A variable concept for the preparation of branched glycosyl phosphatidyl inositol anchors

Article abstract of DOI:10.1021/jo026380j

A variable concept for the synthesis of branched glycosyl phosphatidyl inositol (GPI) anchors was established. Its efficiency could be shown by the successful synthesis of the GPI anchor of rat brain Thy-1 and of the scrapie prion protein both in the water soluble 1c and lipidated form 1a. Retrosynthesis led to building blocks 2-6 of which 5 could be further disconnected to building blocks 7-9. Trichloroacetimidate 5 was built up in a straightforward manner starting from glycosyl acceptor 9 using known glycosyl donors 7 and 8. The carbohydrate backbone was then assembled by glycosylation of pseudodisaccharide acceptor 6 with donor 5. To ensure high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. Successive deprotection and introduction of the various phosphate residues gave the fully protected GPI anchors. Catalytic hydrogenation and acid-catalyzed cleavage of the Boc protecting groups afforded the target molecules, which could be fully structurally assigned.

Full text of DOI:10.1021/jo026380j

A Va r ia ble Con cep t for th e P r ep a r a tion of Br a n ch ed Glycosyl
P h osp h a tid yl In ositol An ch or s
Klaus Pekari and Richard R. Schmidt*
Fachbereich Chemie, Universita¨t Konstanz, Fach M725, D-78457 Konstanz, Germany
richard.schmidt@uni-konstanz.de
Received August 31, 2002
A variable concept for the synthesis of branched glycosyl phosphatidyl inositol (GPI) anchors was
established. Its efficiency could be shown by the successful synthesis of the GPI anchor of rat brain
Thy-1 and of the scrapie prion protein both in the water soluble 1c and lipidated form 1a .
Retrosynthesis led to building blocks 2-6 of which 5 could be further disconnected to building
blocks 7-9. Trichloroacetimidate 5 was built up in a straightforward manner starting from glycosyl
acceptor 9 using known glycosyl donors 7 and 8. The carbohydrate backbone was then assembled
by glycosylation of pseudodisaccharide acceptor 6 with donor 5. To ensure high stereoselectivity
and good yields in the glycosylation reactions, anchimeric assistance was employed. Successive
deprotection and introduction of the various phosphate residues gave the fully protected GPI
anchors. Catalytic hydrogenation and acid-catalyzed cleavage of the Boc protecting groups afforded
the target molecules, which could be fully structurally assigned.
In tr od u ction
or to function as second messengers, e.g. in insulin-
mediated signal transduction processes.7 Therefore, to
perform biological studies elucidating the functions of
GPI anchors, it seems to be an important objective to
have access to structurally homogeneous GPI anchors
and their derivatives. For the total synthesis of GPI
anchors, a combination of lipid, phosphate, and oligosac-
charide chemistry is required. This has been successfully
carried out for a ceramide-containing GPI anchor of
yeast8,9 and for the acylglycerol-containing GPI anchors
of Trypanosoma brucei10,11 and rat brain Thy-1.12
Glycosyl phosphatidyl inositol (GPI) anchors are a class
of naturally occurring glycolipids that link proteins and
glycoproteins via their C-terminus to eukaryotic cell
membranes. The first full structural assignment of a GPI
anchor was published in 1988 by Ferguson et al.1,2 Since
then quite a few anchors have been characterized,
allowing the definition of a common core structure, which
is highlighted in Scheme 1.3, This common core struc-
4
ture, present in a wide variety of species, is conserved
during evolution while there are a lot of species-specific
variations in the branching groups of the glycan residue
(R2-R4). Additional ethanolaminephosphate groups (R1)
on the central mannose residue seem to be specific for
higher eukaryotes. Another modification site is the lipid
anchor where various structures can be found.
The function of GPI anchors has been extensively
discussed and there is evidence that, besides the obvious
one of anchoring proteins to membranes, there are a lot
of different functions of GPI anchors and/or metabolites
of them. For example, GPI anchoring of a protein seems
to signal transport to certain areas of the cell mem-
brane.5,6 One of the most controversial aspects of GPI
anchors is their ability to mediate signaling mechanisms
The aim of this work was the development of a highly
variable synthetic strategy for the preparation of branched
GPI anchors. This strategy should also allow the attach-
ment of peptide residues and (naturally occurring) pro-
teins to the anchor. All GPI syntheses published so far
do not take into consideration the attachment of a peptide
residue nor the variability required for the preparation
of practically all types of GPI anchors. We focused on 4,6-
branched GPI anchors as there are several prominent
examples found in nature. In a first approach we syn-
thesized the fully phosphorylated pseudohexasaccharide
(7) Caro, H.-H.; Martin-Lomas, M.; Bernabe´, M. Carbohydr. Res.
1993, 240, 119-131.
(8) Mayer, T. G.; Kratzer, B.; Schmidt, R. R. Angew. Chem. 1994,
106, 2289-2293; Angew. Chem., Int. Ed. Engl. 1994, 33, 2177-2181.
(9) Mayer, T. G.; Schmidt, R. R. Eur. J . Org. Chem. 1999, 1153-
1165.
(10) Murakata, C.; Ogawa, T. Carbohydr. Res. 1992, 234, 75-91.
Murakata, C.; Ogawa, T. Carbohydr. Res. 1992, 235, 95-114. The
enantiomer of the naturally occurring inositol moiety was selected in
this synthesis.
(11) Baeschlin, D. K.; Chaperon, A. R.; Green, L. G.; Hahn, M. E.;
Ince, S. J .; Ley, S. V. Chem. Eur. J . 2000, 6(1), 172-186.
(12) Campbell. A. S.; Fraser-Reid, B. J . Am. Chem. Soc. 1995, 117,
10387-10388. The 1H NMR spectrum of the material indicates the
presence of some byproduct.
(1) Ferguson, M. A. J .; Homans, S. W.; Dwek, R. A.; Rademacher,
T. W. Science 1988, 239, 753-759.
(2) Homans, S. W.; Ferguson, M. A. J .; Dwek, R. A.; Rademacher,
T. W.; Anand, R.; Williams, A. F. Nature 1988, 333, 269-272.
(3) Frankhauser, C.; Homans, S. W.; Oates, J . E.; McConville, M.
J .; Desponds, C.; Conzelmann, A.; Ferguson, M. A. J . J . Biol. Chem.
1993, 268, 26365-26374.
(4) Striepen, B.; Zinecker, C. F.; Damm, J . B. L.; Melgers, P. A. T.;
Gerwing, G. J .; Koolen, M.; Vliegenthart, J . F. G.; Dubremetz, J .-F.;
Schwarz, R. T. J . Mol. Biol. 1997, 266, 797-813.
(5) Rothberg, K. G.; Heuser, J . E.; Donzell, W. C.; Ying, Y.-S.;
Glenney, J . R.; Anderson, G. W. Cell 1992, 68, 673-682.
(6) Varma, R.; Mayor, S. Science 1992, 394, 802-805.
10.1021/jo026380j CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/25/2003
J . Org. Chem. 2003, 68, 1295-1308
1295

Pekari and Schmidt
SCHEME 1. Gen er a l Str u ctu r e of GP I An ch or s (EA, eth a n ola m in e; P , p h osp h a te; DAG, d ia cylglycer ol)
of the GPI anchor of Toxoplasma gondii.13 We now
present the synthesis of the more demanding GPI anchor
of rat brain Thy-1 which is identical with one subtype of
the GPI anchors of the scrapie prion protein.14
anchimeric assistance in the glycosylation reaction, thus
yielding selective â-glycoside formation. Known mannosyl
donors 7 and 8 were chosen as they provide anchimeric
assistance in the glycosylation step as well as offer the
possibility to install different building blocks on the
terminal carbohydrate position.
One aim of the synthesis was having the potential to
introduce a peptide moiety at the terminal ethanolamine
residue. As the C-terminal amino acid residue of the
naturally occurring peptide attached to the GPI anchor
is cysteine, amino acid attachment has to take place after
the hydrogenolytic removal of the benzyl ether protecting
groups. Therefore, all amino functions, except the acces-
sible one, had to be protected in a hydrogenation stable
manner, i.e., by using Boc protection.
The introduction of the lipid anchor was planned at a
very late stage in the synthesis as after that reaction the
physical properties of the compounds often lead to
difficult separation of byproducts. In general, also the
phosphate groups should be introduced at a very late
stage in the synthesis, thus providing the possibility of
varying these building blocks, for example, to introduce
different biological markers. With all the above-men-
tioned prerequisites in mind, the following synthetic
concept was established.
Syn t h esis of P h osp h or a m id it e Bu ild in g Block s
2-4. The use of the cyanoethyl protecting group in
building blocks 2-4 allowed the selective deprotection
of the phosphate residues on the per-O-benzylated oli-
gosaccharides. This deprotection resulted in the loss of
the chirality on the phosphorus atom and therefore
simplified further analysis.
This GPI anchor consists of four different R-mannosyl
residues, of which one is mono-, one is di-, one is tri-,
and one is tetrasubstituted. It also contains an N-
acetylgalactopyranose, a glucopyranosamine, two other
free amino functions, which should be differentiated in
the case of the introduction of a peptide, three different
phosphate esters, and nineteen free hydroxy functions.
In a first approach we accomplished a highly conver-
gent synthesis of the GPI anchor of rat brain Thy-1.15
As this synthesis allowed neither the intended high
variability nor the introduction of a peptide we decided
to perform a different route. Main emphasis was now put
on high variability in the synthesis and ready accessibil-
ity of building blocks which were already developed and
optimized in earlier syntheses.
Resu lts a n d Discu ssion
Target molecule 1 is disconnected at positions A-C
leading to phosphorus ester building blocks 2-4 and the
pseudoheptasaccharide residue. This carbohydrate back-
bone was then disconnected at position D yielding
building blocks 5 and 6, which could further be discon-
nected at positions E-F to give known building blocks 7
and 89 and disaccharide 9.
The main emphasis in the synthesis of pentasaccharide
5 was placed on central mannose c, which should allow
selective access to all hydroxy functions. By slight
modification of the synthesis developed in this paper,
access to 3,6-branched GPI anchors should also be
possible. In both types of GPI anchors there can be
attached additional residues in the 2-position of that
particular mannose. The trichloroacetyl group was chosen
as the amino protecting group in galactosamine residue
d in order to (i) increase the electron deficiency of the
amino function, thus avoiding side reactions in the
glycosylation reactions, and (ii) take advantage of the
The preparation of lipid phosphate building block 4
started from readily available 3-O-benzyl-sn-glycerol
(Scheme 3).16 Activation of the primary hydroxy function
of the glycerol residue with dibutylstannane and intro-
duction of the octadecyl chain gave compound 10 with
the secondary hydroxy function unprotected. Ester for-
mation of this hydroxy group with octadecanoyl chloride
in the presence of triethylamine gave intermediate 11,
which was hydrogenated over Pd/C to yield compound
12. Then, compound 12 was subjected to phosphoramidite
formation as described by Bannwarth and Treziak17 to
form phosphoramidite reagent 4. N-Benzyloxycarbonyl
(13) Pekari, K.; Tailler, D.; Weingart, R.; Schmidt, R. R. J Org.
Chem. 2001, 66, 7432-7442.
(14) Stahl, N.; Baldwin, M. A.; Hecker, R.; Pan, K.-M.; Burlingame,
A. L.; Prusiner, S. B. Biochemistry 1992, 31, 5043-5053.
(15) Tailler, D.; Ferrie`res, V.; Pekari, K.; Schmidt, R. R. Tetrahedron
Lett. 1999, 40, 679-682.
(16) Hirth, G.; Barner, R. Helv. Chim. Acta 1982, 65, 1059-1084.
1296 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
SCHEME 2. Retr osyn th esis
SCHEME 3. Syn th esis of Lip id Bu ild in g Block 4
SCHEME 4. P r ep a r a tion of Bu ild in g Block 18
and N-tert-butyloxycarbonyl protected ethanolamine phos-
phoramidites 2 and 3 were prepared as described ear-
lier.13
Syn th esis of Bu ild in g Block 5. The synthesis of
pentasaccharide trichloroacetimidate 5 (see Schemes 4
and 5) is based on the results of the previously reported
synthesis of the GPI anchor of T. gondii.13
N-trichloroacetyl-tri-O-benzyl-galactosyl trichloroacetimi-
date 1613 under BF3‚OEt2 catalysis at -40 °C in toluene
as solvent gave selectively â-disaccharide 17 in good
yield.13 Removal of the p-methoxybenzyl group under
oxidative conditions afforded disaccharide acceptor 18.
To increase the overall yield and to reduce the time for
the preparation of disaccharide 18 starting from com-
pound 13, another pathway was developed. After allyla-
tion of compound 13 to diallyl compound 14, the p-meth-
oxybenzylidene acetal was cleaved under acidic conditions
Starting from known mannose derivative 1313 allyla-
tion (f14) and subsequent reductive opening of the
p-methoxybenzylidene acetal gave diallyl intermediate
15 (see Scheme 4). Glycosylation of 15 with known
(17) Bannwarth, W.; Treziak, A. Helv. Chim. Acta 1987, 70, 175-
186.
J . Org. Chem, Vol. 68, No. 4, 2003 1297

Pekari and Schmidt
SCHEME 5. P r ep a r a tion of P en ta sa cch a r id e
Tr ich lor oa cetim id a te 5
mannosyl trichloroacetimidate 7 in the final glycosylation
reaction, other carbohydrate building blocks could be
used, thus giving access to structurally different GPI
anchors. All R-mannosylation reactions were completely
stereoselective and very high yielding due to the anchi-
meric assistance of the 2-O-acetyl group of the donor
building blocks. Full structural assignment of 26 could
be performed by a combination of 1H, 13C, HMQC, DQF-
COSY, and ROESY spectroscopy (see Table 1). The
determination of the anomeric configuration of the man-
nopyranoses was performed according to Bock and Pe-
1
tersen.18 J C,H coupling constants (obtained from proton
undecoupled HMQC) greater than 170 Hz refer to R-link-
1
ages (e.g., in pentasaccharide 26: J C,H-1c ) 170.7 Hz,
1J C,H-1e ) 173.2 Hz,1J C,H-1f ) 174.5 Hz,1J C,H-1g ) 172.1
1
Hz) while the â-galactosidic linkage has a J C,H-1c value
of 164.1 Hz.
