20 as a colorless syrup (1.5 g, 71%). [R]D -2.1
1
(c 0.8, CHCl3). H NMR (CDCl3, 300 MHz): δ 7.29-7.37 (m,
10 H), 5.90-6.00 (m, 1 H), 5.30 (dd, J ) 17.2, 1.6 Hz, 1 H),
5.18 (d, J ) 10.3 Hz, 1 H), 4.88 (d, J ) 11.3 Hz, 1 H), 4.71-
4.78 (m, 3 H), 4.38 (dd, J ) 12.8, 5.5 Hz, 1 H), 4.30 (dd, J )
5.7, 3.9 Hz, 1 H), 4.22 (dd, J ) 12.7, 5.9 Hz, 1 H), 4.03 (t, J )
6.9 Hz, 1 H), 3.81 (t, J ) 8.0 Hz, 1 H), 3.70 (dd, J ) 8.0, 3.9
Hz, 1 H), 3.61 (dd, J ) 9.7, 7.0 Hz, 1 H), 3.45 (dd, J ) 9.8, 8.0
Hz, 1 H), 2.64 (br, 1 H), 1.30-1.84 (m, 10 H).
(-)-6-O-Allyl-3,4,5-tri-O-benzyl-myo-inositol (22).51 Benz-
ylation of 20 (1.5 g, 3.13 mmol) following the same procedure
described above gave 21 in >99% yield. The product was
directly used in the next step. Thus, after it was dissolved in
MeOH-CH2Cl2 (1:3, 116 mL), acetyl chloride (0.25 mL) was
added. The mixture was stirred at room temperature for 2 h,
and then triethylamine (0.96 mL) was added. The solution was
concentrated, and column chromatography of the residue
produced 22 as a colorless syrup (1.37 g, 89%). [R]D -31 (c 1.0,
1
CHCl3) (lit.51 [R]D -35 (c 1.5, CHCl3)). H NMR (CDCl3, 300
MHz): δ 7.29-7.42 (m, 15 H), 5.92-6.01 (m, 1 H), 5.28 (dd, J
) 17.6, 1.6 Hz, 1 H), 5.18 (dd, J ) 10.4, 1.7 Hz, 1 H), 4.80-
4.93 (m, 4 H), 4.74 (d, J ) 11.7 Hz, 1 H), 4.70 (d, J ) 11.7 Hz,
1 H), 4.39-4.46 (m, 1 H), 4.25-4.30 (m, 1 H), 4.22 (t, J ) 2.8
Hz, 1 H), 3.94 (t, J ) 9.5 Hz, 1 H), 3.72 (t, J ) 9.5 Hz, 1 H),
3.40-3.48 (m, 3 H), 2.54 (br, 2 H). 13C NMR (CDCl3, 50 MHz):
δ 138.7, 138.6, 137.8, 135.0, 128.6, 128.4, 128.0, 128.0, 128.0,
127.9, 127.7, 117.3, 83.2, 81.7, 81.0, 80.1, 76.0, 75.7, 74.4, 72.8,
71.7, 69.3.
(-)-6-O-Allyl-1-O-(p-methoxybenzyl)-3,4,5-tri-O-benzyl-
myo-inositol (23). A solution of 22 (1.36 g, 2.78 mmol) and
Bu2SnO (0.83 g, 3.34 mmol) in toluene (100 mL) was refluxed
under N2 with azeotropic removal of water for 2.5 h. The
reaction mixture was then concentrated to dryness under
vacuum. To the residue was added DMF (30 mL), CsF (1.06
g, 7.05 mmol), NaI (0.7 g, 4.17 mmol), and 4-methoxbenzyl
chloride (0.61 mL, 4.17 mmol) at -10 °C. After being stirred
at -10 °C for 1 h, the reaction mixture was allowed to warm
to room temperature and stirred for 16 h. The solution was
then diluted by ethyl acetate, washed with water, dried over
Na2SO4, and concentrated. Column chromatography of the
residue afforded 23 (1.59 g, 94%). Its spectroscopic data were
exactly the same as those of the product obtained from the
previous synthetic route.
(-)-6-O-Allyl-2-O-hexadecanoyl-1-O-(p-methoxybenzyl)-
3,4,5-tri-O-benzyl-myo-inositol (24). To a solution of 23 (1.59
g, 2.6 mmol), palmitic acid (0.9 g, 3.5 mmol), and DMAP (50
mg, 0.4 mmol) in dry DCM (20 mL) was added DCC (0.72 g,
3.5 mmol) at room temperature. The mixture was stirred under
argon for 1 day, and the reaction was monitored with TLC.
When the reaction was over, the solution was concentrated in
a vacuum. Column chromatography of the residue gave 24 as
4-O-Allyl-2-azido-3,6-di-O-benzyl-2-deoxy-D-glucopyra-
nosyl Fluoride (10).38 To the solution of 28 (2.3 g, 6.1 mmol)
in dry DCM (100 mL) was added DAST (1.22 mL, 8.2 mmol)
under argon with stirring at 0 °C. The mixture was allowed
to warm to room temperature and stirred for another 30 min.
