Glycosyl phosphatidylinositol (GPI) anchor synthesis based on versatile building blocks - Total synthesis of a GPI anchor of yeast

Article abstract of DOI:10.1002/(SICI)1099-0690(199905)1999:5<1153::AID-EJOC1153>3.0.CO;2-S

For the design of a synthesis of target molecule 1 the retrosynthetic analysis yielded building blocks 2-5, of which ceramide 2-phosphite derivative 2 and aminoethyl phosphite derivative 5 are known. The generation of α-glucosaminyl (1→6)inositol building

Full text of DOI:10.1002/(SICI)1099-0690(199905)1999:5<1153::AID-EJOC1153>3.0.CO;2-S

FULL PAPER
Glycosyl Phosphatidylinositol (GPI) Anchor Synthesis Based on Versatile
Building Blocks – Total Synthesis of a GPI Anchor of Yeast[1]
Thomas G. Mayer[a] and Richard R. Schmidt*[a]
Keywords: Carbohydrates / Phospholipids / Glycolipids / Sphingosines / Ceramides / Ceramides-1-phosphates /
Glycosylation
For the design of a synthesis of target molecule 1 the
retrosynthetic analysis yielded building blocks 2–5, of which
removal of the anomeric O-acetyl group and treatment with
CCl3CN/DBU afforded 4. Glycosylation of 3 with donor 4 led
ceramide 2-phosphite derivative 2 and aminoethyl phosphite to pseudohexasaccharide 31 in high yield. Replacement of
derivative 5 are known. The generation of α-glucosaminyl
(1Ǟ6)inositol building block was based on
the O-acyl groups by O-benzyl groups and then exchange of
the menthyloxycarbonyl group by an O-acetyl group gave 36
which enabled regioselective attachment of 2 and 5. To this
end, the 6e-O-silyl group was removed and then the
aminoethyl phosphate residue was attached with reagent 5
to give 38 in high yield. 1a-O-Deacetylation and then
reaction with 2 afforded 40 as fully protected 1 which was
liberated in two steps; treatment with acid removed all acid
3
pseudodisaccharide 6 which was selectively benzoylated at
6b-O and then selectively benzylated at 3b-O to give 3. The
synthesis of tetramannosyl building block 4 started from
known ortho ester derivative 8 which was transformed into
versatile mannosyl donors 13 and 18 and into acceptor 22.
Reaction of 13 with 22 gave α-disaccharide 23, deacetylation
and then mannosylation with 18 gave trisaccharide 25; labile protective groups and finally catalytic hydrogenation
ensuing deacetylation and mannosylation with 13 gave
tetrasaccharide 27; deallylation, acetylation, regioselective
afforded the desired GPI anchor 1 which could be fully
structurally assigned.
Glycosylphosphatidylinositols (GPIs) are a class of nat- acetylcholinesterase[8] and human folate-binding protein,[9]
urally occurring glycophospholipids that anchor the C ter- and sn-1,2-diacylglycerol in torpedo acetylcholinesterase.[10]
mini of proteins and glycoproteins to the membrane of eu- In addition, the inositol residue can be palmitoylated. In
karyotic cells.[2,3] The first structure of a member of this GPI anchors of yeast (Saccharomyces cerevisiae), ceramide-
family, the Typanosoma brucei variant surface glycoprotein containing phytosphingosine was found as membrane
(VSG) was reported by Ferguson et al. in 1988.[4,5] Since anchor instead of glycerolipids.[11,12] The typical structure
then various GPI anchors have been characterized, thus of a yeast GPI anchor is shown in Scheme 2 (1).
leading to a general structure as shown in Scheme 1 (frame)
The role of GPI-anchored proteins as enzymes, in inter-
which was conserved during evolution of the various spec- actions with bioactive factors, and in cell-cell recognition
ies. The occurrence of additional carbohydrate (R2, R3, has been extensively discussed.[2,3,13] Moreover, there is evi-
and/or R4) or ethanolamine phosphate (R1) side chains is dence that metabolites derived from GPI anchors or struc-
species-specific.[2,3] Their is also a variation in the mem- turally related compounds are mediators of regulatory pro-
brane-anchoring lipid structures which depend on species cesses.[13] For instance, GPI-derived fragments are thought
and on cell type. For instance, in the case of glycerolipids to participate in signal transduction which is triggered by
(variation of the lipid anchor) the lipids are made up of sn- insulin.[14] Therefore, the GPI anchors themselves and par-
1,2-dimyristoylglycerol in T. brucei VSGs,[6] sn-1-stearyl-2- tial structures thereof are gaining in significance. Thus, the
lysoglycerol in T. brucei procyclic acidic repetitive protein,[7] challenging chemical synthesis of structurally homogeneous
sn-1-alkyl-2-acylglycerol in bovine and human erythrocyte GPI anchors and their derivatives is an important objective
Scheme 1
in order to perform biological studies. We report here de-
tails on the previously communicated first total synthesis
of a GPI anchor,[1,15] namely the ceramide-containing GPI
anchor 1 of yeast (Scheme 2). The required combination of
[a]
Fakultät für Chemie, Universität Konstanz, Fach M 725,
D-78457 Konstanz, Germany
Eur. J. Org. Chem. 1999, 1153Ϫ1165
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0505Ϫ1153 $ 17.50ϩ.50/0
1153

T. G. Mayer, R. R. Schmidt
FULL PAPER
Scheme 2. R ϭ Bn; Z ϭ BnOCO
lipid, phosphate, and oligosaccharide chemistry has been O-benzylation with benzyl bromide in the presence of silver
meanwhile successfully employed to the synthesis of partial oxide gave the desired building block 3 in good overall
1
structures[16Ϫ26] and to the total synthesis of diacylglycerol- yield. The structure of 3 was confirmed by the H-NMR
based GPI anchors of Trypanosoma brucei and rat brain data.
Thy-1.[15,26Ϫ29]
The strategy, which we developed in order to synthesize
1, is convergent and highly versatile. The target molecule 1
was disconnected at positions AϪC , thus affording build-
ing blocks 2؊5, which due to proper protection are widely
useful in GPI-anchor synthesis.[28] Slight protective-group
modifications, especially at O-3 and/or O-4 of mannose resi-
due c, will yield a great variety of structurally different GPI
anchors. Our aim is to demonstrate the efficiency of this
strategy with respect to the synthesis of the individual
building blocks and their coupling in order to provide tar-
get molecule 1.
Synthesis of Building Blocks 2؊5
The ceramide-containing building block 2, which con-
tains phytosphingosine as nitrogen base, was prepared start-
ing from the readily available azido derivative of phytos-
phingosine;[30] this was recently already reported in de-
tail.[21]
The synthesis of the α-glucosaminyl(1Ǟ6)inositol build-
ing block 3 was based on readily available derivative 6[21]
Scheme 3
(Scheme 3) which on benzoylation with benzoyl cyanide in
the presence of triethylamine afforded at Ϫ60°C regioselec-
The synthesis of tetramannosyl building block 4 started
tively 6b-O-benzoyl derivative 7. Ensuing regioselective 3b- from known ortho ester 8 (Scheme 4a) which is readily
1154
Eur. J. Org. Chem. 1999, 1153Ϫ1165

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
available from -mannose;[31] also O-deacetylation (Ǟ 9), tetrabutylammonium fluoride (TBAF) in acetic acid/THF
benzylation (Ǟ 10), subsequent acid-catalyzed ortho ester gave 6-O-unprotected mannoside 22 which served as man-
opening and then O-acetylation (Ǟ 11α,β) followed essen- nosyl residue c. Its structure was confirmed with the help
1
tially published procedures.[32,33] Selective 1-O-deacety- of the H-NMR data of derivative 22a.
lation with ammonium carbonate in DMF afforded 12α,β
in high yield. Ensuing treatment with trichloroacetonitrile
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) afforded almost exclusively α-trichloroacetimidate
13 (α/β ഠ 17:1) in 93% yield; 13 provides mannosyl residues
d and f in the construction of tetramannosyl building block
4. The 2-O-acetyl group in 13 promotes the desired α-selec-
tive O-glycosylations.
Scheme 4b
Scheme 4a. R ϭ Bn
The c and e mannosyl residues were prepared starting
from intermediate 9. Regioselective 6-O-silylation with tert-
butyldiphenylsilyl chloride (TBDPS-Cl) in the presence of
imidazole afforded compound 14 (Scheme 4b); the structure
1
of 14 was confirmed with the help of the H-NMR data of
the trichloroacetyl isocyanate addition product 14a which
exhibited the expected downfield shifts for the 3-H and 4-
H signals (δ ϭ 5.29, 5.60). Treatment of 14 with benzyl
bromide in the presence of sodium hydride gave 3,4-di-O-
benzyl derivative 15. Acid-catalyzed ortho ester cleavage
and then treatment with acetic anhydride and pyridine af-
forded 1,2-di-O-acetyl derivative 16α,β. Selective 1-O-de-
acetylation with hydrazinium acetate in DMF afforded
Scheme 4c. R ϭ Bn
Reaction of mannosyl donor 13 with acceptor 22 in the
17α,β which on treatment with trichloroacetonitrile in the presence of TMSOTf as the catalyst and ether as solvent
presence of DBU furnished in very high overall yield tri- gave at room temperature α(1Ǟ6)-linked disaccharide 23 in
chloroacetimidate 18 (α,β ഠ 12:1), which served as e man- high yield (Scheme 4d). Cleavage of the 2d-O-acetyl group
nosyl residue. The c mannosyl residue, required for tetrasac- under Zemplen conditions[34] afforded glycosyl acceptor 24;
´
charide 4, was prepared from 18 in a straightforward reac- its reaction with mannosyl donor 18 under standard con-
tion sequence. Reaction of 18 with allyl alcohol in the pres- ditions afforded trisaccharide 25 in 92% yield. Cleavage of
´
ence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) the 2e-O-acetyl group under Zemplen conditions led to 26
as catalyst afforded allyl α-mannoside 19 in practically as acceptor which on treatment with 13 as mannosyl donor
quantitative yield (Scheme 4c). 2-O-Deacetylation under furnished again under standard conditions α(1Ǟ2)-α(1Ǟ2)-
Zemplen conditions[34] gave 20; ensuing treatment with allyl α(1Ǟ6)-linked tetramannosyl derivative 27 in 91% yield.
´
bromide in the presence of sodium hydride gave 1,2-di-O- Thus, a very high overall yield in the α-selective linkage of
allyl-mannoside 21. Removal of the 6-O-TBDPS group with the four mannosyl residues cϪf could be verified in only
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1155

T. G. Mayer, R. R. Schmidt
FULL PAPER
five steps. The 1c- and 2c-O-allyl groups were then removed
by treatment with the Wilkinson catalyst and ensuing hy-
drolysis (Ǟ 28α,β). Treatment with acetic anhydride in pyri-
dine afforded O-acetyl derivative 29α,β (α/β ϭ 6:1). Regiose-
lective removal of the 1-O-acetyl group with ammonium
carbonate in DMF furnished 30α,β which gave on treat-
ment with trichloroacetonitrile in the presence of DBU the
desired tetramannosyl donor 4 in very high overall yield;
the structure of 4 was assigned on the basis of the 1H-NMR
data (J1c,2c ϭ 1.4, J1d,2d ഠ J1f,2f < 1 Hz).
Construction of Target Molecule 1
In order to arrive at the final goal, glycosyl donor 4 was
first treated with acceptor 3; under standard glycosylation
conditions, i.e. with TMSOTf as catalyst in ether as solvent
at room temperature, the desired (pseudo) hexasaccharide
31 was obtained in almost quantitative yield (Scheme 5a).
It was structurally assigned with the help of DQF-COSY,
HMQC and HMBC experiments (see Experimental Sec-
tion); formation of the α linkage was confirmed by the C,H-
coupling constants (INEPT): 169.51 > JC,H > 177.30 Hz.
The three different types of O-acyl groups in 31 (O-acetyl,
O-benzoyl, and O-menthyloxycarbonyl) can be readily dis-
tinguished: Methanolysis in the presence of potassium cy-
anide as catalyst led to removal of the two O-acetyl groups
yielding 32 accompanied by some 33, where also the ben-
zoyl group was removed; extension of the reaction time gave
essentially only desired 33. Treatment of 33 with benzyl bro-
mide in the presence of sodium hydride led to introduction
of three additional O-benzyl groups furnishing 34 in 86%
yield. Methanolysis of the menthyloxycarbonyl group was
accomplished with potassium carbonate in methanol, af-
fording 1a-O-unprotected compound 35, which on treat-
ment with acetic anhydride in pyridine led to 1a-O-acetyl
derivative 36. This measure was undertaken to ease removal
of the 1a-O protective group after introduction of the 6e-
O-aminoethyl phosphate moiety. To this end, 36 was treated
with TBAF in the presence of acetic acid in THF as the
solvent leading to the 6e-O-unprotected compound 37. Re-
action with phosphite derivative 5 in the presence of tetra-
Scheme 4d. R ϭ Bn
The N-benzyloxycarbonyl (Z)-protected aminoethyl
phosphite compound 5 was prepared according to pub-
lished procedures[15]. Thus, the synthesis of all building
blocks required for the construction of target molecule 1
could be very successfully performed.
Scheme 5a. R ϭ Bn
1156
Eur. J. Org. Chem. 1999, 1153Ϫ1165

