A lysoganglioside/poly-L-glutamic acid conjugate as a picomolar inhibitor of influenza hemagglutinin

Article abstract of DOI:10.1002/(SICI)1521-3773(19980619)37:11<1524::AID-ANIE1524>3.0.CO;2-D

Based on the principle of a multivalent interaction, the amphiphilic polymer 1, present in solution as an aggregate (see below right), is able to inhibit infection with the influenza virus. After recognition of a specific sialyllactose epitope through hemagglutinin (HA) on the virus surface, the sphingosine residues and the fluorescent tag form a stable complex with HA through hydrophobic interactions. Polymer 1 shows in vitro inhibitory activity 10⁶-fold greater than that of sialyllactose. PGA = polyglutamic acid.

Full text of DOI:10.1002/(SICI)1521-3773(19980619)37:11<1524::AID-ANIE1524>3.0.CO;2-D

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sites lead to similar results. The HOMO region corresponds to
lone pairs of the oxygen atoms bonded to the zeolite lattice.
The LUMO is located on the Ag ion and corresponds to the
Keywords: silver ´ UV/Vis spectroscopy ´ zeolites
‡
[1] D. W. Breck, Zeolite Molecular Sieves, Wiley, New York, 1974.
empty 5s orbital. On the six-ring, two transitions from the lone
Â
Ê
[2] M. Ralek, P. Jíru, O. Grubner, H. Beyer, Collect. Czech. Chem.
Commun. 1962, 27, 142.
‡
‡
pairs of two oxygen atoms to the Ag 5s orbital [Ag (5s)
O(n)] are found (340, 380 nm). The third band at 405 nm is a
transition from the oxygen atoms corresponding to the
[3] T. Sun, K. Seff, Chem. Rev. 1994, 94, 857, and references therein.
[4] a) L. R. Gellens, W. J. Mortier, R. A. Schoonheydt, J. B. Uytterhoeven,
J. Phys. Chem. 1981, 85, 2783; b) J. Texter, R. Kellerman, T.
Gonsiorowski, ibid. 1986, 90, 2118; c) H. G. Karge, Stud. Surf. Sci.
Cat. 1982, 12, 103.
[5] a) G. Calzaferri, R. Rytz, J. Phys. Chem. 1995, 99, 12141; b) G.
Calzaferri, R. Rytz, M. Brändle, ICON-EDiT, Extended Hückel
Molecular Orbital Calculations; available under http://iacrs1.unibe.ch
(130.92.11.3), 1997; c) A. Kunzmann, Dissertation, Universität Bern,
1996.
‡
connections to the neighboring a cage. If the Ag ion is
‡
coordinated by water only, the Ag (5s) O(n) transition is
found below 250 nm. The first ionization energy of water is
12.6 eV, which means that the HOMO lies at 12.6 eV. For
silicates, however, it is found at 10.7 eV, which explains the
long-wavelength situation of the LMCT.[6] The coordination
‡
of only one water molecule to a Ag ion which is coordinated
[6] a) G. Calzaferri, R. Hoffmann, J. Chem. Soc. Dalton Trans. 1991, 917;
b) D. L. Griscom, J. Non-Cryst. Solids 1977, 24, 155.
to zeolite oxygen atoms leads to a destabilization or a raising
‡
of the Ag (5s) level, and therefore to a blue shift of the LMCT
Â
[7] P. Laine, R. Seifert, R. Giovanoli, G. Calzaferri, New J. Chem. 1997, 21,
453.
transitions.
[8] a) J. W. Li, K. Pfanner, G. Calzaferri, J. Phys. Chem. 1995, 99, 2119;
b) H. S. Sherry, H. F. Walton, ibid. 1967, 71, 1457.
We were able to show that activation at room temperature
of silver-containing zeolite A with very low degrees of
exchange already leads to a yellow coloring, and that the
absorption of light and therefore the impression of yellow
coloring are reversible with respect to the desorption/adsorp-
tion of water. The dependency of the absorption bands on the
degree of exchange can be explained with a simple model of
the cation distribution. Quantum-chemical calculations show
that electronic transitions from the lattice oxygen atom to the
silver ion are allowed, that these transitions are in the visible
range, and that they experience a blue shift if coordinated
with water. Hence, an observation first described 35 years ago
has been quantified and explained in a noncontradictory way.
