Synthesis of labeled glycosyl phosphatidyl inositol (GPI) anchors

Article abstract of DOI:10.1002/(sici)1099-0690(199910)1999:10<2563::aid-ejoc2563>3.0.co;2-i

The exploration of the molecular and structural basis for the sorting of GPI-anchored proteins is based on labeled partial structures of GPI's which can be incorporated into the GPI anchor biosynthesis and cellular transport systems. To this end, from mannosyl donor 6 and the D-glucosaminyl-(1→6)D- myo-inositol derivative 7 as acceptor, the pseudotrisaccharide 8 was prepared. Compound 8 was transformed into the GPI partial structures 5a,b which contain the pseudotrisaccharide ligated to two different phosphatidyl residues. Compounds 5a,b have Boc protection at the 2- amino group of the glucosamine residue (2b-position) and a free amino group at the 6b-position. The 6b-amino group was used for the ligation of the 3-(7-nitrobenzofurazan-4- yl)aminopropanoyl group as a fluorescent label, the 5-azido-2-nitrobenzoyl and 4-azidophenylaminothiocarbonyl groups as photolabels, and the 4-azido-2- hydroxybenzoyl group as a radiolabel after the introduction of radioactive iodine by an electrophilic aromatic substitution. Thus, after acid-catalyzed removal of the protective groups, the unprotected target molecules 1-4 were obtained.

Full text of DOI:10.1002/(sici)1099-0690(199910)1999:10<2563::aid-ejoc2563>3.0.co;2-i

FULL PAPER
Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors
Thomas G. Mayer,[a] Ralf Weingart,[a] Florian Münstermann,[a] Toshinari Kawada,[a]
Teymuras Kurzchalia,[b] and Richard R. Schmidt*[a]
Keywords: Carbohydrates / Phospholipids / Glycolipids / Diacylglycerolphosphates / Glycophosphoinositol anchors
The exploration of the molecular and structural basis for the
amino group of the glucosamine residue (2b-position) and a
sorting of GPI-anchored proteins is based on labeled partial free amino group at the 6b-position. The 6b-amino group was
structures of GPIЈs which can be incorporated into the GPI
anchor biosynthesis and cellular transport systems. To this
used for the ligation of the 3-(7-nitrobenzofurazan-4-yl)-
aminopropanoyl group as a fluorescent label, the 5-azido-2-
end, from mannosyl donor 6 and the
-myo-inositol derivative as acceptor, the pseudotri-
saccharide 8 was prepared. Compound 8 was transformed radiolabel after the introduction of radioactive iodine by an
D-glucosaminyl-(1Ǟ6)- nitrobenzoyl and 4-azidophenylaminothiocarbonyl groups as
D
7
photolabels, and the 4-azido-2-hydroxybenzoyl group as a
into the GPI partial structures 5a,b which contain the
pseudotrisaccharide ligated to two different phosphatidyl
electrophilic aromatic substitution. Thus, after acid-catalyzed
removal of the protective groups, the unprotected target
residues. Compounds 5a,b have Boc protection at the 2- molecules 1–4 were obtained.
Many cell surface proteins are anchored by glycosyl labels were desirable for the biological studies; therefore
phosphatidylinositols (GPIЈs) to the cell surface mem- compounds 1؊4 were chosen as target molecules.
brane.[1][2] This protein modification is involved in intra-
molecular signaling and seems to act as an apical targeting
signal for proteins in epithelial cells.[3] However, the mecha-
nism by which the GPI anchors confer the ability to sort
membrane proteins is unknown, and the importance of spe-
cialized membrane domains in this process remains to be
elucidated.[4][5]
Labeled GPI partial structures should be important tools
for the exploration of the cellular and molecular basis for
the sorting of GPI-anchored proteins because their fate can
be followed in the endocytic and exocytic pathways of both
polarized and nonpolarized cells.[5][6] Based on the bio-
synthesis of GPI anchors,[1,2,7] compound A (Scheme 1),
with one mannosyl residue at the glucosaminylinositol
phosphatide moiety, seemed to be a particularly versatile
intermediate for these studies as it is present in almost all
species-specific variations of the GPI anchor biosynthesis.
Thus, it should be incorporated into the GPI anchor bio-
synthesis and cellular transport systems. For the label at-
tachment, the 6-position of the glucosamine residue (6b-
position) was chosen because this position is at a reasonable
distance from the membrane surface and from the site of
further elongation of the GPI anchor. Therefore, modifi-
cation at the 6b-position of A should be tolerated by the
biological systems involved in GPI anchor metabolism and
transport. In order to generate a firm connection between
the 6b-position and the label, replacement of the 6b-
hydroxy group by an amino group (Ǟ B) was performed,
Scheme 1. Target molecules
Synthesis of the 6b-Amino-6-deoxy GPI Anchor
Intermediates 5a,b
For the synthesis of compounds 1؊4 intermediate B is
thus providing a convenient means for attaching the labels not useful because the presence of two amino groups will
by amide bonds. Fluorescent labels, photolabels and radio- prevent regioselective label attachment at the 6b-position.
Additionally, the sensitivity of the envisaged labels to hy-
drogenolytic conditions requires that protective groups
which are cleaved by hydrogenolysis are removed prior to
label attachment. The known stability of the labels to mild
acid treatment, led to the choice of compounds 5a,b
[a]
Fakultät Chemie, Universität Konstanz, Fach M 725,
D-78465 Konstanz, Germany
[b]
Max-Delbrück-Centrum für molekulare Medizin,
Robert-Rössle-Str. 10, D-13122 Berlin-Buch, Germany
Eur. J. Org. Chem. 1999, 2563Ϫ2571
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/1010Ϫ2563 $ 17.50ϩ.50/0
2563

R. R. Schmidt et al.
FULL PAPER
(Scheme 2) as decisive intermediates for the label attach-
ment. Compounds 5a,b were also selected as targets be-
cause readily accessible building blocks, employed in our
previous successful GPI anchor syntheses,[8Ϫ11] can be also
utilized here. For the lipidic part two different chain lengths
(C16, C14) were chosen because cellular uptake and trans-
port are highly dependent on the lipid composition.[12]
Scheme 2. Decisive intermediates
The synthesis of the required α-glucosaminyl(1Ǟ6)inosi-
tol building block 7 (Scheme 3), which permits regioselec-
tive access to the 4b- and also 6b- and 1a-O-positions, was
performed as previously described.[11] For the attachment
of the mannosyl residue the known donor 6[8][11] was selec-
ted. The efficiency of the synthesis of 6 could be improved
by the direct and almost quantitative transformation of
3,4,6-tri-O-benzyl-1,2-O-[1-(1R)-methoxyethylidene]-β--
mannopyranose[13] into 2-O-acetyl-3,4,6-tri-O-benzyl-α.β-
-mannopyranose,[8,11,14] the precursor for the synthesis of
6. Reaction of 6 with 7 as acceptor in diethyl ether as sol-
vent, in the presence of tin(II)trifluoromethanesulfonate as
catalyst, afforded the desired α-linked pseudotrisaccharide
8 in 91% yield [1H NMR: δ ϭ 5.47 (d, 1 H, J1,2 ϭ 1.7 Hz,
1c-H), 5.33 (d, 1 H, J1,2 ϭ 3.6 Hz, 1b-H)]. The 2-O-acetyl
group could be selectively removed by treatment with meth-
ylamine in ethanol (Ǟ 9). Subsequent 2c-O-benzylation
with benzyl bromide in the presence of NaH as base af-
forded compound 10 in high yield. Next, the 2b-azido
group was transformed into the amino function by treat-
ment with propanedithiol in pyridine/water in the presence
of catalytic amounts of triethylamine,[15] and the ensuing
reaction with di-tert-butyl-dicarbonate (Boc2O)[16] afforded
the desired N-Boc protected derivative 11.
Scheme 3. Synthesis of 5a, b
The regioselective removal of the 6b-O-benzoyl group
from 11 turned out to be a critical step. Treatment with
potassium cyanide as base, in methanol, gave the desired
6b-O-deprotected compound 12. However, the reaction had
potassium carbonate as base, in methanol, thus providing
the 1a-O-unprotected compound 14.
Attachment of the phosphatidyl residues to 14 was per-
to be carefully monitored and stopped after 50% com- formed with the phosphitamides 15a,b, which were readily
pletion otherwise partial loss of the menthyloxycarbonyl obtained from bis(diisopropylamino)cyanoethoxyphos-
(Mntoc) group also occurred. After chromatography, 12 phane[18] and 1,2-di-O-palmitoyl- or 1,2-di-O-myristoyl-sn-
could be isolated in 46% yield and 40% of the starting mate- glycerol.[19] Reaction of 14 with 15a,b in the presence of
rial 11 could be recovered. For the azide introduction at the tetrazole followed by oxidation with tert-butylhydroper-
6b-position, a modified Mitsunobu reaction was em- oxide led to the corresponding phosphotriesters, which
ployed:[17] treatment of the diisopropylazodicarboxylate gave, on treatment with dimethylamine in ethanol, the dies-
(DIAD)/triphenylphosphane-activated 6b-hydroxy group ters 16a and 16b. Hydrogenolysis with palladium hydroxide
with the zinc azide pyridine complex[17] as azide group do- on carbon as catalyst (PearmanЈs catalyst[20]), in a mixture
nor afforded directly the 6b-azido compound 13. The men- of dichloromethane/methanol/water as solvent, led to clean
thyloxy carbonyl group was then removed by addition of O-debenzylation and to concomitant liberation of the am-
2564
Eur. J. Org. Chem. 1999, 2563Ϫ2571

Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors
FULL PAPER
ino group in the 6b-position, thus providing target mol- tography on silica gel with a mixture of butyl alcohol/etha-
ecules 5a and 5b in high yields.
nol/aqueous ammonia as eluent, 1a and 1b were obtained
in satisfactory yields. The structures could be fully assigned
based on DQF-COSY and HMBC NMR correlations (see
Experimental Section).
Attachment of the Labels to 5a,b
The 5-azido-2-nitrobenzoyl (ANB) group is an excellent
crosslinker because photoactivation is possible at
320Ϫ350 nm and the reactivity of the generated arylnitrene
intermediate is very high.[22] Therefore the activated 5-az-
ido-2-nitrobenzoate derivative 20 (Scheme 5) was prepared
following literature procedures.[22] Reaction of 20 with 5a
in DMF as solvent and in the presence of triethylamine led
to amide bond formation affording 21a in high yield. Ensu-
ing de-O-cyclohexylidenation and removal of the N-Boc
group was performed as described above furnishing the tar-
get molecule 2a in 64% yield. The structural assignment was
again based on DQF-COSY and HMBC correlations.