Normal workup of the reaction afforded an anomeric mixture
(R:â ca. 1:6) of 6-O-acetyl-4-O-allyl-2-azido-3,6-di-O-benzyl-2-
deoxy-D-glucopyranosyl fluoride (29, >99%). This crude prod-
uct was directly treated with NaOMe (0.6 mmol) in MeOH (30
mL) for deacetylation. The solution was stirred at room
temperature for 2 h and neutralized with Amberlyst ion-
exchange resin. The mixture was concentrated to give the
1
a colorless syrup (2.2 g, >99%). [R]D -11.5 (c 1.0, CHCl3). H
NMR (CDCl3, 300 MHz): δ 7.29-7.37 (m, 17 H), 6.88 (d, J )
8.5 Hz, 2 H), 5.92-6.06 (m, 1 H), 5.84 (t, J ) 2.6 Hz, 1 H),
5.32 (dd, J ) 17.1, 1.5 Hz, 1 H), 5.17 (d, J ) 10.4 Hz, 1 H),
4.85-4.90 (m, 3 H), 4.79 (d, J ) 10.7 Hz, 1 H), 4.74 (d, J )
(52) Ding, Y.; Shen, J.; Zhou, S. Chin. Chem. Lett. 1993, 4, 565-
569.
(53) Koshida, S.; Suda, Y.; Fukui, Y.; Ormsby, J.; Sobel, M.;
Kusumoto, S. Tetrahedron Lett. 1999, 40, 5725-5729.
4026 J. Org. Chem., Vol. 68, No. 10, 2003

First Total Synthesis of a GPI-Anchored Peptide
crude 4-O-allyl-2-azido-3-O-benzyl-2-deoxy-D-glucopyranosyl
fluoride (30). Finally, 30 was benzylated according to the
procedure described above to afford 10 (1.95 g, 75%, R:â 1:6).
This inseparable anomeric mixture was directly used in the
next step of the reaction. 1H NMR (CDCl3, 300 MHz) of
deoxy-â-D-glucopyranosyl)-2-O-hexadecanoyl-1-O-(p-methoxy-
benzyl)-3,4,5-tri-O-benzyl-myo-inositol (â-isomer): [R]D -18 (c
1
1.0, CHCl3). H NMR (CDCl3, 300 MHz): δ 7.24-7.44 (m, 27
H), 6.84 (d, J ) 8.7 Hz, 2 H), 5.81 (t, J ) 2.7 Hz, 1 H), 3.78 (s,
3 H), 2.79 (d, J ) 2.0 Hz, 1 H), 2.28 (dt, J ) 7.4, 2.8 Hz, 2 H),
1.57 (m, 2 H), 0.90 (t, J ) 6.5 Hz, 3 H). FABMS: calcd for
C71H89N3O12 1175, found 1175.
1
R-isomer: δ 5.64 (dd, J1,F ) 52.8 Hz, 1 H). H NMR (CDCl3,
300 MHz) of â-isomer: δ 5.03 (dd, J1,F ) 52.8 Hz, 1 H).
Ethyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-l-r-D-mannopy-
ranosyl)-(1f6)-2,3,4-tri-O-benzyl-1-thio-r-D-mannopyra-
noside (40). To the solution of 35 (2.53 g, 5.0 mmol) in DCM
(20 mL) was added 1.0 M trimethylsilyl chloride (7.5 mL) at 0
°C. After being stirred for 1 h, the reaction mixture was
concentrated in a vacuum to give the glycosyl chloride 8 as
avsyrup that was directly used without further purification.
After the mixture of 7 (2.1 g, 4.25 mmol), AgOTf (2.6 g, 10
mmol), and 4 Å MS (5 g) in dry DCM (30 mL) was stirred at
room temperature for 1 h and then cooled to -40 °C, a solution
of 8 in DCM (10 mL) was added. After the reaction mixture
was warmed to room temperature and stirred overnight, it was
filtered off through Celite, and the resulting solution was
washed with brine, dried, and concentrated. Column chroma-
tography of the residue gave 40 as a colorless syrup (3.46 g,
6-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-4-O-2-propenyl-
r-D-glucopyranosyl)-2-O-hexadecanoyl-1-O-(p-methoxy-
benzyl)-3,4,5-tri-O-benzyl-myo-inositol (31). After the mix-
ture of silver triflate (206 mg, 0.8 mmol), hafnocene dichloride
(190 mg, 0.5 mmol), and MS 4 Å (1.5 g) in diethyl ether (5
mL) was stirred at room temperature for 0.5 h and then cooled
to -15 °C, a solution of 11 (180 mg, 0.22 mmol) and 10 (143
mg, 0.33 mmol) in dry ether (2 mL) was introduced. The
mixture was allowed to warm to room temperature and stirred
overnight. Then, DCM (30 mL) was added. The mixture was
filtered through a Celite pad, and the filtrated was washed
with aq NaHCO3 and brine, dried with Na2SO4, and finally
concentrated. The crude product was carefully purified by silica
gel column to give 31 as a colorless syrup (106 mg, 40%), as
well as the â-isomer (79 mg, 30%). 31: [R]D +51 (c 1.0, CHCl3).
1H NMR (CDCl3, 300 MHz): δ 7.21-7.44 (m, 27 H), 6.85 (d, J
) 8.6 Hz, 2 H), 5.93 (bs, 1 H), 5.72-5.85 (m, 1 H), 5.65 (d, J
) 3.8 Hz, 1 H), 4.49-5.15 (m, 12 H), 4.40 (d, J ) 9.4 Hz, 1 H),
4.23 (d, J ) 11.9 Hz, 1 H), 4.16 (dd, J ) 12.6, 5.5 Hz, 1 H),
3.85-4.05 (m, 5 H), 3.80 (s, 3 H), 3.52-3.61 (m, 3 H), 3.47 (t,
J ) 9.4 Hz, 1 H), 3.08-3.21 (m, 3 H), 2.40 (t, J ) 7.1 Hz, 2 H),
1.62 (m, 2 H), 1.17-1.40 (m, 24 H), 0.89 (t, J ) 6.6 Hz, 3 H).