A Lysoganglioside/Poly-l-glutamic Acid
Conjugate as a Picomolar Inhibitor of Influenza
Hemagglutinin**
Hiroshi Kamitakahara, Takashi Suzuki, Noriko
Nishigori, Yasuo Suzuki, Osamu Kanie,* and
Chi-Huey Wong*
Experimental Section
Chemically pure zeolite was produced and characterized by a known
procedure.[7] For the examinations, 80 mg of zeolite were freshly exchanged
each time with 0.1m AgNO3 (Merck, Titrisol). At degrees of exchange up to
Cell surfaces are often covered with oligosaccharides that
play important roles in many cellular recognition events,
including infection, cancer metastasis, and other intercellular
adhesion processes.[1] Hemagglutinins (HAs) of influenza
virus are typical examples of such receptor molecules. These
trimeric proteins are capable of binding to sialylated oligo-
saccharides on the epithelial cell surface.[2] The minimum
‡
six Ag ions, the zeolite was placed in 5 mL of H2O, and a calculated
amount of AgNO3 was added and suspended for 15 min. Quantitative
‡
uptake of the Ag ions can be assumed.[8] The zeolite was washed with
water twice. The zeolite stored at 92% humidity was assumed to have
27 H2O molecules per formula unit.[1] To obtain samples fully exchanged
‡
with Ag ions, the zeolite was suspended twice in 20 mL of AgNO3 solution
for 15 min and washed three times with 15 mL of H2O. The exchanged
zeolite was put into 2 mL of H2O and transferred to a quartz ampoule
(cylinder with a height of 2 cm and a radius of 0.75 cm) which was
connected to a HV-Flansch Adapter in a gas-tight way. The ampoule was
connected to the pump in a horizontal position so that the zeolite could be
deposited evenly at the bottom of the ampoule. The extra water was
vaporized at 10 mbar (2 ± 3 h). For activation, the turbo pump (Alcatel) was
connected until the final pressure of the apparatus was achieved (48 ± 72 h,
1 ± 2 Â 10 7 mbar). After activation at room temperature, temperature-
treated samples were gradually brought to the desired temperature
( Æ 58C) with an adjustable heat pistol. The heating rate was chosen such
that the pressure within the apparatus did not exceed 5 Â 10 6 mbar, to
avoid irreversible brown color changes. The ampoule with the sample
sufficiently adhered to the bottom was separated from the pump with a
burner under gas-tight conditions and transferred to the spectrometer. UV/
Vis spectra were measured as diffuse reflection spectra at room temper-
ature with a Perkin Elmer Lambda 14 spectrometer with an integration
sphere (Labshere RSA-PE-20). Before they were graphically represented,
the automatically collected data were converted using the Kubelka ± Munk
formula.
[*] Dr. O. Kanie, Prof. Dr. C.-H. Wong,[‡] Dr. H. Kamitakahara
Frontier Research Program
The Institute of Physical and Chemical Research (RIKEN)
2-1 Hirosawa, Wako-shi, Saitama 351-01 (Japan)
Fax : (‡ 81)48-467-3620
E-mail: kokanee@postman.riken.go.jp
Dr. T. Suzuki, N. Nishigori, Prof. Dr. Y. Suzuki
Department of Biochemistry, School of Pharmaceutical Science
University of Shizuoka (Japan)
‡
[ ] Permanent address:
Department of Chemistry
The Scripps Research Institute
10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax : (‡ 1)619-784-2409
E-mail: wong@scripps.edu
[**] We thank the NMR laboratory staff and Dr. Sadamu Kurono
1
(RIKEN) for the H NMR spectroscopic and mass spectral analyses.
We are grateful to Dr. Yoshitaka Nagai and Dr. Tomoya Ogawa
(RIKEN) for their continued support and encouragement. This
research was supported in part by the Science and Technology Agency
of the Japanese Government.
Received: December 10, 1997 [Z11249IE]
German version: Angew. Chem. 1998, 110, 1604 ± 1606
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structure involved in this recognition is either a-Neu5Ac-
(2 !3)-b-Gal or a-Neu5Ac-(2 !6)-b-Gal as the nonreducing
terminal saccharide, depending on the strain of the virus.[3]
The HAs are also known to help trigger fusion between the
viral membrane and a lysosomal-lipid bilayer through changes
in their conformations.[4] Thus, inhibition of either or both of
these processes could prevent viral infection. We report here
the synthesis of polyvalent inhibitors that contain a hydro-
phobic moiety designed to disturb the fusion mechanism
initiated by the protein ± carbohydrate recognition process.