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
Conclusion
The ready accessibility of all starting materials, the high
regio- and diastereoselectivities, and the high yields ob-
tained in all reaction steps, including the glycosylation reac-
tions which were carried out with trichloroacetimidates,[36]
provide an excellent basis for further successful syntheses in
this field. The versatility of the building blocks described in
this paper has been meanwhile demonstrated in their suc-
cessful utilization to the synthesis of T. brucei P3 and Thy-
1 GPI anchors.[28]
Experimental Section
General: Solvents were purified in the usual way; boiling range of
petroleum ether: 35Ϫ60°C. Ϫ Melting points are uncorrected. Ϫ
Optical rotations: PerkinϪElmer polarimeter 241 MC; 1-dm cell,
temperature 20°C. Ϫ Thin-layer chromatography (TLC): Plastic
sheets, silica gel 60 F254 (Merck; layer thickness 0.2 mm). Ϫ Col-
umn chromatography: Kieselgel 60 (Merck; 0.063Ϫ0.200 mm). Ϫ
Flash chromatography: Silica gel (J. T. Baker, particle size 40 mm).
1
Ϫ H NMR: Bruker AC 250 (250 MHz) Cryospec, Bruker DRX
600 (600 MHz), internal standard tetramethylsilane (TMS). Ϫ 31P
NMR: Jeol JNM-GX 400; external standard 85% phosphoric acid.
Ϫ FAB MS: Finnigan MAT 312/AMD 5000; matrix NBOH (ϭ 3-
nitrobenzyl alcohol). Ϫ Elemental analyses: Heraeus CHN-O-Ra-
pid.
O-(2-Azido-6-O-benzoyl-2-deoxy-␣-
2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyloxycarbonyl-
D
-glucopyranosyl)-(1Ǟ6)-
-myo-
D
inositol (7): A solution of benzoyl cyanide (0.79 g, 5.78 mmol) in
dry acetonitrile (10 mL) was added dropwise within 1 h to a solu-
tion of crude 6[21] (4.1 g, 5.78 mmol) in dry acetonitrile/diethyl
ether/triethylamine (10:7:3, 200 mL) at Ϫ60°C under nitrogen. The
mixture was stirred for 2 h at Ϫ60°C. Within 2 h, the mixture was
warmed up to room temp. and concentrated in vacuo. The residue
was purified by flash chromatography with toluene/ethyl acetate
(5:1) to yield 7 (3.2 g,, 68%) as a colorless foam which was lyophil-
ized from dioxane.Ϫ TLC (petroleum ether/ethyl acetate, 6:4): Rf ϭ
0.45; (toluene/ethyl acetate, 3:1): Rf ϭ 0.26. Ϫ [α]D ϭ ϩ14 (c ϭ 1,
1
chloroform). Ϫ H NMR (250 MHz; CDCl3): δ ϭ 0.72Ϫ0.75 (d,
3J ϭ 6.9 Hz, 3 H, CH3), 0.80Ϫ1.10 (2 d, m, 9 H, 2 CH3, HMnt),
1.40Ϫ1.76 (m, 24 H, 20 Hcycloh., 4 HMnt), 1.86Ϫ2.12 (m, 2 H, 2
HMnt), 2.84 (d, 3J3b,OH ϭ 2.6 Hz, 1 H, OH), 3.20 (dd, 3J1b,2b ϭ 3.6,
Scheme 5b. R ϭ Bn
3
3
3J2b,3b ϭ 10.3 Hz, 1 H, 2b-H), 3.38 (ddd, J4b,OH ϭ 4.0, J3b,4b
ϭ
3
3
3J4b,5b ϭ 9.5 Hz, 1 H, 4b-H), 3.53 (dd, J4a,5a ϭ 10.8, J5a,6a ϭ 8.5
zole as activating agent and then oxidation with tert-butyl
hydroperoxide gave Z-protected aminoethyl phosphate de-
rivative 38 in 81% yield (Scheme 5b). Now, the 1a-O-acetyl
3
Hz, 1 H, 5a-H), 3.60(d, J4b,OH ϭ 3.9 Hz, 1 H, OH), 3.93Ϫ4.11
(m, 4 H, 4a-, 6a-, 3b-, 5b-H), 4.33Ϫ4.41 (m, 2 H, 6b-, 3a-H),
4.45Ϫ4.58 (m, 2 H, HMnt, 2a-H), 4.89Ϫ4.95 (m, 2 H, 6bЈ-, 1a-H),
´
group was removed under Zemplen conditions (Ǟ 39), thus
3
5.29 (d, J1b,2b ϭ 3.6 Hz, 1 H, 1b-H), 7.41Ϫ7.61 (m, 3 H, m, p-
permitting attachment of the ceramide 1-phosphate moiety.
To this end, reaction of 39 with phosphite amide derivative
2 in the presence of tetrazole followed by oxidation with
tert-butyl hydroperoxide was carried out, leading to the cor-
responding phosphotriester, which gave with dimethylamine
in ethanol diester 40 in 60% overall yield. Simultaneous
cleavage of the O-isopropylidene and O-cyclohexylidene
groups with glycol in the presence of camphersulfonic acid
(CSA) as the catalyst (Ǟ 41) followed by hydrogenolysis of
the O-benzyl groups and the azido group with PearlmanЈs
catalyst[35] afforded target molecule 1 which was charac-
COPh), 8.02Ϫ8.06 (m, 2 H, OCOPh). Ϫ C42H59N3O13 (813.94):
calcd. C 61.98, H 7.31, N 5.16; found C 61.49, H 7.44, N 5.72.
O-(2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-␣-
syl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyl-
oxycarbonyl- -myo-inositol (3): To a solution of 7 (10 g, 12.3 mmol)
D-glucopyrano-
D
in dry dichloromethane (35 mL) freshly prepared Ag2O (10 g),
˚
powdered molecular sieves (3 A, 7 g), and benzyl bromide (1.68
mL, 14.1 mmol) were added. After stirring for 18 h at room temp.,
the mixture was filtered through Celite, concentrated in vacuo, and
chromatographed on silica gel (toluene/ethyl acetate, 10:1). MPLC
(toluene/ethyl acetate, 15:1) of the residue in order to remove the
terized with the help of NMR and MS data (Experimental). 4-O-benzyl isomer gave pure 3 (8.1 g, 73%) as a colorless foam. Ϫ
Eur. J. Org. Chem. 1999, 1153Ϫ1165 1157

T. G. Mayer, R. R. Schmidt
FULL PAPER
TLC (petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.51; (toluene/ethyl
the residue by flash chromatography (petroleum ether/ethyl acetate,
acetate, 10:1): Rf ϭ 0.38. Ϫ [α]D ϭ ϩ35 (c ϭ 0.5, chloroform). Ϫ 7:3) gave oily 12 (1.15 g, 84%) as an anomeric mixture (α/β ϭ
1H NMR (400 MHz; CDCl3): δ ϭ 0.68Ϫ0.70 (d, 3J ϭ 7.3 Hz, 3 6.5:1). Ϫ TLC (petroleum ether/ethyl acetate, 7:3): Rf ϭ 0.25 (12α);
H, CH3), 0.78Ϫ1.02 (2 d, m, 9 H, 2 CH3, HMnt), 1.28Ϫ1.68 (m, 24
H, 20 Hcycloh., 4 HMnt), 1.85Ϫ1.89 (m, 1 H, HMnt), 1.98Ϫ2.03 (m,
Rf ϭ 0.17 (12β). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 2.14 (s,
COCH3), 2.19 (s, COCH3), 3.42Ϫ3.78 (m), 3.99Ϫ4.09 (m),
1 H, HMnt), 3.04 (d, 3J4b,OH ϭ 4.4 Hz, 1 H, OH), 3.27 (dd, 3J1b,2b ϭ 4.45Ϫ4.86 (m, CH2Ph, 1-H), 5.19 (br. s, 1-H), 5.35 (dd, 3J1,2 ϭ 1.9,
3
3
3
3.7, J2b,3b ϭ 10.3 Hz, 1 H, 2b-H), 3.49Ϫ3.56 (m, 2 H, 5a-, 4b-H),
3J2,3 ϭ 3.2 Hz, 2-H), 5.44 (dd, J1,2 ϭ 1, J2,3 ϭ 2 Hz, 2-H). Ϫ
3.82 (dd, 3J2b,3b ϭ 3J3b,4b ϭ 9.5 Hz, 1 H, 3b-H), 3.94 (dd, 3J3a,4a
ϭ
C29H32O7 (492.57): calcd. C 70.71, H 6.55; found C 70.03, H 6.27.
3
7.3, J4a,5a ϭ 10.3 Hz, 1 H, 4a-H), 4.32Ϫ4.36 (m, 2 H, 3a-, 6b-H),
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣/β-D-mannopyranosyl) Trichloro-
4.45 (dd, 3J ϭ 4.4, 3J ϭ 3J ϭ 11.0 Hz, 1 H, HMnt), 4.51 (dd,
acetimidate (13): A cooled solution of 12 (1.4 g, 2.84 mmol) in dry
dichloromethane (35 mL) and trichloroacetonitrile (1.4 mL) was
treated with 140 µL of DBU and stirred for 1 h at 0°C under nitro-
gen. The reaction mixture was diluted with dichloromethane and
extracted subsequently with satd. aqueous NH4Cl solution and
water. The organic solution was dried (Na2SO4) and concentrated
to dryness. Flash chromatography (petroleum ether/ethyl acetate,
8:2 with 1% triethylamine) gave 13 (1.69 g, 93%) as a colorless foam
in a α/β ratio of 17:1. Ϫ TLC (petroleum ether/ethyl acetate, 8:2
3
3
3J1a,2a ϭ 4.4, J2a,3a ϭ 6.6 Hz, 1 H, 2a-H), 4.79 (dd, J5b,6b ϭ 2.9,
3Jgem ϭ 12.5 Hz, 1 H, 6bЈ-H), 4.84 (s, 2 H, CH2Ph), 4.91 (dd,
3J6a,1a ϭ J1a,2a ϭ 4.9 Hz, 1 H, 1a-H), 5.24 (d, J1b,2b ϭ 3.7 Hz, 1
H, 1b-H), 7.17Ϫ7.39 (m, 7 H, Ph, m-COPh), 7.49Ϫ7.53 (m, 1 H,
p-COPh), 7.96Ϫ7.98 (m, 2 H, o-COPh). Ϫ C49H65N3O13 (904.06):
calcd. C 65.09, H 7.25, N 4.65; found C 64.77, H 7.26, N 4.73.
3
3
3,4,6-Tri-O-acetyl-1,2-O-[1-(R)-methoxyethylidene]-β-D-mannopy-
ranose (8): Compound 8 was prepared as described in ref.[31]
Ϫ
1
TLC (petroleum ether/ethyl acetate, 1:1): Rf ϭ 0.59. Ϫ 1H NMR
(250 MHz; CDCl3): δ ϭ 1.72 (s, 3 H, CMe), 2.03 (s, 3 H, COCH3),
2.05 (s, 3 H, COCH3), 2.10 (s, 3 H, COCH3), 3.25 (s, 3 H, OMe),
with 1% Et3N): Rf ϭ 0.26. Ϫ 13α: H NMR (250 MHz; CDCl3):
δ ϭ 2.18 (s, 3 H, COCH3), 3.68Ϫ4.09 (m, 5 H), 4.47Ϫ4.89 (m, 6
3
3
H, 3 CH2Ph), 5.50 (dd, 1 H, J1,2 ϭ J2,3 ϭ 2.3 Hz, 2-H), 6.31 (d,
3
3
3
1 H, J1,2 ϭ 1.9 Hz, 1-H), 7.16Ϫ7.35 (m, 15 H, Ph), 8.68 (s, 1 H,
3.62Ϫ3.69 (m, 1 H, 5-H), 4.12 (dd, 1 H, J5,6 ϭ 2.7, J6,6Ј ϭ 12.1
NH). Ϫ 13β: 1H NMR (250 MHz; CDCl3): δ ϭ 5.38 (dd, 1 H,
3
3
Hz, 6-H), 4.22 (dd, 1 H, J5,6Ј ϭ 4.9, J6,6Ј ϭ 12.2 Hz, 6Ј-H), 4.59
3
3J1,2
ϭ
3J2,3 ϭ 2.0 Hz, 2-H), 5.67 (d, 1 H, J1,2 ϭ 2.0 Hz, 1-H),
3
3
3
(dd, 1 H, J1,2 ϭ 2.6, J2,3 ϭ 3.9 Hz, 2-H), 5.12 (dd, 1 H, J2,3
ϭ
3
3
3
8.54 (s, 1 H, NH). Ϫ C31H32Cl3NO7 (636.95): calcd. C 58.46, H
5.06, N 2.20; found C 58.30, H 5.30, N 2.00.
4.0, J3,4 ϭ 9.9 Hz, 3-H), 5.28 (dd, 1 H, J3,4 ϭ J4,5 ϭ 9.7 Hz, 4-
H), 5.47 (d, 1 H, 3J1,2 ϭ 2.6 Hz, 1-H). Ϫ C15H22O10 (362.33): calcd.
C 49.72, H 6.12; found C 49.73, H 6.03.
6-O-tert-Butyldiphenylsilyl-1,2-O-[-1-(R)-methoxyethylidene]-β-D-
mannopyranose (14): tert-Butylchlorodiphenylsilane (4 mL, 15.28
mmol) and imidazole (1.5 g, 22.62 mmol) were added to a cooled
solution of crude 9 (3 g, 12.71 mmol) in dry N,N-dimethylformam-
ide (50 mL) under nitrogen. After stirring for 24 h at 4°C, the
solution was diluted with water and extracted several times with
dichloromethane. The combined organic extracts were sub-
sequently washed with satd. aqueous NH4Cl solution, and satd.
aqueous NaCl solution, dried with Na2SO4, and concentrated in
vacuo until a white precipitate appeared. On cooling, 14 crys-
tallized as a colorless powder which was filtered off. The mother
liquor was concentrated to dryness and purified by flash chroma-
tography (petroleum ether/ethyl acetate, 1:1) to yield 14 as a syrup
which was crystallized from diethyl ether/petroleum ether (10:1).
Total yield was 3.8 g (64%); m.p. 161°C. Ϫ TLC (petroleum ether/
ethyl acetate, 1:3): Rf ϭ 0.35 Ϫ [α]D ϭ ϩ12 (c ϭ 1, chloroform).
1,2-O-[1-(R)-Methoxyethylidene]-β-D-mannopyranose (9): Com-
pound 9 was prepared as described previously.[32] Crude 9 was di-
rectly used for further preparations.
3,4,6-Tri-O-benzyl-1,2-O-[1-(R)-methoxyethylidene]-β-D-mannopy-
ranose (10): Compound 10 was prepared according to the pro-
cedure described by Ponpipom.[32] Ϫ TLC (petroleum ether/ethyl
1
acetate, 7:3): Rf ϭ 0.42. Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.74
(s, 3 H, CMe), 3.28 (s, 3 H, OMe), 3.38Ϫ3.44 (m, 1 H, 5-H),
3
3
3.67Ϫ3.73 (m, 3 H, 6-, 6Ј-, 3-H), 3.92 (dd, 1 H, J4,5 ϭ J5,6 ϭ 9.3
Hz, 4-H), 4.39 (dd, 1 H, 3J1,2 ϭ 2.6, 3J2,3 ϭ 3.9 Hz, 2-H), 4.51Ϫ4.92
3
(m, 6 H, 3 CH2Ph), 5.34 (d, 1 H, J1,2 ϭ 2.5 Hz, 1-H), 7.21Ϫ7.41
(m, 15 H, Ph). Ϫ C30H34O7 (506.59): calcd. C 71.13, H 6.76; found
C 71.16, H 6.72.
1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-␣/β-D-mannopyranose
(11):
1
Compound 10 was prepared as described previously.[32] A small
amount of the anomeric mixture was separated for the determi-
nation of the physical data. Ϫ 11α: colorless oil. Ϫ TLC (petroleum
ether/ethyl acetate, 7:3): Rf ϭ 0.45. Ϫ 1H NMR (250 MHz; CDCl3):
δ ϭ 2.07 (s, 3 H, COCH3), 2.16 (s, 3 H, COCH3), 3.67Ϫ3.99 (m, 5
H, 3-, 4-, 5-, 6-, 6Ј-H), 4.48Ϫ4.88 (m, 6 H, 3 CH2Ph), 5.36 (dd, 1
Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.06 (s, 9 H, tBu), 1.68 (s, 3
3
3
H, CMe), 2.50 (d, 1 H, J ϭ 7.4 Hz, OH), 2.81 (d, 1 H, J ϭ 2.3
Hz, OH), 3.24Ϫ3.30 (m, 1 H), 3.31 (s, 3 H, OMe), 3.71Ϫ3.79 (m,
3
3
1 H, 5-H), 3.87Ϫ3.97 (m, 3 H), 4.50 (dd, 1 H, J1,2 ϭ 2.6, J2,3
ϭ
3
4.1 Hz, 2-H), 5.43 (d, 1 H, J1,2 ϭ 2.5 Hz, 1-H), 7.35Ϫ7.72 (m, 10
H, Ph). Ϫ C25H34O7Si (474.62): calcd. C 63.27, H 7.22; found C
63.17, H 7.20.
3
3
3
H, J1,2 ϭ J2,3 ϭ 2.5 Hz, 2-H), 6.12 (d, 1 H, J1,2 ϭ 2 Hz, 1-H),
7.15Ϫ7.37 (m, 15 H, Ph). Ϫ 11β: colorless oil. Ϫ TLC (petroleum
ether/ethyl acetate, 7:3): Rf ϭ 0.36. Ϫ 1H NMR (250 MHz; CDCl3):
δ ϭ 2.09 (s, 3 H, COCH3), 2.22 (s, 3 H, COCH3), 3.54Ϫ3.98 (m, 5
H, 3-, 4-, 5-, 6-, 6Ј-H), 4.48Ϫ4.99 (m, 6 H, 3 CH2Ph), 5.61 (dd, 1
H, 3J1,2 ϭ 0.99, 3J2,3 ϭ 2.2 Hz, 2-H), 5.74 (d, 1 H, 3J1,2 ϭ 0.99 Hz,
1-H), 7.11Ϫ7.38 (m, 15 H, Ph). C31H34O8 (534.60): calcd. C 69.65,
H 6.41; found C 68.76, H 6.28.
6-O-tert-Butyldiphenylsilyl-1,2-O-[1-(R)-methoxyethylidene]-3,4-
di-O-trichloroacetylcarbamoyl-β-D-mannopyranose (14a): To a solu-
tion of diol 14 (10 mg) in chloroform (1 mL) trichloroacetyl isocy-
anate (1 drop) was added and the mixture was characterized by 1H-
1
NMR studies. Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.04 (s, 9 H,
tBu), 1.75 (s, 3 H, CMe), 3.31 (s, 3 H, OCH3), 3.61Ϫ3.66 (m, 1 H,
3
3
5-H), 3.78 (dd, 1 H, J5,6 ϭ 3.3, J6,6Ј ϭ 11.5 Hz, 6-H), 3.88 (dd, 1
3
3
3
H, J5,6Ј ϭ 2.7, J6,6Ј ϭ 11.5 Hz, 6Ј-H), 4.71 (dd, 1 H, J1,2 ϭ 2.5,
2-O-Acetyl-3,4,6-tri-O-benzyl-␣/β-D-mannopyranose (12): A solu-
3J2,3 ϭ 4.0 Hz, 2-H), 5.29 (dd, 1 H, J2,3 ϭ 4.0, J3,4 ϭ 9.9 Hz, 3-
H), 5.54 (d, 1 H, 3J1,2 ϭ 2.5 Hz, 1-H), 5.60 (dd, 1 H, 3J3,4 ϭ 3J4,5 ϭ
9.7 Hz, 4-H), 7.29Ϫ7.71 (m, 10 H, Ph), 8.25 (s, 1 H, NH), 8.59 (s,
1 H, NH).
3
3
tion of compound 11 (1.96 g, 3.67 mmol) and hydrazinium acetate
(0.41 g, 4.42 mmol) in dry N,N-dimethylformamide (40 mL) was
heated for 2 h at 50°C under nitrogen. After cooling to room temp.,
water (250 mL) was added and the mixture was extracted twice
with ethyl acetate. The combined organic extracts were washed with
water, dried (Na2SO4), and concentrated in vacuo. Purification of
3,4-Di-O-benzyl-6-O-tert-butyldiphenylsilyl-1,2-O-[1-(R)-meth-
oxyethylidene]-β-D-mannopyranose (15): Sodium hydride (170 mg,
1158
Eur. J. Org. Chem. 1999, 1153Ϫ1165