From the various fluorophores which have been em-
ployed for labeling experiments, the 7-nitrobenzofurazan
(NBD) moiety seemed to be particularly interesting because
it combines compactness with high sensitivity.[21] In order
to increase the distance to the GPI anchor structure a β-
alanine spacer was inserted. Thus, commercially available 4-
chloro-7-nitro-benzofurazan was treated with β-alanine in
the presence of sodium hydrogencarbonate to give the sub-
stitution product 17 (Scheme 4). Activation of the acid moi-
ety by treatment with N-hydroxysuccinimide (NHS) in the
presence of the water-soluble carbodiimide EDC as con-
densing agent gave 18. Reaction of 5a or 5b with 18 in
DMF in the presence of triethylamine as base led to attach-
ment of the NBD residue affording compounds 19a and
19b. Their treatment with camphorsulfonic acid (CSA) in
the presence of ethylene glycol, in order to facilitate cyclo-
hexylidene cleavage, and then with trifluoroacetic acid
(TFA) and ensuing neutralization with triethylamine af-
forded the target molecules 1a and 1b. After flash chroma-
Scheme 5. Synthesis of 2a and 3a
Isothiocyanate addition to amino groups has been exten-
sively employed for label attachment to peptides and pro-
Scheme 4. Synthesis of 1a, b
Eur. J. Org. Chem. 1999, 2563Ϫ2571
2565

R. R. Schmidt et al.
FULL PAPER
teins. Therefore, direct reaction of 5a with 4-azidophenyl immobilized chloramine T (IODO BEADS)[25]; the FAB-
isothiocyanate[23] was investigated. In DMF as solvent and MS data of the product indicated that target molecule 4a
in the presence of triethylamine the 4-azidophenyl thiourea had been generated [m/z ϭ 1420 (M. Ϫ Hϩ), 1407 (M. Ϫ
derivative 22a was obtained, which exhibits a shoulder in Hϩ Ϫ N), 1392 (M. Ϫ Hϩ Ϫ N2)].
the UV absorption spectrum at 320 nm, which can be used
Successful biological studies have been most recently per-
for the photoactivation. Compound 22a was immediately formed with target molecule 1a,[26] which confirmed the
deprotected as described above to afford the target molecule utility of the conceptual approach to cellular sorting studies
3a in 48% overall yield. Compound 3a could also be fully with the help of the target molecules described in this paper.
structurally assigned.
Iodine isotopes are often chosen as radioactive labels for
biological studies because they can be readily introduced
Experimental Section
into phenols by electrophilic aromatic substitution at the
General: Solvents were purified in the usual way; boiling range of
position ortho to the hydroxy group; an additional advan-
petroleum ether: 35Ϫ65°C. Ϫ Optical rotations: PerkinϪElmer
polarimeter MC; 1 dm cell. Ϫ Thin-layer chromatography: Plastic
tage of this choice is that γ-radiation of 125I with a half life
of 60 days makes this radiolabel readily detectable.[24]
A
sheets, silica gel 60 F254 (Merck; layer thickness 0.2 mm). Ϫ Flash
chromatography: Silica gel (J.T. Baker particle size 40 µm). Ϫ
1H 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 Elemental analyses: Heraeus CHN-O-Rapid.
photoreactive compound which can be radiolabeled with
125I is 4-azido salicylic acid. On activation with NHS this
salicylic acid gave intermediate 23 (Scheme 6), which under-
went reaction with 5a in the presence of triethylamine and
led to the amide derivative 24a. The removal of all protec-
tive groups under the conditions described above furnished
the desired 6b-(4-azido-2-hydroxybenzoylamido-6-deoxy)
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣/β-
acetimidate (6): 3,4,6-Tri-O-benzyl-1,2-O-[1-(1R)-methoxyethylid-
D-mannopyranosyl)trichloro-
compound 25a. Compounds 24a and 25a were fully charac- ene]-β--mannopyranose[13] (21.4 g) was stirred for 2 h in 60%
acetic acid (950 mL) and then the acetic acid was removed by evap-
oration. Flash chromatography of the residue yielded 2-O-acetyl-
3,4,6-tri-O-benzyl-α.β--mannopyranose[8,11,14] (20.8 g, 98%). An
anomeric ratio of α:β (6.5:1) was determined by NMR spec-
troscopy. TLC (petroleum ether/ethyl acetate, 2:1): Rf ϭ 0.28 (α-
terized by NMR spectroscopy and mass spectrometry. Di-
rect iodination of 25a was performed on a small scale with
1
anomer), Rf ϭ 0.18 (β-anomer). Ϫ H NMR (250 MHz, CDCl3):
δ ϭ 2.14 (s, COCH3), 2.19 (s, COCH3), 3.42Ϫ3.78 (m), 3.99Ϫ4.09
(m), 4.45Ϫ4.86 (m, CH2Ph, 1-H), 5.19 (br. s, 1-H), 5.35 (dd, 3J1,2 ϭ
3
3
3
1.9 Hz, J2,3 ϭ 3.2 Hz, 2-H), 5.44 (dd, J1,2 ϭ 1 Hz, J2,3 ϭ 2 Hz,
2-H), 7.13Ϫ7.35 (m, 15 H, Ph). Ϫ C29H32O7 (492.6): calcd. C 70.71,
H 6.55; found C 70.03, H 6.27. Ϫ This compound was transformed
into 6 as previously described.[11]
O-(2-O-Acetyl-3,4,6-tri-O-benzyl-␣-
(2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-␣-
(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyloxycarbonyl-
-myo-inositol (8): To a solution of compounds 7[11] (3.4 g,
D
-mannopyranosyl)-(1Ǟ4)-O-
D
-glucopyranosyl)-
D
3.76 mmol) and 6 (3.4 g, 5.34 mmol) in dry diethyl ether were ad-
ded powdered molecular sieves and the mixture stirred for 15 min
under argon. Tin(II) trifluoromethanesulfonate (63 mg, 0.15 mmol)
was then added and the reaction was stopped after 20 min by neu-
tralization with solid NaHCO3. After evaporation of the solvent,
the residue was purified by flash chromatography (petroleum ether/
ethyl acetate, 6:1) to yield 8 (4.7 g, 91%). Ϫ TLC (petroleum ether/
ethyl acetate, 4:1): Rf ϭ 0.50. Ϫ [α]D ϭ ϩ52 (c ϭ 1, CHCl3). Ϫ 1H
NMR (250 MHz, CDCl3): δ ϭ 0.74Ϫ0.77 (d, 3J ϭ 6.9 Hz, 3 H,
CH3), 0.81Ϫ1.08 (2 d, m, 9 H, 2 CH3, 3 HMnt), 1.30Ϫ1.78 (m, 24
H, 20 HCycloh., 4 HMnt), 1.88Ϫ2.09 (m, 2 H, 2 HMnt), 1.98 (s, 3 H,
3
COCH3), 3.37Ϫ3.43 (m, 2 H, 2b-, 6c-H), 3.60 (dd, J4a,5a
ϭ
3
3
10.8 Hz, J5a,6a ϭ 8.7 Hz, 1 H, 5a-H), 3.68 (dd, J5c,6cЈ ϭ 2.9 Hz,
Jgem ϭ 10.6 Hz, 1 H, 6cЈ-H), 3.75 (m 1 H, 5c-H), 3.89Ϫ3.93 (m 3
H, 3c-, 4c-H), 3.96Ϫ4.04 (m 3 H, 4a-, 3b-, 4b-H), 4.09 (dd, 3J1a,6a ϭ
3
2.9 Hz, J5a,6a ϭ 8.6 Hz, 1 H, 6a-H), 4.15Ϫ4.20 (m, 2 H, 0.5
CH2Ph, 5b-H), 4.37Ϫ4.61( m, 7 H, 1.5 CH2Ph, 2a-, 3a-, 6b-H,
HMnt), 4.67Ϫ4.77 (m, 3 H, CH2Ph, 6bЈ-H), 4.82 (d, Jgem ϭ 10.7 Hz,
1 H, 0.5 CH2Ph), 4.93 (d, Jgem ϭ 10.7 Hz, 1 H, 0.5 CH2Ph), 4.96
(dd, 3J1a,2a ϭ 3J1a,6a ϭ 3.8 Hz, 1 H, 1a-H), 5.33 (d, 3J1b,2b ϭ 3.6 Hz,
3
1 H, 1b-H), 5.47 (d, J1c,2c ϭ 1.7 Hz, 1 H, 1c-H), 5.54 (br. s, 1 H,
Scheme 6. Synthesis of 4a
2c-H), 7.06Ϫ7.48 (m, 22 H, 4 Ph, m-COPh), 7.55Ϫ7.59 (m, 1 H,
Eur. J. Org. Chem. 1999, 2563Ϫ2571
2566

Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors
FULL PAPER
1
3
p-COPh), 8.03Ϫ8.06 (m, 2 H, o-COPh). Ϫ C78H95N3O19 (1378.6): CHCl3). Ϫ H NMR (250 MHz, CDCl3): δ ϭ 0.72Ϫ0.75 (d, J ϭ
calcd. C 67.96, H 6.95, N 3.05; found C 67.91, H 6.96, N 3.33.
6.9 Hz, 3 H, CH3), 0.78Ϫ1.07 (2 d, m, 9 H, 2 CH3, 3 HMnt),
1.26Ϫ1.77 [s, m, 33 H, C(CH3)3, 20 HCycloh., 4 HMnt], 1.89Ϫ2.05
(m, 2 H, 2 HMnt), 3.52Ϫ4.82 (m, 29 H), 4.93 (bt, 1 H, 1a-H), 5.17
(d, 3J1b,2b ϭ 3.0 Hz, 1 H, 1b-H), 5.29 (s, 1 H, 1c-H), 7.08Ϫ7.34 (m,
25 H, 5 Ph), 7.49Ϫ7.58 (m, 3 H, m-, p-COPh), 8.03Ϫ8.06 (m, 2 H,
o-COPh). Ϫ C88H109NO20 (1500.8): calcd. C 70.43, H 7.32, N 0.9;
found C 70.42, H 7.53, N 1.0
O-(3,4,6-Tri-O-benzyl-␣- -mannopyranosyl)-(1Ǟ4)-O-(2-azido-6-
D
O-benzoyl-3-O-benzyl-2-deoxy-␣- -gluco-pyranosyl)-(1Ǟ6)-
D
2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyloxycarbonyl- -myo-
D
inositol (9): Compound 8 (3.1 g, 2.25 mmol) was dissolved in a
solution of methylamine (33%) in dry ethanol (15 mL) and then
stirred until the reaction was complete (2 h, monitored by TLC).