Reduction of the N-trichloroacetyl group to the N-acetyl
group (f27) was performed under neutral reaction
conditions as developed by J acquinet et al.19 Deacetyla-
tion of the terminal 2-O-acetyl group (f28) and benzy-
lation of the free hydroxy functions gave benzylated
pentasaccharide 29. In this three-step sequence it is
important to transform the trichloroacetyl group prior
to O-benzylation as otherwise also N-benzylation occurs.
Transformation of the 1,2-di-O-allyl function in compound
29 to 2-O-phenoxyacetyl-protected trichloroacetimidate
5 was performed similar to a protocol used in an earlier
GPI total synthesis of our group:9 deprotection of the allyl
protecting groups via isomerization with Wilkinson’s
catalyst and subsequent cleavage of the enol ethers with
iodine gave pentasaccharide 30, which was diacylated
with phenoxyacetyl (PA) chloride to give compound 31.
Selective removal of the anomeric phenoxyacetyl group
(f32) by treatment with (NH4)2CO3 in DMF at 50 °C for
2 days and trichloroacetimidate formation under stan-
dard conditions afforded pentasaccharide trichloroace-
timidate 5 in very good overall yield.
Syn th esis of Bu ild in g Block 6. To allow for the
introduction of a peptide moiety at residue f, the amino
function of the pseudodisaccharide building block re-
quires liberation and N-Boc-protection. Therefore, known
pseudodisaccharide 3313 was transformed to 6 in 5 steps
in good overall yield (see Scheme 6).
In a first sequence of reactions, protection of the free
hydroxy function as phenoxyacetyl ester (f34), removal
of the p-methoxybenzyl function with ceric(IV) am-
monium nitrate (f35), and benzoylation with benzoyl
cyanide/triethylamine in dichloromethane led to fully
protected pseudodisaccharide 36. The benzoyl ester was
chosen as the protecting group for the 1-position of the
inositol moiety as it can be quantitatively cleaved without
monitoring the cleavage reaction. This seemed to be
important as very small changes in polarity of the totally
assembled molecule at later stages complicated monitor-
ing of the reaction products and purification in the
synthesis. Subsequent removal of the phenoxyacetyl
protecting group under mild basic conditions (f37),
reduction of the azido function (f38), and subsequent
selective N-Boc protection of the free amino group af-
(f19) and the primary hydroxy function was selectively
protected by formation of the monochloroacetyl ester
(f20). Glycosylation as described above (f21) and
subsequent removal of the ester function by treatment
with methylamine led to disaccharide 18 in 58% overall
yield compared to 36% overall yield of the former
pathway. Access to 3,6-branched GPI anchors is easily
possible by using a mannose c building block similar to
13 having a p-methoxybenyl group at O-3 and a 4,6-O-
benzylidene acetal protecting group.
Starting from disaccharide acceptor 18, the pentasac-
charide backbone was built up in a very straightforward
manner (see Scheme 5): R-selective glycosylation of 18
with mannosyl trichloroacetimidate 7 as donor (f22),
deprotection of the 2-O-acetyl function (f23), and sub-
sequent glycosylation with mannosyl donor 8 led to
tetrasaccharide 24. Again, deprotection of the 2-O-acetyl
group (f25) and R-mannosylation with trichloroacetimi-
date 7 finally led to pentasaccharide 26. Instead of using
(18) Bock, K.; Petersen, C. J . Chem. Soc., Perkin Trans. 2 1974,
293-297.
(19) Blattner, G.; Beau, J .-M.; J acquinet, J .-C. Carbohydr. Res. 1994,
260, 189-202.
1298 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
TABLE 1. Cor r ela tion Ta ble of Com p ou n d 26 (13C a n d 1H Ch em ica l Sh ifts a t 150.9 a n d 600 MHz, Resp ectively)
position
1
2
3
4
5
6
c
d
e
f
97.3/4.71
99.2/4.97
98.3/4.83
99.9/5.35
99.8/5.07
74.8/3.68
55.9/3.93
72.7/4.09
75.2/4.08
68.7/5.57
78.0/3.85
77.4/3.98
80.4/3.87
79.5/3.94
78.6/3.99
74.0/4.01
72.0/3.75
74.8/3.71
74.1/4.14
74.0/3.90
70.1/3.68
73.3/3.50
72.1/3.78
73.1/3.73
71.8/3.86
66.3/3.77 + 3.57
68.2/3.51 + 3.30
69.5/3.62
62.9/4.03 + 3.87
68.5/3.58 + 3.47
g
TABLE 2. Cor r ela tion Ta ble of Com p ou n d 39 (13C a n d 1H Ch em ica l Sh ifts a t 150.9 a n d 600 MHz, Resp ectively)
position
1
2
3
4
5
6
a
b
c
d
e
f
75.1/5.05
98.6/5.33
98.1/5.29
100.8/4.65
99.3/4.55
100.2/5.28
99.6/5.22
74.1/4.21
54.5/3.92
70.2/5.48
52.4/4.26
72.7/4.07
74.4/4.10
74.7/3.86
80.8/3.61
81.5/3.48
75.4/3.75
81.5/3.48
80.1/3.80
/3.88
81.9/4.16
73.3/3.83
72.9/4.01
71.9/3.49
75.4/3.42
74.3/4.13
74.7/4.00
79.7/3.49
70.6/3.99
70.8/3.58
73.5/3.52
72.5/3.70
73.1/3.65
72.1/3.81
75.1/4.45
68.6/3.30
66.4/3.80 + 3.16
68.9/3.48 + 3.30
70.9/3.63 + 3.49
62.8/4.01 + 3.82
68.9/3.56 + 3.48
g
/3.88
TABLE 3. Cor r ela tion Ta ble of Com p ou n d 1c (13C a n d 1H Ch em ica l Sh ifts a t 150.9 a n d 600 MHz, Resp ectively)
position
1
2
3
4
5
6
a
b
c
d
e
f
75.3/4.07
94.7/5.54
98.9/5.35
101.2/4.43
98.3/5.03
100.3/5.19
101.6/4.98
71.1/4.12
53.3/3.30
73.6/4.43
52.1/3.85
78.6/3.92
77.7/4.05
69.6/3.99
70.0/3.49
69.7/4.02
67.6/3.96
70.0/3.70
89.6/3.88
89.4/3.90
69.9/3.76
71.8/3.62
75.8/3.68
76.9/3.79
67.2/3.86
66.5/3.61
65.9/3.71
66.3/3.56
72.2/3.34
70.2/4.11
70.8/3.79
74.9/3.66
72.9/3.63
71.7/3.80
72.8/3.69
76.9/3.83
59.7/3.78 + 3.74
66.3/3.84 + 3.67
66.3/3.84 - 3.67
66.3/3.84 - 3.67
64.2/4.04
g
66.3/3.84 - 3.67
SCHEME 6. P r ep a r a tion of Bu ild in g Block 6
forded pseudodisaccharide 6 in good overall yield. Thus,
the syntheses of all required building blocks could be very
successfully performed.
Con str u ction of Ta r get Molecu les 1a -1c (see
Sch em e 7). To construct the carbohydrate backbone of
the GPI anchor, glycosyl acceptor 6 was treated with
pentasaccharide trichloroacetimidate 5 under standard
glycosylation conditions, i.e., with TMSOTf as catalyst
in dry diethyl ether as solvent at room temperature. The
desired R-connected pseudoheptasaccharide 39 was se-
lectively obtained in excellent yield. The configuration
1
of all glycosidic linkages could be assigned by the J C,H
1
coupling constants: R-linkages for J C,H-1b ) 174.4 Hz,
1
1
1J C,H-1c ) 176.2 Hz, J C,H-1e ) 173.4 Hz, J C,H-1f ) 174.4
1
Hz, J C,H-1g ) 170.7 Hz and the â-galactosidic linkage
1
with J C,H-1d ) 160.5 Hz (for correlation data of all other
13C/1H pairs see Table 2).
Removal of the silyl protecting group with acetic acid
buffered TBAF at 40 °C in THF over several days gave
intermediate 40. Phosphorylation with benzyloxycarbonyl
protected ethanolamine phosphoramidite 210 and im-
mediate removal of the phenoxyacetyl group while elimi-
nating the cyanoethyl group on the phosphate group led
to monophosphorylated compound 41 with the 2c-hydroxy
function already deprotected. Due to the choice of the
cyanoethyl protecting group on the phosphate residue
only one diastereomer of compound 41 was obtained.
Subsequent phosphorylation at the O-2c-position with
N-Boc-protected phosphoramidite building block 320 (f42)
and deprotection of the benzoyl group on the inositol
moiety with sodium methanolate in methanol overnight
gave advanced intermediate 43 in excellent yield. To
ensure the integrity of the backbone, compound 43 was
phosphorylated with dibenzyl N,N-diisopropylphosphora-
midite to give fully phosphorylated compound 44. Depro-
tection by hydrogenation (f45) and subsequent treat-
(20) Watanabe, Y.; Sofue, S.; Ozaki, S.; Hirata, M. J . Chem. Soc.,
Chem. Commun. 1996, 15, 1815-1816.
J . Org. Chem, Vol. 68, No. 4, 2003 1299

Pekari and Schmidt
3). The anomeric configurations could be proven by 1J C,H
SCHEME 7. P r ep a r a tion of th e Ca r boh yd r a te
Ba ck bon e
1
coupling constants (1J C,H-1b ) 178.1 Hz, J C,H-1c ) 175.7
1
1
1
Hz, J C,H-1e ) 173.3 Hz, J C,H-1f ) 174.5 Hz, J C,H-1g
)
172.1 Hz, all of them being R-linked; 1J C,H-1d ) 162.6 Hz,
â-linked). The phosphorus signals, corresponding to the
three phosphate esters, were obtained at δ ) -2.36,
-2.61, and -3.15 ppm.
To arrive at the final goal, i.e., the lipidated GPI
anchor, advanced intermediate 44 was lipidated by using
phosphoramidite building block 4 under standard condi-
tions with tetrazole activation in dichloromethane as
solvent (see Scheme 8). Oxidation with tert-butyl hydro-
peroxide, instead of m-chloroperbenzoic acid as in the
previous phosphorylations, and elimination of the cya-
noethyl group by treatment with dimethylamine gave the
fully protected GPI anchor 46 of rat brain Thy-1 and of
scrapie prion protein, respectively. Removal of the Boc-
protecting groups (f47) by treatment with TFA and Et3-
SnH as scavenger followed by hydrogenation with Pearl-
man’s catalyst afforded the desired fully deprotected GPI-
anchor 1a . To allow for the introduction of a peptide
residue at the GPI anchor, selectively deprotected GPI-
anchor 1b was synthesized by hydrogenation of inter-
mediate 45 with Pearlman’s catalyst.
1
The comparison of the anomeric region of the H NMR
of compounds 1c and 1a with that of the naturally
occurring GPI anchor glycan, which was obtained from
natural sources by Ferguson et al.,1,2 showed great
similarities (see Figure 1); the compound isolated from
natural sources was not lipidated and contained an
impurity. The pattern of the anomeric proton signals in
the 1H NMR spectrum of the synthetic, water soluble,
GPI anchor glycan 1c (bottom left side in Figure 1) is in
good accordance with that of the isolated GPI anchor
glycan (top in Table 1). Even the lipidated GPI anchor
1
1a has a H spectroscopic pattern in the anomeric region
very similar to that of the water-soluble GPI (bottom
right side in Figure 1). The downfield shift of the 1c
proton in lipidated compound 1a can be easily explained
by the short distance between this proton and the lipid
anchor.
Con clu sion
In summary, a highly variable concept for the synthesis
of branched GPI anchors could be established. It is based
on versatile building blocks which are readily accessible
and permitted high regio- and stereoselectivity in all
reaction steps. The efficiency of this concept could be
shown by the synthesis of the 4,6-branched GPI anchor
of rat brain Thy-1 and scrapie prion protein, which was
synthesized in the water-soluble form as well as in the
lipidated form. The synthetic concept further allows the
attachment of a peptide moiety or different biological
markers to the GPI anchor. Therefore, in principle the
synthesis of a large number of branched or linear GPI
anchors or derivatives of them can be easily performed
by using this synthetic strategy.
Exp er im en ta l Section
ment with TFA afforded the fully phosphorylated water
soluble GPI anchor of rat brain Thy-1 1c.
Full structural assigment could be obtained by means
of one- and two-dimensional NMR techniques (see Table
Solvents were purified in the usual way; the boiling range
of petroleum ether was 35-65 °C. Melting points are uncor-
1
rected. H NMR were recorded at 250 and 600 MHz with an
1300 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
SCHEME 8. P r ep a r a tion of th e GP I An ch or of Ra t Br a in Th y-1
internal standard of tetramethylsilane (TMS). FAB MS were
recorded with the NBOH () 3-nitrobenzyl alcohol) matrix.
3-O-Ben zyl-1-O-octa d ecyl-sn -glycer ol (10), 3-O-Ben zyl-
2-O-octa d eca n olyl-1-O-octa d ecyl-sn -glycer ol (11), a n d
2-O-Octa d eca n oyl-1-O-octa d ecyl-sn -glycer ol (12). A mix-
ture of 3-O-benzyl-sn-glycerol (6.65 g, 36.74 mmol)16 and
dibutyltin oxide (9.60 g, 38.58 mmol) in 250 mL of dry toluene
was stirred under reflux with azeotropic removal of water for
2 h. The mixture was then cooled and concentrated to dryness
to give a white solid, which was mixed with petroleum ether,
stirred for 15 min, and filtered to furnish 14.7 g of the
dibutylstannylene-acetal. A 7.35-g sample of this compound,
1-bromooctadecane (6.2 g, 18.63 mmol), and tetrabutylammo-
nium iodine (TBAI) (6.57 g, 17.8 mmol) were stirred in 200
mL of dry DMF at 140 °C for 3.5 h and then concentrated.
The residue was dissolved in ethyl acetate, and the organic
solution was washed with brine and water, dried (MgSO4), and
concentrated to give an oil (f10). TLC [petroleum ether/ethyl
acetate (5:1)] Rf 0.35. A mixture of this material, triethylamine
(6.3 mL, 41.40 mmol), and stearoyl chloride (13.8 mL, 41.40
mmol) was stirred in 30 mL of dry CH2Cl2 at room temperature
overnight then diluted with ethyl acetate, washed with brine
and water, dried (MgSO4), and concentrated (f11). The
residue was dissolved in 50 mL of THF/MeOH 1:1 and some
Pd/C was added. This mixture was stirred under H2 for 1 h
and concentrated. Flash chromatography (petroleum ether/
ethyl acetate 9:1 f 7:1) afforded compound 12 (3.7 g, 6.05
mmol, 33%) as a white solid. TLC [petroleum ether/ethyl
acetate (9:1)] Rf 0.25; [R]D -2.5 (c ) 1.0, CDCl3); 1H NMR (250
MHz, CDCl3) δ 0.88 (m, 6H, 2 Me), 1.18-1.40 (m, 58H, CH2),
1.53-1.66 (m, 4H, CH2), 2.21 (m, 1H, OH), 2.36 (m, 2H, CH2-
COO), 3.41-3.49 (m, 2H, CH2CH2O), 3.56-3.68 (m, 2H, 1/1′-
H), 3.81 (m, 2H, 3/3′-H), 5.00 (m, 1H, 2-H). Anal. Calcd for
C
39H78O4 (611.17): C 76.66, H 12.87. Found: C 76.59, H 13.08.