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
7.09 mmol) was cautiously added to a cooled solution of 14 (1.4 g,
2.95 mmol) in dry N,N-dimethylformamide (15 mL) under nitro-
gen. After 30 min, benzyl bromide (0.85 mL, 7.09 mmol) was added
dropwise within 10 min. The solution was warmed up to room
temp. and stirred overnight. The reaction mixture was diluted with
methanol (1 mL) and water (150 mL) and extracted twice with ethyl
acetate. The combined organic extracts were dried with Na2SO4,
concentrated in vacuo, and flash-chromatographed (petroleum
ether/ethyl acetate, 6:1) to yield 15 (1.53 g, 79%) as a colorless
ous NH4Cl solution and water. The organic solution was dried
(Na2SO4) and concentrated in vacuo. Flash chromatography
(petroleum ether/ethyl acetate, 9:1 with 1% triethylamine) gave 18
(0.27 g, 89%) as a colorless foam in a α/β ratio of 12:1. Ϫ TLC
(petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.54. Ϫ 18α: 1H NMR
(250 MHz; CDCl3): δ ϭ 1.08 (s, 9 H, tBu), 2.19 (s, 3 H, COCH3),
3
3
3.88Ϫ4.10 (m, 4 H), 4.24 (dd, 1 H, J3,4 ϭ J4,5 ϭ 9.7 Hz, 4-H),
3
3
4.58Ϫ4.97 (m, 4 H, 2 CH2Ph), 5.50 (dd, 1 H, J1,2 ϭ J2,3 ϭ 3.1
3
Hz, 2-H), 6.33 (d, 1 H, J1,2 ϭ 1.9 Hz, 1-H), 7.18Ϫ7.75 (m, 20 H,
1
syrup. Ϫ TLC (petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.5. Ϫ Ph), 8.65 (s, 1 H, NH). Ϫ 18β: H NMR (250 MHz; CDCl3): δ ϭ
1
[α]D ϭ ϩ28 (c ϭ 1, chloroform). Ϫ H NMR (250 MHz; CDCl3): 5.37 (dd, 1 H, 2-H), 5.57 (d, 1 H, 1-H), 8.28 (s, 1 H, NH). Ϫ
δ ϭ 1.07 (s, 9 H, tBu), 1.80 (s, 3 H, CMe), 3.24Ϫ3.30( m, 1 H, 5-
C40H44Cl3NO7Si (785.23): calcd. C 61.18, H 5.65, N 1.78; found C
61.03, H 5.71, N 1.77.
3
H), 3.32 (s, 3 H, OCH3), 3.75 (dd, 1 H, J2,3 ϭ 4.0, 3J3,4 ϭ 9.3 Hz,
3
3
3-H), 3.89 (dd, 1 H, J5,6 ϭ 1.9, J6,6Ј ϭ 11.1 Hz, 6-H), 3.99 (dd, 1
Allyl 2-O-Acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-D-
3
3
3
H, J5,6Ј ϭ 3.0, J6,6Ј ϭ 11.2 Hz, 6Ј-H), 4.17 (dd, 1 H, J3,4
ϭ
mannopyranoside (19): To a mixture of trichloroacetimidate 18
3J4,5 ϭ 9.4 Hz, 4-H), 4.40 (dd, 1 H, J1,2 ϭ 2.4, J2,3 ϭ 4.0 Hz, 2-
3
3
˚
(16.0 g, 20.4 mmol), powdered molecular sieves (3 A), and dry allyl
3
H), 4.76Ϫ5.04 (m, 4 H, 2 CH2Ph), 5.35 (d, 1 H, J1,2 ϭ 2.3 Hz, 1-
alcohol (4 mL, 58.7 mmol) in dry diethyl ether (200 mL) was added
TMSOTf (0.5 mL, 2.7 mmol) under nitrogen. After stirring for 15
min at room temp., triethylamine was added and the solvent was
evaporated in vacuo. Flash chromatography (petroleum ether/ethyl
H), 7.22Ϫ7.79 (m, 20 H, Ph). Ϫ C39H46O7Si (654.87): calcd. C
71.53, H 7.08; found C 71.23, H 7.13.
1,2-Di-O-acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣/β-
D-mannopyranose (16): To a mixture of acetic acid (30 mL), water acetate, 8:2) of the residue gave 19 (13.0 g, 94%) as a colorless
(30 mL), and acetone (60 mL) 15 (1.4 g, 2.14 mmol) was added and
stirred for 1 h at room temp. The clear solution was concentrated in
vacuo. The residue was codistilled with toluene. The residue was
dissolved in dry pyridine/acetic anhydride (1:1, 10 mL) and stirred
for 7 h at room temp. Evaporation of the solvents, codistillation
with toluene and flash chromatography with petroleum ether/ethyl
acetate (8:2) gave 16 (1.41 g, 96%) as an anomeric mixture (α/β ϭ
5:1). Ϫ TLC (petroleum ether/ethyl acetate, 9:1): Rf ϭ 0.18 (16α),
syrup. Ϫ TLC (petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.65,
[α]D ϭ ϩ31 (c ϭ 1, chloroform). Ϫ H NMR (250 MHz; CDCl3):
1
δ ϭ 1.08 [s, 9 H, C(CH3)3], 2.14 (s, 3 H, COCH3), 3.69Ϫ3.79 (m,
1 H, 5-H), 3.87Ϫ4.18 (m, 6 H), 4.54Ϫ4.76 (m, 3 H, 1.5 CH2Ph),
3
4.89 (d, J1,2 ϭ 1.7 Hz, 1 H, 1-H), 4.92 (d, Jgem ϭ 10.8 Hz, 1 H,
3
0.5 CH2Ph), 5.14Ϫ5.28 (m, 2 H, CH2CHϭCH2), 5.40 (dd, J1,2
ϭ
1.8, 3J2,3 ϭ 2.6 Hz, 1 H, 2-H), 5.78Ϫ5.92 (m, 1 H, CH2CHϭCH2),
7.16Ϫ7.44 (m, 16 H, 2 Ph, m, p-Ph2Si), 7.69Ϫ7.77 (m, 4 H, o-
Rf ϭ 0.10 (16β). Ϫ The anomeric mixture was separated by flash Ph2Si). Ϫ C41H48O7Si (680.91): calcd. C 72.32, H 7.11; found C
chromatography (petroleum ether/ethyl acetate, 9:1). Ϫ 16α: [α]D ϭ
ϩ28 (c ϭ 1, chloroform). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ
1.08 (s, 9 H, tBu), 2.04 (s, 3 H, COCH3), 2.16 (s, 3 H, COCH3),
71.91, H 7.15.
Allyl 3,4-Di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-D-mannopyra-
noside (20): To a solution of 19 (10 g, 14.7 mmol) in dry methanol
(150 mL) satd. sodium methoxide solution in methanol (0.5 mL)
was added. After stirring for 6 h at room temp., the solution was
neutralized with Amberlite IR 120 (Hϩ). Evaporation of the sol-
vent in vacuo and flash chromatography with petroleum ether/ethyl
acetate (6:1) gave 20 (9 g, 95%) as a colorless foam.Ϫ TLC (petro-
leum ether/ethyl acetate, 8:2): Rf ϭ 0.40, [α]D ϭ ϩ28 (c ϭ 2, chloro-
form). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 1.06 [s, 9 H, C(CH3)3],
3
3
3.71Ϫ3.76 (m, 1 H, 5-H), 3.85Ϫ3.90 (dd, 1 H, J5,6 ϭ 1.5, J6,6Ј
ϭ
11.4 Hz, 6-H), 3.98Ϫ4.08 (m, 2 H, 6Ј-, 3-H), 4.23 (dd, 1 H, 3J3,4
ϭ
3J4,5 ϭ 9.7 Hz, 4-H), 4.56Ϫ4.98 (m, 4 H, 2 CH2Ph), 5.37 (dd, 1 H,
3
3
3J1,2 ϭ 2.2, J2,3 ϭ 3.1 Hz, 2-H), 6.15 (d, 1 H, J1,2 ϭ 2.0 Hz, 1-
H), 7.18Ϫ7.77 (m, 20 H, Ph). Ϫ C40H46O8Si (682.88): calcd. C
70.35, H 6.79; found C 69.57, H 6.63.
2-O-Acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣/β-D-
3
mannopyranose (17): A solution of compound 16 (1.39 g, 2.09
mmol) and hydrazinium acetate (0.23 g, 1.50 mmol) in dry N,N-
dimethylformamide (30 mL) was heated for 4 h at 50°C under ni-
2.44 (d, J2,OH ϭ 2.2 Hz, 1 H, OH), 3.72Ϫ4.22 (m, 8 H), 4.56 (d,
Jgem ϭ 10.7 Hz, 1 H, 0.5 CH2Ph), 4.71 (s, 2 H, CH2Ph), 4.85 (d,
Jgem ϭ 10.7 Hz, 1 H, 0.5 CH2Ph), 4.96 (s, 1 H, 1-H), 5.16Ϫ5.29
trogen. After cooling to room temp., water (200 mL) was added (m, 2 H, CH2CHϭCH2), 5.80Ϫ5.93 (m, 1 H, CH2CHϭCH2),
and the mixture was extracted twice with ethyl acetate. The com-
bined organic extracts were washed with water, dried (Na2SO4),
and concentrated in vacuo. Purification of the residue by flash
chromatography (petroleum ether/ethyl acetate, 8:2) gave 17 (1.01
g, 77%, colorless foam) as an anomeric mixture (α/β ϭ 12:1). Ϫ
TLC (petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.25 (17α); Rf ϭ
7.14Ϫ7.42 (m, 16 H, 2 Ph, m, p-Ph2Si), 7.69Ϫ7.75 (m, 4 H, o-
Ph2Si). Ϫ C39H46O6Si (638.87): calcd. C 73.32, H 7.26; found C
73.28, H 7.29.
Allyl
2-O-Allyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-D-
mannopyranoside (21): Sodium hydride (0.4 g, 16.6 mmol) was cau-
tiously added to a cooled solution of 20 (8.5 g, 13.3 mmol) in dry
N,N-dimethylformamide (75 mL) under nitrogen. After 30 min, al-
lyl bromide (3.52 mL, 26.6 mmol) was added dropwise within 15
min. The solution was warmed up to room temp. and then stirred
for 2 h. After concentration in vacuo (0.1 bar), water was added,
and the mixture was extracted twice with ethyl acetate. The com-
bined organic extracts were dried with Na2SO4, concentrated in
vacuo, and flash-chromatographed (petroleum ether/ethyl acetate,
9:1) to give 21 (8.1 g, 90%) as a colorless syrup. Ϫ TLC (petroleum
ether/ethyl acetate, 8:2): Rf ϭ 0.67. Ϫ [α]D ϭ ϩ31 (c ϭ 4, chloro-
1
0.15 (17β). Ϫ 17α: H NMR (250 MHz; CDCl3): δ ϭ 1.08 (s, 9 H,
tBu), 2.14 (s, 3 H, COCH3), 2.68 (d, 1 H, 3J ϭ 3.6 Hz, OH),
3.83Ϫ4.16 (m, 5 H), 4.55Ϫ4.96 (m, 4 H, 2 CH2Ph), 5.22 (dd, 1 H,
3J1,2 ϭ 1.9, 3J1,OH ϭ 3.5 Hz, 1-H), 5.37 (dd, 1 H, 3J1,2 ϭ 2.1, 3J2,3 ϭ
2.5 Hz, 2-H), 7.18Ϫ7.75 (m, 20 H, Ph). Ϫ C38H44O7Si (640.85):
calcd. C 71.22, H 6.92; found C 71.01, H 6.86.
O-(2-O-Acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣/β-D-
mannopyranosyl) Trichloroacetimidate (18): A cooled solution of 17
(0.25 g, 0.382 mmol) in dry dichloromethane (5 mL) and trichlo-
roacetonitrile (0.15 mL) was treated with 10 µL of DBU and stirred form). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 1.06 [s, 9 H, C(CH3)3]
for 1 h at 0°C under nitrogen. The reaction mixture was diluted
with dichloromethane and extracted subsequently with satd. aque-
3.68Ϫ3.74 (m, 1 H, 5-H), 3.44 (br. s, 1 H), 3.91Ϫ3.99 (m, 5 H),
4.11Ϫ4.28 (m, 3 H), 4.54 (d, Jgem ϭ 10.7 Hz, 1 H, 0.5 CH2Ph),
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1159