One problem associated with carbohydrate ± receptor in-
teractions is their relatively low binding affinity. A possible
approach to increase the affinity is to use multivalent ligands
with specific monomeric carbohydrate ligands.[5] For this
reason, functionalyzed polymers,[5d, 6, 7] peptides,[8] dendri-
mers,[9] or liposomes that contain sugars[10] have recently
attracted interest, and some have been shown to exhibit
higher affinities to receptors.[5] Various types of polymers that
incorporate sialic acid residues have also been synthesized in
an attempt to inhibit the influenza virus.[5d, 7] Because this
virus has both HA and neuraminidase (NA) on its surface,
polymers with sialic acid residues bound through noncleav-
able linkages have emerged as stable and potent inhibitors of
HA. Alternatively, introduction of a second functionality to
provide additional affinity for the target or nearby molecules
might increase the affinity. Hydrophobic groups, for example,
could be used to enhance binding affinity by their interaction
with hydrophobic amino acid side chains and/or the mem-
brane; the latter interaction might in turn interfere with the
conformational change of the membrane-fusion domain of
HA even after the first inhibition process has had its effect.
An infection by influenza virus could thus be inhibited at two
different stages: first during initial binding, and later in the
course of HA-mediated membrane fusion.
To test the hypothesis that hydrophobic groups in a polymer
might contribute to enhanced binding and disruption of the
virus, we chose the molecule lysoganglioside GM3 (lyso-GM3),
a-Neu5Ac-(2 !3)-b-Gal-(1 !4)-b-Glc-(1 !)-sphingosine,[11]
which differs from the ganglioside GM3 by the absence of the
stearic acid residue. This was then conjugated with the
polymer poly-l-glutamic acid (PGA). The reason for our
choice of lyso-GM3 is that GM3 is the simplest ganglioside
recognized by HA[2]. The amino functionality of the sphingo-
sine is suitable for connection to the polymer. The amphiphilic
nature of sphingosine, which is directly attached to the
oligosaccharide moiety, could help present the epitope unit to
the surface of the synthetic polymer by the formation of either
intra- (folded) or intermolecular aggregates (Figure 1). PGA
(degree of polymerization ˆ 540) was selected because of its
low toxicity, low immunogenicity, and biodegradability.[12]
Furthermore, its length is sufficient to meet the criteria for
the ªchain lockº strategy to be effective, as described above.
Another advantage of this strategy is that, because we chose
the natural form of the oligosaccharide as ligand, galactosyl
residues exposed on the virus surface after cleavage of sialic
acids by NA may lead to galactose-mediated endocytosis. We
describe here the synthesis of a lyso-GM3/poly-l-glutamic
acid conjugate and its inhibitory effect on HAs that bind the
a-sialyl-(2 !3)-galactosyl residue.
Figure 1. Schematic representation of lyso-GM3-PGA, which exists in a
stretched form in methanol and in either folded or aggregated forms in
aqueous media.
The synthesis (Scheme 1) begins with derivatization of l-
lysine, which was used as a spacer between lyso-GM3 and the
polymer backbone and as the trifunctional linker for incor-
poration of a fluorescent dye. The latter was expected to
facilitate subsequent quantification and visualization of the
disruption process if the polymer± virus complexes were
indeed taken up by macrophages. The copper chelate of l-
lysine was used to protect the e-amino functionality with a 9-
fluorenylmethyloxylcarbonyl (Fmoc) group under basic con-
ditions (58.2%). The fluorescent tag, a 4,4-difluoro-5,7-
dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl (BODY-
PYFLC3) group,[13] was then attached to the a-amino
functionality (87%). Compound 3 was next transformed into
active ester 4, which without further purification was coupled
with lyso-GM3 (5) to provide condensate 6[14] (quantitative
yield based on thin-layer chromatography with fluorescence
detection). The Fmoc group was removed under standard
conditions, although it was found that prolongation of the
reaction resulted in a poor yield because of decomposition of
the fluorescent tag. The coupling reaction between 7 and poly-
l-glutamic acid (PGA) activated as its succinimidyl ester (8)
proceeded smoothly to give, after treatment with sodium
bicarbonate and purification by gel-permeation chromatog-
raphy (GPC) over a Superose-12-pg column (Pharmacia
Biotech), a polymer that contained lyso-GM3 (9).[15] The lyso-
GM3 (or sialic acid) content was calculated on the basis of
absorption at 508 nm (BODIPY; in MeOH) and was deter-
mined to have an average value of 44.6 nmol per mg of
polymer (0.72 mol%).