(2-Cya n oeth oxy)(2-O-octa d eca n oyl-1-O-octa d ecyl-sn -
glycer yl)(d iisop r op yla m in o)p h osp h in e (4). Compound 12
(500 mg, 820 µmol), 2-cyanoethoxybis(diisopropylamino)-
phosphine (285 µL, 902 µmol) and tetrazole (28 mg, 180 µmol)
were dried in a vacuum for 2 h. After addition of 5.0 mL of
dry CH2Cl2 the solution was stirred under argon at room
temperature for 90 min. The solution was diluted with CH2-
Cl2, washed with saturated NaHCO3 solution, and dried
(MgSO4) and the solvent was removed. Flash chromatography
(petroleum ether/ethyl acetate 30:1) afforded compound 4 (600
mg, 705 mmol, 86%) as a colorless wax. TLC [petroleum ether/
ethyl acetate (18:2)] Rf 0.80; 1H NMR (250 MHz, CDCl3) δ 0.89
J . Org. Chem, Vol. 68, No. 4, 2003 1301

Pekari and Schmidt
F IGURE 1. 1H spectra of the anomeric region of the rat brain Thy-1 GPI (top, naturally occurring compound; bottom left, synthetic
GPI anchor glycan 1c; bottom right, synthetic GPI anchor 1a ; spectra of the synthetic GPI anchors were recorded at 293 K in
D2O and D2O/d4-MeOH, respectively).
(t, 6H, 2Me), 1.18 (d, 6H, Me), 1.20 (d, 6H, Me), 1.23-1.40 (m,
58H, CH2), 1.51-1.64 (m, 4H, CH2), 2.31 (t, 2H, CH2COO),
3.37-3.50 (m, 2H, CH2O), 3.58 (d, 2H, 1/1′-H), 3.61-3.84 (m,
4H, Me2CH), 4.61-4.76 (m, 2H, CH2Ph), 5.12-5.16 (m, 1H,
2-H), 7.24-7.37 (m, 5H, Ph).
NaHCO3 solution and water and dried (MgSO4). Removal of
the solvent and flash chromatography (petroleum ether/ethyl
acetate 3:1) afforded compound 15 (3.73 g, 7.91 mmol, 68%)
and allyl 2-O-allyl-3-O-benzyl-4-O-(4-methoxybenzyl)-R-D-man-
nopyranoside (1.1 g, 2.25 mmol, 20%), both as colorless foams.
15: TLC [petroleum ether/ethyl acetate (2:1)] Rf 0.50; [R]D
+11.0 (c 1.6, CDCl3); 1H NMR (250 MHz, CDCl3) δ 2.52 (d,
J OH ) 1.9 Hz, 1H, 4-OH), 3.68-3.81 (m, 5H), 3.72 (s, 3H, OMe),
3.93-4.03 (m, 2H, allyl), 4.10-4.25 (m, 3H, allyl), 4.50 (d, J H,H
) 11.3 Hz, 1H, PMB), 4.55 (d, J H,H ) 11.3 Hz, 1H, PMB), 4.60
(d, J H,H ) 12.2 Hz, 1H, CH2Ph), 4.72 (d, J H,H ) 12.2 Hz, 1H,
CH2Ph), 4.90 (br s, 1H, 1-H), 5.15-5.31 (m, 4H, allyl), 5.89
(m, 2H, allyl), 6.82-6.88 (m, 2H, PMB), 7.22-7.40 (m, 7H, Ph);
HMQC data (13C (150.9 MHz)/1H (600 MHz))) 97.4/4.90 (1c),
74.1/3.74 (2c), 79.5/3.73 (3c), 71.4/3.72 (4c), 70.1/3.71 (5c), 72.1/
4.13 (6c). Anal. Calcd for C27H34O7 (470.61): C 68.90, H 7.30.
Found: C 68.81, H 7.33. Other isomer: TLC [petroleum ether/
ethyl acetate (3:1)] Rf 0.20; [R]D +22.8 (c 1.9, CDCl3); 1H NMR
(250 MHz, CDCl3) δ 1.99 (t, J OH ) 6.6 Hz, 1H, 6-OH), 3.60-
3.98 (m, 8H), 3.79 (s, 3H, OMe), 4.10-4.28 (m, 3H), 4.56 (d,
J H,H ) 12.0 Hz, 1H), 4.72 (br s, 2H, CH2Ph), 4.82-4.88 (m,
2H, 1-H, CH2Ph), 5.15-5.33 (m, 4H, allyl), 6.80-6.88 (m, 2H,
Allyl 2-O-Allyl-3-O-b en zyl-4,6-O-(4-m et h oxyb en zyli-
d en e)-r-D-m a n n op yr a n osid e (14). Compound 1313 (15 g, 35
mmol) was dissolved with allyl bromide (3.9 mL, 4.6 mmol) in
160 mL of DMF and sodium hydride (1.09 g, 4.5 mmol) was
added in portions. After 1 h some methanol was added and
the solvent was removed. Flash chromatography (petroleum
ether/ethyl acetate 4:1) of the residue afforded compound 14
(15.6 g, 33 mmol, 95%) as a syrup. TLC [petroleum ether/ethyl
1
acetate (3:1)] Rf 0.60; [R]D +38 (c 2.5, CDCl3); H NMR (250
MHz, CDCl3) δ 3.82 (s, 3H, OMe), 3.76-4.18 (m, 5H, 4-H, allyl,
6′,5,2-H), 4.12-4.35 (m, 5H, 3 × allyl, 6,3-H), 4.69 (d, J H,H
)
1
12.2 Hz, 1H, CH2Ph), 4.85 (d, J H,H ) 1.5 Hz, J CH ) 171.8 Hz,
1H, 1-H), 4.86 (d, J H,H ) 12.2 Hz, 1H, CH2Ph), 5.16-5.34 (m,
4H, allyl), 5.59 (s, 1H, benzylidene), 5.81-6.01 (m, 2H, allyl),
6.85-6.91 (m, 2H, PMB), 7.26-7.44 (m, 8H); HMQC data (13C
(150.9 MHz)/1H (600 MHz)) 98.7/4.85 (1c), 76.5/3.8 (2c), 79.0/
4.17 (3c), 76.2/3.96 (4c), 64.2/3.81 (5c), 68.7/4.22 + 3.85 (6c).
Anal. Calcd for C27H32O7 (468.59): C 69.20, H 6.90. Found: C
69.34, H 6.94.
PMB), 7.18-7.41 (m, 7H, Ph). Anal. Calcd for
C27H34O7
(470.61): C 68.90, H 7.30. Found: C 68.56, H 7.39.
Allyl 2-O-Allyl-3-O-ben zyl-6-O-(4-m eth oxyben zyl)-r-D-
m a n n op yr a n osid e (15). Compound 14 (5.4 g, 11.5 mmol) and
sodium cyanoborohydride (3.62 g, 57.6 mmol) were dissolved
with some MS 4 Å in 80 mL of dry DMF and cooled to -20 °C.
After addition of 7.8 mL of TFA in 40 mL of dry DMF the
mixture was stirred at -20 °C under argon for 3 days. After
neutralization with triethylamine the reaction mixture was
dissolved with ethyl acetate and washed with saturated
Allyl (3,4,6-Tr i-O-ben zyl-2-deoxy-2-tr ich lor oacetam ido-
â-D-ga la ctop yr a n osyl)-(1f4)-2-O-a llyl-3-O-ben zyl-6-O-(4-
m eth oxyben zyl)-r-D-m a n n op yr a n osid e (17). Acceptor 14
(2.38 g, 5.1 mmol) and trichloroacetimidate 1613 (3.93 g, 5.3
mmol) were dissolved in 45 mL of dry toluene under argon
and cooled to -60 °C. BF3‚OEt2 (500 µL) was added 5 times
every 30 min. After 1 h the temperature was raised to -40
°C. Neutralization with triethylamine, removal of the solvent,
1302 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
and flash chromatography (petroleum ether/ethyl acetate 6:1
f 5:1 f 2:1) afforded disaccharide 17 (3.9 g, 3.72 mmol, 73%)
as a colorless syrup. For TLC: One drop of the reaction
mixture has to be rapidly dissolved in one drop of triethy-
lamine. TLC [petroleum ether/ethyl acetate (3:1)] Rf 0.45; [R]D
(2.2 g, 6.3 mmol, 22%). TLC [petroleum ether/ethyl acetate (1:
1)] Rf 0.65; [R]D +10 (c 1.0, CDCl3); 1H NMR (250 MHz, CDCl3)
δ 2.38 (d, J OH ) 2.0 Hz, 1H, OH), 3.70-3.84 (m, 3H), 3.90 (dd,
J 2,3 ) 2.4 Hz, J 3,4 ) 9.2 Hz, 1H, 3c-H), 3.93-4.04 (m, 1H),
4.08-4.22 (m, 5H), 4.41-4.48 (m, 2H), 4.55 (d, J H,H ) 11.6 Hz,
1H, CH2Ph), 4.72 (d, J H,H′ ) 11.6 Hz, 1H, CH2Ph), 4.90 (d, J 1,2
) 1.1 Hz, 1H, 1c-H), 5.13-5.32 (m, 4H, allyl), 5.82-5.97 (m,
2H, allyl), 7.31-7.38 (m, 5H). Anal. Calcd for C21H27O7Cl
(426.93): C 59.08, H 6.39. Found: C 59.19, H 6.35.
1
+18 (c 1.1, CDCl3); H NMR (250 MHz, CDCl3) δ 3.36-3.49
(m, 2H), 3.50-3.60 (m, 1H), 3.62-3.78 (m, 7H), 3.80-4.00 (m,
5H), 4.02-4.20 (m, 4H), 4.20-4.64 (m, 8H), 4.76-4.88 (m, 4H),
5.06-5.28 (m, 4H), 5.72-5.95 (m, 2H, allyl), 6.66 (d, J NH
)
10.6 Hz, 1H, NH), 6.80-6.85 (m, 2H, PMB), 7.18-7.35 (m,
23H, Ph); HMQC data (13C (150.9 MHz)/1H (600 MHz)) 97.6/
4.85 (1c), 75.3/3.71 (2c), 78.2/3.85 (3c), 74.6/4.10 (4c), 70.9/3.73
(5c), 69.0/3.65 (6c), 99.2/4.81 (1d), 55.9/3.94 (2d), 78.2/3.85 (3d),
72.1/3.94 (4d), 73.4/3.42 (5d), 68.2/3.55 + 3.38 (6d). Anal. Calcd
for C58H62O12NCl3 (1047.54): C 66.50, H 5.98, N 1.34. Found:
C 66.46, H 5.94, N 1.16.
Allyl (3,4,6-Tr i-O-ben zyl-2-deoxy-2-tr ich lor oacetam ido-
â-D-galactopyr an osyl)-(1f4)-2-O-allyl-3-O-ben zyl-r-D-m an -
n op yr a n osid e (18). (a ) F r om 17: Disaccharide 17 (2.87 g,
2.74 mmol) and ceric(IV)ammonium nitrate (7.47 g, 12.7 mmol)
were stirred in 220 mL of CH3CN/toluene/H2O 50/45/5 at 0 °C
for half an hour and at room temperature for 2 h. The reaction
mixture was diluted with ethyl acetate, washed with saturated
NaHCO3 solution, dried over Na2SO4 ,and evaporated. Flash
chromatography (petroleum ether/ethyl acetate 3:1 f 2:1)
afforded compound 18 (1.91 g, 2.06 mmol, 75%) as colorless
foam.
Allyl
(3,4,6-Tr i-O-b en zyl-2-d eoxy-2-t r ich lor oa cet -
a m id o-â-D-ga la ctop yr a n osyl)-(1f4)-2-O-a llyl-3-O-ben zyl-
6-O-m on och lor oa cetyl-r-D-m a n n op yr a n osid e (21). Com-
pound 20 (5.0 g, 11.3 mmol) and trichloroacetimidate 16 (9.6
g, 13.0 mmol) were dissolved in 140 mL of dry toluene under
argon and cooled to -40 °C. BF3‚OEt2 solution (6 mL, 0.2 N
solution in dry toluene) was added 5 times every 30 min. After
1 h the temperature was raised to -30 °C. Neutralization with
triethylamine, removal of the solvent, and flash chromatog-
raphy (petroleum ether/ethyl acetate 6:1 f 5:1 f 2:1) of the
remaining residue afforded compound 21 (9.8 g, 9.6 mmol,
85%) as a colorless solid. TLC [petroleum ether/ethyl acetate
(3:1)] Rf 0.39; [R]D +21 (c 1.7, CDCl3); 1H NMR (250 MHz,
CDCl3) δ 3.32-3.39 (m, 1H), 3.48-3.62 (m, 2H), 3.70-4.75 (m,
22H), 4.81-4.92 (m, 2H), 5.20-5.32 (m, 5H), 5.70-5.98 (m,
2H, allyl), 6.96 (d, J NH ) 6.7 Hz, 1H, NH), 7.17-7.38 (m, 20H,
Ph). Anal. Calcd for C50H55O13Cl4N (1019.86): C 58.88, H 5.45,
N 1.37. Found: C 58.52, H 5.64, N 1.20.
(b) F r om 21: Compound 21 (720 mg, 706 µmol) was
dissolved in 2 mL of dry methanol and 2 mL of dry diethyl
ether and 1 mL of MeNH2 solution (33% in dry ethanol) was
added. The solvent was removed after 15 min and subsequent
flash chromatography (petroleum ether/ethyl acetate 1:1) of
the residue afforded compound 18 (617 mg, 665 µmol, 88%)
as a colorless foam.