After evaporation and flash chromatography (petroleum ether/ethyl
acetate, 4:1) compound 9 (2.77 g, 92%) was obtained as a colorless
foam. Ϫ TLC (petroleum ether/ethyl acetate, 7:3): Rf ϭ 0.65. Ϫ
O-[2,3,4,6-Tetra-O-benzyl-␣-
zyl-2-N-(tert-butoxycarbonyl)amino-2-deoxy-␣-
syl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyloxy-
carbonyl- -myo-inositol (12): Compound 11 (0.9 g, 0.60 mmol) was
D-mannopyranosyl)-(1Ǟ4)-O-(3-O-ben-
D
-glucopyrano-
[α]D ϭ ϩ61 (c ϭ 1, CHCl3). Ϫ 1H NMR (250 MHz, CDCl3): δ ϭ
D
3
0.74Ϫ0.77 (d, J ϭ 6.9 Hz, 3 H, CH3), 0.80Ϫ1.08 (2 d, m, 9 H, 2 dissolved in a mixture of dry diethyl ether and methanol (30 mL
CH3, 3 HMnt), 1.26Ϫ1.79 (m, 24 H, 20 HCycloh., 4 HMnt), 1.90Ϫ2.07 1:1); then potassium cyanide (0.45 g) was added and the mixture
3
(m, 2 H, 2 HMnt), 2.19 (d, J2c,OH ϭ 2.9 Hz, 1 H, OH), 3.37Ϫ3.48
was heated to 40°C. After 50% conversion, the reaction was
(m, 2 H, 2b-, 6c-H), 3.56Ϫ3.84 (m, 4 H), 3.92Ϫ4.23 (m, 8 H), stopped (7 h, monitored by TLC) by careful separation of the pot-
4.38Ϫ4.77 (m, 11 H, 3 CH2Ph, 2a-, 3a-, 6b-, 6bЈ-H, HMnt), assium cyanide by filtration through Celite. The solvents were eva-
3
4.92Ϫ4.99 (m, 2 H, 0.5 CH2Ph, 1a-H), 5.34 (d, J1b,2b ϭ 3.5 Hz, 1 porated and flash chromatography [petroleum ether/ethyl acetate,
3
H, 1b-H), 5.39 (d, J1c,2c ϭ 1.4 Hz, 1 H, 1c-H), 7.09Ϫ7.44 (m, 22 (5:1) Ǟ(4:1)] yielded compound 12 (384 mg, 46%). Starting mate-
H, 4 Ph, m-COPh), 7.51Ϫ7.57 (m, 1 H, p-COPh), 8.03Ϫ8.07 (m, 2 rial (360 mg, 40%) was also recovered. TLC (petroleum ether/ethyl
H, o-COPh). Ϫ C76H93N3O18 (1336.6): calcd. C 68.29, H 7.01, N acetate, 4:1): Rf ϭ 0.23. Ϫ [α]D ϭ ϩ17 (c ϭ 1, CHCl3). Ϫ 1H NMR
3.14; found C 67.96, H 7.04, N 3.29.
O-(2,3,4,6-Tetra-O-benzyl-␣- -mannopyranosyl)-(1Ǟ4)-O-(2-
(250 MHz, CDCl3): δ ϭ 0.73Ϫ0.76 (d, 3J ϭ 6.9 Hz, 3 H, CH3),
0.79Ϫ1.13 (2 d, m, 9 H, 2 CH3, 3 HMnt), 1.24Ϫ1.77 [s, m, 33 H,
C(CH3)3, 20 HCycloh., 4 HMnt], 1.89Ϫ2.00 (m, 1 H, HMnt),
2.10Ϫ2.19 (m, 1 H, HMnt), 3.12 (bt, 1 H, OH), 3.50Ϫ4.13 (m, 17
H), 4.40Ϫ4.81 (m, 12 H, 4 CH2Ph, NHCOO, 2a-, 3a-, HMnt), 4.94
D
azido-6-O-benzoyl-3-O-benzyl-2-deoxy-␣- -glucopyranosyl)-(1Ǟ6)-
D
2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-menthyloxycarbonyl- -myo-
D
inositol (10): To a solution of compound 9 (2.2 g, 1.65 mmol) in dry
dimethylformamide (5 mL) was added in portions sodium hydride
(55 mg, 2.4 mmol) at 0°C. After hydrogen evolution stopped benzyl
bromide (0.59 mL, 4.95 mmol) was added and the mixture warmed
to room temperature. If some starting material remained after 24 h
the solution was stirred at 40°C for a few more hours. The excess
of sodium hydride was destroyed with small amounts of ethyl acet-
ate and the reaction mixture was concentrated at 35°C under high
vacuum. Flash chromatography of the residue (petroleum ether/
ethyl acetate, 9:1) gave compound 10 (2.0 g, 85%). Ϫ TLC (petro-
3
(bt, 1 H, 1a-H), 5.11 (d, J1b,2b ϭ 3.3 Hz, 1 H, 1b-H), 5.29 (br. s,
1 H, 1c-H), 7.09Ϫ7.37 (m, 25 H, 5 Ph). Ϫ FAB-MS (positive-mode,
matrix: 3-nitrobenzyl alcohol with NaI): m/z ϭ 1419 [M ϩ Naϩ].
Ϫ C81H105NO19 (1396.72).
O-(2,3,4,6-Tetra-O-benzyl-␣-
azido-3-O-benzyl-2-N-(tert-butoxy-carbonyl)amino-2,6-di-deoxy-␣-
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-
menthyloxycarbonyl- -myo-inositol (13): Zinc azide dipyridine com-
plex[17] (0.39 g, 2.55 mmol) was suspended in a solution of com-
D-mannopyranosyl)-(1Ǟ4)-O-[6-
D
D
leum ether/ethyl acetate, 4:1): Rf ϭ 0.50. Ϫ [α]D ϭ ϩ39 (c ϭ 0.86, pound 12 (0.47 g, 0.34 mmol) and triphenylphosphane (0.89 g,
1
3
CHCl3). Ϫ H NMR (250 MHz, CDCl3): δ ϭ 0.74Ϫ0.77 (d, J ϭ 3.4 mmol) in dry toluene (5 mL). Diisopropyl azodicarboxylate
6.9 Hz, 3 H, CH3), 0.82Ϫ1.08 (2 d, m, 9 H, 2 CH3, 3 HMnt), (DIAD, 0.68 mL, 3.4 mmol) was added dropwise while stirring.
1.34Ϫ1.78 (m, 24 H, 20 HCycloh., 4 HMnt), 1.88Ϫ2.08 (m, 2 H, 2 The mixture was heated to 50°C for 30 min to accelerate the reac-
3
3
HMnt), 3.41 (dd, J1b,2b ϭ 3.7 Hz, J2b,3b ϭ 9.5 Hz, 1 H, 2b-H), tion. The heterogeneous mixture was filtered through Celite and
3.54Ϫ4.78 (m, 26 H), 4.93Ϫ5.00 (m, 2 H, 0.5 CH2Ph, 1a-H), the Celite was washed several times with toluene. The solvent was
5.36Ϫ5.37 (m, 2 H, 1b-, 1c-H), 7.11Ϫ7.49 (m, 27 H, 5 Ph, m- removed under reduced pressure and flash chromatography (petro-
COPh), 7.50Ϫ7.56 (m, 1 H, p-COPh), 8.04Ϫ8.07 (m, 2 H, o-
leum ether/ethyl acetate, 4:1) yielded compound 13 (0.42 g, 87%)
COPh). Ϫ C83H99N3O18 (1426.7): calcd. C 69.88, H 6.99, N 2.95; as a colorless foam. TLC (petroleum ether/ethyl acetate, 4:1): Rf ϭ
found C 69.79, H 7.02, N 3.54.
0.40. Ϫ [α]D ϭ ϩ35 (c ϭ 0.33, CHCl3). Ϫ 1H NMR (250 MHz,
CDCl3): δ ϭ 0.73Ϫ0.76 (d, J ϭ 6.9 Hz, 3 H, CH3), 0.86Ϫ1.13 (2
3
O-(2,3,4,6-Tetra-O-benzyl-␣-
D
-mannopyranosyl)-(1Ǟ4)-O-[-6-
d, m, 9 H, 2 CH3, 3 HMnt), 1.25Ϫ1.75 [s, m, 33 H, C(CH3)3, 20
HCycloh., 4 HMnt], 1.88Ϫ1.98 (m, 1 H, HMnt), 2.06Ϫ2.15 (m, 1 H,
HMnt), 3.45Ϫ4.22 (m, 17 H), 4.40Ϫ4.84 (m, 12 H, 4 CH2Ph,
NHCOO, 2a-, 3a-, HMnt), 4.91 (bt, 1 H, 1a-H), 5.18 (m, 2 H, 1b-
H, 1c-H), 7.09Ϫ7.32 (m, 25 H, 5 Ph). Ϫ FAB-MS (positive-mode,
matrix: 3-nitrobenzyl alcohol with NaI): m/z ϭ 1445 [M ϩ Naϩ].
Ϫ C81H104N4O18 (1421.73).
O-benzoyl-3-O-benzyl-2-N-(tert-butoxycarbonyl)amino-2-deoxy-␣-
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-O-(1R)-
menthyloxycarbonyl- -myo-inositol (11): Compound 10 (1 g,
D
D
0.701 mmol) was dissolved in pyridine (50 mL), water (7 mL) and
propanedithiol (1.5 mL). An alkaline pH was established by ad-
dition of triethylamine and the mixture was stirred until the free
amine had formed (16 h). TLC (petroleum ether/ethyl acetate, 7:3):
Rf ϭ 0.19. The solution was diluted with 150 mL of toluene/etha-
O-(2,3,4,6-Tetra-O-benzyl-␣-D-mannopyranosyl)-(1Ǟ4)-O-[6-
nol (5:1) and concentrated to 25% of its volume at 30°C. This azido-3-O-benzyl-2-N-(tert-butoxycarbonyl)-amino-2,6-di-deoxy-␣-
procedure was repeated three times in order to coevaporate water.
Di-tert-butyldicarbonate (0.76 g, 3.50 mmol) was then added and
the mixture stirred for a few hours at room temperature. The solu-
tion was concentrated and coevaporated with toluene. Flash chro-
matography (petroleum ether Ǟ petroleum ether/ethyl acetate, 7:1)
yielded compound 11 (0.95 g, 90%) as colorless foam. TLC (petro-
D-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-D-myo-
inositol (14): Potassium carbonate (1.0 g) was suspended in a solu-
tion of compound 13 (0.42 g, 0.30 mmol) in dry methanol (25 mL)
and the mixture was heated to 60°C under vigorous stirring. After
the reaction was complete (1Ϫ2 h, TLC monitoring) the solvent
was removed, the residue was mixed with water and ethyl acetate
leum ether/ethyl acetate, 5:1): Rf ϭ 0.32. Ϫ [α]D ϭ ϩ27 (c ϭ 1, and the organic layer separated. The aqueous layer was then
Eur. J. Org. Chem. 1999, 2563Ϫ2571 2567

R. R. Schmidt et al.
FULL PAPER
washed again with ethyl acetate. The combined organic layers were
dried with Na2SO4 and the solvent evaporated. Flash chromatogra-
phy (petroleum ether/ethyl acetate, 4:1) yielded compound 14
diluted with chloroform and washed with half-saturated sodium
hydrogencarbonate solution. The aqueous layer was extracted once
more with chloroform, and the organic layer was dried (Na2SO4)
(0.35 g, 94%). TLC (petroleum ether/ethyl acetate, 4:1): Rf ϭ 0.13. and concentrated. Purification by flash chromatography yielded
1
Ϫ [α]D ϭ ϩ67 (c ϭ 0.5, CHCl3). Ϫ H NMR (250 MHz, CDCl3): compounds 16a,b.