Synthetic polymer 9 was unique in the sense that the green
luminescence observed in MeOH disappeared in water,
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in fluorescence (lem ˆ 512,
lex ˆ 480 nm) and a slight shift
in the absorption maximum
(lmax 504.6 and 507.8 nm in
MeOH and H2O, respective-
ly; Figure 3). Neither a shift in
emission wavelength nor ad-
ditional peaks were found to
arise from excimer interac-
tions. The BODIPY fluoro-
phore is known to be insensi-
tive to the pH value and
largely independent of its en-
vironment; however, it can
form aggregates, which results
in self-quenching in aqueous
media. The observed self-
quenching is not a result of a
high concentration of fluoro-
phore incorporated into the
polymer, because quenching
did not occur for the free
fluorophore under the same
conditions. The lyso-GM3
content was also consistent
with the quantities of materi-
als used in the condensation
of lyso-GM3-Lys(BODIPY)-
NH2 (7) with activated PGA
(8). We therefore conclude
that the synthetic polymer 9
exists in a stretched form in
methanol and as a folded or
aggregated form in aqueous
media.
We investigated the inhib-
itory activity of the polymer
toward influenza A/PR/8/34
(H1N1) with an enzyme-
linked immunosorbent assay
(ELISA).[16] The IC50 value
for 9 was 1.9 Â 10 12 m based
on the lyso-GM3 content (or
7.5 Â 10 12 m based on the sial-
ic acid content; estimated
from the UV absorption of
the fluorophore). For compar-
ison, the IC50 values for GM3,
lyso-GM3 (5), and sialyllac-
tose (the trisaccharide portion of GM3) were also determined
(1 Â 10 , 3 Â 10 , and 1.5 Â 10 7 m, respectively). Sialyl-PGA
was much less active than 9. PGA showed no inhibition up to a
concentration of 1 Â 10 9 m; compound 9 was also assayed at
this concentration (Table 1).
We have thus developed a new strategy for the inhibition of
influenza virus, and the process itself can now be studied with
the aid of an incorporated fluorophore. Experimental evi-
dence was obtained to support the ªchain lockº mechanism
(Figure 4). Enhancement in the fluorescence emission of
Scheme 1. a) 1. CuCO3Cu(OH)2 ´ H2O, H2O; 2. Fmoc-O-Su, 10% Na2CO3, 1,2-dimethoxyethane, 58.2%;
b) BODIPYFLC3-O-Su, 10% Na2CO3, MeOH, 87%; c) HO-Su, 1,3-diisopropylcarbodiimide, CH2Cl2;
d) Et3N, MeOH/DMF, quantitative; e) piperidine, DMF, 34%; f) DMF/MeOH. Su ˆ succinimidyl; m ˆ 270,
n:m ꢀ 1.5:100.
whereupon the solution acquired a faint orange color, even
though BODYPYFLC3 itself displays a green luminescence
in both of these media as well as in NaOAc solution (c ꢀ
10 nmolmL 1; Figure 2). Quenching of the fluorescence and
the observed shift in the absorption maximum were attributed
to folding or aggregation in water caused by the hydro-
phobicity of sphingosine and the BODIPY fluorophore. The
lyso-GM3 content of the polymer may cause the fluorophore
in the synthetic polymer to be highly localized and thus able to
interact in the ground state, evidenced by an 83.3% decrease
9
9
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Figure 4. Schematic representation of the ªchain lockº inhibition strategy.
A) The aggregated polymeric inhibitor with a specific ligand, in this case
sialyllactose, approaches the receptor, here hemagglutinin (HA). B) The
aggregated polymer interacts with the receptor, whereby the fluorescence
intensity increases. Emission from the exposed BODIPY fluorophore is
greater than the energy loss caused by energy transfer to a Trp residue at
the binding site. C) Hydrophobic interaction occurs to cause formation of a
stable complex. The hydrophobic groups in HA, including the aromatic
residues, lead to a further decrease in the fluorescence emission of
BODIPY as a result of energy transfer. The bar graphs (bottom) indicate
the relative fluorescence intensity (lem ˆ 510, lex ˆ 480 nm) of the assay
medium. The emission intensity of the mixture at t ˆ 0 min was set to be
100%. See Figure 1 for an explanation of the symbols. LD ˆ lipid double
layer of the virus.