TLC [petroleum ether/ethyl acetate (2:1)] Rf 0.11; [R]D +47
(c 1.5, CDCl3); 1H NMR (250 MHz, CDCl3) δ 2.04 (br s, 1H,
OH), 3.39 (dd, J 5,6 ) 5.1 Hz, J 6,6 ) 9.1 Hz, 1H, 6d-H), 3.68
(m, 3H, 5c,5d,6′d-H), 3.72-3.77 (m, 3H, 2c,6c,6′c-H), 3.86-4.76
(m, 16H, see table), 4.84-4.90 (m, 2H, 1c-H), 5.04-5.29 (m,
Allyl (2-O-Acetyl-3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n o-
syl)-(1f6)-[(3,4,6-tr i-O-ben zyl-2-d eoxy-2-tr ich lor oa ceta -
m id o-â-D-ga la ctop yr a n osyl)-(1f4)]-2-O-a llyl-3-O-ben zyl-
r-D-m a n n op yr a n osid e (22). Compound 18 (2.78 g, 3.0 mmol)
and trichloroacetimidate 7 (2.29 g, 3.6 mmol) [120] were
dissolved in 31 mL of dry diethyl ether and 4.6 mL of dry CH2-
Cl2 under argon and cooled to -10 °C (NaCl/ice). After addition
of a 0.1 N TMSOTf solution (1.5 mL), stirring at -10 °C for
10 min, and neutralization with triethylamine, the solvent was
removed. Flash chromatography (petroleum ether/ethyl acetate
7:2) of the residue afforded compound 22 (4.0 g, 2.9 mmol, 95%)
as a colorless foam. TLC [petroleum ether/ethyl acetate (2:1)]
Rf 0.52; [R]D +26 (c 1.0, CDCl3); 1H NMR (250 MHz, CDCl3) δ
2.12 (s, 3H, OAc), 3.05-3.16 (m, 2H), 3.32 (m, 1H), 3.47-4.14
(m, 22H), 4.17-4.55 (m, 8H), 4.61-4.93 (m, 7H, 1e-H, 1c-
5H, CH2Ph, 1d-H), 5.71-5.95 (m, 2H, allyl), 6.90 (d, J NH
)
7.4 Hz, 1H, NH), 7.20-7.35 (m, 20H, Ph); HMQC data (13C
(150.9 MHz)/1H (600 MHz)) 97.7/4.84 (1c), 74.8/3.75 (2c), 78.4/
3.88 (3c), 73.2/4.19 (4c), 71.9/3.61 (5c), 62.2/3.76 (6c), 99.0/5.09
(1d), 56.2/3.97 (2d), 77.3/4.14 (3d), 71.9/3.97 (4d), 73.3/3.55 (5d),
68.3/3.55 + 3.39 (6d). Anal. Calcd for C48H54O11Cl3N (927.38):
C 62.61, H 5.88, N 1.51. Found: C 62.08, H 5.91, N 1.25.
H), 5.02-5.28 (m, 5H, 1d-H), 5.34 (dd, J 1,2 ) 1.9 Hz, J 2,3
5.0 Hz, 1H, 2e-H), 5.71-5.89 (m, 2H, allyl), 7.04 (d, J NH
)
)
7.04 Hz, 1H, NH), 7.10-7.36 (m, 35H, Ph); HMQC data (13C
(150.9 MHz)/1H (600 MHz)) 97.2/4.83 (1c), 74.7/3.74 (2c), 78.3/
3.92 (3c), 74.1/4.03 (4c), 70.1/3.79 (5c), 66.5/3.82 (6c), 99.0/5.12
(1d), 56.3/3.92 (2d), 77.2/4.10 (3d), 72.0/3.87 (4d), 73.3/3.53 (5d),
68.2/3.58 + 3.35 (6d), 97.0/4.94 (1e), 68.3/5.43 (2e), 78.3/3.99
(3e), 74.3/3.85 (4e), 71.3/3.90 (5e), 69.0/3.72 (6e). Anal. Calcd
for C77H84O17Cl3N (1401.97): C 65.96, H 6.05, N 1.00. Found:
C 65.92, H 6.14, N 0.99.
Allyl 2-O-Allyl-3-O-ben zyl-r-D-m a n n op yr a n osid e (19).
Compound 14 (15.5 g, 33 mmol) and CSA (420 mg, 1.6 mmol)
were dissolved in 70 mL of diethyl ether and 70 mL of
methanol. After stirring for 1.5 h and removal of the solvent,
flash chromatography (petroleum ether/ethyl acetate 1:1 f 2:3)
afforded compound 19 (11.5 g, 33 mmol, qu.) as a colorless
solid. TLC [petroleum ether/ethyl acetate (1:1)] Rf 0.2; [R]D +23
(c 2.0, CDCl3); 1H NMR (250 MHz, CDCl3) δ 2.15 (br s, 1H,
OH), 2.43 (br s, 1H, OH), 3.61-3.69 (m, 1H), 3.70-3.86 (m,
Allyl (3,4,6-Tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-(1f6)-
[(3,4,6-tr i-O-ben zyl-2-d eoxy-2-tr ich lor oa ceta m id o-â-D-ga -
lactopyr an osyl)-(1f4)]-2-O-allyl-3-O-ben zyl-r-D-m an n opy-
r a n osid e (23). Compound 21 (4 g, 2.85 mmol) was dissolved
in 0.1 N NaOMe solution (15 mL) and 21 mL of dry diethyl
ether. After removal of the solvent and flash chromatography
(petroleum ether/ethyl acetate 2:1) compound 22 (3.88 g, 2.85
mmol, qu.) was obtained as a colorless foam. TLC [petroleum
4H), 3.88-4.02 (m, 2H), 4.13-4.24 (m, 3H), 4.57 (d, J H,H
)
11.6 Hz, 1H, CH2Ph), 4.74 (d, J H,H‘ ) 11.6 Hz, 1H, CH2Ph),
4.91 (d, J 1,1 ) 1.2 Hz, 1H, 1c-H), 5.17-5.33 (m, 4H, allyl),
5.83-5.99 (m, 2H, allyl), 7.28-7.39 (m, 5H, Ph). Anal. Calcd
for C19H26O6 (350.45): C 63.20, H 7.58 (+1/2H2O). Found: C
63.95, H 7.46.
1
ether/ethyl acetate (2:1)] Rf 0.20; [R]D +40 (c 1.5, CDCl3); H
NMR (250 MHz, CDCl3) δ 3.30-3.40 (m, 1H), 3.48-3.55 (m,
2H), 3.65-4.14 (m, 19H), 4.17-4.84 (m, 15H), 4.98-5.28 (m,
6H), 5.72-5.93 (m, 2H, allyl), 6.97 (d, J NH ) 7.2 Hz, 1H, NH),
Allyl 2-O-Allyl-3-O-ben zyl-6-O-m on och lor oa cetyl-r-D-
m a n n op yr a n osid e (20). Compound 19 (10 g, 28.5 mmol) and
imidazole (4.2 g, 62 mmol) were dissolved under argon in 115
mL of dry CH2Cl2 and monochloroacetyl chloride (2.5 mL, 31
mmol) was added. The solvent was removed after 1 h and flash
chromatography (petroleum ether/ethyl acetate 3:1) of the
residue afforded compound 20 (9.1 g, 20.4 mmol, 75%) as a
colorless syrup. All side products were collected and treated
with 1 mL of MeNH2 solution (33% in dry ethanol) in 25 mL
of CH2Cl2. Evaporation of the solvent and flash chromatogra-
phy (petroleum ether/ethyl acetate 2:1) afforded compound 19
7.12-7.37 (m, 35H, Ph). Anal. Calcd for
C75H82O16Cl3N
(1359.93): C 66.24, H 6.09, N 1.03. Found: C 66.03, H 6.23,
N 0.93.
Allyl (2-O-Acetyl-3,4-d i-O-ben zyl-6-O-ter t-bu tyld ip h e-
n ylsilyl-r-D-m a n n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-
r-D -m a n n op yr a n osyl)-[(3,4,6-t r i-O-b e n zyl-2-d e oxy-2-
t r ich lor oa cet a m id o-â-D-ga la ct op yr a n osyl)-(1f4)]-2-O-
a llyl-3-O-ben zyl-r-D-m a n n op yr a n osid e (24). Compound 23
J . Org. Chem, Vol. 68, No. 4, 2003 1303

Pekari and Schmidt
(3.88 g, 2.85 mmol) and trichloroacetimidate 8 (2.80 g, 3.56
mmol) were dissolved in 30 mL of dry diethyl ether under
argon. At 0 °C a 0.1 N TMSOTf solution (3.8 mL) was added.
After stirring at 0 °C for 15 min, neutralization with triethy-
lamine, and removal of the solvent, flash chromatography
(petroleum ether/ethyl acetate 4:1) afforded compound 24 (5.43
g, 2.73 mmol, 96%) as a colorless foam. TLC [petroleum ether/
d eoxy-â-D-ga la ctop yr a n osyl)-(1f4)]-2-O-a llyl-3-O-ben zyl-
r-D-m a n n op yr a n osid e (27). Compound 26 (1.5 g, 620 µmol)
was dissolved in 47 mL of dry toluene and Bu3SnH (740 µL,
2.79 mmol) and AIBN (30 mg) were added. After 25 min of
vigorous stirring at room temperature under an argon stream
the solution was rapidly heated to 110 °C; after 25 min again
500 µL of Bu3SnH and some AIBN were added, after an
additional 15 min again 200 µL of Bu3SnH and some AIBN.
After removal of the solvent and immediate flash chromatog-
raphy (petroleum ether/ethyl acetate 5:1 f 5:2) of the residue
compound 27 (1.18 g, 505 µmol, 81%) was obtained as a
colorless foam. TLC [petroleum ether/ethyl acetate (5:2)] Rf
0.25; [R]D +22 (c 1.7, CDCl3); 1H NMR (250 MHz, CDCl3) δ
1
ethyl acetate (3:1)] Rf 0.55; [R]D +24 (c 1.5, CDCl3); H NMR
(250 MHz, CDCl3) δ 1.08 (s, 9H, tBu), 2.10 (s, 3H, OAc), 3.50-
4.12 (m, 24H), 4.15-4.37 (m, 4H), 4.39-4.49 (m, 5H), 4.56-
4.68 (m, 7H), 4.72-4.85 (m, 6H, 1c-H, 1e-H), 4.88-5.19 (m,
7H, 1f-H, 1d-H), 5.49 (br s, 1H, 2f-H), 5.66-5.85 (m, 2H,
allyl), 6.93 (d, J NH ) 6.8 Hz, 1H, NH), 7.11-7.39 (m, 51H, Ph),
7.67-7.76 (m, 4H, TBDPS); HMQC data (13C (150.9 MHz)/1H
(600 MHz)) 97.4/4.76 (1c), 75.0/3.70 (2c), 78.1/3.86 (3c), 74.1/
4.04 (4c), 70.2/3.68 (5c), 66.4/3.78 + 3.57 (6c), 99.2/4.97 (1d),
56.0/3.94 (2d), 77.6/3.97 (3d), 72.0/3.73 (4d), 73.4/3.52 (5d), 68.4/
3.52 + 3.32 (6d), 98.4/4.82 (1e), 73.1/4.08 (2e), 80.4/3.90 (3e),
74.6/3.75 (4e), 72.1/3.79 (5e), 69.6/3.62 (6e), 98.9/5.18 (1f), 68.9/
5.49 (2f), 78.0/4.02 (3f), 73.7/4.20 (4f), 73.0/3.74 (5f), 62.7/4.09
+ 3.87 (6f). Anal. Calcd for C113H124O22Cl3NSi (1982.82): C
68.44, H 6.32, N 0.71. Found: C 68.39, H 6.40, N 0.49.
t
1.03 (s, 9H, Bu), 1.68 (s, 3H, NAc), 2.13 (s, 3H, OAc), 3.03-
5.16 (m, 65H, see table, 4.70: 1c-H, 4.88: 1e-H, 4.88: 1d-
H, 5.09: 1g-H), 5.34 (br s, 1H, 1f-H), 5.58 (br s, 1H, 2g-H),
5.63-5.83 (m, 2H, allyl), 6.19 (d, J NH ) 7.6 Hz, 1H, NH), 7.01-
7.35 (m, 66H, Ph), 7.63-7.78 (m, 4H, TBDPS); HMQC data
(13C (150.9 MHz)/1H (600 MHz)) 97.9/4.70 (1c), 75.5/3.65 (2c),
78.6/3.84 (3c), 75.3/3.97 (4c), 70.2/3.67 (5c), 66.3/3.79 + 3.67
(6c), 101.5/4.88 (1d), 54.4/3.86 (2d), 79.1/3.79 (3d), 72.4/3.63
(4d), 73.6/3.44 (5d), 68.9/3.47 + 3.23 (6d), 98.6/4.88 (1e), 73.2/
4.14 (2e), 80.3/3.90 (3e), 75.5/3.50 (4e), 72.6/3.76 (5e), 70.6/3.63
+ 3.53 (6e), 100.4/5.34 (1f), 75.6/4.07 (2f), 79.7/3.92 (3f), 74.5/
4.14 (4f), 73.6/3.71 (5f), 63.2/4.02 + 3.84 (6f), 100.2/5.09 (1g),
69.1/5.57 (2g), 79.0/3.99 (3g), 74.5/3.89 (4g), 72.2/3.89 (5g), 68.9/
3.61 + 3.90 (6g). Anal. Calcd for C140H155O27NSi (2312.05): C
72.72, H 6.77, N 0.61. Found: C 72.73, H 6.83, N 0.47.
Allyl (3,4-Di-O-ben zyl-6-O-ter t-bu tyld ip h en ylsilyl-r-D-
m an n opyr an osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m an n opy-
r a n osyl)-(1f6)-[(3,4,6-t r i-O-b en zyl-2-d eoxy-2-t r ich lor o-
a cet a m id o-â-D-ga la ct op yr a n osyl)-(1f4)]-2-O-a llyl-3-O-
ben zyl-r-D-m a n n op yr a n osid e (25). Compound 24 (5.2 g,
2.62 mmol) was dissolved in 8.1 mL of dry diethyl ether and
8.1 mL of dry methanol under argon. After addition of sodium
(200 mg, 870 µmol), the solution was stirred at room temper-
ature for 30 min and the solvent was removed. Flash chro-
matography (petroleum ether/ethyl acetate 3:1) afforded com-
pound 25 (4.9 g, 2.49 mmol, 95%) as a colorless foam. TLC
[petroleum ether/ethyl acetate (3:1)] Rf 0.43; [R]D +27 (c 1.0,
Allyl (3,4,6-Tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-(1f2)-
(3,4-di-O-ben zyl-6-O-ter t-bu tyldiph en ylsilyl-r-D-m an n opy-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a c e t a m id o -3,4,6-t r i-O -b e n zy l-2-d e o x y -â-D -
g a la c t o p y r a n o s y l)-(1f4)]-2-O -a lly l-3-O -b e n zy l-r-D -
m a n n op yr a n osid e (28). Compound 27 (890 mg, 385 µmol)
was dissolved in 3.8 mL of dry methanol and 2.7 mL of dry
diethyl ether under argon. After addition of sodium (10 mg,
435 µmol) and stirring at room temperature for 1 h, the solvent
was removed. After flash chromatography (petroleum ether/
ethyl acetate 5:2) compound 28 (780 mg, 342 µmol, 89%) was
obtained as a colorless foam. TLC [petroleum ether/ethyl
t
CDCl3); 1H NMR (250 MHz, CDCl3) δ 1.06 (9s, H, Bu), 3.28-
5.26 (m, 54H), 5.66-5.84 (m, 2H, allyl), 6.89 (d, J NH ) 6.8 Hz,
1H, NH), 7.09-7.42 (m, 51H, Ph), 7.67-7.76 (m, 4H, TBDPS).