T. G. Mayer, R. R. Schmidt
FULL PAPER
1
4.71 (s, 2 H, CH2Ph), 4.90 (d, Jgem ϭ 10.7 Hz, 1 H, 0.5 CH2Ph),
7:3): Rf ϭ 0.14. Ϫ [α]D ϭ ϩ59 (c ϭ 0.5, methanol). Ϫ H NMR
3
4.92 (br. s, 1 H, 1-H), 5.13Ϫ5.35 (m, 4 H, 2 CH2CHϭCH2), (250 MHz; CDCl3): δ ϭ 2.39 (d, J2d,OH ϭ 2.6 Hz, 1 H, OH),
5.80Ϫ6.03 (m, 2 H, 2 CH2CHϭCH2), 7.12Ϫ7.43 (m, 16 H, 2 Ph, 3.55Ϫ3.95 (m, 13 H), 4.07Ϫ4.19 (m, 3 H), 4.45Ϫ4.84 (m, 9 H, 4.5
m, p-Ph2Si), 7.69Ϫ7.76 (m, 4 H, o-Ph2Si). Ϫ C42H50O6Si (678.94): CH2Ph), 4.85 (d, J1c,2c ϭ 1.6 Hz, 1 H, 1c-H), 4.92 (d, Jgem ϭ 11.1
3
3
calcd. C 74.30, H 7.42; found C 74.22, H 7.57.
Hz, 1 H, 0.5 CH2Ph), 5.04 (d, J1d,2d ϭ 1.5 Hz, 1 H, 1d-H),
5.13Ϫ5.33 (m, 4 H, 2 CH2CHϭCH2), 5.77Ϫ6.00 (m, 2 H, 2
CH2CHϭCH2), 7.12Ϫ7.39 (m, 25 H, 5 Ph). Ϫ C53H60O11 (873.05):
calcd. C 72.91, H 6.93; found C 72.71, H 6.99.
Allyl 2-O-Allyl-3,4-di-O-benzyl-␣- -mannopyranoside (22): A solu-
D
tion of 21 (13 g, 19.1 mmol) in dry tetrahydrofuran was treated
with tetrabutylammonium fluoride (1.1  solution in THF, 40 mL,
44 mmol) and dry acetic acid (2.5 mL) at 0°C under nitrogen. Stir-
ring was continued for 4 d at room temp. After addition of satd.
aqueous NaHC3 solution and water, the mixture was extracted
twice with dichloromethane. The combined organic extracts were
dried (MGSO4) and concentrated in vacuo. Purification of the
crude product by flash chromatography with petroleum ether/ethyl
acetate (7:3) gave 7.74 g (92%) of 22 as a colorless oil. Ϫ TLC
(petroleum ether/ethyl acetate, 7:3): Rf ϭ 0.16. Ϫ [α]D ϭ ϩ47 (c ϭ
Allyl
␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣-
pyranosyl)-(1Ǟ6)-2-O-allyl-3,4-di-O-benzyl-␣-
O-(2-O-Acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
-manno-
-manno-
D
D
D
pyranoside (25): To a mixture of 24 (10.9 g, 12.2 mmol), 18 (12.6
g, 16.0 mmol), and powdered molecular sieves (3 A) in dry diethyl
˚
ether (200 mL) TMSOTf (0.4 mL, 2.2 mmol) was added under
nitrogen. After stirring for 20 min at room temp., solid NaHCO3
was added and stirring was continued for 15 min. The solvent was
evaporated in vacuo and the residue was purified by MPLC (tolu-
ene/ethyl acetate, 20:1) to yield 25 (17.2 g, 92%) as a colorless foam.
1
1, chloroform). Ϫ H NMR (250 MHz; CDCl3): δ ϭ 2.39 (br. s, 1
H, OH), 3.62Ϫ3.97 (m, 7 H), 4.09Ϫ4.26 (m, 3 H), 4.63 (d, Jgem
10.8 Hz, 1 H, 0.5 CH2Ph), 4.70 (s, 2 H, CH2Ph), 4.86 (d, J1,2
ϭ
ϭ
Ϫ TLC (petroleum ether/ethyl acetate, 8:2): Rf ϭ 0.51. Ϫ [α]D
ϭ
ϩ34 (c ϭ 2, chloroform). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ
1.07 [s, 9 H, C(CH3)3], 2.12 (s, 3 H, COCH3), 3.49Ϫ3.94 (m, 14
H), 4.03Ϫ4.19 (m, 7 H), 4.36Ϫ4.74 (m, 11 H, 5.5 CH2Ph), 4.78 (d,
3J1c,2c ϭ 1.2 Hz, 1 H, 1c-H), 4.82Ϫ4.95 (m, 3 H, 1.5 CH2Ph), 4.86
(br. s, 1 H, 1d-H), 5.07Ϫ5.29 (m, 4 H, 2 CH2CHϭCH2), 5.22 (br.
s, 1 H, 1e-H), 5.53 (br. t, 1 H, 2e-H), 5.71Ϫ5.92 (m, 2 H, 2
CH2CHϭCH2), 7.10Ϫ7.42 (m, 41 H, 7 Ph, m,p-Ph2Si), 7.66Ϫ7.75
(m, 4 H, o-Ph2Si). Ϫ C91H102O17Si (1495.88): calcd. C 73.07, H
6.87; found C 72.36, H 6.96.
1.6 Hz, 1 H, 1-H), 4.92 (d, Jgem ϭ 10.8 Hz, 1 H, 0.5 CH2Ph),
5.14Ϫ5.32 (m, 4 H, 2 CH2CHϭCH2), 5.77Ϫ5.99 (m, 2 H, 2
CH2CHϭCH2), 7.21Ϫ7.39 (m, 10 H, 2 Ph). Ϫ C26H32O6 (440.54):
calcd. C 70.89, H 7.32; found C 70.78, H 7.38.
Allyl 2-O-Allyl-3,4-di-O-benzyl-6-O-trichloroacetylcarbamoyl-␣-D-
mannopyranoside (22a): To a solution of 22 (10 mg) in [D]chloro-
form (1 mL) trichloroacetyl isocyanide (1 drop) was added and the
reaction mixture was characterized by 1H-NMR studies. Ϫ 1H
NMR (250 MHz; CDCl3): δ ϭ 3.77Ϫ4.01 (m, 5 H), 4.12Ϫ4.20 (m,
3 H), 4.47 (m, 2 H, 6-, 6Ј-H), 4.61 (d, Jgem ϭ 11.0 Hz, 1 H, 0.5
CH2Ph), 4.67 (d, Jgem ϭ 11.6 Hz, 1 H, 0.5 CH2Ph), 4.73 (d, Jgem ϭ
Allyl 2-O-Allyl-3,4-di-O-benzyl-O-(3,4-di-O-benzyl-6-O-tert-butyl-
diphenylsilyl-␣-
D-mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣-
3
11.6 Hz, 1 H, 0.5 CH2Ph), 4.88 (d, J1,2 ϭ 1.8 Hz, 1 H, 1-H), 4.94
D-mannopyranosyl)-(1Ǟ6)-␣-
D-mannopyranoside (26): To a solution
(d, Jgem ϭ 11.0 Hz, 1 H, 0.5 CH2Ph), 5.17Ϫ5.33 (m, 4 H, 2
CH2CHϭCH2), 5.79Ϫ6.00 (m, 2 H, 2 CH2CHϭCH2), 7.27Ϫ7.41
(m, 10 H, 2 Ph), 8.38 (s, 11 H, NH).
of 25 (17.0 g, 11.5 mmol) in dry methanol/diethyl ether (300 mL,
5:1) satd. sodium methoxide solution in methanol (0.5 mL) was
added. After stirring for 5 h at room temp., the solution was neu-
tralized with Amberlite IR 120 (Hϩ). Evaporation of the solvents in
vacuo and flash chromatography with petroleum ether/ethyl acetate
(8:2) gave 26 (15.6 g, 94%) as a colorless foam. Ϫ TLC (petroleum
ether/ethyl acetate, 8:2): Rf ϭ 0.33. Ϫ [α]D ϭ ϩ41 (c ϭ 0.5, meth-
anol). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 1.05 [s, 9 H, C(CH3)3],
Allyl
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣-D-mannopyranosyl)-
(1Ǟ6)-2-O-allyl-3,4-di-O-benzyl-α-D-mannopyranoside (23): To a
mixture of 13 (14.3 g, 22.5 mmol), 22 (7.9 g, 17.9 mmol) and pow-
˚
dered molecular sieves (3 A) in dry diethyl ether (200 mL) TMSOTf
(0.4 mL, 2.21 mmol) was added under nitrogen. After stirring for
20 min at room temp., triethylamine was added and the solvent
was evaporated in vacuo. The residue was redissolved in toluene/
petroleum ether (1:1) and on cooling trichloroacetamide crys-
tallized as a white powder which was filtered off. Concentration in
vacuo and flash chromatography (petroleum ether/ethyl acetate, 9:1
Ǟ 8:2) gave 23 (14.2 g, 86%) as a colorless oil. Ϫ TLC (petroleum
ether/ethyl acetate, 8:2): Rf ϭ 0.28. Ϫ [α]D ϭ ϩ50 (c ϭ 1, meth-
anol). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 2.13 (s, 3 H, COCH3),
3.56Ϫ4.00 (m, 12 H), 4.07Ϫ4.18 (m, 3 H), 4.40Ϫ4.74 (m, 8 H, 4
3
2.34 (d, J2e,OH ϭ 3.0 Hz, 1 H, OH), 3.48Ϫ4.20 (m, 22 H),
4.36Ϫ4.74 (m, 11 H, 5.5 CH2Ph), 4.78Ϫ4.89 (m, 5 H, 1.5 CH2Ph,
1c-, 1d-H), 5.07Ϫ5.30 (m, 5 H, 4 CH2CHϭCH2, 1e-H), 5.70Ϫ5.97
(m, 2 H, 2 CH2CHϭCH2), 7.13Ϫ7.39 (m, 41 H, 7 Ph, m,p-Ph2Si),
7.67Ϫ7.75 (m, 4 H, o-Ph2Si). Ϫ C89H100O16Si (1453.84): calcd. C
73.53, H 6.93; found C 73.51, H 6.93.
Allyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣-
(1Ǟ2)-O-(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-
pyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -mannopyrano-
syl)-(1Ǟ6)-2-O-allyl-3,4-di-O-benzyl-␣- -mannopyranoside (27): To
D
-mannopyranosyl)-
D
-manno-
D
3
CH2Ph), 4.83Ϫ4.94 (m, 2 H, CH2Ph), 4.86 (d, J1a,2a ϭ 1.8 Hz, 1
D
3
H, 1a-H), 4.95 (d, J1b,2b ϭ 1.7 Hz, 1 H, 1b-H), 5.13Ϫ5.34 (m, 4
a mixture of 13 (7.7 g, 12.09 mmol), 26 (14.5 g, 9.97 mmol), and
3
3
H, 2 CH2CHϭCH2), 5.47(dd, J1b,2b ϭ 1.9, J2b,3b ϭ 2.9 Hz, 1 H,
2b-H), 5.77Ϫ6.00 (m, 2 H, 2 CH2CHϭCH2), 7.09Ϫ7.40 (m, 25 H,
5 Ph). Ϫ C55H62O12 (915.09): calcd. C 72.41, H 6.63; found C
71.84, H 6.73.
˚
powdered molecular sieves (3 A) in dry diethyl ether (175 mL) was
added TMSOTf (40 mL, 0.22 mmol) under nitrogen. After stirring
for 15 min at room temp., triethylamine was added in order to
neutralize the reaction mixture. The solvent was evaporated in va-
cuo and the residue was filtered through silica gel (petroleum ether/
Allyl 2-O-Allyl-3,4-di-O-benzyl-O-(3,4,6-tri-O-benzyl-␣-
D-manno-
pyranosyl)-(1Ǟ6)-␣-
D-mannopyranoside (24): To a solution of 23 ethyl acetate, 6:1). Purification of the crude product by MPLC with
(12.5 g, 13.7 mmol) in dry methanol (150 mL) satd. sodium meth-
oxide solution in methanol (0.5 mL) was added. After stirring over-
night at room temp., the solution was neutralized with Amberlite
IR 120 (Hϩ). Evaporation of the solvent in vacuo and flash chro-
matography with petroleum ether/ethyl acetate (7:3) gave 24 (11.3
g, 94%) as a colorless oil. Ϫ TLC (petroleum ether/ethyl acetate,
toluene/ethyl acetate (25:1) gave 27 (17.5 g, 91% as a colorless foam.
Ϫ TLC (toluene/ethyl acetate, 10:1): Rf ϭ 0.49. Ϫ [α]D ϭ ϩ31 (c ϭ
0.5, methanol). Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.03 [s, 9 H,
1
C(CH3)3], 2.12 (s, 3 H, COCH3), 3.47Ϫ3.68 (m, 7 H), 3.72 (dd,
3
3J1c,2c ϭ J2c,3c ϭ 2.0 Hz, 1 H, 2c-H), 3.74Ϫ4.10 (m, 17 H), 4.13
(br. t, 1 H, 2e-H), 4.16 (br. t, 1 H, 2d-H), 4.27Ϫ4.70 (m, 16 H, 8
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1160