13C NMR (CDCl3, 50 MHz): δ 173.4, 159.4, 138.6, 138.2, 138.0,
137.9, 137.6, 135.1, 130.3, 129.3, 128.6, 128.6, 128.6, 128.6,
128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2,
128.2, 128.2, 128.2, 128.0, 127.9, 127.6, 116.0, 113.8, 97.5, 81.8,
80.8, 79.8, 79.3, 78.4, 78.2, 76.2, 75.8, 75.3, 75.2, 73.5, 73.3,
72.1, 71.5, 70.1, 67.6, 65.5, 63.0, 55.3, 34.4, 32.0, 29.8, 29.6,
29.5, 29.4, 29.1, 25.3, 22.8, 14.2. 6-O-(2-Azido-3,6-di-O-benzyl-
2-deoxy-4-O-2-propenyl-â-D-glucopyranosyl)-2-O-hexadecanoyl-
1-O-(p-methoxybenzyl)-3,4,5-tri-O-benzyl-myo-inositol (â-iso-
mer): [R]D -15 (c 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz): δ
7.19-7.43 (m, 27 H), 6.81 (d, J ) 8.7 Hz, 2 H), 5.81-5.97 (m,
1 H), 5.79 (m, 1 H), 3.76 (s, 3 H), 3.25-5.32 (m, 28 H), 2.22
(m, 2 H), 1.60 (m, 2 H), 1.14-1.40 (m, 24 H), 0.89 (t, J ) 6.6
Hz, 3 H). 13C NMR (CDCl3, 50 MHz): δ 173.2, 159.2, 138.6,
138.5, 138.3, 137.9, 137.7, 134.5, 130.4, 129.4, 117.9, 117.4,
113.7, 101.0, 109.7, 83.4, 83.0, 81.8, 78.4, 78.1, 77.9, 77.6, 76.2,
75.8, 75.6, 75.0, 74.5, 74.0, 73.4, 72.5, 72.1, 70.5, 68.4, 67.4,
65.4, 59.9, 55.3, 34.4, 32.0, 29.8, 29.6, 29.6, 29.4, 29.1, 25.2,
22.8, 14.2. FABMS: calcd for C74H93N3O12 1215.6, found
1215.7.
6-O-(2-Azido-3,6-di-O-benzyl-2-deoxy-r-D-glucopyrano-
syl)-2-O-hexadecanoyl-1-O-(p-methoxybenzyl)-3,4,5-tri-
O-benzyl-myo-inositol (6). To the solution of 31 (a mixture
of R,â-isomers; 1.3 g, 1.07 mmol) in acetic acid (12 mL) and
water (13 drops) were added PdCl2 (0.32 g, 1.8 mmol) and
NaOAc (0.32 g) at room temperature. After 18 h of stirring,
the reaction mixture was concentrated in a vacuum. The
residue was dissolved in ethyl acetate, and the solution was
washed with aq NaHCO3, brine, and water. The organic
solution was dried over Na2SO4 and then concentrated to
dryness. Flash chromatography of the crude product gave 6
(0.53 g) and its â-isomer (0.46 g). 6: [R]D +27.6 (c 1.0, CHCl3).
1H NMR (CDCl3, 300 MHz): δ 7.19-7.47 (m, 27 H), 6.87 (d, J
) 8.1 Hz, 2 H), 5.95 (br, 1 H), 5.67 (d, J ) 3.6 Hz, 1 H), 3.46-
4.09 (m, 8 H), 3.81 (s, 3 H), 3.30 (t, J ) 3.7 Hz, 2 H), 3.17 (dd,
J ) 10.1, 3.4 Hz, 1 H), 2.41 (t, J ) 7.2 Hz, 2 H), 2.11 (d, J )
3.5 Hz, 1 H), 1.64 (m, 2 H), 1.15-1.40 (m, 24 H), 0.91 (t, J )
6.6 Hz, 3 H). 13C NMR (CDCl3, 50 MHz): δ 173.4, 159.5, 138.5,
138.3, 138.2, 138.1, 137.6, 130.4, 129.3, 128.6, 128.5, 128.4,
128.3, 128.2, 128.0, 127.9, 127.7, 127.6, 113.8, 97.5, 81.8, 81.0,
79.3, 79.2, 78.4, 77.7, 76.1, 75.8, 75.0, 74.8, 73.5, 72.2, 71.6,
69.6, 69.2, 65.5, 62.6, 55.3, 34.5, 32.0, 29.8, 29.7, 29.6, 29.5,
29.4, 29.1, 25.3, 22.8, 14.2. 6-O-(2-Azido-3,6-di-O-benzyl-2-
1
82%). [R]D +66 (c 1.0, CHCl3). H NMR (CDCl3, 600 MHz): δ
7.38 (d, J ) 7.2 Hz, 2 H), 7.21-7.32 (m, 26 H), 7.12 (dd, J )
7.2, 1.8 Hz, 2 H), 5.47 (dd, J ) 3.0, 2.4 Hz, 1 H), 5.33 (s, 1 H),
4.94 (d, J ) 1.2 Hz, 1 H), 4.91 (d, J ) 11.4 Hz, 1 H), 4.84 (d,
J ) 10.8 Hz, 1 H), 4.71 (d, J ) 12.6 Hz, 1 H), 4.63-4.66 (m, 3
H), 4.55 (s, 2 H), 4.48 (d, J ) 10.8 Hz, 1 H), 4.42-4.45 (m, 3
H), 4.08 (ddd, J ) 9.6, 5.4, 1.8 Hz, 1 H), 3.93 (dd, J ) 9.6, 3.6
Hz, 1 H), 3.82-3.90 (m, 5 H), 3.76 (ddd, J ) 9.6, 3.6, 1.8 Hz,
1 H), 3.69 (dd, J ) 9.6, 3.6 Hz, 1 H), 3.67 (dd, J ) 10.8, 1.8
Hz, 1 H), 3.58 (dd, J ) 10.8, 1.8 Hz, 1 H), 2.45-2.58 (m, 2 H),
2.14 (s, 3 H), 1.19 (t, J ) 7.2 Hz, 3 H). FABMS: calcd for
C62H76NO13S 1074, found 1074.