δ ϭ 1.25Ϫ1.78 [s, m, 29 H, C(CH3)3, 20 HCycloh], 2.61 (br. s, 1 H,
O-(2,3,4,6-Tetra-O-benzyl-␣-
azido-3-O-benzyl-2-N-(tert-butoxycarbonyl)amino-2,6-dideoxy-␣-
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-2,3-
bis{(1-oxohexadecyl)-oxy}propyldimethylammonium phosphate]-
D-mannopyranosyl)-(1Ǟ4)-O-[6-
OH), 3.40Ϫ3.60 (m, 4 H), 3.70Ϫ4.12 (m, 12 H), 4.25Ϫ4.84 (m, 13
H, 5 CH2Ph, NHCOO, 2a-, 3a-H), 5.11 (br. s, 1 H, 1b-H), 5.22
(br.s, 1 H, 1c-H), 7.15Ϫ7.34 (m, 25 H, 5 Ph). Ϫ C70H86N4O16
(1239.5): calcd. C 67.83, H 6.99, N 4.52; found C 67.51, H 7.05,
N 4.65.
D
D
-
myo-inositol (16a): Treatment of 15a (400 mg) according to the gen-
eral procedure afforded 16a (495 mg, 79%) after flash chromatog-
raphy (toluene Ǟ toluene/acetone, 9:1 Ǟ toluene/acetone, 5:3 Ǟ
toluene/acetone, 1:2) [TLC (toluene/acetone, 1:1): Rf ϭ 0.10Ϫ0.20].
General Procedure for the Synthesis of Compounds 15a,b: (S)-1,2-
Di-O-acylglycerols (0.70 mmol) and (2-cyanoethoxy)bis(diisoprop-
ylamino)phosphane[18] were dissolved in dry acetonitrile/dichloro-
1
Ϫ [α]D ϭ ϩ25 (c ϭ 0.5, CHCl3). Ϫ H NMR [250 MHz, CDCl3/
methane (15 mL, 2:3). After addition of tetrazole (30 mg, CDOD3 (1:1)]: δ ϭ 0.83Ϫ0.90 (t, 3J ϭ 6.4 Hz, 6 H, 2 CH3),
0.42 mmol) the mixture was stirred for 4 h. The solution was di-
luted with dichloromethane and washed with saturated sodium hy-
drogencarbonate solution, and water. The organic layer was dried
(Na2SO4), the solvents were removed and then fast flash chroma-
tography (petroleum ether/ethyl acetate, 4:1) was performed in or-
der to purify compounds 15a,b.
1.22Ϫ1.75 [m, 84 H, C(CH3)3, 20 HCycloh., 26 CH2, CH3NH3ϩ],
2.17Ϫ2.40 (m, 4 H, 2 COCH2), 3.25Ϫ4.92 (m, 32 H,), 5.21Ϫ5.36
(m, 3 H, 1b-, 1c-, 2 g-H), 7.09Ϫ7.35 (m, 25 H, 5 Ph). Ϫ 31P NMR
[161.7 MHz, CDCl3/CD3OD (1:1)]: δ ϭ Ϫ0.90. Ϫ FAB-MS (posi-
tive-mode, matrix: 3-nitrobenzyl alcohol with NaI): m/z ϭ 1915 [M.
Ϫ Naϩ]Naϩ. Ϫ [C105H152N4O23P]Ϫ (1869.34).
[(R)-2,3-Bis{(1-oxohexadecyl)oxy}propyloxy](2-cyanoethoxy)-
(diisopropylamino)phosphane (15a): Treatment of (S)-glycerine-1,2-
dipalmitate[19] (400 mg) according to the general procedure af-
O-(2,3,4,6-Tetra-O-benzyl-␣-
azido-3-O-benzyl-2-N-(tert-butoxycarbonyl)amino-2,6-di-deoxy-␣-
-gluco-pyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-2,3-
D-mannopyranosyl)-(1Ǟ4)-O-[6-
D
forded 15a (470 mg, 87%) as a waxy solid. TLC (petroleum ether/ bis-{(1-oxotetradecyl)-oxy}propyldimethylammonium phosphate]-
1
ethyl acetate, 4:1): Rf ϭ 0.67. Ϫ [α]D ϭ Ϫ5 (c ϭ 4, CHCl3). Ϫ H
D-myo-inositol (16b): Treatment of 15b (371 mg) according to the
3
NMR (250 MHz, CDCl3): δ ϭ 0.81Ϫ0.86 (t, J ϭ 6.2 Hz, 6 H, 2
general procedure afforded 16b (437 mg, 88%) after flash chroma-
CH3), 1.11Ϫ1.15 [2 d, m, 12 H, 2 CH(CH3)2], 1.17Ϫ1.29 (m, 48 H, tography (toluene/acetone, 9:1 Ǟ toluene/acetone, 1:1 Ǟ toluene/
1
24 CH2), 1.51Ϫ1.61 (m, 4 H, 2 COCH2CH2), 2.22Ϫ2.31 (m, 4 H,
acetone, 1:2) [TLC (toluene/acetone, 1:1): Rf ϭ 0.10Ϫ0.20]. Ϫ H
2 COCH2), 2.56Ϫ2.61 (t, 3J ϭ 6.4 Hz, 2 H, CH2CN), 3.48Ϫ3.85 NMR [250 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 0.82Ϫ0.90 (t, 3J ϭ
[m, 6 H, 2 CH(CH3)2, OCH2, 1-, 1Ј-H], 4.07Ϫ4.17 (m, 1 H, 3 -H), 6.4 Hz, 6 H, 2 CH3), 1.15Ϫ1.75 [m, 76 H, C(CH3)3, 20 HCycloh., 22
4.25Ϫ4.34 (m, 1 H, 1Ј-H), 5.11Ϫ5.20 (m, 1 H, 2-H). Ϫ 31P NMR CH2, CH3NH3ϩ], 2.18Ϫ2.30 (m, 4 H, 2 COCH2), 3.25Ϫ4.92 (m,
[161.7 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 150.05, 150.19. Ϫ FAB-
32 H,), 5.18Ϫ5.37 (m, 3 H, 1b-, 1c-, 2 g-H), 7.08Ϫ7.35 (m, 25 H,
MS (positive-mode, matrix: 3-nitrobenzyl alcohol): m/z ϭ 808 [M 5 Ph). Ϫ FAB-MS (positive-mode, matrix: 3-nitrobenzyl alcohol
ϩ Kϩ]. Ϫ C44H85N2O6P (769.14).
with NaI): m/z ϭ 1859 [M. Ϫ Naϩ]Naϩ. Ϫ [C101H144N4O23P]Ϫ
(1813.23)
[(R)-2,3-Bis{(1-oxotetradecyl)oxy}propyloxy](2-cyanoethoxy)-
(diisopropylamino)phosphane (15b): Treatment of (S)-glycerine-1,2-
dimyristate[19] (359 mg) according to the general procedure af-
General Procedure for the Synthesis of Compounds 5a,b: Pearlman
catalyst [Pd(OH)2 on charcoal, 20 mg] was added to a solution of
forded 15b (261 mg, 52%) as a waxy solid. TLC (petroleum ether/ compound 16a,b (80 µm) in methanol (4 mL), dichloromethane
1
ethyl acetate, 4:1): Rf ϭ 0.67. Ϫ H NMR (250 MHz, CDCl3): δ ϭ (4 mL), and water (0.1 mL), and the mixture was stirred under a
3
0.85Ϫ0.90 (t, J ϭ 6.2 Hz, 6 H, 2 CH3), 1.15Ϫ1.20 [2 d, m, 12 H, hydrogen atmosphere. The reaction was monitored by TLC [5a,b:
2 CH(CH3)2], 1.22Ϫ1.35 (m, 40 H, 20 CH2), 1.55Ϫ1.68 (m, 4 H, 2
(chloroform/methanol, 4:1), Rf ϭ 0.28] at 5 min intervals. If the
COCH2CH2), 2.27Ϫ2.36 (m, 4 H, 2 COCH2), 2.60Ϫ2.67 (t, J ϭ reaction did not start within 20 min more Pearlman catalyst
6.4 Hz, 2 H, CH2CN), 3.53Ϫ3.90 [m, 6 H, 2 CH(CH3)2, OCH2, (20 mg) was added. At the end of the reaction (0.5Ϫ2 h) triethyl-
3
1-, 1Ј-H], 4.11Ϫ4.22 (m, 1 H, 3 -H), 4.29Ϫ4.40 (m, 1 H, 1Ј-H), amine was added until pH ϭ 7 was reached. The mixture was then
5.13Ϫ5.23 (m, 1 H, 2-H). Ϫ 31P NMR (161.7 MHz, CDCl3): δ ϭ
149.90, 150.13. Ϫ FAB-MS (positive-mode, matrix: 3-nitrobenzyl
filtered through Celite. The Celite was washed thoroughly with
chloroform/methanol (3:2). Clean 5a,b was obtained directly after
alcohol with NaI): m/z ϭ 735 [M ϩ Naϩ]. Ϫ C40H77N2O6P evaporation of the solvents. For the following reaction the com-
(713.03).
pound was dissolved in dimethylformamide (4 mL) and triethyl-
amine (0.1 mL).
General Procedure for the Synthesis of Compounds 16a,b: Com-
pound 14 (340 mg, 0.274 mmol), phosphitamides 15a,b O-(␣-
D
-Mannopyranosyl)-(1Ǟ4)-O-[6-amino-2-N-(tert-butoxy-
carbonyl)amino-2,6-di-deoxy-α- -glucopyranosyl]-(1Ǟ6)-2,3:4,5-
di-O-cyclohexylidene-1-[(R)-2,3-bis{(1-oxohexadecyl)oxy}-
-myo-inositol (5a): Treat-
(0.520 mmol) and tetrazole (50 mg, 0.71 mmol) were dried in the
reaction flask under high vacuum for 1 h, then dry dichlorometh-
ane (8 mL) was added and the mixture was stirred for 2 h to obtain propyldimethylammonium phosphate]-
D
D
the phosphite [TLC (petroleum ether/ethyl acetate, 4:1): Rf ϭ 0.38].