Figure 2. Solutions of lyso-GM3-PGA (9) and BODIPY fluorophore in
different media: BODIPYFLC3 in 1) NaOAc, 2) water, and 3) MeOH, as
well as solutions of lyso-GM3-PGA in 4) MeOH and 5) water, all at 268C.
The upper photograph (A) was taken with ambient light; for the lower
photograph (B) the solutions were irradiated with UV light (365 nm).
quenching in a folded state. The emission intensity then
decreased upon further incubation at 108C, perhaps explained
by energy transfer to the aromatic amino acid residues of HA.
When the tryptophane residue was irradiated (lex ˆ 287 nm),
emission from BODIPY (lem 510 nm) was also observed,
indicative of energy transfer between BODIPY and the Trp
residues.[17] The tight binding may be a consequence of initial
multivalent interactions between 9 and the virus, followed by
a hydrophobic interaction between the ligand and the
receptor. Alternatively, the hydrophobic interaction may
occur first. Both GM3 and lyso-GM3 might form liposomes
and micelles and exhibit a multivalent effect, but not as
effectively as 9.
Figure 3. A) UV/Vis (0.1 mgmL 1) and B) fluorescence emission spectra
(80 mgmL 1, lex ˆ 480 nm) of solutions of lyso-GM3-PGA (9) in 1) MeOH,
2) 75% MeOH, 3) 50% MeOH, 4) 25% MeOH, and 5) water. A ˆ ab-
sorbance, Ir ˆ relative fluorescence intensity.
Received: December 12, 1997 [Z11259IE]
German version: Angew. Chem. 1998, 110, 1607 ± 1611
Table 1. Inhibitory activity of synthetic polymer 9 and related compounds
with respect to the influenza virus A/PR/8/34(H1N1).
Keywords: amphiphiles ´ gangliosides ´ molecular recogni-
tion ´ receptors ´ sialic acids
Compound
IC50 [m][a]
Compound
IC50 [m][a]
12
9
9
9
1.9 Â 10
7.5 Â 10
1.0 Â 10
lyso-GM3 5
sialyllactose
PGA
3 Â 10
12[b]
9
7
1.5 Â 10
[c]
GM3
±
[1] A. Varki, Glycobiology 1993, 3, 97 ± 130.
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b) D. C. Wiley, I. A. Wilson, J. J. Skehel, ibid. 1981, 289, 373 ± 378.
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Guiyun, T. Suzuki, T. Kobayashi, Y. Kimura, A. Yamada, K.
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A. Hasegawa, ibid. 1992, 189, 121 ± 131.
[a] Relative to the sialyllactose content. [b] Relative to the sialic acid
content. [c] No inhibition.
BODIPY (lem 510, lex 480 nm) was observed immediately
after the addition of polymer 9 to a virus solution, which
suggests an unfolding of the polymer after an initial self-
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‡
‡
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[M2Na H] : 1362, found: 1362; calcd for [M‡Na] : 1340, found:
1340; negative ions: calcd for [M H] : 1316, found 1316.
[15] The average molecular weight (Mr) was estimated to be 87 kDa based
Å
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on the lyso-GM3 content (0.72%; m ˆ 536.1, n ˆ 3.9) and the degree of
Å
polymerization (DP ˆ 540) of PGA. Mr of 9 estimated by GPC to be
336 kDa (Waters Ultrahydrogel Liner column (7.8 Â 300 mm), elution
with 0.1m NaNO3, flow rate 1 mLmin 1, UV detection at 500 nm and
408C). Millenium 2010 with GPC software was used to analyze the
data. Polyethylene oxides were employed as standards and detected
by refractive index. The GPC result is apparently erroneous with
Å
respect to the number of introduced lyso-GM3 groups(Mr(GPC)/
Å
Mr(UV) ˆ 3.86), perhaps as a result of aggregation.
[16] The inhibitory activity of polymer 9 toward influenza virus A/PR/8/34
(H1N1) was determined with an ELISA. Thus, an ethanol solution
with 1 nmol of GM3 was added to each well of 96-microwell plates.