Anal. Calcd for C111H122O21Cl3NSi (1940.79): C 68.69, H 6.35,
N 0.72. Found: C 68.56, H 6.27, N 0.57.
Allyl (2-O-Acetyl-3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n o-
syl)-(1f2)-(3,4-d i-O-ben zyl-6-O-ter t-bu tyld ip h en ylsilyl-r-
D-m a n n op yr a n osyl)-(1f2)-(3,4,6-t r i-O-b en zyl-r-D-m a n -
n o p y r a n o s y l)-(1f6)-[(3,4,6-t r i-O -b e n zy l-2-d e o x y -2-
t r ich lor oa cet a m id o-â-D-ga la ct op yr a n osyl)-(1f4)]-2-O-
a llyl-3-O-ben zyl-r-D-m a n n op yr a n osid e (26). Compound 25
(5.1 g, 2.62 mmol) and trichloroacetimidate 7 (1.84 g, 2.88
mmol) [120] were dissolved in 32 mL of dry diethyl ether under
argon. At room temperature a 0.1 N TMSOTf solution (2.5 mL,
0.25 mmol) was added. After stirring for 15 min, neutralization
with triethylamine, and removal of the solvent, flash chroma-
tography (petroleum ether/ethyl acetate 4:1) afforded com-
pound 26 (5.9 g, 2.42 mmol, 92%) as a colorless foam. TLC
[petroleum ether/ethyl acetate (3:1)] Rf 0.48; [R]D +22 (c 1.7,
1
acetate (3:2)] Rf 0.55; [R]D +26 (c 2.5, CDCl3); H NMR (250
t
MHz, CDCl3) δ 1.04 (s, 9H, Bu), 1.66 (s, 3H, OAc), 2.43 (br s,
1H, OH), 3.19-3.25 (m, 1H), 3.38-4.22 (m, 35H), 4.23-4.92
(m, 24H), 4.93-5.16 (m, 5H), 5.22 (br s, 1H), 5.37 (br s, 1H),
5.59-5.82 (m, 2H, allyl), 6.19 (d, J NH ) 7.4 Hz, 1H, NH), 7.05-
7.40 (m, 66H, Ph), 7.65-7.80 (m, 4H, TBDPS). Anal. Calcd
for C138H153O26NSi (2270.01): C 73.01, H 6.81, N 0.62. Found:
C 72.96, H 6.95, N 0.53.
Allyl (2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl)-
(1f2)-(3,4-d i-O-b en zyl-6-O-ter t-b u t yld ip h en ylsilyl-r-D-
m an n opyr an osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m an n opy-
r a n osyl)-(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-
â-D-ga la ct op yr a n osyl)-(1f4)]-2-O-a llyl-3-O-b en zyl-R-D-
m a n n op yr a n osid e (29). Compound 28 (760 mg, 335 µmol)
was dissolved in 1.8 mL of dry DMF under argon and benzyl
bromide (44 µL, 368 µmol) and sodium hydride (9 mg) were
added. After the mixture was stirred for 40 min some methanol
was added and the solvent removed. Flash chromatography
(petroleum ether/ethyl acetate 5:2) afforded compound 29 (583
mg, 247 µmol, 74%) besides some N-O-dibenzylated compound
(82 mg, 94 µmol, 10%), both as colorless foams. 29: TLC
[petroleum ether/ethyl acetate (3:2)] Rf 0.65; [R]D +21 (c 1.9,
1
t
CDCl3); H NMR (250 MHz, CDCl3) δ 1.04 (s, 9H, Bu), 2.11
(s, 3H, OAc), 3.28-5.16 (m, 65H, 1g-H, 1d-H, 1e-H, 1c-H),
5.36 (br s, 1H, 1f-H), 5.57 (br s, 1H, 2g-H), 5.63-5.83 (m,
2H, allyl), 6.91 (d, J NH ) 6.7 Hz, 1H, NH), 7.03-7.53 (m, 66H,
Ph), 7.67-7.77 (m, 4H, TBDPS); HMQC data (13C (150.9 MHz)/
1H (600 MHz)) 97.3/4.71 (1c), 74.8/3.68 (2c), 78.0/3.85 (3c), 74.0/
4.01 (4c), 70.1/3.68 (5c), 66.3/3.77 + 3.57 (6c), 99.2/4.97 (1d),
55.9/3.93 (2d), 77.4/3.98 (3d), 72.0/3.75 (4d), 73.3/3.50 (5d), 68.2/
3.51 + 3.30 (6d), 98.3/4.83 (1e), 72.7/4.09 (2e), 80.4/3.87 (3e),
74.8/3.71 (4e), 72.1/3.78 (5e), 69.5/3.62 (6e), 99.9/5.35 (1f), 75.2/
4.08 (2f), 79.5/3.94 (3f), 74.1/4.14 (4f), 73.1/3.73 (5f), 62.9/4.03
+ 3.87 (6f), 99.8/5.07 (1g), 68.7/5.57 (2g), 78.6/3.99 (3g), 74.0/
3.90 (4g), 71.8/3.86 (5g), 68.5/3.58 + 3.47 (6g). Anal. Calcd for
1
t
CDCl3); H NMR (250 MHz, CDCl3) δ 1.00 (s, 9H, Bu), 1.66
(s, 3H, OAc), 3.18-3.24 (m, 1H), 3.38-4.18 (m, 36H), 4.20-
4.93 (m, 26H), 4.93-5.15 (m, 4H), 5.25 (br s, 1H), 5.37 (br s,
1H), 5.58-5.80 (m, 2H, allyl), 6.18 (d, J NH ) 7.3 Hz, 1H, NH),
7.00-7.38 (m, 71H, Ph), 7.65-7.78 (m, 4H, TBDPS). Anal.
Calcd for C145H159O26NSi (2360.14): C 73.79, H 6.80, N 0.59.
Found: C 73.67, H 6.72, N 0.46. N-O-Dibenzyl compound: TLC
[petroleum ether/ethyl acetate (3:2)] Rf 0.75; [R]D +24 (c 1.1,
C
140H152O27Cl3NSi (2415.37): C 69.61, H 6.36, N 0.58. Found:
C 69.16, H 6.36, N 0.47.
Allyl (2-O-Acetyl-3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n o-
syl)-(1f2)-(3,4-d i-O-ben zyl-6-O-ter t-bu tyld ip h en ylsilyl-r-
D-m a n n op yr a n osyl)-(1f2)-(3,4,6-t r i-O-b en zyl-r-D-m a n -
n op yr a n osyl)-(1f6)-[(2-a cet a m id o-3,4,6-t r i-O-b en zyl-2-
1
t
CDCl3); H NMR (250 MHz, CDCl3) δ 0.98 (s, 9H, Bu), 1.60
(s, 3H, OAc), 3.20-4.18 (m, 33H), 4.20-4.87 (m, 30H), 4.98-
1304 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
5.18 (m, 5H), 5.25-5.32 (m, 2H), 5.39 (br s, 1H), 5.60-5.85
(m, 2H, allyl), 7.00-7.32 (m, 76H, Ph), 7.62-7.78 (m, 4H,
TBDPS). Anal. Calcd for C152H165O26NSi (2450.27): C 74.50,
H 6.80, N 0.57. Found: C 74.54, H 6.76, N 0.45.
3.04 (m, 1H), 3.32-4.91 (m, 59H), 5.12-5.23 (m, 2H), 5.28 (br
s, 1H), 6.47 (d, J NH ) 7.6 Hz, 1H, NH), 6.72-6.90 (m, 2H, PA),
7.02-7.40 (m, 74H, Ph), 7.65-7.78 (m, 4H, TBDPS). Anal.
Calcd for C147H157O28NSi (2414.14): C 72.59, H 6.60, N 0.58
(+H2O). Found: C 72.16, H 6.46, N 0.46. 5: TLC [petroleum
(2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-
(3,4-d i-O-ben zyl-6-O-ter t-bu tyld ip h en ylsilyl-r-D-m a n n o-
pyr an osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m an n opyr an osyl)-
(1f6)-[(2-a cet a m id o-3,4,6-t r i-O-b en zyl-2-d eoxy-â-D-ga -
la ctop yr a n osyl)-(1f4)]-3-O-ben zyl-r/â-D-m a n n op yr a n ose
(30r/â). Compound 29 (590 mg, 250 µmol) and Rh(PPh3)3Cl
(65 mg, 70 µmol) were dissolved in 19 mL of solvent (toluene/
EtOH/H2O 20/10/1) and refluxed for 14 h. After cooling the
solution was filtered through a pad of Celite and evaporated,
and the remaining residue was dissolved in 29 mL of THF/
H2O 4/1. I2 (130 mg, 1 mmol) was added and after 30 min the
solution was diluted with ethyl acetate and washed with
Na2S2O3 solution (20% in H2O). The aqueous fraction was
reextracted three times with ethyl acetate, the combined
organic fractions were dried over MgSO4, and the solvent was
removed. Flash chromatography (petroleum ether/ethyl acetate
2:1 f 1:1) of the remaining residue afforded compound 30r/â
(498 mg, 218 µmol, 87%) as a colorless foam. TLC [petroleum
ether/ethyl acetate (2:1)] Rf 0.1; [R]D 16 (c 1.5, CDCl3); 1H NMR
1
ether/ethyl acetate (4:3)] Rf 0.85; [R]D +15 (c 2.3, CDCl3); H
t
NMR (250 MHz, CDCl3) δ 1.00 (s, 9H, Bu), 1.62 (s, 3H, OAc),
3.20-3.25 (m, 1H), 3.48-4.95 (m, 58H), 5.09 (d, J ) 8.2 Hz,
1H), 5.40 (br s, 1H, 1-H), 5.48 (br s, 1H, 2-H), 6.10 (d, J NH
)
8.0 Hz, 1H, NH), 6.14 (d, J 1,2 ) 1.8 Hz, 1H, 1c-H), 6.69-6.90
(m, 2H, PA), 7.01-7.40 (m, 74H, Ph), 7.66-7.77 (m, 4H,
TBDPS), 8.62 (s, 1H, dNH). Anal. Calcd for C149H157O28Cl3N2-
Si (2558.51): C 69.94, H 6.20, N 1.10. Found: C 69.55, H 6.14,
N 1.02.
(2-Azid o-3,6-d i-O-ben zyl-2-d eoxy-4-p h en oxya cetyl-r-D-
glu copyr an osyl)-(1f6)-2,3,4,5-tetr a-O-ben zyl-1-O-(4-m eth -
oxyben zyl)-m yo-in ositol (34). Compound 3313 (500 mg, 486
µmol) was dissolved in 6 mL of pyridine and cooled to 0 °C.
Phenoxyacetyl chloride (500 µL, 3.6 mmol) was added, and the
solution was allowed to warm to room temperature and stirred
overnight. After coevaporation with toluene, the residue was
dissolved in CH2Cl2, some silica gel was added, and the solvent
was removed. Flash chromatography (petroleum ether/ethyl
acetate 9:2) afforded compound 34 (470 mg, 403 µmol, 83%)
as a colorless foam. TLC [petroleum ether/ethyl acetate (3:1)]
Rf 0.55; [R]D +42 (c 1.8, CDCl3); 1H NMR (250 MHz, CDCl3) δ
2.74 (dd, J ) 2.8 Hz, J ) 11.0 Hz, 1H), 3.21 (dd, J ) 2.6 Hz,
J ) 11.1 Hz, 1H), 3.32 (dd, J ) 3.7 Hz, J ) 10.4 Hz, 1H), 3.39
(dd, J ) 2.2 Hz, J ) 9.8 Hz, 1H), 3.43 (dd, J ) 9.2 Hz, 1H),
3.48 (dd, J ) 2.0 Hz, J ) 9.6 Hz, 1H), 3.81 (s, 3H, OMe), 3.82-
4.35 (m, 8H), 4.35-5.10 (m, 13H), 5.25 (dd, J ) 9.4 Hz, 1H),
5.79 (d, J 1,2 ) 3.6 Hz, 1H, 1b-H), 6.72-6.76 (m, 2H, PMB),
6.84-6.88 (m, 2H, PMB), 6.93-6.99 (m, 1H, PA), 7.13-7.42
(m, 34H, Ph). Anal. Calcd for C70H71O13N3 (1162.44): C 72.32,
H 6.17, N 3.62. Found: C 72.26, H 6.12, N 3.15.
t
(250 MHz, CDCl3) δ 1.06 (s, 9H, Bu), 1.73 (s, 3H, OAc), 2.35
(br s, 1H, OH), 3.18-3.24 (m, 1H), 3.35-4.93 (m, 57H), 5.07
(d, J ) 8.5, 1H), 5.33 (br s, 1H), 5.37 (br s, 1H), 6.31 (d, J NH
)
7.2 Hz, 1H, NH), 7.05-7.40 (m, 71H, Ph), 7.63-7.67 (m, 4H,
TBDPS). Anal. Calcd for C139H151O26NSi (2280.00): C 72.61,
H 6.72, N 0.61 (+1/2H2O). Found: C 72.34, H 6.88, N 0.45.