After the reaction was complete, tert-butylhydroperoxide (2 mL, 3
 in toluene) was added and oxidation to the phosphate occurred
within 1.5 h [TLC (petroleum ether/ethyl acetate, 4:1): Rf ϭ 0.06].
Subsequently the mixture was concentrated to a volume of 2 mL.
ment of 16a (150 mg) according to the general procedure afforded
5a (81 mg, 89%). TLC (chloroform/methanol, 4:1), Rf ϭ 0.28. Ϫ
1H NMR [250 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 0.83Ϫ0.92 (t, 3J ϭ
6.4 Hz, 6 H, 2 CH3), 1.21Ϫ1.81 [m, 81 H, C(CH3)3, 20 HCycloh., 26
3
CH2], 2.27Ϫ2.38 (m, 4 H, 2 COCH2), 3.07 (dd, J1b,2b ϭ 3.5 Hz,
A solution of dimethylamine (33% in dry ethanol,5 mL) was added 3J2b,3b ϭ 8.0 Hz, 1 H, 2b-H), 3.40Ϫ4.53 (m, 21 H,), 5.19Ϫ5.25 (m,
and stirred for 30 min to cleave the cyanoethoxy group [TLC (tolu-
ene/acetone, 1:1): Rf ϭ 0.45]. The concentrated mixture (1 mL) was [161.7 MHz, CDCl3/CDOD3 (1:1)]: δ ϭ Ϫ1.24. Ϫ FAB-MS [nega-
2 H, 1c-, 2 g-H), 5.35 (d, 3J1b,2b ϭ 3.6 Hz, 1 H, 1b-H). Ϫ 31P NMR
2568
Eur. J. Org. Chem. 1999, 2563Ϫ2571

Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors
FULL PAPER
tive-mode, matrix: 3-nitrobenzyl alcohol/glycerol (1:1)]: m/z ϭ 1392
[MϪ]. Ϫ C70H125N2O23P (1393.73).
ethylammonium phosphate]-
D
-myo-inositol (19a): Treatment of 5a
(81 mg) according to the general procedure afforded 19a (72 mg,
67%) as an amorphous red solid. TLC (ethyl acetate/methanol,
4:1): Rf ϭ 0.08. Ϫ [α]D ϭ ϩ86 (c ϭ 1, CHCl3). Ϫ 1H NMR
O-(␣-
onyl)amino-2,6-di-deoxy-␣-
cyclohexylidene-1-[(R)-2,3-bis{(1-oxotetradecyl)oxy}propyldimethyl-
ammonium phosphate]- -myo-inositol (5b): Treatment of 16b
D-Mannopyranosyl)-(1Ǟ4)-O-[6-amino-2-N-(tert-butoxycarb-
D
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-
3
[600 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 0.87Ϫ0.90 (t, J ϭ 7.1 Hz,
6 H, 2 CH3), 1.24Ϫ1.77 [m, 90 H, C(CH3)3, 20 HCycloh., 26 CH2,
(CH3CH2)3NHϩ], 2.29Ϫ2.33 (m, 4 H, 2 COCH2), 2.70Ϫ2.85 (m, 2
H, CH2NH], 3.14Ϫ3.18 [m, 6 H, (CH3CH2)3NHϩ], 3.48Ϫ4.58 (m,
24 H), 5.24Ϫ5.31 (m, 3 H, 1b-, 1c-, 2 g-H), 6.40 (br. s, 1 H, 5Ј-H),
D
(145 mg) according to the general procedure afforded 5b (96 mg,
89%). TLC (chloroform/methanol, 4:1), Rf ϭ 0.28. Ϫ 1H NMR
3
[250 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 0.84Ϫ0.92 (t, J ϭ 6.4 Hz,
3
8.52 (d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 6Ј-H). Ϫ 31P NMR [161.7 MHz,
6 H, 2 CH3), 1.21Ϫ1.81 [m, 73 H, C(CH3)3, 20 HCycloh., 22 CH2],
2.28Ϫ2.36 (m, 4 H, 2 COCH2), 3.07 (dd, 3J1b,2b ϭ 3.5 Hz, 3J2b,3b ϭ
8.0 Hz, 1 H, 2b-H), 3.40Ϫ4.53 (m, 21 H,), 5.17Ϫ5.30 (m, 2 H,
1c-, 2g-H), 5.37 (d, 3J1b,2b ϭ 3.6 Hz, 1 H, 1b-H). Ϫ FAB-MS [posi-
tive-mode, matrix: 3-nitrobenzyl alcohol/glycerol (1:1)]: m/z ϭ 1338
[Mϩ]. Ϫ C66H117N2O23P (1337.62).
CDCl3/CD3OD (1:1)]: δ ϭ 0.008. Ϫ FAB-MS [negative-mode, ma-
trix: 3-nitrobenzyl alcohol/glycerol (1:1)]: m/z ϭ 1627 [M• Ϫ]. Ϫ
[C79H130N6O27P]Ϫ (1626.89).
O-(␣-
1Ј,3Ј-diazol-4Ј-yl)amino]-1-oxopropyl}amino-2-N-(tert-butoxy-
carbonyl)amino-2,6-di-deoxy-␣- -glucopyranosyl]-(1Ǟ6)-2,3:4,5-
di-O-cyclohexylidene-1-[(R)-2,3-bis{(1-oxotetradecyl)oxy}-
propyltriethylammonium phosphate]- -myo-inositol (19b): Treat-
D-Mannopyranosyl)-(1Ǟ4)-O-[6-{3-[N-(7Ј-nitrobenz-2Ј-oxa-
D
3-[N-(7Ј-Nitrobenz-2Ј-oxa-1Ј,3Ј-diazol-4Ј-yl)amino]propanic
Acid
(17): β-Alanine (0.23 g, 2.5 mmol) and sodium hydrogencarbonate
(0.63 g, 7.5 mmol) were dissolved in water (5 mL). After addition
of a solution of 4-chloro-7-nitro-benzofurazane (0.5 g, 2.5 mmol)
in methanol (20 mL) the mixture was stirred for 1 h at 50°C. The
mixture was then cooled to room temperature and the pH was ad-
justed to 1 by the addition of 2  hydrochloric acid (2Ϫ3 mL).
After evaporation of the solvents, the product was crystallized by
addition of water. The crystals were dissolved in sodium hydrogen-
carbonate solution (0.25 g in 5 mL water) and crystallized once
more by addition of 2  hydrochloric acid (2Ϫ3 mL) to yield pure
compound 17 (0.39 g, 61%) as a brown solid. m.p.: > 199°C, de-
composition. Ϫ TLC (chloroform/methanol, 4:1): Rf ϭ 0.32. Ϫ 1H
D
ment of 5b (78 mg) according to the general procedure afforded
19b (58 mg, 63%) as an amorphous red solid. TLC (ethyl acetate/
1
methanol, 4:1): Rf ϭ 0.08. Ϫ H NMR [250 MHz, CDCl3/CD3OD
3
(1:1)]: δ ϭ 0.87Ϫ0.90 (t, J ϭ 6.6 Hz, 6 H, 2 CH3), 1.23Ϫ1.75 [m,
82 H, C(CH3)3, 20 HCycloh., 22 CH2, (CH3CH2)3NHϩ], 2.29Ϫ2.35
(m, 4 H, 2 COCH2), 2.75Ϫ2.85 (m, 2 H, CH2NH), 3.14Ϫ3.23 [m,
6 H, (CH3CH2)3NHϩ], 3.48Ϫ4.46 (m, 24 H), 5.26Ϫ5.31 (m, 3 H,
3
1b-, 1c-, 2 g-H), 6.38Ϫ6.42 (d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 5Ј-H),
3
8.53Ϫ8.56 (d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 6Ј-H). Ϫ FAB-MS (positive-
mode, matrix: 3-nitrobenzyl alcohol with NaI): m/z ϭ 1594 [M ϩ
Naϩ]. Ϫ C75H123N6O27P (1570.78).
3
NMR [250 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 2.80 (t, J ϭ 6.7 Hz,
3
2 H, COCH2), 3.85 (bt, 2 H, CH2NH), 6.33 (d, J5Ј,6Ј ϭ 8.7 Hz, 1
General Procedure for the Synthesis of Compounds 1a,b: Compound
19a,b (26.0 µmol) was dissolved in a mixture of dichloromethane/
acetonitrile, (4 mL, 1:1) and ethanediol (135 µl). The solution was
acidified to pH ϭ 1 with camphor-10-sulfonic acid and stirred for
4 h under argon. The tert-butoxycarbonyl group was cleaved on
addition of trifluoroacetic acid (1 mL). After 2 h, triethylamine was
added until the pH was alkaline. The mixture was then concen-
trated under high vacuum. Ethanediol was removed by flash chro-
matography (n-butyl alcohol/ethanol/conc. ammonia solution/
water, 30:30:2:2) and pure compound 1a,b was obtained after elu-
tion with n-butyl alcohol/ethanol/conc. ammonia solution/
water ϭ 30:30:5:5.
3
H, 5Ј-H), 8.54 (d, J5Ј,6Ј ϭ 8.7 Hz, 1 H, 6Ј-H). Ϫ EI-MS (positive-
mode, 70 eV, 195°C): m/z ϭ 252 [Mϩ. Ϫ radical]. Ϫ C9H8N4O5
(252.19).
N-[2-{N-(7Ј-Nitrobenz-2Ј-oxa-1Ј,3Ј-diazol-4Ј-yl)amino}ethyl-
carbonyloxy]succinimide (18): Compound 17 (250 mg, 1.0 mmol)
and N-hydroxysuccinimide (138 mg, 1.2 mmol) were dissolved in a
mixture of dry dioxane (5 mL) and dry dichloromethane (5 mL).
After addition of N-(3-dimethylaminopropyl)-NЈ-ethyl-carbodiim-
ide hydrochloride (230 mg, 1.2 mmol) the mixture was stirred for
2 h. The solvents were then removed and the residue was poured
into a half-saturated sodium chloride solution and ethyl acetate.
After separation of the organic layer the aqueous layer was ex-
tracted once more with ethyl acetate. The dried organic layers
(Na2SO4) were concentrated and purified by flash chromatography
(ethyl acetate) to yield 18 (210 mg, 61%) as a red orange powder.
m.p.: > 188°C, decomposition. Ϫ TLC (ethyl acetate): Rf ϭ 0.68.