Ethyl O-(3,4,6-Tri-O-benzyl-l-r-D-mannopyranosyl)-
(1f6)-2,3,4-tri-O-benzyl-1-thio-r-D-mannopyranoside (41).
A solution of sodium (46 mg, 2.0 mmol) in methanol (20 mL)
was added to a stirred solution of 40 (3.46 g, 3.57 mmol) in
DCM (3 mL). After being stirred at room temperature for 1 h,
the reaction mixture was diluted with ethyl acetate and
washed with water. The organic layer was dried and concen-
trated. The residue was purified with a silica gel column to
give 41 as a colorless syrup (3.0 g, 91%). [R]D +471.0 (c 1.0,
1
CHCl3). H NMR (CDCl3, 600 MHz): δ 7.39 (d, J ) 7.2 Hz, 2
H), 7.24-7.34 (m, 26 H), 7.16 (dd, J ) 7.2, 1.8 Hz, 2 H), 5.34
(s, 1 H), 5.07 (d, J ) 1.8 Hz, 1 H), 4.92 (d, J ) 10.8 Hz, 1 H),
4.83 (d, J ) 10.8 Hz, 1 H), 4.70 (d, J ) 12.0 Hz, 1 H), 4.67 (d,
J ) 12.0 Hz, 1 H), 4.63 (d, J ) 12.0 Hz, 1 H), 4.62 (d, J ) 12.0
Hz, 1 H), 4.59 (s 2 H), 4.56 (d, J ) 12.0 Hz, 1 H), 4.52 (d, J )
10.8 Hz, 1 H), 4.49 (d, J ) 10.8 Hz, 1 H), 4.47 (d, J ) 12.0 Hz,
1 H), 4.12 (dd, J ) 3.6, 1.2 Hz, 1 H), 4.09 (ddd, J ) 10.2, 4.8,
1.2 Hz, 1 H), 3.83-3.96 (m, 6 H), 3.77 (ddd, J ) 9.6, 3.6, 1.8
Hz, 1 H), 3.73 (dd, J ) 12.0, 1.8 Hz, 1 H), 3.68 (dd, J ) 10.8,
4.2 Hz, 1 H), 3.61 (dd, J ) 10.8, 1.8 Hz, 1 H), 2.49-2.60 (m, 2
H), 1.22 (t, J ) 7.2 Hz, 3 H). 13C NMR (CDCl3, 75 MHz): δ
138.6, 138.5, 138.3, 138.2, 138.1, 137.9, 128.6, 128.5, 128.4,
128.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 99.6, 81.8, 80.4,
79.7, 76.6, 75.1, 75.0, 74.8, 74.2, 73.4, 72.2, 72.1, 71.8, 71.5,
71.1, 68.9, 68.1, 66.3, 25.4, 15.1. HRFABMS: calcd for C56H62O10
926.4064, found 926.4034; calcd for C56H62O10Na (M + Na+)
949.3961, found 949.3945.
Ethyl O-(6-O-Acetyl-2,3,4-tri-O-benzyl-r-D-mannopy-
ranosyl)-(1f2)-O-(3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-
(1f6)-2,3,4-tri-O-benzyl-1-thio-r-D-mannopyranoside (42).
To the solution of 7 (1.07 g, 2.0 mmol) in DCM (20 mL) was
added 33% HBr in HOAc (1.0 mL) at 0 °C. After being stirred
at room temperature for 2 h, the reaction mixture was washed
with saturated NaHCO3 solution and brine and then dried and
concentrated in a vacuum to give the glycosyl bromide 9 as a
syrup that was directly used without further purification. After
the mixture of 41(1.0 g, 1.08 mmol), AgOTf (0.52 g, 2.0 mmol),
and 4 Å MS (2 g) in dry DCM (20 mL) was stirred at room
J. Org. Chem, Vol. 68, No. 10, 2003 4027

Xue et al.
temperature for 1 h, it was cooled to -40 °C. To this mixture
was added the solution of 8 in DCM (5 mL). The reaction
mixture was then warmed to room temperature and stirred
overnight. The solution was filtered off, washed with brine,
dried, and concentrated. Column chromatography of the
residue afforded 42 as a colorless syrup (0.8 g, 53%). [R]D +48.6
1.25 (m, 24 H), 0.86 (t, J ) 7.2 Hz, 1 H), 0.85 (s, 9 H), 0.02 (s,
3 H), 0.00 (s, 3 H). MALDI-TOF-MS: calcd for C158H187N3O27-
Si 2586, found 2586.