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
3
3
3
CH2Ph), 4.76 (d, J1c,2c ϭ 1.5 Hz, 1 H, 1c-H), 4.79Ϫ4.93 (m, 4 H,
3J1c,2c < 1, J2c,3c ϭ 2.8 Hz, 1 H, 2c-H), 5.30 (d, J1e,2e < 1 Hz, 1
3
3
2 CH2Ph), 4.84 (br. s, 1 H, 1d-H), 5.05Ϫ5.27 (m, 4 H, 2 CH2CHϭ
CH2), 5.10 (br. s, 1 H, 1f-H), 5.36 (br. s, 1 H, 1e-H), 5.58 (dd,
H, 1e-H), 5.56 (dd, J1f,2f ϭ 1.8, J2f,3f ϭ 2.9 Hz, 1 H, 2f-H),
7.08Ϫ7.37 (m, 56 H, 10 Ph, m,p-Ph2Si), 7.67Ϫ7.76 (m, 4 H, o-
3J1f,2f
ϭ
3J2f,3f ϭ 1.8 Hz, 1 H, 2f-H), 5.71Ϫ5.92 (m, 2 H, 2 Ph2Si). Ϫ C114H124O23Si (1890.31): calcd. C 72.44, H 6.61; found
CH2CHϭCH2), 7.03Ϫ7.36 (m, 56 H, 10 Ph, m,p-Ph2Si), C 72.11, H 6.57.
7.66Ϫ7.77(m, 4 H, o-Ph2Si). Ϫ FAB MS (positive ion mode, ma-
O-[2-O-Acetyl-O-(2-O-acetyl-3,4,6-tri-O-benzyl-␣-D-mannopyra-
trix: NBOH with NaI): m/z ϭ 1951 [M ϩ Naϩ]. Ϫ C118H130O22Si
(1928.40): calcd. C 73.50, H 6.79; found C 73.52, H 6.80.
nosyl)-(1Ǟ2)-O-(3,4-di-O-benzyl-6-O-tert-butyldiphenylsi-
lyl-␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ6)-3,4-di-O-benzyl-␣- -mannopyranosyl]
D
D
D
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣-
O-(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-
syl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -mannopyranosyl)-
(1Ǟ6)-3,4-di-O-benzyl-␣/β- -mannopyranose (28): A solution of 27
D
-mannopyranosyl)-(1Ǟ2)-
Trichloroacetimidate (4): A solution of 30 (4.0 g, 2.12 mmol) in
dry dichloromethane (30 mL) and trichloroacetonitrile (4 mL) was
treated with DBU (1Ϫ2 drops) and stirred for 1.5 h at room temp.
The solvents were evaporated in vacuo, and the residue was purified
by flash chromatography (toluene/ethyl acetate, 20:1 with 1% tri-
ethylamine) to yield 4 (3.97 g, 92%) as a colorless foam. Ϫ TLC
(petroleum ether/ethyl acetate, 5:1, with 1% triethylamine): Rf ϭ
D
-mannopyrano-
D
D
(6.0 g, 3.11 mmol) in a mixture of toluene (25 mL), ethanol (13
mL) and water (1.2 mL) was heated to 95°C. Then WilkinsonЈs
catalyst (0.5 g, 0.54 mmol) was added and the red solution was
stirred under reflux for 5 h. The reaction was monitored by TLC
and color. After cooling, the yellow reaction mixture was concen-
trated in vacuo. The residue was redissolved in toluene/ethanol
(5:1) and again concentrated in vacuo. Flash chromatography of
the syrup (toluene/ethyl acetate, 9:1 Ǟ 5:1) gave 28 (4.3 g, 75%) as
an anomeric mixture (α:β ϭ 4:1). Ϫ TLC (toluene/ethyl acetate,
1
0.19. Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.03 [s, 9 H, C(CH3)3],
2.03 (s, 3 H, COCH3), 2.12 (s, 3 H, COCH3), 3.55Ϫ4.12 (m, 22 H),
4.27Ϫ4.93 (m, 20 H, 10 CH2Ph), 4.85 (br. s, 1 H, 1d-H), 5.09 (d,
3
3J1f,2f < 1 Hz, 1 H, 1f-H), 5.37 (d, J1e,2e < 1 Hz, 1 H, 1e-H), 5.48
3
3
(dd, J1c,2c ϭ 1.9, J2c,3c ϭ 2.9 Hz, 1 H, 2c-H), 5.58 (br. t, 1 H, 2f-
3
1
H), 6.14 (d, J1c,2c ϭ 1.4 Hz, 1 H, 1c-H), 7.08Ϫ7.35 (m, 56 H, 10
10:1): Rf ϭ 0.08. Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.09 [s, 9
H, C(CH3)3], 2.12 (s, 3 H, COCH3), 2.26 (d, 3J ϭ 1.3 Hz, 1 H,
OH), 2.65 (d, 3J ϭ 2.5 Hz, 0.5 H, OH), 3.46Ϫ4.17 (m, 28 H),
4.26Ϫ4.68 (m, 16 H, 8 CH2Ph), 4.74Ϫ4.98 (m, 6 H, 2 CH2Ph,
1c-, 1d-H), 5.11 (s, 1 H, 1f-H), 5.32 (s, 0.8 H, 1e-H), 5.38 (s, 0.2 H,
1e-H), 5.58 (br. t, 1 H, 2f-H), 7.08Ϫ7.34 (m, 56 H, 10 Ph, m,p-
Ph2Si), 7.67Ϫ7.77 (m, 4 H, o-Ph2Si). Ϫ C112H122O22Si (1848.7):
calcd. C 72.78, H 6.65; found C 72.68, H 6.66.
Ph, m,p-Ph2Si), 7.67Ϫ7.77 (m, 4 H, o-Ph2Si), 8.63 (s, 1 H, NH). Ϫ
C
116H124Cl3NO23Si (2034.69): calcd. C 68.48, H 6.14, N 0.69;
found C 68.80, H 6.09, N 0.77.
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣-
D
-mannopyranosyl)-(1Ǟ2)-
-manno-
-mannopyra-
-mannopyranosyl)-
(1Ǟ4)-O-(2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-␣- -gluco-
pyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyl-
oxycarbonyl- -myo-inositol (31): To a mixture of 3 (444 mg, 0.491
O-(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-
pyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣-
nosyl)-(1Ǟ6)-O-(2-O-acetyl-3,4-di-O-benzyl-␣-
D
D
D
D
Acetyl-2-O-acetyl-O-(2-O-acetyl-3,4,6-tri-O-benzyl-␣-
pyranosyl)-(1Ǟ2)-O-(3,4-di-O-benzyl-6-O-tert-butyldiphenyl-
silyl-␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ6)-3,4-di-O-benzyl-␣,β- -mannopyranose
D-manno-
D
D
D
mmol), 4 (1.19 g, 0.585 mmol), and powdered molecular sieves (3
D
˚
A) in dry diethyl ether (15 mL) a solution of TMSOTf in dry di-
(29): A solution of 28 (3.8 g, 2.06 mmol) in dry pyridine/acetic
anhydride (1:1, 30 mL) was stirred overnight at room temp. When
the reaction was completed, the solution was concentrated in va-
cuo, codistilled with toluene, and flash-chromatographed on silica
gel (toluene/ethyl acetate, 15:1) to gave 29 (3.8 g, 96%, α:β ϭ 3.5:1)
as a colorless foam. Ϫ TLC (toluene/ethyl acetate, 10:1): Rf ϭ 0.38.
ethyl ether (17 µL, 0.094 mmol TMSOTf in 0.1 mL dry diethyl
ether) was added under nitrogen. After stirring for 30 min at room
temp., solid NaHCO3 was added and stirring was continued for 15
min. The solvent was evaporated in vacuo and the residue was fil-
tered through silica gel with the mixed solvent toluene/ethyl acetate
(20:1). MPLC (toluene/ethyl acetate, 25:1) of the crude mixture
yielded 31 (1.24 g, 91%) as a colorless oil which was lyophilized
from dioxane. Ϫ TLC (toluene/ethyl acetate, 20:1): Rf ϭ 0.48. Ϫ
Ϫ
1H NMR (250 MHz; CDCl3): δ ϭ 1.03 [s, 9 H, C(CH3)3],
1.93Ϫ2.12 (several s, 9 H, 3 COCH3), 3.43Ϫ4.16 (m, 22 H),
4.28Ϫ4.73 (m, 16 H, 8 CH2Ph), 4.77Ϫ4.92 (m, 5 H, 2 CH2Ph, 1d-
1
[α]D ϭ ϩ41 (c ϭ 1, chloroform). Ϫ H NMR (600 MHz; CDCl3):
3
H), 5.09 (d, J1f,2f ϭ 1.5 Hz, 1 H, 1f-H), 5.33Ϫ5.35 (m, 2 H, 2c-,
3
δ ϭ 0.75Ϫ0.77 (d, J ϭ 6.9 Hz, 3 H, CH3), 0.83Ϫ0.88 (2 d, m, 7
1e-H), 5.57 (br. t, 1 H, 2f-H), 5.70 (s, 0.2 H, β-1c-H), 6.01 (d,
3J1c,2c ϭ 2.0 Hz, 0.8 H, α-1c-H), 7.00Ϫ7.38 (m, 56 H, 10 Ph, m,p-
Ph2Si), 7.66Ϫ7.76 (m, 4 H, o-Ph2Si). Ϫ C116H126O24Si (1932.34):
calcd. C 72.10, H 6.57; found C 71.93, H 6.59.
H, 2 CH3, HMnt), 0.96Ϫ1.07 [s, m, 11 H, C(CH3)3, 2 HMnt],
1.25Ϫ1.75 (m, 24 H, 20 Hcycloh., 4 HMnt), 1.85 (s, 3 H, COCH3),
1.90Ϫ2.05 (m, 2 H, 2 HMnt), 2.11 (s, 3 H, COCH3), 6.92Ϫ7.37 (m,
64 H, 11 Ph, m,p-Ph2Si, m,p-COPh), 7.67Ϫ7.75 (m, 4 H, o-Ph2Si),
7.91Ϫ7.93 (m, 2 H, o-COPh). Signal assignment based on DQF-
COSY and HMQC, connection of the sugar moieties based on
2-O-Acetyl-O-(2-O-acetyl-3,4,6-tri-O-benzyl-␣-
syl)-(1Ǟ2)-O-(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ6)-3,4-di-O-benzyl-␣/β- -mannopyranose
(30): A solution of 29 (0.8 g, 0.41 mmol) in N,N-dimethylformam-
ide (8 mL) was treated with ammonium carbonate (1 g) at 40°C. H), 3.73 (dd, J3c,4c
D-mannopyrano-
1
D
D
HMBC experiments: H NMR (600 MHz): δ ϭ 3.28 (6c-H), 3.30
D
(6d-H), 3.31 (2b-H), 3.40 (dd, 6dЈ-H), 3.50 (5d-H), 3.51 (6f-H), 3.60
(dd, J4a,5a ϭ 10.8, J5a, 6a ϭ 9.0 Hz, 5a-H), 3.66 (6fЈ-H), 3.69 (5e-
3
3
3
ϭ
3J4c,5c ϭ 9.3 Hz, 4c-H), 3.78 (4d-H), 3.79
The reaction was monitored by TLC. After 4 d the mixture was
concentrated in vacuo (0.1 bar), and flash-chromatographed on sil-
(5c-H), 3.84 (3d-H), 3.84 (6e-H), 3.85 (6cЈ-H), 3.86 (4b-H), 3.88
(5f-H), 3.91 (3c-H), 3.94 (3e-H), 3.94 (4f-H), 3.98 (4a-H), 4.00 (3f-
3
3
ica gel (toluene/ethyl acetate, 13:1) to yield 30 (0.67 g, 86%) as an H), 4.00 (6eЈ-H), 4.01 (3b-H), 4.11 (dd, J1a,6a ϭ 3.2, J5a,6a ϭ 9.0
anomeric mixture (α/β ϭ 6:1). Ϫ TLC (petroleum ether/ethyl acet- Hz, 6a-H), 4.12 (2e-H), 4.15 (4e-H), 4.17 (2d-H), 4.19 (5b-H), 4.33
1
3
3
ate, 7:3): Rf ϭ 0.45 (30α) and Rf ϭ 0.28 (30β). Ϫ H NMR (250 (6b-H), 4.40 (3a-H), 4.58 (dd, J1a,2a ϭ 3.2, J2a,3a ϭ 7.5 Hz, 2a-
3
3
MHz; CDCl3): δ ϭ 1.09 [s, 9 H, C(CH3)3], 2.03Ϫ2.12 (several s, 6 H), 4.61 (1d-H), 4.64 (6bЈ-H), 4.97 (dd, J1a,2a ϭ J1a,6a ϭ 3.2 Hz,
3
H, 2 COCH3), 3.36Ϫ4.02 (m, 21 H), 4.10Ϫ4.12 (m, 2 H, 2e-, 2d- 1a-H), 5.09 (s, 1f-H), 5.30 (d, J1b,2b ϭ 3.5 Hz, 1b-H), 5.36 (s, 1e-
H), 4.25Ϫ4.74 (m, 17 H, 8.5 CH2Ph), 4.80Ϫ4.91 (m, 5 H, 1.5 H), 5.42 (s, 1c-H), 5.53 (2c-H), 5.58 (2f-H). Ϫ 13C NMR (150.9
3
CH2Ph, 1c-, 1d-H), 5.09 (d, J1f,2f ϭ 1.5 Hz, 1 H, 1f-H), 5.25 (dd, MHz): δ ϭ 62.94 (C-6e), 63.13 (C-2b), 63.33 (C-6b), 65.68 (C-6c),
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1161