O-(α-
(7Ј-nitrobenz-2Ј-oxa-1Ј,3Ј-diazol-4Ј-yl)amino]-1-oxopropyl}amino-α-
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-2,3-
bis{(1-oxohexadecyl)oxy}propyl hydrogen phosphate]- -myo-inositol
D-Mannopyranosyl)-(1Ǟ4)-O-[2-amino-2,6-di-deoxy-6-{3-[N-
D
D
1
(1a): Treatment of 19a (45 mg) according to the general procedure
afforded 1a (19 mg, 50%) as an orange solid. TLC (n-butyl alcohol/
ethanol/conc. ammonia/water, 4:4:1:1): Rf ϭ 0.24. Ϫ 1H NMR
[600 MHz, CDCl3/CD3OD/D2O (1:1:0.15)]: δ ϭ 0.87Ϫ0.90 (2 t,
3J ϭ 7.1 Hz, 6 H, 2 CH3), 1.25Ϫ1.35 (m, 48 H, 24 CH2), 1.57Ϫ1.65
(m, 4 H, 2 COCH2CH2), 2.31Ϫ2.37 (2 t, 4 H, 2 COCH2), 2.74 (t,
Ϫ H NMR [250 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 2.79 (s, 4 H, 4
HSucc.), 3.07 (t, 3J ϭ 6.6 Hz, 2 H, COCH2), 3.88 (bt, 2 H, CH2NH),
3
3
6.29 (d, J5Ј,6Ј ϭ 8.7 Hz, 1 H, 5Ј-H), 8.45 (d, J5Ј,6Ј ϭ 8.7 Hz, 1 H,
6Ј-H). Ϫ EI-MS (positive-mode, 70 eV, 220°C): m/z ϭ 349 [Mϩ. Ϫ
radical]. Ϫ C13H11N5O7 (349.26).
General Procedure for the Synthesis of Compounds 19a,b: To a solu-
tion of compound 5a,b (58 µmol) in dimethylformamide (4 mL)
and triethylamine (0.1 mL) was added compound 18 (81 mg, 234
µmol). The solution was stirred for 1 h and then the solvents were
evaporated under high vacuum. The residue was purified by flash
chromatography (ethyl acetate/methanol, 4:1) to yield compound
19a,b.
2 H, 3J ϭ 6.8 Hz, CH2NH), 3.20 (dd, J1b,2b ϭ 3.8 Hz, J2b,3b
ϭ
3
3
3
10.7 Hz, 1 H, 2b-H), 3.24 (dd, Jgem ϭ 13 Hz, J5b,6b ϭ 8.0 Hz, 1
H, 6b-H), 3.36 (dd, 3J4a,5a ϭ 3J5a,6a ϭ 9.3 Hz, 1 H, 5a-H), 3.46 (dd,
3J3a,4a ϭ 9.2 Hz 1 H, 3a-H), 3.48 (dd, J3b,4b
ϭ
3J4b,5b ϭ 9.5 Hz,
3
3
1 H, 4b-H), 3.64 (dd, J3c,4c
ϭ
3J4c,5c ϭ 7.8 Hz, 1 H, 4c-H), 3.66
3
(3J3a,4a ϭ 9.2 Hz, J4a,5a ϭ 9.3 Hz 1 H, 4a-H), 3.67 (3J4c,5c
ϭ
7.8 Hz, 1 H, 5c-H), 3.72 (3J3c,4c ϭ 7.8 Hz, 1 H, 3c-H), 3.74 (Jgem ϭ
11 Hz, 1 H, 6c-H), 3.76 (bd, Jgem ϭ 13 Hz, 1 H, 6bЈ-H), 3.87
O-(α-
1Ј,3Ј-diazol-4Ј-yl)amino]-1-oxopropyl}amino-2-N-(tert-butoxycarb-
onyl)amino-2,6-di-deoxy-␣-
D-Mannopyranosyl)-(1Ǟ4)-O-[6-{3-[N-(7Ј-nitrobenz-2Ј-oxa-
3
3
(Jgem ϭ 11 Hz, 1 H, 6cЈ-H), 3.96 (dd, J1a,6a ϭ 9.5 Hz, J5a,6a
ϭ
D
-glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O- 9.3 Hz 1 H, 6a-H), 3.98 (1 H, 2c-H), 3.99 (3J2b,3b ϭ 10.7 Hz,
cyclohexylidene-1-[(R)-2,3-bis{(1-oxohexadecyl)oxy}propyltri-
3J2b,3b ϭ 9.5 Hz, 1 H, 3b-H), 4.03 (1 H, 1 g-,1gЈ-H), 4.10 (bt, 1 H,
Eur. J. Org. Chem. 1999, 2563Ϫ2571
2569

R. R. Schmidt et al.
FULL PAPER
3
2a-H), 4.19 (1 H, 1a-H), 4.21 (1 H, 3 g-H), 4.29 (m, J4b,5b
ϭ
sulfonic acid and stirred for 4 h under argon. The tert-butoxycar-
bonyl group was cleaved on addition of trifluoroacetic acid
9.5 Hz, 3J5b,6b ϭ 8.0 Hz,1 H, 5b-H), 4.42 (1 H, 3gЈ-H), 5.25 (s, 1 H,
1c-H), 5.28 (1 H, 2 g-H), 5.49 (d, 3J1b,2b ϭ 3.8 Hz, 1 H, 1b-H), 6.42 (0.5 mL). After 2 h, triethylamine was added until the pH was alka-
3
3
(d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 5Ј-H), 8.56 (d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 6Ј- line and the mixture was concentrated under high vacuum. Purifi-
H). Ϫ 31P NMR [161.7 MHz, CDCl3/CD3OD/D2O (1:1:0.15)]: δ ϭ cation by flash chromatography [n-butyl alcohol/ethanol/conc. am-
Ϫ0.95. Ϫ FAB-MS [negative-mode, matrix: 3-nitrobenzyl alcohol/
glycerol (1:1)]: m/z ϭ 1365 [MϪ]. Ϫ [C62H107N6O25P]Ϫ (1367.53).
monia solution/water (30:30:5:5) Ǟ (30:30:8:8)] yielded compound
2a (7.0 mg, 64%) as an amorphous solid. TLC (n-butyl alcohol/
1
ethanol/conc. ammonia solution/water, 2:2:1:0.3): Rf ϭ 0.45. Ϫ H
O-(␣-
D-Mannopyranosyl)-(1Ǟ4)-O-[2-amino-2,6-di-deoxy-6-{3-[N-
NMR [600 MHz, CDCl3/CD3OD (1:1)]: δ ϭ 0.88Ϫ0.90 (t, 3J ϭ
6.9 Hz, 6 H, 2 CH3), 1.23Ϫ1.34 (m, 48 H, 24 CH2), 1.57Ϫ1.64 (m,
4 H, 2 COCH2CH2), 2.30Ϫ2.37 (m, 4 H, 2 COCH2), 3.24 (dd,
(7Ј-nitrobenz-2Ј-oxa-1Ј,3Ј-diazol-4Ј-yl)amino]-1-oxopropyl}amino-␣-
D-
g l u c o p y r a n o s y l ] - ( 1 Ǟ 6 ) - 2 , 3 : 4 , 5 - d i - O - c y c l o h e x y l i -
dene-1-[(R)-2,3-bis{(1-oxotetradecyl)oxy}propyl hydrogen phosphate]-
3
3
3J1b,2b ϭ 3.8 Hz, J2b,3b ϭ 9.9 Hz, 1 H, 2b-H), 3.34 (dd, J4a,5a
ϭ
D-myo-inositol (1b): Treatment of 19b (41 mg) according to the gen-
3J5a,6a ϭ 9.2 Hz, 1 H, 5a-H), 3.44 (dd, J2a,3a ϭ 2.8 Hz, J3a,4a
ϭ
3
3
eral procedure afforded 1b (13 mg, 38%) as an orange solid. TLC
(n-butyl alcohol/ethanol/conc. ammonia solution/water, 4:4:1:1):
Rf ϭ 0.24. Ϫ 1H NMR [250 MHz, CDCl3/CDOD3/D2O (1:1:0.15)]:
9.2 Hz 1 H, 3a-H), 3.58 (1 H, 6b-H), 3.59 (1 H, 4b-H), 3.62
3
3
3
(3J3a,4a ϭ J4a,5a ϭ 9.2 Hz 1 H, 4a-H), 3.66 (dd, J3c,4c ϭ J4c,5c
ϭ
7.8 Hz, 1 H, 4c-H), 3.74 (3J4c,5c ϭ 7.8 Hz, 1 H, 5c-H), 3.74 (1 H,
3
δ ϭ 0.87Ϫ0.90 (2 t, J ϭ 7.1 Hz, 6 H, 2 CH3), 1.25Ϫ1.35 (m, 40
6c-H), 3.77 (3J3c,4c ϭ 7.8 Hz, 1 H, 3c-H), 3.89 (1 H, 6bЈ-H), 3.89
H, 20 CH2), 1.55Ϫ1.70 (m, 4 H, 2 COCH2CH2), 2.30Ϫ2.40 (2 t, 4
H, 2 COCH2), 2.72Ϫ2.77 (t, 2 H, 3J ϭ 6.8 Hz, CH2NH), 3.15Ϫ4.35
(m, 24 H), 5.25Ϫ5.30 (m, 2 H, 1c-, 2-H), 5.48 (d, 1 H 1b-H),
3
3
(1 H, 6cЈ-H), 3.96 (dd, J1a,6a ϭ 9.4 Hz, J5a,6a ϭ 9.2 Hz 1 H, 6a-
H), 4.02 (1 H, 1 g-,1gЈ-H), 4.03 (1 H, 2c-H), 4.04 (3J2b,3b ϭ 9.9 Hz,1
3
3
H, 3b-H), 4.09 (dd, J1a,2a ϭ 2.8 Hz, J2a,3a ϭ 2.8 Hz, 1 H, 2a-H),
3
3
6.42Ϫ6.46 (d, J5Ј,6Ј ϭ 8.8 Hz, 1 H, 5Ј-H), 8.56Ϫ8.60 (d, J5Ј,6Ј
ϭ
4.18 (3J1a,2a ϭ 2.8 Hz, J1a,6a ϭ 9.4 Hz, 1 H, 1a-H), 4.20 (1 H, 3 g-
3
8.8 Hz, 1 H, 6Ј-H). Ϫ FAB-MS [negative-mode, matrix: 3-nitro-
H), 4.40 (1 H, 3gЈ-H), 4.41 (1 H, 5b-H), 5.26 (1 H, 2 g-H), 5.27 (s,
benzyl alcohol/glycerol (1:1)]: m/z
ϭ
1310 [MϪ].
Ϫ
1 H, 1c-H), 5.55 (d, 3J1b,2b ϭ 3.8 Hz, 1 H, 1b-H), 7.28Ϫ7.31 (d, dd,
[C58H99N6O25P]Ϫ (1311.42).