O-(2,3,4-Tri-O-benzyl-r-D-mannopyranosyl)-(1f2)-O-
(3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-(1f6)-O-(2,3,4-
tri-O-benzyl-r-D-mannopyranosyl)-(1f4)-O-(2-azido-3,6-
di-O-benzyl-2-deoxy-r-D-glucopyranosyl)-(1f6)-2-O-
hexadecanoyl(palmitoyl)-1-O-(p-methoxybenzyl)-3,4,5-
tri-O-benzyl-myo-inositol (45). After 44 (600 mg, 232 µmol)
was dissolved in DCM (15 mL) under nitrogen, TBAF (1 M
solution in THF, 2.24 mL, 2.24 mmol) and acetic acid (130 µL,
2.28 mmol) were added to the solution at 0 °C. The mixture
was warmed to room temperature and stirred at 35-40 °C for
72 h. After removal of the solvent in a vacuum, the residue
was purified by flash column chromatography to give 45 (545
mg, 95%). [R]D +39.0 (c 1.0, CHCl3). 1H NMR (CDCl3, 600
MHz): δ 7.05-7.34 (m, 72 H), 6.84 (d, J ) 8.4 Hz, 2 H), 5.93
(t, J ) 2.4 Hz, 1 H), 5.69 (d, J ) 3.6 Hz, 1 H), 5.24 (d, J ) 1.8
Hz, 1 H), 5.03 (d, J ) 1.8 Hz, 1 H), 4.97 (d, J ) 10.8 Hz, 1 H),
4.93 (d, J ) 12.0 Hz, 1 H), 4.91 (d, J ) 12.0 Hz, 1 H), 4.90 (d,
J ) 10.2 Hz, 1 H), 4.88 (d, J ) 1.2 Hz, 1 H), 4.87 (d, J ) 11.4
Hz, 1 H), 4.78 (d, J ) 11.4 Hz, 1 H), 4.75 (d, J ) 11.4 Hz, 1 H),
4.73 (d, J ) 12.0 Hz, 1 H), 4.70 (d, J ) 10.8 Hz, 1 H), 4.64 (d,
J ) 10.2 Hz, 1 H), 4.62 (d, J ) 11.4 Hz, 1 H), 4.59 (d, J ) 11.4,
1 H), 4.56 (d, J ) 12.0, 1 H), 4.54 (d, J ) 12.0, 1 H), 4.52 (d,
J ) 12.0 Hz, 1 H), 4.49 (d, J ) 12.0 Hz, 1 H), 4.48 (s, 2 H),
4.47 (d, J ) 12.0 Hz, 1 H), 4.44 (d, J ) 11.4 Hz, 1 H), 4.21 (d,
J ) 12.6 Hz, 1 H), 4.13 (d, J ) 12.6 Hz, 1 H), 3.97 (t, J ) 9.6
Hz, 1 H), 3.78 (s, 3 H), 3.38 (t, J ) 9.0 Hz, 2 H), 3.29 (dd, J )
11.4, 3.6 Hz, 1 H), 3.17 (dd, J ) 10.2, 3.6 Hz, 1 H), 2.38 (m, 2
H), 1.61 (m, 2 H), 1.26 (m, 24 H), 0.88 (t, J ) 7.2 Hz, 1 H).
MALDI-TOF-MS: calcd for C152H173N3O27 2472, found 2495
(M + Na+).
O-(2,3,4-Tri-O-benzyl-r-D-mannopyranosyl)-(1f2)-
O-(3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-(1f6)-O-
(2,3,4-tri-O-benzyl-r-D-mannopyranosyl)-(1f4)-O-{2-(benzyl-
oxycarbonyl)amino-3,6-di-O-benzyl-2-deoxy-r-D-glucopy-
ranosyl}-(1f6)-2-O-hexadecanoyl-1-O-(p-methoxybenzyl)-
3,4,5-tri-O-benzyl-myo-inositol (47). To the mixture of 45
(120 mg, 48.5 µmol) and dibenzyl dicarbonate (140 mg, 0.49
mmol) in methanol and DCM (1:2, 20 mL) was added trieth-
ylphosphine (72 µL, 0.49 mmol) under nitrogen at 0 °C. The
reaction mixture was then warmed to room temperature and
stirred at room temperature overnight. After the removal of
solvents in a vacuum, the residue was purified by flash
chromatography to afford 47 (88 mg, 70%). TLC: Rf 0.50
(acetone, hexane, and DCM 4:10:2). 1H NMR (CDCl3, 600 MHz,
25 °C) major isomer: δ 6.67 (d, J ) 7.8 Hz, 2 H), 5.89 (s, 1 H),
5.67 (d, J ) 10.2 Hz, 1 H), 5.44 (d, J ) 3.0 Hz, 1 H), 5.21 (s,
1 H), 5.09 (d, J ) 11.4 Hz, 1 H), 5.05 (s, 1 H), 4.83 (s, 1 H),
3.58 (s, 3 H), 2.28 (m, 2 H), 1.61 (m, 2 H), 1.25 (m, 24 H), 0.88
(t, J ) 7.2 Hz, 1 H). 1H NMR (CDCl3, 600 MHz, 25 °C) minor
isomer: δ 6.72 (d, J ) 7.8 Hz, 2 H), 5.92 (s, 1 H), 5.27 (d, J )
10.2 Hz, 1 H), 5.23 (s, 1 H). 1H NMR (CDCl3, 600 MHz, 50
°C): 6.69 (d, J ) 6.6 Hz, 2 H), 5.87 (s, 1 H), 5.64 (d, J ) 9.0
Hz, 1 H), 5.43 (d, J ) 3.6 Hz, 1 H), 5.20 (s, 1 H), 5.05 (d, J )
12.0 Hz, 1 H), 5.02 (s, 1 H), 4.82 (s, 1 H), 3.59 (s, 3 H), 2.27
(m, 2 H), 1.56 (m, 2 H), 1.25 (m, 24 H), 0.88 (t, J ) 7.2 Hz, 1
H). MALDI-TOF-MS: calcd for C160H181NO29 2580, found 2603
(M + Na+).