T. G. Mayer, R. R. Schmidt
68.44 (C-6f), 68.58 (C-2c), 68.71 (C-2f), 68.71 (C-5b), 68.71 (C-6d), acetate, 20:1) showed complete turnover. After about 24 h, ethyl
FULL PAPER
71.75 (C-5c), 71.89 (C-5f), 72.00 (C-5d), 72.40 (C-2d), 73.14 (C-4c),
73.14 (C-5e), 74.04 (C-4f), 74.14 (C-4e), 74.32 (C-4d), 74.97 (C-2e),
acetate was added in order to destroy excess of NaH. The solvents
were evaporated in vacuo (0.1 bar) and the residue was purified by
75.54 (C-4b),76.14 (C-3a), 76.36 (C-1a), 76.36 (C-5a), 76.55 (C-6a), MPLC (petroleum ether/ethyl acetate, 5:1) to give 34 (1.07 g, 86%)
77.11 (C-4a), 78.64 (C-3f), 78.68 (C-3c), 79.51 (C-3d), 79.64 (C-3e), as a colorless foam, which was lyophilized from dioxane. Ϫ TLC
80.32 (C-3b), 96.25 (C-1b), 98.69 (C-1d), 99.30 (C-1c), 99.82 (C-1f), (toluene/ethyl acetate, 20:1): Rf ϭ 0.48. Ϫ [α]D ϭ ϩ28 (c ϭ 1,
1
99.90 (C-1e), 154.15 (OCOO), 165.84 (OCOPh), 169.72 (OCOMe),
170.06 (OCOMe). Ϫ PD MS (positive-ion mode; matrix: nitrocellu-
chloroform). Ϫ H NMR (400 MHz; CDCl3): δ ϭ 0.76Ϫ0.78 (d,
3J ϭ 6.6 Hz, 3 H, CH3), 0.84Ϫ1.12 [m, 18 H, 2 CH3, C(CH3)3, 3
lose with NaI): m/z ϭ 2801 [M ϩ Naϩ]. Ϫ C163H187N3O35Si HMnt], 1.20Ϫ1.80 (m, 24 H, 20 Hcycloh., 4 HMnt), 1.91Ϫ2.13 (m, 2
3
3
(2776.35): calcd. C 70.52, H 6.79, N 1.51; found C 70.40, H 6.78, H, 2 HMnt), 3.27 (dd, J1b,2b ϭ 3.6 Hz, J2b,3b ϭ 10.2 Hz, 1 H, 2b-
N 1.90.
H), 3.31Ϫ3.39 (m, 2 H, 2 6-H), 3.45Ϫ4.01 (m, 25 H), 4.04Ϫ4.13
(m, 4 H, 6a-, 2d-H), 4.18Ϫ4.67 (m, 26 H, 11 CH2Ph, 3a-, 2a-, 2-
H, HMnt), 4.77 (s, 1 H, 1d-H), 4.77Ϫ4.92 (m, 6 H, 3 CH2Ph), 4.99
O-(3,4,6-Tri-O-benzyl-␣-
D
-mannopyranosyl)-(1Ǟ2)-O-(3,4-di-O-
-mannopyranosyl)-(1Ǟ2)-O-
-mannopyranosyl)-(1Ǟ6)-O-(3,4-di-O-
-mannopyranosyl)-(1Ǟ4)-O-(2-azido-6-O-benzoyl-3-O-
benzyl-2-deoxy-␣- -glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclo-
hexylidene-1-O-(1R)-menthyloxycarbonyl- -myo-inositol (33) and
O-(3,4,6-Tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4-di-O-
benzyl-6-O-tert-butyldiphenylsilyl-␣- -mannopyranosyl)-(1Ǟ2)-O-
(3,4,6-tri-O-benzyl-␣- -manno-pyranosyl)-(1Ǟ6)-O-(3,4-di-O-
benzyl-␣- -mannopyranosyl)-(1Ǟ4)-O-(2-azido-3-O-benzyl-2-
deoxy-␣- -glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-
O-(1R)-menthyloxycarbonyl- -myo-inositol (32): A suspension of 31
benzyl-6-O-tert-butyldiphenylsilyl-␣-
(3,4,6-tri-O-benzyl-␣-
benzyl-␣-
D
3
(dd, J1a,2a ϭ 3J6a,1a ϭ 3.3 Hz, 1 H, 1a-H), 5.26Ϫ5.28 (m, 3 H, 1b-
D
H, 2 1-H), 5.34 (s, 1 H, 1-H), 6.96Ϫ7.34 (m, 76 H, 14 Ph, m, p-
Ph2Si), 7.65Ϫ7.74 (m, 4 H, o-Ph2Si). Ϫ FAB MS (positive-ion
mode; matrix: NBOH with NaI): m/z ϭ 2882 [M ϩ Naϩ]. Ϫ
C173H197N3O32Si (2858.55): calcd. C 72.69, H 6.95, N 1.47; found
C 72.69, H 6.98, N 2.11.
D
D
D
D
D
D
O-(2,3,4,6-Tetra-O-benzyl-␣-
di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-
(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ6)-O-
(2,3,4-tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-
O-benzyl-2-deoxy-␣- -glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-
cyclohexylidene- -myo-inositol (35) and 1-O-Acetyl-O-(2,3,4,6-
tetra-O-benzyl-␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4-di-O-benzyl-6-
O-tert-butyldiphenylsilyl-␣- -mannopyranosyl)-(1Ǟ2)-O-(3,4,6-tri-
O-benzyl-␣- -mannopyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣-
mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣-
glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-
D
-mannopyranosyl)-(1Ǟ2)-O-(3,4-
D
D
-mannopyranosyl)-
D
D
D
D
(2.4 g, 0.864 mmol) and KCN (0.9 g) in dry diethyl ether/methanol
(30 mL, 1:1) was vigorously stirred at 35°C. The progress of the
reaction was monitored by TLC (toluene/ethyl acetate, 3:1). After
36 h, the reaction mixture, containing about 50% triol 32 (Rf ϭ
0.56) and about 50% diol 33 (Rf ϭ 0.63), was concentrated in va-
cuo, and the residue was chromatographed on silica gel (toluene/
ethyl acetate, 10:1). The crude mixture was purified by MPLC
(toluene/ethyl acetate, 7:1) in order to remove diol 33 (0.97 g, 45%)
to yield triol 32 (1.05 g, 47%) as a colorless oil which was lyophil-
D
D
D
D
D
D
D
-
-
D-myo-inositol
(36): A mixture of 34 (0.41 g, 0.143 mmol) and K2CO3 (0.5 g) in
dry methanol/diethyl ether (1:1, 30 mL) was vigorously stirred for
about 24 h at 40°C. The reaction was monitored by TLC [toluene/
ethyl acetate, 20:1; Rf(35) ϭ 0.22]. After concentration in vacuo,
water was added, and the mixture was extracted twice with ethyl
acetate. The combined organic solutions were dried (Na2SO4) and
concentrated in vacuo to give crude 35 as a colorless oil which was
of sufficient purity for the preparation of 36. Crude 35 was dis-
solved in dry pyridine/acetic anhydride (1:1, 5 mL). After stirring
overnight at room temp., the solution was concentrated in vacuo
(0.1 bar). Flash chromatography (toluene/ethyl acetate, 25:1) of the
residue yielded 36 (319 mg, 82%) as a colorless foam which was
lyophilized from dioxane. Ϫ 35: [α]D ϭ ϩ35 (c ϭ 2, chloroform).
1
ized from dioxane. Ϫ 33: [α]D ϭ ϩ43 (c ϭ 1, chloroform). Ϫ H
NMR (250 MHz; CDCl3): δ ϭ 0.67Ϫ0.70 (d, 3J ϭ 6.9 Hz, 3 H,
CH3), 0.76Ϫ1.02 [m, 18 H, 2 CH3, C(CH3)3, 3 HMnt], 1.19Ϫ1.71
(m, 24 H, 20 Hcycloh., 4 HMnt), 1.81Ϫ2.03 (m, 3 H, 2 HMnt, OH),
2.37 (m, 1 H, OH), 3.20Ϫ3.29 (m, 3 H), 3.35Ϫ4.91 (m), 5.15 (s, 1
H, 1-H), 5.21Ϫ5.23 (m, 2 H, 1-, 1b-H), 5.34 (m, 1 H, 1-H),
6.87Ϫ7.32 (m, 64 H, 11 Ph, m,p-Ph2Si, m,p-COPh), 7.59Ϫ7.69 (m,
4 H, o-Ph2Si) 7.83Ϫ7.87 (m, 2 H, o-COPh). Ϫ 32: [α]D ϭ ϩ36 (c ϭ
1, chloroform). Ϫ 1H NMR (250 MHz; CDCl3): δ ϭ 0.76Ϫ0.79 (d,
3J ϭ 6.9 Hz, 3 H, CH3), 0.81Ϫ1.14 [m, 18 H, 2 CH3, C(CH3)3, 3
HMnt], 1.28Ϫ1.78 (m, 24 H, 20 Hcycloh., 4 HMnt), 1.88Ϫ2.16 (m, 3
H, 2 HMnt, OH), 2.39Ϫ2.44 (m, 2 H, 2 OH), 3.23 (dd, 3J1b,2b ϭ 3.6
1
Ϫ H NMR (250 MHz; CDCl3): δ ϭ 0.99 [s, 9 H, C(CH3)3], 2.75
3
Hz, J2b,3b ϭ 9.7 Hz, 1 H, 2b-H), 3.42Ϫ4.21 (m, 32 H), 4.27Ϫ4.92
3
3
3
(d, J1a,OH ϭ 2.5 Hz, 1 H, OH), 3.28 (dd, J1b,2b ϭ 3.5, J2b,3b
ϭ
3
(m, 26 H, 11 CH2Ph, 3a-, 2a-, 1d-H, HMnt), 4.97 (dd, J1a,2a
ϭ
9.7 Hz, 1 H, 2b-H), 3.31Ϫ3.39 (m, 2 H), 3.46Ϫ3.54 (m, 3 H),
3.60Ϫ4.13 (m, 27 H), 4.18Ϫ4.67 (m, 26 H, 11.5 CH2Ph, 3a-, 2a-H,
3J6a,1a ϭ 3.2 Hz, 1 H, 1a-H), 5.14 (d, 3J1b,2b ϭ 3.6 Hz, 1 H, 1b-H),
5.20Ϫ5.22 (m, 2 H, 2 1-H), 5.39 (br. s, 1 H, 1-H), 7.01Ϫ7.39 (m,
61 H, 11 Ph, m,p-Ph2Si), 7.67Ϫ7.77 (m, 4 H, o-Ph2Si). Ϫ 33:
C159H183N3O33Si (2692.28): calcd. C 70.93, H 6.85, N 1.56; found
C 70.38, H 6.86, N 1.50. Ϫ 32: C152H179N3O32Si (2588.17): calcd.
C 70.54, H 6.97, N 1.62; found C 70.38, H 6.94, N 2.01.
3
2-H), 4.74Ϫ4.94 (m, 6 H, 2.5 CH2Ph, 1d-H), 5.10 (d, J1b,2b ϭ 3.5
Hz, 1 H, 1b-H), 5.29Ϫ5.31 (m, 2 H, 2 1-H), 5.35 (br. s, 1 H, 1-H),
6.96Ϫ7.37 (m, 76 H, 14 Ph, m,p-Ph2Si), 7.64Ϫ7.75 (m, 4 H, o-
Ph2Si). Ϫ 36: TLC (toluene/ethyl acetate, 15:1): Rf ϭ 0.45. Ϫ [α]D ϭ
1
ϩ23 (c ϭ 0.5, chloroform). Ϫ H NMR (400 MHz; CDCl3): δ ϭ
O-(2,3,4,6-Tetra-O-benzyl-␣-
di-O-benzyl-6-O-tert-butyldiphenylsilyl-␣-
(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ6)-O-
(2,3,4-tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-
O-benzyl-2-deoxy-␣- -glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclo-
hexylidene-1-O-(1R)-menthyloxycarbonyl- -myo-inositol (34): So-
dium hydride (90 mg, 3.75 mmol) was cautiously added to a cooled
solution of 32 (1.13 g, 0.437 mmol) in dry N,N-dimethylformamide
(7.5 mL) under nitrogen. After 30 min, benzyl bromide (1.2 mL,
D
-mannopyranosyl)-(1Ǟ2)-O-(3,4-
0.99 [s, 9 H, C(CH3)3], 1.24Ϫ1.67 (m, 20 H, 20 Hcycloh.), 2.10 (s, 3
H, COCH3), 3.25 (dd, J1b,2b ϭ 3.5, J2b,3b ϭ 9.6 Hz, 1 H, 2b-H),
3.31Ϫ3.39 (m, 2 H, 2 6-H), 3.46Ϫ4.12 (m, 29 H), 4.18Ϫ4.66 (m,
3
3
D
-mannopyranosyl)-
D
D
26 H, 11.5 CH2Ph, 3a-, 2a-, 2-H), 4.76Ϫ4.91 (m, 6 H, 2.5 CH2Ph,
3
D
1d-H), 5.13 (dd, J1a,2a
ϭ
3J6a,1a ϭ 3.7 Hz, 1 H, 1a-H), 5.23 (d,
D
3J1b,2b ϭ 3.6 Hz, 1 H, 1b-H), 5.27Ϫ5.28 (m, 2 H, 2 1-H), 5.34 (br.
s, 1 H, 1-H), 6.96Ϫ7.31 (m, 76 H, 14 Ph, m,p-Ph2Si), 7.65Ϫ7.74
(m, 4 H, o-Ph2Si). Ϫ 35: C162H179N3O30Si (2676.28): calcd. C 72.70,
H
6.74, N 1.57; found C 72.90, H 6.61, N 1.65. Ϫ 36:
10.11 mmol) was added dropwise within 10 min. The solution was C164H181N3O31Si (2718.32): calcd. C 72.46, H 6.71, N 1.55; found
warmed up to room temp. and stirred until TLC (toluene/ethyl
1162
C 72.12, H 6.73, N 2.01.
Eur. J. Org. Chem. 1999, 1153Ϫ1165