P h en oxya cetyl (2,3,4,6-Tetr a -O-ben zyl-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4-d i-O-b en zyl-6-O-ter t-b u t yld ip h en yl-
silyl-r-D-m a n n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-
m a n n opyr a n osyl)-(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-
2-d eoxy-â-D-ga la ct op yr a n osyl)-(1f4)]-3-O-b en zyl-2-O-
p h en oxya cetyl-r-D-m a n n op yr a n ose (31r/â). Compound 30
(510 mg, 224 µmol) was dissolved in 5 mL of pyridine,
phenoxyacetyl chloride (180 mL, 1.3 mmol) was added at 0
°C, and the solution was warmed to room temperature and
stirred for 14 h. After coevaporation with toluene for 3 times,
the remaining residue was put on silica gel by dissolving in
CH2Cl2, adding some silica gel, and evaporation. Flash chro-
matography (petroleum ether/ethyl acetate 2:1 f 1:1) afforded
compound 31r/â (460 mg, 180 µmol, 81%) as a colorless foam.
TLC [petroleum ether/ethyl acetate (3:2)] Rf 0.60 R, 0.33 â; 1H
(2-Azid o-3,6-d i-O-ben zyl-2-d eoxy-4-p h en oxya cetyl-r-D-
glu cop yr a n osyl)-(1f6)-2,3,4,5-tetr a -O-ben zyl-m yo-in osi-
tol (35). Compound 34 (510 mg, 439 µmol) was dissolved in
39 mL of CH3CN/toluene/H2O 91/5/4 and cooled to 0 °C. After
addition of ceric(IV)ammonium nitrate (1.22 g, 2.2 mmol) and
stirring at 0 °C for 30 min the solution was allowed to warm
to room temperature and stirred for another 90 min. The
mixture was diluted with ethyl acetate and washed three times
with saturated NaHCO3 solution, and the combined organic
fractions were dried over MgSO4. After removal of the solvent
flash chromatography (petroleum ether/ethyl acetate 3:1)
afforded compound 35 (439 mg, 421 µmol, 96%) as a colorless
foam. TLC [petroleum ether/ethyl acetate (3:1)] Rf 0.32; [R]D
t
NMR (250 MHz, CDCl3) 31r, δ 1.00 (s, 9H, Bu), 1.65 (s, 3H,
OAc), 3.30-4.95 (m, 61H), 5.20 (br s, 1H), 5.31 (dd, J ) 2.2
Hz, J ) 5.6 Hz, 1H), 5.38 (br s, 1H), 6.07 (d, J 1,2 ) 1.9 Hz, 1H,
1-H), 6.64 (d, J NH ) 7.7 Hz, 1H, NH), 6.60-7.40 (m, 81H, Ph),
7.68-7.80 (m, 4H, TBDPS). Anal. Calcd for C155H163O30NSi
(2548.28): C 73.05, H 6.46, N 0.55. Found: C 72.76, H 6.35,
N 0.45.
1
+34 (c 15, CDCl3); H NMR (250 MHz, CDCl3) δ 2.86 (d, J )
3.1 Hz, J ) 10.9 Hz, 1H), 2.93 (d, J OH ) 7.6 Hz, 1H, OH), 3.12
(dd, J ) 3.0 Hz, J ) 10.9 Hz, 1H), 3.36-3.55 (m, 3H), 3.60-
3.69 (m, 1H), 3.89-4.17 (m, 8H), 4.33 (d, J H,H ) 11.1 Hz, 1H,
CH2Ph), 4.53-4.82 (m, 7H), 4.90-5.08 (m, 3H), 5.26 (dd, J )
9.5 Hz, 1H), 5.57 (d, J 1,2 ) 3.6 Hz, 1H, 1b-H), 6.73-6.77 (m,
2H, PA), 6.93-6.99 (m, 1H, PA), 7.16-7.40 (m, 32H, Ph). Anal.
Calcd for C62H63O12N3 (1042.28): C 71.44, H 6.10, N 4.03.
Found: C 71.34, H 6.08, N 3.55.
(2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-
(3,4-di-O-ben zyl-6-O-ter t-bu tyldiph en ylsilyl-r-D-m an n opy-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a c e t a m id o -3,4,6-t r i-O -b e n zy l-2-d e o x y -â-D -
ga la ctop yr a n osyl)-(1f4)]-3-O-ben zyl-2-O-p h en oxya cetyl-
r-D-m a n n op yr a n ose (32) a n d O-((2,3,4,6-Tetr a -O-ben zyl-
r-D-m a n n op yr a n osyl)-(1f2)-(3,4-d i-O-b e n zyl-6-O-ter t-
bu tyld ip h en ylsilyl-r-D-m a n n op yr a n osyl)-(1f2)-(3,4,6-tr i-
O-ben zyl-r-D-m an n opyr an osyl)-(1f6)-[(2-acetam ido-3,4,6-
t r i-O-b en zyl-2-d eoxy-â-D-ga la ct op yr a n osyl)-(1f4)]-3-O-
ben zyl-2-O-ph en oxyacetyl-r-D-m an n opyr an osyl) Tr ich lo-
r oa cetim id a te (5). Compound 31 (1.6 g, 628 µmol) was
dissolved in 60 mL of DMF and stirred with (NH4)2CO3 (1.6 g,
20.5 mmol) at 50 °C for 2 h. After removal of the solvent (f32)
the residue was dissolved in 20 mL of CH2Cl2 and trichloro-
acetonitrile (500 µL, 5 mmol) and 2 drops of DBU were added.
After 45 min the solvent was removed and flash chromatog-
raphy (petroleum ether/ethyl acetate 2:1) afforded compound
5r/â (1.22 g, 477 µmol, 76% over 2 steps) as a colorless foam.
32: TLC [petroleum ether/ethyl acetate (4:3)] Rf 0.5; 1H NMR
(250 MHz, CDCl3) δ 1.01 (s, 9H, tBu), 1.77 (s, 3H, OAc), 2.95-
(2-Azid o-3,6-d i-O-ben zyl-2-d eoxy-4-p h en oxya cetyl-r-D-
glu copyr an osyl)-(1f6)-1-O-ben zoyl-2,3,4,5-tetr a-O-ben zyl-
m yo-in ositol (36). Compound 35 (360 mg, 315 µmol) and
benzoyl cyanide (53 mg, 360 mol) were dissolved in 3.2 mL of
dry CH2Cl2 under argon and 1.3 mL of dry triethylamine was
added. After stirring for 2 h the solution was diluted with ethyl
acetate, washed with saturated NaHCO3 solution, and dried
over MgSO4, and then the solvent was removed. Flash chro-
matography (petroleum ether/ethyl acetate 7:2) afforded com-
pound 36 (318 mg, 277 µmol, 88%) as a colorless foam. TLC
[petroleum ether/ethyl acetate (2:1)] Rf 0.70; [R]D +17 (c 2.4,
1
CDCl3); H NMR (250 MHz, CDCl3) δ 2.59 (dd, J ) 2.2 Hz, J
) 11.0 Hz, 1H), 3.07 (dd, J ) 2.4 Hz, J ) 11.0 Hz, 1H), 3.23
(dd, J ) 3.7 Hz, J ) 10.3 Hz, 1H), 3.55 (t, J ) 9.4 Hz, 1H),
3.66 (dd, J ) 2.1 Hz, J ) 9.9 Hz, 1H), 3.78-3.86 (m, 1H), 3.78-
J . Org. Chem, Vol. 68, No. 4, 2003 1305

Pekari and Schmidt
4.05 (m, 3H), 4.12-4.25 (m, 3H), 4.34-4.52 (m, 3H), 4.62-
4.75 (m, 5H), 4.76-5.02 (m, 3H), 5.11-5.29 (m, 3H), 5.41 (d,
J 1,2 ) 3.7 Hz, 1H, 1b-H), 6.70-6.79 (m, 2H, PA), 6.93-7.00
(m, 1H, PA), 7.12-7.39 (m, 32H, Ph), 7.41-7.51 (m, 2H, Bz),
7.52-7.65 (m, 1H, Bz), 8.01-8.05 (m, 2H, Bz). Anal. Calcd for
(m, 4H, TBDPS), 8.00-8.05 (m, 2H, Bz); HMQC data (13C
(150.9 MHz)/1H (600 MHz)) 75.1/5.05 (1a), 74.1/4.21 (2a), 80.8/
3.61 (3a), 81.9/4.16 (4a), 79.7/3.49 (5a), 75.1/4.45 (6a), 98.6/
5.33 (1b), 54.5/3.92 (2b), 81.5/3.48 (3b), 73.3/3.83 (4b), 70.6/
3.99 (5b), 68.6/3.30 (6b), 98.1/5.29 (1c), 70.2/5.48 (2c), 75.4/3.75
(3c), 72.9/4.01 (4c), 70.8/3.58 (5c), 66.4/3.80 + 3.16 (6c), 100.8/
4.65 (1d), 52.4/4.26 (2d), 81.5/3.48 (3d), 71.9/3.49 (4d), 73.5/
3.52 (5d), 68.9/3.48 + 3.30 (6d), 99.3/4.55 (1e), 72.7/4.07 (2e),
80.1/3.80 (3e), 75.4/3.42 (4e), 72.5/3.70 (5e), 70.9/3.63 + 3.49
(6e), 100.2/5.28 (1f), 74.4/4.10 (2f), -/3.88 (3f), 74.3/4.13 (4f),
73.1/3.65 (5f), (6f), 99.6/5.22 (1g), 74.7/3.86 (2g), -/3.88 (3g),
74.7/4.00 (4g), 72.1/3.81 (5g), 68.9/3.56 + 3.48 (6g). Anal. Calcd
C
69H67O13N3 (1146.39): C 72.29, H 5.90, N 3.67. Found: C
72.22, H 5.88, N 3.42.
(2-Azid o-3,6-d i-O-ben zyl-2-d eoxy-r-D-glu cop yr a n osyl)-
(1f6)-1-O-ben zoyl-2,3,4,5-tetr a-O-ben zyl-myo-in ositol (37).
Compound 36 (234 mg, 204 µmol) was stirred at 0 °C for 15
min in 5 mL of MeNH2 solution (33% in dry ethanol) and the
solvent was rapidly removed at room temperature. Flash
chromatography (petroleum ether/ethyl acetate 4:1) afforded
compound 37 (197 mg, 194 µmol, 96%) as ca olorless foam.
TLC [petroleum ether/ethyl acetate (5:2)] Rf 0.62; [R]D +5.8 (c
for
C213H226O40N2Si (3482.50): C 73.46, H 6.55, N 0.80.
Found: C 73.23, H 6.50, N 0.65. MALDI-Tof-MS [positive
mode, matrix: 4-nitroaniline with NaI in MeOH] m/z 3504 [M
+ Na]+.
1
1.2, CDCl3); H NMR (250 MHz, CDCl3) δ 2.00 (d, J OH ) 3.2
Hz, 1H, OH), 3.05-3.20 (m, 3H), 3.52-3.72 (m, 4H), 3.80-
3.91 (m, 1H), 4.18-4.26 (m, 3H), 4.36-4.49 (m, 2H), 4.65-
4.87 (m, 8H), 4.96-5.20 (m, 3H), 5.36 (d, J 1,2 ) 3.8 Hz, 1H,
1b-H), 7.14-7.36 (m, 30H, Ph), 7.43-7.49 (m, 2H, Bz), 7.56-
7.63 (m, 1H, Bz), 8.02-8.05 (m, 2H, Bz). Anal. Calcd for
(2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-
(3,4-d i-O-ben zyl-r-D-m a n n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-
ben zyl-r-D-m a n n op yr a n osyl)-(1f6)-[(2-a ceta m id o-3,4,6-
tr i-O-ben zyl-2-d eoxy-â-D-ga la ctop yr a n osyl)-(1f4)]-(3-O-
ben zyl-2-O-p h en oxya cetyl-r-D-m a n n op yr a n osyl)-(1f4)-
(3,6-d i-O-b e n zyl-2-N -(t er t -b u t yloxyca r b on yl)a m in o-2-
d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-tetr a -O-ben zyl-
1-O-ben zoyl-m yo-in ositol (40). Compound 39 (190 mg, 55
µmol) was dissolved in 2 mL of THF, 440 µL (440 µmol) of
TBAF solution (1 N in THF), and 25 µL (433 µmol, 8 equiv) of
acetic acid. After stirring at 45 °C for 2 to 6 days, removal of
the solvent at room temperature, and flash chromatography
(petroleum ether/ethyl acetate 3:1 f 2:1) compound 40 (137
mg, 43 µmol, 75%) was obtained, which could be lyophilized
from dioxane. TLC [petroleum ether/ethyl acetate (2:1)] Rf 0.52;
[R]D +15 (c 0.9, CDCl3); 1H NMR (250 MHz, CDCl3) δ 1.09 (s,
C
61H61O11N3 (1012.25): C 72.37, H 6.09, N 4.15. Found: C
72.31, H 5.97, N 3.71.
(2-Azid o-3,6-d i-O-ben zyl-2-N-(ter t-bu tyloxyca r bon yl)-
a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-1-O-ben zoyl-
2,3,4,5-tetr a -O-ben zyl-m yo-in ositol (6). Compound 37 (25
mg, 242 µmol) was dissolved in 5 mL of pyridine/water 4/1 and
propanedithiol (500 µL, 5 mmol) and 10 drops of triethylamine
were added. After stirring at 45 °C for 2 days in a tightly closed
flask (free amine: TLC [petroleum ether/ethyl acetate (5:2)]
Rf 0.05) the solution was coevaporated three times with dry
toluene and the residue dissolved in 3 mL of dry toluene. Boc2O
(370 mg, 1.7 mmol) was added, the solution was stirred at room
temperature for 4 h, and the solvent was removed. Flash
chromatography (petroleum ether/ethyl acetate 3:1) afforded
compound 6 (215 mg, 199 µmol, 82%) as a colorless foam. TLC
[petroleum ether/ethyl acetate (5:2)] Rf 0.38; [R]D +12 (c 1.6,
CDCl3); 1H NMR (250 MHz, CDCl3) δ 1.16 + 1.13 (s, 9H, tBu),
2.07 (d, J OH ) 3.0 Hz, 1H, OH), 3.18-3.25 (m, 1H, 6b-H),
3.30-3.52 (m, 3H, 3b,5a,6′b-H), 3.58-3.75 (m, 2H, 3a,4b-H),
3.80-4.00 (m, 2H, 2b,5b-H), 4.14-4.32 (m, 3H, 2a,4a-H),
4.41-4.72 (m, 9H), 4.75-4.85 (m, 2H), 4.95-5.08 (m, 3H, 1a-
H), 5.33 (d, J 1,2 ) 3.6 Hz, 1H, 1b-H), 7.03-7.35 (m, 30H, Ph),
7.38-7.48 (m, 2H, Bz), 7.50-7.61 (m, 1H, Bz), 7.95-8.05 (m,
2H, Bz); HMQC data (13C (150.9 MHz)/1H (600 MHz)) 75.5/
5.03 (1a), 74.3/4.20 (2a), 80.8/3.61 (3a), 81.9/4.17 (4a), 81.5/
3.47 (5a), 74.4/4.48 (6a), 98.7/5.33 (1b), 53.4/3.86 (2b), 80.6/
3.38 (3b), 70.9/3.70 (4b), 70.5/3.96 (5b), 69.0/3.33 + 3.23 (6b).