The solvent was evaporated at 378C, and the remaining binding sites
on the wells were blocked for 12 h at 48C with 200 mL of phosphate-
buffered saline (PBS; 0.15m NaCl, 8.1mm Na2HPO4, 1.5mm NaH2-
PO4) that contained 2% bovine serum albumin (BSA). The wells were
then washed five times with PBS. A series of dilutions, each by a factor
of two, was prepared from a solution of the polymer (200 pmol) in
0.2% BSA-PBS. These dilute solutions were preincubated at 48C for
2 h with 50 mL of the influenza virus suspension (32 hemagglutinin
units (HAU) and then introduced into the wells. The plates were
incubated at 48C for 12 h, washed five times with PBS, and then
incubated for 2 h at 48C with a 1000-fold diluted 0.2% BSA-PBS
solution of 50 mL of anti-influenza virus antibodies and treated at 48C
for 2 h with horse radish peroxidase (HRP)-conjugated protein A
diluted 1000-fold with solution A. The virions bound to GM3 and
immobilized in the wells were detected with o-phenylenediamine
(OPD) solution that contained 4 mg of OPD and 0.01% H2O2 in
100mm of phosphate buffer (adjusted to pH 5.0 with citric acid). The
reactions were terminated by addition of 4n H2SO4, and viral binding
activities in the form of color development were determined at 492 nm
(reference wavelength 630 nm). Serial dilutions (dilution factor two)
of sialyl lactose (2 mmol), PGA (800 pmol), and lyso-GM3 (20 nmol)
were tested as controls in the manner described.
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[17] The HA trimer of H1 serotype influenza virus contains a total of
15 Trp residues.
Assembly of DNA/Fullerene Hybrid
Materials**
Alan M. Cassell, Walter A. Scrivens, and
James M. Tour*
There are biochemical approaches to molecular structures
of precisely defined dimensions ranging from 1 nm to 10 cm in
length. Conversely, the synthesis of precisely defined unnatu-
ral molecular architectures beyond 25 nm in length is often
unattainable due to solubility, material through-put, and
characterization constraints.[1] Therefore, as nanotechnolog-
ical needs advance, syntheses could rely upon self-assembling
strategies using natural scaffolds as templates for the con-
struction of synthetic nanostructures.[2] DNA is particularly
[11] A. Hasegawa, N. Suzuki, F. Kozawa, H. Ishida, M. Kiso, J. Carbohydr.
Chem. 1996, 15, 639 ± 648.
[12] H. Hirabayashi, M. Nishikawa, Y. Takakura, M. Hashida, Pharm. Res.
1996, 13, 880 ± 884.
[13] R. P. Haugland in Molecular Probes (Ed.: M. T. Z. Spence), Molecular
Probes, Eugene, OR, USA, 1992.
[14] Rf ˆ 0.27 (CHCl3/CH3OH/15mm CaCl2 (60/35/8, v/v/v), 1H NMR
(600 MHz, CD3OD): d ˆ 7.34 (s, 1H, BODIPY), 6.93 (d, 1H,
BODIPY), 6.26 (d, 1H, BODIPY), 6.13 (s, 1H, BODIPY), 3.13 (m,
2H, BODIPY-CH2), 2.62 (t, 2H, BODIPY-CH2), 2.42 (s, 3H,
BODIPY-CH3), 2.19 (s, 3H, BODIPY-CH3), 4.2 ± 4.3 (1H, Ca-H),
3.03 (m, 2H, Ce-H), 1.5 ± 1.65 (m, 4H, Cb-H, Cg-H), 1.69 (m, 2H, Cd-
H), 4.21 (d, 1H, J ˆ 8.1 Hz, Glc-H1), 4.32 (d, 1H, J ˆ 8.1 Hz, Gal-H1),
3.93 ± 3.95 (1H, Gal-H3), 3.80 (br. s, 1H, Gal-H4), 2.81 (dd, 1H,
Neu5Ac-H3eq.), 1.54 (1H, Neu5Ac-H3ax.), 1.91 (3H, Neu5Ac-
NHCOCH3), 5.32 ± 5.38 (m, 1H, sphingosine, olefinic H4), 5.56 ±
5.59 (m, 1H, sphingosine, olefinic H5), 1.19 (16H, sphingosine-
CH2), 0.80 (sphingosine-CH3); ESI-MS positive ions: calcd for
[*] Prof. J. M. Tour, Dr. A. M. Cassell, Dr. W. A. Scrivens
Department of Chemistry and Biochemistry
University of South Carolina
Columbia, SC 29208 (USA)
Fax: (‡1)803-777-9521
E-mail: tour@psc.sc.edu
[**] This research work was supported by the Defense Advanced
Research Projects Agency and the Office of Naval Research.
1528
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
1433-7851/98/3711-1528 $ 17.50+.50/0
Angew. Chem. Int. Ed. 1998, 37, No. 11