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
1-O-Acetyl-O-(2,3,4,6-Tetra-O-benzyl-␣-
D
-mannopyranosyl)-
reaction mixture was poured onto a satd. aqueous NaHCO3 solu-
tion and extracted several times with dichloromethane. The com-
bined extracts were dried (Na2SO4) and concentrated in vacuo. The
residue was purified by flash chromatography with petroleum ether/
ethyl acetate (7:3 Ǟ 6:4) to yield 38 (182 mg, 81%) as a colorless
foam. Ϫ TLC (petroleum ether/ethyl acetate, 6:4): Rf ϭ 0.38, Ϫ
(1Ǟ2)-O-(3,4-di-O-benzyl-␣-
tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣-
-mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣-
-glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene- -myo-
D-mannopyranosyl)-(1Ǟ2)-O-(3,4,6-
D
D
D
D
inositol (37): A solution of 36 (160 mg, 58.9 µmol) in dry tetra-
hydrofuran (2 mL) was treated with TBAF (1.1  solution in THF, [α]D
0.4 mL, 440 mmol) and dry acetic acid (26 µL, 440 µmol) at 0°C MHz;
ϭ
ϩ30 (c
ϭ
0.33, chloroform).
1.27Ϫ1.69
Ϫ
1H NMR (250
(m, 20 H,
CDCl3):
δ
ϭ
under nitrogen. Stirring was continued for about 2 d at 40°C until
TLC showed complete disapperance of the substrate. After ad-
dition of satd. aqueous NaHC3 solution and ice, the mixture was
extracted twice with dichloromethane. The combined organic ex-
tracts were dried (Na2SO4), concentrated in vacuo, and filtered
through silica gel (toluene/ethyl acetate, 10:1). MPLC (toluene/ethyl
20 Hcycloh.), 2.08Ϫ2.18 (m, 2 H, CH2CN), 2.11 (s, 3 H, COCH3),
3.20Ϫ4.08 (m, 39 H), 4.18Ϫ4.63 (m, 25 H, 11.5 CH2Ph, 3a-, 2a-
H), 4.75Ϫ4.90 (m, 6 H, 2.5 CH2Ph, 1d-H), 5.00Ϫ5.04 (m, 3 H, 1.5
CH2Ph), 5.14 ( dd, 3J1a,2a ϭ 3J6a,1a ϭ 3.4 Hz, 1 H, 1a-H), 5.19Ϫ5.30
(m, 4 H, 1b-H, 2 1-H, NHCOO), 7.06Ϫ7.34 (m, 75 H, 15 Ph). Ϫ
31P NMR (161.7 MHz; CDCl3): δ ϭ Ϫ0.31, Ϫ0.60. Ϫ FAB MS
acetate, 10:1) of the crude product yielded 37 (129 mg, 89%) as a (positive-ion mode; matrix: NBOH with NaI): m/z ϭ 2814 [M ϩ
colorless foam which was lyophilized from dioxane. Ϫ TLC (tolu-
ene/ethyl acetate, 12:1): Rf ϭ 0.38. Ϫ [α]D ϭ ϩ47 (c ϭ 1, chloro-
form). Ϫ H NMR (250 MHz; CDCl3): δ ϭ 1.31Ϫ1.79 (m, 20 H,
Naϩ].
O-(2,3,4,6-Tetra-O-benzyl-␣-
di-O-benzyl-6-{2-[N-(benzyloxycarbonyl)amino]ethyl
monium
benzyl-␣-
mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣-
glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-
(39): To a solution of 38 (130 mg, 46.59 µmol) in dry methanol/
diethyl ether (1:1, 3 mL), satd. sodium methoxide solution in meth-
anol (5 drops) was added. After stirring for 3 h under TLC control,
the solution was neutralized with Amberlite IR 120 (Hϩ). Evapor-
ation of the solvents in vacuo and chromatography (chloroform/
methanol 100:0 Ǟ 100:7 with 1% triethylamine) yielded 39 (125
mg, 96%) as triethylammonium salt. Ϫ TLC (chloroform/meth-
D
-mannopyranosyl)-(1Ǟ2)-O-[3,4-
triethylam-
D-mannopyranosyl]-(1Ǟ2)-O-(3,4,6-tri-O-
1
20 Hcycloh.), 2.11 (s, 3 H, COCH3), 3.31 (dd, 3J1b,2b ϭ 3.5, 3J2b,3b ϭ
9.5 Hz, 1 H, 2b-H), 3.44Ϫ3.49 (m, 2 H, 2 6-H), 3.52Ϫ4.07 (m, 29
H), 4.16 (br. t, 1 H, 2-H), 4.22Ϫ4.66 (m, 25 H, 11.5 CH2Ph, 3a-,
2a-H), 4.78Ϫ4.91 (m, 5 H, 2.5 CH2Ph), 4.92 (br. s, 1 H, 1d-H),
phosphate}-␣-
-mannopyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣-
D
D
-
-
D
D-myo-inositol
3
3
5.10 (s, 1 H, 1-H), 5.14 (dd, J1a,2a ϭ J6a,1a ϭ 3.6 Hz, 1 H, 1a-H),
3
5.21 (br. s, 1 H, 1-H), 5.26 (d, J1b,2b ϭ 3.5 Hz, 1 H, 1b-H), 5.29
(br. s, 1 H, 1-H), 7.02Ϫ7.35 (m, 70 H, 14 Ph). Ϫ C148H163N3O31
(2479.92): calcd. C 71.68, H 6.62, N 1.69; found C 71.49, H 6.51,
N 1.96.
2-[N-(Benzyloxycarbonyl)amino]ethanol: This compound was pre-
pared according to the procedure described by Rose.[37] Flash chro-
matography (petroleum ether/ethyl acetate, 6:4 Ǟ 1:1, Rf ϭ 0.15)
of the crude product gave pure product.
1
anol, 9:1): Rf ϭ 0.48. Ϫ [α]D ϭ ϩ25 (c ϭ 0.33, chloroform). Ϫ H
NMR (250 MHz; CDCl3/CD3OD, 3:1): δ ϭ 1.04 [t, J ϭ 7.4 Hz,
3
9 H, (CH3CH2)3NHϩ], 1.27Ϫ1.80 (m, 20 H, 20 Hcycloh.), 2.73 [q,
3J ϭ 7.3 Hz, 6 H, (CH3CH2)3NHϩ], 3.18Ϫ4.68 (m, 61 H),
4.72Ϫ5.03 (m, 10 H, 4.5 CH2Ph, 1d-H), 5.20Ϫ5.23 (m, 3 H, 1b-H,
2 1-H), 5.36 (br. s, 1 H, 1-H), 7.04Ϫ7.38 (m, 75 H, 15 Ph). Ϫ
31P NMR (161.7 MHz; CDCl3/CD3OD, 3:1): δ ϭ 4.9. Ϫ FAB MS
(negative-ion mode; matrix: NBOH): m/z ϭ 2694 [MϪ].
{2-[N-(Benzyloxycarbonyl)amino]ethoxy}(2-cyanoethoxy)-
(diisopropylamino)phosphane (5): To a solution of 2-[N-(benzyloxy-
carbonyl)amino]ethanol (690 mg, 3.54 mmol) and 2-cyanoethyl-
N,N,NЈ,NЈ-tetraisopropylphosphorodiamidite (1.6 g, 5.31 mmol) in
dry acetonitrile (15 mL) 1H-tetrazole (75 mg, 1.06 mmol) was ad-
ded. After stirring overnight at room temp., the mixture was diluted
with satd. aqueous NaHCO3 solution and extracted twice with di-
chloromethane. The combined organic extracts were dried
(Na2SO4) and concentrated in vacuo. Flash chromatography (tolu-
ene/ethyl acetate, 10:1 with 1% triethylamine) of the residue yielded
5 (1.23 g, 88%) as a wax. Physical data were in agreement with
those reported by Ogawa et al.[15] Ϫ 1H NMR (250 MHz; CDCl3):
δ ϭ 1.13Ϫ1.20 [2 d, 2 3J ϭ 6.8 Hz, 12 H, 2 CH(CH3)2], 2.61 (t,
3J ϭ 6.3 Hz, 2 H, OCH2CH2CN), 3.39Ϫ3.46 (q, 3J ϭ 3J ϭ 6.2
Hz, 2 H, OCH2CH2NHCOO), 3.52Ϫ3.89 [m, 6 H, 2 CH(CH3)2,
OCH2CH2NHCOO, OCH2CH2CN], 5.12 (s, 2 H, CH2Ph), 5.23
(br. t, 1 H, NHCOO), 7.31Ϫ7.38 (m, 5 H, Ph).
O-(2,3,4,6-Tetra-O-benzyl-␣-
di-O-benzyl-6-{2-[N-(benzyloxycarbonyl)amino]ethyl sodium phos-
phate}-␣- -mannopyranosyl}-(1Ǟ2)-O-(3,4,6-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣- -gluco-
pyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(2S,3S,4R)-2-N-
(hexacosanoyl)amino-3,4-O-isopropylideneoctadecan-3,4-diol-1-yl
sodium phosphate]-D-myo-inositol (40): To a solution of 39 (135 mg,
48.28 µmol), phosphitamide 2 (200 mg, 213.9 µmol), and powdered
molecular sieves (3 A) in dry dichloromethane/acetonitrile (1:1, 6
mL) was added 1H-tetrazole (15 mg, 213.9 µmol) under nitrogen.
The reaction was monitored by TLC chloroform/methanol, 9:1;
Rf(39) ϭ 0.39; Rf(phosphite) ϭ 0.43]. After stirring for 6 h at room
D-mannopyranosyl)-(1Ǟ2)-O-[3,4-
D
D
D
D
˚
1-O-Acetyl-O-(2,3,4,6-Tetra-O-benzyl-␣-D-mannopyranosyl)-
(1Ǟ2)-O-[3,4-di-O-benzyl-6-{2-[N-(benzyloxycarbonyl)amino]ethyl temp., tert-butyl hydroperoxide (3  solution in toluene, 1 mL) was
2-cyanoethyl phosphate}-␣-
O-benzyl-␣- -mannopyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣-
mannopyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣-
glucopyranosyl)-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-
(38): To a mixture of alcohol 37 (200 mg, 80.65 µmol), phos-
D
-mannopyranosyl]-(1Ǟ2)-O-(3,4,6-tri-
added in order to oxidize the phosphorus(III) species. The mixture
was stirred for 2 h at room temp. After addition of brine and satd.
aqueous NaHCO3 solution, the mixture was extracted three times
with dichloromethane. The combined organic extracts were dried
(Na2SO4), concentrated in vacuo, and redissolved in a mixture of
D
D
-
-
D
D-myo-inositol
phoroamidite 5 (200 mg, 505.8 µmol), and powdered molecular dichloromethane (3 mL) and ethanolic dimethylamine solution
˚
sieves (3 A) in dry acetonitrile (4 mL) 1H-tetrazole (11 mg, 157.1 (33% in ethanol, 3 mL). After stirring for 1 h at room temp., the
µmol) was added under nitrogen. After stirring for 1.5 h at room
mixture was concentrated in vacuo. The residue was chromato-
temp., tert-butyl hydroperoxide (3  solution in toluene, 0.3 mL) graphed on silica gel (chloroform/methanol, 100:0 Ǟ 100:2 Ǟ
was added. The progress of the coupling reaction and of the oxi-
dation was monitored by TLC [petroleum ether/ethyl acetate, 6:4;
Rf(37) ϭ 0.74; Rf(phosphite) ϭ 0.68; Rf(38) ϭ 0.38]. After 2 h, the
100:3) to yield diphosphate 40. The disodium salt of 40 (101 mg,
60%) was obtained by treating a solution of 40 in chloroform/meth-
anol (1:1.4 mL) with ion-exchange resin (Amberlite IR 120, Naϩ
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1163