3
3
4J4Ј,6Ј ϭ 2.5 Hz, J3Ј,4Ј ϭ 8.6 Hz, 2 H, 4Ј-,6Ј-H), 8.18 (d, J3Ј,4Ј
ϭ
8.6 Hz, 1 H, 3Ј-H). Ϫ 31P NMR (161.7 MHz, CDCl3/CD3OD/D2O,
1:1:0.15): δ ϭ 0.02. Ϫ FAB-MS [negative-mode, matrix: 3-nitro-
benzyl alcohol/glycerol (1:1)]: m/z
O-(␣-
benzoyl)amino-2-N-(tert-butoxycarbonyl)amino-2,6-di-deoxy-␣-
glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-2,3-bis-
{(1-oxohexadecyl)oxy}propyltriethylammonium phosphate]- -myo-
D-Mannopyranosyl)-(1Ǟ4)-O-[6-N-(5Ј-azido-2Ј-nitro
D
-
ϭ 1322 [M. Ϫ Ϫ
Hϩ].
C60H103N6O24P (1323.47).
D
inositol (21a): To a solution of compound 5a in dimethylformam-
ide/triethylamine (1 mL, 19.8 mg, 14.2 µmol) was added N-(5-az-
ido-2-nitrobenzoyloxy)succinimide[22] (13 mg, 42.6 µmol) and the
mixture stirred for 2 h at room temperature. The solution was then
concentrated under high vacuum. Flash chromatography loroform
Ǟ chloroform/methanol (9:1), 1% triethylamine] yielded the tri-
ethylammonium salt of compound 21a (16 mg, 81%) as an
amorphous solid. TLC (chloroform/methanol, 4:1): Rf ϭ 0.59. Ϫ
[α]D ϭ ϩ22 (c ϭ 1, CHCl3). Ϫ 1H NMR [600 MHz, CDCl3/
CD3OD (1:1)]: δ ϭ 0.87Ϫ0.90 (t, 3J ϭ 7.1 Hz, 6 H, 2 CH3),
O-(␣-
amino]thiocarbonyl}amino-2-N-(tert-butoxycarbonyl)amino-2,6-di-
deoxy-␣- -glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-
[(R)-2,3-bis{(1-oxohexadecyl)oxy}propyltriethylammonium phos-
phate] -myo-inositol (22a): To a solution of 5a in dimethylformam-
D-Mannopyranosyl)-(1Ǟ4)-O-[6-{[N-(4Ј-azidophenyl)-
D
D
ide/triethylamine (0.73 mL, 14.4 mg, 10.3 µmol) was added 4-azido-
phenyl isothiocyanate[23] (4.5 mg, 26 µmol) and the mixture was
stirred for 30 minutes at room temperature. The reaction was
quenched by methylamine and the solvents removed under high
vacuum. Flash chromatography loroform Ǟ chloroform/methanol
(9:1), 1% triethylamine] yielded crude compound 22a as its triethyl-
ammonium salt which was pure enough for the following reaction.
1.25Ϫ1.85 [m, 90 H, C(CH3)3, 20 HCycloh.
,
26 CH2,
(CH3CH2)3NHϩ], 2.30 (t,
4
H, COCH2), 3.13Ϫ3.17 (q,
2
CH3CH2)3NHϩ), 3.49 (dd, 3J4a,5a ϭ 10.3 Hz, 3J5a,6a ϭ 8.6 Hz, 1 H,
5a-H), 3.54 (dd, 3J3b,4b ϭ 3J4b,5b ϭ 9.9 Hz 1 H, 4b-H), 3.59 (3Jgem ϭ
12.7 Hz, 1 H, 6b-H), 3.60 (3J1b,2b ϭ 3.2 Hz, 1 H, 2b-H), 3.62 (1 H,
5c-H), 3.73 (1 H, 3c-H), 3.75 (1 H, 4c-H), 3.75 (1 H, 6c-H), 3.76
(1 H, 3b-H), 3.89 (1 H, 6bЈ-H), 3.94 (1 H, 6cЈ-H), 3.97 (1 H, 5b-
H), 3.99 (1 H, 1 g-H), 3.99 (1 H, 2c-H), 4.07 (1 H, 1gЈ-H), 4.10
TLC (chloroform/methanol, 4:1): Rf
[161.7 MHz, CDCl3/CD3OD (1:1:0.15)]: δ ϭ Ϫ1.20.
ϭ 0.57. Ϫ
31P NMR
O-(␣- -Mannopyranosyl)-(1Ǟ4)-O-[2-amino-6-{[N-(4Ј-azido-
phenyl)amino]thiocarbonyl}amino-2,6-di-deoxy-␣- -gluco-
pyranosyl]-(1Ǟ6)-1-[(R)-2,3-bis{(1-oxohexadecyl)oxy}propyl hydro-
gen phosphate]- -myo-inositol (3a): Compound 22a (14 mg, 8.3
D
D
D
3
(3J3a,4a ϭ 7.3 Hz, J4a,5a ϭ 10.3 Hz 1 H, 4a-H), 4.13 (1 H, 3 g-H),
µmol) was dissolved in a mixture of dichloromethane/acetonitrile,
(2 mL, 1:1) and ethanediol (40 µl). The solution was acidified to
pH ϭ 1 with camphor-10-sulfonic acid and stirred for 4 h under
argon. The tert-butoxycarbonyl group was cleaved on addition of
trifluoroacetic acid (0.5 mL). After 2 h, triethylamine was added
until the pH was alkaline. The mixture was then concentrated un-
der high vacuum. Purification by flash chromatography [n-butyl
3
3
4.24 (m, J1a,2a ϭ 6.3 Hz, J1a,6a ϭ 3.2 Hz, 1 H, 1a-H), 4.28 (dd,
3J1a,6a ϭ 3.2 Hz, J5a,6a ϭ 8.6 Hz 1 H, 6a-H), 4.35 (dd, J2a,3a
ϭ
3
3
3J3a,4a ϭ 7.3 Hz 1 H, 3a-H), 4.47 (1 H, 3gЈ-H), 4.47 (3J1a,2a
ϭ
3
6.3 Hz, J2a,3a ϭ 3.2 Hz, 1 H, 2a-H), 5.22 (1 H, 2 g-H), 5.26 (d,
3J1b,2b ϭ 3.2 Hz, 1 H, 1b-H), 5.32 (s, 1 H, 1c-H), 7.20 (d, J4Ј,6Ј
ϭ
4
4
3
2.6 Hz,1 H, 6Ј-H), 7.20 (dd, J4Ј,6Ј ϭ 2.6 Hz, J3Ј,4Ј ϭ 8.9 Hz, 1 H,
4Ј-H), 8.17 (d, J3Ј,4Ј
ϭ 8.9 Hz, 1 H, 3Ј-H). Ϫ
31P NMR
3
alcohol/ethanol/conc. ammonia solution/water (30:30:2:2)
Ǟ
[161.7 MHz, CDCl3/CD3OD (1:1)]: δ ϭ Ϫ1.11. Ϫ FAB-MS (nega-
tive-mode, matrix: 3-nitrobenzyl alcohol): m/z ϭ 1582 [MϪ]. Ϫ
[C77H126N6O26P]Ϫ (1582.84).
(30:30:7:7)] yielded compound 3a (6.5 mg, 48% for two steps) as
an amorphous solid. The compound is thermolabile and was stored
at 4°C. TLC (n-butyl alcohol/ethanol/conc. ammonia/water,
2:2:1:0.3): Rf ϭ 0.46Ϫ1H NMR [250 MHz, CDCl3/CD3OD/D2O
O-(␣-
nitrobenzoyl)amino-2,6-di-deoxy-␣-
2,3-bis{(1-oxohexadecyl)oxy}propyl hydrogen phosphate]-
D
-Mannopyranosyl)-(1Ǟ4)-O-[2-amino-6-N-(5Ј-azido-2Ј-
-glucopyranosyl]-(1Ǟ6)-1-[(R)-
-myo-
3
D
(1:1:0.15)]: δ ϭ 0.81Ϫ0.88 (t, J ϭ 7.4 Hz, 6 H, 2 CH3), 1.18Ϫ1.31
D
(m, 48 H, 24 CH2), 1.50Ϫ1.62 (m, 4 H, 2 COCH2CH2), 2.24Ϫ2.34
(2 t, 4 H, 2 COCH2), 3.16Ϫ4.5 (m, 22 H), 5.19Ϫ5.27 (m, 2 H,
inositol (2a): Compound 21a (14 mg, 8.3 µmol) was dissolved in a
mixture of dichloromethane/acetonitrile (2 mL, 1:1) and ethanediol
(40 µl). The solution was acidified to pH ϭ 1 with camphor-10-
3
1c-, 2 g-H), 5.49 (d, J1b,2b ϭ 4.0 Hz, 1 H 1b-H), 7.04 (d, 3J ϭ
9.5 Hz, 2 H, 2 HAr), 7.49 (d, 3J ϭ 9.5 Hz, 2 H, 2 HAr). Ϫ 31P NMR
2570
Eur. J. Org. Chem. 1999, 2563Ϫ2571

Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors
[161.7 MHz, CDCl3/CD3OD/D2O (1:1:0.1)]: δ ϭ 0.41. Ϫ FAB-MS 20 min. After preparative TLC [n-butyl alcohol/ethanol/conc. am-
FULL PAPER
[positive-mode, matrix: 3-nitrobenzyl alcohol/glycerol (1:1) mit
NaI]: m/z ϭ 1310 [M ϩ Hϩ], 1332 [M ϩ Naϩ], 1354 [MNa ϩ
Naϩ]. Ϫ C60H105N6O21PS (1309.55).
monia solution/water (2:2:0.5:1.5)] the silica gel containing the product
was scratched off and suspended in chloroform/methanol/water
(1:1:0.15). After the silica gel was removed by centrifugation, the pres-
ence of compound 4a in the solution was determined by FAB-MS.
TLC (n-butyl alcohol/ethanol/conc. ammonia/water, 2:2:0.5:1.5): Rf ϭ
0.58. Ϫ FAB-MS [negative-mode, matrix: 3-nitrobenzyl alcohol/gly-
cerol (1:1)]: m/z ϭ 1392 [M. Ϫ Hϩ. Ϫ N2], 1407 [M. Ϫ Hϩ. Ϫ N],
1420 [M. Ϫ Hϩ]. Ϫ C60H103IN5O23P (1420.37).