Anal. Calcd for C66H71O13N (1086.38): C 72.96, H 6.60, N 1.29.
Found: C 72.67, H 6.52, N 1.06.
t
9H, Bu), 1.66 (s, 3H, NAc), 3.20-5.15 (m, 83H), 5.28-5.34
(m, 2H), 5.46 (br s, 1H), 6.12 (d, J NH ) 8.3 Hz, 1H, NH), 6.53-
6.62 (m, 2H, PA), 6.75-6.82 (m, 1H, PA), 6.87-7.51 (m, 103H,
Ph), 8.01-8.04 (m, 2H, Bz). Anal. Calcd for C197H208O40N2
(3244.07): C 72.93, H 6.48, N 0.86. Found: C 72.71, H 6.38,
N 0.60. MALDI-TOF-MS [positive mode, matrix: 4-nitroa-
niline with NaI in MeOH] m/z 3266 [M + Na]+.
Tr iet h yla m m on iu m (2,3,4,6-Tet r a -O-b en zyl-r-D-m a n -
n opyr an osyl)-(1f2)-(3,4-di-O-ben zyl-6-O-(2-(N-ben zyloxy-
ca r bon yl)a m in oeth yl-p h osp h on a to)-r-D-m a n n op yr a n o-
syl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m an n opyr an osyl)-(1f6)-
[(2-a cet a m id o-3,4,6-t r i-O-b en zyl-2-d eoxy-â-D-ga la ct op y-
r a n osyl)-(1f4)]-(3-O-ben zyl-r-D-m a n n op yr a n osyl)-(1f4)-
(3,6-d i-O-b e n zyl-2-N -(t er t -b u t yloxyca r b on yl)a m in o-2-
d e oxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-t e t r a -O-b e n -
zyl-1-O-ben zoyl-m yo-in ositol (41). Compound 40 (125 mg,
39 µmol), compound 2 (92 mg, 230 µmol), and tetrazole (16
mg, 230 µmol) were dried in a vacuum for 2 h. After addition
of 2.5 mL of dry CH2Cl2, the solution was stirred at room
temperature under argon for 3 h and MCPBA (70%) (56 mg,
230 µmol) was added. The solution was stirred for another 2
h and then 2 mLof MeNH2 solution (33% in dry ethanol) was
added. After stirring for an additional hour, removal of the
solvent, and flash chromatography (4 g silica gel, toluene/ethyl
acetate 95:5 f 9:1 f 8:2 f 7:3 f 6:4 f 1:1 f 1:2) compound
41 (96 mg, 29 µmol, 74%) was obtained as a colorless foam.
(2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl)-(1f2)-
(3,4-di-O-ben zyl-6-O-ter t-bu tyldiph en ylsilyl-r-D-m an n opy-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a c e t a m id o -3,4,6-t r i-O -b e n zy l-2-d e o x y -â-D -
galactopyr an osyl)-(1f4)]-(3-O-ben zyl-2-O-ph en oxyacetyl-
r-D-m a n n op yr a n osyl)-(1f4)-(3,6-d i-O-b en zyl-2-N-(ter t-
b u t yloxyca r b on yl)a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-
(1f6)-2,3,4,5-tetr a-O-ben zyl-1-O-ben zoyl-myo-in ositol (39).
Compound 6 (90 mg, 83 µmol) and trichloroacetimidate 5 (230
mg, 90 µmol) were dissolved under argon in 1.3 mL of dry CH2-
Cl2 and cooled to 0 °C. After addition of a 0.1 N TMSOTf
solution (85 µL, 8.5 µmol) and stirring at 0 °C for 1 h the
solution was neutralized with triethylamine. Removal of the
solvent and flash chromatography (petroleum ether/ethyl
acetate 3:1) with subsequent MPLC afforded compound 39 (213
mg, 61 µmol, 74%), which could be lyophilized from dioxane.
TLC [HPTLC, petroleum ether/ethyl acetate (2:1)] Rf 0.63; [R]D
1
TLC [toluene/ethyl acetate (1:1)] Rf 0.4; H NMR (250 MHz,
t
CDCl3/d4-MeOH 2:1) δ 1.11 (s, 9H, Bu), 1.83 (s, 3H, NAc),
2.61-2.73 (m, 2H), 3.18-5.37 (m, 88H), 7.05-7.61 (m, 103H,
Ph), 8.01-8.04 (m, 2H, Bz); HMQC data (13C (150.9 MHz)/1H
(600 MHz)) 74.8/5.08 (1a), 73.7/4.23 (2a), 80.4/3.63 (3a), 81.4/
4.13 (4a), 79.7/3.50 (5a), -/4.47 (6a), 98.0/5.32 (1b), 54.2/3.79
(2b), 80.3/3.48 (3b), 73.8/4.02 (4b), 70.5/4.04 (5b), 68.4/3.48 (6b),
101.4/5.10 (1c), 68.7/3.86 (2c), 77.3/3.67 (3c), (4c), 71.3/3.64 (5c),
101.5/4.65 (1d), 52.1/4.31 (2d), 81.2/3.51 (3d), 71.8/3.62 (4d),
98.7/4.89 (1e), 73.1/4.11 (2e), 80.1/3.90 (3e), 74.8/3.65 (4e), 71.8/
3.80 (5e), 100.0/5.16 (1f), (73.5/4.182f), 79.0/3.87 (3f), 74.2/4.01
(4f), 98.8/5.19 (1g), 74.5/3.84 (2g), 79.2/3.86 (3g), 74.2/4.01 (4g);
31P NMR (242.94 MHz, CDCl3) δ -1.43. Anal. Calcd for
C199H214O43N3P (3367.13): C 69.70, H 6.30, N 1.22. Found: C
1
+10 (c 1.0, CDCl3); H NMR (250 MHz, CDCl3) δ 0.98 (s, 9H,
tBu), 1.11 (s, 9H, Boc), 1.58 (s, 3H, NAc), 3.10-5.08 (m, 81H),
5.20-5.35 (m, 4H, 1b-H, 1c-H, 1f-H, 1g-H), 5.50 (m, 1H,
2c-H), 6.12 (d, J NH ) 9.2 Hz, 1H, NH), 6.51-6.58 (m, 2H, PA),
6.76-6.85 (m, 1H, PA), 6.90-7.55 (m, 109H, Ph), 7.62-7.73
1306 J . Org. Chem., Vol. 68, No. 4, 2003

Branched Glycosyl Phosphatidyl Inositol Anchors
69.92, H 6.60, N 1.28. MALDI-Tof-MS [positive mode, matrix:
4-nitroaniline with NaI in MeOH] m/z 3390 [M + Na]+, 3411
[M - H + 2Na]+, 3562 [M - H + 2Na + NaI]+.
f 50:4.5) afforded compound 44 (62 mg, 16 µmol, 90%.), which
could be lyophilized from dioxane. TLC [CH2Cl2/MeOH (9/1)]
Rf 0.52; 1H NMR (250 MHz, magic) δ 1.27 (s, 9H, tBu), 1.41 (s,
t
9H, Bu), 1.61 (s, 3H, NAc), 2.61-2.73 (m, 4H), 3.08-5.38 (m,
Tr iet h yla m m on iu m (2,3,4,6-Tet r a -O-b en zyl-r-D-m a n -
n op yr a n osyl)-(1f2)-(3,4-d i-O-b en zyl-6-O-(2-(N-b en zyl-
oxyca r b on yl)a m in oet h yl-p h osp h on a t o)-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-ga la c-
top yr a n osyl)-(1f4)]-(3-O-ben zyl-2-O-(2-(N-ter t-bu tyloxy-
ca r bon yl)a m in oeth yl-p h osp h a te)-r-D-m a n n op yr a n osyl)-
(1f4)-(3,6-di-O-ben zyl-2-N-(ter t-bu tyloxycar bon yl)am in o-
2-deoxy-r-D-glu copyr an osyl)-(1f6)-2,3,4,5-tetr a-O-ben zyl-
1-O-ben zoyl-m yo-in ositol (42). Compound 41 (60 mg, 18
µmol), phosphoramidite 3 (38 mg, 106 µmol), and tetrazole (7.5
mg, 105 µmol) were dried in a vacuum for 2 h. After addition
of 1 mL of dry CH2Cl2 the solution was stirred at room
temperature under argon for 2.5 h (TLC: toluene/ethyl acetate
95H), 6.95-7.41 (m, 110H, Ph). C213H237O50N4P3 (3746.45):
MALDI-TOF-MS [positive mode, matrix: 4-nitroaniline with
NaI] m/z 3785 [M + K]+, 3806 [M - H+ + Na + K]+.
r-D-Man n opyr an osyl-(1f2)-(6-O-(am in oeth ylph osph on -
a t o)-r-D-m a n n op yr a n osyl)-(1f2)-r-D-m a n n op yr a n osyl-
(1f6)-[(2-a ce t a m id o-2-d e oxy-â-D -ga la ct op yr a n osyl)-
(1f4)]-(2-O-(2-(N-ter t-bu tyloxycar bon yl)am in oeth ylph os-
ph on ato)-r-D-m an n opyr an osyl)-(1f4)-(2-N-(ter t-bu tyloxy-
ca r bon yl)a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-m yo-
in osit-1-yl P h osp h a te (45). Compound 44 (17 mg, 4.5 µmol)
was dissolved in 2 mL of CH2Cl2/MeOH 1:4, 10 mg of
Pd(OH)2/C (20%) was added, and the solution was stirred
under H2 at room temperature for 8 h. If necessary the catalyst
was removed, the solvent evaporated, and the residue dis-
solved in H2O and hydrogenated again. During hydrogenation,
the pH value was controlled to be within the range of 4-6.
The solution was filtered through a 45 µm syringe filter,
neutralized with NH4HCO3, and purified by P4 gel chroma-
tography (l ) 40 cm, d ) 1.6 cm, 0.03 M NH4HCO3, 1 mL/
min). Compound 45 (7 mg, 4 µmol, 95%) was obtained, which
could be lyophilized from water. 1H NMR (250 MHz, D2O) δ
1.28 (s, 18H, tBu), 1.92 (s, 3H, NAc), 3.05-3.30 (m, 4H), 3.32-
3.98 (m, 45H), 4.39-4.48 (m, 2H, 2c-H, 1d-H), 4.88 (br s,
1H, 1g-H), 4.93 (br s, 1H, 1e-H), 5.13 (br s, 1H, 1f-H), 5.31
(br s, 2H, 1b-H, 1c-H); HMQC data (13C (150.9 MHz)/1H (600
MHz)) 75.0/3.97 (1a), 71.4/4.28 (2a), 70.5/3.50 (3a), 72.2/3.59
(4a), 73.5/3.35 (5a), 76.4/3.76 (6a), 96.6/5.38 (1b), 55.3/3.61 (2b),
72.0/3.87 (3b), 77.2/3.59 (4b), 71.2/4.03 (5b), 99.3/5.39 (1c), 74.2/
4.40 (2c), 68.0/3.93 (3c), 76.4/3.76 (4c), 71.2/3.79 (5c), 66.8/3.83
+ 3.65 (6c), 101.5/4.43 (1d), 52.7/3.84 (2d), 70.6/3.70 (3d), 67.8/
3.86 (4d), 75.3/3.63 (5d), 98.7/5.00 (1e), 79.0/3.91 (2e), 70.1/
3.91 (3e), 67.0/3.61 (4e), 73.3/3.62 (5e), 100.8/5.20 (1f), 78.2/
4.04 (2f), 70.1/3.91 (3f), 66.4/3.74 (4f), 72.2/3.79 (5f), 64.7/4.04
(6f), 102.1/4.97 (1g), 70.0/3.99 (2g), 70.4/3.77 (3g), 66.8/3.55 (4g),
73.2/3.68 (5g); 31P NMR (242.94 MHz, CDCl3) δ -0.21, -0.56,
-1.50. C58H105O48N4P3 (1719.58): MALDI-TOF-MS [positive
mode, matrix: R-cyano-hydroxyzimt-sa¨ure with NaI] m/z 1718
[M]+, 1741 [M + Na]+, 1761 [M + 2Na - H]+.
r-D-Man n opyr an osyl-(1f2)-(6-O-(am in oeth yl-ph osph o-
n a to)-r-D-m a n n op yr a n osyl)-(1f2)-r-D-m a n n op yr a n osyl-
(1f6)-[(2-acetam ido-2-deoxy-â-D-galactopyr an osyl)-(1f4)]-
2-O-(a m in oe t h yl-p h osp h on a t o)-r-D-m a n n op yr a n osyl-
(1f4)-(2-a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-m yo-
in osit-1-yl P h osp h a te (1c). Compound 45 (7 mg, 4.1 µmol)
was stirred in 900 µL of TFA and 300 µL of H2O at room
temperature for 5 h. After dilution with water and lyophiliza-
tion, the residue was purified by P4 gel chromatography (l )
40 cm, d ) 1.6 cm, 0.03 M NH4HCO3, 1 mL/min). Compound
1c (5.5 mg, 3.2 mol, 85%) was obtained, which could be
lyophilized from water. 1H NMR (250 MHz, D2O) δ 1.92 (s,
3H, NAc), 3.05-3.14 (m, 4H), 3.15-4.03 (m, 45H), 4.30-4.37
(m, 2H, 2d-H, 2c-H), 4.89 (br s, 1H, 1g-H), 4.93 (br s, 1H,
1e-H), 5.11 (br s, 1H, 1f-H), 5.25 (br s, 1H, 1c-H), 5.43 (br
s, 1H, 1b-H); HMQC data (13C (150.9 MHz)/1H (600 MHz))
75.3/4.07 (1a), 71.1/4.12 (2a), 70.0/3.49 (3a), 71.8/3.62 (4a), 72.2/
3.34 (5a), 76.9/3.83 (6a), 94.7/5.54 (1b), 53.3/3.30 (2b), 69.7/
4.02 (3b), 75.8/3.68 (4b), 70.2/4.11 (5b), 59.7/3.78 + 3.74 (6b),
98.9/5.35 (1c), 73.6/4.43 (2c), 67.6/3.96 (3c), 76.9/3.79 (4c), 70.8/
3.79 (5c), 66.3/3.84 + 3.67 (6c), 101.2/4.43 (1d), 52.1/3.85 (2d),
70.0/3.70 (3d), 67.2/3.86 (4d), 74.9/3.66 (5d), 66.3/3.84-3.67
(6d), 98.3/5.03 (1e), 78.6/3.92 (2e), 89.6/3.88 (3e), 66.5/3.61 (4e),
72.9/3.63 (5e), 66.3/3.84-3.67 (6e), 100.3/5.19 (1f), 77.7/4.05
(2f), 89.4/3.90 (3f), 65.9/3.71 (4f), 71.7/3.80 (5f), 64.2/4.04 (6f),
101.6/4.98 (1g), 69.6/3.99 (2g), 69.9/3.76 (3g), 66.3/3.56 (4g),
72.8/3.69 (5g), 66.3/3.84-3.67 (6g); 31P NMR (242.94 MHz,
CDCl3) δ -2.36, -2.61, -3.15 (calibrated with H3PO4). Anal.