T. G. Mayer, R. R. Schmidt
FULL PAPER
form). Ϫ TLC (chloroform/methanol, 9:1): Rf ϭ 0.32, Ϫ [α]D
ϭ
glycerine, 1:1): m/z ϭ 1872 [M ϩ Hϩ]. Ϫ PD MS (positive-ion
mode, matrix: nitrocellulose with NaI): m/z ϭ 1939 [M2Ϫ ϩ 3 Naϩ].
ϩ43 (c ϭ 2.5, chloroform). Ϫ 1H NMR (250 MHz; CDCl3/
CD3OD, 1:1): δ ϭ 0.76Ϫ0.82 (t, 3J ϭ 6.5 Hz, 6 H, 2 CH3),
1.12Ϫ1.82 (m, 98 H, CMe2, 20 Hcyclohex., 36 CH2), 2.07 (t, 3J ϭ 7.2
Hz, 2 H, COCH2), 3.25Ϫ4.87 (m), 4.71 (1d-H), 4.92 (br. s, 1 H, 1-
Acknowledgments
3
H), 5.02 (br. s, 1 H, 1-H), 5.14 (s, 1 H, 1-H), 5.48 (d, J1b,2b ϭ 3.3
Hz, 1 H, 1b-H), 7.01Ϫ7.30 (m, 75 H, 15 Ph). Ϫ 31P NMR (161.7
MHz; CDCl3/CD3OD, 1:1): δ ϭ Ϫ0.36, Ϫ3.90. Ϫ FAB MS (nega-
tive-ion mode, matrix: NBOH with NaI): m/z ϭ 3514 [M2Ϫ ϩ Naϩ]
and 3664 [M2Ϫ ϩ NaI ϩ Naϩ].
This work was supported by the Deutsche Forschungsgemein-
schaft and the Fonds der Chemischen Industrie. T. G. M. gratefully
acknowledges a fellowship from the Landesgraduiertenförderung
Baden-Württemberg. We are grateful to Dr. A. Geyer for DQF-
COSY, HMQC, and HMBC experiments.
O-(2,3,4,6-Tetra-O-benzyl-␣-
di-O-benzyl-6-{2-[N-(benzyloxycarbonyl)amino]ethyl triethylammo-
nium phosphate}-␣- -mannopyranosyl]-(1Ǟ2)-O-(3,4,6-tri-O-ben-
zyl-␣- -mannopyranosyl)-(1Ǟ6)-O-(2,3,4-tri-O-benzyl-␣- -manno-
pyranosyl)-(1Ǟ4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-␣- -gluco-
pyranosyl)-(1Ǟ6)-1-[(2S,3S,4R)-2-N-(hexacosanoyl)amino-3,4-dihy-
droxy-1-yl triethylammonium phosphate]- -myo-inositol (41): To a
D-mannopyranosyl)-(1Ǟ2)-O-[3,4-
[1]
D
For a preliminary publication of a part of this work, see: T. G.
Mayer, B. Kratzer, R. R. Schmidt, Angew. Chem. 1994, 106,
2289Ϫ2293; Angew. Chem. Int. Ed. Engl. 1994, 33, 2177Ϫ2181.
D
D
D
[2]
M. A. J. Ferguson, A. F. Williams, Annu. Rev. Biochem. 1988,
57, 285Ϫ320; M. A. J. Ferguson, Curr. Opin. Struct. Biol. 1991,
1, 522Ϫ529.
D
[3]
R. Thomas, R. A. Dwek, T. W. Rademacher, Biochemistry 1990,
solution of 40 (50 mg, 14.14 µmol) and ethylene glycol (0.1 mL)
in dry acetonitrile/dichloromethane (1:1, 3 mL) (1S)-camphor-10-
sulphonic acid (20 mg) was added to adjust the pH to 1. After
stirring at room temp. for 4 h, the reaction was completed. The
solution was neutralized by addition of triethylamine and then con-
centrated in vacuo (0.1 mbar). Brine and water were added and the
mixture was extracted three times with chloroform. The combined
organic solutions were dried (MgSO4) and concentrated in vacuo.
Chromatography (chloroform/methanol, 100:0 Ǟ 100:8 Ǟ 9:1 with
1% triethylamine) of the residue gave oily 41 (31 mg, 63%) as
bis(triethylammonium) salt. Ϫ TLC (chloroform/methanol, 8:2):
29, 5413Ϫ5422; G. A. M. Cross, Annu. Rev. Cell. Biol. 1990, 6,
1Ϫ39; M. C. Field, Glycoconjugate J. 1992, 9, 155Ϫ159; A. M.
Tartakoff, N. Singh, Trends Biochem. Sci. 1992, 17, 470Ϫ473.
[4]
M. A. J. Ferguson, S. W. Homans, R. A. Dwek, T. W. Rade-
macher, Science 1988, 239, 753Ϫ759.
[5]
S. W. Homans, M. A. J. Ferguson, R. A. Dwek, T. W. Rade-
macher, R. Anand, A. F. Williams, Nature 1988, 333, 269Ϫ272.
[6]
M. A. J. Ferguson, G. A. M. Cross, J. Biol. Chem. 1985, 260,
14547Ϫ14555.
[7]
M. C. Field, G. A. M. Cross, J. Biol. Chem. 1991, 266,
8392Ϫ8400.
[8]
W. L. Roberts, T. L. Rosenberry, J. Biol. Chem. 1988, 263,
18776Ϫ18784.
1
[9]
Rf ϭ 0.48. Ϫ [α]D ϭ ϩ39 (c ϭ 2, chloroform). Ϫ H NMR (250
C. A. Luhrs, B. C. Slomiany, J. Biol. Chem. 1989, 264,
3
21446Ϫ21449.
MHz; CDCl3/CD3OD, 3:1): δ ϭ 0.86Ϫ0.92 (t, J ϭ 6.9 Hz, 6 H, 2
[10]
P. Butikofer, F. A. Knypers, C. Shackleton, J. Biol. Chem. 1990,
CH3), 1.17 [t, 3J ϭ 7.2 Hz, 18 H, (CH3CH2)3NHϩ], 1.21Ϫ1.63 (m,
265, 18983Ϫ18987.
3
3
[11]
72 H, 36 CH2), 2.17 (t, J ϭ 7.5 Hz, 2 H, COCH2), 2.91 [q, J ϭ
7.3 Hz, 12 H, (CH3CH2)3NHϩ], 3.14Ϫ4.61 (m, 65 H), 4.69Ϫ4.99
(m, 11 H, 5 CH2Ph, 1d-H), 5.21 (s, 1 H, 1-H), 5.23 (br. s, 1 H, 1-
A. Conzelmann, A. Puoti, R. L. Lester, C. Desponds, EMBO
J. 1992, 11, 457.466; C. Fankhauser, S. W. Homans, J. E.
Thomas Oates, M. J. McConville, C. Desponds, A. Conzel-
mann, M. A. J. Ferguson, J. Biol. Chem. 1993, 268,
26365Ϫ26374.
3
H), 5.31 (s, 1 H, 1-H), 5.48 (d, J1b,2b ϭ 3.0 Hz, 1 H, 1b-H),
7.03Ϫ7.35 (m, 75 H, 15 Ph). Ϫ 31P NMR (161.7 MHz; CDCl3/
CD3OD, 3:1): δ ϭ 4.19, 4.92.
[12]
A ceramide anchor was also found for D-discoideum “Con-
tactside-A” protein: J. Stadler, G. Bauer, EMBO J. 1989, 8,
371Ϫ377.
[13]
O-(␣-
phosphate)-␣-
(1Ǟ6)-O-(␣-
glucopyranosyl)-(1Ǟ6)-1-[(2S,3S,4R)-2-N-(hexacosanoyl)amino-
3,4-dihydroxyoctadecan-1-yl hydrogen phosphate]- -myo-inositol
D
-Mannopyranosyl)-(1Ǟ2)-O-[6-(2-aminoethyl hydrogen
-mannopyranosyl]-(1Ǟ2)-O-(␣- -mannopyranosyl)-
-mannopyranosyl)-(1Ǟ4)-(2-amino-2-deoxy-␣-
M. J. McConville, M. A. J. Ferguson, Biochem. J. 1993, 294,
D
D
305Ϫ324.
[14]
A. R. Saltiel, J. A. Fox, P. Sherline, P. Cuatrecasas, Science 1986,
D
D-
233, 967Ϫ972; G. Romero, L. Luttrell, A. Rogol, K. Zeller, E.
Hewlett, J. Larner, Science 1988, 240, 509Ϫ511; H.-H. Caro,
D
´
M. Martin-Lomas, M. Bernabe, Carbohydr. Res. 1993, 240,
(1): To a solution of 42 (25 mg, 7.15 µmol) in chloroform/methanol/
water (1:1:9.25, 3 mL) PearlmanЈs catalyst (8 mg) was added and
the mixture was stirred at room temp. for 2 h under hydrogen (1
bar). Then methanol (0.25 mL) and water (0.25 mL) were added
and hydration was continued overnight. After evaporation of the
solvents in vacuo (0.1 mbar), the residue was chromatographed on
silica gel (ethanol/n-butanol/30% aqueous NH3/water, 2:2:1.05 Ǟ
2:2:1:1.5) to give GPI anchor 1 (9 mg, 70%). Ϫ TLC (EtOH/
nBuOH/conc. NH3/H2O, 2:2:1.5:1): Rf ϭ 0.60. Ϫ 1H NMR (250
MHz; [D6]DMSO/D2O, 40:1, T ϭ 60°C): δ ϭ 4.86, 4.91, 5.02, 5.16
(d, J < 1 Hz, s, s, d J < 1 Hz, 1c, d, e, f-H), 5.42 d (J ഠ 3 Hz, 1b-
H). Ϫ Data based on DQF-COSY and NOESY experiments in
SDS micelles: δ ϭ 4.18 (1a-H), 4.07 (2a-H), 3.56 (3a-H), 3.59 (4a-
H), 3.42 (5a-H), 3.84 (6a-H); 5.44 (d, J1b,2b ഠ 3 Hz, 1b-H), 3.24
(2b-H), 4.00 (3b-H), 3.64 (4b-H), 4.11 (5b-H), 3.70, 3.79 (6b- and
6bЈ-H), 5.15 (s, 1c-H), 4.02 (2c-H), 3.76 (3c-H), 3.70, 3.79 (6c-, 6cЈ-
H), 5.00 (s, 1d-H), 3.94 (2d-H); 5.22 (s, 1e-H), 4.05 (2e-H), 3.90
(3e-H), 3.73 (4e-H), 4.97 (s, 1f-H), 3.99 (2f-H), 3.77 (3f-H). Ϫ 31P
NMR (161.7 MHz; [D6]DMSO, 60°C): δ ϭ 0.79, 1.41. Ϫ FAB MS
(negative-ion mode, matrix: NBOH/glycerine, 1:1): m/z ϭ 1868
[M2Ϫ ϩ Hϩ]. Ϫ FAB MS (positive-ion mode, matrix: NBOH/
119Ϫ131.
[15]
[16]
In the published synthesis of the Trypanosoma crucei GPI
anchor the enantiomer of the naturally occurring inositol moi-
ety was selected: C. Murakata, T. Ogawa, Carbohydr. Res. 1992,
234, 75Ϫ91; Carbohydr. Res. 1992, 235, 95Ϫ114.
R. R. Mootoo, P. Konradsson, B. Fraser-Reid, J. Am. Chem.
Soc. 1989, 111, 8540Ϫ8542; U. E. Udodong, R. Madsen, C.
Roberts, B. Fraser-Reid, J. Am. Chem. Soc. 1993, 115,
7886Ϫ7887; A. Asarpan, B. Fraser-Reid, J. Org. Chem. 1996,
61, 2401Ϫ2406.
[17]
[18]
[19]
R. Verduyn, C. J. J. Elie, C. E. Dreef, G. A. van der Marcl, J.
H. van Boom, Recl. Trav. Chim. Pays-Bas 1990, 109, 591Ϫ593.
R. Plourde, M. dЈAlarcao, A. R. Saltiel, J. Org. Chem. 1992,
57, 2606Ϫ2610.
A. Zapata, M. Martin-Lomas, Carbohydr. Res. 1992, 234,
93Ϫ106; N. Khiar, M. Martin-Lomas, J. Org. Chem. 1995, 60,
7017Ϫ7021.
S. V. Ley, L. L. Young, Synlett 1992, 997Ϫ998; G. J. Boons, P.
Grice, R. Leslie, S. V. Ley, L. L. Young, Tetrahedron Lett. 1993,
34, 8523Ϫ8526.
B. Kratzer, T. G. Mayer, R. R. Schmidt, Tetrahedron Lett. 1993,
34, 6881Ϫ6884; Eur. J. Org. Chem. 1998, 291Ϫ298.
A. Y. Frantova, A. E. Stepanov, A. S. Bushnev, E. N. Zvonkova,
V. I. Slwets, Tetrahedron Lett. 1992, 33, 3539Ϫ3542.
T. Ziegler, R. Dettmann, M. Duszenko, V. Kolb, Carbohydr.
Res. 1996, 295, 7Ϫ23.
[20]
[21]
[22]
[23]
1164
Eur. J. Org. Chem. 1999, 1153Ϫ1165

Total Synthesis of a GPI Anchor of Yeast
FULL PAPER
226, 219Ϫ231; H. B. Boren, G. Ekborg, K. Ekland, Acta Chem.
Scan. 1973, 27, 2639Ϫ2644.
[24]
´
M. M. Silva, J. Cleophac, A. Benicio, M. V. Almeida, J.-M.
Delaumeny, A. S. Machado, S. D. Gero, Synlett 1996, 764Ϫ766.
[32]
[33]
[25]
M. M. Ponpipom, Carbohydr. Res. 1977, 59, 311Ϫ317.
Direct transformation of 10 into 12α,β with silica gel for flash
chromatography could be also accomplished: V. Ferrieres, un-
published results.
P. J. Garegg, P. Konradsson, S. Oscarson, K. Ruda, Tetrahedron
1997, 53, 17727Ϫ17734.
[26]
T. G. Mayer, Dissertation, Universität Konstanz, 1996.
[27]
A. S. Campbell, B. Fraser-Reid, J. Am. Chem. Soc. 1995, 117,
[34]
[35]
´
G. Zemplen, Ber. Dtsch. Chem. Ges. 1927, 60, 1554Ϫ1564.
10387Ϫ10388.
L. F. Fieser, M. Fieser, Reagents for Organic Synthesis, John
Wiley and Sons Inc., New York, 1967, vol. 1, p. 782.
R. R. Schmidt, Angew. Chem. 1986, 98, 213Ϫ236; Angew.
Chem. Int. Ed. Engl. 1986, 25, 212Ϫ235; R. R. Schmidt, W.
Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50, 21Ϫ123.
W. G. Rose, J. Am. Chem. Soc. 1947, 69, 1384Ϫ1387.
Received November 2, 1998
[28]
D. Tailler, V. Ferrieres, K. Pekari, R. R. Schmidt, Tetrahedron
Lett. 1999, 40, 679Ϫ682.
S. V. Ley, XIXth International Carbohydrate Symp., San Diego,
Aug. 1998, Abstract BK 006.
R. R. Schmidt, T. Maier, Carbohydr. Res. 1988, 174, 169Ϫ179.
E. Fischer, R. Oetker, Ber. Dtsch. Chem. Ges. 1913, 46,
[36]
[37]
[29]
[30]
[31]
4029Ϫ4040; K. J. Kaur, O. Hindsgaul, Carbohydr. Res. 1992,
[O98490]
Eur. J. Org. Chem. 1999, 1153Ϫ1165
1165