1
(c 1.0, CHCl3). H NMR (CDCl3, 300 MHz): δ 7.15-7.40 (m,
45 H), 5.39 (s, 1 H), 5.14 (s, 1 H), 4.91-4.94 (m, 3 H), 4.84 (d,
J ) 12.0 Hz, 1 H), 2.56 (m, 2 H), 2.01 (s, 3 H), 1.23 (t, J ) 7.2
Hz, 3 H). FABMS: calcd for C85H92O16S 1400.6, found 1400.6.
Ethyl O-(2,3,4-Tri-O-benzyl-6-O-tert-butyldimethylsi-
lyl-r-D-mannopyranosyl)-(1f2)-O-(3,4,6-tri-O-benzyl-r-D-
mannopyranosyl)-(1f6)-2,3,4-tri-O-benzyl-1-thio-r-D-man-
nopyranoside (5). A solution of sodium (2.3 mg, 0.1 mmol)
in methanol (5 mL) was added to a stirred solution of 42 (0.8
g, 0.6 mmol) in DCM (10 mL). After being stirred at room
temperature for 3 h, the reaction mixture was diluted with
ethyl acetate and washed with water. The organic layer was
dried with Na2SO4 and concentrated to give the crude ethyl
O-(2,3,4-tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-O-(3,4,6-tri-
O-benzyl-R-D-mannopyranosyl)-(1f6)-2,3,4-tri-O-benzyl-1-thio-
R-D-mannopyranoside (43), which was directly used in the next
step. To the solution of 43, DMAP (22 mg, 0.2 mmol), and
imidazole (82 mg, 1.2 mmol) in dry DMF (15 mL) was added
TBDMSCl (121 mg, 0.8 mmol) at 0 °C. After the mixture was
warmed to room temperature and stirred for 3 h, it was diluted
with 50 mL of ethyl acetate, washed with brine, dried over
Na2SO4, and concentrated in a vacuum. Finally, the residue
was purified with a silica gel column to give 5 (810 mg, 92%).
[R]D +25.6 (c 1.0, CHCl3). 1H NMR (CDCl3, 600 MHz): δ 7.05-
7.35 (m, 45 H), 5.29 (s, 1 H), 5.14 (d, J ) 1.2 Hz, 1 H), 4.88 (d,
J ) 10.8 Hz, 1 H), 4.84 (d, J ) 10.8 Hz, 1 H), 4.81 (d, J ) 1.2
Hz, 1 H), 4.75 (d, J ) 10.8 Hz, 1 H), 4.65 (d, J ) 12.0 Hz, 1 H),
4.56-4.58 (m, 3 H), 4.49 (s, 2 H), 4.35-4.46 (m, 9 H), 4.08 (t,
J ) 2.4 Hz, 1 H), 4.04 (m, 1 H), 3.93 (t, J ) 9.6 Hz, 1 H), 3.77-
3.87 (m, 8 H), 3.64-3.72 (m, 4 H), 3.58 (dd, J ) 11.4, 4.2 Hz,
1 H), 3.55 (dd, J ) 10.8, 1.8 Hz, 1 H), 3.49 (dd, J ) 11.4, 1.8
Hz, 1 H), 2.40-2.55 (m, 2 H), 1.15 (t, J ) 7.2 Hz, 3 H), 0.83 (s,
9 H), 0.01 (s, 3 H), 0.00 (s, 3 H). FABMS: calcd for C89H104O15-
SSi 1472, found, 1472, 1495 (M + Na+).
O-(2,3,4-Tri-O-benzyl-6-O-tert-butyldimethylsilyl-r-D-
mannopyranosyl)-(1f2)-O-(3,4,6-tri-O-benzyl-r-D-man-
nopyranosyl)-(1f6)-O-(2,3,4-tri-O-benzyl-r-D-mannopy-
ranosyl)-(1f4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-r-D-
glucopyranosyl)-(1f6)-2-O-hexadecanoyl(palmitoyl)-1-
O-(p-methoxybenzyl)-3,4,5-tri-O-benzyl-myo-inositol (44).
After the mixture of MS 4A (3.0 g), 5 (720 mg, 0.488 mmol),
and 6 (410 mg, 0.348 mmol) in DCM and ethyl ether (1:1, 20
mL) was stirred at room temperature for 1 h and then cooled
to 0 °C, NIS (220 mg, 0.97 mmol) was added. The mixture was
stirred for another 30 min and cooled to -10 °C, whereupon
TfOH (6.1 µL, 0.035 mmol) in DCM (3 mL) was added. The
reaction mixture was warmed to 0 °C and stirred for 30 min.