Calcd for C48H89O44N4P3 (1519.32): EI-MS (matrix: 2% HOAc/
t
(1:1) 0.4 f 0.55). Then BuOOH solution (3 N in dry toluene,
35 µL, 105 µmol) was added, the solution was stirred again
for 2 h, and 1 mL of MeNH2 solution (33% in dry ethanol) was
added. After stirring for an additional hour, removal of the
solvent, and flash chromatography (3 g silica gel, toluene/ethyl
acetate/MeOH 4:1:0 f 2:1:0 f 1:1:0 f 1:2:0 f 1:3:0 f 1:4:0
f 1:3:1) compound 42 (49 mg, 14 µmol, qu.) was obtained as
1
a colorless foam. TLC [CH2Cl2/MeOH (9/1)] Rf 0.53; H NMR
t
(250 MHz, magic, NEt3 salt) δ 1.11 (s, 9H, Bu), 1.38 (s, 9H,
tBu), 1.91 (s, 3H, NAc), 2.61-2.73 (m, 4H), 3.08-5.38 (m, 90H),
7.05-7.61 (m, 103H, Ph), 8.01-8.04 (m, 2H, Bz); 31P NMR
(242.94MHz,CDCl3)δ-3.07,-3.49(not calibrated).C206H228O48N4P2
(3590.32): MALDI-Tof-MS [positive mode, matrix: 4-nitroa-
niline with NaI in MeOH] m/z 3613 [M + Na]+, 3635 [M +
2Na]+, 3657 [M + 3Na]+, 3807 [M + 2Na + NaI]+.
Bistr ieth ylam m on iu m (2,3,4,6-Tetr a-O-ben zyl-r-D-m an -
n op yr a n osyl)-(1f2)-(3,4-d i-O-b en zyl-6-O-(2-(N-b en zyl-
oxyca r b on yl)a m in oet h yl-p h osp h on a t o)-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-ga la c-
top yr a n osyl)-(1f4)]-(3-O-ben zyl-2-O-(2-(N-ter t-bu tyloxy-
ca r bon yl)a m in oeth yl-p h osp h on a to)-r-D-m a n n op yr a n o-
syl)-(1f4)-(3,6-d i-O-ben zyl-2-N-(ter t-bu tyloxyca r bon yl)-
a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-t et r a -
O-ben zyl-m yo-in ositol (43). Compound 42 (55 mg, 15 µmol)
was dissolved in 500 µL of a freshly prepared NaOMe solution
(0.1 N in dry MeOH, 50 µmol) and 200 µL of dry CH2Cl2 under
argon and stirred at 40 °C for 24 h. Flash chromatography
(CH2Cl2/MeOH 94:6 f 9:1) afforded compound 43 (44 mg, 12
µmol, 78%), which could be lyophilized from dioxane. TLC
[CH2Cl2/MeOH (9:1)] Rf 0.53; 1H NMR (250 MHz, magic) δ 1.23
t
t
(s, 9H, Bu), 1.31 (s, 9H, Bu), 1.61 (s, 3H, NAc), 2.81-2.91
(m, 4H), 3.00-5.48 (m, 90H), 7.03-7.41 (m, 100H, Ph); 31P
NMR (242.94 MHz, CDCl3) δ -0.58, -0.97 (calibrated).
C
199H224O47N4P2 (3486.21): MALDI-Tof-MS [positive mode,
matrix: 4-nitroaniline with NaI] m/z 3509 [M + Na]+, 3525
[M + K]+, 3547 [M - H + Na + K]+.
Tr ist r iet h yla m m on iu m (2,3,4,6-Tet r a -O-b en zyl-r-D-
m a n n op yr a n osyl)-(1f2)-(3,4-d i-O-ben zyl-6-O-(2-(N-ben -
zyloxyca r bon yl)a m in oeth yl-ph osph on a to)-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-ga la c-
top yr a n osyl)-(1f4)]-(3-O-ben zyl-2-O-(2-(N-ter t-bu tyloxy-
ca r bon yl)a m in oeth yl-p h osp h on a to)-r-D-m a n n op yr a n o-
syl)-(1f4)-(3,6-d i-O-ben zyl-2-N-(ter t-bu tyloxyca r bon yl)-
a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-t et r a -
O-ben zyl-m yo-in osit-1-yl-(d i-O-ben zyl) P h osp h a te (44).
Compound 43 (65 mg, 19 µmol), dibenzyl-N,N-diisopropyl-
phosphoramidite (35 µL, 131 µmol), and tetrazole (16 mg, 228
µmol) were dried in a vacuum for 1 h. After addition of 750
µL of dry CH2Cl2 the solution was stirred at room temperature
t
under argon for 3 h, BuOOH solution (5.5 N in nonane, 20
µL, 110 µmol) was added, and the solution was again stirred
for 35 min. Removal of the solvent and subsequent flash
chromatography (3 g silica gel, CH2Cl2/MeOH 50:1.5 f 50:3
J . Org. Chem, Vol. 68, No. 4, 2003 1307

Pekari and Schmidt
MeOH 9:1) 1521 [M + H]+, MALDI-TOF-MS (positive mode,
NBOH) 1518 [M]+, 1540 [M + Na]+, 1562 [M + 2Na - H]+.
Tr ist r iet h yla m m on iu m (2,3,4,6-Tet r a -O-b en zyl-r-D-
m a n n op yr a n osyl)-(1f2)-(3,4-d i-O-ben zyl-6-O-(2-(N-ben -
zyloxyca r bon yl)a m in oeth yl-ph osph on a to)-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-ga la c-
top yr a n osyl)-(1f4)]-(3-O-ben zyl-2-O-(2-(N-ter t-bu tyloxy-
ca r bon yl)a m in oeth yl-p h osp h on a to)-r-D-m a n n op yr a n o-
syl)-(1f4)-(3,6-d i-O-ben zyl-2-N-(ter t-bu tyloxyca r bon yl)-
a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-t et r a -
O-ben zyl-1-O-(2-O-octadecan oyl-1-O-octadecyl-sn -glycer yl-
p h osp h on a to)-m yo-in ositol (46). Compound 44 (25 mg, 7.2
µmol), phosphoramidite 4 (30 mg, 37 µmol), and tetrazole (3
mg, 35 µmol) were dried in a vacuum. After addition of 350
µL of dry CH2Cl2 the solution was stirred at room temperature
(1f6)-[(2-acetam ido-2-deoxy-â-D-galactopyr an osyl)-(1f4)]-
(2-O-(2-a m in oe t h yl-p h osp h on a t o)-r-D-m a n n op yr a n o-
syl)-(1f4)-(2-am in o-2-deoxy-r-D-glu copyr an osyl)-(1f6)-1-
O-(2-O-oct a d e ca n oyl-1-O-oct a d e cyl-sn -glyce r yl-p h os-
p h on a to)-m yo-in ositol (1a ). Compound 47 (16 mg, 4 µmol)
was dissolved in 1.3 mL of BuOH, 0.5 mL of MeOH, and 200
µL of H2O, 10 mg of Pd(OH)2/C was added, and the suspension
was stirred under H2 for 8 h. The suspension was filtered
through a 45 µm syringe filter, neutralized with NH4HCO3,
and purified by P4 gel chromatography (l ) 40 cm, d ) 1.6
n
cm, 10% PrOH in 0.03 M NH4HCO3, 1 mL/min). Compound
1a (5 mg, 2.4 µmol, 60%) was obtained, which could be
lyophilized from dioxane/water. 1H NMR (250 MHz, D2O) δ
0.88-0.97 (m, 6H), 1.18-1.80 (m, 66H), 2.10 (s, 3H, NAc),
2.70-4.26 (m, 83H), 4.50-4.52 (m, 2H, GalNAc-g, Man-c(2)),
5.04 (br s, 1H, Man-f), 5.10 (br s, 1H, Man-d), 5.24 (br s, 1H,
Man-e), 5.39 (br s, 1H, Man-c), 5.45 (br s, 1H, GlcN-b).
t
under argon for 2.5 h. Then BuOOH solution (5.5 N in nonane,
7 µL, 110 µmol) was added and the solution was again stirred
for 1 h. A 2.5 mL Me2NH solution (33% in dry ethanol) and 2
mL of CH2Cl2 were added. After 1 h the solvent was removed
and the residue subjected to flash chromatography (9 g silica
gel, CH2Cl2/MeOH 100:1 f 50:1 f 50:1.5 f 50:2 f 50:2.5 f
50:3 f 50:3.5). Compound 46 (25 mg, 6.1 µmol, 83%.) was
obtained, which could be lyophilized from dioxane/water. TLC
[CH2Cl2/MeOH (9/1)] Rf 0.50; 1H NMR (250 MHz, magic) δ 0.87
C87H165O47N4P3 (2112.47): MALDI-TOF-MS [positive mode,
matrix: 4-nitroaniline with NaI] m/z 2136 [M + Na]+, 2175
[M + Na + K]+.
Tr iet h yla m m on iu m (2,3,4,6-Tet r a -O-b en zyl-r-D-m a n -
n op yr a n osyl)-(1f2)-(3,4-d i-O-b en zyl-6-O-(2-(N-b en zyl-
oxyca r b on yl)a m in oet h yl-p h osp h on a t o)-r-D-m a n n op y-
ranosyl)-(1f2)-(3,4,6-tri-O-benzyl-r-D-mannopyranosyl)-(1f6)-
[(2-a cet a m id o-3,4,6-t r i-O-b en zyl-2-d eoxy-â-D-ga la ct op y-
r a n osyl)-(1f4)]-(3-O-ben zyl-(2-O-a m in oeth yl-p h osp h on -
a t o)-r-D -m a n n op yr a n osyl)-(1f4)-(3,6-d i-O-b e n zyl-2-
a m in o-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-t et r a -
O-ben zyl-1-O-(2-O-octadecan oyl-1-O-octadecyl-sn -glycer yl-
p h osp h on a to)-m yo-in ositol (1b). Compound 45 (49 mg, 11
µmol) was dissolved in 1.5 mL of BuOH, 1 mL of MeOH, and
0.5 mL of THF and 10 mg Pd(OH)2/C was added. The
suspension was stirred under H2 for 8 h, then filtered through
t
(t, 6H, CH3), 1.43-1.10 (m, 69H), 1.41 (s, 9H, Bu), 1.47-1.63
(m, 4H), 1.67 (s, 3H, NAc), 1.64-1.73 (m, 2H), 2.21-2.41 (m,
4H),2.78-5.48(m,97H),6.95-7.41(m,100H,Ph).C238H301O53N4P3
(4159.34): MALDI-TOF-MS [positive mode, matrix: 4-nitroa-
niline with NaI] m/z 4237 [M - H+ + 2K]+.
Bistr ieth ylam m on iu m (2,3,4,6-Tetr a-O-ben zyl-r-D-m an -
n op yr a n osyl)-(1f2)-(3,4-d i-O-b en zyl-6-O-(2-(N-b en zyl-
oxyca r b on yl)a m in oet h yl-p h osp h on a t o)-r-D-m a n n op y-
r a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-D-m a n n op yr a n osyl)-
(1f6)-[(2-a ceta m id o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-ga la c-
top yr a n osyl)-(1f4)]-(3-O-ben zyl-2-O-(2-a m in oeth yl-p h os-
p h on a to)-r-D-m a n n op yr a n osyl)-(1f4)-(2-a m in o-3,6-d i-O-
ben zyl-2-d eoxy-r-D-glu cop yr a n osyl)-(1f6)-2,3,4,5-tetr a -
O-ben zyl-1-O-(2-O-octadecan oyl-1-O-octadecyl-sn -glycer yl-
p h osp h on a to)-m yo-in ositol (47). Compound 46 (24 mg, 5.8
µmol) was stirred in 1.5 mL of CH2Cl2/TFA/TES 9:1:0.5 for
1.5 h and evaporated at room temperature. Flash chromatog-
raphy (2 g silica gel, CH2Cl2/MeOH 50:1 f 50:3 f 50:6 f 40:
20 f 20:20 f 20:40) afforded compound 47 (21 mg, 5.2 µmol,
90%), which could be lyophilized from dioxane/water. TLC
[CH2Cl2/MeOH (9:1)] Rf 0.46; 1H NMR (250 MHz, magic) δ
0.78-1.73 (m, 75H), 2.17-2.28 (m, 4H), 2.89-5.58 (m, 97H),
6.95-7.44 (m, 100H, Ph). C228H285O49N4P3 (3959.08): MALDI-
TOF-MS [positive mode, matrix: 4-nitroaniline with NaI] m/z
4018 [M - H + Na + K]+, 4034 [M - H + 2K]+.
a
45 µm syringe filter, neutralized with NH4HCO3, and
purified by P4 gel chromatography (l ) 40 cm, d ) 1.6 cm,
10% nPrOH in 0.03 m NH4HCO3, 1 mL/min). Compound 1b
(18.8 mg, 8.2 µmol, 75%) was obtained, which could be
lyophilized from dioxane/water. C97H181O49N4P3 (2280.73):
MALDI-TOF-MS [positive mode, matrix: 4-nitroaniline with
NaI] m/z 2304 [M + Na]+, 2320 [M + K]+.
Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft and Fonds der Che-
mischen Industrie. K. Pekari is grateful for a Fonds
Stipendium.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
all described compounds as well as selected HMQC and mass
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
r-D-Man n opyr an osyl-(1f2)-(6-O-(am in oeth ylph osph on -
a t o)-r-D-m a n n op yr a n osyl)-(1f2)-r-D-m a n n op yr a n osyl-
J O026380J
1308 J . Org. Chem., Vol. 68, No. 4, 2003