O-(␣-
benzoyl)amino)-2-N-(tert-butoxycarbonyl)amino-2,6-dideoxy-
␣- -glucopyranosyl]-(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-
2,3-bis{(1-oxohexadecyl)-oxy}propyltriethylammonium phosphate]-
-myo-inositol (24a): To a solution of compound 5a in dimethylfor-
D-Mannopyranosyl)-(1Ǟ4)-O-[6-(N-(4Ј-azido-2Ј-hydroxy-
D
D
mamide/triethylamine (1 mL, 19.8 mg, 14.2 µmol) was added N-(4-
azido-2-hydroxybenzoyloxy)succinimide (23)[24] (9.8 mg, 36 µmol)
and the mixture stirred for 1.5 h at room temperature. The solution
was then concentrated under high vacuum. Flash chromatography
(ethyl acetate /methanol, 5:1, 1% triethylamine) yielded the triethyl-
ammonium salt of compound 24a (17 mg, 74%) as an amorphous
solid. TLC (chloroform/methanol, 4:1): Rf ϭ 0.19. Ϫ [α]D ϭ ϩ26
(c ϭ 1, CHCl3). Ϫ 1H NMR [600 MHz, CDCl3/CD3OD (1:1)]: δ ϭ
0.87Ϫ0.90 (2 t, 6 H, 2 CH3), 1.20Ϫ1.85 [m, 90 H, C(CH3)3, 20
HCycloh., 26 CH2, (CH3CH2)3NHϩ], 2.29Ϫ2.33 (2 t, 4 H, 2
COCH2), 3.12Ϫ3.16 [q, 3J ϭ 7.3 Hz, 6 H, CH3CH2)3NHϩ],
3.17Ϫ3.22 (m, 1 H, 6b-H), 3.46Ϫ3.55 (m, 2 H, 4b-, 5a-H),
3.60Ϫ3.81 (m, 6 H, 2b-, 3b-, 3c-, 4c-, 5c-, 6c-H), 3.93Ϫ4.04 (1 H,
5b-, 2c-, 6cЈ-H), 4.05Ϫ4.09 (m, 2 H, 1 g-, 1gЈ-H), 4.13Ϫ4.24 (1 H,
4a-, 6bЈ-, 3 g-H), 4.32Ϫ4.45 (m, 5 H, 1a-, 2a-, 3a-, 6a-, 3gЈ-H), 5.24
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinsch-
aft, the Fonds der Chemischen Industrie, and the Human Frontier
Science Program. 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.
[1]
M. A. J. Ferguson, A. F. Williams, Ann. Rev. Biochem. 1998,
57, 285Ϫ320; M. A. J. Ferguson, Curr. Opin. Struct. Biol. 1991,
1, 522Ϫ529; M. J. McConville, M. A. J. Ferguson, Biochem. J.
1993, 294, 30Ϫ204.
[2]
R. Thomas, R. A. Dwek, T. W. Rademacher, Biochemistry 1990,
29, 5413Ϫ5422; G. A. M. Cross, Ann. 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.
(1 H, 2 g-H), 5.35 (s, 1 H, 1c-H), 5.44 (s 1 H, 1b-H), 6.58 (d,
3
3J5Ј,6Ј ϭ 8.6 Hz, 1 H, 5Ј-H), 6.62 (s, 1 H, 3Ј-H), 7.94 (d, J5Ј,6Ј
ϭ
8.5 Hz, 1 H, 6Ј-H). Ϫ 31P NMR [161.7 MHz, CDCl3/CD3OD
(1:1)]: δ ϭ Ϫ1.47. Ϫ FAB-MS [negative-mode, matrix: 3-nitro-
M. P. Lisanti, E. Rodriguez-Boulan, Trends Biochem. Sci. 1990,
[3]
15, 113Ϫ118; K. Simons, A. Wandinger-Ness, Cell 1990, 62,
207Ϫ210.
benzyl alcohol/glycerol (1:1)]: m/z
ϭ
1553 [M•Ϫ].
Ϫ
[4]
K.G. Rothberg, J. E. Heuser, W. C. Donzell, Y.-S. Ying, J. R.
[C77H127N5O25P]Ϫ (1553.84).
Glenney, R. G. W. Anderson, Cell 1992, 68, 673Ϫ682.
[5]
For recent work addressing these questions see: R. Varma, S.
O-(␣-
hydroxybenzoyl)amino-2,6-di-deoxy-␣-
[(R)-2,3-bis{(1-oxohexadecyl)-oxy}propyl hydrogen phosphate]-
D
-Mannopyranosyl)-(1Ǟ4)-O-[2-amino-6-N-(4Ј-azido-2Ј-
Mayor, Nature 1998, 394, 802Ϫ805.
[6]
D
-glucopyranosyl]-(1Ǟ6)-1-
S. Mayor, K. G. Rothberg, F. R. Maxfield, Science 1994, 264,
1948Ϫ1951.
D
-
[7]
V. L. Stevens, Biochem. J. 1995, 310, 361Ϫ370.
myo-inositol (25a): Compound 24a (10 mg, 6.0 µmol) was dissolved
in a mixture of dichloromethane/acetonitrile, (2 mL, 1:1) and
ethanediol (40 µl). The solution was acidified to pH ϭ 1 with cam-
phor-10-sulfonic acid and stirred for 4 h under argon. The tert-
butoxycarbonyl group was cleaved on addition of trifluoroacetic
acid (0.5 mL). After 2 h, triethylamine was added until the pH was
alkaline. The mixture was then concentrated under high vacuum.
Purification by flash chromatography [n-butyl alcohol/ethanol/
conc. ammonia solution/water (30:30:3:3)] yielded compound 25a
(4.1 mg, 53%) as a white solid. TLC (n-butyl alcohol/ethanol/conc.
ammonia/water, 30:30:7:7): Rf ϭ 0.18. Ϫ 1H NMR [250 MHz,
[8]
T. G. Mayer, B. Kratzer, R. R. Schmidt, Angew. Chem. 1994,
106, 2289Ϫ2293; Angew. Chem. Int. Ed. Engl. 1994, 33,
2177Ϫ2181.
[9]
B. Kratzer, T. G. Mayer, R. R. Schmidt, Tetrahedron Lett. 1993,
34, 6881Ϫ6884; Eur. J. Org. Chem. 1998, 291Ϫ298.
[10]
D. Tailler, V. Ferrieres, K. Pekari, R. R. Schmidt, Tetrahedron
Lett. 1999, 40, 679Ϫ682.
[11]
T. G. Mayer, R. R. Schmidt, Eur. J. Org. Chem. 1999,
1153Ϫ1165.
[12]
D. Jeckel, A. Karrenbauer, R. Birk, R. R. Schmidt, F. Wieland,
FEBS Lett. 1990, 261, 155Ϫ157; A. Karrenbauer, D. Jeckel, W.
Just, R. Birk, R. R. Schmidt, J. E. Rothman, F. Wieland, Cell
1990, 63, 259Ϫ267.
[13]
3
M. M. Ponpipom, Carbohydr. Res. 1977, 59, 311Ϫ317.
CDCl3/CD3OD/D2O (1:1:0.15)]: δ ϭ 0.86Ϫ0.91 (t, J ϭ 7.2 Hz, 6
[14]
V. Ferrieres, Universität Konstanz, 1995.
H, 2 CH3), 1.22Ϫ1.36 (m, 48 H, 24 CH2), 1.52Ϫ1.66 (m, 4 H, 2
[15]
U. Greilich, R. Brescello, K.-H. Jung, R. R. Schmidt, Liebigs
3
COCH2CH2), 2.29Ϫ2.38 (2 t, 4 H, 2 COCH2), 3.24 (dd, J1b,2b
ϭ
Ann. 1996, 663Ϫ672; H. Baylay, D. N. Standring, J. R. Knowles,
Tetrahedron Lett. 1978, 3633Ϫ3634.
3
4.0 Hz, J2b,3b ϭ 10.2 Hz, 1 H, 2b-H), 3.28Ϫ4.5 (m, 21 H),
5.20Ϫ5.29 (m, 1 H, 2 g-H), 5.29 (d, J1c,2c ϭ 1.5 Hz, 1 H, 1c-H),
5.55 (d, J1b,2b ϭ 4.0 Hz 1 H, 1b-H), ), 6.58 (d, J3Ј,5Ј ϭ 2.2 Hz,
[16]
3
B. M. Pope, Y. Yamamoto, D. S. Tarbell, Org. Synth. Coll. Vol.
VI 1988, 418Ϫ421; and ref. therein.
3
4
[17]
M. Viaud, P. Rollin, Synthesis 1990, 130Ϫ133.
4
3
1 H, 3Ј-H), 6.66 (dd, J3Ј,5Ј ϭ 2.2 Hz, J5Ј,6Ј ϭ 8.6 Hz, 1 H, 5Ј-H),
[18]
W. Bannwarth, A. Trzeciak, Helv. Chim. Acta 1987, 70,
7.87 (d, J5Ј,6Ј ϭ 8.6 Hz, 1 H, 6Ј-H). Ϫ 31P NMR [161.7 MHz,
3
175Ϫ183.
[19]
R. J. How, T. Malkin, J. Chem. Soc. 1951, 2663Ϫ2667.
CDCl3/CD3OD/D2O (1:1:0.15)]: δ ϭ 0.37. Ϫ FAB-MS [negative-
mode, matrix: 3-nitrobenzyl alcohol/glycerol (1:1)]: m/z ϭ 1292 [M.
Ϫ Hϩ]. Ϫ C60H104N5O23P (1294.48)
[20]
T. Stauch, U. Greilich, R. R. Schmidt, Liebigs Ann. 1995,
2101Ϫ2111.
[21]
L. Johnson, S. Lagerkrist, P. Lindroth, M. Ahnoff, K. Mar-
tinsson, Anal. Chem. 1982, 54, 939Ϫ942.
O-(␣-
hydroxy-3Ј-iodobenzoyl)amino-2,6-di-deoxy-␣-
(1Ǟ6)-2,3:4,5-di-O-cyclohexylidene-1-[(R)-2,3-bis{(1-
D
-Mannopyranosyl)-(1Ǟ4)-O-[2-amino-6-N-(4Ј-azido-2Ј-
[22]
R. V. Lewis, M. F. Roberts, E. A. Dennis, W. S. Allison, Bio-
D
-glucopyranosyl]-
chemistry 1977, 16, 5650Ϫ5654.
[23]
H. Sigrist, P. Zahler, FEBS Lett. 1980, 113, 307Ϫ311.
[24]
J. R. Knowles, Acc. Chem. Res. 1972, 5, 155Ϫ160.
oxohexadecyl)-oxy}propyl hydrogen phosphate]-D-myo-inositol (4a):
[25]
Commercially available from Pierce Co.
IODO-BEAD[25] was added to a solution of compound 25a (0.2 mg)
[26]
T. V. Kurzchalia, private communication.
2Ϫ
in chloroform/methanol/0.1  sodium iodode in H2PO4Ϫ/HPO4
-
Received May 10, 1999
[O99262]
buffer in an Eppendorf cup and the mixture was allowed to stand for
Eur. J. Org. Chem. 1999, 2563Ϫ2571
2571