Finally, triethylamine was added to quench the reaction. The
molecular sieves were filtered off and the solution was diluted
with ethyl ether. The organic layer was separated, washed,
dried, and concentrated. Column chromatography of the
residue afforded 44 as a colorless syrup (810 mg, 90%). [R]D
+44.8 (c 1.0, CHCl3). 1H NMR (CDCl3, 600 MHz): δ 7.03-
7.36 (m, 72 H), 6.82 (d, J ) 9.0 Hz, 2 H), 5.91 (t, J ) 2.4 Hz,
1 H), 5.68 (d, J ) 3.6 Hz, 1 H), 5.21 (s, 1 H), 5.12 (d, J ) 1.2
Hz, 1 H), 4.95 (d, J ) 10.8 Hz, 1 H), 4.93 (d, J ) 11.4 Hz, 1 H),
4.91 (d, J ) 10.2 Hz, 1 H), 4.88 (d, J ) 10.2 Hz, 1 H), 4.86 (d,
J ) 10.8 Hz, 1 H), 4.72-4.75 (m, 4 H), 4.68 (d, J ) 10.8 Hz, 1
H), 4.63 (d, J ) 9.6 Hz, 1 H), 4.62 (d, J ) 10.8 Hz, 1 H), 4.59
(d, J ) 10.8 Hz, 1 H), 4.52 (d, J ) 11.4 Hz, 1 H), 4.51 (d, J )
12.0 Hz, 1 H), 4.48 (d, J ) 12.0 Hz, 1 H), 4.46 (d, J ) 12.0 Hz,
1 H), 4.17 (d, J ) 12.0 Hz, 1 H), 3.93 (t, J ) 9.6 Hz, 1 H), 3.78
(dd, J ) 9.6, 3.0 Hz, 1 H), 3.76 (s, 3 H), 3.73-3.75 (m, 2 H),
3.69 (t, J ) 2.4 Hz, 1 H), 3.66 (t, J ) 9.6 Hz, 1 H), 3.51 (dd, J
) 9.6, 3.0 Hz, 1 H), 3.38 (m, 1 H), 3.29 (dd, J ) 10.8 Hz, 1 H),
3.14 (d, J ) 10.2, 3.6 Hz, 1 H), 2.34 (m, 2 H), 1.58 (m, 2 H),
O-{2,3,4-Tri-O-benzyl-6-O-[benzyl-[N-(9-fluorenyl-
methoxycarbonyl)]aminoethylphosphonato]-r-D-man-
nopyranosyl}-(1f2)-O-(3,4,6-tri-O-benzyl-r-D-mannopy-
ranosyl)-(1f6)-O-(2,3,4-tri-O-benzyl-r-D-mannopyranosyl)-
(1f4)-O-(2-benzyloxycarbonylamino-3,6-di-O-benzyl-2-
deoxy-r-D-glucopyranosyl)-(1f6)-2-O-hexadecanoyl-1-O-
(p-methoxybenzyl)-3,4,5-tri-O-benzyl-myo-inositol (48).
To a solution of 47 (70 mg, 27 µmol) and phosphitamide 4 (0.19
mmol, 7 equiv) in dry DCM and acetonitrile (3:1, 6 mL) was
added 1H-tetrazole (0.19 mmol, 0.41 mL of 0.47 M in CH3-
CN). The mixture was stirred at room temperature for 6 h,
and then tert-butyl hydroperoxide (0.50 mmol, 0.1 mL of 5 M
4028 J. Org. Chem., Vol. 68, No. 10, 2003

First Total Synthesis of a GPI-Anchored Peptide
in decane) was added at -20 °C. The solution was warmed to
room temperature and stirred for another 1.5 h and finally
concentrated. Column chromatography of the residue gave 48
(68 mg, 84%) as a mixture of two diastereoisomers. TLC: Rf
0.44 (acetone, hexane, and DCM 4:10:2). 1H NMR (CDCl3, 600
MHz): δ 7.73 (d, J ) 7.8 Hz, 2 H), 7.55 (br, 2 H), 7.35 (t, J )
7.2 Hz, 1 H), 6.66 (d, J ) 5.4 Hz, 2 H), 5.89 (s, 1 H), 5.74 (t, J
) 9.6 Hz, 1 H/2), 5.73 (t, J ) 9.6 Hz, 1 H/2), 5.43 (br, 1 H),
5.28 (m, 1 H), 5.21 (br, 1 H), 3.55 (s, 3 H/2), 3.54 (s, 3 H/2),
2.27 (m, 2 H), 1.55 (m, 2 H), 1.25 (m, 24 H), 0.88 (t, J ) 7.2
Hz, 1 H). 31P NMR (CDCl3): δ -0.70. MALDI-TOF-MS: calcd
for C184H203N2O34P 3015, found 3038 (M + Na+).
O-{6-O-[N-(L-Prolinyl-L-serinyl)aminoethyl]phospho-
nato-r-D-mannopyranosyl}-(1f2)-O-(r-D-mannopyrano-
syl)-(1f6)-O-(r-D-mannopyranosyl)-(1f4)-O-(2-amino-2-
deoxy-r-D-glucopyranosyl)-(1f6)-2-O-hexadecanoyl-myo-
inositol (1). After 49 (24 mg, 7.5 µmol) was dissolved in 2 M
of dimethylamine in THF (4 mL), the reaction mixture was
stirred at room temperature for 1 h, which was followed by
concentration in a vacuum. Column chromatography of the
residue gave the free amine 50 (19 mg, 6.38 µmol, 85%): Rf
0.33 (DCM and methanol 10:1). The mixture of 50 (19 mg) and
Pd/C (50 mg, 10%) in CHCl3, MeOH, and H2O (4 mL, 10:10:3)
was then stirred in H2 atmosphere for 1 d. The reaction
mixture was filtered off through a Celite pad with a mixture
of MeOH, H2O, and CHCl3 (5:5:1) as the eluent. The filtrate
was concentrated in a vacuum to afford the synthetic target 1
(8 mg, 91%) as white powder. 1H NMR (D2O, 600 MHz, 35
°C): δ 5.36 (br, 2 H), 5.19 (s, 1 H), 5.07 (s, 1 H), 4.98 (s, 1 H),
O-{2,3,4-Tri-O-benzyl-6-O-[benzyl-[N-[N-(9-fluorenyl-
methoxycarbonyl)-L-prolinyl-L-serinyl]aminoethyl]phos-
phonato]-r-D-mannopyranosyl}-(1f2)-O-(3,4,6-tri-O-benz-
yl-r-D-mannopyranosyl)-(1f6)-O-(2,3,4-tri-O-benzyl-r-D-
mannopyranosyl)-(1f4)-O-(2-benzyloxycarbonylamino-
3,6-di-O-benzyl-2-deoxy-r-D-glucopyranosyl)-(1f6)-2-O-
hexadecanoyl-1-O-(p-methoxybenzyl)-3,4,5-tri-O-benzyl-
myo-inositol (49). Compound 48 (60 mg, 20 µmol) was
dissolved in a 2 M solution of dimethylamine in THF (5 mL).
The reaction mixture was stirred at room temperature for 1 h
and then concentrated. The residue was dissolved and con-
centrated several times with chloroform as solvent to remove
the excessive dimethylamine. The resulting crude product 3
(TLC: Rf 0.40; EtOAc, hexane, DCM, and methanol 3:3:3:1)
was directly used in the next step. Thus, after the crude 3,
HOBt (13.5 mg, 100 µmol), and 2 (34 mg, 80 µmol) were
dissolved in DCM and DMF (10 mL, 10:1), to the solution was
added EDC (19.2 mg, 100 µmol) at 0 °C under nitrogen
atmosphere. The reaction mixture was then warmed to room
temperature and stirred overnight, which was followed by
concentration in a vacuum. Column chromatography of the
residue gave 49 (38.4 mg, 60%). TLC: Rf 0.54 (EtOAc, hexane,
1
4.43 (t, J ) 5.4 Hz, 1 H), 4.40 (t, J ) 7.2 Hz, 1 H). H NMR
(CD3OD, 600 MHz, 35 °C): δ 5.41 (s, 1 H), 5.34 (s, 1 H), 5.22
(s, 1 H), 5.13 (s, 1 H), 4.98 (s, 1 H), 4.45 (br, 1 H), 4.38 (br, 1
H). HMQC (CD3OD, 13C 150 MHz, 1H 600 MHz, 35 °C): δ 75.6/
5.41 (Ino-2), 97.9/5.34 (Glc-1), 102.8/5.22 (Man-1), 100.0/5.13
(Man-1), 104.2/4.98 (Man-1), 57.6/4.45 and 4.38 (Pro and Ser
R-C/H). 31P NMR (CD3OD): δ 1.72. MALDI-TOF-MS: calcd
for C57H102N3O32P 1372, found 1372, 1395 (M + Na+), 1434
(M + Na + K+).
Acknowledgment. The authors acknowledge the
Research Corporation (Research Innovation Award RI
0663) and the donors of the Petroleum Research Fund,
administered by the American Chemical Society (ACS
PRF grant 37743-G), for their financial support of this
research. The authors are also grateful to Dr. S. Chen
and Mr. H. Faulk for the MS measurements.
1
DCM, and methanol 3:3:3:1). H NMR (CDCl3, 600 MHz): δ
7.73 (m, 2 H), 7.53 (m, 2 H), 7.36 (t, J ) 7.2 Hz, 1 H), 6.66 (d,
J ) 8.4 Hz, 2 H), 5.90 (br, 1 H), 5.74 (m, 1 H), 5.43 (d, J ) 3.6
Hz, 1 H), 5.20 (br, 1 H), 5.12 (br, 1 H), 5.08 (d, J ) 12.0 Hz, 1
H), 4.73 (s, 1 H), 3.57 (s, 3 H), 2.28 (m, 2 H), 2.12 (m, 2 H),
1.94 (m, 1 H), 1.84 (m, 1 H), 1.56 (m, 2 H), 1.25 (m, 24 H),
0.88 (t, J ) 7.2 Hz, 1 H). HMQC (13C 150 MHz, 1H 600 MHz):
100.6/5.20 (Man-1), 99.3/5.43 (Glu-1), 99.2/5.12 (Man-1), 99.2/
4.73 (Man-1), 65.3/5.90 (Ino-2). 31P NMR (CDCl3): δ 3.04.
MALDI-TOF-MS: calcd for C193H216N3O37P 3198, found 3221
(M + Na+).
Supporting Information Available: 1H NMR spectra of
all new compounds, as well as selected 13C NMR, HQMC NMR,
and MS spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO034213T
J. Org. Chem, Vol. 68, No. 10, 2003 4029